RU2816871C2 - CONTROLLED EXPRESSION OF TRANSGENES USING CLOSED-END DNA VECTORS (ceDNA) - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: present invention relates to use of genetic constructs containing DNA with closed ends (ceDNA vectors) for expression control. Invention discloses a method for maintaining, stabilizing or increasing the level of expression of a target transgene in a cell or in a subject due to at least one sequential introduction of an additional dose of the ceDNA vector after the initial introduction.

EFFECT: ceDNA vector as disclosed in the present invention can be re-introduced ("repeated dose" or "booster" introduction) to continue expression of the target protein in a subject, wherein the second dose increases the level of expression of the target protein or prolongs it, wherein such repeated administration does not cause an immune response sufficient to prevent achieving expression of the target protein.

35 cl, 11 ex, 6 tbl, 11 dwg

Description

Cross reference to related applications

[0001] This application claims priority under 35 USC §119(e) based on US Provisional Application No. 62/633882, filed February 22, 2018; No. 62/633757, filed February 22, 2018; No. 62/633795, filed February 22, 2018; and No. 62/746762, filed October 17, 2018, the contents of each of which are incorporated herein by reference in their entirety.

[0002] This application includes a sequence listing filed electronically in ASCII format and is incorporated herein by reference in its entirety. The ASCII copy in question, created on February 21, 2019, is named 080170-091100-WOPT_SL.txt and is 116872 bytes in size.

TECHNICAL FIELD

[0003] This invention relates to the field of gene therapy, including capsidless vectors for the controlled expression of a transgene or isolated polynucleotides in a subject or cell. The technology described herein relates to methods for controlled expression of a transgene in vivo from closed-end capsidless DNA (ccDNA) vectors, where the level of expression can be maintained at a desired level for a given time or increased with one or more subsequent administrations (e.g., boost or repeat dose).

BACKGROUND OF THE ART

[0004] The goal of gene therapy is to improve clinical outcomes for patients suffering from genetic mutations or acquired diseases caused by aberrations in the gene expression profile. Gene therapy includes the treatment or prevention of medical conditions caused by defective genes or abnormal regulation or expression, such as underexpression or overexpression, that can lead to a disorder, disease, cancer, or the like. For example, treating, preventing, or ameliorating a disease or disorder caused by a defective gene may be accomplished by delivering corrective genetic material to a patient, modifying or silencing the defective gene, or delivering a therapeutic antibody, for example, resulting in therapeutic expression of said genetic material in said patient.

[0005] The basis of gene therapy is the introduction of a transcription cassette with an active gene product (sometimes called a transgene), which, for example, can lead to a positive gain-of-function effect, a negative loss-of-function effect, or other outcome. Gene therapy can also be used to treat a disease or cancer caused by other factors. Treatment of monogenic disorders in humans can be achieved by delivery and expression of a normal gene in target cells. Delivery and expression of the corrective gene into the target cells (target cells) of the patient can be carried out using numerous methods, including the use of genetically engineered viruses and viral vectors for gene delivery. Among the numerous viral-derived vectors available (eg, recombinant retrovirus, recombinant lentivirus, recombinant adenovirus, etc.), recombinant adeno-associated virus (rAAV) is gaining popularity as a multi-purpose vector for gene therapy.

[0006] Adeno-associated viruses (AAV) belong to the Parvoviridae family; more specifically, they constitute the genus Dependoparvovirus. AAV-derived vectors (i.e., recombinant AAV (rAAV) or AAV vectors) are a promising method of delivering genetic material because (i) they are capable of infecting (transducing) a wide range of nondividing and dividing cell types, including myocytes and neurons; (ii) they lack viral structural genes, which provides a reduction in host cell responses to viral infection, such as interferon-mediated responses; (iii) wild-type viruses are considered non-pathogenic to humans; (iv) unlike wild-type AAV, which is capable of integration into the host cell genome, replication-defective AAV vectors lack the rep gene and usually persist as episomes, which limits the risk of insertional mutagenesis or genotoxicity; and (v) compared to other vector systems, AAV vectors are generally considered to be relatively weak immunogens and accordingly do not elicit a significant immune response (see ii), thus allowing vector DNA persistence and potentially long-term expression of therapeutic transgenes.

[0007] However, the use of AAV particles as a vector for gene delivery has a number of serious disadvantages. One of the major disadvantages associated with rAAV is the limited viral packaging capacity of approximately 4.5 kb. heterologous DNA (Dong et al., 1996; Athanasopoulos et al., 2004; Lai et al., 2010), as a result of which the use of AAV vectors is limited by their ability to encode proteins smaller than 150,000 Da. A second disadvantage is that, as a result of the prevalence of wild-type AAV infection in the population, candidates for rAAV gene therapy must be screened for the presence of neutralizing antibodies that eliminate the vector from the patient. A third disadvantage relates to the immunogenicity of the capsid, which prevents re-administration in patients not excluded from primary treatment. The patient's immune system may respond to the vector, which essentially acts as a "booster" agent, stimulating the immune system to generate high titers of anti-AAV antibodies, precluding future treatment. Some recent reports have raised concerns about immunogenicity in high-dose situations. Another notable disadvantage is the relatively slow onset of AAV-mediated gene expression, given that single-stranded AAV DNA must be converted to double-stranded DNA before expression of the heterologous gene.

[0008] In addition, conventional AAV capsid virions are produced by introducing a plasmid or plasmids containing the AAV genome, rep genes and cap genes (Grimm et al., 1998). However, such capsid-enclosed viral AAV vectors have been found to ineffectively transduce certain types of cells and tissues, and these capsids also induce an immune response.

[0009] In addition, traditional gene therapy vectors for transgene delivery (eg, adeno-associated virus (AAV), adenovirus, lentiviral vectors, etc.) are typically limited to single administration of the vector to patients, driven in part by the patient's immune response to viral proteins. In addition, sustained, long-term transgene expression typically requires an initial high titer injection, which can lead to harmful side effects. Traditional viral vectors for gene therapy are essentially unsuitable due to the lack of stable long-term expression of the transgene. In addition, the range of transgenic genetic material suitable for delivery in such viral vectors is limited by the packaging capacity of the viral capsid proteins (eg, about 4.5 kb for AAV), precluding delivery of larger transgenes for therapy. Accordingly, the use of conventional adeno-associated virus vectors (AAV vectors) for gene therapy is limited due to single administration to patients (due to the patient's immune response), a limited range of transgenic genetic material suitable for delivery in AAV vectors, due to the minimal capacity of the viral packaging (about 4.5 kb), and slow AAV-mediated gene expression.

[0010] In addition, a number of reports have raised concerns about the use of too high a dose of the viral vector. Most viral vectors are also of concern due to the immunogenicity associated with AAV.

[0011] Accordingly, there is a need in the art for technology that allows for the use of multiple doses of a vector for gene therapy. Additionally, there is a need in the art for methods of controlling gene expression from a gene therapy vector with minimal off-target effects, and there remains an important unmet need for controlled recombinant DNA vectors with improved production and/or expression characteristics. Additionally, as will be appreciated by a skilled physician, the ability to titrate transgene expression/dose is desirable to tailor gene therapy based on a subject's particular set of symptoms and/or severity of disease and further to minimize side effects or toxicity.

BRIEF DESCRIPTION OF THE INVENTION

[0012] The invention described herein relates to a capsidless DNA vector with covalently closed ends (referred to herein as a “closed-end DNA vector” or “ccDNA vector”) for controlled expression of a transgene in a cell, for example, for treatment diseases. In particular, the technology described herein relates to capsidless closed-end DNA (ccDNA) vectors for controlled transgene expression, including, without limitation, any of sustained transgene expression, long-term controlled transgene expression, dose-dependent and/or titratable transgene expression , and re-dosing the transgene using the vectors described herein. Accordingly, the methods disclosed herein allow gene therapy to be personalized throughout an individual's life to express a transgene at a level that meets the individual's needs by maintaining the expression level of the transgene at a predetermined level for a predetermined period of time or, alternatively, increasing expression transgene in a dose-dependent manner through one or more administrations after the initial primary administration, thereby controlling the level of expression of the transgene according to the desired level of expression or the desired range of expression levels based on the concentration of the cccDNA vector upon repeated dosing, allowing for a controlled and specific increase expression of the transgene in a cell or subject.

[0013] Accordingly, in some embodiments, a cccDNA vector, as described herein, can be repeatedly administered (also referred to herein as a “re-dosing” or “booster”) to ensure that the level of transgene expression is maintained at a predetermined level in over a predetermined period of time, or to increase the level of expression of the transgene above the previous level of expression that was achieved after the first or previous administration of the cccDNA vector, wherein the second administration or booster administration does not cause an immune response that interferes with the expression of the transgene without causing an immune response to the vector itself influencing transgene expression or the immune response is less than reintroduction of a viral vector containing viral proteins, including, but not limited to, a viral vector containing a capsid such as a parvovirus or lentivirus.

[0014] Without being limited by theory, we point out that studies show that long-term transgene expression when using conventional AAV viral vectors decreases over time. One way to achieve long-lasting transgene expression using conventional AAV vectors has traditionally been to increase the titer and dose of AAV vector delivered upon initial administration. However, it is known that too high a titer or dose of AAV vector can lead to various side effects. Additionally, as described above, reintroduction of the AAV viral vector is traditionally not possible due to immune responses. Methods to avoid an immune response to repeated administration of AAV vectors typically require reintroduction of an AAV vector with a different capsid configuration compared to the AAV vector administered during any previous administration. However, such strategies may still pose significant risks to the subject by inducing an immune response to the AAV vector and potentially harmful effects.

[0015] The invention described herein relates to a method for controlled expression of a transgene, including long-term expression, using a closed-end DNA (ccDNA) vector. This document demonstrates that the cccDNA vector can be titrated to increase transgene expression levels, for example, by repeating or reintroducing the cccDNA vector. In addition, the expression level of the transgene can be maintained for a long time and if there is any drop in expression levels, repeated dosing of the cccDNA vector can be used to maintain the desired level or even increase the level of transgene expression if desired by the subject and/or or a disease or disorder being treated.

[0016] Accordingly, provided herein are methods of administering cccDNA vectors to maintain and/or increase the level of expression of a transgene from the cccDNA vector in a cell or subject, which level of expression may be maintained or increased by one or more subsequent administrations (e.g. repeat dose or booster administration). In the event that the expression of a cccDNA-delivered transgene is reduced for any reason, repeated dosing of the cccDNA vector can restore or maintain the desired expression of the transgene at the desired level. Also provided herein are methods for personalized gene therapy such that the level of expression of a transgene expressed by a cccDNA vector can be increased gradually or stepwise with one or more administrations after an initial priming administration (eg, at time 0), thereby optimizing ( eg, by titration) the level of expression of the transgene to the desired level of expression or to a value within the desired range of expression levels required by the subject.

[0017] Accordingly, in some embodiments, the cccDNA vectors and methods disclosed herein can be used to titrate or cause an increase in the level of expression of a transgene by the ccDNA vector, wherein the level of transgene expression can be titrated in a dose-dependent manner using one or more subsequent administrations ( e.g., dose-dependent repeat dosing or booster administration). As such, the methods disclosed herein allow gene therapy to be personalized throughout an individual's life to express a transgene at a level that meets the individual's needs by maintaining the level of transgene expression at a predetermined level or, alternatively, increasing transgene expression in a dose-dependent manner by one or more administrations after the initial priming administration (e.g., at time 0), thereby controlling the level of transgene expression to a desired expression level or a desired range of expression levels based on the concentration of the cccDNA vector upon repeated dosing, allowing for controlled and specific increase in transgene expression in a cage or in a subject.

[0018] Accordingly, in some embodiments, a cccDNA vector as described herein may be reintroduced (also referred to herein as a “boost” or “booster”) to maintain transgene expression at a predetermined level for a predetermined period of time, or to increase the expression level of the transgene above the previous expression level that was achieved upon the first or previous administration of the cccDNA vector.

[0019] Accordingly, one aspect of the technology described herein relates to the use of a cccDNA vector in methods for controlled expression of a transgene, for example, in a method for modulating the expression levels of a transgene, or for controlled increase in the level of expression of a transgene, or for dose-dependent expression of the level of a transgene in cell or in a subject, wherein the cDNA vector contains at least one heterologous nucleotide sequence (for example, a transgene) operably linked to a promoter and located between two inverted terminal repeat (ITR) sequences, wherein the ITR sequences may be asymmetric or symmetric, or substantially symmetrical, as those terms are defined herein, wherein at least one of the ICPs comprises a functional end resolution site and a Rep binding site, and optionally a heterologous nucleic acid sequence encodes a transgene, and wherein the vector is not enclosed within a viral capsid.

[0020] In some embodiments, the cccDNA vector, as described herein, is a capsidless linear duplex DNA molecule formed from a continuous strand of complementary DNA with covalently closed ends (a linear, contiguous, non-capsidated structure) that contains two inverted terminal repeat (ITR) sequences flanking a transgene that is operably linked to a promoter or other regulatory switch as described herein. The 5'-ICP and 3'-ICP may have the same symmetrical three-dimensional organization relative to each other (i.e., symmetrical or substantially symmetrical), or, alternatively, the 5'-ICP and 3' ICP may have different three-dimensional organization relative to each other. friend (i.e., asymmetric ICPs) as those terms are defined herein. In addition, ICPs may belong to the same or different serotypes. In some embodiments, the cccDNA vector may contain ICP sequences that have a symmetrical three-dimensional spatial organization such that their structure has the same shape in geometric space, or have the same A, C-C', and B-B' loops in three-dimensional space (i.e., they are the same or are mirror images of each other). In some embodiments, one ICP may belong to one AAV serotype, and the other ICP may belong to a different AAV serotype.

[0021] Accordingly, certain aspects of the technology described herein relate to a cDNA gene editing vector that contains ICP sequences selected from any of: (i) at least one wild-type (WT) ICP and at least one modified inverted terminal repeat (ITR) of AAV (eg, asymmetric modified ITR); (ii) two modified ICPs, wherein the mod-ICP pair has a different three-dimensional spatial organization relative to each other (for example, asymmetric modified ICPs), or (iii) a symmetric or substantially symmetrical pair of DT-DT ICPs, with each ICP-DT having the same three-dimensional spatial organization, or (iv) a symmetric or substantially symmetrical pair of modified ICPs, each mod-ICP having the same three-dimensional spatial organization. The cccDNA vectors disclosed herein can be produced in eukaryotic cells and are accordingly free of prokaryotic DNA modifications and bacterial endotoxin contamination in insect cells.

[0022] In some embodiments, the methods and cDNA vectors as described herein enable a personalized genetic drug approach, i.e. titrating the increase in transgene expression by repeated dosing in a concentration-dependent manner. It is envisaged that the increase in transgene expression can be achieved in a dose-dependent manner, in a stepwise manner, using repeated dosing, thereby increasing the level of transgene expression by a predetermined or defined amount with each repeated dosing. This allows for a controlled increase in transgene expression in a dose-dependent manner and can be done gradually. Accordingly, 1, 2, 3, 4, 5 or 6 or more than 6 repeated doses of a specified amount of cccDNA may be administered to each time increase the expression level of the transgene by a certain amount to achieve a desired level or a desired range of expression levels higher than the level of expression achieved at the previous administration or before such repeated dosing.

[0023] Accordingly, in some embodiments, a method of regulating transgene expression in a host is provided, comprising: (i) administering to the host a sufficient amount of a cccDNA vector, as described herein, comprising a nucleic acid cassette containing at least one transgene operably linked with a promoter located between flanking inverted terminal repeats (ITRs) for expressing measurable levels of the transgene, the transgene encoding the desired protein; and (ii) administering at least a second dose of a cccDNA vector containing at least one transgene or modified transgene between the flanking ICPs to (i) continue expression of the desired protein at a predetermined level for a predetermined period of time, or (ii) modulating the expression of the desired protein to a predetermined level, wherein the second administration of the cDNA vector does not cause an immune response that interferes with the expression of the desired protein.

[0024] One aspect of the technology described herein relates to the use of a cccDNA vector in a method of maintaining a desired level of transgene expression in a cell, comprising: (a) introducing into the cell at a first time a first dose of the cccDNA vector to achieve expression of the transgene from the cccDNA vector, and (b) introducing into the cell at a second time point another dose of the same or a different cccDNA vector to increase the expression level of the transgene to a desired level or to compensate for any decrease in the level of transgene expression after the initial introduction of the cccDNA vector. It should be understood that such a gradual increase in transgene expression allows the dosage to be titrated in a subject to the level desired by such subject. In some embodiments, use of a cccDNA vector in a method for maintaining the expression level of a transgene in a cell ensures that the transgene is expressed at the desired expression level for at least 42 days. In some embodiments, the cccDNA vector expresses the transgene at the desired expression level for at least 84 days. In some embodiments, the cccDNA vector expresses the transgene at the desired expression level for at least 132 days.

[0025] In some embodiments, a cccDNA vector used in the methods described herein, for example, in a method for maintaining expression of a transgene in a cell and/or treating a subject with a disease, is administered in combination with a pharmaceutically acceptable carrier and/or excipient. In some embodiments, the cDNA vector is administered at a second time point after at least 30 days, or at least 60 days, or 60-90 days, or 90-120 days, or about 3-6 months, or 6-12 months , or 1-2 years, or 2-3 years, after the first point in time.

[0026] In addition to repeatedly dosing the cccDNA vector to simply increase transgene expression levels if expression levels have decreased over time (e.g., to continue transgene expression at a desired predetermined level), in some embodiments, methods and compositions for reintroducing cccDNA vectors can increase transgene levels in a dose-dependent manner, i.e. repeated introduction of a certain amount of cDNA vector can cause a certain increase in the level of transgene expression. In other words, using arbitrary units for illustrative purposes only, a single dose of ccDNA when reintroduced will result in a 10% increase in transgene expression over the previous level, and a 2-fold dose of ccDNA vector will result in a 20% increase in transgene expression. compared to the previous level, and half (0.5) dose of cccDNA will increase the level of transgene expression by 5% compared to the previous level.

[0027] Accordingly, in one embodiment, a cccDNA vector disclosed herein for controlled expression of a transgene can be used to increase the level of expression of a transgene in a cell or subject in a controlled manner. For example, the level of transgene expression can be increased by one or more subsequent administrations (eg, repeat dosing or boosting) of the cccDNA vector.

[0028] Another aspect of this technology relates to a method of increasing the expression of a transgene in a cell, for example, to increase the level of expression of the transgene above the previous level of expression that was achieved by prior introduction of cccDNA, the method comprising: (a) introduction into the cell at a first time point a priming dose of the cccDNA vector to achieve expression of the transgene, and (b) introducing into the cell at a second time point a dose of the cccDNA vector to increase the level of expression of the transgene compared to the level of transgene expression achieved after introduction of the cccDNA vector at the first time point, or for increasing the level of expression of the transgene to achieve the desired level of expression.

[0029] In all aspects described herein, the cccDNA vector that is administered at any time point (e.g., at the first, second, third time point, etc.) is administered in combination with a pharmaceutically acceptable carrier, and optionally can be administered with a carrier such as particles, liposomes or lipid nanoparticles (LNPs). In all aspects described herein, the cccDNA vector that is administered at any of the first, second, or any subsequent time point is administered in combination with a pharmaceutically acceptable carrier.

[0030] In all aspects described herein, a cccDNA vector used in methods of controlled transgene expression as described herein, for example, in a method of maintaining transgene expression, or controlled increase of transgene expression, or dose-dependent transgene expression, and/or treating a subject with a disease, said cccDNA vector, which is administered at any of the first, second, or any subsequent time point, is administered in combination with a pharmaceutically acceptable carrier and/or excipient.

[0031] In some embodiments in which there is more than one administration of cccDNA (e.g., at a second or any subsequent time point), the second time point or any subsequent time point is at least 10 days later, or 10-30 days, or at least 30 days, or 30-60 days, at least 60 days, or 60-90 days, or 90-120 days, or about 3-6 months, or 6-12 months, or at least a year after administration ccDNA vector at the first time point or at a previous time point.

[0032] In some embodiments, the cccDNA vector that is introduced at the first, second, or any subsequent time point is the same cccDNA vector containing the same transgene or a modified transgene, and in alternative embodiments, a cccDNA vector that introduced at the first, second, or any subsequent time point, is another cDNA vector containing the same transgene or a modified transgene, for example, another cDNA vector with a different promoter operably linked to the same transgene or a modified transgene. In some embodiments, the promoter is an inducible or repressible promoter. The transgene may also be part of a regulatory switch, as described herein.

[0033] In some embodiments of the cccDNA vector used in the controlled transgene expression methods described herein, the cccDNA vector that is administered at the first, second, or any subsequent time point is the same cccDNA vector containing that same transgene, or modified transgene. In alternative embodiments, the cccDNA vector that is introduced at the first, second, or any subsequent time point is another cccDNA vector containing the same transgene or a modified transgene, for example, without limitation, in cases where a different cccDNA vector has a different a promoter operably linked to the same transgene, or to a modified transgene, or to a different transgene. For illustrative purposes only, the cccDNA vector that is introduced at the first time point may contain a transgene and a first promoter or regulatory switch, and the cccDNA that is introduced at the second or subsequent time points may contain the same or a modified transgene and a second promoter or regulatory switch. switch. wherein the first and second promoter (or regulatory switch) are different promoters or different regulatory switches. Examples of control switches are described in this document.

[0034] In some embodiments, use of a cccDNA vector in methods of controlled transgene expression as described herein, for example, in a method of maintaining transgene expression, or controlled increase of transgene expression, or dose-dependent expression of a transgene and/or treatment of a subject with a disease, may optionally including the step of introducing into the cell, at one or more time points after the second time point, an additional dose of the cccDNA vector to increase the level of transgene expression compared to the level of transgene expression achieved after introduction of the cccDNA vector at the second time point or at the previous time point , or to increase the level of expression of the transgene to maintain a desired level of sustained expression, wherein the composition administered at one or more time points after the second time point contains a cccDNA vector as described herein.

[0035] In some embodiments, the cccDNA vector used in the controlled transgene expression methods disclosed herein allows expression of the transgene in a therapeutically effective amount.

[0036] In some embodiments, increasing the predetermined dose of cccDNA vector administered at the second time point or any subsequent time point increases the level of expression of the transgene in the cell and/or subject. In some embodiments, a predetermined dose of the cccDNA vector administered to the cell or subject at the second time point or at a subsequent time point is determined by a dose response for the cccDNA vector to achieve the desired level of transgene expression in the cell or subject.

[0037] In some embodiments, the predetermined dose of cccDNA vector administered at the second or any subsequent time point is an amount that is 2-10 times greater than the dose of ccDNA vector administered at the first time point. In some embodiments, the predetermined dose of cccDNA vector administered at the second or any subsequent time point is an amount that increases transgene expression by at least 3-fold, or at least 5-fold, or at least 10-fold , or 2-15-fold, or 2-20-fold, or more than 20-fold, compared to the transgene expression achieved after the introduction of cccDNA at the first time point or at the previous time point. In some embodiments, the desired level of transgene expression achieved following administration of the composition at one or more time points after the second time point is a therapeutically effective amount of the transgene.

[0038] Aspects of the present invention relate to methods for producing a cccDNA vector used in controlled transgene expression methods as described herein, for example, in a method for maintaining transgene expression, or controlled increase in transgene expression, or dose-dependent transgene expression, and/or treatment a subject with a disease. In all aspects, a non-capsid non-viral DNA vector (ccDNA vector) for controlled transgene expression is derived from a plasmid (referred to herein as a "ccDNA plasmid") containing a polynucleotide expression construct template containing, in this order: a first 5' inverted terminal repeat (eg, ICP AAV); heterologous nucleic acid sequence; and 3'-ICP (e.g., AAV ICP), wherein the 5'-ICP and 3'-ICP may be asymmetric to each other or symmetrical (e.g., ICP-DT or modified symmetric ICP) as defined herein .

[0039] An cccDNA vector used in methods for controlled transgene expression as described herein (e.g., in a method for maintaining transgene expression levels and/or controlled increases in transgene expression levels, or dose-dependent transgene expression levels) can be produced by a number of methods that will known to one skilled in the art upon reading this disclosure. For example, the expression construct polynucleotide template used to generate the ccDNA vectors of the present invention may be a ccDNA plasmid (e.g., see Figure 4 B), a ccDNA bacmid, and/or a ccDNA baculovirus. In one embodiment, the ccDNA is the plasmid contains a restriction cloning site (for example, SEQ ID NO: 123 and/or 124, operably located between the indicated ICPs, into which an expression cassette can be inserted, containing, for example, a promoter operably linked to a transgene, for example, a reporter gene and/or therapeutic gene). In some embodiments, the cDNA vectors are derived from a polynucleotide template (e.g., cDNA plasmid, ccDNA bacmid, ccDNA baculovirus) containing symmetric or asymmetric ICPs (modified ICPs or wild-type (WT) ICPs).

[0040] In a permissive host cell, in the presence of, for example, Rep, a polynucleotide template having at least two ICPs is replicated to produce cccDNA vectors. The production of a cDNA vector takes place in two stages: firstly, excision (“release”) of the template from the matrix backbone (for example, cDNA plasmid, ccDNA bacmid, ccDNA baculovirus genome, etc.) by means of Rep proteins and, second, Rep-mediated replication of the excised cccDNA vector. The Rep proteins and Rep-binding sites of various AAV serotypes are well known to those of ordinary skill in the art. One of ordinary skill in the art will appreciate that the Rep protein should be selected from a serotype that binds to and replicates a nucleic acid sequence based on at least one functional ICP. For example, if a replication competent ICP is of serotype AAB 2, the corresponding Rep will be of an AAB serotype that works with that serotype, such as an AAV2 ICP with Rep AAB2 or AAB4, but not with Rep AAB5, which is unable to do so. After replication, the cccDNA vector with covalently closed ends continues to accumulate in permissive cells, and the ccDNA vector is preferably sufficiently stable over time in the presence of the Rep protein under standard replication conditions, for example, to accumulate at an amount of at least 1 pg/cell. preferably at least 2 pg/cell, preferably at least 3 pg/cell, more preferably at least 4 pg/cell, even more preferably at least 5 pg/cell.

[0041] Accordingly, one aspect of the invention relates to a method of producing a cccDNA vector for use in controlled transgene expression methods as described herein, comprising the steps of: a) incubating a population of host cells (eg, insect cells) bearing a polynucleotide expression template construct (e.g., cccDNA plasmid, ccDNA bacmid, and/or ccDNA baculovirus) lacking sequences encoding the viral capsid, in the presence of the Rep protein, under conditions effective to, and for a period of time sufficient to induce production of the cccDNA vector in host cells, wherein the host cells do not contain sequences encoding the viral capsid; and b) collecting and isolating the cDNA vector from the host cells. The presence of the Rep protein induces replication of the ICP-modified vector polynucleotide to produce a cDNA vector in the host cell. However, viral particles (eg, AAV virions) are not expressed. Thus, there are no size restrictions imposed by virions.

[0042] The presence of a cccDNA vector suitable for controlled expression of a transgene as described herein isolated from host cells can be confirmed by digestion of DNA isolated from the host cell with a restriction enzyme having a single recognition site on the ccDNA vector , and analysis of digested DNA material on denaturing and non-denaturing gels to confirm the presence of characteristic bands of linear and continuous DNA, as opposed to linear and discontinuous DNA.

[0043] In another embodiment of this aspect and all other aspects presented herein, the transgene expressed in a controlled manner from the cccDNA vector is a therapeutic transgene, e.g., a protein of interest, including, but not limited to, a receptor, a toxin, a hormone , an enzyme or cell surface protein. In another embodiment of this aspect and all other aspects provided herein, the protein of interest is a receptor. In another embodiment of this aspect and all other aspects provided herein, the protein of interest is an enzyme. Representative examples of target genes and proteins of interest are described in detail in the application and treatment sections of this document.

[0044] In some embodiments, this claim may be defined by any of the following paragraphs:

[0045] A method for regulating transgene expression in a subject, comprising: (a) administering to the subject a sufficient amount of non-viral capsid-free closed-ended DNA (ccDNA) containing a nucleic acid cassette containing at least one transgene operably linked to a promoter between flanking inverted ends repeats (IKR) to express a measurable level of the transgene, wherein the transgene encodes a desired protein for treating a disease; and (b) titrating the cccDNA vector by administering to the subject at least a second dose of the cccDNA vector containing at least one transgene between the flanking ICPs to ensure transgenic expression of the desired protein at a predetermined level for a predetermined period of time, or to increase transgenic expression of the desired protein to a predetermined level.

[0046] In some embodiments, the subject is examined at a predetermined time after step (a), for example, at least 30 days, or at least 60 days, or from 60 to 90 days, or more than 90 days after step ( a), to determine the titrated dose. For example, in some embodiments, the subject is assessed to determine the subject's disease state beyond stage (a) and/or the level of the desired protein expressed by the cccDNA vector in the subject. In some embodiments, the disease state assessment is an assessment of at least one disease symptom in the subject. In some embodiments, if the subject's disease state has remained stable or has not improved, or when the subject's disease state has decreased, for example, compared to the disease state at the time of the first administration of the cccDNA vector or at any time before step (a), the subject is administered a second dose of the cccDNA vector according to step (b). The disease status for any given disease can be determined by a physician or person skilled in the art, and includes assessment of one or more clinical symptoms and/or disease biomarkers, including protein biomarkers, microRNA (miRNA) and mRNA biomarkers, and the like. In some In embodiments, if the subject's transgene expression level has decreased from a predetermined level or has decreased from a therapeutically effective amount, the subject is administered a second dose of the cccDNA vector in accordance with step (b). In some embodiments, the expression level of the transgene is determined by measuring the level of the transgene (e.g., measuring the protein level or mRNA levels) expressed from the cccDNA vector in a biological sample obtained from the subject. In some embodiments, the biological sample is selected from a blood sample, plasma, synovial fluid, cerebrospinal fluid (CSF), saliva, or tissue biopsy sample. In some embodiments in which the cccDNA vector expresses a transgene encoding a desired protein or therapeutic gene and a reporter protein, the level of the transgene can be determined by measuring the desired reporter protein expressed from the cccDNA vector in vivo using methods well known to those of ordinary skill in the art. this field of technology. In some embodiments, titrating the cccDNA vector determines the level of transgene expressed by the cccDNA vector and administering a second dose of the cccDNA vector to the subject to adjust or modulate expression of the transgene to a predetermined desired level.

[0047] Another aspect of the technology described herein relates to a method of regulating transgene expression in a subject, comprising: (a) administering to the subject a sufficient amount of a non-viral capsid-free closed-ended DNA (ccDNA) vector containing a nucleic acid cassette containing at least at least one transgene operably linked to a promoter between flanking inverted terminal repeats (ITRs) to express a measurable level of the transgene, the transgene encoding a desired protein; and (b) administering to the subject at least a second dose of a cccDNA vector containing at least one transgene or a modified transgene between the flanking ICPs to (i) continue expression of the desired protein at a predetermined level for a predetermined period of time, or (ii) modulating the expression of the desired protein to a predetermined level.

[0048] In all aspects of the present invention, a second administration of the cccDNA vector to a subject does not elicit an immune response sufficient to prevent the predetermined level of expression of the desired protein from being achieved.

[0049] In some embodiments, the cccDNA vector is administered to a subject upon a first administration, or a second administration, or any subsequent administration, in combination with a pharmaceutically acceptable carrier.

[0050] In some embodiments, the second administration of the cccDNA vector occurs at a time when the level of transgene expression decreases from the desired predetermined level, for example, in some embodiments, the second administration occurs after at least about 30 days, or at least about 60 days, or at least about 90 days, after the first administration. When a subject is administered more than two doses of the cccDNA vector, each repeat dose (eg, 3rd, 4th, 5th, 6th, and subsequent repeat doses) is administered at a time when the level of transgene expression decreases or falls from the desired level a predetermined level achieved by the previous administration, for example, in some embodiments, each repeat administration is at least about 30 days, or at least about 60 days, or at least about 90 days after the previous administration of the cDNA vector.

[0051] In some embodiments, the method includes administering at least three or more administrations of the cccDNA vector to a subject, and where at least three administrations of the cccDNA vector are administered, none of the administrations induces an immune response to the cccDNA vector that prevents achieving a given level of expression of the desired protein.

[0052] In some embodiments, the cccDNA vector is administered to a subject on a periodic schedule, such as every 2 months, every 3 months, every 6 months, every 12 months, every 18 months, and the like.

[0053] In some embodiments, the second administration is designed to increase the level of expression of the desired protein, for example, to prolong the expression of the desired protein at a predetermined expression level.

[0054] In all aspects of the present invention, the transgene encodes a therapeutic protein, and the desired level of expression of the transgene is a therapeutically effective amount of the therapeutic protein. In some embodiments, the transgene is a genetic drug selected from any of: a nucleic acid, an inhibitor, a peptide or polypeptide, an antibody or antibody fragment, a fusion protein, an antigen, an antagonist, an agonist, an RNAi molecule, etc. In some embodiments, the desired protein or therapeutic protein is an inhibitor protein, such as, without limitation, an antibody or antigen binding fragment or fusion protein. In some embodiments, a desired protein or therapeutic protein replaces a defective protein or a protein that is not expressed or is expressed at low levels. In some embodiments, the transgene is under the control of a regulatory switch, as defined herein.

[0055] In some embodiments, the cccDNA vector contains a promoter that is an inducible or repressible promoter.

[0056] In some embodiments, the cccDNA vector that is administered at the first, second, or any subsequent time point is the same type of cccDNA vector containing the same transgene or a modified transgene. For example, in other words, the same cDNA vector is administered to a subject multiple times, which is comparable to administering the same serotype of viral vector to a subject multiple times.

[0057] In some embodiments, the cccDNA vector that is administered to a subject upon a second administration or any subsequent administration thereafter (eg, a repeat dose) has a different promoter operably linked to the same transgene or a modified transgene than the promoter in a cccDNA vector introduced at an earlier time point or at an earlier injection.

[0058] In some embodiments, the cccDNA vector that is administered at the first administration, or at the second administration, or at any subsequent administration thereafter, contains two inverted terminal repeat (ITR) sequences that are AAV ICRs and may be, for example, , AAV-2 or any IKP selected from Table 1 or AAV1, AAV3, AAV4, AAV5, AAV 5, AAV7, AAV8, AAV9, AAV10, AAV 11, AAV12, AAVrh8, AAVrh10, AAV-DJ and AAV-DJ8. In some embodiments, at least one ICP contains a functional end resolution site and a Rep binding site. In some embodiments, the flanking ICPs in the cccDNA vector introduced at the first injection, or the second injection, or any subsequent injection thereafter, are symmetrical, either substantially symmetrical or asymmetrical, as defined herein. In some embodiments, one or both of the ICPs are wild type, or both of the ICPs are wild type. In some embodiments, the flanking ICPs are from different viral serotypes. In some embodiments, when both flanking ICPs are wild type, they can be selected from any AAV serotype as listed in Table 1.

[0059] In some embodiments, the flanking CPIs in the cccDNA vector, upon the first injection, or the second injection, or any subsequent injection thereafter, may contain a sequence selected from the sequences in Tables 2, 4A, 4B, or 5 herein.

[0060] In some embodiments, at least one of the ICPs in the cDNA vector administered upon the first administration, or the second administration, or any subsequent administration thereafter, is altered relative to the wild-type AAV ICP sequence by deletion, addition, or substitution , which influence the overall three-dimensional conformation of the ICP. In some embodiments, one or both ICPs in the cccDNA vector administered at the first administration, or the second administration, or any subsequent administration thereafter, is derived from an AAV serotype selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAB8, AAV9, AAV10, AAV11 and AAV12.

[0061] In some embodiments, one or both of the ICPs in the cDNA vector administered at the first administration, or the second administration, or any subsequent administration thereafter, are synthetic. In some embodiments, one or both of the ICPs are non-wild type ICPs, or both of the ICPs are non-wild type.

[0062] In some embodiments, one or both of the ICPs in the cccDNA vector administered at the first administration, or the second administration, or any subsequent administration thereafter, is modified by deletion, insertion, and/or substitution in at least one of the ICP regions , selected from A, A', B, B', C, C', D and D'. In some embodiments, the deletion, insertion, and/or substitution removes all or part of the stem-loop structure typically formed by regions A, A', B, B', C, or C'. In some embodiments, one or both of these ICPs are modified by a deletion, insertion, and/or substitution that removes all or a portion of the stem-loop structure typically formed by regions B and B'. In some embodiments, one or both of these ICPs are modified by a deletion, insertion, and/or substitution that removes all or a portion of the stem-loop structure typically formed by regions C and C'. In some embodiments, one or both of these ICPs are modified by a deletion, insertion, and/or substitution that removes a portion of the stem-loop structure typically formed by regions B and B' and/or a portion of the stem-loop structure typically formed by regions C and C'. In some embodiments, one or both of these ICPs comprise a single stem-loop structure in a region typically comprising a first stem-loop structure formed by regions B and B' and a second stem-loop structure formed by regions C and C'. In some embodiments, one or both of these ICPs comprise one stem and two loops in a region typically comprising a first stem-loop structure formed by regions B and B' and a second stem-loop structure formed by regions C and C'.

[00563] In some embodiments, both ICPs in the cccDNA vector administered at the first injection, or the second injection, or any subsequent administration thereafter, are altered in a manner that results in an overall three-dimensional symmetry where the ICPs are inverted with respect to each other.

[0064] In some embodiments, the cccDNA vector administered upon the first administration, or the second administration, or any subsequent administration thereafter, comprises at least one heterologous nucleotide sequence under the control of at least one regulatory switch, such as at least one a regulatory switch selected from a binary regulatory switch, a small molecule regulatory switch, a password-controlled regulatory switch, a nucleic acid-based regulatory switch, a post-transcriptional regulatory switch, radiation-controlled or ultrasound-controlled regulatory switch, hypoxia-mediated regulatory switch, inflammatory response regulatory switch, activated by shifting the control switch and the kill switch. The control switches are disclosed in more detail later in this document.

[0065] In some embodiments, the cccDNA vector that is administered at the first administration, or the second administration, or any subsequent administration thereafter, is administered to a subject with a disease or disorder selected, for example, from cancer, an autoimmune disease, a neurodegenerative disorder, hypercholesterolemia, acute organ rejection, multiple sclerosis, postmenopausal osteoporosis, skin diseases, asthma or hemophilia. In some embodiments, the subject with cancer has a solid tumor, soft tissue sarcoma, lymphoma, and leukemia. In some embodiments, the subject has an autoimmune disease, such as one selected from rheumatoid arthritis and Crohn's disease. In some embodiments, the subject has a skin disorder, such as one selected from psoriasis and atopic dermatitis. In some embodiments, the subject has a neurodegenerative disorder, such as Alzheimer's disease, amyotrophic lateral sclerosis (ALS), Parkinson's disease, Huntington's disease.

[0066] In some embodiments, the method disclosed herein further includes administering to the subject, at one or more time points after the second time point, a dose of a cccDNA vector to increase the level of expression of the heterologous nucleic acid sequence (e.g., a transgene) relative to the level of expression of the transgene achieved after introduction of the cccDNA vector at a second time point or at a previous time point, or to increase the level of expression of the transgene to achieve the desired level of expression.

[0067] In some embodiments, the predetermined dose of the cccDNA vector administered to the subject at the second or any subsequent time point is an amount that is 2-10 times greater than the dose of the cccDNA vector composition administered at the first time point. In some embodiments, the predetermined dose of the cccDNA vector composition administered at the second or any subsequent time point is an amount that increases transgene expression by at least 3-fold, or at least 5-fold, or at least 10-fold times, or 2 to 15 times, or from 2 to 20 times, compared with the level of transgene expression after the introduction of the cDNA vector at the first time point or during the previous administration. In some embodiments, a predetermined dose of the cccDNA vector administered at the second administration or at a second time point is determined by a dose response for the cccDNA vector to achieve the desired level of transgene expression in the cell.

[0068] These and other aspects of the invention are described in more detail below.

BRIEF DESCRIPTION OF GRAPHIC MATERIALS

[0069] The embodiments of the present invention briefly set forth above and described in more detail below will be understood with reference to the illustrative embodiments of the present invention shown in the accompanying drawings. However, the accompanying drawings illustrate only exemplary embodiments of the present invention and therefore should not be construed as limiting the scope thereof, since the disclosure may permit other equally effective embodiments.

[0070] Figure 1A shows a typical example of the structure of a cccDNA vector for controlled expression of a transgene, as disclosed herein, containing asymmetric ICPs. In this embodiment, a typical cccDNA vector comprises an expression cassette containing a CAG promoter, a WPRE (woodchuck hepatitis virus post-transcriptional regulatory element), and a BGHpA (bovine growth hormone polyadenylation signal). An open reading frame (ORF) encoding a luciferase transgene can be inserted into the cloning site (R3/R4) between the CAG promoter and the WPRE. The expression cassette is flanked by two inverted terminal repeats (ITRs) - wild-type AAV2 ITRs upstream (at the 5' end) and a modified ITR downstream (at the 3' end) of the expression cassette, thus the two ITRs flanking the expression cassette are asymmetrical to each other. regarding a friend.

[0071] Figure 1B shows a typical example of the structure of a cccDNA vector for controlled transgene expression, as disclosed herein, containing asymmetric ICPs with an expression cassette containing a CAG promoter, WPRE and BGHpA. An open reading frame (ORF) encoding a luciferase transgene can be inserted into the cloning site between the CAG promoter and the WPRE. The expression cassette is flanked by two inverted terminal repeats (ITRs) - a modified ITR upstream (at the 5' end) and a wild-type ITR downstream (at the 3' end) of the expression cassette.

[0072] Figure 1C shows a typical example of the structure of a cccDNA vector for controlled transgene expression, as disclosed herein, containing asymmetric ICPs, with an expression cassette containing an enhancer/promoter, a transgene, a post-transcriptional element (WPRE), and a poly(A) signal. . The open reading frame (ORF) allows insertion of the transgene into the cloning site between the CAG promoter and the WPRE. The expression cassette is flanked by two inverted terminal repeats (ITRs), which are asymmetrical relative to each other - a modified IRT to the left of (at the 5' end) and a modified IRT to the right of (at the 3' end) the expression cassette, with the 5' ITR and 3'-ICPs are both modified ICPs, but have different modifications (ie, they do not have the same modifications).

[0073] Figure 1D shows a typical example of the structure of a cccDNA vector for controlled expression of a transgene, as disclosed herein, containing symmetrical modified ICPs or substantially symmetrical modified ICPs as defined herein, with an expression cassette containing a CAG promoter, WPRE and BGHpA. The open reading frame (ORF) encoding the luciferase transgene is inserted into the cloning site between the CAG promoter and the WPRE. The expression cassette is flanked by two modified inverted terminal repeats (ITRs), the 5' modified ITR and the 3' modified ITR being symmetrical or substantially symmetrical.

[0074] Figure 1E shows a typical example of the structure of a cccDNA vector for controlled expression of a transgene, as disclosed herein, containing symmetrical modified ICPs or substantially symmetrical modified ICPs as defined herein, with an expression cassette containing an enhancer/promoter, transgene, posttranscriptional element (WPRE) and poly(A) signal. The open reading frame (ORF) allows insertion of the transgene into the cloning site between the CAG promoter and the WPRE. The expression cassette is flanked by two modified inverted terminal repeats (ITRs), the 5' modified ITR and the 3' modified ITR being symmetrical or substantially symmetrical.

[0075] Figure 1F shows a typical example of the structure of a cccDNA vector for controlled expression of a transgene, as disclosed herein, containing symmetric ICP-WT or substantially symmetric ICP-WT, as defined herein, with an expression cassette containing a CAG promoter , WPRE and BGHpA. The open reading frame (ORF) encoding the luciferase transgene is inserted into the cloning site between the CAG promoter and the WPRE. The expression cassette is flanked by two wild-type inverted terminal repeats (ITR-WT), with the 5' ICT-WT and 3' ICT-WT being symmetrical or substantially symmetrical.

[0076] Figure 1G shows a typical example of the structure of a cccDNA vector for controlled expression of a transgene, as disclosed herein, containing symmetrical modified ICPs or substantially symmetrical modified ICPs as defined herein, with an expression cassette containing an enhancer/promoter, transgene, posttranscriptional element (WPRE) and poly(A) signal. The open reading frame (ORF) allows insertion of the transgene into the cloning site between the CAG promoter and the WPRE. The expression cassette is flanked by two wild-type inverted terminal repeats (ITR-WT), with the 5' ICT-WT and 3' ICT-WT being symmetrical or substantially symmetrical.

[0077] Figure 2A shows the T-shaped stem-loop structure of the left ICP of wild type AAB2 (SEQ ID NO: 52) with the A-A' arm, B-B' arm, C-C' arm designated two Rep-binding sites (RBE and RBE'), as well as a specified terminal resolution site (trs). RBE contains a series of 4 duplex tetramers that are believed to interact with either Rep 78 or Rep 68. In addition, RBE' is also thought to interact with the Rep complex assembled on a wild-type ICP or a mutated ICP construct. Regions D and D' contain transcription factor binding sites and other conserved structures. Figure 2B shows the putative Rep-catalyzed nicking and ligation activities of the wild-type left ICP (SEQ ID NO: 53), including the T-shaped stem-loop structure of the wild-type AAB2 left ICP labeled arm A-A', arm B-B', arm C-C', two Rep-binding sites (RBE and RBE'), as well as the indicated terminal resolution site (trs) and region D and D' containing several transcription factor binding sites and other conserved structures.

[0078] Figure 3A shows the primary structure (polynucleotide sequence) (left) and secondary structure (right) of the RBE-containing regions of arm A-A' and arms C-C' and B-B' of the left ICP of wild-type AAB2 (SEQ ID NO: 54). Figure 3B shows a typical example of a mutated ICP (also called modified ICP) sequence for the left ICP. Shown are the primary structure (left) and predicted secondary structure (right) of the RBE-containing portion of arm A-A', arm C, and arm B-B' of a representative example of a mutated left IKP (IKP-1, left) (SEQ ID NO: 113) . Figure 3C shows the primary structure (left) and secondary structure (right) of the RBE-containing portion of the A-A' loop and arms B-B' and C-C' of the right ICP of wild-type AAB2 (SEQ ID NO: 55). FIG. 3D shows a typical example of a right modified ICP. Shown are the primary structure (left) and predicted secondary structure (right) of the RBE-containing portion of the A-A' arm, arms B-B', and C of a representative example of a mutant right IKP (IKP-1, right) (SEQ ID NO: 114). Any combination of left and right ICPs (eg, AAV2 or other viral serotype ICPs, or synthetic ICPs) as described herein can be used. Each of the polynucleotide sequences in FIGS. 3A-3D refers to sequences used in a plasmid or bacmid/baculovirus genome used to produce cccDNA as described herein. Also, each of FIGS. 3A-3D includes corresponding predicted cccDNA secondary structures derived from the configurations of the cccDNA vector in the plasmid or bacmid/baculovirus genome and predicted Gibbs free energy values.

[0079] FIG. 4A is a schematic representation of the upstream process for producing baculovirus-infected insect cells (BIICs) that are suitable for producing a cccDNA vector for controlled transgene expression in the manner schematically illustrated in FIG. 4B. FIG. 4B an example of a method for producing cccDNA is schematically illustrated, and FIG. 4C shows a biochemical method and a process for confirming the production of cccDNA vector. FIG. 4D and FIG. 4E are schematic diagrams describing the process of identifying the presence of cccDNA in DNA collected from cell pellets obtained during the cccDNA production process of Fig. 4B. FIG. 4D is a schematic representation of the expected bands for a typical example of cccDNA , undigested or digested with a restriction endonuclease, and then subjected to electrophoresis on a native or denaturing gel. The leftmost diagram corresponds to a native gel and shows several bands indicating that in duplex and uncut form, ccDNA exists in at least monomeric and dimeric states, which appear as a smaller, faster-migrating monomer and a slower-migrating, smaller-sized dimer. 2 times greater than that of the monomer. The second schematic from the left demonstrates that when cccDNA is cut by a restriction endonuclease, the original bands disappear and faster migrating bands (eg, smaller) appear that correspond to the expected fragment sizes remaining after digestion. Under denaturing conditions, the original duplex DNA is single-stranded and migrates as an object twice the size of that observed on the native gel because the complementary strands are covalently linked. Accordingly, in the second schematic from the right, the digested ccDNA shows a band distribution similar to that observed on the native gel, however, the bands migrate as fragments that are twice the size of their equivalents in the native gel. The rightmost diagram demonstrates that uncut cccDNA under denaturing conditions migrates as a single-stranded open ring and therefore the bands observed are twice as large as under native conditions when the ring is not open. In this figure, “t.p.o.” used to indicate the relative size of nucleotide molecules, based, depending on the context, on either the length of the nucleotide chain (for example, for single-stranded molecules observed under denaturing conditions) or the number of base pairs (for example, for double-stranded molecules observed under native conditions) . Figure 4E shows DNA having a discontinuous structure. cDNA can be cut by a restriction endonuclease that has a single recognition site on the cDNA vector to produce two DNA fragments of different sizes (1 kb and 2 kb) under both neutral and denaturing conditions. Figure 4E also shows cccDNA having a linear and continuous structure. The cccDNA vector can be cut by a restriction endonuclease and generates two DNA fragments that migrate as 1 kb. and 2 kb. under neutral conditions, but under denaturing conditions, these chains remain associated and form single chains that migrate as 2 kb. and 4 kb.

[0080] Figure 5 shows an illustrative denaturing gel run of representative examples of cccDNA vectors with (+) or without (-) endonuclease digestion (EcoRI for ccDNA constructs 1 and 2; BamH1 for ccDNA constructs 3 and 4; SpeI for cccDNA constructs 5 and 6 and XhoI for cccDNA constructs 7 and 8). Constructs 1-8 are described in Example 1 of international application PCT/US18/49996, which is incorporated herein by reference in its entirety. The dimensions for stripes marked with an asterisk are defined and shown at the bottom of the drawing.

[0081] FIG. 6 is a graph showing the effect of repeated dosing (ie, boosting) to increase the expression level of a transgene from a cDNA luciferase expression vector present in a composition containing liposomes. Luciferase expression was measured after administration of the cccDNA vector as described in Example 6, and later reintroduction of the cccDNA vector derived from the cccDNA vector on day 84 or 87. Luciferase expression was assessed and detected in all three groups at least up to 132 days (the longest period of time was assessed). Figure 6 shows that at or near day 80, the level of transgene expression in mice receiving 1 mg/kg cccDNA vector in the presence of liposomes (LNP-ccDNA) decreases slightly. Reintroduction of the cccDNA vector in the presence of liposomes at day 84 or day 87 can be used to continue transgene expression at the desired preset level (data not shown) or to increase the expression level of the cccDNA transgene to a level higher than that achieved by the previous cccDNA injection -vector. This paper demonstrates a 7-fold increase in expression over the previous level of transgene expression by administering 3 mg/kg LNP-cccDNA vector (LNPceDNA), or a 17-fold increase in expression over the previous level of transgene expression by administering 10 mg/kg LNP-ccDNA vector compositions.

[0082] Figure 7 presents the results of the experiments described in Example 7 and, in particular, shows IVIS images obtained for mice treated with LNP-poly(C) control (leftmost mouse) and four LNP-treated mice. cDNA-luciferase (all mice except the leftmost one). Four mice treated with cccDNA showed significant fluorescence in the liver region.

[0083] Figure 8 shows the results of the experiment described in Example 8. Dark spots indicate the presence of protein derived from the cDNA-expressed transgene and demonstrate the association of the introduced LNP-ccDNA with hepatocytes.

[0084] Figures 9A-9B show the results of the eye studies described in Example 9. Figure 9A shows representative IVIS images of a JetPEI®-ccDNA-luciferase-injected rat eye (top left), compared to that eye the same rat without injection (top right), or the eye of a rat with an injection of luciferase plasmid DNA (bottom left), and the eye of the same rat without injection (bottom right). Figure 9 B shows a plot of the day-by-day brightness observed in the treated eyes or the corresponding untreated eyes in each of the experimental groups. The cccDNA-treated rats showed prolonged significant fluorescence (and therefore luciferase transgene expression) for 99 days, which was in sharp contrast with rats receiving the luciferase plasmid, which showed minimal relative fluorescence (and therefore luciferase transgene expression).

[0085] Figures 10 and 10B show the results of the persistence and repeat dosing studies of cccDNA in Rag2 mice described in Example 10. Figure 10A shows a plot of total flux versus time observed in LNP-ccDNA-Luc-treated c57bl/6 mice wild type or Rag2 mice. Figure 10B shows a graph showing the effect of repeat dosing on luciferase transgene expression levels in Rag2 mice, whereby increased stable expression was observed after repeat dosing (arrow indicates time of boost).

[0086] Figure 11 shows data from the cDNA luciferase expression studies in the experimental mice described in Example 11, showing the total flux in each group of mice over the course of the studies. High levels of unmethylated CpG correlated with lower total flux observed in mice over time, whereas liver-specific promoter usage correlated with prolonged stable expression of the transgene from the cccDNA vector for at least 77 days.

DETAILED DESCRIPTION OF THE INVENTION

[0087] Described herein are methods and compositions comprising novel capsidless, covalently closed-end DNA (ccDNA) vectors for controlled expression of a transgene, for example, to provide sustained expression of a desired transgene at a desired time and for a predetermined period of time, or to modulating the expression level of the transgene (including increasing the level of expression) in a cell, in vivo or in vitro, and the level of expression of the transgene can be increased by at least one (i.e., one or more) subsequent administrations (for example, boost administration or repeated dose).

[0088] The cccDNA vector and methods described herein allow the expression level of the transgene to be maintained in vitro and in vivo in a host cell or subject, i.e. maintain expression at the desired level or stop any decline in expression level by at least one repeat administration (herein also referred to as a repeat dose or boost) at some point in time after the initial administration.

[0089] The cccDNA vector and methods disclosed herein allow the level of transgene expression to be increased from pre-existing levels in vitro and in vivo, i.e. increase expression to or above the desired level, or increase the expression level to a value within the desired expression range, by at least one repeat administration (herein also referred to as a repeat dose or boost) at some point in time after the initial administration.

[0090] In other words, the expression of the transgene expressed by the cccDNA can be increased above the level from the previous administration. If the previous administration was an initial dose (ie, a priming dose), then a repeat dose at a second time point can be used to increase the level of transgene expression. Likewise, if the previous administration was a second administration (i.e., a booster dose), then an additional booster dose can be used to increase the level of transgene expression to a level higher than the previous booster dose, or to a desired level or range expression values. Thus, the technology, methods and cccDNA vector disclosed herein can be used to gradually, in a controlled manner, increase the expression level of a transgene to a desired level of expression. This stepwise and gradual increase in the level of transgene expression is advantageous for treating a subject because it allows the level of transgene expression in a particular individual to be titrated based on the needs of the subject and/or the effectiveness of the cccDNA vector and/or the expressed transgene (eg, genetic drug) in the subject, without the risk of administering an excessively high initial dose, higher than actually necessary, and/or without the immune complications associated with other AAV-based vectors.

Definitions

[0091] Unless otherwise defined herein, scientific and technical terms used in connection with this invention have the meanings commonly understood by those skilled in the art to which this invention pertains. It should be understood that the present invention is not limited to the specific methodology, protocols and reagents, etc. described herein and is therefore subject to variations. The terminology used herein is intended to describe specific embodiments only and is not intended to limit the scope of the invention, which is defined solely by the claims. Definitions of common immunology and molecular biology terms can be found in the following sources: The Merck Manual of Diagnosis and Therapy, 19th ed., published by Merck Sharp & Dohme Corp., 2011 (ISBN 978-0-911910-19-3); Robert S. Porter et al. (ed.), Fields Virology, 6th ed., published by Lippincott Williams & Wilkins, Philadelphia, PA, USA (2013), Knipe, D.M. and Howley, P.M. (ed.), The Encyclopedia of Molecular Cell Biology and Molecular Medicine, published by Blackwell Science Ltd., 1999-2012 (ISBN 9783527600908); and Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by VCH Publishers, Inc., 1995 (ISBN 1-56081-569-8); Immunology, Werner Luttmann, published by Elsevier, 2006; Janeway's Immunobiology, Kenneth Murphy, Allan Mowat, Casey Weaver (eds.), Taylor & Francis Limited, 2014 (ISBN 0815345305, 9780815345305); Lewin's Genes XI, published by Jones & Bartlett Publishers, 2014 (ISBN-1449659055); Michael Richard Green and Joseph Sambrook, Molecular Cloning: A Laboratory Manual, 4th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., USA (2012) (ISBN 1936113414); Davis et al., Basic Methods in Molecular Biology, Elsevier Science Publishing, Inc., New York, USA (2012) (ISBN 044460149X); Laboratory Methods in Enzymology: DNA, Jon Lorsch (ed.) Elsevier, 2013 (ISBN 0124199542); Current Protocols in Molecular Biology (CPMB), Frederick M. Ausubel (ed.), John Wiley and Sons, 2014 (ISBN 047150338X, 9780471503385), Current Protocols in Protein Science (CPPS), John E. Coligan (ed.), John Wiley and Sons, Inc., 2005; and Current Protocols in Immunology (CPI) (John E. Coligan, ADA M Kruisbeek, David H Margulies, Ethan M Shevach, Warren Strobe, (eds.) John Wiley and Sons, Inc., 2003 (ISBN 0471142735, 9780471142737), contents which are incorporated herein by reference in their entirety.

[0092] As used herein, the terms “heterologous nucleotide sequence” and “transgene” are used interchangeably and refer to a nucleic acid of interest (other than a nucleic acid encoding a capsid polypeptide) that is included in and can be delivered and expressed by a cccDNA vector , as described in this document. Transgenes of interest include, but are not limited to, nucleic acids encoding polypeptides, preferably therapeutic (eg, for medical, diagnostic or veterinary purposes) or immunogenic polypeptides (eg, for vaccines). In some embodiments, the nucleic acids of interest include nucleic acids that are transcribed into therapeutic RNA. Transgenes included for use in cccDNA vectors of the invention include, but are not limited to, those expressing or encoding one or more polypeptides, peptides, ribozymes, aptamers, peptide nucleic acids, siRNAs, RNAi, microRNAs, lncRNAs (long non-coding RNAs), antisense oligos - or polynucleotides, antibodies, antigen-binding fragments, or any combination thereof. A transgene may be a "genetic drug" and includes any of: inhibitor, nucleic acid, oligonucleotide, silencing nucleic acid, microRNA, RNAi, antagonist, agonist, polypeptide, peptide, antibody or antibody fragments, fusion proteins or variants thereof, epitopes , antigens, aptamers, ribosomes, etc. The transgene used herein in the cccDNA vector is not limited in size.

[0093] The term “genetic drug” as disclosed herein refers to any DNA structure or nucleic acid sequence that can be used to treat or prevent a disease or disorder in a subject.

[0094] As used herein, the terms "expression cassette" and "transcription cassette" are used interchangeably and refer to a linear stretch of nucleic acids that includes a transgene operably linked to one or more promoters or other regulatory sequences sufficient to direct transcription of the transgene, but does not include capsid coding sequences, other vector sequences, or inverted terminal repeat regions. The expression cassette may further comprise one or more cis-acting sequences (eg, promoters, enhancers, or repressors), one or more introns, and one or more post-transcriptional regulatory elements.

[0095] As used herein, the term “terminal repeat” or “CT” includes any viral terminal repeat or synthetic sequence that contains at least one minimum required origin of replication and a region containing a palindromic hairpin structure. The Rep binding sequence (RBS) (also called RBE (Rep binding element)) and the terminal resolution site (TRS) together constitute the "minimum required origin of replication" and, accordingly, the terminal repeat (TRS) contains at least one RBS and at least one TRS. Terminal repeats that are inversely complementary to each other within a given segment of a polynucleotide sequence are usually called “inverted terminal repeats,” or “ITRs.” In the context of a virus, ICPs mediate viral replication, packaging, integration, and proviral release. It has surprisingly been discovered by the present inventors that terminal repeats that are not reverse complementary throughout their entire length can still perform traditional ICP functions and thus the term ICP herein refers to a PC in a cccDNA genome or a cccDNA vector. , which is capable of mediating the replication of the cDNA vector. One skilled in the art will appreciate that cccDNA vectors of complex configuration may contain more than two pairs of ICPs or asymmetric ICPs. The ICP may be an AAV ICP or a non-AAV ICP, or may be derived from an AAV ICP or a non-AAV ICP. For example, the ICP may be from a virus in the Parvoviridae family, which includes parvoviruses and dependoviruses (e.g., canine parvovirus, bovine parvovirus, murine parvovirus, porcine parvovirus, human parvovirus B-19), or the SV40 hairpin serving as the SV40 origin of replication may be used as an ICP that can be further modified by truncation, substitution, deletion, insertion and/or addition. The Parvoviridae family of viruses consists of two subfamilies: Parvovirinae, which infect vertebrates, and Densovirinae, which infect invertebrates. Dependoparvoviruses include the adeno-associated virus (AAV) family of viruses that are capable of replication in vertebrate hosts, including, but not limited to, human, primate, bovine, canine, equine, and sheep species.

[0096] The terms "polynucleotide" and "nucleic acid", used interchangeably herein, refer to the polymeric form of nucleotides of any length, whether ribonucleotides or deoxyribonucleotides. Thus, the term includes single-, double- or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or a polymer comprising purine and pyrimidine bases or other natural, chemically or biochemically modified, non-natural or derivatized nucleotide bases. "Oligonucleotide" generally refers to polynucleotides containing from about 5 to about 100 nucleotides of single- or double-stranded DNA. However, for the purposes of this invention, there is no upper limit on the length of an oligonucleotide. Oligonucleotides are also known as “oligomers” or “oligos” and can be isolated from genes or chemically synthesized by methods known in the art. The terms “polynucleotide” and “nucleic acid” should be understood to include, for the purposes of the described embodiments, single-stranded (such as sense or antisense) and double-stranded polynucleotides.

[0097] As used herein, the term “nucleic acid construct” refers to a nucleic acid molecule, single-stranded or double-stranded, that is isolated from a naturally occurring gene, or is modified to contain nucleic acid segments in a manner not found in nature, or is synthetic. The term "nucleic acid construct" is synonymous with the term "expression cassette" when the nucleic acid construct contains control sequences necessary for expression of the coding sequence of the present invention. An "expression cassette" includes a DNA coding sequence operably linked to a promoter.

[0098] By "hybridizable" or "complementary" or "substantially complementary" is meant that the nucleic acid (eg, RNA) includes a nucleotide sequence that allows it to bind non-covalently, i.e. form Watson-Crick base pairs and/or G/U base pairs, undergo "annealing" or "hybridize" with another nucleic acid in a sequence-specific, antiparallel manner (i.e., the nucleic acid specifically binds to a complementary nucleic acid) in suitable in vitro and/or in vivo conditions of temperature and ionic strength of the solution. As is known in the art, standard Watson-Crick base pairing includes adenine (A) pairing with thymidine (T), adenine (A) pairing with uracil (U), and guanine (G) pairing with cytosine (C). In addition, it is also known in the art that when two RNA molecules (eg, dsRNA) are hybridized, a guanine (G) base is paired with a uracil (U). For example, G/U base pairing is partially responsible for the degeneracy (i.e., redundancy) of the genetic code in the context of the base pairing of tRNA anti-codons with codons in mRNA. In the context of the present invention, the guanine (G) of the protein-binding segment (dsRNA duplex) of the DNA-targeting RNA molecule of the present invention is considered to be complementary to uracil (U), and vice versa. Thus, when a G/U base pair can be obtained at a given nucleotide position with the protein binding segment (dsRNA duplex) of the DNA-targeting RNA molecule of the present invention, this position is not considered to be non-complementary, but instead is considered to be complementary.

[0099] The terms "peptide", "polypeptide" and "protein" are used interchangeably herein and refer to the polymeric form of amino acids of any length, which may include encoded and non-encoded amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides having modified peptide backbones.

[00100] A DNA sequence that "encodes" a particular RNA, or protein gene product, is a DNA nucleic acid sequence that is transcribed into a particular RNA and/or protein. A DNA polynucleotide may encode RNA (mRNA) that is translated into a protein, or a DNA polynucleotide may encode RNA that is not translated into a protein (eg, tRNA, rRNA, or DNA-targeting RNA; also called “non-coding” RNA or “ncRNA”).

[00101] As used herein, the term “genomic safe harbor gene” or “safe harbor gene” refers to a gene or loci into which a nucleic acid sequence can be inserted such that the sequence can integrate and function in a predictable manner (e.g., to express a protein, of interest) without significant negative consequences for endogenous gene activity or cancer development. In some embodiments, a safe harbor gene also represents loci or a gene at which the inserted nucleic acid sequence can be expressed efficiently and at higher levels than at a non-safe harbor site.

[00102] As used herein, the term “gene delivery” refers to the process by which foreign DNA is transferred into host cells for gene therapy use.

[00103] As used herein, the term “terminal repeat” or “CT” includes any viral terminal repeat or synthetic sequence that contains at least one minimum required origin of replication and a region containing a palindromic hairpin structure. The Rep binding sequence (RBS) (also called RBE (Rep binding element)) and the terminal resolution site (TRS) together constitute the "minimum required origin of replication" and thus the terminal repeat (TRS) contains at least one RBS and at least one TRS. Terminal repeats that are inversely complementary to each other within a given segment of a polynucleotide sequence are usually called “inverted terminal repeats,” or “ITRs.” In the context of a virus, ICPs mediate viral replication, packaging, integration, and proviral release. It has surprisingly been discovered by the present inventors that terminal repeats that are not reverse complementary throughout their entire length can still perform traditional ICP functions and thus the term ICP herein refers to a PC in a cccDNA genome or a cccDNA vector. , which is capable of mediating the replication of the cDNA vector. One skilled in the art will appreciate that cccDNA vectors of complex configuration may contain more than two pairs of ICPs or asymmetric ICPs. The ICP may be an AAV ICP or a non-AAV ICP, or may be derived from an AAV ICP or a non-AAV ICP. For example, the ICP may be from a virus in the Parvoviridae family, which includes parvoviruses and dependoviruses (e.g., canine parvovirus, bovine parvovirus, murine parvovirus, porcine parvovirus, human parvovirus B-19), or the SV40 hairpin serving as the SV40 origin of replication may be used as an ICP that can be further modified by truncation, substitution, deletion, insertion and/or addition. The Parvoviridae family of viruses consists of two subfamilies: Parvovirinae, which infect vertebrates, and Densovirinae, which infect invertebrates. Dependoparvoviruses include the adeno-associated virus (AAV) family of viruses that are capable of replication in vertebrate hosts, including, but not limited to, human, primate, bovine, canine, equine, and sheep species. For convenience, in this document the ICP located 5' from (upstream) the expression cassette in a cccDNA vector is referred to as the "5'-ICP" or "left ICP", and the ICP located 3' from (downstream) the expression cassette in the cDNA vector is designated as “3'-IKP” or “right IKP”.

[00104] "Wild-type ICP" or "WT ICP" refers to a naturally occurring ICP sequence from an AAV or other dependovirus that retains, for example, Rep binding activity and Rep nicking ability. Nucleotide sequences of ICP-WT from any AAV serotype may differ slightly from the canonical natural sequence due to degeneracy or drift of the genetic code and, therefore, IKP-DT sequences suitable for use in this invention include IKP-DT sequences resulting from natural changes occurring during production (for example, replication errors).

[00105] As used herein, the term “substantially symmetrical WT ICPs” or “substantially symmetrical WT ICP pair” refers to a pair of WT ICPs in a single cccDNA genome or ccDNA vector that are both wild-type ICPs having reverse complementary sequences along their entire length. For example, an ICP may be considered a wild-type sequence even if it has one or more nucleotides that deviate from the canonical natural sequence, provided that these changes do not affect the properties and overall three-dimensional structure of the sequence. In some aspects, deviant nucleotides are conserved sequence changes. As one non-limiting example, a sequence having a degree of sequence identity of at least 95%, 96%, 97%, 98%, or 99% to the canonical sequence (measured, for example, using BLAST at default settings), and also having symmetric three-dimensional spatial organization with another ICP-DT, so that their three-dimensional structures have the same shape in geometric space. Essentially symmetrical ICP-DTs have identical loops A, C-C' and B-B' in three-dimensional space. An essentially symmetric IKP-WT can be functionally confirmed as wild type (WT) by determining that it has a functional Rep binding site (RBE or RBE') and a terminal resolution site (trs) that binds to the corresponding Rep protein. further test other functions, including transgene expression under permissive conditions.

[00106] As used herein, the phrases "modified ICP" or "mod-ICP" or "mutant ICP" are used interchangeably and refer to an ICP that has a mutation in at least one or more nucleotides compared to the WT ICP from the same serotype. The mutation may result in a change in one or more of the regions A, C, C', B, B' in the ICP, and may result in a change in three-dimensional spatial organization (ie, its three-dimensional structure in geometric space). compared with the three-dimensional spatial organization of ICP-DT of the same serotype.

[00107] As used herein, the term “asymmetric ICPs,” also referred to as “asymmetric ICP pairs,” refers to a pair of ICPs in the same cccDNA genome or ccDNA vector that are not reverse complements in their full length. The sequence difference between the two ICPs may be due to nucleotide addition, deletion, truncation, or point mutation. In one embodiment, one ICP of a pair may be a wild-type AAV sequence and the other a non-wild-type or synthetic sequence. In another embodiment, neither ICP of the pair is a wild-type AAV sequence, and the sequences of the two ICPs are different from each other. For convenience, in this document the ICP located 5' from (upstream) the expression cassette in a cccDNA vector is referred to as the "5'-ICP" or "left ICP", and the ICP located 3' from (downstream) the expression cassette in the cDNA vector is designated as “3'-IKP” or “right IKP”. As one non-limiting example, in an asymmetric pair, the ICPs do not have a symmetrical three-dimensional spatial organization with their partner ICPs, and their three-dimensional structures have different shapes in geometric space. In other words, an asymmetric pair of ICPs has different overall geometric structures, i.e. they have different organization of their A, C-C' and B-B' loops in three-dimensional space (for example, one ICP may have a short C-C' arm and/or a short B-B' arm compared to a related ICP). The sequence difference between two ICPs may be due to addition, deletion, truncation, or point mutation of one or more nucleotides. In one embodiment, one PKI of an asymmetric PKI pair may be a wild-type AAV PKI sequence and the other PKI a modified PKI as defined herein (eg, a non-wild type or synthetic PKI sequence). In another embodiment, neither of the ICPs of an asymmetric pair of ICPs is a wild-type AAV sequence, and the two ICPs are modified ICPs that have different shapes in geometric space (ie, a different overall geometric structure). In some embodiments, one mod ICP of an asymmetric ICP pair may have a short C-C' arm, and the other ICP may have a different modification (e.g., a single arm or a short B-B' arm, etc.), such that they have a different three-dimensional spatial organization compared to the related asymmetric mod-ICP.

[00108] As used herein, the term “symmetrical ICPs” refers to a pair of ICPs in a single cccDNA genome or cccDNA vector that are mutated or modified relative to wild-type dependoviral ICP sequences and are reverse complementary throughout their entire length. None of the ICPs are the wild-type AAV2 ICP sequence (ie, they are modified ICPs, also called mutant ICPs), and may differ from the wild-type ICP sequence due to addition, deletion, substitution, truncation, or point mutation of nucleotides. For convenience, in this document the ICP located 5' from (upstream) the expression cassette in a cccDNA vector is referred to as the "5'-ICP" or "left ICP", and the ICP located 3' from (downstream) the expression cassette in the cDNA vector is designated as “3'-IKP” or “right IKP”.

[00109] As used herein, the term “substantially symmetric modified ICPs” or “substantially symmetric mod-ICP pair” refers to a pair of modified ICPs in a single ccDNA genome or ccDNA vector that both have reverse complementary sequences throughout their entire length. For example, a modified ICP may be considered substantially symmetrical even if it has some nucleotide sequences that deviate from the reverse complementary sequence, as long as these changes do not affect the properties and overall shape. As one non-limiting example, a sequence having a degree of sequence identity of at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% to the canonical sequence (measured using BLAST at default settings) also has a symmetrical three-dimensional spatial organization with its related modified ICP such that their three-dimensional structures have the same shape in geometric space. In other words, an essentially symmetrical pair of modified ICPs has the same organization of loops A, C-C' and B-B' in three-dimensional space. In some embodiments, ICPs from a mod-ICP pair may have different reverse complementary nucleotide sequences, but still have the same symmetrical three-dimensional spatial organization, i.e. both ICPs have mutations that result in the same overall three-dimensional shape. For example, one ICP (e.g., 5'-ICP) in a mod-ICP pair may belong to one serotype and the other ICP (e.g., 3'-ICP) to a different serotype, but both may have the same corresponding mutations (e.g., if 5'-IKP has a deletion in region C, then a related modified 3'-IKP of another serotype has a deletion at the corresponding position in region C'), so that the specified modified pair of IKP has the same symmetrical three-dimensional spatial organization. In such embodiments, each ICP in a pair of modified ICPs may belong to different serotypes (e.g., AAB1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12), such as a combination of AAB2 and AAB6, wherein the modification of one ICP is displayed in the corresponding position of the related ICP of another serotype. In one embodiment, a pair of substantially symmetrical modified ICPs refers to a pair of modified ICPs (mod-ICPs), provided that the difference in nucleotide sequences between the ICPs does not affect the properties or overall shape and they have substantially the same shape in three-dimensional space. As a non-limiting example, a mod-ICP having a degree of sequence identity of at least 95%, 96%, 97%, 98%, or 99% to a canonical mod-ICP, as determined by standard means well known to those skilled in the art, such as BLAST (Basic Local Alignment Search Tool) or BLASTN, with default settings, also has a symmetrical 3D spatial organization, so that their 3D structures have the same shape in geometric space. An essentially symmetric mod-IKP pair has identical A, C-C' and B-B' loops in three-dimensional space, e.g., if a modified IKP in an essentially symmetric mod-IKP pair has a deletion of the C-C' arm, then the cognate mod-IKP has a corresponding deletion loops C-C', and also has a similar three-dimensional structure of the remaining A and B-B' loops with the same shape in geometric space with its related mode-ICP.

[00110] The term "flanking" refers to the relative position of one nucleic acid sequence with respect to another nucleic acid sequence. In general, in the ABC sequence, A and C flank B. The same is true for the AxBxC arrangement. Thus, the flanking sequence precedes or follows the flanking sequence, but need not be adjacent to, or immediately adjacent to, the flanking sequence. In one embodiment, the term flanking refers to the terminal repeats at each end of a linear duplex ccDNA vector.

[00111] As used herein, the term “ccDNA genome” refers to an expression cassette that further includes at least one inverted terminal repeat region. The cccDNA genome may further comprise one or more spacer regions. In some embodiments, the cccDNA genome is included as an intermolecular duplex DNA polynucleotide in a plasmid or viral genome.

[00112] As used herein, the term “ccDNA spacer region” refers to an intervening sequence that separates functional elements in a specified cccDNA vector or ccDNA genome. In some embodiments, cccDNA spacer regions hold two functional elements at the required distance for optimal functionality. In some embodiments, the cDNA spacer regions provide or increase the genetic stability of the cDNA genome within, for example, a plasmid or baculovirus. In some embodiments, cccDNA spacer regions facilitate prepared genetic manipulation of the cccDNA genome by providing convenient locations for cloning sites and the like. For example, in some aspects, an oligonucleotide "polylinker" containing multiple restriction endonuclease sites or a non-open reading frame sequence designed so , so that it does not contain known protein (e.g., transcription factor) binding sites, can be placed in the cccDNA genome to separate cis-acting factors, e.g., with insertions of 6-mer, 12-mer, 18-mer, 24-mer, 48 -mer, 86-mer, 176-mer, etc., between the end resolution site and the 5'-located transcription regulatory element. Likewise, a spacer may be included between the polyadenylation signal sequence and the 3'-terminal resolution site.

[00113] As used herein, the terms “Rep binding site,” “Rep binding element,” “RBE,” and “RBS” are used interchangeably and refer to the binding site of a Rep protein (e.g., Rep 78 AAB or Rep 68 AAB) that upon binding of the Rep protein, allows the Rep protein to exert its site-specific endonuclease activity against the sequence including the RBS. The RBS sequence and its reverse complementary sequence together form one RBS. RBS sequences are known in the art and include, for example, 5'-GCGCGCTCGCTCGCTC-3' (SEQ ID NO: 60), which is the RBS sequence identified in AAB2. Any known RBS sequence can be used in embodiments of the present invention, including other known AAB RBS sequences and other known natural or synthetic RBS sequences. Without being bound by any theory, it is believed that the nuclease domain of the Rep protein binds to the duplex nucleotide sequence GCTC and, accordingly, direct binding and stable assembly of the two known Rep proteins of AAB on the duplex oligonucleotide 5'-(GCGC)(GCTC)(GCTC) occurs. (GCTC)-3' (SEQ ID NO: 60). In addition, soluble aggregated conformers (i.e., an indefinite number of interconnected Rep proteins) dissociate and bind to oligonucleotides that contain Rep binding sites. Each Rep protein interacts with both the nitrogenous bases and the phosphodiester backbone of each chain. Interactions with nitrogenous bases provide sequence specificity, whereas interactions with the phosphodiester backbone are nonspecific or less sequence specific and stabilize the protein-DNA complex.

[00114] As used herein, the terms "terminal resolution site" and "TRS" are used interchangeably and refer to the region in which Rep forms a tyrosine phosphodiester bond with a 5'-thymidine to form a 3'-OH, which serves as a substrate for elongation DNA using a cellular DNA polymerase, such as DNA polymerase delta or DNA polymerase epsilon. Alternatively, the Rep-thymidine complex may participate in a coordinated ligation reaction. In some embodiments, the TRS spans at least the unpaired thymidine base. In some embodiments, the nicking efficiency of the TRS can be controlled, at least in part, by the distance between it and the RBS within a single molecule. When the acceptor substrate is complementary to the ICP, the resulting product is an intramolecular duplex. TRS sequences are known in the art and include, for example, 5'-GGTTGA-3' (SEQ ID NO: 61), which is a hexanucleotide sequence identified in AAB2 . Any known TRS sequence can be used in embodiments of the present invention, including other known AAB TRS sequences and other known natural or synthetic TRS sequences, such as AGTT (SEQ ID NO: 62), GGTTGG (SEQ ID NO: 63), AGTTGG ( SEQ ID NO: 64), AGTTGA (SEQ ID NO: 65) and other motifs such as RRTTRR (SEQ ID NO: 66).

[00115] As used herein, the term “cDNA plasmid” refers to a plasmid that contains the cDNA genome as an intermolecular duplex.

[00116] As used herein, the term “ccDNA bacmid” refers to an infectious baculovirus genome comprising a cccDNA genome as an intermolecular duplex capable of propagating in E. coli as a plasmid and thus serving as a shuttle vector for the baculovirus.

[00117] As used herein, the term “ccDNA baculovirus” refers to a baculovirus that contains the cccDNA genome as an intermolecular duplex within the baculovirus genome.

[00118] As used herein, the terms “ccDNA-baculovirus-infected insect cell” and “ccDNA-BIIC” are used interchangeably and refer to an invertebrate animal host cell (including, without limitation, an insect cell (e.g., an Sf9 cell)) infected cDNA baculovirus.

[00119] As used herein, the term “closed-end DNA vector” refers to a capsidless DNA vector with at least one covalently closed end, wherein at least a portion of the vector has an intramolecular duplex structure.

[00120] As used herein, the terms “ccDNA vector” and “ccDNA” are used interchangeably and refer to a closed-ended DNA vector containing at least one terminal palindrome. In some embodiments, the cccDNA contains two covalently closed ends.

[00121] As defined herein, “reporters” refer to proteins that can be used to provide detectable readouts. Reporters typically produce a measurable signal, such as fluorescence, color, or luminescence. The coding sequences of reporter proteins encode proteins whose presence in a cell or organism can be easily observed. For example, fluorescent proteins cause a cell to fluoresce when excited by light of a specific wavelength, luciferases cause the cell to catalyze a reaction that generates light, and enzymes such as β-galactosidase convert a substrate into a colored product. Typical examples of reporter polypeptides useful for experimental or diagnostic purposes include, but are not limited to, β-lactamase, β-galactosidase (LacZ), alkaline phosphatase (ALP), thymidine kinase (TK), green fluorescent protein (GFP) and other fluorescent proteins, chloramphenicol acetyltransferase (ChAT), luciferase and others, well known in the art.

[00122] As used herein, the term "effector protein" refers to a polypeptide that provides a detectable readout, either, for example, as a reporter polypeptide, or, more preferably, as a cell-killing polypeptide, such as a toxin, or a cell-sensing agent. to killing by a specific agent or in the absence of a specific agent. Effector proteins include any protein or peptide that directly targets or damages the DNA and/or RNA of a host cell. For example, effector proteins may include, but are not limited to, a restriction endonuclease that targets a host cell DNA sequence (genomic or extrachromosomal element), a protease that cleaves a target polypeptide essential for cell survival, a DNA gyrase inhibitor, and a ribonuclease-type toxin. In some embodiments, effector protein expression controlled by a synthetic biological circuit as described herein may participate as a factor in another synthetic biological circuit, thereby expanding the range and complexity of the response of the biological circuit system.

[00123] Transcription regulators refer to transcriptional activators and repressors that either activate or repress transcription of a gene of interest. Promoters are regions of nucleic acid that initiate transcription of a particular gene. Transcription activators typically bind in the vicinity of transcriptional promoters and recruit RNA polymerase to directly initiate transcription. Repressors bind to transcriptional promoters and sterically interfere with the initiation of transcription by RNA polymerase. Other transcriptional regulators can serve as either an activator or a repressor depending on where they bind and the conditions in the cell and environment. Non-limiting examples of classes of transcription regulators include, but are not limited to, homeodomain proteins, zinc finger proteins, forkhead proteins, and leucine zipper proteins.

[00124] As used herein, a “repressor protein” or “inducer protein” is a protein that binds to a regulatory sequence element and represses or activates, respectively, the transcription of sequences operably associated with said regulatory sequence element. Preferred repressor and inducer proteins as described herein are sensitive to the presence or absence of at least one administered agent or environmental input. Preferred proteins, as described herein, are modular in form and contain, for example, detachable DNA-binding and binding input agents, or elements or domains responsive thereto.

[00125] As used herein, the term “carrier” includes any and all possible solvents, dispersion media, bases, coatings, diluents, antibacterial and antifungal agents, isotonic and absorption retarding agents, buffers, carrier solutions, suspensions, colloids, etc. n. The use of such media and agents for pharmaceutically active substances is well known in the art. Additional active ingredients may also be included in these compositions. The expression "pharmaceutically acceptable" refers to molecular entities and compositions that do not result in a toxic, allergic or similar adverse reaction when administered to a host.

[00126] As used herein, the term “input agent responsive domain” is a transcription factor domain that binds or otherwise responds to a condition or input agent by causing the DNA binding fusion domain attached thereto to respond to the presence of the condition or input impact. In one embodiment, the presence of a condition or input leads to a conformational change in the responsive domain of the input agent or its fusion protein, which modifies the transcription-modulating activity of the transcription factor.

[00127] The term "in vivo" refers to assays or processes that occur in or within an organism, such as a multicellular animal. In some aspects described herein, the method or use may be referred to as occurring "in vivo" when using a single cell organism such as a bacterium. The term "ex vivo" refers to methods and applications that are carried out using a living cell with an intact membrane located outside the body of a multicellular animal or plant, for example, explants, cultured cells, including primary cells and cell lines, transformed cell lines, and extracted tissues or cells, including blood cells, etc. The term "in vitro" refers to assays and methods that do not require the presence of a cell with an intact membrane, such as those performed on cell extracts, and may refer to the introduction of a programmable synthetic biological circuit into a non-cellular system, such as a medium containing no cells or cellular systems, such as cell extracts.

[00128] As used herein, the term “promoter” refers to any nucleic acid sequence that regulates the expression of another nucleic acid sequence by directing transcription of said nucleic acid sequence, which may be a heterologous protein-encoding target gene or RNA. Promoters can be constitutive, inducible, repressible, tissue specific, or any combination thereof. A promoter is a control region of a nucleic acid sequence by which the initiation and level of transcription of the remainder of the nucleic acid sequence is controlled. The promoter may also contain genetic elements in which the binding of regulatory proteins and molecules, such as RNA polymerase, and other transcription factors, can occur. In some embodiments of the aspects described herein, the promoter may drive the expression of a transcription factor that regulates the expression of the promoter itself, or another promoter used in another modular component of the synthetic biological circuits described herein. The promoter sequence will contain a transcription initiation site, as well as protein binding domains responsible for binding RNA polymerase. Eukaryotic promoters often, but not always, contain TATA boxes and CAT boxes. Various promoters, including inducible promoters, can be used to drive the expression of transgenes in the cDNA vectors described herein. The promoter sequence may be limited at its 3' end by the transcription initiation site and extend in an upstream direction (towards the 5' end), including the minimum number of bases or elements necessary to initiate transcription at levels detected above background.

[00129] As used herein, the term “enhancer” refers to a cis-acting regulatory sequence (e.g., 50-1500 base pairs) that binds one or more proteins (e.g., activator proteins or transcription factor) to enhance transcriptional activation of a nucleic acid sequence acids. Enhancers can be located up to 1,000 base pairs upstream of the start site of the gene or downstream of the start site of the gene they regulate. An enhancer can be located in the intronic region or in the exonic region of an unrelated gene.

[00130] A promoter can be said to drive the expression or direct transcription of the nucleic acid sequence that it regulates. The expressions "operably linked", "operably located", "operably linked", "under control" and "under transcriptional control" indicate that the promoter is in the correct functional position and/or orientation relative to the nucleic acid sequence that it regulates to control the initiation of transcription and/or expression of that sequence. "Inverted promoter" as used herein refers to a promoter whose nucleic acid sequence is reversed so that the coding strand becomes non-coding and vice versa. Inverted promoter sequences can be used in various embodiments to regulate the state of a switch. Additionally, in various embodiments, a promoter may be used in combination with an enhancer.

[00131] A promoter can be naturally associated with a gene or sequence, which can be achieved by isolating 5' non-coding sequences located upstream of the coding segment and/or exon of the gene or sequence. Such a promoter may be called "endogenous". Likewise, in some embodiments, an enhancer may be naturally associated with a nucleic acid sequence and located either after or before the specified sequence.

[00132] In some embodiments, a coding segment of a nucleic acid is placed under the control of a “recombinant promoter” or a “heterologous promoter,” both terms referring to a promoter that is not normally associated with the coding sequence of the nucleic acid to which it is operably linked in its natural environmental conditions. A recombinant or heterologous enhancer refers to an enhancer that is not normally associated with a given nucleic acid sequence in its natural environment. Such promoters or enhancers may include promoters or enhancers of other genes; promoters or enhancers isolated from any other prokaryotic, viral or eukaryotic cell; and synthetic promoters or enhancers that are not “naturally occurring”, i.e. contain various elements of different transcriptional regulatory regions and/or expression-altering mutations introduced using genetic engineering methods known in the art. In addition to obtaining promoter and enhancer nucleic acid sequences synthetically, promoter sequences can be obtained using recombinant cloning and/or nucleic acid amplification technology, including PCR, in relation to the synthetic biological circuits and modules disclosed herein (see, for example, US Patent No. 4683202, US Patent No. 5928906, each of which is incorporated herein by reference). In addition, it is also possible to use control sequences that direct transcription and/or expression of sequences in non-nuclear organelles such as mitochondria, chloroplasts, and the like.

[00133] As described herein, an “inducible promoter” is a promoter that is characterized by initiation or enhancement of transcriptional activity in the presence of, under the influence of, or upon contact with, an inducer or inducing agent. An “inducer” or “inducing agent” as defined herein can be a normally endogenous or exogenous compound or protein that is administered so as to have the activity of inducing the transcriptional activity of an inducible promoter. In some embodiments, the inducer or inducing agent, i.e. the chemical, compound, or protein may itself be produced by the transcription or expression of a nucleic acid sequence (ie, the inducer may be an inducer protein expressed by another component or module), which may itself be under the control of an inducible promoter. In some embodiments, the inducible promoter is induced in the absence of certain agents, such as a repressor. Examples of inducible promoters include, but are not limited to, tetracycline, metallothioneine, ecdysone, mammalian viruses (eg, adenovirus late promoter and murine mammary tumor virus long terminal repeat (MMTV-LTR)) and other steroid-responsive promoters, rapamycin-responsive promoters, etc. .P.

[00134] The terms "DNA regulatory sequences", "control elements" and "regulatory elements", used interchangeably herein, refer to transcriptional and translational control sequences such as promoters, enhancers, polyadenylation signals, terminators, protein degradation signals, etc. .p. that mediate and/or regulate transcription of a non-coding sequence (eg, DNA-targeting RNA) or coding sequence (eg, site-directed modifying polypeptide or Cas9/Csn1 polypeptide) and/or regulate translation of the encoded polypeptide.

[00135] The term "operably coupled" refers to an arrangement in close proximity in which the described components are in a relationship that allows them to function as intended. For example, a promoter is operably linked to a coding sequence if the promoter affects its transcription or expression. An "expression cassette" includes a heterologous DNA sequence that is operably linked to a promoter or other regulatory sequence sufficient to direct transcription of the transgene in the cDNA vector. Suitable promoters include, for example, tissue-specific promoters. Promoters may also be derived from AAV.

[00136] As used herein, the term “subject” refers to a human or animal being treated, including prophylactic treatment, with the cccDNA vector of the present invention. Typically, said animal is a vertebrate animal, such as, but not limited to, a primate, rodent, pet or game animal. Primates include, but are not limited to, chimpanzees, cynomolgus monkeys, coatats, and macaques such as rhesus monkeys. Rodents include mice, rats, marmots, ferrets, rabbits and hamsters. Domestic and game animals include, but are not limited to, cows, horses, pigs, deer, bison, buffalo, felines such as the domestic cat, canid species such as dogs, foxes, wolves, bird species such as chickens, emus, ostriches , and fish such as trout, catfish and salmon. In certain embodiments of the aspects described herein, the subject is a mammal, such as a primate or a human. The subject may be male or female. In addition, the subject may be an infant or child. In some embodiments, the subject may be a newborn or unborn subject, for example, an in utero subject. Preferably, the subject is a mammal. Said mammal may be a human, non-human primate, mouse, rat, dog, cat, horse or cow, but is not limited to these examples. Mammals other than humans can be advantageously used as subjects that represent animal models of diseases and disorders. In addition, the methods and compositions described herein can be used in domesticated animals and/or pets. A person can be of any age, gender, belong to any race or ethnic group, for example, he can be Caucasian (white), Asian, African, black, African American, Afro-European, Hispanic, Middle Eastern, etc. In some embodiments, the subject may be a patient or other subject in a clinical setting. In some embodiments, the subject is already undergoing treatment. In some embodiments, the subject is an embryo, fetus, newborn, infant, child, adolescent, or adult. In some embodiments, the subject is a human fetus, a newborn, an infant, a child, an adolescent, or an adult. In some embodiments, the subject is an animal embryo or a non-human embryo or a non-human primate embryo. In some embodiments, the subject is a human embryo.

[00137] As used herein, the term “host cell” includes any type of cell that is susceptible to transformation, transfection, transduction, or the like, using a nucleic acid construct or cDNA expression vector of the present invention. By way of non-limiting examples, the host cell may be an isolated primary cell, pluripotent stem cells, CD34 ⁺ cells), induced pluripotent stem cells, or any of a number of immortalized cell lines (eg, HepG2 cells). Alternatively, the host cell may be an in situ or in vivo cell in a tissue, organ, or organism.

[00138] The term "exogenous" refers to a substance present in a cell other than its natural source. The term “exogenous” as used herein can refer to a nucleic acid (e.g., a nucleic acid encoding a polypeptide) or polypeptide that has been introduced through a human process into a biological system, such as a cell or organism, in which it usually undetectable, and introduction of the nucleic acid or polypeptide into such a cell or organism is desirable. Alternatively, "exogenous" may refer to a nucleic acid or polypeptide that has been introduced through a human-related process into a biological system, such as a cell or organism, in which it is present in relatively small quantities, with an increase in the amount of the nucleic acid or polypeptide in a cell or organism is desired, for example, to create ectopic expression or levels. In contrast, the term "endogenous" refers to a substance that is native to a biological system or cell.

[00139] The term "sequence identity" refers to the affinity between two nucleotide sequences. For the purposes of this invention, the degree of sequence identity between two deoxyribonucleotide sequences is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, supra), implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000 above), preferably version 3.0.0 or later. The optional parameters used are a gap opening penalty of 10, a gap expansion penalty of 0.5, and the substitution matrix EDNAFULL (EMBOSS NCBI NUC4.4 version). The Needle output parameter, designated "longest identity" (obtained by using the -nobrief parameter), is applied as a percentage identity and is calculated as follows: (Identical deoxyribonucleotides x 100) / (Alignment length - Total number of gaps in alignment). The length of the alignment is preferably at least 10 nucleotides, preferably at least 25 nucleotides, more preferably at least 50 nucleotides, and most preferably at least 100 nucleotides.

[00140] The term "homology" or "homologous" as used herein is defined as the percentage of nucleotide residues that are identical to nucleotide residues in the corresponding sequence on the target chromosome after sequence alignment and the introduction of gaps, if necessary, to achieve maximum percentage sequence identity . Alignment to determine percent homology of nucleotide sequences can be accomplished in a variety of ways known to those skilled in the art, for example, using publicly available computer software such as the BLAST, BLAST-2, ALIGN, ClustalW2, or Megalign (DNASTAR) applications. Those skilled in the art can determine appropriate parameters for sequence alignment, including any algorithms necessary to achieve maximum alignment over the entire length of the sequences being compared. In some embodiments, a nucleic acid sequence (e.g., a DNA sequence), such as a homologous arm, is considered “homologous” when the sequence is at least 70%, at least 75%, at least 80%, at least 85 %, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more identical to the corresponding native or unedited nucleic acid sequence (eg, genomic sequence) of the host cell.

[00141] The term “heterologous” as used herein means a nucleotide or polypeptide sequence that is not found in the native nucleic acid or protein, respectively. A heterologous nucleic acid sequence can be linked to a naturally occurring nucleic acid sequence (or variant thereof) (eg, by genetic engineering) to produce a chimeric nucleotide sequence encoding a chimeric polypeptide. A heterologous nucleic acid sequence can be linked to a variant polypeptide (eg, by genetic engineering) to produce a nucleotide sequence encoding the variant fusion polypeptide.

[00142] A "vector" or "expression vector" is a replicon, such as a plasmid, bacmid, phage, virus, virion or cosmid, to which another DNA segment can be attached, i.e. "insertion" to cause the attached segment to replicate in the cell. A vector may be a nucleic acid construct designed for delivery into a host cell or for transfer between different host cells. As used herein, a vector may be viral or non-viral in origin and/or final form, however, for purposes of this invention, “vector” generally refers to a cccDNA vector as that term is used herein. The term "vector" covers any genetic element that is capable of replication when associated with appropriate control elements and that can transfer gene sequences into cells. In some embodiments, the vector may be an expression vector or a recombinant vector.

[00143] As used herein, the term “expression vector” refers to a vector that directs the expression of RNA or polypeptide from sequences linked to transcriptional control sequences in the vector. The expressed sequences will often, but not necessarily, be heterologous to the cell. The expression vector may contain additional elements, for example, the expression vector may have two replication systems, allowing it to be maintained in two organisms, for example, human cells for expression and a prokaryotic host for cloning and amplification. The term “expression” refers to cellular processes involved in the production of RNA and proteins and, as appropriate, the secretion of proteins, including, where applicable, but not limited to, for example, transcription, transcript processing, translation and protein folding, modification and processing. "Expression products" include RNA transcribed from a gene and polypeptides produced by translation of mRNA transcribed from the gene. The term "gene" means a nucleic acid sequence that is transcribed (DNA) into RNA in vitro or in vivo when operably linked to appropriate regulatory sequences. A gene may or may not include regions preceding and following the coding region, such as 5'-untranslated (5'-UTR) or "leader" sequences and 3'-UTR or "trailer" sequences, as well as intervening sequences (introns) between individual coding segments (exons).

[00144] By “recombinant vector” is meant a vector that contains a heterologous nucleic acid sequence, or “transgene,” that is capable of expression in vivo. It should be understood that the vectors described herein, in some embodiments, can be combined with other suitable compositions and therapies. In some embodiments, the vector is episomal. The use of a suitable episomal vector provides a means of maintaining the subject's nucleotide of interest in multicopy extrachromosomal DNA, thereby eliminating the potential effects of chromosome integration.

[00145] As used herein, the expression “genetic disease” refers to a disease, partially or wholly, directly or indirectly, caused by one or more abnormalities in the genome, especially a condition present from birth. The abnormality may be a mutation, insertion, or deletion. The abnormality may affect the coding sequence of a gene or its regulatory sequence. The genetic disease may be, but is not limited to, DMD (Duchenne muscular dystrophy), hemophilia, cystic fibrosis, Huntington's chorea, familial hypercholesterolemia (LDL receptor defect), hepatoblastoma, Wilson's disease, congenital hepatic porphyria, inherited liver metabolic disorders, Lesch-Nyhan syndrome, sickle cell anemia, thalassemia, xeroderma pigmentosum, Fanconi anemia, retinitis pigmentosa, ataxia-telangiectasia, Bloom's syndrome, retinoblastoma and Tay-Sachs disease.

[00146] As used herein, the term “biomarker” means any analyte or composition that can be used to identify a condition (eg, a disease) or the status of a condition (eg, stage of a disease) in a subject or sample. The biomarker, in some examples disclosed herein, may be a gene whose expression characteristics can be used to identify a condition or the status of a condition in a subject or sample. In other examples, the biomarker may be a gene product. In some embodiments, the term “biomarker” refers to a polypeptide expressed endogenously in an individual or detected or sequestered in a biological sample from an individual.

[00147] By "gene product" is meant a transcript (eg, mRNA), nucleic acid (eg, microRNA), or protein. Thus, biomarkers are disclosed herein, the presence, absence, or relative amount of which can be used to identify a condition or the status of a condition in a subject or sample. In one specific example, a biomarker may be a gene product whose presence or absence in a subject is characteristic of the subject having or not having a particular neurodegenerative disease, having a particular risk of developing the disease (eg, a neurodegenerative disease), or being at a particular stage of the disease. In yet another example, a biomarker may be a gene product that, when increased or decreased, indicates a particular disease state, a particular risk for developing a disease, or a particular stage of a disease. In another example, a biomarker may be a group of different gene products, the presence or absence of which indicates that a subject has or does not have a particular disease, is at a particular risk of developing a disease, or is at a particular stage of a disease. In a further example, a biomarker may be a group of gene products whose pattern of increased or decreased expression characterizes a particular disease or lack thereof. In addition, a biomarker may be a gene product or group of gene products whose expression pattern is characteristic of the presence or absence of a disease, or a particular prognosis or outcome of a disease. The biomarker used herein may be a surrogate for other clinical trials. The biomarkers identified herein can be measured to determine levels, expression, activity, or to detect variants. As used throughout this document when discussing detection of expression or activity levels, it is intended that this may reflect variants of a given biomarker. Variants include amino acid or nucleic acid variants, or post-translationally modified variants.

[00148] As used herein, the term “biological sample” refers to a cell or population of cells, or a quantity of tissue or fluid from a subject. Most often the sample is taken from a subject, but the term "biological sample" can also refer to cells or tissue analyzed in vivo, i.e. without extraction from the subject. Often a "biological sample" will contain cells from the subject, but the term can also refer to non-cellular biological material, such as non-cellular fractions of blood, saliva or urine, which can be used to measure gene expression or protein expression levels. Biological samples include, but are not limited to, tissue biopsies, scrapings (eg, buccal scrapings), whole blood, plasma, serum, urine, saliva, cell culture, or cerebrospinal fluid. Biological samples also include tissue biopsies, cell culture. A biological or tissue sample may refer to a sample of tissue or fluid collected from an individual, including, but not limited to, blood, plasma, serum, tumor biopsy, urine, stool, sputum, cerebrospinal fluid, pleural fluid, nipple aspirates, lymph fluid, external skin, respiratory tract, intestinal and genitourinary tracts, tears, saliva, breast milk, cells (including, but not limited to, blood cells), tumors, organs, and samples of in vitro cell culture components. In some embodiments, the sample is material taken from a resection, bronchoscopic biopsy, or core needle biopsy of a primary or metastatic tumor, or a cell block from pleural fluid. In addition, fine needle aspirates are used. Samples can be either paraffin-embedded or frozen tissue. The sample can be obtained by collecting a sample of cells from a subject, but can also be prepared using previously isolated cells (eg, isolated from another person), or by analyzing the level of transgene expression from a cccDNA vector in vivo. Biological sample also refers to a sample of tissue or fluid collected from an individual, including, but not limited to, blood, plasma, serum, tumor biopsy, urine, stool, sputum, cerebrospinal fluid, pleural fluid, nipple aspirates, lymph fluid, external skin, respiratory tract, intestinal and genitourinary tracts, tears, saliva, breast milk, cells (including, but not limited to, blood cells), tumors, organs, and samples of in vitro cell culture components. In some embodiments, the biological samples may be prepared, for example, the biological samples may be fresh, fixed, frozen, or paraffin-embedded.

[00149] As used herein, the term “blood sample” or “blood” includes, without limitation, whole blood, serum, or plasma. In some embodiments, the whole blood sample is further processed into serum or plasma samples. This term also includes a mixture of the above samples.

[00150] As used herein, the term “inhibitor” refers to any agent or entity that results in inhibition of the biological activity of proteins. "Reduction" or "inhibition", when used in the context of the level of gene activity, refers to a decrease in the level of protein or nucleic acid, or biological activity in a cell, cell extract or cell supernatant. For example, such inhibition may be due to decreased binding of the polypeptide to its endogenous ligand, or noncompetitive binding of the inhibitor to the polypeptide to reduce catalytic activity or affinity for the target ligand, etc. or alternatively, decreased RNA stability, transcription or translation, increased protein degradation, or RNA interference. Preferably, the reduction is at least about 5%, at least about 10%, at least about 25%, at least about 50%, at least about 75%, at least about 80%, or even at least at least approximately 90% of the level of expression or activity under the conditions of the control experiment. As used herein, the term “inhibition”, referring to inhibition of the activity of topoisomerase I protein or variants thereof, does not necessarily mean complete inhibition of expression and/or activity. Rather, the expression or activity of the protein, polypeptide, or polynucleotide is inhibited to a certain extent and/or for a time sufficient to produce the desired effect.

[00151] The terms “lower,” “reduced,” “decreased,” or “decreased,” or “inhibited,” are used herein generally to denote a reduction of a statistically significant amount. However, for the avoidance of doubt, “lower,” “reduced,” “reduced,” or “decreased,” or “inhibited” means a reduction of at least 10% relative to the reference level, e.g., a reduction of at least about 20 %, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90%, or a reduction of up to and including 100% (i.e., to the absence level compared to the reference sample), or any reduction in the range of 10-100% compared to the reference level.

[00152] The terms "increased", "increase" or "amplify", or "higher" are used herein to mean an increase by a statically significant amount; for the avoidance of any doubt, the terms "increased", "increase" or "intensify", or "higher" mean an increase of at least 10% above the reference level, such as an increase of at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least at least about 90%, or an increase of up to and including 100%, or any increase in the range of 10-100% above the reference level, or at least about 2 times, or at least about 3 times, or at least at least about 4 times, or at least about 5 times, or at least about 10 times, or any increase in the range of 2 times to 10 times or more compared to the reference level.

[00153] By “increasing” the expression or activity of a gene or protein is meant a positive change in the level or activity of a protein or polypeptide or nucleic acid in a cell, cell extract or cell supernatant. For example, such an increase may be due to increased RNA stability, transcription or translation, or decreased protein degradation. Preferably, such increase is at least 5%, at least about 10%, at least about 25%, at least about 50%, at least about 75%, at least about 80%, at least about 100%, at least about 200%, or even about 500% or more, compared to the level of expression or activity under control conditions.

[00154] As used herein, the term “comprising” or “comprises” is used to refer to compositions, methods and their respective components that are essential to the method or composition but allow for the inclusion of unspecified elements, whether essential or not.

[00155] As used herein, the term “consisting essentially of” refers to those elements that are necessary for a given embodiment. This term allows for the presence of elements that do not significantly affect the essential and novel or functional characteristics of such an embodiment. The use of the word “comprising” indicates inclusion rather than limitation.

[00156] The term “consisting of” refers to compositions, methods and their respective components as described herein, which exclude any element not specified in this embodiment description.

[00157] As used herein, the term “consisting essentially of” refers to those elements that are necessary for a given embodiment. This term allows for the presence of additional elements that do not significantly affect the essential and novel or functional characteristics of this embodiment of the invention.

[00158] As used in this specification and the accompanying claims, the singular forms, including those accompanied by the qualifier “specified,” include references to the plural forms unless the context clearly requires otherwise. Thus, for example, references to “method” include one or more methods and/or steps of the type described herein and/or those that will become apparent to those skilled in the art upon reading the present invention, and so on. Likewise, the word “or” is presumed to include “and” unless the context clearly requires otherwise. Although methods and materials similar or equivalent to those described herein may be used in the practice or testing of this invention, suitable methods and materials are described below. The expression “for example” is used herein to describe a non-limiting example. So, the abbreviation “for example.” is synonymous with the term "for example".

[00159] Except in working examples or as otherwise specified, all numbers expressing quantities of ingredients or reaction conditions according to this invention should in all cases be considered as modified by the term "about". The term “about” when used as a percentage may mean ±1%. The present invention is further explained in more detail in the following examples, but the scope of the present invention is not limited to them.

[00160] The groups of alternative elements or embodiments of the invention disclosed herein are not to be construed as limitations. Each group member may be referred to and stated individually or in any combination with other group members or other elements presented herein. One or more members of the group may be included or excluded from the group for reasons of convenience and/or patentability. Upon any such inclusion or deletion herein, the specification is deemed to contain the group modified so as to satisfy the condition of a written description of all Markush groups used in the appended claims.

[00161] In some embodiments of any of the aspects, the invention described herein does not relate to the process of cloning humans, processes for modifying the genetic identity of human germline, the use of human embryos for industrial or commercial purposes, or processes for modifying the genetic identity of animals that may cause them suffering without any significant medical benefit to the person or animal, as well as the animals resulting from such processes.

[00162] Definitions of other terms are provided herein in the description of various aspects of the invention.

[00163] All patents and other publications, including literature references, issued patents, published patent applications and contemporaneous patent applications, mentioned in this application are expressly incorporated herein by reference for purposes of description and disclosure, e.g. , methodologies described in such publications that may be used in connection with the technology described in this document. These publications are presented solely by reason of their disclosure prior to the filing date of this application. Nothing in this regard should be construed as an admission that the inventors have no rights in an earlier invention with respect to such disclosure by reason of prior invention or for any other reason. All statements regarding dates or representations regarding the contents of these documents are based on information available to the applicants and do not constitute an admission in any way as to the accuracy of the dates or contents of these documents.

[00164] The description of embodiments of the invention is not to be construed as exhaustive or limiting of the invention to the precise form disclosed. Although specific embodiments and examples of the disclosure have been described herein for illustrative purposes, various equivalent modifications are possible within the scope of the disclosure, as will be understood by those skilled in the relevant art. For example, although the steps or functions of a method are presented in the order shown, in alternative embodiments the functions may be performed in a different order, or the functions may be performed substantially simultaneously. The principles of the invention provided herein may be applied to other procedures or methods as appropriate. Various implementations described herein may be combined to provide additional implementations. Aspects of the disclosure may be modified, as necessary, to utilize the compositions, functions and concepts of the above references and uses to provide additional embodiments of the invention. In addition, for reasons of biological functional equivalence, some changes in protein structure may be made without affecting the biological or chemical action in a qualitative or quantitative sense. These and other changes may be made to the disclosure in light of the detailed description. All such modifications are considered to be included within the scope of the appended claims.

[00165] Specific elements of any of the above embodiments may be combined or replaced with elements of other embodiments. In addition, although the advantages associated with some embodiments of the invention have been described in the context of these embodiments, other embodiments may also demonstrate such advantages, and not all embodiments must necessarily demonstrate such advantages to fall within the scope of the disclosure.

[00166] The technology described herein is further illustrated by the following examples, which should in no way be construed as further limitations. It should be understood that the present invention is not limited to the specific methodology, protocols and reagents, etc. described herein and is therefore subject to variations. The terminology used herein is intended to describe specific embodiments only and is not intended to limit the scope of the invention, which is defined solely by the claims.

I. Method for controlled transgene expression using cccDNA vector

[00167] Described herein are methods for introducing cccDNA vectors in vivo or in vitro that allow either (i) sustained expression of transgene levels over a predetermined period of time (i.e., maintenance of transgene expression levels), or (ii) increasing the expression level of the transgene in a dose-dependent manner, the method comprising at least one (eg, one or more) sequential administrations (eg, "booster" administration or "repeat dose") of the cccDNA vector.

[00168] Accordingly, the technology described herein relates to the administration of a cccDNA vector in vivo, with the level of transgene expression being maintained at a desired level or increased by one or more subsequent administrations (eg, boost or repeat dosing). The cccDNA vectors disclosed herein provide the ability to increase the level of transgene expression above previous levels in vitro and in vivo. Increasing the level of the transgene can be achieved to reach or exceed the desired level, or to increase the level of expression to a value within the desired range of expression values, through at least one boost (also referred to herein as a “boost”) at any given time. after initial administration. In some cases, the desired increase must correct the natural decline in these levels so that the final dose provides a return to the previously desired level, for example, a steady-state dose level. This use of repeated dosing or repeated dosing of a cccDNA vector disclosed herein to increase the level of transgene expression is possible because the cccDNA vector does not have a capsid to elicit a host immune response (eg, the vector is not immunogenic) and, thus provides significant advantages over current AAV vector technology, in which repeat doses are not possible due to immune responses or immunity to AAV in the host from prior exposure to AAV or, for example, from initial administration of the AAV vector. Thus, to achieve high levels of expression using rAAV vectors, an initial high dose is usually required, and repeated doses are either impossible or ineffective due to problems associated with the immune response. In contrast, one or more repeated administrations of a cccDNA vector disclosed herein can be performed to increase the level of expression of the transgene, for example, to increase the expression level to a desired expression level, or above a desired threshold level, or to values within a desired range. range of expression levels.

[00169] Accordingly, this invention relates to methods and cccDNA vectors for controlled expression of a transgene for any of: (i) maintaining or stabilizing the expression level of the transgene at a desired level for a predetermined period of time by reintroducing the ccDNA vector into one or more time points, (ii) increasing the level of transgene expression by reintroducing the cccDNA vector at one or more time points, or (iii) dose-dependent expression of the transgene as a result of introducing the cccDNA vector in a dose-dependent manner (ie, titrated transgene expression). As described herein, cccDNA vectors are unique compared to other viral vectors in that the cccDNA vector can be administered to a subject repeatedly, for example, over a short period of time (eg, several months) or over a long period of time ( for example, over several years), allowing controlled expression of the transgene, allowing gene therapy to be tailored to the needs of the subject.

A. Increased or dose-dependent controlled expression of the transgene

(i) Controlled transgene expression: sustained expression of the transgene by repeated introduction of the cccDNA vector

[00170] One aspect of the technology described herein relates to the use of a cccDNA vector disclosed herein in a method of maintaining or stabilizing the expression level of a transgene in a cell, comprising: (a) introducing into the cell, at a first time point, a primary dosing the cccDNA vector to achieve expression of the transgene, and (b) introducing into the cell at a second time point a dose of the cccDNA vector to compensate for any decrease in the level of transgene expression following administration of the cccDNA vector at the first time point.

[00171] As shown in Figure 6, the expression level of the transgene expressed by the cccDNA can be achieved and maintained (i.e., maintained) for at least 42 days, or at least 60 days, or at least 80 days, and can be increased to the desired predetermined level of expression by administering a repeat dose at the second or subsequent time points after the initial priming administration (eg, at time 0). Figure 6 also demonstrates that the level of expression of the cccDNA-expressed transgene can be achieved and maintained for at least 42 days, or at least 60 days, and can be increased in a dose-dependent manner by administering a repeat dose containing a certain amount of cccDNA- vector, at the second or subsequent time points after the initial priming administration (eg, at time 0).

[00172] Expression of the transgene typically occurs within about 7-20 days after a repeat dose and maintains that level of expression (i.e., is sustained expression) of the transgene after a boost for at least about 30 days, or about 60 days , or about 90 days, or about 120 days, or longer than 120 days after administration of the second dose (ie, a repeat dose, or administration at a second or subsequent time points).

[00173] Another aspect of the technology described herein relates to a method of treating a disease in a subject, or alternatively, a method of controlled expression of a transgene in a subject, comprising: (a) administering to the subject, at a first time point, a priming dose of a composition comprising cccDNA -vector, as described herein, to achieve expression of the transgene at a first level (or in a first amount), and (b) administering to the subject, at a second time point, a dose or amount of cccDNA vector to maintain the level of transgene expression at a desired sustained level expression, the level of sustained expression being a higher level of expression compared to the level of expression of the transgene that is achieved by the initial (ie, priming) administration of the cccDNA vector alone at the first time point, thereby treating the disease in the subject.

[00174] Accordingly, in some embodiments, a cccDNA vector, as described herein, can be reintroduced (also referred to herein as a “re-dosing”) to maintain a desired level of transgene expression, wherein the cccDNA vector contains two ICP sequences ( for example, a symmetric ICP pair or an asymmetric ICP pair as described herein) flanking a transgene polynucleotide sequence operably linked to a promoter.

[00175] In some embodiments, use of a cccDNA vector in a method for maintaining the expression level of a transgene in a cell ensures that the transgene is expressed at the desired expression level for at least 42 days. In some embodiments, the cccDNA vector expresses the transgene at the desired expression level for at least 84 days. In some embodiments, the cccDNA vector expresses the transgene at the desired expression level for at least 132 days. For illustrative purposes only, a cccDNA vector produced by the methods disclosed in Example 1 can maintain the level of transgene expression in a murine model using CD-1 IGS mice at a higher level from days 7-28 after administration compared to similar DNA vectors with closed ends obtained by other methods and tested in the same mouse model.

[00176] In some embodiments, the increased transgene expression is not dose-dependent or not completely dose-dependent, but is sustained expression because increased transgene expression is typically observed in days 7-20 after a booster dose and that level of increased transgene expression persists after a booster dose. for at least about 30 days, or at least about 60 days, or at least about 90 days, or at least about 120 days, or longer than about 120 days, after administration of the second dose (i.e. repeat dose or administration at a second or subsequent time points).

[00177] Specific dose-response relationships for a given cccDNA vector or composition disclosed herein can be determined by means well known to those skilled in the art, including, for example, those described in Example 6. The ccDNA vector described herein, can be titrated by administering additional doses of the cccDNA vector at one or more time points after the initial administration as necessary to achieve the desired level of expression. More than one, 2, 3, 4, 5, or 6 or more repeated administrations may be performed to titrate transgene expression levels to or near the desired level. As a rule, each repeated administration is carried out approximately 7 days before the onset of the observed decrease in transgene expression after the previous administration. Essentially, repeated administrations serve to titrate the expression level of the transgene to a desired level or, in other words, repeated administrations allow the expression level of the transgene to be maintained within a desired range of expression levels, e.g., a desired range that is therapeutic for treating a disease or disorder, or within therapeutic window composition.

[00178] In some embodiments, the cccDNA vector described herein can be used to adjust the level of expression of a transgene in vivo, in which the level of expression is increased from a prior level (where the prior level is the level after primary priming or booster dosing) to desired level of expression, or within a desired range of expression levels, or above a desired threshold level in vivo, wherein the increased level of expression is maintained or maintained for about 30-60 days, or for about 40-70 days, or for about 50-50 days. 80 days, or within about 60-90 days, or within about 70-100 days, or within about 80-110 days, or within about 40-120 days, or longer than 120 days after administration of a repeat dose of the cccDNA vector .

[00179] In some embodiments, a cccDNA vector used in the methods described herein, for example, in a method for maintaining expression of a transgene in a cell and/or treating a subject with a disease, is administered in combination with a pharmaceutically acceptable carrier and/or excipient. In some embodiments, the cDNA vector is administered at a second time point after at least 30 days, or at least 60 days, or 60-90 days, or 90-120 days, or about 3-6 months, or 6-12 months , or 1-2 years, or 2-3 years, after the first point in time.

(ii) Controlled transgene expression: increasing transgene expression by reintroducing the cccDNA vector

[00180] In addition to administering repeated doses of the cccDNA vector to simply increase transgene expression levels if expression levels have decreased over time (e.g., to continue or maintain transgene expression at a desired predetermined level), in some embodiments, methods and compositions for repeated administration of the cccDNA vector can increase the level of the transgene in a dose-dependent manner, i.e. Repeated dosing of a certain amount of cDNA vector can cause a certain increase in the level of transgene expression. In other words, using arbitrary units for illustrative purposes only, a single dose of ccDNA when reintroduced will result in a 10% increase in the level of transgene expression over the previous level, and a 2-fold dose of the ccDNA vector will result in a 20% increase in the level of the transgene. compared to the previous level, and half (0.5) dose of cccDNA will increase the level of transgene expression by 5% compared to the previous level.

[00181] Accordingly, in one embodiment, a cccDNA vector disclosed herein for controlled expression of a transgene can be used to increase the level of expression of a transgene in a cell or subject in a controlled manner. For example, the level of transgene expression can be increased by one or more subsequent administrations (eg, repeat dosing or boosting) of the cccDNA vector.

[00182] cccDNA vectors, as described herein, allow for a dose-dependent or concentration-dependent effect of administering a repeat dose of cccDNA at one or more time points after the initial priming to increase the level of transgene expression by a certain amount in vivo. In some embodiments, increasing expression of a transgene by a certain amount may bring the in vivo expression level of the transgene to or above a desired threshold value (or to a predetermined level) or to a value within a desired range of expression levels, wherein the desired threshold value or desired range of levels expression exceeds the level of transgene expression after the previous administration (ie, the initial priming administration or the previous repeated dose administration).

[00183] Figure 6 demonstrates that it is possible to easily increase the level of transgene expression after re-dosing (ie, repeat or boost) the cccDNA vector to a subject in vivo. In particular, FIG. 6 demonstrates different magnitudes of increase in transgene expression following administration of varying concentrations of cccDNA vector to a subject upon repeated dosing in vivo. Specifically, a repeat dose concentration of 3 mg/ml cccDNA provided a 7-fold increase in transgene expression, and a repeat dose concentration of 10 mg/kg cccDNA resulted in a 17-fold increase in transgene expression compared to the expression level without a repeat dose, with a dose-dependent effect. Accordingly, the technology described herein relates to at least two administrations of cccDNA to a subject in vivo, the second or subsequent administrations resulting in a dose-dependent increase in the expression level of the transgene by a desired amount, and in some embodiments, achieving a desired range of expression levels, or a desired level of expression, or a threshold level of expression compared to the level of transgene expression achieved with prior administration of the cccDNA vector, or without repeated dosing, with a dose-dependent effect. Thus, increasing the level of transgene expression is achieved by one or more repeated doses with a dose-dependent effect to increase the level of transgene expression in a controlled manner, i.e. to titrate transgene expression based on the dose (or amount) of cccDNA upon repeated dosing.

[00184] In other words, repeated dosing with a dose-dependent effect disclosed herein increases the level of expression of the transgene. Increased transgene expression is dose-dependent and sustained expression, i.e. expression of the transgene at a higher level (due to repeated dosing with a dose-dependent effect) is maintained for a certain period of time, or does not decrease, or does not fall below the level of expression observed without repeated dosing.

[00185] Typically, each dose-dependent booster dose is administered approximately 7 days prior to the desired increase in transgene expression. In essence, repeated doses with a dose-dependent effect serve to titrate the level of expression of the transgene to a desired level, or a desired range of expression levels, or, in other words, repeated doses with a dose-dependent effect provide an increase in the level of expression of the transgene by a certain amount above the previous level of expression, and this increase may occur with at least one dose-dependent repeat dose or, alternatively, with a gradual increase with more than one dose-dependent boost, resulting in the transgene increasing in a controlled, titrated manner until expressed at a level that is within a desired range of expression levels, for example, in a desired range that is therapeutic for treating a disease or disorder.

[00186] In some embodiments, the desired range of expression level or the desired range of expression level of the transgene may be a therapeutically effective amount of the transgene to effectively treat or reduce a symptom of a disease. Accordingly, in some embodiments, to achieve such a therapeutically effective amount of the transgene, the expression level of the transgene can be increased using one or more repeated dosing administrations, as described herein, to gradually increase levels to a therapeutically effective amount of the transgene. Thus, in some embodiments, a priming dose of a cccDNA vector that expresses a transgene at a low level of expression (i.e., in a subtherapeutically effective amount) may be administered to a subject, and one or more repeat dosing may be administered to the subject over a period of time to increasing the level of expression until the desired therapeutic effect is achieved. This strategy allows the subject's body to adapt to the level of expressed transgene and effectively allows the level of expression of the transgene to be titrated or adjusted (in this case, by gradually increasing) the level of expression of the transgene to achieve the desired therapeutic goal or effect and/or prevent drug overdose and/or side effects due to transgene overexpression. Alternatively, in some embodiments, the subject may be administered a priming dose of a cccDNA vector that expresses a transgene at a desired level of expression when the subject is an infant or child, typically at a low level of expression, and one or more repeat dosing administrations with a dose-dependent effect may be administered to a subject over a period of time as the subject grows to increase expression levels in order to maintain a therapeutic effect.

B. Choice of timing and quantity of cccDNA vector for repeated administration

[00187] The expression level of a transgene from a cccDNA vector, as described herein, can be increased from the previous level (i.e., the expression level achieved at the initial priming administration on day 0 or the previous booster dose) by repeated administration (i.e. e. (repeated dosing) of the cccDNA vector at one or more time points after the initial administration.Typically, repeated dosing to increase the expression level to the desired level or desired range of expression levels is carried out approximately 7 days or more than 7 days before the desired increase in expression. As an illustrative example, if an increase in the level of expression of the transgene is desired approximately 30 days after the previous administration (ie, priming or previous booster dosing), then the booster dose can be administered after 28 days or earlier. Likewise, if the increase in transgene expression is desirable approximately 90 days (or approximately 3 months) after the previous administration (i.e. priming or previous booster dosing), then the booster dose may be administered on or about 83 or 84 days so that the expression level of the transgene to the desired level or to a value within the desired range of expression levels occurs after 90 days or so, the desired the level or desired range of expression levels exceeds the level of expression of the transgene achieved after previous administration.

[00188] As described herein, since the methods and cDNA vectors described herein allow for a personalized genetic drug approach, i.e. gradual titration of transgene expression levels with repeated doses to gradually increase expression levels, it is envisaged that 1, 2, 3, 4, 5 or 6 or more than 6 repeated doses may be administered over a period of time to increase transgene expression levels to the desired level level or to a desired range of expression levels that exceed the level of expression achieved after the previous administration or before such repeated dosing. The magnitude of the gradual increase in the level of transgene expression with each administration of a repeated dose may be the same, i.e. each repeated dose may increase the transgene expression level by approximately 10% of the previous expression level, or may vary, i.e. the first booster dose may increase expression by about 10% of the previous expression level, and the second booster dose can increase expression by about 20% of the previous expression level. Typically, each repeat dose is administered approximately 7 days before the desired increase in transgene expression. Essentially, repeated doses serve to titrate the expression level of the transgene to a desired level or range of expression levels, or, in other words, repeated doses allow the expression level of the transgene to increase relative to the previous expression level. and such enhancement may be accomplished gradually through one or more repeated dosing such that the transgene is expressed at a level that is within a desired range of expression levels, eg, a desired range that is therapeutic for treating a disease or disorder.

[00189] In some embodiments, a repeat dose to increase transgene expression is administered after at least about 20 days, or at least about 30 days, or at least about 40 days, or at least about 50 days, or at least at least about 60 days, or about 60-90 days, or about 90-120 days, or about 120-150 days, after prior administration (eg, an initial priming administration on day 0 or a prior booster administration) of the cccDNA composition.

[00190] In some embodiments, the technology described herein relates to repeated dosing of cccDNA to increase transgene expression in vivo, wherein transgene expression may be increased through one or more repeated dosing (i.e., re-dosing or boosting) of the composition cccDNA. In some embodiments, the dosage (or amount) of cccDNA vector at a booster dose at the second or subsequent time points is the same as or different from the dose (i.e., amount) of cccDNA at a prior dosing, or at the immediately preceding booster dosing (initial priming administration on day 0 or preceding a repeat dose). As one example, if the amount of cccDNA administered at the first time point is 1 mg/kg, then the amount when repeated dosing at the second or subsequent time points may be 1 mg/kg, or less than 1 mg/kg, or more than 1 mg/kg. mg/kg. In some embodiments, the repeat dose is an amount selected from any of the following: about 2 mg/kg, about 3 mg/kg, about 4 mg/kg, about 5 mg/kg, about 6 mg/kg , about 7 mg/kg, about 8 mg/kg, about 9 mg/kg, about 10 mg/kg, or about 2-5 mg/kg, or 5-10 mg/kg, or 10-15 mg/kg, or more than 15 mg/kg.

[00191] In some embodiments, to increase the expression level of a transgene through one or more booster doses (i.e., sequential booster doses to gradually increase the expression level of the transgene), each booster dose may be the same, i.e. each repeated dose may increase the level of transgene expression by approximately 10% compared to the previous level of expression, or may be different, i.e. the first booster dose may increase expression by about 10% of the previous expression level, and the second booster dose can increase expression by about 20% of the previous expression level.

[00192] In some embodiments, when more than one booster dose is administered, the magnitude of the increase in transgene expression with each booster dose may be the same, i.e. each repeated dose administered may increase the level of transgene expression by approximately 10% of the previous expression level, or may vary, i.e. the first booster dose may increase expression by about 10% of the previous expression level, and the second booster dose can increase expression by about 20% of the previous expression level. In some embodiments, when more than one booster dose is administered, the magnitude of the increase in transgene expression with each booster dose may be the same, i.e. Each repeated dose administered may increase the level of transgene expression by approximately 1, or 2, or 3 times, etc., from the previous expression level, or may vary, i.e. a first booster dose may increase expression to about 2 times the previous expression level, and a second booster dose can increase expression to about 6 times the previous expression level, or about 6 times the expression level achieved with the initial priming administration.

[00193] In some embodiments, the cccDNA vector that is introduced into a cell or subject upon the second or any subsequent administration (e.g., repeat dosing) is the same cccDNA vector that was introduced upon the initial administration (i.e., i.e. upon first administration at time 0). In some embodiments, the cDNA vector may be the same cDNA vector. For illustrative purposes only, when viral vectors, such as AAV vectors, are reintroduced, they will typically differ in serotype from those previously introduced. In contrast, the cccDNA vector, when reintroduced, is the same as the previously introduced cccDNA vector, i.e. The cccDNA vector does not change, which is equivalent to repeated administration of the same AAV serotype.

[00194] While equivalent to repeated administration of the same serotype cccDNA vector, in some embodiments the cccDNA vector that is administered on the second or any subsequent administration (e.g., repeat doses) after the initial priming administration is different, for example, a different pair of ICPs, a different promoter operably linked to the transgene, a different transgene or a modified transgene, etc. In some embodiments, the gene of the transgene is the same, or may be a modified transgene.

[00195] In some embodiments, the intervals between the first administration (i.e., priming administration) of the cccDNA vector and the second dose administration (i.e., at the second time point or at any subsequent time point thereafter, e.g., the 3rd 4th, 5th 6th, 7th, 8th, 9th, 10th, etc.) can be at least 30 days, or at least 60 days, or at least 80 days , or from 60 to 90 days, or from 90 to 120 days, or about 2-3 months, or about 3-6 months, or about 6-12 months, or about 1 year, or 1-2 years, or about 2 years, or 2-3 years, or approximately 3 years, or 3-4 years, or approximately 5 years, or 5-6 years, or 5-10 years, or 10-20 years, etc., after previous administration (i.e. priming dose or previous administration of a repeat dose). As described herein, in one embodiment, a repeat dose to increase transgene expression is administered approximately 7 days or more than 7 days (eg, 8-10 or 14 days) before the increase in expression is desired.

[00196] For purposes of illustration only, if the amount of cccDNA vector when initially administered on day 0 is set to an arbitrary unit of 1, then the amount of cccDNA vector when reintroduced at the second, third, fourth, fifth, sixth time points can be selected from 2 -x, 3x, 4x, 5x, 6x, 7x, 8x, 9x, 10x, 11x, 12x, 13x, 14x , 15-fold or 15-20-fold or 20-50-fold (or more) increased amount of cccDNA vector when initially administered on day 0.

[00197] In some embodiments, if the amount of cccDNA vector upon initial dosing on day 0 results in an expression level that is set to an arbitrary unit of 1, then the amount of cccDNA vector upon repeated dosing on day 2, third, fourth, fifth, sixth time points may represent the amount of cDNA vector that results in an increase in the level of transgene expression by at least 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold , 11 times, 12 times, 13 times, 14 times, 15 times, or 15-20 times, or 20-50 times (or more) compared to the level of transgene expression when the cDNA vector was initially administered on day 0.

[00198] In some embodiments, the re-dose is administered in the same manner or by the same route of administration as the initial administration of cccDNA on day 0. In some embodiments, the re-dosage is administered in a different manner or by a different route of administration than the initial administration of cccDNA on day 0. 0. In some embodiments, when the initial administration (i.e., priming administration at time 0) is followed by one or more repeat doses (i.e., booster administrations), administration may be by intravenous, intranasal, or intramuscular administration, or any other medically appropriate method by administering a composition containing the cccDNA vector.

[00199] The cccDNA vector described herein may be administered to a subject at a first time point (e.g., upon initial administration, e.g., day 0), and at some time after the first time point, if necessary or desired . The cccDNA vector described herein can be administered at a second time point to titrate transgene levels at a desired level (eg, above an efficacy threshold), or within a desired range of expression levels (eg, within a therapeutic window of the composition). It is contemplated that a subject may be administered more than one dose of the cccDNA vector as described herein, for example, a repeat administration may be administered at any one or more of: a second time point, a third time point, a fourth time point, etc. . It is contemplated that additional doses may be administered to maintain a desired level of transgene expression (ie, to maintain or maintain a constant level of transgene expression). The intervals between the first and second or any two consecutive repeat doses should not be the same.

C. Predetermined Transgene Expression Levels

[00200] In some embodiments, a predetermined transgene expression level (also referred to as a desired expression level range or desired transgene expression level range) may represent a therapeutically effective amount of the transgene to effectively treat or reduce a symptom of a disease. Accordingly, in some embodiments, to achieve such a therapeutically effective amount of transgene, the level of transgene expression may be maintained and/or the level of transgene expression may be increased, as described herein, using one or more repeated dosing administrations, as described herein, to gradually increase levels to a therapeutically effective amount of transgene. Thus, in some embodiments, a subject may be administered a priming dose of a cccDNA vector that expresses a transgene at a desired expression level, typically a low expression level (i.e., a subtherapeutically effective amount), and one or more repeat doses with a dose-dependent effect may be administered to a subject over a period of time to increase expression levels until the desired therapeutic effect is achieved. Such a strategy allows the subject's body to adapt to the level of expression of the transgene and allows for effective titration or regulation (in this case, increase) of the level of expression of the transgene with at least one repeated dose with a dose-dependent effect or more (i.e., with two or more increments) to achieve the desired therapeutic goal or effect and/or prevent drug overdose and/or side effects due to overexpression of the transgene. Alternatively, in some embodiments, the subject may be administered a priming dose of a cccDNA vector that expresses a transgene at a desired level of expression when the subject is an infant or child, typically at a low level of expression, and one or more repeat dosing administrations with a dose-dependent effect may be administered to a subject over a period of time as the subject grows to increase expression levels in order to maintain a therapeutic effect. Likewise, in some embodiments, a priming dose of a cccDNA vector that expresses a transgene at a desired level of expression in the subject may be administered to a subject, and one or more repeated dosing administrations with a dose-dependent effect may be administered to the subject over a period of time as the subject gains weight, etc. .d., to increase the level of expression in order to maintain the therapeutic effect.

[00201] Thus, the cccDNA vector as described herein is administered to a subject at a first time point (e.g., at initial administration, e.g., day 0), and some time after the first time point, the cccDNA vector is as described herein, is administered at a second time point to increase the expression level of the transgene to a predetermined level of transgene expression (e.g., to a desired level or within a desired range of expression levels), wherein the predetermined level of transgene expression is higher than the level of transgene expression before reintroducing doses. In some embodiments, when the predetermined level of transgene expression is not necessarily a therapeutically effective amount of transgene, it is contemplated that the subject may be administered more than one repeat dose of a cccDNA vector with a dose-dependent effect, as described herein, at any one or more of : a second time point, a third time point, a fourth time point, etc., wherein the transgene is increased to a predetermined level of expression of the transgene. It is contemplated that repeated doses with a dose-dependent effect can be administered to increase the expression level of the transgene by a certain amount that depends on the dose of cccDNA at repeated dosing, and in some embodiments can be used to gradually increase expression levels to achieve an expression level that represents is a therapeutically effective amount of a transgene (eg, is a level that produces a desired therapeutic effect or reduces one or more symptoms of a disease or disorder).

[00202] Although repeated dosing results in a dose-dependent increase in transgene expression, in some embodiments the effect of repeated dosing is synergistic, i.e. the degree of increase in transgene expression exceeds the sum of the values of individual injections. For example, as demonstrated in Figure 6, increasing the amount of cccDNA vector by 3-fold upon repeated administration resulted in a more than 3-fold increase in transgene expression; and a 10-fold increase in the amount of cccDNA vector upon repeated administration resulted in a more than 10-fold increase in transgene expression.

[00203] In particular embodiments described herein, for initial administration, it may be desirable to select a low dose of cccDNA on the dose-response curve (i.e., at the first time point, e.g., day 0) and optionally gradually increase dose (i.e. the level of transgene expression) through one or more repeated doses with a dose-dependent effect on the second, third, fourth, etc. administrations to produce a therapeutic effect while preventing the occurrence of adverse side effects or intolerance to the composition. In one embodiment, the dose-response relationship for a given cccDNA vector is used to determine and/or estimate the optimal dose for effective treatment of a given disease, the value of which is far from the limits of the therapeutic window of the composition. Thus, titrating the dose of cccDNA vectors as described herein, using an initial priming administration (eg, on day 0) and gradually increasing repeat doses with a dose-dependent effect at subsequent time points can provide maximum therapeutic effect of the expressed transgene, while also minimizing side effects or unwanted toxicity.

[00204] In vivo and/or in vitro assays may optionally be used to determine optimal dosage ranges of the cccDNA vector for repeated administrations to achieve a given level of transgene expression. The exact dose of the cDNA vector upon initial priming administration (eg, at time 0), and each re-administration thereafter, will also depend on the route of administration and the severity of the condition, and should be determined by the judgment of one skilled in the art and taking into account the circumstances. each subject. Effective doses can be extrapolated from dose-response curves obtained from in vitro test systems or animal models.

[00205] The cccDNA vector for controlled transgene expression is administered in sufficient quantities to transfect cells of the desired tissue and to ensure sufficient levels of transgene expression and excessive adverse events. Conventional and pharmaceutically acceptable routes of administration include, but are not limited to, those described below in the Introduction section, such as direct delivery to the target organ (eg, intraportal delivery to the liver), oral delivery, inhalation (including intranasal and intratracheal delivery), intraocular , intravenous, intramuscular, subcutaneous, intradermal, intratumoral delivery; and other parenteral routes of administration. Routes of administration can be combined, if necessary.

[00206] The dosage amount of cccDNA vector upon initial priming administration (e.g., at time 0), and upon each reintroduction thereafter, for controlled transgene expression, required to achieve a particular "therapeutic effect" will vary depending on several factors, including, without limitation: the route of administration of the nucleic acid, the level of gene or RNA expression required to achieve a therapeutic effect, the specific disease or disorder being treated, and the stability of the gene(s), RNA product(s), or resulting protein(s) expressed. . One skilled in the art can readily determine a dosage range of a cccDNA vector for treating a patient having a particular disease or disorder based on the above factors as well as other factors well known in the art.

[00207] The dosing schedule may be adjusted to provide an optimal therapeutic response. For example, the oligonucleotide may be administered repeatedly, for example, multiple doses may be administered daily, or the dose may be proportionally reduced based on the therapeutic situation dictated. One skilled in the art will readily be able to determine suitable dosages and administration schedules for the oligonucleotides of this invention, both for administration to cells and for administration to subjects.

[00208] A "therapeutically effective dose" will fall within a relatively wide range of values, which can be determined through clinical trials, and will depend on the specific application (nerve cells will require very small amounts, while systemic injections will require significant amounts). For example, for direct in vivo injection into human skeletal or cardiac muscle, the therapeutically effective dose would range from about 1 μg to 100 g of cccDNA vector. If exosomes or microparticles are used to deliver the cccDNA vector, the therapeutically effective dose can be determined experimentally but is expected to deliver between 1 μg and approximately 100 g of vector. In addition, a therapeutically effective dose is an amount of cccDNA vector that expresses an amount of the transgene sufficient to cause an effect in a subject that results in a reduction in one or more symptoms of the disease, but does not cause significant off-target or unwanted side effects.

[00209] Formulation of pharmaceutically acceptable excipients and carrier solutions is well known to those skilled in the art, as is the development of suitable dosing and treatment regimens for use of the specific compositions described herein in various treatment regimens.

[00210] For in vitro transfection, the effective amount of ccDNA vector delivered to cells (1×10 ⁶ cells) will be on the order of 0.1-100 μg of ccDNA vector, preferably 1 to 20 μg, and more preferably from 1 to 15 µg, or 8 to 10 µg. Larger cccDNA vectors require higher doses. If exosomes or microparticles are used, the effective in vitro dose can be determined experimentally, but must deliver generally the same amount of cccDNA vector.

[00211] Without being bound by any particular theory, the absence of the typical antiviral immune response elicited by administration of a cccDNA vector as described herein (ie, the absence of capsid components) allows repeated administration of the cccDNA vector to a host. In some embodiments, the number of times the heterologous nucleic acid is administered to a subject is 2 to 10 times (eg, 2, 3, 4, 5, 6, 7, 8, 9, or 10 times). In some embodiments, the cDNA vector is delivered to a subject more than 10 times.

[00212] In particular embodiments, more than one administration (e.g., two, three, four, or more administrations) may be used to achieve the desired level of gene expression over time periods of varying lengths, such as daily, weekly, monthly, annually, etc. d.

[00213] In some embodiments, a transgene encoded by a cccDNA vector for controlled transgene expression, as described herein, can be regulated by a regulatory switch, an inducible or a repressible promoter, such that it is expressed in a subject for at least 1 hours, at least 2 hours, at least 5 hours, at least 10 hours, at least 12 hours, at least 18 hours, at least 24 hours, at least 36 hours, at least 48 hours, at least 72 hours, at least 1 week, at least 2 weeks, at least 1 month, at least 2 months, at least 6 months, at least 12 months/one year, at least 2 years , at least 5 years, at least 10 years, at least 15 years, at least 20 years, at least 30 years, at least 40 years, at least 50 years or more. In one embodiment, expression can be achieved by reintroducing the cDNA vectors described herein at predetermined or desired intervals. Alternatively, controlled expression from a cccDNA vector as described herein may further include gene editing system components (e.g., CRISPR/Cas, TALENs (transcription activator-like effector nucleases), zinc finger endonucleases, etc. .), to enable the insertion of one or more nucleic acid sequences encoding a transgene to substantially permanently treat or “cure” a disease. Such cccDNA vectors containing gene editing components are disclosed in international application PCT/US18/64242 and may include 5' and 3' homologous arms (for example, the sequences SEQ ID NO: 151-154, or sequences with at least 40 %, 50%, 60%, 70%, or 80% homology thereto) to insert a nucleic acid encoding a transgene into a safe harbor region such as but not including the albumin gene or the CCR5 gene.

[00214] The duration of treatment depends on the subject's clinical progress and responsiveness to therapy. Provides continuous administration of relatively low maintenance doses after an initial higher therapeutic dose.

II. Personalized gene therapy

[00215] As described herein, the methods and cDNA vectors described herein allow for a personalized genetic drug approach, i.e. dose-dependent titration of transgene expression levels using repeated dosing, for example, in a stepwise manner with one or more repeat doses to increase transgene expression levels by a specified amount with each repeat dose. As such, repeated doses can be used to gradually titrate the level of transgene expression. Accordingly, in some embodiments, 1, 2, 3, 4, 5, or 6 or more repeat doses with a dose-dependent effect may be administered to maintain and/or increase the level of transgene expression by a certain amount each time (i.e., at each repeated dosing) to increase the expression level to a desired level or to a desired range of expression levels that is higher than the expression level achieved at the previous administration or before such repeated dosing.

[00216] The ability of a skilled practitioner to titrate the dose of a composition containing cccDNA vectors as described herein based on the dose-response relationship for the vector is useful for treating disease in a variety of ways. At a minimum, a skilled practitioner can increase the dose of the cDNA vector when increasing the effect (eg, transgene expression) is desired. Alternatively, one skilled in the art may select a dose that is known to produce a level of expression that is therapeutic based on prior knowledge of the dose-response relationship for the cccDNA composition. In some embodiments, it may be desirable to select a relatively high dose on the dose-response curve, for example, to accelerate treatment of a subject having severe symptoms of a given disease. In contrast, it is generally desirable to select a low dose on the dose-response curve and, optionally, gradually increase the dose to produce a therapeutic effect while preventing the occurrence of unwanted side effects or intolerance to the composition. In one embodiment, the dose-response relationship for a given cccDNA vector is used to determine and/or estimate the optimal dose for effective treatment of a given disease, the value of which is in the central part of the therapeutic window for the composition. Thus, dose titration of cccDNA vectors as described herein maximizes the therapeutic effect of the expressed transgene while also minimizing side effects or unwanted toxicity.

[00217] By way of illustration only, subjects with cystic fibrosis may have different degrees of disease severity and/or respond differently to the same level of CFTR1 gene transgene expression and/or have lower or higher than normal clearance of the drug, and therefore gradual increase in the expression of the CFTR1 transgene by one or more repeated injections of a cccDNA vector containing the CFTR1 transgene allows a gradual increase in the level of expression of the CFTR1 transgene, i.e. to a level of expression that effectively reduces one or more symptoms of cystic fibrosis in that particular subject. Previously, such a personalized approach or titration method to increase transgene expression levels was either ineffective and/or impossible due to the immune responses associated with other virus-based vectors, such as AAV vectors.

[00218] In some embodiments, repeated dosing with a dose-dependent effect provides a controlled increase in the level of transgene expression and, therefore, the methods and compositions disclosed herein enable a personalized drug approach to gene therapy. By way of illustration only, subjects with cystic fibrosis may have different degrees of disease severity and/or respond differently to the same level of CFTR1 transgene expression and/or have lower or higher than normal drug clearance and therefore, the controlled increase in expression of the CFTR1 transgene by administering one or more repeated doses with a dose-dependent effect of the cccDNA vector containing the CFTR1 transgene allows for a controlled increase in the expression level of the CFTR1 transgene, and in some embodiments, the controlled increase in expression can be brought to the level of expression, which effectively reduces one or more symptoms of cystic fibrosis in that particular subject. Previously, this personalized or titration approach to dose-dependently increase transgene expression was ineffective and/or impossible due to immune responses associated with other viral or AAV vectors.

[00219] As described herein, in some embodiments, the subject is examined at a specific point in time after the first administration of the cccDNA vector, for example, at any point in time after at least 30 days, or at least 60 days, or from 60 to 90 days, or more than 90 days, after the first administration of the cDNA vector, to determine the titration dose. For example, in some embodiments, the subject is examined to determine the disease state of the subject after the first administration of the cccDNA vector and/or the level of the transgene expressed by the cccDNA vector in the subject.

[00220] In some embodiments, the disease state assessment is an assessment of at least one disease symptom in the subject. Disease status for any particular disease can be determined by a physician or person skilled in the art and includes assessment of one or more clinical symptoms and/or disease biomarkers, including protein biomarkers, microRNA and mRNA biomarkers, and other molecular profiling systems.

[00221] In some embodiments, an assessment of a subject's disease state may be performed using molecular profiling in combination with clinical characterization of the patient, such as observations made by a physician (e.g., such as an International Classification of Diseases code, and dates of determination of such codes), results laboratory tests, x-rays, biopsy results, statements made by the patient, and any other medical information that a physician would normally rely on to make a diagnosis for a particular disease. Methods for determining a disease state based on molecular profiling in a subject are disclosed in US Patent and Patent Application Nos. 7167734, 9372193, 9383365, 2006/0224191, 2011/0172501, 2009/0104596, 2009/0023149, all of which are incorporated herein in their entirety. .

[00222] In some embodiments, the methods described herein can be used to titrate a cccDNA vector into a subject for a personalized medical intervention for a specific disease.

[00223] Studying changes in various biomarkers provides information about the condition of the subject from which the biomarkers are obtained. Understanding how biomarkers change (eg, increase, decrease, do not change) with disease progression allows, by taking measurements of one biological sample at a time, to confirm (eg, presence or absence of disease), typify disease, and characterize disease states (eg , early stage or "onset", or late stage or "recovery").

[00224] In some embodiments, biomarkers can be assessed to assess a disease progression state, such as onset or recovery, based on the level of each of the biomarkers as well as their trend (increase, decrease, or persistence) over time. Profiling biomarkers of a subject's disease state can also be combined with other methods, such as stable isotope ratios in natural respiration (eg, US Pat. No. 5,912,178), to assess whether a person is healthy or in a disease state. Disease states are identified by measuring changes in levels of biomarkers and, in particular, multiple biomarkers interrelated within a biological pathway associated with a disease state. In some embodiments, a specific disease state can be characterized by detecting and analyzing complex signals in NMR spectra to identify biomarkers whose levels change as the disease progresses. This initial assessment of the disease state allows one to “fingerprint” the dynamic changes associated with disease progression and helps assess the current state of development and disease process. Once a disease state is identified, administration of the cDNA vector can be tailored and/or titrated to the subject so as to shorten the duration of the disease.

[00225] In some embodiments, a disease state can be assessed by analyzing a biomarker profile in a biological sample obtained from a subject. The specific biomarkers measured are determined from analysis of the underlying biochemical pathways underlying the disease and the associated host immune response. In one embodiment, a standard biomarker profile is obtained from a healthy person and from a person with a disease. Comparison of a biomarker profile from a biological sample with a standard biomarker profile (healthy and diseased) allows positive identification of a disease state. Optionally, a second biological sample is collected from the patient at a second time point or at a time of disease progression to determine trends in the biomarker profile (eg, which biomarkers change between the first and second samples), thereby providing additional information about the disease state or the patient's condition. In some embodiments, a standard biomarker profile is assessed at one or more of the following time points: before the first administration of the cccDNA vector, after at least 30 days, or at least 60 days, or from 60 to 90 days, or after more than 90 days after the first administration of the cccDNA vector, or at least 30 days, or at least 60 days, or from 60 to 90 days, or more than 90 days after the second administration (for example, a repeat dose) of the cccDNA vector, or after at least 30 days, or at least 60 days, or from 60 to 90 days, or more than 90 days after any subsequent administrations (eg, repeated doses) of the cccDNA vector.

[00226] Protein biomarkers for diabetes, Alzheimer's disease and cancer have been identified. (See, for example, US Patent Nos. 7125663; 7097989; 7074576; and 6925389, which are incorporated herein in their entirety). Methods for detecting protein biomarkers such as mass spectrometry and specific antibody binding can also be used. High-throughput microarray expression analysis methods can be used for mRNA biomarkers as well as focused arrays and qPCR for multiple relevant genes to identify stress-related genes, see for example WO2007106685A2. DNA microarrays have been used to measure gene expression in tumor samples from patients and to aid diagnosis. Gene expression can reveal whether a patient has cancer, its type, stage and origin, and test for the involvement of genetic mutations. Gene expression may even play a role in predicting the effectiveness of chemotherapy. Over the past decades, the US National Cancer Institute (NCI) has tested the effects of compounds, including chemotherapy agents, on limiting the growth of 60 human cancer cell lines. NCI also measured gene expression in these 60 cancer cell lines using DNA microarrays. Various studies have examined the relationship between gene expression and compound effect using NCI datasets. Critical time in finding effective chemotherapy for cancer patients is often lost due to a trial-and-error approach. In addition, cancer cells often develop resistance to previously effective therapy. In such situations, patient outcome can be significantly improved by early identification of such resistance.

[0001] In some embodiments, the expression level of a biomarker of a disease state is determined by measuring the level of mRNA transcribed from the gene(s), by determining the level of the protein product of the gene(s), or by determining the level of biological activity of the protein product of the gene(s). In some embodiments, the level of a biomarker (including microRNA biomarkers) of a disease state is measured using quantitative reverse transcription-polymerase chain reaction (qRT-PCR). Such methods for measuring gene expression products, such as protein levels, include ELISA (enzyme-linked immunosorbent assay), Western blotting and immunoprecipitation, immunofluorescence using detection reagents such as antibody or protein binding agents. Alternatively, the peptide may be detected in a subject by administering to the subject a labeled antibody against the peptide and other types of detection agents. For example, the antibody may be labeled with a radioactive marker, the presence and location of which in a subject is determined by standard imaging techniques.

[0002] In some embodiments, gene expression products can be determined by measuring the level of messenger RNA (mRNA) expression of a disease biomarker. Such molecules can be isolated, prepared, or amplified from a biological sample, such as whole blood or plasma, such as platelet-rich plasma. Detection of mRNA expression is known to those skilled in the art and includes, for example, but is not limited to, PCR procedures, RT-PCR, Northern blotting, differential gene expression, RNA protection assays, microarray analysis, hybridization techniques, etc. In some embodiments, the mRNA level may be measured using quantitative RT-PCR. In general, the PCR procedure describes a method of gene amplification that involves (i) sequence-specific hybridization of primers to specific genes or sequences in a sample or nucleic acid library, (ii) subsequent amplification involving multiple cycles of annealing, extension and denaturation using thermostable DNA -polymerase, and (iii) screening the PCR products for the presence of a band of the correct size. The primers used are oligonucleotides of sufficient length and suitable sequence to initiate polymerization, i.e. each primer is specifically designed to be complementary to the genomic locus strand that is to be amplified. In an alternative embodiment, the mRNA level of the gene expression products described herein can be determined using reverse transcription (RT) PCR and quantitative RT-PCR (qRT-PCR) or real-time PCR methods. RT-PCR and qRT-PCR methods are well known in the art.

[00227] In some embodiments, methods for measuring one or more biomarkers of a disease state or the level of transgene expression from a cccDNA vector may be selected from any of: immunohistochemical (IHC) analysis and/or microarray analysis, comparative genomic hybridization (CGH) microarrays, single nucleotide polymorphism (SNP) microarrays, fluorescence in situ hybridization (FISH), in situ hybridization (ISH) and proteomic analysis.

[00228] As used herein, the term “microarray” means a device used in any manner capable of quantifying, at a time, one or more of the oligonucleotides of interest, such as DNA or RNA, or analogs thereof. One typical example of a class of microarrays consists of DNA probes attached to the surface of glass or quartz. Many microarrays, such as those made by Affymetrix, use multiple probes to detect the expression of a single gene. The DNA microarray may contain oligonucleotide probes, which are, for example, full-length cDNA complementary to RNA, or cDNA fragments that hybridize with part of the RNA. Typical RNAs include mRNAs, microRNAs, and miRNA precursors. Typical microarrays also include a “nucleic acid microarray” having a plurality of substrate-bound nucleic acids, with hybridization to each of the plurality of bound nucleic acids being determined separately. The substrate may be solid or porous, flat or non-flat, uniform or distributed. Typical nucleic acid microarrays include all devices so designated in Schena (ed.), DNA Microarrays: A Practical Approach (Practical Approach Series), Oxford University Press (1999); Nature Genet. 21(1)(suppl.):1-60 (1999); and Schena (ed.), Microarray Biochip: Tools and Technology, Eaton Publishing Company/BioTechniques Books Division (2000). In addition, examples of nucleic acid microarrays may include a substrate-associated plurality of nucleic acids that are placed on multiple beads rather than on a single flat support, as described in particular in Brenner et al., Proc. Natl. Acad. Sci. USA 97(4):1665-1670 (2000). Examples of nucleic acid microarrays are given in US patents No. 6391623, 6383754, 6383749, 6380377, 6379897, 6376191, 6372431, 6351712, 6344316, 6316193, 6312906, 6309828 , 6309824, 6306643, 6300063, 6287850, 6284497, 6284465, 6280954, 6262216, 6251601, 6245518, 6263287, 6251601, 6238866, 6228575, 6214587, 6203989, 6171797, 6103474, 6083726, 6054274, 6040138, 608372 6, 6004755, 6001309, 5958342, 5952180, 5936731, 5843655, 5814454, 5837196, 5436327, 5412087 and 5405783, which are incorporated herein by reference.

[00229] Exemplary microarrays may also include “peptide microarrays” or “protein microarrays” having a plurality of substrate-bound polypeptides, wherein the binding of any oligonucleotide, peptide or protein to the plurality of bound polypeptides may be separately determined. Alternatively, the peptide microarray may have a variety of coupling agents, including, but not limited to, monoclonal antibodies, polyclonal antibodies, phage display coupling agents, yeast two-hybrid assay coupling agents, aptamers, which can specifically detect the binding of specific oligonucleotides, peptides, or proteins. Examples of peptide matrices are given in international patent publications No. WO 02/31463, WO 02/25288, WO 01/94946, WO 01/88162, WO 01/68671, WO 01/57259, WO 00/61806, WO 00/54046, WO 00/47774, WO 99/40434, WO 99/39210 and WO 97/42507, and US Patent Nos. 6,268,210, 5,766,960 and 5,143,854, incorporated herein by reference.

[00230] In some embodiments, when the subject's disease state remains stable or has not improved, or when the subject's disease state has worsened, for example, compared to the disease state at the time of first administration of the cccDNA vector or at any point in time prior to administration of the cccDNA vector , the subject is administered a second dose of the cccDNA vector, wherein, for example, in some embodiments, the amount of cccDNA vector administered is a titrated dose.

[00231] In alternative embodiments, if the expression level of a transgene in a subject has decreased from a predetermined level or has decreased from a therapeutically effective amount, for example, has fallen from an initial level of transgene expression after the first administration of the cccDNA vector, the subject is administered a second dose of the cccDNA vector, wherein for example, in some embodiments, the amount of cccDNA vector administered is a titrated dose. In some embodiments, the expression level of a transgene is determined by measuring the level of the transgene (e.g., measuring protein levels and/or mRNA levels) expressed from a cccDNA vector in a biological sample obtained from a subject. In some embodiments, the biological sample is selected from any of: blood, plasma, synovial fluid, cerebrospinal fluid (CSF), saliva, or a tissue biopsy sample.

[00232] In some embodiments, when the cccDNA vector expresses a reporter protein in addition to a transgene encoding a desired protein or therapeutic gene, the level of the transgene can be determined by measuring the level of the reporter protein expressed from the cccDNA vector in vivo using methods generally known specialists in this field of technology. In some embodiments, titrating the cccDNA vector determines the level of transgene expressed by the cccDNA vector and administering a second dose of the cccDNA vector to the subject to adjust or modulate expression of the transgene to a predetermined desired level.

III. cccDNA vector as a whole

[00233] Embodiments of the invention are based on methods and compositions containing linear closed-end duplex vectors (ccDNA) that can express a transgene, as described herein. cccDNA vectors, as described herein, are not limited in size, allowing, for example, all components required for transgene expression to be expressed from a single vector. The cccDNA vector is preferably duplex, eg, self-complementary, in at least part of the molecule, such as an expression cassette (eg, the ccDNA is not a double-stranded circular molecule). The cccDNA vector has covalently closed ends and is thus resistant to exonuclease hydrolysis (eg by exonuclease I or exonuclease III), for example, within an hour at 37°C. In some embodiments, the cDNA vector, as described herein, is translocated into the nucleus, where expression of a transgene in the cDNA vector, such as a genetic drug transgene, can occur. In some embodiments, a cccDNA vector, as described herein, is translocated into the nucleus, where expression of a transgene, for example, a genetic drug transgene located between two ICPs, can occur.

[00234] Typically, the cccDNA vector disclosed herein contains, in the 5' to 3' direction: the adeno-associated virus (AAV) first inverted terminal repeat (ITR), a nucleotide sequence of interest (e.g., an expression cassette, as described in this document), and a second AAV ICP. The IKP sequences are selected from any of: (i) at least one wild-type IKP (IKP-WT) and at least one modified AAV inverted terminal repeat (mod-IKP) (eg, asymmetric modified IKP); (ii) two modified ICPs, wherein the pair of mod-ICPs have a different three-dimensional spatial organization relative to each other (for example, asymmetric modified ICPs), or (iii) a pair of symmetric or substantially symmetrical DT-DT ICPs, each of the ICP-DTs having the same three-dimensional spatial organization, or (iv) pairs of symmetric or substantially symmetric modified ICPs, each of the mod-ICPs having the same three-dimensional spatial organization.

[00235] This document covers methods and compositions containing a cccDNA vector, which may further include a delivery system, such as, but not limited to, a liposomal nanoparticle delivery system. Non-limiting examples of liposome nanoparticle systems suitable for use are disclosed herein. In some aspects, the present invention relates to a lipid nanoparticle comprising cccDNA and an ionizable lipid. For example, the composition of lipid nanoparticles that are manufactured and loaded with a cccDNA vector obtained by this method is disclosed in international application PCT/US2018/050042, filed September 7, 2018, which is incorporated herein.

[00236] cccDNA vectors, as described herein, do not have the packaging restrictions imposed by limited space within the viral capsid. cccDNA vectors provide a viable eukaryotic-produced alternative to prokaryotic-produced plasmid DNA vectors, as opposed to encapsulated AAV genomes. This allows the insertion of control elements, such as regulatory switches as described herein, large transgenes, multiple transgenes, etc.

[00237] FIGS. 1A-1E show diagrams of non-limiting examples of cccDNA vectors, or corresponding ccDNA plasmid sequence. cccDNA vectors are capsidless and can be obtained from a plasmid encoding, in this order: a first ICP, an expression cassette containing the transgene, and a second ICP. An expression cassette may include one or more regulatory sequences that enable and/or control expression of a transgene, for example, where the expression cassette may contain one or more of the following, in that order: enhancer/promoter, reporter ORF (transgene), post-transcriptional regulatory element (eg, WPRE) and polyadenylation and termination signal (eg, poly(A) BGH).

[00238] The expression cassette may also contain an internal ribosome entry site (IRES) and/or a 2A element. Cis-regulatory elements include, but are not limited to, promoter, riboswitch, insulator, microRNA-regulated element, post-transcriptional regulatory element, tissue-specific and cell-specific promoter and enhancer. In some embodiments, the ICP may act as a promoter for the transgene. In some embodiments, said cccDNA vector contains additional components for regulating the expression of said transgene, for example, regulatory switches, which are described herein in the "Regulatory Switches" section, for controlling and regulating the expression of said transgene, and may include, if necessary, a regulatory switch, which is a “kill switch” that allows controlled cell death of the cell containing the cccDNA vector.

[00239] Said expression cassette may contain more than 4000 nucleotides, 5000 nucleotides, 10000 nucleotides or 20000 nucleotides, or 30000 nucleotides, or 40000 nucleotides, or 50000 nucleotides, or any range between about 4000-10000 nucleotides or 10000-5000 0 nucleotides , or more than 50,000 nucleotides. In some embodiments, the expression cassette may contain a transgene having a length in the range of 500 to 50,000 nucleotides. In some embodiments, the expression cassette may contain a transgene having a length ranging from 500 to 75,000 nucleotides. In some embodiments, the expression cassette may contain a transgene having a length in the range of 500 to 10,000 nucleotides. In some embodiments, the expression cassette may contain a transgene having a length in the range of 1000 to 10,000 nucleotides. In some embodiments, the expression cassette may contain a transgene having a length in the range of 500 to 5000 nucleotides. cccDNA vectors do not have the size restrictions characteristic of encapsidated AAV vectors, which allows delivery of a large expression cassette to ensure efficient transgene expression. In some embodiments, the cDNA vector lacks specific prokaryotic methylation.

[00240] The cccDNA expression cassette may include, for example, an exogenous expressed sequence (e.g., an open reading frame) or a transgene encoding a protein that is either absent, inactive, or deficient in activity in the recipient subject, or a gene that encodes a protein having a desired biological or therapeutic effect. The transgene may encode a gene product that may function to correct the expression of a defective gene or transcript. In principle, the expression cassette can include any gene encoding a protein, polypeptide or RNA that is either reduced or absent due to mutation, or that provides a therapeutic benefit when overexpression is considered within the scope of the invention.

[00241] The expression cassette may contain any transgene useful for treating a disease or disorder in a subject. The cccDNA vector can be used to deliver and express any gene of interest in a subject, including, without limitation, polypeptide-encoding nucleic acids or non-coding nucleic acids (e.g., RNAi, mature microRNAs, etc.), as well as exogenous genes and nucleotide sequences, including viral sequences in the genome of subjects, for example, HIV virus sequences and the like. Preferably, the cDNA vector disclosed herein is used for therapeutic purposes (for example, for medical, diagnostic or veterinary applications), or immunogenic polypeptides. In some embodiments, the cccDNA vector is suitable for expression in a subject of any gene of interest, which includes one or more polypeptides, peptides, ribozymes, peptide nucleic acids, siRNA, RNAi, antisense oligonucleotides, antisense polynucleotides, or RNA (encoding or non-coding, e.g. siRNAs, short hairpin RNAs (shRNAs), microRNAs and their antisense analogs (eg, antagomir (antagoMiR)), antibodies, antigen-binding fragments, or any combination thereof.

[00242] The expression cassette may also encode polypeptides, sense or antisense oligonucleotides, or RNAs (coding or non-coding; e.g., siRNAs, short hairpin RNAs, microRNAs, and their antisense analogues (e.g., antagomir)). Expression cassettes may include an exogenous sequence encoding a reporter protein to be used for experimental or diagnostic purposes, such as β-lactamase, β-galactosidase (LacZ), alkaline phosphatase, thymidine kinase, green fluorescent protein (GFP), chloramphenicol acetyltransferase (ChAT), luciferase and others well known in the art.

[00243] The sequences present in the expression cassette, expression construct of the cDNA vector described herein, can be codon optimized for the target host cell. As used herein, the term “codon-optimized” or “codon optimization” refers to the process of modifying a nucleic acid sequence to enhance expression in cells of a vertebrate animal of interest, such as a mouse or human, by replacing at least one, more than one , or a significant number of codons from a native sequence (e.g., a prokaryotic sequence) to codons that are more frequently or most frequently used in the genes of that vertebrate. Different species exhibit a particular affinity for certain codons of a particular amino acid. Typically, codon optimization does not change the amino acid sequence of the original translated protein. Optimized codons can be determined using, for example, the Gene Forge® custom gene synthesis and codon optimization platform from Aptagen (Aptagen, Inc., 2190 Fox Mill Rd. Suite 300, Herndon, Va. 20171) or other publicly available database.

[00244] In some embodiments, the transgene expressed by a cccDNA controlled expression vector as described herein is a therapeutic gene. In some embodiments, the therapeutic gene is an antibody, or an antibody fragment, or an antigen-binding fragment thereof, or a fusion protein. In some embodiments, the antibody or fusion protein thereof is an activating antibody or a neutralizing antibody, or an antibody fragment, etc. In some embodiments, the cDNA vector for controlled gene expression comprises an antibody or fusion protein, as described in International Patent PCT/US19 /18016, filed February 14, 2019, which is incorporated herein by reference.

[00245] In particular, a therapeutic gene is one or more therapeutic agents, including, without limitation, protein(s), polypeptide(s), peptide(s), enzyme(s), antibodies, antigen binding fragments, as well as the variants and/or active moieties, for use in the treatment, prevention and/or alleviation of one or more symptoms of a disease, dysfunction, lesion and/or disorder. Representative examples of therapeutic genes are described herein in the section entitled “Method of Treatment.”

[00246] There are many structural features of cccDNA vectors that differ from plasmid-based expression vectors. cccDNA vectors may have one or more of the following features: lack of parent (i.e., uninserted) bacterial DNA, lack of a prokaryotic origin of replication, self-sufficiency, i.e. they do not require any sequences other than the two ICPs, including Rep and terminal resolution binding sites (RBS and TRS), and exogenous sequence between the ICPs, the presence of hairpin-forming ICP sequences, and the absence of bacterial-type DNA methylation or any other methylation , considered abnormal in the mammalian host. In general, it is preferred that the present vectors do not contain any prokaryotic DNA, but it is possible to insert some prokaryotic DNA as an exogenous sequence, by way of non-limiting example, in the promoter or enhancer region. Another important feature that distinguishes cccDNA vectors from plasmid expression vectors is that cccDNA vectors are single-stranded linear DNA with closed ends, whereas plasmids are always double-stranded DNA.

[00247] cccDNA vectors produced by the methods presented herein preferably have a linear and continuous structure, rather than a discontinuous structure, as determined by a restriction enzyme digestion assay (Figure 4D). A linear and continuous structure is considered more stable when exposed to cellular endonucleases and is also less likely to undergo recombination and cause mutagenesis. Thus, a cccDNA vector with a linear and continuous structure is the preferred implementation option. A continuous linear single-stranded cccDNA vector with an intramolecular duplex may have covalently linked terminal ends, without sequences encoding AAV capsid proteins. Such cDNA vectors are structurally different from plasmids (including the cDNA plasmids described herein), which are circular duplex nucleic acid molecules of bacterial origin. The complementary strands of plasmids can be separated after denaturation to form two nucleic acid molecules, while cccDNA vectors, on the contrary, although they have complementary strands, represent a single DNA molecule and therefore remain a single molecule even in a denatured state. In some embodiments, cccDNA vectors, as described herein, can be produced without prokaryotic DNA base methylation, unlike plasmids. Accordingly, cccDNA vectors and ccDNA plasmids differ both in structure (in particular, linear or circular), and in the methods used to obtain and purify these different objects (see below), as well as in the nature of DNA methylation, which occurs in a prokaryotic manner in cDNA plasmids and in the eukaryotic type in the cDNA vector.

[00248] There are several advantages to using a cccDNA vector as described herein over plasmid-based expression vectors, such advantages include, but are not limited to, the following: 1) Plasmids contain bacterial DNA sequences and are subject to specific prokaryotic methylation, e.g. methylation with the formation of 6-methyladenosine and 5-methylcytosine, while the capsid-free sequences of the AAV vector are of eukaryotic origin and are not subject to specific prokaryotic methylation; as a result, capsidless AAV vectors will be less likely to induce inflammatory and immune responses compared to plasmids; 2) if plasmids require the presence of a resistance gene during production, then ccDNA vectors do not; 3) if the circular plasmid is not delivered to the nucleus when introduced into the cell and requires an excess load to avoid degradation by cellular nucleases, then ccDNA vectors contain viral cis-elements, i.e. ICPs confer nuclease resistance and can be engineered to target and deliver to the nucleus. It has been proposed that the minimum defining elements required for ICP function are the Rep binding site (RBS; 5'-GCGCGCTCGCTCGCTC-3' (SEQ ID NO: 60) for AAB2) and the terminal resolution site (TRS; 5'-AGTTGG -3' (SEQ ID NO: 64) for AAB2) plus a variable palindromic sequence enabling hairpin formation; and 4) cccDNA vectors do not have the overrepresentation of CpG dinucleotides often observed in plasmids of prokaryotic origin, which are known to bind to a member of the Toll-like receptor family, inducing a T-cell-mediated immune response. In contrast, transduction with capsidless AAV vectors, disclosed herein can provide effective targeting of cell and tissue types that are difficult to transduce with standard AAV virions using a variety of delivery reagents.

IV. ICP

[00249] As disclosed herein, cccDNA vectors for controlled transgene expression comprise a transgene or heterologous nucleic acid sequence located between two inverted terminal repeat (ITR) sequences, wherein the ITR sequences may be an asymmetric ICT pair or a symmetric or substantially symmetric pair ICP as these terms are defined in this document. The cccDNA vector, as described herein, may contain ICP sequences that are selected from any of: (i) at least one wild-type (WT) ICP and at least one modified inverted terminal repeat (mod-IRT) AAV ( for example, asymmetric modified ICP); (ii) two modified ICPs, wherein the mod-ICP pair has a different three-dimensional spatial organization relative to each other (for example, asymmetric modified ICPs), or (iii) a pair of symmetric or substantially symmetrical DT-DT ICPs, in which each ICP-DT has the same three-dimensional spatial organization, or (iv) pairs of symmetric or substantially symmetric modified ICPs, wherein each mod-ICP has the same three-dimensional spatial organization, wherein the methods of this invention may further include a delivery system, such as, but not limited to, a delivery system based on liposomal nanoparticles.

[00250] In some embodiments, the ICP sequence may be taken from viruses of the Parvoviridae family, which includes two subfamilies: Parvovirinae, which infect vertebrates, and Densovirinae, which infect insects. The subfamily Parvovirinae (called "parvoviruses") includes the Dependovirus genus, which requires co-infection with a helper virus, such as an adenovirus or herpesvirus, for productive infection under most conditions. The Dependovirus genus includes adeno-associated virus (AAV), which typically infects humans (eg , serotypes 2, 3A, 3B, 5 and 6) or primates (eg, serotypes 1 and 4), and related viruses that infect other warm-blooded animals (eg, bovine, canine, equine and ovine adeno-associated viruses). Parvoviruses and other members of the family Parvoviridae are described generally in Kenneth I. Berns, "Parvoviridae: The Viruses and Their Replication," Chapter 69 in FIELDS VIROLOGY (3d Ed. 1996).

[00251] Although the ICPs provided for illustration herein in the Description and Examples are AAV2-DT ICPs, one skilled in the art will recognize that, as stated above, ICPs from any known parvovirus, such as dependovirus, can be used. such as AAV (for example, from the genome of AAV1, AAV2, AAV3, AAV4, AAV5, AAV 5, AAV7, AAV8, AAV9, AAV10, AAV 11, AAV12, AAVrh8, AAVrh10, AAV-DJ and AAV-DJ8. For example, NCBI: NC 002077; NC 001401; NC001729; NC001829; NC006152; NC 006260; NC 006261), chimeric ICPs or ICPs from any synthetic AAV. In some embodiments, AAV can infect warm-blooded animals, such as avian adeno-associated viruses (AAV), bovine (BAAV), canine, equine, and ovine adeno-associated viruses. In some embodiments, said ICP is derived from parvovirus B19 (GenBank Accession No. NC 000883), small mouse virus (MVM) (GenBank Accession No. NC 001510); goose parvovirus (GenBank accession no. NC 001701); snake parvovirus 1 (GenBank accession no. NC 006148). In some embodiments, the 5'-ICP-WT may be from one serotype and the 3'-ICP-WT from a different serotype, as described herein.

[00252] One of ordinary skill in the art will know that ICP sequences are uniformly arranged and contain a double-stranded Holliday structure, which is typically a T-shaped or Y-shaped hairpin structure (see, for example, Fig. 2A and Fig. 3A), with each ICP formed by two palindromic arms or loops (B-B' and C-C') embedded in a larger palindromic arm (A-A'), and a single-stranded sequence D (where the order of these palindromic sequences determines the direction ICP orientation). See, for example, for a description of the structural analysis and comparison of ICP sequences from different AAV serotypes (AAV1-AAV6) in Grimm et al., J. Virology, 2006; 80(1); 426-439; Yan et al., J. Virology, 2005; 364-379; Duan et al., Virology 1999; 261; 8-14. One of ordinary skill in the art can readily determine WT ICP sequences from any AAV serotype for use in a cccDNA vector or cccDNA plasmid based on the example AAV2 ICP sequences provided herein. See, for example, comparison of ICP sequences from different AAV serotypes (AAB1-AAV6 and avian AAV (AAAV) and bovine AAV (BAAV)), described in Grimm et al., J. Virology, 2006; 80(1); 426-439; where the % identity of the left ICP of AAV2 and the left ICP from other serotypes is given: AAV-1 (84%), AAV-3 (86%), AAV-4 (79%), AAV-5 (58%) AAV-6 (left IKP) (100%) and AAV-6 (right IKP) (82%).

A. Symmetrical ICP pairs

[00253] In some embodiments, the cccDNA vector, as described herein, contains, in the 5' to 3' direction: the first inverted terminal repeat (ITR) of an adeno-associated virus (AAV), a nucleotide sequence of interest (e.g., an expression cassette as described herein) and a second AAV ICP, wherein the first ICP (5'-ICP) and the second ICP (3'-ICP) are symmetrical or substantially symmetrical with respect to each other, i.e. The cccDNA vector may contain ICP sequences that have a symmetrical three-dimensional spatial organization such that their structure has the same shape in geometric space, or has the same A, C-C' and B-B' loops in three-dimensional space. In such an embodiment, the pair of symmetrical ICPs or the pair of substantially symmetrical ICPs may be modified ICPs (eg, mod-ICPs) that are not wild-type ICPs. A mod-IKP pair may have identical sequences that have one or more modifications compared to the wild-type IKP and are reverse complementary (inverted) to each other. In alternative embodiments, a pair of modified ICPs are substantially symmetrical as defined herein, i.e. a pair of modified ICPs may have different sequences but a similar or identical symmetrical three-dimensional shape.

[00254] (i) Wild type ICP

[00255] In some embodiments, symmetric ICPs or substantially symmetric ICPs are wild type (ICP-WT), as described herein. That is, both ICPs have a wild-type sequence, but do not have to be WT ICPs belonging to the same AAV serotype. Thus, in some embodiments, one ICP-WT may belong to one AAV serotype, and another ICP-WT may belong to a different AAV serotype. In such an embodiment, the ICP-DT pair is substantially symmetrical as defined herein, i.e. they can have one or more conservative nucleotide modifications while maintaining a symmetrical three-dimensional spatial organization.

[00256] Accordingly, as described herein, cccDNA vectors for controlled transgene expression contain a transgene or heterologous nucleic acid sequence located between two flanking wild-type inverted terminal repeat (ITR-WT) sequences that are either reverse complementary (inverted) relative to each other, or, alternatively, are substantially symmetrical relative to each other - i.e. the ICP-DT pair has a symmetrical three-dimensional spatial organization. In some embodiments, the wild-type ICP sequence (e.g., AAV ICP-DT) contains a functional Rep binding site (RBS; e.g., 5'-GCGCGCTCGCTCGCTC-3' for AAV2, SEQ ID NO: 60) and a functional terminal resolution site (TRS) ; e.g. 5'-AGTT-3', SEQ ID NO: 62).

[00257] In one aspect, cccDNA vectors for controlled transgene expression can be derived from a vector polynucleotide that encodes a heterologous nucleic acid operably located between two wild-type inverted terminal sequences (ICT-WT) (eg, AAV ICT-WT). That is, both ICPs have a wild-type sequence, but do not have to be WT ICPs belonging to the same AAV serotype. Thus, in some embodiments, one ICP-WT may belong to one AAV serotype, and another ICP-WT may belong to a different AAV serotype. In such an embodiment, the ICP-DT pair is substantially symmetrical as defined herein, i.e. they can have one or more conservative nucleotide modifications while maintaining a symmetrical three-dimensional spatial organization. In some embodiments, 5'-ICP-WT is from one AAV serotype, and 3'-ICP-WT is from the same or a different AAV serotype. In some embodiments, the 5'-ICP-DT and 3'-ICP-DT are mirror images of each other, i.e. they are symmetrical. In some embodiments, the 5'-ICP-WT and the 3'-ICP-WT are from the same AAV serotype.

[00258] Wild type (WT) ICPs are well known. In one embodiment, the two ICPs are of the same serotype AAB2. In some embodiments, wild-type (WT) sequences from other serotypes can be used. There are a number of serotypes that are homologous, for example, AAV2, AAV4, AAV6, AAV8. In one embodiment, closely homologous ICPs (eg, ICPs with a similar loop structure) may be used. In another embodiment, an ICP-DT AAB with a greater degree of variation may be used, for example, AAB2 and AAV5, and in yet another embodiment, an ICP may be used that is essentially related to the DT, i.e. it has the basic loop structure characteristic of DT, but contains some conservative nucleotide substitutions that do not change or affect its properties. When using ICP-DT from the same viral serotype, one or more regulatory sequences can be additionally used. In certain embodiments, the regulatory sequence is a regulatory switch that allows the activity of the cccDNA to be modulated.

[00259] In some embodiments, one aspect of the technology described herein relates to a cccDNA vector comprising at least one heterologous nucleotide sequence operably positioned between two wild-type inverted terminal repeat (WT) sequences, wherein the ICT -DTs may be of the same serotype, different serotypes, or be essentially symmetrical with respect to each other (i.e., have a symmetrical three-dimensional spatial organization such that their structure has the same shape in geometric space or has the same A, C-C' and B-B'-loops in three-dimensional space). In some embodiments, symmetrical WT-ICPs contain a functional end resolution site and a Rep binding site. In some embodiments, the heterologous nucleic acid sequence encodes a transgene and wherein the vector is not enclosed within a viral capsid.

[00260] In some embodiments, the ICP-DTs are the same, but inversely complementary to each other. For example, the sequence AACG in the 5'-ICP may correspond to CGTT (ie, reverse complement) at the corresponding site in the 3'-ICP. In one example, the 5'-ICP-DT sense strand contains the sequence ATCGATCG, and the corresponding 3'-ICP-DT sense strand contains CGATCGAT (ie, the reverse complement of ATCGATCG). In some embodiments, the ICP-WT cccDNA further comprises an end resolution site and a replication protein binding site (RPS) (sometimes referred to as a replication protein binding site), such as a Rep binding site.

[00261] Typical ICP-WT sequences for use in cDNA vectors for controlled transgene expression containing ICP-WT are shown in Table 2 of this document, which shows ICP-WT pairs (5'-ICP-WT and 3'-ICP- DT).

[00262] As a typical example, the present invention provides a cccDNA vector containing a promoter operably linked to a transgene (e.g., a heterologous nucleic acid sequence), with or without a regulatory switch, wherein the cccDNA is devoid of capsid proteins and: (a) is produced from a cccDNA plasmid (e.g., see FIGS. 1F-1G) that encodes IKP-DT, each IKP-DT having the same number of base pairs of intramolecular duplexes in its hairpin secondary configuration (preferably excluding deletion of any terminal AAA or TTT loop in this configuration compared to such reference sequences), and (b) identified as cccDNA using the cccDNA identification assay by agarose gel electrophoresis with native gel and denaturing conditions described in Example 1.

[00263] In some embodiments, the flanking ICP-DTs are substantially symmetrical to each other. In this embodiment, the 5'-ICP-WT may be from one AAV serotype and the 3'-ICP-WT from a different AAV serotype, such that the ICP-WT are not identical reverse complements. For example, 5'-IKP-WT may belong to AAV2, and 3' IKP-WT may belong to another serotype (eg, AAV1, 3, 4, 5, 6, 7, 8, 9, 10, 11 and 12). In some embodiments, the ICP-DT may be selected from two different parvoviruses selected from any of: AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, snake parvovirus (e.g. , royal python parvovirus), bovine parvovirus, caprine parvovirus, avian parvovirus, canine parvovirus, equine parvovirus, shrimp parvovirus, porcine parvovirus or insect AAV. In some embodiments, this ICP-DT combination is a combination of the ICP-DT from AAB2 and AAV6. In one embodiment, the ICP-DTs are substantially symmetrical when one of them is inverted with respect to another ICP that is at least 90% identical, at least 95% identical, at least 96%... 97%. .. 98%... 99%... 99.5%, including all intermediate values, and has the same symmetrical three-dimensional spatial organization. In some embodiments, the ICP-DT pair is substantially symmetrical in that they have a symmetrical three-dimensional spatial organization, for example, having the same three-dimensional organization of the A, C-C', B-B', and D arms. In one embodiment, a substantially symmetrical ICP-DT pair is inverted with respect to each other and is at least 95% identical, at least 96%... 97%... 98%... 99%.... 99.5%, including all intermediate values, and one ICP-DT retains the Rep binding site (RBS) 5'-GCGCGCTCGCTCGCTC-3' (SEQ ID NO: 60) and the terminal resolution site (trs). In some embodiments, a substantially symmetrical ICP-DT pair is inverted with respect to each other, and is at least 95% identical, at least 96%... 97%... 98%... 99%.. .. 99.5%, including all intermediate values, and one ICP-DT retains the Rep binding site (RBS) 5'-GCGCGCTCGCTCGCTC-3' (SEQ ID NO: 60) and the terminal resolution site (trs), in addition to a variable palindromic sequence that allows the formation of a secondary hairpin structure. Homology can be determined by standard tools well known in the art, such as BLAST (Basic Local Alignment Search Tool), BLASTN, with default settings.

[00264] In some embodiments, the ICP structural element can be any structural element that is involved in the functional interaction of the ICP with the large Rep protein (eg, Rep 78 or Rep 68). In some embodiments, said structural element provides selectivity for the interaction of ICP with the large Rep protein, i.e. determines, at least in part, which Rep protein functionally interacts with ICP. In other embodiments, the structural element physically interacts with a large Rep protein upon binding of said Rep protein to the ICP. Each structural element may be, for example, an ICP secondary structure, an ICP nucleotide sequence, an intermediate sequence between two or more elements, or a combination of any of the above. In one embodiment, said structural elements are selected from the group consisting of arm A and arm A', arm B and arm B', arm C and arm C', arm D, a Rep-binding site (RBE) and RBE' (i.e. e. sequence complementary to RBE) and a terminal resolution site (trs).

[00265] By way of example only, Table 1 provides examples of ICP-DT combinations.

[00266] Table 1: Illustrative examples of ICP-DT combinations of the same serotype, or different serotypes, or different parvoviruses. The above order does not indicate the position of the IKP, for example, “AAB1, AAV2” means that ccDNA may contain IKP-DT AAV1 at the 5' position and IKP-DT AAV2 at the 3' position or, conversely, IKP-DT AAB2 at the 5' position and IKP-DT AAV1 at position 3'. Abbreviations: AAV serotype 1 (AAV1), AAV serotype 2 (AAV2), AAV serotype 3 (AAV3), AAV serotype 4 (AAV4), AAV serotype 5 (AAV5), AAV serotype 6 (AAV6), AAV serotype 7 (AAV7) , AAV serotype 8 (AAV8), AAV serotype 9 (AAV9), AAV serotype 10 (AAV10), AAV serotype 11 (AAV11) or AAV serotype 12 (AAV12); genome AABrh8, AABrh10, AAV-DJ and AAV-DJ8 (for example, NCBI: NC 002077; NC 001401; NC001729; NC001829; NC001829; NC006152; NC 006260; NC 006261), ICP of warm-blooded animals (avian A AB (AAAB), bullish AAB (bovine AAV) (BAAV), canine, equine and sheep AAV), parvovirus B19 ICP (GenBank, accession no. NC 000883), mouse small virus (MVM) (GenBank, accession no. NC 001510); geese: goose parvovirus (GenBank accession no. NC 001701); snakes: snake parvovirus 1 (GenBank accession no. NC 006148).

[00267] Table 1

AAV1, AAV1 AAV2, AAV2 AAV3, AAV3 AAV4, AAV4 AAV5, AAV5 AAV1, AAV2 AAV2, AAV3 AAV3, AAV4 AAV4, AAV5 AAV5, AAV6 AAV1, AAV3 AAV2, AAV4 AAV3, AAV5 AAV4, AAV6 AAV5, AAV7 AAV1, AAV4 AAV2, AAV5 AAV3, AAV6 AAV4, AAV7 AAV5, AAV8 AAV1, AAV5 AAV2, AAV6 AAV3, AAV7 AAV4, AAV8 AAV5, AAV9 AAV1, AAV6 AAV2, AAV7 AAV3, AAV8 AAV4, AAV9 AAV5, AAV10 AAV1, AAV7 AAV2, AAV8 AAV3, AAV9 AAV4, AAV10 AAV5, AAV11 AAV1, AAV8 AAV2, AAV9 AAV3, AAV10 AAV4, AAV11 AAV5, AAV12 AAV1, AAV9 AAV2, AAV10 AAV3, AAV11 AAV4, AAV12 AAB5, AABrh8 AAV1, AAV10 AAV2, AAV11 AAV3, AAV12 AAV4, AAVrh8 AAV5, AAVrh10 AAV1, AAV11 AAV2, AAV12 AAB3, AABrh8 AAV4, AAVrh10 AAV5, AAV13 AAV1, AAV12 AAV2, AAVrh8 AAV3, AAVrh10 AAV4, AAV13 AAV5, AAV-DJ AAB1, AABrh8 AAV2, AAVrh10 AAV3, AAV13 AAV4, AAV-DJ AAV5, AAV-DJ8 AAV1, AAVrh10 AAV2, AAV13 AAB3, AAV-DJ AAV4, AAV-DJ8 AAV5, BIRD AAV1, AAV13 AAV2, AAV-DJ AAV3, AAV-DJ8 AAV4, BIRD AAB5, BULLISH AAV1, AAV-DJ AAV2, AAV-DJ8 AAV3, BIRD AAB4, BULLISH AAV5, CANINE AAB1, AAV-DJ8 AAV2, BIRD AAB3, BULLISH AAV4, CANINE AAV5, HORSES AAV1, BIRD AAB2, BULLISH AAV3, CANINE AAV4, HORSES AAV5, GOAT AAB1, BULLISH AAV2, CANINE AAV3, HORSES AAV4, GOAT AAV5, SHRIMP AAV1, CANINE AAV2, HORSES AAV3, GOAT AAB4, SHRIMP AAV5, PIGS AAV1, HORSES AAV2, GOAT AAB3, SHRIMP AAV4, PIGS AAV5, INSECTS AAV1, GOAT AAB2, SHRIMP AAV3, PIGS AAV4, INSECTS AAB5, SHEEP AAB1, SHRIMP AAV2, PIGS AAV3, INSECTS AAB4, SHEEP AAV5, B19 AAV1, PIGS AAV2, INSECTS AAB3, SHEEP AAV4, B19 AAB5, MVM AAV1, INSECTS AAV2, SHEEP AAV3, B19 AAB4, MVM AAV5, GEESE AAB1, SHEEP AAV2, B19 AAB3, MVM AAV4, GEESE AAV5, SERPENT AAV1, B19 AAB2, MVM AAV3, GEESE AAV4, SERPENT ° AAB1, MVM AAV2, GEESE AAV3, SERPENT ° ° AAV1, GEESE AAV2, SERPENT ° ° ° AAV1, SERPENT ° ° ° ° AAV6, AAV6 AAV7, AAV7 AAV8, AAV8 AAV9, AAV9 AAV10, AAV10 AAV6, AAV7 AAV7, AAV8 AAV8, AAV9 AAV9, AAV10 AAV10, AAV11 AAV6, AAV8 AAV7, AAV9 AAV8, AAV10 AAV9, AAV11 AAV10, AAV12 AAV6, AAV9 AAV7, AAV10 AAV8, AAV11 AAV9, AAV12 AAV10, AAVrh8 AAV6, AAV10 AAV7, AAV11 AAV8, AAV12 AAB9, AABrh8 AAB10, AABrh10 AAV6, AAV11 AAV7, AAV12 AAB8, AABrh8 AAV9, AAVrh10 AAV10, AAV13 AAV6, AAV12 AAB7, AABrh8 AAV8, AAVrh10 AAV9, AAV13 AAB10, AAV-DJ AAV6, AAVrh8 AAV7, AAVrh10 AAV8, AAV13 AAV9, AAV-DJ AAB10, AAV-DJ8 AAV6, AAVrh10 AAV7, AAV13 AAV8, AAV-DJ AAV9, AAV-DJ8 AAV10, BIRD AAV6, AAV13 AAV7, AAV-DJ AAB8, AAV-DJ8 AAV9, BIRD AAB10, BULLISH AAV6, AAV-DJ AAV7, AAV-DJ8 AAV8, BIRD AAB9, BULLISH AAV10, CANINE AAV6, AAV-DJ8 AAV7, BIRD AAB8, BULLISH AAV9, CANINE AAV10, HORSES AAV6, BIRD AAB7, BULLISH AAV8, CANINE AAV9, HORSES AAV10, GOAT AAB6, BULLISH AAV7, CANINE AAV8, HORSES AAV9, GOAT AAB10, SHRIMP AAV6, CANINE AAV7, HORSES AAV8, GOAT AAV9, SHRIMP AAV10, PIGS AAV6, HORSES AAV7, GOAT AAB8, SHRIMP AAV9, PIGS AAV10, INSECTS AAV6, GOAT AAB7, SHRIMP AAV8, PIGS AAV9, INSECTS AAB10, SHEEP AAB6, SHRIMP AAV7, PIGS AAV8, INSECTS AAB9, SHEEP AAV10, B19 AAV6, PIGS AAV7, INSECTS AAB8, SHEEP AAV9, B19 AAB10, MVM AAV6, INSECTS AAB7, SHEEP AAV8, B19 AAB9, MVM AAV10, GEESE AAB6, SHEEP AAV7, B19 AAB8, MVM AAV9, GEESE AAV10, SERPENT AAV6, B19 AAB7, MVM AAV8, GEESE AAV9, SERPENT ° AAB6, MVM AAV7, GEESE AAV8, SERPENT ° ° AAV6, GEESE AAV7, SERPENT ° ° ° AAV6, SERPENT ° ° ° ° AAV11, AAV11 AAV12, AAV12 AABrh8, AABrh8 ААВrh10, ААВrh10 AAV13, AAV13 AAV11, AAV12 AAV12, AAVrh8 ААВrh8, ААВrh10 AABrh10, AAV13 AAV13, AAV-DJ AAV11, AAVrh8 AAV12, AAVrh10 AABrh8, AAV13 ААВrh10, ААВ-DJ AAV13, AAV-DJ8 AAV11, AAVrh10 AAV12, AAV13 ААВrh8, ААВ-DJ ААВrh10, ААВ-DJ8 AAV13, BIRD AAV11, AAV13 AAV12, AAV-DJ ААВrh8, ААВ-DJ8 AABrh10, BIRD AAB13, BULLISH AAV11, AAV-DJ AAV12, AAV-DJ8 AABrh8, BIRD ААВrh10, BULLISH AAV13, CANINE AAV11, AAV-DJ8 AAV12, BIRD AABrh8, BULLISH AABrh10, CANINE AAV13, HORSES AAV11, BIRD AAB12, BULLISH AABrh8, CANINE ААВrh10, HORSES AAV13, GOAT AAB11, BULLISH AAV12, CANINE ААВrh8, HORSES AABrh10, GOAT AAV13, SHRIMP AAV11, CANINE AAV12, HORSES AABrh8, GOAT AABrh10, PRAWN AAV13, PIGS AAV11, HORSES AAV12, GOAT AABrh8, SHRIMP AABrh10, PIGS AAV13, INSECTS AAV11, GOAT AAV12, SHRIMP AABrh8, PIGS AABrh10, INSECTS AAV13, SHEEP AAB11, SHRIMP AAV12, PIGS AABrh8, INSECTS AABrh10, SHEEP AAV13, B19 AAV11, PIGS AAV12, INSECTS AABrh8, SHEEP ААВrh10, B19 AAV13, MVM AAV11, INSECTS AAB12, SHEEP ААВrh8, B19 ААВrh10, MVM AAV13, GEESE AAB11, SHEEP AAV12, B19 ААВrh8, MVM ААВrh10, GEESE AAV13, SERPENT AAV11, B19 AAV12, MVM ААВrh8, GEESE ААВrh10, SERPENT AAV11, MVM AAV12, GEESE ААВrh8, SERPENT AAV11, GEESE AAV12, SERPENT AAV11, SERPENT AAB-DJ, AAV-DJ AAV-DJ8, AVVDJ8 BIRD, BIRD BULLY, BULLY DOGGY, DOGGY AAB-DJ, AAV-DJ8 AAV-DJ8, BIRD BIRD, BULL BULL, DOG DOGS, HORSES AAV-DJ, BIRD AAB-DJ8, BULLISH BIRD, CANINE BULL, HORSES DOG, GOAT AAB-DJ, BULLISH AAV-DJ8, DOG BIRD, HORSES BULL, GOAT DOG, PRAWN AAV-DJ, DOG AAB-DJ8, HORSES BIRD, GOAT BUFF, PRAWN DOGS, PIGS AAV-DJ, HORSES AAV-DJ8, GOAT BIRD, PRAWN COWS, PIGS CANINE, INSECTS AAV-DJ, GOAT AAB-DJ8, SHRIMP POULTRY, PIGS BULL, INSECTS CANINE, SHEEP AAB-DJ, SHRIMP AAV-DJ8, PIGS BIRD, INSECTS BULL, SHEEP DOG, B19 AAV-DJ, PIGS AAB-DJ8, INSECTS BIRD, SHEEP BULLISH, B19 CANINE, MVM AAV-DJ, INSECTS AAB-DJ8, SHEEP BIRD, B19 BULLISH, MVM DOG, GEESE AAB-DJ, SHEEP AAB-DJ8, B19 BIRD, MVM BULL, GOOSE DOG, SNAKE AAB-DJ, B19 AAB-DJ8, MVM BIRD, GEESE BULL, SNAKE ° AAV-DJ, MVM AAV-DJ8, GEESE BIRD, SNAKE ° ° AAV-DJ, GUSEY AAV-DJ8, SERPENT ° ° ° AAV-DJ, SERPENT ° ° ° ° HORSES, HORSES GOAT, GOAT SHRIMP, PRAWN PIGS, PIGS INSECTS, INSECTS HORSES, GOAT GOAT, PRAWN PRAWNS, PIGS PIGS, INSECTS INSECTS, SHEEP HORSES, PRAWNS GOAT, PIGS PRAWNS, INSECTS PIGS, SHEEP INSECTS, B19 HORSES, PIGS GOAT, INSECTS SHRIMP, SHEEP PIGS, B19 INSECTS, MVM HORSES, INSECTS GOAT, SHEEP SHRIMP, B19 PIGS, MVM INSECTS, GEESE HORSES, SHEEP GOAT, B19 SHRIMP, MVM PIGS, GEESE INSECTS, SNAKES HORSES, B19 GOAT, MVM SHRIMP, GEESE PIGS, SNAKES ° HORSES, MVM GOAT, GEESE SHRIMP, SNAKE ° ° HORSES, GEESE GOAT, SERPENT ° ° ° HORSES, SNAKES ° ° ° ° SHEEP, SHEEP B19, B19 MVM, MVM GEESE, GEESE SNAKE, SNAKE SHEEP, B19 B19, MVM MVM, GEESE GEESE, SNAKE ° SHEEP, MVM B19, GEESE MVM, SERPENT ° ° SHEEP, GEESE B19, SERPENT ° ° ° SHEEP, SNAKE ° ° ° °

[00268] By way of example only, Table 2 shows sequences of typical ICP-DTs from several different AAV serotypes.

[00269] Table 2

[00270] In some embodiments, the IKP-DT nucleotide sequence may be modified (e.g., by modifying 1, 2, 3, 4, or 5 or more nucleotides, or any range thereof), wherein the modification is a substitution with a complementary nucleotide . for example, G to C and vice versa, and T to A and vice versa.

[00271] In certain embodiments of the present invention, the synthetically produced cDNA vector does not have an ICP-DT consisting of a nucleotide sequence selected from any of: SEQ ID NO: 1, 2, 5-14. In alternative embodiments of the present invention, if the cccDNA vector has a WT ICP comprising a nucleotide sequence selected from any of: SEQ ID NO: 1, 2, 5-14, then the flanking ICP is also wild type (WT), and The cccDNA vector contains a regulatory switch, for example, as described herein and in international application PCT/US18/49996 (for example, see Table 11 in PCT/US18/49996). In some embodiments, the cccDNA vector contains a regulatory switch as described herein, and the selected WT-ICP has a nucleotide sequence selected from any member of the group consisting of: SEQ ID NO: 1, 2, 5-14.

[00272] The cccDNA vector described herein may include ICP-DT structures that retain the functional portions of RBE, trs, and RBE'. Figure 2A and Figure 2B demonstrate, using wild-type ICP for illustration, one possible mechanism for the functioning of the trs site in part of the wild-type ICP structure of the ccDNA vector. In some embodiments, the cccDNA vector contains one or more functional ICP-DT polynucleotide sequences that contain a Rep-binding site (RBS; 5'-GCGCGCTCGCTCGCTC-3' (SEQ ID NO: 60) for AAB2) and a terminal resolution site (TRS) ; 5'-AGTT (SEQ ID NO: 62)). In some embodiments, at least one ICP-DT is functional. In alternative embodiments, when the cccDNA vector contains two ICP-WTs that are substantially symmetrical to each other, at least one ICP-WT is functional and at least one ICP-WT is non-functional.

B. Modified ICPs (mod-ICPs) in general for cccDNA vectors for controlled transgene expression containing asymmetric ICP pairs or symmetric ICP pairs

[00273] As described herein, the cccDNA vector may contain a symmetric ICP pair or an asymmetric ICP pair. In both cases, one or both of the ICPs can be modified ICPs - the difference is that in the first case (i.e., symmetric mod-ICPs) said mod-ICPs have the same three-dimensional spatial organization (i.e., they have the same configurations A -A', C-C' and B-B' arms), while in the second case (i.e. asymmetric mod-ICPs) these mod-ICPs have different three-dimensional spatial organization (i.e., they have different A-A configurations ', C-C' and B-B'-shoulders).

[00274] In some embodiments, a modified ICP is an ICP that is modified by deletion, insertion, and/or substitution from a wild-type ICP sequence (eg, an AAV ICP). In some embodiments, at least one of the ICPs in the cccDNA vector contains a functional Rep binding site (RBS; e.g., 5'-GCGCGCTCGCTCGCTC-3' for AAB2, SEQ ID NO: 60) and a functional terminal resolution site (TRS; e.g., 5'-AGTT-3', SEQ ID NO: 62). In one embodiment, at least one of the CPIs is a non-functional CPI. In one embodiment, each of the different or modified ICPs is not wild-type ICP from different serotypes.

[00275] Specific changes and mutations in ICPs are described in detail herein, but in the context of "altered" or "mutated" or "modified" ICPs, these definitions indicate that nucleotides have been inserted, deleted and/or substituted relative to those belonging to wild type, reference or original ICP sequence. The altered or mutated ICP may be a recombinant ICP. As used herein, the term “recombinant” refers to the human manipulation aspect. For example, a polypeptide is considered "recombinant" when at least one aspect of the polypeptide, such as its sequence, has been manipulated by humans to be different from the aspect in which it exists in nature.

[00276] In some embodiments, the mod-ICP may be synthetic. In one embodiment, the synthetic ICP is based on ICP sequences from more than one AAV serotype. In another embodiment, the synthetic ICP does not include an AAV-based sequence. In yet another embodiment, the synthetic ICP retains the structure of the ICP described above, although it has only some or no AAV sequences. In some aspects, a synthetic ICP may interact preferentially with wild-type Rep or with Rep of a particular serotype, or, in some cases, will not be recognized by wild-type Rep and will only be recognized by mutated Rep.

[00277] One skilled in the art can determine the corresponding sequence in other serotypes by known methods. For example, by determining whether the change is in region A, A', B, B', C, C', or D, and identifying the corresponding region in a different serotype. BLAST® (Basic Local Alignment Search Tool) or other homology alignment programs can be used with default settings to determine the corresponding sequence. The present invention further provides populations and pluralities of cccDNA vectors for controlled transgene expression containing mod-ICPs from a combination of different AAV serotypes, i.e. one mod-ICP may belong to one AAV serotype, and another mod-ICP may belong to a different serotype. Without being bound by any theory, in one embodiment, one ICP may be taken from or based on the AAV2 ICP sequence, and the other cDNA vector ICP may be taken from or be based on any one or more serotype ICP sequences 1 AAV (AAV1), serotype 4 AAV (AAV4), serotype 5 AAV (AAV5), serotype 6 AAV (AAV6), serotype 7 AAV (AAV7), serotype 8 AAV (AAV8), serotype 9 AAV (AAV9), serotype 10 AAV (AAV10), AAV serotype 11 (AAV11) or AAV serotype 12 (AAV12).

[00278] Any parvovirus ICP can be used as an ICP or as a base ICP for modification. Preferably, the parvovirus is a dependovirus. More preferably, it is an AAV. Serotype selection may be based on tissue tropism of the serotype. AAB2 has tropism for a wide range of tissues, AAB1 predominantly targets neural tissue and skeletal muscle, and AAV5 predominantly targets neural tissue, retinal pigmented epithelium and photoreceptors. AAB6 primarily targets skeletal muscle and lungs. AAB8 primarily targets the liver, skeletal muscle, heart, and pancreatic tissue. AAB9 is predominantly targeted to the liver, skeletal muscle and lung tissue. In one embodiment, the modified ICP is based on the AAB2 ICP.

[00279] More specifically, the ability of a structural element to operably interact with a particular large Rep protein can be altered by modifying the structural element. For example, the nucleotide sequence of the structural element may be modified compared to the wild-type ICP sequence. In one embodiment, a structural element (e.g., arm A, arm A', arm B, arm B', arm C, arm C', arm D, RBE, RBE', and trs) of the ICP may be removed and replaced with a wild-type structural element from another parvovirus. For example, the replacement structure may be taken from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, snake parvovirus (eg, royal python parvovirus), bovine parvovirus, parvovirus goat, avian parvovirus, canine parvovirus, equine parvovirus, shrimp parvovirus, porcine parvovirus or insect AAV. For example, the ICP may be an AAB2 ICP, and the A or A' arm or RBE may be replaced by a structural element from AAB5. In another example, the ICP may be an AAB5 ICP, and arms C or C', RBE, and trs may be replaced by a structural element from AAB2. In another example, the AAB ICP may be an AAB5 ICP in which arms B and B' are replaced by arms B and B' of the AAB2 ICP.

[00280] By way of example only, Table 3 provides examples of modifications of at least one nucleotide (e.g., deletions, insertions, and/or substitutions) in regions of modified ICPs, where X indicates modification of at least one nucleic acid (e.g., deletion , insertion and/or substitution) in the indicated compartment relative to the corresponding wild-type ICP. In some embodiments, any modification of at least one nucleotide (e.g., deletion, insertion, and/or substitution) in any of the C and/or C' and/or B and/or B' regions preserves three consecutive T nucleotides (i.e. .TTT) in at least one terminal loop. For example, if the modification results in any of: a single arm ICP (for example, a single C-C' arm or a single B-B' arm), or a modified C-B' arm or a C'-B arm, or ICP with two arms, containing at least one truncated arm (for example, truncated arm C-C' and/or truncated arm B-B'), then in at least the specified single arm, or in at least one of the arms of the ICP with two arms (in which one arm may be truncated), three consecutive T nucleotides (i.e., TTT) are retained in at least one terminal loop. In some embodiments, the C-C' truncated arm and/or the B-B' truncated arm contains three consecutive T nucleotides (i.e., TTT) in the terminal loop.

[00281] Table 3: Examples of combinations of modifications to at least one nucleotide (e.g., deletions, insertions, and/or substitutions) in different regions or arms B-B' and C-C' of the ICP (X indicates a nucleotide modification, e.g., addition , deletion or substitution of at least one nucleotide in the specified region).

Area B Region B' Area C Region C' X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X X

[00282] In some embodiments, a mod-ICP for use in a cccDNA vector containing an asymmetric ICP pair or a symmetric mod-ICP pair as described herein may contain any one of the combinations of modifications listed in Table 3, as well as a modification at least one nucleotide in any of one or more regions selected from: between A' and C, between C and C', between C' and B, between B and B', and between B' and A. In some embodiments , any modification of at least one nucleotide (eg, deletion, insertion and/or substitution) in regions C or C' or B or B' still retains the terminal loop of the stem-loop structure. In some embodiments, any modification of at least one nucleotide (e.g., deletion, insertion, and/or substitution) between C and C' and/or B and B' preserves three consecutive T nucleotides (i.e., TTT) at least in one end loop. In alternative embodiments, any modification of at least one nucleotide (e.g., deletion, insertion, and/or substitution) between C and C' and/or B and B' preserves three consecutive nucleotides of A (i.e., AAA) in at least one end loop. In some embodiments, a modified ICP for use with the present invention may comprise any one of the combinations of modifications set forth in Table 3, as well as modification of at least one nucleotide (e.g., deletion, insertion, and/or substitution) in any one or more regions , selected from: A', A and/or D. For example, in some embodiments, a modified ICP for use in this invention may contain any one of the combinations of modifications listed in Table 3, as well as modification of at least one nucleotide (for example, deletion, insertion, and/or substitution) in region A. In some embodiments, a modified ICP for use in this invention may contain any one of the combinations of modifications listed in Table 3, as well as a modification of at least one nucleotide (e.g., deletion, insertion and/or replacement) in area A'. In some embodiments, a modified ICP for use with the present invention may comprise any one of the combinations of modifications set forth in Table 3, as well as modification of at least one nucleotide (e.g., deletion, insertion, and/or substitution) in the A and/or A region '. In some embodiments, a modified ICP for use in this invention may contain any one of the combinations of modifications listed in Table 3, as well as a modification of at least one nucleotide (e.g., deletion, insertion, and/or substitution) in region D.

[00283] In one embodiment, the nucleotide sequence of the building block may be modified (e.g., by modifying 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 17, 18, 19 or 20 or more nucleotides or any range of values) to obtain a modified structural element. Examples of specific modifications of the ICP according to one embodiment are provided herein (e.g., SEQ ID NO: 3, 4, 15-47, 101-116, or 165-187, or shown in FIGS. 7A-7B of PCT/US2018/064242, filed December 6, 2018 (for example, SEQ ID NO: 97-98, 101-103, 105-108, 111-112, 117-134, 545-54 in PCT/US2018/064242). In some embodiments, the ICP may be modified (for example, by modifying 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 or more nucleotides or any range of specified values).In other embodiments, the ICP may be at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, according to at least 98%, at least 99% or more, sequence identity with one of the modified ICPs from SEQ ID NO: 3, 4, 15-47, 101-116 or 165-187, or the RBE-containing portion of the A-A' arm and arms C-C' and B-B' according to SEQ ID NO: 3, 4, 15-47, 101-116 or 165-187, or those shown in Tables 2-9 (i.e. SEQ ID NO: 110-112, 115-190, 200-468) of international application PCT/US18/49996, which is incorporated herein by reference in its entirety.

[00284] In some embodiments, a modified ICP may, for example, comprise removal or deletion of all or part of an arm A-A', or all or part of an arm B-B', or all or part of an arm C-C', or , alternatively, removing 1, 2, 3, 4, 5, 6, 7, 8, 9 or more base pairs forming the stem of the loop, while maintaining the presence of a capping loop at the end of the stem (e.g., one arm) (e.g., see IKP-21 in Fig. 7A of application PCT/US2018/064242, filed December 6, 2018). In some embodiments, the modified ICP may comprise removing 1, 2, 3, 4, 5, 6, 7, 8, 9, or more base pairs from the B-B' arm. In some embodiments, the modified ICP may comprise removing 1, 2, 3, 4, 5, 6, 7, 8, 9 or more base pairs from the C-C' arm (see, for example, ICP-1 in Figure 3B or ICP- 45 in Fig. 7A of application PCT/US2018/064242, filed December 6, 2018). In some embodiments, the modified ICP may include deleting 1, 2, 3, 4, 5, 6, 7, 8, 9, or more base pairs from the C-C' arm and deleting 1, 2, 3, 4, 5, 6, 7, 8, 9 or more base pairs from arm B-B'. Any combination of base pair deletions is contemplated, for example, 6 base pairs in the C-C' arm and 2 base pairs in the B-B' arm may be deleted. As an illustrative example, FIG. 3B shows an example of a modified ICP with a deletion of at least 7 base pairs from each of the C portion and the C' portion, a nucleotide substitution in the loop between the C and C' region, and a deletion of at least one base pair from each of region B and region B', so that the modified ICP contains two arms, at least one of which (for example, C-C') is truncated. In some embodiments, the modified ICP also contains at least one base pair deletion from each of the B region and the B' region such that the B-B' arm is also truncated relative to the wild type ICP.

[00285] In some embodiments, the modified ICP may have from 1 to 50 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50) nucleotide deletions relative to the full-length wild-type ICP sequence. In some embodiments, the modified ICP may have 1 to 30 nucleotide deletions relative to the full-length ICP-DT sequence. In some embodiments, the modified ICP has 2 to 20 nucleotide deletions relative to the full-length wild-type ICP sequence.

[00286] In some embodiments, the modified ICP does not contain any nucleotide deletions in the RBE-containing portion of the A or A' regions so as not to interfere with DNA replication (eg, Rep protein binding to the RBE or nicking at the end resolution site). In some embodiments, a modified ICP suitable for use in the present invention has one or more deletions in the B, B', C, and/or C region, as described herein.

[00287] In some embodiments, a synthetically produced cccDNA vector containing a symmetric pair of ICPs or an asymmetric pair of ICPs comprises a regulatory switch as described herein and at least one modified ICP selected from having a nucleotide sequence selected from any from the group consisting of: SEQ ID NO: 3, 4, 15-47, 101-116 or 165-187.

[00288] In another embodiment, the structure of the structural element may be modified. For example, a structural element (has) a changed stem height and/or number of nucleotides in a loop. For example, the stem height may be about 2, 3, 4, 5, 6, 7, 8 or 9 nucleotides or more, or any range of values. In one embodiment, the stem may be about 5 nucleotides to about 9 nucleotides in height and operably interact with Rep. In another embodiment, the stem may be about 7 nucleotides in height and operably interact with Rep. In another example, the loop may be 3, 4, 5 , 6, 7, 8, 9 or 10 nucleotides or more, or any range thereof.

[00289] In another embodiment, the number of GAGY binding sites or GAGY-related binding sites within an RBE or extended RBE may be increased or decreased. In one example, an RBE or extended RBE may contain 1, 2, 3, 4, 5, or 6 or more GAGY binding sites, or any range thereof. Each GAGY binding site may independently be the exact GAGY sequence or a sequence similar to GAGY, provided that the sequence is sufficient for binding of the Rep protein.

[00290] In another embodiment, the gap between two elements (such as, but not limited to, an RBE and a hairpin) may be changed (eg, increased or decreased) to change the functional interaction with the large Rep protein. For example, the gap may be approximately 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or 21 nucleotides or more, or any range thereof.

[00291] The cccDNA vector described herein may include an ICP structure that is modified from the wild-type AAB2 ICP structure disclosed herein but still retains the functional portion of RBE, trs, and RBE'. Figure 2A and Figure 2B demonstrate one of the possible mechanisms of operation of the trs site in part of the ICP structure of the wild type ccDNA vector. In some embodiments, the cccDNA vector contains one or more functional ICP polynucleotide sequences that contain a Rep binding site (RBS; 5'-GCGCGCTCGCTCGCTC-3' (SEQ ID NO: 60) for AAB2) and a terminal resolution site (TRS; 5 '-AGTT (SEQ ID NO: 62)). In some embodiments, at least one ICP (wild type or modified ICP) is functional. In alternative embodiments, when the cccDNA vector contains two modified ICPs that are different from or asymmetrical with respect to each other, at least one modified ICP is functional and at least one modified ICP is non-functional.

[00292] In some embodiments, the modified ICP (eg, left or right ICP) of the synthetically produced cccDNA vector described herein has modifications in the loop arm, truncated arm, or spacer. Examples of ICP sequences having modifications in the loop arm, truncated arm or spacer are listed in Table 2 (ie SEQ ID NO: 135-190, 200-233); Table 3 (eg SEQ ID NO: 234-263); Table 4 (eg SEQ ID NO: 264-293); Table 5 (eg, SEQ ID NO: 294-318 herein); Table 6 (eg SEQ ID NO: 319-468); and Tables 7-9 (for example, SEQ ID NO: 101-110, 111-112, 115-134) or Table 10A or 10B (for example, SEQ ID NO: 9, 100, 469-483, 484-499) of the international application PCT/US18/49996, which is incorporated herein by reference in its entirety.

[00293] In some embodiments, the modified ICP for use in a cccDNA vector containing an asymmetric ICP pair or a symmetric mod-ICP pair is selected from any or a combination of ICPs listed in Tables 2, 3, 4, 5, 6, 7. 8. 9 and 10A-10B of application PCT/US18/49996, which is incorporated herein by reference in its entirety.

[00294] Additional examples of modified ICPs for use in a cccDNA vector containing an asymmetric ICP pair or a symmetric mod-ICP pair in each of the above classes are presented in Tables 4A and 4B. The predicted secondary structures of the right modified ICPs shown in Table 4A are presented in Fig. 7A of international application PCT/US2018/064242, filed December 6, 2018, and the predicted secondary structures of the left modified ICPs shown in Table 4B are presented in Fig. 7B international application PCT/US2018/064242, filed December 6, 2018, which is incorporated herein by reference in its entirety.

[00295] Table 4A and Table 4B provide examples of right and left modified ICPs.

[00296] Table 4A: Examples of Modified Right ICPs These examples of modified right ICPs may contain the RBE GCGCGCTCGCTCGCTC-3′ (SEQ ID NO: 60), the ACTGAGGC spacer (SEQ ID NO: 69), the complement spacer GCCTCAGT (SEQ ID NO: 70) and RBE' (ie, complement of RBE) GAGCGAGCGAGCGCGC (SEQ ID NO: 71).

Table 4A: Examples of right modified ICPs IKP construct Subsequence SEQ ID NO: IKP-18 right AGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCGCACGCCCGGGTTTCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAGCTGCCTGCAGG 15 IKP-19 right AGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGACGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAGCTGCCTGCAGG 16 IKP-20 right AGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGGCGCCTCAGTGAGCGAGCGAGCGCGCAGCTGCCTGCAGG 17 IKP-21 right AGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCTTTGCCTCAGTGAGCGAGCGAGCGCGCAGCTGCCTGCAGG 18 IKP-22 right AGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACAAAGTCGCCCGACGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAGCTGCCTGCAGG 19 IKP-23 right AGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGAAAATCGCCCGACGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAGCTGCCTGCAGG 20 IKP-24 right AGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGAAACGCCGACGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAGCTGCCTGCAGG 21 IKP-25 right AGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCAAAGCCCGACGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAGCTGCCTGCAGG 22 IKP-26 right AGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGGTTTCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAGCTGCCTGCAGG 23 IKP-27 right AGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGTTTCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAGCTGCCTGCAGG 24 IKP-28 right AGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCGTTTCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAGCTGCCTGCAGG 25 IKP-29 right AGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCTTTGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAGCTGCCTGCAGG 26 IKP-30 right AGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCTTTGGCGGCCTCAGTGAGCGAGCGAGCGCGCAGCTGCCTGCAGG 27 IKP-31 right AGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCTTTGCGGCCTCAGTGAGCGAGCGAGCGCGCAGCTGCCTGCAGG 28 IKP-32 right AGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGTTTCGGCCTCAGTGAGCGAGCGAGCGCGCAGCTGCCTGCAGG 29 IKP-49 right AGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGGCTCAGTGAGCGAGCGAGCGCGCAGCTGCCTGCAGG thirty IKP-50 right AGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAGCTGCCTGCAGG 31

[00297] TABLE 4B: Examples of modified left ICPs. These examples of left-handed modified ICPs may contain the RBE GCGCGCTCGCTCGCTC-3' (SEQ ID NO: 60), spacer ACTGAGGC (SEQ ID NO: 69), spacer complement GCCTCAGT (SEQ ID NO: 70) and complement RBE (RBE') GAGCGAGCGAGCGCGC (SEQ ID NO: 71).

Table 4B: Examples of modified left ICPs IKP-33 left CCTGCAGGCAGCTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGGAAACCCGGGCGTGCGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCT 32 IKP-34 left CCTGCAGGCAGCTGCGCGCTCGCTCGCTCACTGAGGCCGTCGGGCGACCTTTGGTCGCCCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCT 33 IKP-35 left CCTGCAGGCAGCTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCAAAGCCCGGGCGTCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCT 34 IKP-36 left CCTGCAGGCAGCTGCGCGCTCGCTCGCTCACTGAGGCGCCCGGGCGTCGGGCGACCTTTGGTCGCCCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCT 35 IKP-37 left CCTGCAGGCAGCTGCGCGCTCGCTCGCTCACTGAGGCAAAGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCT 36 IKP-38 left CCTGCAGGCAGCTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCAAAGCCCGGGCGTCGGGCGACTTTGTCGCCCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCT 37 IKP-39 left CCTGCAGGCAGCTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCAAAGCCCGGGCGTCGGGCGATTTTCGCCCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCT 38 IKP-40 left CCTGCAGGCAGCTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCAAAGCCCGGGCGTCGGGCGTTTCGCCCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCT 39 IKP-41 left CCTGCAGGCAGCTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCAAAGCCCGGGCGTCGGGCTTTGCCCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCT 40 IKP-42 left CCTGCAGGCAGCTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGGAAACCCGGGCGTCGGGCGACCTTTGGTCGCCCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCT 41 IKP-43 left CCTGCAGGCAGCTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGAAACCGGGCGTCGGGCGACCTTTGGTCGCCCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCT 42 IKP-44 left CCTGCAGGCAGCTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGAAACGGGCGTCGGGCGACCTTTGGTCGCCCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCT 43 IKP-45 left CCTGCAGGCAGCTGCGCGCTCGCTCGCTCACTGAGGCCGCCCAAAGGGCGTCGGGCGACCTTTGGTCGCCCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCT 44 IKP-46 left CCTGCAGGCAGCTGCGCGCTCGCTCGCTCACTGAGGCCGCCAAAGGCGTCGGGCGACCTTTGGTCGCCCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCT 45 IKP-47 left CCTGCAGGCAGCTGCGCGCTCGCTCGCTCACTGAGGCCGCAAAGCGTCGGGCGACCTTTGGTCGCCCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCT 46 IKP-48 left CCTGCAGGCAGCTGCGCGCTCGCTCGCTCACTGAGGCCGAAACGTCGGGCGACCTTTGGTCGCCCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCT 47

[00298] In one embodiment, the cccDNA vector contains, in the 5' to 3' direction: the first inverted terminal repeat (ITR) of an adeno-associated virus (AAV), a nucleotide sequence of interest (e.g., an expression cassette as described herein ), and the second ICP of AAV, and the first ICP (5'-ICP) and the second ICP (3'-ICP) are asymmetric relative to each other, i.e. they have different three-dimensional spatial configurations relative to each other. As an exemplary embodiment, the first ICP may be a wild-type ICP and the second ICP may be a mutated or modified ICP, or vice versa, where the first ICP may be a mutated or modified ICP and the second ICP may be a wild-type ICP. In some embodiments, the first ICP and the second ICP are both mod ICPs, but have different sequences, or have different modifications, and thus are not the same modified ICPs and have different 3D spatial configurations. In other words, a cccDNA vector with asymmetric ICPs contains ICPs in which any changes in one ICP relative to the WT ICP are not reflected in the other ICP; or, alternatively, wherein the asymmetric ICPs are an asymmetric pair of modified ICPs that may have different sequences and different three-dimensional shapes relative to each other. Typical examples of asymmetric ICPs in the cDNA vector and for use in the preparation of the cDNA plasmid are presented in Tables 4A and 4B.

[00299] In an alternative embodiment, the synthetically produced ccDNA vector contains two symmetrical mod-ICPs, i.e. both ICPs have the same sequence, but are reverse complements (inverted) of each other. In some embodiments, the symmetric mod-ICP pair contains at least one or any combination of deletions, insertions, or substitutions relative to a wild-type ICP sequence from the same AAV serotype. Additions, deletions or substitutions in a symmetric ICP are the same, but inversely complementary to each other. For example, an insertion of 3 nucleotides into the C region of a 5'-ICP will be mapped to an insertion of 3 reverse complementary nucleotides into the corresponding region of the C-region of a 3'-ICP. For purposes of illustration only, if in a 5'-ICP the addition is AACG, then in a 3'-ICP the addition will be CGTT at the corresponding site. For example, if the sense strand of the 5' ICP is ATCGATCG with the addition of AACG between G and A, the resulting sequence would be ATCGAACGATCG (SEQ ID NO: 51). The corresponding sense strand of the 3'-ICP is CGATCGAT (reverse complement ATCGATCG) with the addition of CGTT (i.e. reverse complement AACG) between T and C, resulting in the sequence CGATCGTTCGAT (SEQ ID NO: 49) (reverse complement ATCGAACGATCG) ( SEQ ID NO: 51).

[00300] In alternative embodiments, the modified ICP pair is substantially symmetrical as defined herein, i.e. a modified pair of ICPs may have different sequences, but have a similar or identical symmetrical three-dimensional shape. For example, one modified ICP may belong to one serotype and another modified ICP may belong to a different serotype, but they have the same mutation (eg, insertion, deletion, or nucleotide substitution) in the same region. In other words, for illustrative purposes only, a 5' mod-IKP may be taken from AAB2 and have a deletion in the C region, and a 3' mod-IKP may be taken from AAB5 and have a corresponding deletion in the C' region, and provided that The 5' mod-ICP and the 3' mod-ICP have the same or symmetrical three-dimensional spatial organization and will be included for use in this invention as a modified ICP pair.

[00301] In some embodiments, a substantially symmetric mod-ICP pair has identical A, CC', and B-B' loops in three-dimensional space, for example, if a modified ICP in a substantially symmetric mod-ICP pair has a C-C arm deletion ', then the cognate mod-IKP has a corresponding deletion of the C-C' loop, and also has a similar three-dimensional structure of the remaining A and B-B' loops of the same shape in geometric space compared to its cognate mod-IKP. By way of example only, substantially symmetrical ICPs may have a symmetrical spatial organization such that their structure has the same shape in geometric space. This may occur, for example, when a GC pair is modified, for example, into a CG pair, or vice versa, or an AT pair is modified into a TA pair, or vice versa. Thus, using the above illustrative example of a modified 5'-ICP as ATCGAACGATCG (SEQ ID NO: 51), and a modified 3'-ICP as CGATCGTTCGAT (SEQ ID NO: 49) (i.e. the reverse complement of ATCGAACGATCG (SEQ ID NO: 51)), then these modified ICPs will remain symmetrical if, for example The 5'-ICP has the sequence ATCGAAC C ATCG (SEQ ID NO: 50), where the G in the appendix is modified to C, and the substantially symmetric 3'-ICP has the sequence CGATCGTTCGAT (SEQ ID NO: 49), without corresponding modification in the appendix T to A. In some embodiments, such a pair of modified ICPs is substantially symmetrical because the pair of modified ICPs has symmetrical stereochemistry.

[00302] Table 5 shows examples of symmetric modified PKI pairs (i.e., modified left PKI and symmetric right modified PKI). The bold (red) portion of the sequences identifies partial ICP sequences (ie, loop sequences A-A', C-C', and B-B'), also shown in FIGS. 31A-46B. These examples of modified ICPs may contain the RBE GCGCGCTCGCTCGCTC-3′ (SEQ ID NO: 60), the spacer ACTGAGGC (SEQ ID NO: 69), the spacer complement GCCTCAGT (SEQ ID NO: 70) and RBE' (i.e. the complement RBE) GAGCGAGCGAGCGCGC (SEQ ID NO: 71).

Table 5: Examples of symmetrical modified ICP pairs LEFT modified IKP
(modified 5'-ICP) Symmetrical RIGHT modified IKP
(modified 3'-ICP) SEQ ID NO: 32 (IKP-33, left) CCTGCAGGCAGCTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGGAAACCCGGGCGTGCGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCT SEQ ID NO: 15 (IKP-18, right) AGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCGCACGCCCGGGTTTCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAGCTGCCTGCAGG SEQ ID NO: 33 (IKP-34, left) CCTGCAGGCAGCTGCGCGCTCGCTCGCTCACTGAGGCCGTCGGGCGACCTTTGGTCGCCCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCT SEQ ID NO: 48 (IKP-51, right) AGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGGCTCAGTGAGCGAGCGAGCGCGCAGCTGCCTGCAGG SEQ ID NO: 34 (IKP-35, left) CCTGCAGGCAGCTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCAAAGCCCGGGCGTCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCT SEQ ID NO: 16 (IKP-19, right) AGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGACGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAGCTGCCTGCAGG SEQ ID NO: 35 (IKP-36, left) CCTGCAGGCAGCTGCGCGCTCGCTCGCTCACTGAGGCGCCCGGGCGTCGGGCGACCTTTGGTCGCCCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCT SEQ ID NO: 17 (IKP-20, right) AGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGGCGCCTCAGTGAGCGAGCGAGCGCGCAGCTGCCTGCAGG SEQ ID NO: 36 (IKP-37, left) CCTGCAGGCAGCTGCGCGCTCGCTCGCTCACTGAGGCAAAGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCT SEQ ID NO: 18 (IKP-21, right) AGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCTTTGCCTCAGTGAGCGAGCGAGCGCGCAGCTGCCTGCAGG SEQ ID NO: 37 (IKP-38, left) CCTGCAGGCAGCTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCAAAGCCCGGGCGTCGGGCGACTTTGTCGCCCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCT SEQ ID NO: 19 (IKP-22, right) AGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACAAAGTCGCCCGACGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAGCTGCCTGCAGG SEQ ID NO: 38 (IKP-39, left) CCTGCAGGCAGCTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCAAAGCCCGGGCGTCGGGCGATTTTCGCCCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCT SEQ ID NO: 20 (IKP-23, right) AGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGAAAATCGCCCGACGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAGCTGCCTGCAGG SEQ ID NO: 39 (IKP-40, left) CCTGCAGGCAGCTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCAAAGCCCGGGCGTCGGGCGTTTCGCCCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCT SEQ ID NO: 21 (IKP-24, right) AGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGAAACGCCGACGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAGCTGCCTGCAGG SEQ ID NO: 40 (IKP-41, left) CCTGCAGGCAGCTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCAAAGCCCGGGCGTCGGGCTTTGCCCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCT SEQ ID NO: 22 (IKP-25, right) AGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCAAAGCCCGACGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAGCTGCCTGCAGG SEQ ID NO: 41 (IKP-42, left) CCTGCAGGCAGCTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGGAAACCCGGGCGTCGGGCGACCTTTGGTCGCCCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCT SEQ ID NO: 23 (IKP-26, right) AGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGGTTTCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAGCTGCCTGCAGG SEQ ID NO: 42 (IKP-43, left) CCTGCAGGCAGCTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGAAACCGGGCGTCGGGCGACCTTTGGTCGCCCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCT SEQ ID NO: 24 (IKP-27, right) AGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGTTTCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAGCTGCCTGCAGG SEQ ID NO: 43 (IKP-44, left) CCTGCAGGCAGCTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGAAACGGGCGTCGGGCGACCTTTGGTCGCCCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCT SEQ ID NO: 25 (IKP-28, right) AGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCGTTTCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAGCTGCCTGCAGG SEQ ID NO: 44 (IKP-45, left) CCTGCAGGCAGCTGCGCGCTCGCTCGCTCACTGAGGCCGCCCAAAGGGCGTCGGGCGACCTTTGGTCGCCCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCT SEQ ID NO: 26 (IKP-29, right) AGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCTTTGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAGCTGCCTGCAGG SEQ ID NO: 45 (IKP-46, left) CCTGCAGGCAGCTGCGCGCTCGCTCGCTCACTGAGGCCGCCAAAGGCGTCGGGCGACCTTTGGTCGCCCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCT SEQ ID NO: 27 (IKP-30, right) AGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCTTTGGCGGCCTCAGTGAGCGAGCGAGCGCGCAGCTGCCTGCAGG SEQ ID NO: 46 (IKP-47, left) CCTGCAGGCAGCTGCGCGCTCGCTCGCTCACTGAGGCCGCAAAGCGTCGGGCGACCTTTGGTCGCCCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCT SEQ ID NO: 28 (IKP-31, right) AGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCTTTGCGGCCTCAGTGAGCGAGCGAGCGCGCAGCTGCCTGCAGG SEQ ID NO: 47 (IKP-48, left) CCTGCAGGCAGCTGCGCGCTCGCTCGCTCACTGAGGCCGAAACGTCGGGCGACCTTTGGTCGCCCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCT SEQ ID NO: 29 (IKP-32, right) AGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGTTTCGGCCTCAGTGAGCGAGCGAGCGCGCAGCTGCCTGCAGG

[00303] In some embodiments, a cccDNA vector containing a pair of asymmetric ICPs may contain an ICP with a modification corresponding to any of the modifications to the ICP sequences or partial ICP sequences set forth in any one or more of Tables 4A-4B herein, or sequences 7A-7B of international application PCT/US2018/064242, filed December 6, 2018, which is incorporated herein in its entirety, or disclosed in Tables 2, 3, 4, 5, 6, 7, 8, 9 or 10A-10B of international application PCT/US18/49996, filed September 7, 2018, which is incorporated herein by reference in its entirety.

V. Typical cDNA vectors for controlled transgene expression

[00304] As described above, this invention relates to recombinant cccDNA expression vectors and cccDNA vectors for controlled transgene expression that encode any of: a pair of asymmetric ICPs, a pair of symmetric ICPs, or a pair of substantially symmetric ICPs, as described above. In some embodiments, the present invention provides recombinant cccDNA vectors for controlled transgene expression having ICP and transgene flanking sequences, wherein the ICP sequences are asymmetric, symmetric, or substantially symmetrical with respect to each other as defined herein, and the cccDNA further comprises a nucleotide a sequence of interest (eg, an expression cassette containing a transgene nucleic acid) located between flanking ICPs, wherein said nucleic acid molecule lacks viral capsid protein coding sequences.

[00305] The cccDNA vector for controlled transgene expression may be any cccDNA vector that can be conveniently subjected to recombinant DNA procedures comprising the nucleotide sequence(s) described herein, provided that at least one ICP has been altered . The cccDNA vectors of the present invention are compatible with the host cell into which the cccDNA vector is to be introduced. In certain embodiments, the cDNA vectors may be linear. In some embodiments, the cDNA vectors may exist as an extrachromosomal entity. In certain embodiments, the cDNA vectors of this invention may contain element(s) that allow integration of a donor sequence into the genome of a host cell. As used herein, “transgene” and “heterologous nucleotide sequence” are synonymous.

[00306] Figures 1A-1G show diagrams of the functional components of two non-limiting plasmids useful for producing cccDNA vectors of this invention. FIGS. 1A, 1B, 1D, 1F depict the construction of cccDNA vectors or corresponding ccDNA plasmid sequences. cccDNA vectors are capsidless and can be derived from a plasmid encoding, in this order: a first ICP, a transgene expression cassette, and a second ICP, wherein the first and second ICP sequences are asymmetric, symmetrical, or substantially symmetrical to each other, as defined in this document. cccDNA vectors are capsidless and can be obtained from a plasmid encoding, in this order: a first ICP, the expressed transgene (protein or nucleic acid), and a second ICP, wherein the sequences of the first and second ICP are asymmetric, symmetric, or substantially symmetrical to each other relative to each other, as defined herein. In some embodiments, the transgene expression cassette includes, as appropriate, an enhancer/promoter, one or more homologous arms, a donor sequence, a post-transcriptional regulatory element (e.g., WPRE, e.g., SEQ ID NO: 67), and a polyadenylation and termination signal (e.g., poly (A) BGH, e.g. SEQ ID NO: 68).

[00307] Figure 5 depicts a gel confirming the production of cctDNA from multiple plasmid constructs using the method described in the examples. Preparation of the cDNA vector is confirmed by a representative gel electropherogram as described for FIG. 4A above and in the Examples.

A. Regulatory elements.

[00308] cccDNA vectors, as described herein, containing an asymmetric ICP pair or a symmetric ICP pair, as defined herein, may further contain a particular combination of cis-regulatory elements. Cis-regulatory elements include, but are not limited to, a promoter, a riboswitch, an insulator, a microRNA-regulated element, a post-transcriptional regulatory element, a tissue-specific and cell-specific promoter, and an enhancer. In some embodiments, said ICP may act as a promoter for the transgene. In some embodiments, the cccDNA vector for regulated transgene expression contains additional components to regulate transgene expression, such as regulatory switches, as described herein, to regulate transgene expression, or a kill switch that can kill a cell containing the cccDNA- vector. Control elements, including control switches, that can be used in this invention are described in more detail in international application PCT/US18/49996, which is incorporated herein by reference in its entirety.

[00309] In embodiments, the second nucleotide sequence includes a regulatory sequence and a nucleotide sequence encoding a nuclease. In certain embodiments, the gene regulatory sequence is operably linked to a nucleotide sequence encoding the nuclease. In certain embodiments, the regulatory sequence is useful for controlling the expression of a nuclease in a host cell. In certain embodiments, the regulatory sequence includes a suitable promoter sequence capable of directing transcription of a gene operably linked to the promoter sequence, such as a nucleotide sequence encoding the nuclease(s) of the present invention. In some embodiments, the second nucleotide sequence includes an intronic sequence associated with the 5' end of the nucleotide sequence encoding the nuclease. In some embodiments, an enhancer sequence is provided upstream of a promoter to improve promoter efficiency. In some embodiments, the regulatory sequence includes an enhancer and a promoter, wherein the second nucleotide sequence includes an intronic sequence preceding a nucleotide sequence encoding a nuclease, wherein the intron includes one or more nuclease cleavage sites, and the promoter is operably linked to the nucleotide sequence encoding the nuclease.

[00310] cccDNA vectors produced synthetically or using a cell-based production method as described herein in the Examples may further contain a specific combination of cis-regulatory elements, such as the woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) (eg, SEQ ID NO: 67) and poly(A) BGH (SEQ ID NO: 68). Suitable expression cassettes for use in expression constructs do not have the packaging restrictions imposed by the viral capsid.

(i). Promoters

[00311] One skilled in the art will appreciate that the promoters used in the cDNA vectors of the invention must be tailored to the specific sequences they stimulate. For example, guide RNA may not require a promoter at all, since its function is to form duplex with a specific target sequence on native DNA to carry out a recombination event. In contrast, for a nuclease encoded by a cccDNA vector, a promoter would be beneficial so that it can be expressed efficiently from the vector - and, optionally, in a regulated manner.

[00312] The expression cassettes of this invention include a promoter that can influence overall expression levels as well as cell specificity. For transgene expression, they may include a highly active viral-derived immediate early promoter. Expression cassettes may contain tissue-specific eukaryotic promoters to restrict transgene expression to certain cell types and reduce toxic effects and immune responses resulting from unregulated ectopic expression. In preferred embodiments, the expression cassette may comprise a synthetic regulatory element, such as a CAG promoter (SEQ ID NO: 72). The CAG promoter includes (i) the cytomegalovirus (CMV) early enhancer element, (ii) the promoter, first exon and first intron of the chicken beta-actin gene, and (iii) the splice acceptor of the rabbit beta-globin gene. Alternatively, the expression cassette may comprise an alpha-1 antitrypsin (AAT) promoter (SEQ ID NO: 73 or SEQ ID NO: 74), a liver-specific (LP1) promoter (SEQ ID NO: 75 or SEQ ID NO: 76), or human elongation factor-1 alpha (EF1a) promoter (eg, SEQ ID NO: 77 or SEQ ID NO: 78). In some embodiments, said expression cassette includes one or more constitutive promoters, e.g., a retroviral Rous sarcoma virus (RSV) LTR promoter (optionally with an RSV enhancer), or a cytomegalovirus (CMV) immediate early promoter (optionally with a CMV enhancer, e.g. SEQ ID NO: 79). Alternatively, an inducible promoter, a native transgene promoter, a tissue-specific promoter, or various promoters known in the art can be used.

[00313] Suitable promoters, including those described above, may be derived from viruses and may accordingly be referred to as viral promoters, or they may be derived from any organism, including prokaryotic or eukaryotic organisms. Suitable promoters can be used to drive expression by any RNA polymerase (eg, pol I, pol II, pol III). Exemplary promoters include, but are not limited to, the SV40 early promoter, the murine mammary tumor virus long terminal repeat (LTR) promoter; adenoviral large late promoter (Ad MLP); herpes simplex virus (HSV) promoter, cytomegalovirus (CMV) promoter such as the CMV immediate early (CMVIE) promoter region, Rous sarcoma virus (RSV) promoter, human small nuclear RNA U6 promoter (U6, e.g., SEQ ID NO: 80) (Miyagishi et al., Nature Biotechnology 20, 497-500 (2002)), enhanced U6 promoter (eg, Xia et al., Nucleic Acids Res. 2003 Sep. 1; 31(17)), human H1 promoter (H1) (e.g., SEQ ID NO: 81), CAG promoter, human alpha-1 antitrypsin (HAAT) promoter (e.g., SEQ ID NO: 82), etc. In some embodiments, these promoters are modified to contain a 3′- intron end so that they include one or more nuclease cleavage sites. In some embodiments, the DNA containing the nuclease cleavage site(s) is foreign to the promoter DNA.

[00314] In one embodiment, the promoter used is the native promoter of a gene encoding a therapeutic protein. The promoters and other regulatory sequences of the corresponding genes encoding therapeutic proteins are known and have been characterized. The promoter region used may further include one or more additional regulatory sequences (eg, native), such as enhancers (eg, SEQ ID NO: 79 and SEQ ID NO: 83), including the SV40 enhancer (SEQ ID NO: 126).

[00315] Non-limiting examples of suitable promoters for use in accordance with this invention include the CAG promoter, for example, (SEQ ID NO: 72), the HAAT promoter (SEQ ID NO: 82), the human EF1-α promoter (SEQ ID NO: 77) or a fragment of the EF1a promoter (SEQ ID NO: 78), an IE2 promoter (eg, SEQ ID NO: 84) and a rat EF1-α promoter (SEQ ID NO: 85) or a fragment of the 1E1 promoter (SEQ ID NO: 125).

(ii). Polyadenylation sequences

[00316] A sequence encoding a polyadenylation sequence may be included in a cccDNA vector for controlled expression of a transgene to stabilize the mRNA expressed from the ccDNA vector and to promote nuclear export and translation. In one embodiment, said cDNA vector does not contain a polyadenylation sequence. In other embodiments, said vector includes at least 1, at least 2, at least 3, at least 4, at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 40, at least 45, at least 50 or more adenine dinucleotides. In some embodiments, the polyadenylation sequence comprises about 43 nucleotides, about 40-50 nucleotides, about 40-55 nucleotides, about 45-50 nucleotides, about 35-50 nucleotides, or any range between these values. In some embodiments, in which the cccDNA vector for controlled expression of a transgene may contain two transgenes, for example, in the case of controlled expression of an antibody, the cccDNA vector may contain a nucleic acid encoding a heavy chain of the antibody (for example, an example of a heavy chain is SEQ ID NO: 57) and a nucleic acid encoding an antibody light chain (e.g., an example light chain is SEQ ID NO: 58), and polyadenylation may be present 3' of the first transgene, and an IRES (e.g., SEQ ID NO: 190) located between a first and a second transgene (eg, between a nucleic acid encoding an antibody heavy chain and a nucleic acid encoding an antibody light chain). In some embodiments, a cccDNA vector for controlled transgene expression that encodes more than one transgene (e.g., 2, or 3, or more) may comprise an IRES (internal ribosome entry site) sequence (SEQ ID NO: 190), wherein the sequence The IRES is located, for example, 3' of the polyadenylation sequence, such that a second transgene (for example, an antibody or antigen binding fragment) located 3' of the first transgene is translated and expressed by the same ccDNA vector, so that said ccDNA vector could express two or more transgenes encoded by the cccDNA vector.

[00317] Expression cassettes may include a polyadenylation sequence known in the art, or a variant thereof, such as a naturally occurring sequence isolated from bovine BGHpA (e.g., SEQ ID NO: 68) or from viral SV40pA (e.g., SEQ ID NO: 86), or a synthetic sequence (eg, SEQ ID NO: 87). Some expression cassettes may also include an SV40 late poly(A) signal upstream enhancer (USE) sequence. In some embodiments, USE can be used in combination with SV40pA or a heterologous poly(A) signal.

[00318] Expression cassettes may also include a post-transcriptional element to enhance transgene expression. In some embodiments, a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) is used to enhance transgene expression (eg, SEQ ID NO: 67). Other post-transcriptional processing elements may be used, such as a post-transcriptional element from the herpes simplex virus or hepatitis B virus (HBV) thymidine kinase gene. Secretory sequences can be associated with transgenes, for example, the sequences VH-02 (SEQ ID NO: 88) and VK-A26 (SEQ ID NO: 89), or the IgK signal sequence (SEQ ID NO: 128), the Glu secretory signal sequence ( SEQ ID NO: 188), or the TND secretory signal sequence (SEQ ID NO: 189).

(iii). Nuclear localization sequences

[00319] In some embodiments, the vector encoding the RNA targeting endonuclease contains one or more nuclear localization sequences (NLS), such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more NLS. In some embodiments, one or more NLSs are located at or near the amino terminus, at or near the carboxy terminus, or a combination thereof (e.g., one or more NLSs at the amino terminus and/or one or more NLSs at the carboxy-terminal). If more than one NLS is present, each may be selected independently of the others, such that one NLS will be present in more than one copy and/or in combination with one or more other NLSs present in one or more copies. Non-limiting examples of NLS are given in Table 6.

[00320] Table 6: Nuclear localization signals

SOURCE SUBSEQUENCE SEQ ID NO. SV40 large T antigen PKKKRKV (encoded by the sequence CCCAAGAAGAAGAGGAAGGTG; SEQ ID NO: 91) 90 nucleoplasmin KRPAATKKAGQAKKKK 92 c-myc PAAKRVKLD 93 RQRRNELKRSP 94 hRNPA1 M9 NQSSNFGPMKGGNFGGRSSGPYGGGGQYFAKPRNQGGY 95 IBB domain of importin-alpha RMRIZFKNKGKDTAELRRRRVEVSVELRKAKKDEQILKRRNV 96 fibroid protein T VSRKRPRP 97 PPKKARED 98 human p53 PQPKKKPL 99 c-abl IV mice SALIKKKKKMAP 100 NS1 influenza virus DRLRR 117 PKQKKRK 118 hepatitis delta virus antigen RKLKKKIKKL 119 mouse Mx1 protein REKKKFLKRR 120 human poly(ADP-ribose) polymerase KRKGDEVDGVDEVAKKKSKK 121 Steroid hormone receptor glucocorticoid (human) RKCLQAGMNLEARKTKK 122

B. Additional components of cDNA vectors

[00321] The cccDNA vectors of this invention may contain nucleotides that encode other gene editing components. For example, to select specific gene targeting events, a protective shRNA can be engineered into a microRNA and inserted into a recombinant cDNA vector designed for site-specific integration at a highly active locus, such as the albumin locus. Such embodiments may provide a system for in vivo selection and expansion of gene-modified hepatocytes in any genetic background, such as that described by Nygaard et al., A universal system to select gene-modified hepatocytes in vivo, Gene Therapy, June 8, 2016. cccDNA -vectors of this invention may contain one or more selectable markers that allow the selection of transformed, transfected, transduced or the like cells. A selectable marker is a gene whose product provides resistance to biocides or viruses, resistance to heavy metals, prototrophy to auxotrophs, NeoR, etc. In certain embodiments, positive selection markers, such as NeoR, are included in the donor sequences. Negative selection markers may be included downstream of the donor sequence, for example, an HSV-tk nucleic acid sequence encoding a negative selection marker may be included in the nucleic acid construct downstream of the donor sequence.

[00322] In embodiments, a cccDNA vector for controlled transgene expression obtained using a synthesis process as described herein can be used for gene editing, for example, as disclosed in international application PCT/US2018/064242, filed December 6, 2018 ., which is incorporated herein by reference in its entirety, and may include any one or more of: a 5' homologous arm, a 3' homologous arm, a polyadenylation site located upstream and proximal to the 5' homologous arm. Examples of homologous groups are albumin 5' and 3' homologous arms (SEQ ID NOs: 151 and 152) or CCR5 5' and 3' homologous arms (eg, SEQ ID NOs: 153, 154).

C. Regulatory Switches

[00323] A molecular regulatory switch causes a measurable change in state in response to a signal. Such regulatory switches can be successfully combined with the cccDNA vectors described herein to control the outcome of transgene expression from the cccDNA vector. In some embodiments, the cDNA vector for controlled transgene expression comprises a regulatory switch that serves to fine-tune the expression of the transgene. For example, it may serve to provide a biocontainment function to the cDNA vector. In some embodiments, the switch is a two-position switch that is designed to start or stop (ie, stop) expression of a gene of interest in cccDNA in a controlled and regulated manner. In some embodiments, the switch may activate a kill switch, which may command a cell containing the cccDNA vector to initiate programmed cell death upon activation of the switch. Exemplary examples of regulatory switches provided for use in a cccDNA vector for controlled transgene expression can be used to regulate transgene expression and are described in more detail in international application PCT/US18/49996, which is incorporated herein by reference in its entirety.

(i) Binary control switches

[00324] In some embodiments, the cDNA vector for controlled transgene expression comprises a regulatory switch that can serve to controllably modulate expression of the transgene. For example, an expression cassette located between the ICP of said cDNA vector for controlled expression of a transgene may further comprise a regulatory region, for example, a promoter, cis-element, repressor, enhancer, etc., operably linked to the gene of interest, wherein said regulatory region region is regulated by one or more cofactors or exogenous agents. By way of example only, small molecule switches, or inducible or repressible promoters, can modulate regulatory regions. Non-limiting examples of inducible promoters are hormone inducible or metal inducible promoters. Other examples of inducible promoter/enhancer elements include, but are not limited to, the RU486-inducible promoter, the ecdysone-inducible promoter, the rapamycin-inducible promoter, and the metallothionein promoter.

(ii) Small molecule regulatory switches

[00325] Various small molecule regulatory switches known in the art are known to those skilled in the art and can be combined with the cccDNA vectors disclosed herein to produce a cccDNA vector controlled by a regulatory switch. In some embodiments, the regulatory switch may be selected from any one or combination of the following: an orthogonal ligand/nuclear receptor pair, such as a retinoid receptor variant/LG335 and GRQCIMFI, along with an artificial promoter that controls expression of an operably linked transgene, such as described in: Taylor, et al. BMC Biotechnology 10 (2010): 15; recombinant steroid receptors, for example, a modified C-terminal truncated progesterone receptor that is unable to bind progesterone but binds RU486 (mifepristone) (US Pat. No. 5,364,791); the Drosophila ecdysone receptor and its ecdysteroid ligands (Saez, et al., PNAS, 97(26)(2000), 14512-14517); or a switch controlled by the antibiotic trimethoprim (TMP) as described in: Sando R 3 ^rd ; Nat Methods. 2013, 10(11):1085-8. In some embodiments, said regulatory switch for controlling the transgene, or expressed by the cDNA vector, is a prodrug activation switch, such as described in US Pat. No. 8,771,679 and US Pat. No. 6,339,070.

(iii) Regulatory switches “with password”

[00326] In some embodiments, said control switch may be a "passcode switch" or a "password circuit." Password switches allow for fine-tuned control of expression of a specified transgene from a cccDNA vector for controlled expression of the transgene when specific conditions arise - i.e. combinations of conditions whose presence is necessary for expression and/or repression of the transgene. For example, at least conditions A and B must be present for a transgene to be expressed. A password control switch may have any number of conditions, such as at least 2, or at least 3, or at least 4, or at least 5, or at least 6 or at least 7 or more conditions necessary for transgene expression to occur. In some embodiments, at least 2 conditions are required (eg, conditions A, B), and in some embodiments, at least 3 conditions are required (eg, A, B, and C, or A, B, and D). By way of example only, for gene expression from cccDNA containing a regulatory switch with the password "ABC", conditions A, B and C must be present. Conditions A, B and C may be as follows; condition A represents the presence of a condition or disease, condition B represents a hormonal response, and condition C represents a response to transgene expression. For example, if a transgene edits a defective EPO gene, then condition A is the presence of chronic kidney disease (CKD), condition B is satisfied if there is a hypoxic condition in the subject's kidneys, condition C is the presence of impaired recruitment of erythropoietin-producing cells (EPC) to the kidneys; or, alternatively, impaired HIF-2 activation. Once oxygen levels rise or the required EPO level is reached, the transgene (eg EPO) is turned off again until 3 conditions are met before it turns on again.

[00327] In some embodiments, a password control switch or “password circuit” provided for use in said cccDNA vector for controlled transgene expression comprises hybrid transcription factors (TFs) to increase the range and complexity of environmental signals used to specify conditions of biological containment. Unlike a blocking switch, which triggers cell death when a predetermined condition is met, a "password circuit" allows cell survival or transgene expression provided a specific "password" is present, and can be easily reprogrammed to allow transgene expression and/or cell survival only if there is a preset environmental condition or password.

[00328] Any combination of regulatory switches disclosed herein, for example, small molecule switches, nucleic acid-based switches, small molecule-nucleic acid hybrid switches, post-transcriptional transgene regulatory switches, post-translational regulation, radiation-controlled switches, hypoxia-mediated switches, and other control switches known to those skilled in the art, as described herein, may be used in the password control switch disclosed herein. Regulatory switches contemplated for use are also described in the review article Kis et al., J R Soc Interface. 12: 20141000 (2015), and are summarized in Table 1 of the cited article. In some embodiments, the control switch for use in a password system may be selected from any of the switches shown in Table 11 or a combination thereof.

(iv). Nucleic acid-based regulatory switches to control transgene expression

[00329] In some embodiments, the regulatory switch for controlling the transgene expressed by the cccDNA is based on a nucleic acid-based control mechanism. Typical examples of nucleic acid-based control mechanisms are known in the art and are contemplated for use. For example, such mechanisms include riboswitches such as those disclosed in, for example, US2009/0305253, US2008/0269258, US2017/0204477, WO2018026762A1, US Patent No. 9222093 and European Patent Application EP288071, as well as the review by Villa JK et al. Microbiol Spectr., 2018 May;6(3). Also included are metabolite-responsive transcriptional biosensors such as those described in WO2018/075486 and WO2017/147585. Other mechanisms contemplated in the art include silencing of said transgene by a siRNA or RNA interference (RNAi) molecule (eg, mature microRNA, shRNA). For example, a cccDNA vector for controlled expression of a transgene may contain a regulatory switch that encodes an RNA interference molecule complementary to the transgene expressed by the cccDNA vector. When such RNAi is expressed, even when the transgene is expressed by a cDNA vector, its silencing occurs under the influence of a complementary RNA interference molecule, and in the absence of RNAi expression, when the cDNA vector expresses the specified transgene, such silencing of the transgene due to RNA interference does not occur.

[00330] In some embodiments, said regulatory switch is a tissue-specific self-inactivating regulatory switch, for example, as described in US2002/0022018, wherein the regulatory switch specifically turns off transgene expression at a site where transgene expression may otherwise be detrimental. In some embodiments, said regulatory switch is a recombinase reversible gene expression system, for example, as described in US2014/0127162 and US Patent No. 8324436.

(v). Post-transcriptional and post-translational regulatory switches

[00331] In some embodiments, said regulatory switch for controlling a transgene or gene of interest expressed by a cccDNA vector for controlled expression of the transgene is a post-transcriptional modification system. For example, such a regulatory switch may be an aptazyme riboswitch sensitive to tetracycline or theophylline, as described in US2018/0119156, GB201107768, WO2001/064956A3, EP 2707487 and Beilstein et al., ACS Synth. Biol., 2015, 4 (5), pp.526-534; Zhong et al., Elife. 2016 Nov 2;5. pii: e18858. In some embodiments, it is contemplated that one skilled in the art can encode both the transgene and an inhibitory siRNA that contains a ligand-sensitive (negative switch) aptamer, resulting in a ligand-sensitive positive switch.

(vi). Other examples of control switches

[00332] To control the gene expression of a transgene expressed by a cccDNA vector, any known regulatory switch, including those triggered by environmental changes, can be used in the cccDNA vector. Additional examples include, without limitation; BOC method according to Suzuki et al., Scientific Reports 8; 10051 (2018); genetic code expansion and non-physiological amino acid; radiation- or ultrasonic-controlled positive/negative switches (see, for example, Scott S et al., Gene Ther. 2000 Jul;7(13):1121-5; US Pat. Nos. 5,612,318; 5571797; 5770581; 5817636; and WO1999/ 025385A1 In some embodiments, said regulatory switch is controlled by an implantable system, for example, as disclosed in US Pat. Nos. 7,840,263; US2007/0190028A1, in which gene expression is controlled by one or more forms of energy, including electromagnetic energy, that activates promoters operably associated with the transgene in cccDNA vector.

[00333] In some embodiments, the regulatory switch for use in the cccDNA vector for controlled expression of the transgene is a hypoxia-mediated or stress-activated switch, such as those described in WO1999060142A2, US Pat. Nos. 5,834,306; 6218179; 6709858; US2015/0322410; Greco et al., (2004) Targeted Cancer Therapies 9, S368, as well as FROG, TOAD and NRSE elements and conditionally inducible silencing elements, including hypoxic response elements (HRE), inflammatory response elements (IRE) and shear stress activated elements (SSAE), for example, as disclosed in US Pat. No. 9,394,526. This embodiment is suitable for enabling expression of said transgene from a cccDNA vector for controlled expression of the transgene after ischemia in either ischemic tissues and/or tumors.

(iv). Kill switches

[00334] Other embodiments of the present invention provide a cccDNA vector for controlled transgene expression containing a kill switch. The kill switch, as described herein, allows the cell containing the cccDNA vector to be killed or enter programmed death as a means of permanently eliminating the introduced cccDNA vector from the subject's system. One skilled in the art will appreciate that the use of kill switches in the cccDNA vectors of this invention typically involves targeting said cccDNA vector to a limited number of cells that the subject can tolerate losing, or to a cell type apoptosis of which is desired (for example, on cancer cells). In all aspects, a kill switch as disclosed herein is designed to provide rapid and reliable killing of a cell containing said cccDNA vector in the absence of a survival input or other specific condition. In other words, the kill switch encoded by the cccDNA vector of the present invention can limit the survival of a cell containing the cccDNA vector to an environment defined by specific input signals. Such kill switches serve to provide a biocontainment function if it is desired to either remove the cDNA vector from a subject or ensure that the encoded transgene is not expressed.

VI. Detailed description of the method for producing cccDNA vector

A. Receipt in general

[00335] Certain methods for producing a cccDNA vector for the controlled expression of a transgene containing an asymmetric ICP pair or a symmetric ICP pair, as defined herein, are described in section IV of international application PCT/US18/49996, filed September 7, 2018, which is included incorporated into this document in its entirety by reference. In some embodiments, a cccDNA vector for controlled transgene expression for use in the methods and compositions described herein can be produced using insect cells as described herein. In alternative embodiments, the (ccDNA vector for controlled transgene expression) for use in the methods and compositions described herein can be produced synthetically and, in some embodiments, cell-free, as described in international application PCT/US19/ 14122, filed January 18, 2019, which is incorporated herein by reference in its entirety.

[00336] As described herein, a cccDNA vector for controlled transgene expression can be prepared using a method comprising the steps of: a) incubating a population of host cells (eg, insect cells) carrying a polynucleotide template of the expression construct (eg, cccDNA- plasmid, cccDNA bacmid and/or ccDNA baculovirus) lacking viral capsid coding sequences, in the presence of Rep protein under conditions effective to, and for a period of time sufficient to induce, the production of said ccDNA vector in host cells, wherein said host cells do not contain viral capsid coding sequences; and b) collecting and isolating the cDNA vector from the host cells. The presence of the Rep protein induces replication of the ICP-modified vector polynucleotide to produce a cDNA vector in the host cell. However, viral particles (for example, AAV virions) are not expressed. Thus, there are no size restrictions such as those naturally imposed in AAV or other viral vectors.

[00337] The presence of a cDNA vector isolated from a host cell can be confirmed by digesting DNA isolated from a host cell with a restriction enzyme having one recognition site on the cDNA vector and analyzing said digested DNA material on a non-denaturing gel for confirmation the presence of characteristic bands of linear and continuous DNA that differ from those of linear and discontinuous DNA.

[00338] In yet another aspect, the invention provides for the use of host cell lines having a DNA expression vector polynucleotide template (ccDNA template) stably integrated into its own genome in the production of a non-viral DNA vector, for example, as described in: Lee, L. et al. (2013) Plos One 8(8): e69879. Preferably, Rep is added to host cells at an MOI (multiplicity of infection) of approximately 3. If the host cell line is a mammalian cell line, such as HEK293 cells, then said cell lines may contain a stably integrated polynucleotide vector template, and a second vector , for example, herpesvirus, can be used to introduce the Rep protein into cells, which ensures excision and amplification of cctDNA in the presence of Rep and helper virus.

[00339] In one embodiment, the host cells used to produce the cccDNA vectors described herein are insect cells, and a baculovirus is used to deliver both the polynucleotide that encodes the Rep protein and the nonviral DNA polynucleotide expression construct template. vector for cccDNA, for example, as described in FIGS. 4A-4C and Example 1. In some embodiments, the host cell is modified to express the Rep protein.

[00340] The cccDNA vector is then collected and isolated from the host cells. The timing of collection and extraction of the cccDNA vectors described herein from cells can be selected and optimized to ensure high throughput production of said cccDNA vectors. For example, the collection time may be selected based on cell viability, cell morphology, cell growth, etc. In one embodiment, the cells are cultured under sufficient conditions and harvested sufficiently after infection with the baculovirus to obtain cccDNA vectors, but before the majority of cells begin to die due to toxicity of the baculovirus. These DNA vectors can be isolated using plasmid purification kits such as the Endo-Free Plasmid kits from Qiagen. Other methods developed for isolating plasmids can also be adapted for DNA vectors. In general, any method for purifying nucleic acids can be used.

[00341] DNA vectors can be purified by any DNA purification means known to those skilled in the art. In one embodiment, purified cDNA vectors are obtained as DNA molecules. In another embodiment, purified cDNA vectors are produced in the form of exosomes or microparticles.

[00342] The presence of said cccDNA vector can be confirmed by digesting vector DNA isolated from cells with a restriction enzyme having a single recognition site on the DNA vector and analyzing both digested and undigested DNA material using gel electrophoresis to confirm the presence of characteristic bands of linear and continuous DNA, different from linear and discontinuous DNA. FIG. 4C and FIG. 4D illustrate one embodiment relating to identifying the presence of closed-end cccDNA vectors produced by the methods described herein.

[00343]

B. cDNA plasmid

[00344] A cDNA plasmid is a plasmid used to subsequently produce a cDNA vector. In some embodiments, the cDNA plasmid can be constructed using known techniques to provide at least the following, as operably linked components, in the direction of transcription: (1) a modified 5'-ICP sequence; (2) an expression cassette containing a cis-regulatory element, for example, a promoter, an inducible promoter, a regulatory switch, enhancers, and the like; and (3) a modified 3'-ICP sequence, wherein the 3'-ICP sequence is symmetrical to the 5'-ICP sequence. In some embodiments, the ICP-flanked expression cassette contains a cloning site for introducing an exogenous sequence. This expression cassette replaces the rep and cap coding regions of the AAV genomes.

[00345] In one aspect, a cccDNA vector for controlled transgene expression is derived from a plasmid, referred to herein as a “ccDNA plasmid,” encoding, in this order: an adeno-associated virus (AAV) first inverted terminal repeat (ITR), an expression cassette containing a transgene, and a mutated or modified AAV ICP, wherein said cDNA plasmid lacks the coding sequences of the AAV capsid protein. In alternative embodiments, such a ccDNA plasmid encodes, in that order: a first (or 5') modified or mutated AAV ICP, an expression cassette containing the transgene, and a second (or 3') modified AAV ICP, wherein said ccDNA plasmid lacks coding sequences of the AAV capsid protein, and the 5'- and 3'-ICPs are symmetrical relative to each other. In alternative embodiments, the cccDNA plasmid encodes, in that order: a first (or 5') modified or mutated AAV ICP, an expression cassette containing the transgene, and a second (or 3') mutated or modified AAV ICP, wherein said ccDNA plasmid lacks coding sequences of the AAV capsid protein, and the modified 5'- and 3'-ICPs have the same modifications (i.e., they are reverse complementary or symmetrical relative to each other).

[00346] In a further embodiment, the cDNA plasmid system is devoid of viral capsid protein coding sequences (i.e., it does not contain AAV capsid genes as well as capsid genes of other viruses). Additionally, in a particular embodiment, the cDNA plasmid also lacks Rep AAB protein coding sequences. Accordingly, in a preferred embodiment, the cccDNA plasmid lacks the functional genes cap AAB and rep AAV, GG-3′ for AAV2), as well as the variable palindromic sequence that provides hairpin formation.

[00347] The cDNA plasmid of this invention can be obtained using natural nucleotide sequences from the genomes of any AAV serotypes well known in the art. In one embodiment, the cccDNA plasmid backbone is derived from the genome of AAB1, AAV2, AAV3, AAV4, AAV5, AAV 5, AAV7, AAV8, AAV9, AAV10, AAV 11, AAV12, AAVrh8, AAVrh10, AAV-DJ, and AAV-DJ8. For example, NCBI: NC 002077; NC001401; NC001729; NC001829; NC006152; NC 006260; NC 006261; from the Kotin and Smith virus index ("The Springer Index of Viruses"), available at a link maintained by Springer (web address: oesys.springer.de/viruses/database/mkchapter.asp?virID=42.04.) (note: links URL or database refers to the contents of the URL or database available from the link as of the actual filing date of this application). In a specific embodiment, the cDNA plasmid backbone is derived from the AAB2 genome. In another specific embodiment, the cDNA plasmid backbone is a synthetic backbone engineered to include ICPs at the 5' and 3' ends derived from one of the AAV genomes.

[00348] The cccDNA plasmid may optionally include a selectable or selectable marker for use in creating a cell line producing the cccDNA vector. In one embodiment, the selection marker may be inserted downstream of the ICP sequence 3' (ie, in the 3' direction). In another embodiment, the selection marker may be inserted upstream of the ICP sequence 5' (ie, in the 5' direction). Suitable selection markers include, for example, markers that confer drug resistance. The selection markers may be, for example, a blasticidin S, kanamycin, geneticin, etc. resistance gene. In a preferred embodiment, the drug selection marker is a blasticidin S resistance gene.

[00349] A typical ccDNA (eg, pAAB0) is produced from the pAAB plasmid. A method for producing a rAAV vector may include: (a) delivering a rAAV plasmid into a host cell, as described above, wherein both the host cell and the plasmid are devoid of genes encoding a capsid protein, (b) culturing said host cell under conditions providing the possibility of obtaining the cDNA genome; and (c) collecting cells and isolating the AAV genome produced by said cells.

C. Examples of a method for obtaining cDNA vectors from cDNA plasmids

[00350] This document also provides methods for producing capsidless cccDNA vectors, namely, a method with sufficiently high yield to provide sufficient quantities of vector for in vivo experiments.

[00351] In some embodiments, a method of producing a cccDNA vector for controlled expression of a transgene includes the steps of: (1) introducing a nucleic acid construct containing an expression cassette and two symmetrical ICP sequences into a host cell (e.g., Sf9 cells), (2) optionally producing a clonal cell line, for example, using a selection marker present on a plasmid, (3) introducing a gene encoding Rep (by transfection or infection with a baculovirus carrying said gene) into said insect cell; and (4) collecting said cells and purifying the cDNA vector. The nucleic acid construct containing the expression cassette and the two ICP sequences described above to produce a cccDNA vector for controlled transgene expression may be in the form of a ccDNA plasmid or a bacmid or baculovirus derived from a ccDNA plasmid as described below. The nucleic acid construct can be introduced into a host cell by transfection, viral transduction, stable integration, or other methods known in the art.

D. Cell lines:

[00352] The host cell lines used to generate the cccDNA vector for controlled transgene expression may include insect cell lines derived from Spodoptera frugiperda (fall armyworm), such as Sf9, Sf21 cells, or Trichoplusia ni cells. , or other invertebrate, vertebrate, or other eukaryotic cell lines, including mammalian cells. Other cell lines known to one skilled in the art may also be used, such as HEK293, Huh-7, HeLa, HepG2, HeplA, 911, CHO, COS, MeWo, NIH3T3, A549, HT1 180, monocytes, as well as mature and immature dendritic cells. Host cell lines can be transfected to stably express the cccDNA plasmid to produce a ccDNA vector in high yield.

[00353] cDNA plasmids can be introduced into Sf9 cells by transient transfection using reagents (eg, liposomes, calcium phosphate) or physical means (eg, electroporation) known in the art. Alternatively, stable Sf9 cell lines can be generated with the cccDNA plasmid stably integrated into the genome. Such stable cell lines can be created by incorporating a selection marker into a cccDNA plasmid as described above. If the cccDNA plasmid used to transfect a cell line includes a selection marker, such as an antibiotic, then cells that have been transfected with said ccDNA plasmid and have integrated the ccDNA plasmid DNA into their genomes can be selected by adding said antibiotic to the cell growth media . Resistant cell clones can then be isolated by single cell propagation or colony transfer methods and expanded.

E. Isolation and purification of cDNA vectors

[00354] Examples of the method for preparing and isolating cDNA vectors are described in FIGS. 4A-4E and in the specific examples below. The cccDNA vectors disclosed herein can be derived from a producer cell expressing the AAV Rep protein(s) further transformed with a ccDNA plasmid, ccDNA bacmid, or ccDNA baculovirus. Plasmids useful for producing cccDNA vectors include plasmids comprising one or more Rep proteins and plasmids used to produce cccDNA vector. Exemplary plasmids for producing a cccDNA vector for controlled transgene expression are the plasmid shown in Figure 6B of International Application PCT/US2018/064242, filed December 6, 2018, which is incorporated herein in its entirety. An cccDNA plasmid for producing a ccDNA vector for controlled expression of an antibody is disclosed in FIG. 6A and is SEQ ID NO: 56 of International Application PCT/US19/18016, filed February 14, 2019, which discloses representative examples of ccDNA plasmids for the production of aducanumab. .

[00355] In one aspect, a polynucleotide encoding an AAV Rep protein (Rep 78 or Rep68) is delivered to a producing cell in a plasmid (Rep-plasmid), bacmid (Rep-bacmid), or baculovirus (Rep-baculovirus). Rep plasmid, Rep bacmid and Rep baculovirus can be produced by the methods described above.

[00356] Methods for producing a cDNA vector, which is an example of a cDNA vector, are described herein. Expression constructs used to generate cccDNA vectors of the present invention may be a plasmid (eg, ccDNA plasmids), bacmid (eg, ccDNA-baculovirus), and/or baculovirus (eg, ccDNA-baculovirus). By way of example only, a cDNA vector can be prepared from cells coinfected with a cDNA baculovirus and a Rep baculovirus. Rep proteins produced from Rep baculovirus can replicate cccDNA baculovirus to produce cccDNA vectors. Alternatively, cccDNA vectors can be prepared from cells stably transfected with a construct containing the AAV Rep protein coding sequence (Rep78/52) delivered in Rep plasmids, Rep bacmids or Rep baculovirus. The cccDNA baculovirus can be transiently transfected into cells, replicated by the Rep protein, and produce cccDNA vectors.

[00357] A bacmid (e.g., cccDNA bacmid) can be transfected into permissive insect cells such as Sf9, Sf21 cells, Tni (Trichoplusia ni) cells, High Five cells, and generate a cccDNA baculovirus, which is a recombinant baculovirus comprising sequences containing symmetrical ICPs and an expression cassette. Insect cells can be reinfected with cDNA baculovirus to produce the next generation of recombinant baculovirus. Optionally, this step can be repeated one or more times to obtain a larger quantity of recombinant baculovirus.

[00358] The timing of collection and extraction from cells of the cccDNA vectors described herein can be selected and optimized to ensure high throughput production of said cccDNA vectors. For example, the collection time may be selected based on cell viability, cell morphology, cell growth, etc. Typically, cells can be harvested after sufficient time has elapsed since baculovirus infection to allow production of cDNA vectors (eg, cDNA vectors) but before most cells begin to die due to viral toxicity. cccDNA vectors can be isolated from Sf9 cells using plasmid purification kits such as the ENDO-FREE PLASMID® kits from Qiagen. Other methods developed for plasmid isolation can also be adapted for cDNA vectors. Typically, any nucleic acid purification methods known in the art, as well as commercially available DNA extraction kits, can be used.

[00359] Alternatively, purification can be accomplished by performing an alkaline cell pellet lysis process, centrifuging the resulting lysate, and performing chromatographic separation. As one non-limiting example, this process can be accomplished by loading the supernatant onto an ion exchange column (e.g., SARTOBIND Q®) that retains nucleic acids, followed by elution (e.g., 1.2 M NaCl), and further chromatographic purification. on a gel filtration column (for example, “6 Fast Flow GE”). The capsidless AAV vector is then isolated, for example, by precipitation.

[00360] In some embodiments, purified cDNA vectors may also be provided as exosomes or microparticles. It is known in the art that many cell types release not only soluble proteins, but also complex protein/nucleic acid cargos by shedding membrane microvesicles (Cocucci et al, 2009; EP 10306226.1). Such vesicles include microvesicles (also called microparticles) and exosomes (also called nanovesicles), which contain proteins and RNA as cargo. Microvesicles are formed by direct budding from the plasma membrane, and exosomes are released into the extracellular environment after the fusion of multivesicular endosomes with the plasma membrane. Thus, microvesicles and/or exosomes containing the cccDNA vector can be isolated from cells that have been transduced with the ccDNA plasmid or bacmid or baculovirus generated using the ccDNA plasmid.

[00361] Microvesicles can be isolated by filtration or ultracentrifugation of the culture medium at 20,000×g, and exosomes at 100,000×g. The optimal duration of ultracentrifugation can be determined experimentally and will depend on the specific cell type from which the vesicles are isolated. Preferably, the culture medium is first clarified by low-speed centrifugation (eg at 2000×g for 5-20 minutes), and subjected to centrifugal concentration using, for example, an AMICON® spin column (Millipore, Watford, UK). Microvesicles and exosomes can be further purified by FACS or MACS using specific antibodies that recognize specific surface antigens present on microvesicles and exosomes. Other methods for purifying microvesicles and exosomes include, but are not limited to, immunoprecipitation, affinity chromatography, filtration, and the use of magnetic beads coated with specific antibodies or aptamers. After purification, the vesicles are washed, for example, with phosphate buffered saline. One advantage of using microvesicles or exosomes to deliver cccDNA-containing vesicles is that these vesicles can be targeted to different cell types by incorporating proteins into their membranes that are recognized by specific receptors on the relevant cell types (see also EP 10306226).

[00362] Another aspect of the present invention relates to methods for purifying cccDNA vectors from host cell lines that have the ccDNA construct stably integrated into their genome. In one embodiment, the purified ccDNA vectors are obtained as DNA molecules. In another embodiment, purified cDNA vectors are produced in the form of exosomes or microparticles.

[00363] Figure 5 of International Application PCT/US18/49996 shows a gel confirming the production of cccDNA from a plurality of ccDNA plasmid constructs using the method described in the examples. cccDNA is confirmed by a characteristic banding pattern in the gel, as described with reference to Fig. 4D in the Examples.

VII. Pharmaceutical compositions

[00364] In another aspect, pharmaceutical compositions are provided. The pharmaceutical composition contains a closed-end DNA vector, for example, a cccDNA vector for controlled transgene expression, obtained using a synthesis method as described herein, and a pharmaceutically acceptable carrier or diluent.

[00365] cccDNA vectors, as described herein, can be included in pharmaceutical compositions suitable for administration to a subject for in vivo delivery to cells, tissues or organs of the subject. Typically, the pharmaceutical composition contains a cccDNA vector as described herein and a pharmaceutically acceptable carrier. For example, the cccDNA vectors described herein may be included in a pharmaceutical composition suitable for the desired route of therapeutic administration (eg, parenteral administration). Passive tissue transduction through high-pressure intravenous or intra-arterial infusion, as well as intracellular injection such as intranuclear microinjection or intracytoplasmic injection, are also contemplated. Pharmaceutical compositions for therapeutic purposes can be formulated as a solution, microemulsion, dispersion, liposomes, or other ordered structure that allows for a high concentration of cccDNA vector. Sterile injection solutions can be prepared by incorporating the cccDNA vector formulation in the required quantity into an appropriate buffer with one or a combination of the ingredients listed above, as needed, followed by filter sterilization, including the ccDNA vector that can be prepared to deliver the transgene in the nucleic acid acid into recipient cells, leading to therapeutic expression of a transgene or donor sequence in them. The composition may also include a pharmaceutically acceptable carrier.

[00366] Pharmaceutically active compositions containing a cccDNA vector for controlled expression of a transgene can be formulated to deliver the transgene for various purposes into a cell, for example, into the cells of a subject.

[00367] cccDNA vectors, as described herein, can be included in pharmaceutical compositions suitable for administration to a subject for in vivo delivery to cells, tissues or organs of the subject. Typically, the pharmaceutical composition contains cccDNA vectors as described herein and a pharmaceutically acceptable carrier. For example, the cccDNA vectors described herein can be included in a pharmaceutical composition suitable for the desired route of therapeutic administration (eg, parenteral administration). Passive tissue transduction through high-pressure intravenous or intra-arterial infusion, as well as intracellular injection such as intranuclear microinjection or intracytoplasmic injection, are also contemplated. Pharmaceutical compositions for therapeutic purposes can be formulated as a solution, microemulsion, dispersion, liposomes, or other ordered structure that allows for a high concentration of cccDNA vector. Sterile injectable solutions can be prepared by incorporating the cccDNA vector formulation in the required amount into an appropriate buffer with one or a combination of the ingredients listed above, as needed, followed by filter sterilization.

[00368] Pharmaceutically active compositions containing a cccDNA vector can be formulated to deliver a transgene in nucleic acid to recipient cells, resulting in therapeutic expression of the transgene therein. The composition may also optionally include a pharmaceutically acceptable carrier and/or excipient.

[00369] The compositions and vectors presented herein can be used to deliver a transgene for a variety of purposes. In some embodiments, the transgene encodes a protein or functional RNA that is intended to be used for research purposes, for example, to create a somatic transgenic animal model containing the transgene, for example, to study the function of the transgene product. In another example, the transgene encodes a protein or functional RNA that is intended to be used to create an animal model of a disease. In some embodiments, the transgene encodes one or more peptides, polypeptides, or proteins that are useful for treating or preventing disease states in a mammalian subject. The transgene may be transferred to a patient (eg, expressed in the patient) in an amount sufficient to treat a disease associated with decreased expression, absence of expression, or dysfunction of the gene. In some embodiments, the transgene is a gene editing molecule (eg, a nuclease). In some embodiments, the nuclease is a CRISPR-associated nuclease (Cas nuclease).

[00370] Pharmaceutical compositions for therapeutic purposes generally must be sterile and stable under conditions of manufacture and storage. Sterile injectable solutions can be prepared by incorporating the cccDNA vector formulation in the required amount into an appropriate buffer with one or a combination of the ingredients listed above, as needed, followed by filter sterilization.

[00371] In certain circumstances, it will be desirable to deliver the composition or cccDNA vector as described herein, in pharmaceutical compositions with the appropriate composition described herein, either by subcutaneous, intrapancreatic, intranasal, parenteral, intravenous, intramuscular, intrathecal, systemic administration or orally, intraperitoneally or by inhalation.

[00372] It is specifically contemplated herein that the compositions described herein contain a cccDNA vector for controlled expression of a transgene at some given dose, which is determined by the dose-response relationship of the cccDNA vector, for example, at a “unit dose”, from which when administered, it can be reasonably expected to produce the desired effect or level of expression of the genetic drug in a typical subject.

[00373] Pharmaceutical compositions for therapeutic purposes generally must be sterile and stable under conditions of manufacture and storage. The composition can be formulated as a solution, microemulsion, dispersion, liposomes, or other ordered structure that allows for a high concentration of the cDNA vector. Sterile injectable solutions can be prepared by incorporating the cccDNA vector formulation in the required amount into an appropriate buffer with one or a combination of the ingredients listed above, as needed, followed by filter sterilization.

[00374] The cccDNA vector described herein may be included in a pharmaceutical composition suitable for local, systemic, intra-amniotic, intrathecal, intracranial, intra-arterial, intravenous, intralymphatic, intraperitoneal, subcutaneous, tracheal, interstitial (e.g., intramuscular, intracardiac) , intrahepatic, intrarenal, intracerebral), intrathecal, intravesical, conjunctival (for example, extraorbital, intraorbital, retroorbital, intraretinal, subretinal, choroidal, subchoroidal, intrastromal, intracameral and intravitreal), intracochlear and mucosal, for example, rectal, nasal) administration. Passive tissue transduction through high-pressure intravenous or intra-arterial infusion, as well as intracellular injection such as intranuclear microinjection or intracytoplasmic injection, are also contemplated.

[00375] In some aspects, the methods provided herein include delivering one or more cccDNA vectors, as described herein, into a host cell. Also provided herein are cells produced by such methods and organisms (such as animals, plants or fungi) containing or derived from such cells. Nucleic acid delivery methods may include lipofection, nucleofection, microinjection, biolistics, liposomes, immunoliposomes, polycation or lipid:nucleic acid conjugates, naked DNA, and agent-enhanced DNA uptake. Lipofection is described, for example, in US Pat. Nos. 5,049,386, 4,946,787 and 4,897,355), and lipofection reagents are sold commercially (eg, Transfectam™ and Lipofectin™). Delivery may be to cells (eg, in vitro or ex vivo administration) or to target tissues (eg, in vivo administration).

[00376] Various techniques and methods for delivering nucleic acids into cells are known in the art. For example, compositions of nucleic acids, such as cccDNA, can be formulated in lipid nanoparticles (LNPs), lipidoids, liposomes, lipid nanoparticles, lipoplexes, or core-shell nanoparticles. Typically, LNPs consist of a nucleic acid molecule (e.g., cccDNA), one or more ionizable or cationic lipids (or salts thereof), one or more nonionic or neutral lipids (e.g., phospholipids), a molecule that prevents aggregation (e.g., PEG or a PEG-lipid conjugate) and optionally a sterol (eg cholesterol).

[00377] Another method of delivering nucleic acids, such as cctDNA, into a cell is by conjugating the nucleic acid to a ligand that is internalized by the cell. For example, a ligand can bind to a cell surface receptor and be internalized through endocytosis. A ligand may be covalently linked to a nucleotide in a nucleic acid. Typical examples of conjugates for delivering nucleic acids into cells are described, for example, in WO2015/006740, WO2014/025805, WO2012/037254, WO2009/082606, WO2009/073809, WO2009/018332, WO2006/112872, W O2004/090108, WO2004/091515 and WO2017/177326.

[00378] Nucleic acids such as cctDNA can also be delivered into a cell by transfection. Suitable transfection methods include, but are not limited to, lipid-mediated transfection, cationic polymer-mediated transfection, or calcium phosphate precipitation. Transfection reagents are well known in the art and include, but are not limited to, TurboFect transfection reagent (Thermo Fisher Scientific), Pro-Ject reagent (Thermo Fisher Scientific), TRANSPASS™ protein transfection reagent P (New England Biolabs), Protein Delivery Reagent CHARIOT™ (Active Motif), Protein Transfection Reagent PROTEOJUICE™ (EMD Millipore), 293-Fectin, Lipofectamine (LIPOFECTAMINE™ 2000 "), "LIPOFECTAMINE™ 3000" ("Thermo Fisher Scientific"), "LIPOFECTAMINE™" ("Thermo Fisher Scientific"), lipofectin ("LIPOFECTIN™", "Thermo Fisher Scientific"), "DMRIE-C", "CELLFECTIN ™" (Thermo Fisher Scientific), "OLIGOFECTAMINE™" (Thermo Fisher Scientific), "LIPOFECTACE™", "FUGENE™" (Roche, Basel, Switzerland), "FUGENE™ HD" (Roche) ), TRANSFECTAM™ (Transfectam, Promega, Madison, WI), TFX-10™ (Promega), TFX-20™ (Promega), TFX-50™ ( Promega), transfectin (TRANSFECTIN™, BioRad, Hercules, CA), SILENTFECT™ (Bio-Rad), Effectene™ (Qiagen, Valencia, CA), DC-chol " (Avanti Polar Lipids), "GENEPORTER™" (Gene Therapy Systems, San Diego, California), "DHARMAFECT 1™" (Dharmacon, Lafayette, CO), "DHARMAFECT 2™" (Dharmacon "), "DHARMAFECT 3™" ("Dharmacon"), "DHARMAFECT 4™" ("Dharmacon"), "ESCORT™ III" (Sigma, St. Louis, MO), and "ESCORT™ IV" (" Sigma Chemical Co." Nucleic acids, such as cDNA, can also be delivered into cells by microfluidics techniques known to those skilled in the art.

[00379] cccDNA vectors, as described herein, can also be administered directly into the body to transduce cells in vivo. Administration is by any route commonly used to bring a molecule into direct contact with blood or tissue cells, including, but not limited to, injection, infusion, topical application, and electroporation. Suitable routes for administering such nucleic acids are available and well known to those skilled in the art and, although more than one route may be used to administer a particular composition, a particular route can often provide a faster and more efficient reaction than another route.

[00380] Methods of introducing a nucleic acid vector, for example, a cDNA vector for controlled transgene expression, as described herein, can be introduced into hematopoietic stem cells, for example, by methods described, for example, in US patent No. 5928638.

[00381] cccDNA vectors in accordance with this invention can be added to liposomes for delivery to a cell or target organ in a subject. Liposomes are vesicles that contain at least one lipid bilayer. Liposomes are commonly used as carriers for drug/therapeutic delivery in the context of pharmaceutical development. They work by fusing with the cell membrane and rearranging its lipid structure to deliver a drug or active pharmaceutical ingredient (API). Liposomal compositions for such delivery consist of phospholipids, in particular compounds containing a phosphatidylcholine group, however, these compositions may also include other lipids. Exemplary liposomes and liposome formulations, including, but not limited to, compounds containing polyethylene glycol (PEG) functional groups, are disclosed in International Application PCT/US2018/050042, filed September 7, 2018, and International Application PCT/US2018/064242, filed 6 December 2018, for example, see the section entitled “Pharmaceutical Compositions.”

[00382] Various delivery methods known in the art, or modifications thereof, can be used to deliver cccDNA vectors in vitro or in vivo. For example, in some embodiments, cccDNA vectors are delivered by transiently penetrating the cell membrane using mechanical, electrical, ultrasonic, hydrodynamic, or laser energy in a manner that facilitates entry of DNA into target cells. For example, a cccDNA vector for controlled transgene expression can be delivered by transiently disrupting the cell membrane by pushing the cell through a size-limited channel, or other methods known in the art. In some cases, a single cccDNA vector is administered by direct injection of naked DNA into the skin, thymus, cardiac muscle, skeletal muscle or liver cells. In some cases, the cccDNA vector is delivered using a gene gun. Spherical gold or tungsten particles (1–3 μm in diameter) coated with capsidless AAV vectors can be accelerated to high speed using pressurized gas to penetrate target tissue cells.

[00383] Compositions containing a cccDNA vector for controlled transgene expression and a pharmaceutically acceptable carrier are specifically provided for herein. In some embodiments, the cccDNA vector for controlled transgene expression is formulated with a lipid delivery system composition, such as liposomes, as described herein. In some embodiments, such compositions are administered by any route deemed desirable by one skilled in the art. The compositions may be administered to a subject by various routes, including oral, parenteral, sublingual, transdermal, rectal, transmucosal, topical, inhalation, buccal, intrapleural, intravenous, intraarterial, intraperitoneal, subcutaneous, intramuscular, intranasal, intrathecal, and intraarticular, or their combinations. For veterinary use, the composition may be administered in a reasonably acceptable composition in accordance with normal veterinary practice. The veterinarian can easily determine the dosage schedule and route of administration that is most appropriate for a particular animal. The compositions can be administered using traditional syringes, needleless injection devices, microparticle bombardment gene guns, or other physical methods such as electroporation (“EP”), hydrodynamic methods, or the use of ultrasound.

[00384] In some cases, the cccDNA vector for controlled transgene expression is delivered by hydrodynamic injection, which is a simple and highly effective method for direct intracellular delivery of any water-soluble compounds and particles to internal organs and skeletal muscle throughout the limb.

[00385] In some cases, cccDNA vectors are delivered by ultrasound by creating nanoscopic pores in the membrane to facilitate intracellular delivery of DNA particles to internal organ or tumor cells, so the size and concentration of plasmid DNA is of great importance to the efficiency of the system. In some cases, cccDNA vectors are delivered by magnetofection, using magnetic fields to concentrate particles containing the nucleic acid into target cells.

[00386] In some cases, chemical delivery systems can be used, for example, using nanometer complexes, which involve compaction of negatively charged nucleic acid with polycationic nanometer particles belonging to cationic liposomes/micelles or cationic polymers. Cationic lipids used in this delivery method include, but are not limited to, monovalent cationic lipids, polyvalent cationic lipids, guanidine-containing compounds, cholesterol-derived compounds, cationic polymers (e.g., polyethylenimine, poly-L-lysine, protamine, other cationic polymers ), and lipid-polymer hybrids.

A. Exosomes

[00387] In some embodiments, a cccDNA vector for controlled transgene expression, as disclosed herein, is delivered packaged in exosomes. Exosomes are small membrane vesicles of endocytic origin that are released into the extracellular environment after the fusion of multivesicular bodies with the plasma membrane. Their surface consists of a lipid bilayer from the cell membrane of the donor cell, they contain cytosol from the exosome-producing cell, and they display membrane proteins from the parent cell on the surface. Exosomes are produced by various cell types, including epithelial cells, B and T lymphocytes, mast cells (MCs), and dendritic cells (DCs). Some embodiments include the use of exosomes with a diameter of 10 nm to 1 μm, 20 nm to 500 nm, 30 nm to 250 nm, 50 nm to 100 nm. Exosomes can be isolated for delivery to target cells using either their donor cells or by introducing specific nucleic acids into them. Various approaches known in the art can be used to produce exosomes containing capsidless AAV vectors of this invention.

B. Microparticles/nanoparticles

[00388] In some embodiments, the cDNA vector for controlled transgene expression, as disclosed herein, is delivered using lipid nanoparticles. Typically, lipid nanoparticles contain an ionizable aminolipid (e.g. heptatriakonta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)-butanoate, DLin-MC3-DMA, phosphatidylcholine (1,2-distearoyl-sn- glycero-3-phosphocholine, DSPC), cholesterol and envelope lipid (polyethylene glycol-dimyristol glycerol, PEG-DMG), for example, as described by Tam et al. (2013), Advances in Lipid Nanoparticles for siRNA delivery. Pharmaceuticals 5(3): 498 -507.

[00389] In some embodiments, the lipid nanoparticle has an average diameter of from about 10 nm to about 1000 nm. In some embodiments, the lipid nanoparticle has a diameter of less than 300 nm. In some embodiments, the lipid nanoparticle has a diameter of from about 10 nm to about 300 nm. In some embodiments, the lipid nanoparticle has a diameter of less than 200 nm. In some embodiments, the lipid nanoparticle has a diameter of from about 25 nm to about 200 nm. In some embodiments, a lipid nanoparticle formulation (e.g., a composition containing a plurality of lipid nanoparticles) has a size distribution such that the average size (e.g., diameter) is from about 70 nm to about 200 nm, and more typically, the average size is about 100 nm or less.

[00390] Various lipid nanoparticles known in the art can be used to deliver a cccDNA vector for controlled expression of the transgene disclosed herein. For example, various delivery methods using lipid nanoparticles are described in US Patent Nos. 9404127, 9006417 and 9518272.

[00391] In some embodiments, the cDNA vector for controlled transgene expression disclosed herein is delivered using gold nanoparticles. Typically, the nucleic acid may be covalently associated with the gold nanoparticle or non-covalently associated with the gold nanoparticle (eg, associated through charge-charge interaction), for example, as described by Ding et al. (2014), Gold Nanoparticles for Nucleic Acid Delivery. Mol. Ther. 22(6); 1075-1083. In some embodiments, gold nanoparticle-nucleic acid conjugates are prepared using methods described, for example, in US Pat. No. 6,812,334.

C. Conjugates

[00392] In some embodiments, a cccDNA vector for controlled transgene expression as disclosed herein is conjugated (eg, covalently linked) to an agent that enhances cellular uptake. A "cellular uptake enhancing agent" is a molecule that facilitates the transport of nucleic acid across a lipid membrane. For example, the nucleic acid may be conjugated to a lipophilic compound (e.g., cholesterol, tocopherol, etc.), cell penetrating peptide (CPP) (e.g., penetratin, TAT, Syn1B, etc.), and polyamines (e.g., spermine ). Additional examples of agents that enhance cellular uptake are disclosed, for example, in Winkler (2013), Oligonucleotide conjugates for therapeutic applications. Ther. Deliv. 4(7); 791-809.

[00393] In some embodiments, a cccDNA vector for controlled transgene expression, as described herein, is conjugated to a polymer (eg, a polymer molecule) or a folate molecule (eg, a folic acid molecule). In general, delivery of nucleic acids conjugated to polymers is known in the art, for example, as described in WO2000/34343 and WO2008/022309. In some embodiments, a cccDNA vector for controlled transgene expression, as described herein, is conjugated to a poly(amide) polymer, for example, as described in US Pat. No. 8,987,377. In some embodiments, the nucleic acid described herein is conjugated to a folic acid molecule, as described in US Pat. No. 8,550,455.

[00394] In some embodiments, a cccDNA vector for controlled transgene expression as described herein is conjugated to a carbohydrate, for example, as described in US Pat. No. 8,450,467.

D. Nanocapsules

[00395] Alternatively, nanocapsular cDNA vector compositions may be used for controlled transgene expression, as described herein. In general, substances can be encapsulated in nanocapsules in a stable and reproducible manner. To avoid side effects caused by excessive intracellular loading of polymers, such ultrafine particles (about 0.1 μm in size) must be developed using polymers that can be degraded in vivo. The use of biodegradable polyalkyl cyanoacrylate nanoparticles that meet such requirements is envisaged.

E. Liposomes

[00396] cccDNA vectors in accordance with this invention can be added to liposomes for delivery to a cell or target organ in a subject. Liposomes are vesicles that contain at least one lipid bilayer. Liposomes are commonly used as carriers for drug/therapeutic delivery in the context of pharmaceutical development. They function by fusing with the cell membrane and rearranging its lipid structure to deliver a drug or active pharmaceutical ingredient (API). Liposomal compositions for such delivery consist of phospholipids, in particular compounds containing a phosphatidylcholine group, however, these compositions may also include other lipids.

[00397] The formation and use of liposomes are well known to those skilled in the art. Liposomes with improved serum stability and circulation half-life have been developed (US Patent No. 5,741,516). In addition, various methods for preparing liposomes and liposome-like preparations as potential drug carriers have been described (US Patent Nos. 5,567,434; 5,552,157; 5,565,213; 5,738,868 and 5,795,587).

F. Typical compositions of liposomes and lipid nanoparticles (LNPs)

[00398] cccDNA vectors in accordance with this invention can be added to liposomes for delivery into a cell, for example, into a cell in need of transgene expression. Liposomes are vesicles that contain at least one lipid bilayer. Liposomes are commonly used as carriers for drug/therapeutic delivery in the context of pharmaceutical development. They function by fusing with the cell membrane and rearranging its lipid structure to deliver a drug or active pharmaceutical ingredient (API). Liposomal compositions for such delivery consist of phospholipids, in particular compounds containing a phosphatidylcholine group, however, these compositions may also include other lipids.

[00399] Lipid nanoparticles (LNPs) containing cccDNA are disclosed in International Application PCT/US2018/050042, filed September 7, 2018, and International Application PCT/US2018/064242, filed December 6, 2018, which are incorporated herein in its entirety and is intended for use in the methods and compositions disclosed herein.

[00400] In some aspects, the cDNA-containing lipid nanoparticle is an ionizable lipid.

[00401] Typically, lipid particles are prepared at a ratio of total lipid to cccDNA (w/w) of about 10:1 to 30:1. In some embodiments, the lipid to cccDNA ratio (mass ratio; w/w ratio) may range from about 1:1 to about 25:1, from about 10:1 to about 14:1, from about 3: 1 to about 15:1, about 4:1 to about 10:1, about 5:1 to about 9:1, or about 6:1 to about 9:1. The amount of lipids and cccDNA can be adjusted to provide a desired N/P ratio, for example an N/P ratio of 3, 4, 5, 6, 7, 8, 9, 10 or greater. Typically, the total lipid content of the lipid particle composition may be from about 5 to about 30 mg/ml. Ionizable lipids are also referred to herein as cationic lipids. Typical examples of ionizable lipids are described in international PCT patent publications WO2015/095340, WO2015/199952, WO2018/011633, WO2017/049245, WO2015/061467, WO2012/040184, WO2012/000104, WO2015 /074085, WO2016/081029, WO2017/004143, WO2017/075531, WO2017/117528, WO2011/022460, WO2013/148541, WO2013/116126, WO2011/153120, WO2012/044638, WO2012/054365, WO2011/09096 5, WO2013/016058, WO2012/162210, WO2008/042973, WO2010/ 129709, WO2010/144740, WO2012/099755, WO2013/049328, WO2013/086322, WO2013/086373, WO2011/071860, WO2009/132131, WO2010/048536, WO20 10/088537, WO2010/054401, WO2010/054406, WO2010/054405, WO2010/054384, WO2012/016184, WO2009/086558, WO2010/042877, WO2011/000106, WO2011/000107, WO2005/120152, WO2011/141705, WO2013/12680 3, WO2006/007712, WO2011/038160, WO2005/121348, WO2011/ 066651, WO2009/127060, WO2011/141704, WO2006/069782, WO2012/031043, WO2013/006825, WO2013/033563, WO2013/089151, WO2017/099823, WO20 15/095346 and WO2013/086354, and US patent publications US2016/0311759, US2015/0376115, US2016/0151284, US2017/0210697, US2015/0140070, US2013/0178541, US2013/0303587, US2015/0141678, US2015/0239926, US2016/037622 4, US2017/0119904, US2012/0149894, US2015/0057373, US2013/ 0090372, US2013/0274523, US2013/0274504, US2013/0274504, US2009/0023673, US2012/0128760, US2010/0324120, US2014/0200257, US2015/0203446, US20 18/0005363, US2014/0308304, US2013/0338210, US2012/0101148, US2012/0027796, US2012/0058144, US2013/0323269, US2011/0117125, US2011/0256175, US2012/0202871, US2011/0076335, US2006/0083780, US2013/012333 8, US2015/0064242, US2006/0051405, US2013/0065939, US2006/ 0008910, US2003/0022649, US2010/0130588, US2013/0116307, US2010/0062967, US2013/0202684, US2014/0141070, US2014/0255472, US2014/0039032, US20 18/0028664, US2016/0317458 and US2013/0195920, the contents of which are included in this document is incorporated by reference in its entirety.

[00402] In some embodiments, the ionizable lipid is MC3 (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl-4-(dimethylamino)-butanoate (DLin-MC3- DMA or MC3), having the following structure:

.

VIII. Methods for delivering cccDNA vectors

[00403] In some embodiments, the cccDNA vector for controlled transgene expression can be delivered to the target cell in vitro or in vivo by various suitable methods. Individual cccDNA vectors can be coated or injected. cccDNA vectors can be delivered into cells without the aid of a transfection reagent or other physical means. Alternatively, cccDNA vectors can be delivered using any transfection reagent known in the art or other physical means known in the art that facilitate DNA entry into the cell, such as liposomes, alcohols, polylysine-rich compounds, arginine-rich compounds, calcium phosphate, microvesicles. microinjections, electroporation, etc.

[00404] In contrast, transduction with capsidless AAV vectors disclosed herein can provide effective targeting of cell and tissue types that are difficult to transduce with conventional AAV virions using a variety of delivery reagents.

[00405] In another embodiment, the cccDNA vector for controlled expression of the transgene is introduced into the CNS (eg, the brain or eye). The cccDNA vector for controlled expression of the transgene can be introduced into the spinal cord, brainstem (medulla oblongata, pons), midbrain (hypothalamus, thalamus, epithalamus, pituitary gland, substantia nigra, pineal gland), cerebellum, telencephalon (striatum , cerebrum, including occipital, temporal, parietal and frontal lobes, cortex, basal ganglia, hippocampus and amygdala, limbic system, neocortex, striatum, cerebrum and inferior colliculus.The cDNA vector can also be injected into various areas of the eye such as the retina, cornea and/or optic nerve. The cccDNA vector can be delivered into the cerebrospinal fluid (eg, via lumbar puncture).Additionally, the cccDNA vector can be introduced into the CNS intravascularly in situations where the blood-brain barrier is disrupted (eg, brain tumor or cerebral infarction).

[00406] In some embodiments, the cccDNA vector for controlled transgene expression may be administered to the desired region(s) of the CNS by any route known in the art, including, but not limited to, intrathecal, intraocular, intracerebral, intraventricular, intravenous (e.g., in the presence of a sugar such as mannitol), intranasal, intraauricular, intraocular (eg, intravitreal, subretinal, anterior chamber) and periocular (eg, sub-Tenon's region) delivery, as well as intramuscular delivery with retrograde delivery to motor neurons.

[00407] In some embodiments, the cccDNA vector for controlled transgene expression is administered in a liquid composition by direct injection (eg, stereotactic injection) into the desired region or compartment of the CNS. In other embodiments, the cccDNA vector may be administered by topical application to the desired area or by intranasal administration of an aerosol composition. Administration to the eye can be accomplished by topical application of liquid drops. As a further alternative, the cDNA vector can be administered as a sustained release solid composition (see, for example, US Pat. No. 7,201,898). In additional embodiments, the cccDNA vector can be used for retrograde transport to treat, alleviate, and/or prevent motor neuron-related diseases and disorders (e.g., amyotrophic lateral sclerosis (ALS); spinal muscular atrophy (SMA), etc. ). For example, the cccDNA vector can be delivered to muscle tissue, from which it can migrate into neurons.

IX. Additional applications of cccDNA vectors

[00408] The compositions and cccDNA vectors as described herein can be used to express a target gene or transgene for various purposes. In some embodiments, the resulting transgene encodes a protein or functional RNA that is intended to be used for research purposes, for example, to create a somatic transgenic animal model containing the transgene, for example, to study the function of the transgene product. In another example, the transgene encodes a protein or functional RNA that is intended to be used to create an animal model of a disease. In some embodiments, the resulting transgene encodes one or more peptides, polypeptides, or proteins that are useful for treating, preventing, or alleviating disease states or disorders in a mammalian subject. The resulting transgene can be transferred to a subject (eg, expressed in the subject) in an amount sufficient to treat a disease associated with decreased expression, absence of expression, or dysfunction of the gene. In some embodiments, the resulting transgene may be expressed in a subject in an amount sufficient to treat a disease associated with increased expression, gene product activity, or inappropriate up-regulation of the gene that the resulting transgene suppresses or otherwise causes a decrease in its expression. In other embodiments, the resulting transgene replaces or complements a defective copy of the native gene. One skilled in the art will appreciate that the transgene may not be the open reading frame of a gene that itself must be transcribed; instead, it may be a promoter region or a repressor region of a target gene, and the cDNA vector may modify such a region, resulting in modulation of expression of the gene of interest.

[00409] In some embodiments, the transgene encodes a protein or functional RNA that is intended for use in creating an animal model of a disease. In some embodiments, the transgene encodes one or more peptides, polypeptides, or proteins that are useful for treating or preventing disease states in a mammalian subject. The transgene may be transferred to a patient (eg, expressed in the patient) in an amount sufficient to treat a disease associated with decreased expression, absence of expression, or dysfunction of the gene.

X. Directions for use

[00410] A cccDNA vector for controlled transgene expression, as described herein, can also be used in a method of delivering a nucleotide sequence of interest (eg, a transgene) to a target cell (eg, a host cell). The method, in particular, may be a method of delivering a transgene into a cell of a subject in need thereof and treating a disease of interest. The invention allows for in vivo expression of a transgene, for example, a protein, antibody, nucleic acid such as microRNA, etc., encoded by a cccDNA vector, in a cell of a subject in such a way that a therapeutic effect of transgene expression occurs. Such results are observed with both in vivo and in vitro methods of cDNA vector delivery.

[00411] The invention further provides a method of delivering a transgene into a cell of a subject in need thereof, comprising repeated administration of a cccDNA vector of the invention containing said nucleic acid or transgene of interest to titrate expression of the transgene to a desired level.

[00412] The cccDNA vector nucleic acid(s) are administered in sufficient quantities to transfect cells of the desired tissue and to ensure sufficient levels of gene transfer and expression without undesirable side effects. Conventional and pharmaceutically acceptable routes of administration include, but are not limited to, intravenous (eg, in a liposomal formulation), direct delivery to an organ of choice (eg, intraportal delivery to the liver), intramuscular, and other parenteral routes of administration. Routes of administration may be combined if desired.

[00413] Delivery of a closed-end DNA vector (eg, cDNA vector) is not limited to delivery of replacement genes. For example, conventionally produced (e.g., cell-based or synthetic closed-end DNA vectors) (e.g., cccDNA vectors) as described herein can be used in conjunction with other delivery systems designed to to provide some of the gene therapy. One non-limiting example of a system that can be combined with synthetically produced cccDNA vectors in accordance with the present invention includes systems that individually deliver one or more cofactors or immunosuppressants for efficient gene expression of the transgene.

[00414] The invention also provides a method of treating a disease in a subject, comprising administering to a target cell in need thereof (particularly a muscle cell or tissue) of the subject a therapeutically effective amount of a cccDNA vector, optionally with a pharmaceutically acceptable carrier. Although a cccDNA vector for controlled transgene expression can be introduced along with a carrier, such a carrier is not necessary. The selected cccDNA vector contains a nucleotide sequence of interest useful for treating a disease. In particular, the cccDNA vector may contain a desired exogenous DNA sequence operably linked to control elements capable of directing transcription of the desired polypeptide, protein, or oligonucleotide encoded by the exogenous DNA sequence when administered to a subject. The cccDNA vector can be introduced by any suitable route as described above and elsewhere in this specification.

[00415] The compositions and vectors presented herein can be used to deliver a transgene for a variety of purposes. In some embodiments, the transgene encodes a protein or functional RNA that is intended to be used for research purposes, for example, to create a somatic model of a transgenic animal carrying the transgene, for example, to study the function of the transgene product. In another example, the transgene encodes a protein or functional RNA that is intended to be used to create an animal model of a disease. In some embodiments, the transgene encodes one or more peptides, polypeptides, or proteins that are useful for treating or preventing disease states in a mammalian subject. The transgene may be transferred to a patient (eg, expressed in the patient) in an amount sufficient to treat a disease associated with decreased expression, absence of expression, or dysfunction of the gene.

[00416] In principle, an expression cassette may include a nucleic acid or any transgene encoding a protein or polypeptide that is either reduced or absent due to mutation, or provides a therapeutic benefit when overexpressed, and is considered to be within the scope of the invention. Preferably, no bacterial DNA other than as an insertion is present, and preferably no bacterial DNA is present in the cccDNA compositions of the present invention.

[00417] The cccDNA vector for controlled transgene expression is not limited to one kind of cccDNA vector. In fact, in another aspect, multiple cccDNA vectors containing different transgenes or the same transgene but operably linked to different promoters or cis-regulatory elements can be delivered simultaneously or sequentially to a target cell, tissue, organ or subject. Thus, this strategy could enable gene therapy or the delivery of multiple genes simultaneously. It is also possible to house different parts of the transgene in separate cccDNA vectors (e.g., different domains and/or cofactors required for transgene functionality), which can be introduced simultaneously or at different times, and which can be regulated separately, thereby adding an additional level of expression control transgene Delivery can also be done multiple times and, importantly for gene therapy in a clinical setting, with subsequent increases or decreases in doses given the lack of host immune response against the capsid due to the absence of the viral capsid. It is expected that no reaction against the capsid will occur due to the absence of the capsid.

[00418] The invention also provides a method of treating a disease in a subject, comprising administering to a target cell in need thereof (particularly a muscle cell or tissue) of the subject a therapeutically effective amount of a cccDNA vector as described herein, optionally with a pharmaceutically acceptable carrier. Although the cccDNA vector can be introduced along with a carrier, such a carrier is not necessary. The cDNA vector provided in this invention contains a nucleotide sequence of interest useful for treating a disease. In particular, the cccDNA vector may contain a desired exogenous DNA sequence operably linked to control elements capable of directing transcription of the desired polypeptide, protein, or oligonucleotide encoded by the exogenous DNA sequence when administered to a subject. The cccDNA vector for controlled transgene expression can be introduced by any suitable route, as described above and elsewhere in this document.

XI. Treatment options

[00419] The technology described herein also demonstrates methods for producing, as well as methods for using, the disclosed cccDNA vectors in various ways, including, for example, applications, methodologies, diagnostic procedures and/or gene therapy regimens ex situ, in vitro and in vivo.

[00420] Provided herein is a method of treating a disease or disorder in a subject, comprising administering to a target cell in need thereof (e.g., a muscle cell or tissue, or other type of affected cell) of the subject a therapeutically effective amount of a cccDNA vector, optionally with a pharmaceutical acceptable medium. Although the cccDNA vector can be introduced along with a carrier, such a carrier is not necessary. The cDNA vector provided in this invention contains a nucleotide sequence of interest useful for treating a disease. In particular, the cccDNA vector may contain a desired exogenous DNA sequence operably linked to control elements capable of directing transcription of the desired polypeptide, protein, or oligonucleotide encoded by the exogenous DNA sequence when administered to a subject. The cccDNA vector for controlled transgene expression can be introduced by any suitable route, as described above and elsewhere in this document.

[00421] Disclosed herein are compositions and formulations of cccDNA vectors that include one or more ccDNA vectors of this invention along with one or more pharmaceutically acceptable buffers, diluents, or excipients. Such compositions may be included in one or more diagnostic or therapeutic kits for diagnosing, preventing, treating, or alleviating one or more symptoms of a disease, lesion, disorder, injury, or dysfunction. In one aspect, the disease, lesion, disorder, injury, or dysfunction is a disease, lesion, disorder, injury, or dysfunction in a human.

[00422] Another aspect of the technology described herein provides a method of administering to a subject in need thereof a diagnostically or therapeutically effective amount of a cccDNA vector, the method of introducing into a cell, tissue or organ of the subject in need thereof an amount of the cccDNA vector, as described in this document; performed over a period of time allowing efficient expression of the transgene from the cccDNA vector, thereby providing the subject with a diagnostically or therapeutically effective amount of the protein, peptide, nucleic acid expressed by the cccDNA vector. In an additional aspect, the subject is a person.

[00423] Another aspect of the technology described herein provides a method for diagnosing, preventing, treating, or alleviating at least one or more symptoms of a disease, disorder, dysfunction, lesion, abnormal condition, or injury in a subject. In general, the method includes at least the step of administering to a subject in need thereof one or more disclosed cccDNA vectors, in an amount and for a period of time sufficient to diagnose, prevent, treat, or alleviate one or more symptoms of the disease, disorder, dysfunction , lesion, abnormal condition, or injury in the subject. In an additional aspect, the subject is a person.

[00424] Another aspect is the use of a cccDNA vector for controlled expression of a transgene as a tool for treating or alleviating one or more symptoms of a disease or disease state. There are a number of inherited diseases for which defective genes are known, and which generally fall into two classes: deficiency states, usually enzyme deficiencies, which are generally inherited in a recessive manner, and imbalanced conditions, which may involve regulatory or structural proteins , and which are usually, but not always, inherited in a dominant manner. For diseases with deficiency conditions, cccDNA vectors can be used to deliver transgenes to introduce the normal gene into diseased tissues for replacement therapy, and, in some embodiments, to create animal models of the disease using antisense mutations. In the case of disease states of imbalance, cccDNA vectors can be used to create a disease state in a model system, which can then be used to counteract said disease state. Thus, the cccDNA vectors and methods disclosed herein enable the treatment of genetic diseases. According to this document, treatment of a disease state is carried out by partially or completely eliminating the deficiency or imbalance that causes the disease or increases its severity.

A. Host cells

[00425] In some embodiments, a cccDNA vector for controlled transgene expression delivers the transgene to a host cell of the present invention. In some embodiments, the host cell of the present invention is a human host cell, including, for example, blood cells, stem cells, hematopoietic cells, CD34 ⁺ cells, liver cells, cancer cells, vascular cells, muscle cells, pancreatic cells, nerve cells, ocular or retinal cells, epithelial or endothelial cells, dendritic cells, fibroblasts or any other mammalian cells, including, without limitation, hepatocytes (i.e. liver cells), lung cells, heart cells, pancreatic cells, cells intestine, diaphragm cells, renal cells (ie, kidney cells), nerve cells, blood cells, bone marrow cells, or any one or more selected tissues of the subject for whom gene therapy is to be performed. In one aspect, the host cell of this invention is a human host cell.

[00426] This invention also relates to recombinant host cells, as mentioned above, including cccDNA vectors as described herein. Thus, multiple host cells can be used depending on the purpose, as will be apparent to one skilled in the art. A construct or cDNA vector for controlled transgene expression, including a donor sequence, is introduced into a host cell such that the donor sequence is maintained as a chromosomally integrated element, as previously described. The term "host cell" covers any progeny of a parent cell that is not identical to the parent cell due to mutations occurring during replication. The choice of host cell will largely depend on the donor sequence and its source. The host cell may also be a eukaryote, such as a mammalian, insect, plant or fungal cell. In one embodiment, the host cell is a human cell (eg, primary cells, stem cells, or an immortalized cell line). In some embodiments, a cccDNA vector may be introduced into a host cell ex vivo for controlled expression of the transgene, followed by delivery to the subject after the gene therapy event. The host cell may be any type of cell, such as a somatic cell or a stem cell, an induced pluripotent stem cell, or a blood cell, such as a T cell or B cell, or a bone marrow cell. In certain embodiments, the host cell is an allogeneic cell. For example, T cell genome engineering is useful for cancer immunotherapies, disease modulation such as HIV therapy (eg, receptor knockout such as CXCR4 and CCR5), and immunodeficiency therapies. The targets of immunotherapy may be MHC (major histocompatibility complex) receptors on B cells. In some embodiments, genetically modified host cells, such as bone marrow stem cells, such as CD34 ⁺ cells, or induced pluripotent stem cells, can be transplanted back into the patient to express the therapeutic protein.

B. Typical transgenes and diseases treatable with cccDNA vectors

[00427] cccDNA vectors are also useful for correcting a defective gene. As a non-limiting example, the Duchenne muscular dystrophy DMD gene can be delivered using cccDNA vectors as described herein.

[00428] An cccDNA vector for controlled transgene expression or a composition thereof can be used to treat any inherited disease. As a non-limiting example, a cccDNA vector or composition thereof may be used, for example, to treat transthyretin amyloidosis (ATTR), an orphan disease in which a mutant protein misfolds and aggregates in the nerves, heart, gastrointestinal system, etc. . This document provides that the disease can be treated by deletion of the mutant disease gene (mutTTR) using cccDNA vector systems described herein. Such treatment of hereditary diseases can stop the progression of the disease and can lead to regression of the established disease or a reduction of at least one symptom of the disease by at least 10%.

[00429] In another embodiment, a cccDNA vector for controlled transgene expression can be used to treat ornithine transcarbamylase deficiency (OTC deficiency), hyperammonemia, or other urea cycle disorders that impair the ability of a newborn or child to detoxify ammonia. As with all innate metabolic diseases, the present invention contemplates that even partial restoration of enzyme activity relative to wild-type controls (e.g., at least 20%, at least 30%, at least 40%, at least 50 %, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% or at least 99%) may be sufficient to reduce at least one symptom of OTC and /or improving the quality of life for a subject with OTC deficiency. In one embodiment, a nucleic acid encoding OTC can be inserted downstream of an endogenous albumin promoter to replace the protein in vivo.

[00430] In another embodiment, a cccDNA vector for controlled transgene expression can be used to treat phenylketonuria (PKU) by delivering a nucleic acid sequence encoding the enzyme phenylalanine hydroxylase to reduce the accumulation of dietary phenylalanine, which can be toxic to patients suffering from phenylketonuria. As with all innate metabolic diseases, the present invention contemplates that even partial restoration of enzyme activity relative to wild-type controls (e.g., at least 20%, at least 30%, at least 40%, at least 50 %, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% or at least 99%) may be sufficient to reduce at least one symptom of phenylketonuria and /or improving the quality of life for a subject with PKU. In one embodiment, a nucleic acid encoding a phenylalanine hydroxylase can be inserted downstream of an endogenous albumin promoter to replace the protein in vivo.

[00431] In another embodiment, a cccDNA vector for controlled transgene expression can be used to treat glycogen storage disease (GSD) by delivering a nucleic acid sequence encoding an enzyme to correct aberrant glycogen synthesis or breakdown in subjects with GSD. Non-limiting examples of enzymes that can be delivered or expressed using cccDNA vectors and methods described herein include glycogen synthase, glucose-6-phosphatase, acid alpha-glucosidase, glycogen debranching enzyme, glycogen branching enzyme, muscle glycogen phosphorylase, glycogen phosphorylase liver, muscle phosphofructokinase, phosphorylase kinase, glucose transporter-2 (GLUT-2), aldolase A, beta-enolase, phosphoglucomutase-1 (PGM-1) and glycogenin-1. As with all innate metabolic diseases, the present invention contemplates that even partial restoration of enzyme activity relative to wild-type controls (e.g., at least 20%, at least 30%, at least 40%, at least 50 %, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99%) may be sufficient to reduce at least one symptom of LBP and /or improving the quality of life for the subject with GNP. In one embodiment, a nucleic acid encoding an enzyme for correcting aberrant glycogen storage can be inserted downstream of an endogenous albumin promoter to replace the protein in vivo.

[00432] The cccDNA vectors described herein are also contemplated for use in the treatment of any of Leber's congenital amaurosis (LCA), polyglutamine diseases including polyQ repeats, and alpha-1 antitrypsin deficiency (A1AT). Leber congenital amaurosis is a rare, blinding congenital eye disease that can be caused by a mutation in one of the following genes: GUCY2D, RPE65, SPATA7, AIPL1, LCA5, RPGRIP1, CRX, CRB1, NMNAT1, CEP290, IMPDH1, RD3, RDH12 , LRAT, TULP1, KCNJ13, GDF6 and/or PRPH2. The present invention provides that cccDNA vectors and the compositions and methods described herein can be adapted to deliver one or more VAL-related genes to correct an error in the gene(s) responsible for the symptoms of VAL. Polyglutamine diseases include, but are not limited to: dentarubral atrophy, Huntington's disease, spinal and bulbar muscular atrophy, and spinocerebellar ataxia types 1, 2, 3 (also known as Machado-Joseph disease), 6, 7, and 17. A1AT deficiency is a genetic disorder that causes a defect in the production of alpha-1 antitrypsin, resulting in decreased enzyme activity in the blood and lungs, which in turn can lead to emphysema or chronic obstructive pulmonary disease in affected subjects. This document specifically provides for the treatment of a subject with A1AT deficiency using cccDNA vectors or compositions thereof, as described herein. Provided herein, a cccDNA vector for controlled transgene expression containing a nucleic acid encoding a desired protein for the treatment of VAL, polyglutamine diseases or A1AT deficiency can be administered to a subject in need of treatment.

[00433] In other embodiments, compositions containing a cccDNA vector for controlled transgene expression, as described herein, can be used to deliver, among other things, a viral sequence, a pathogen sequence, a chromosomal sequence, a translocation junction (e.g., a translocation cancer-associated), non-coding RNA gene or disease-associated RNA sequence gene.

[00434] Any nucleic acid or target gene of interest can be delivered or expressed using a cccDNA vector for controlled transgene expression as described herein. Target nucleic acids and genes include, but are not limited to, polypeptide-encoding nucleic acids or non-coding nucleic acids (e.g., RNAi, mature microRNAs, etc.), preferably therapeutic (e.g., for medical, diagnostic or veterinary use) or immunogenic (for example, for vaccines) polypeptides. In some embodiments, the target nucleic acids or genes targeted by the cccDNA vectors described herein encode one or more polypeptides, peptides, ribozymes, peptide nucleic acids, siRNA, RNAi, antisense oligonucleotides, antisense polynucleotides, antibodies, antigen binding fragments , or any combination thereof.

[00435] In particular, a target gene or transgene for expression by a cccDNA vector for controlled transgene expression as disclosed herein may encode, for example, without limitation, protein(s), polypeptide(s), peptide(s), enzyme (s), antibodies, antigen-binding fragments, as well as variants and/or active fragments thereof, for use in the treatment, prevention and/or alleviation of one or more symptoms of a disease, dysfunction, lesion and/or disorder. In one aspect, the disease, dysfunction, injury, impairment, and/or disorder is a human disease, dysfunction, injury, impairment, and/or disorder.

[00436] An expression cassette may also encode polypeptides, sense or antisense oligonucleotides, or RNAs (coding or non-coding; e.g., miRNAs, short hairpin RNAs, microRNAs, and antisense analogues thereof (e.g., antagomir (antagoMiR)). Expression cassettes may include an exogenous sequence , encoding a reporter protein to be used for experimental or diagnostic purposes, such as β-lactamase, β-galactosidase (LacZ), alkaline phosphatase, thymidine kinase, green fluorescent protein (GFP), chloramphenicol acetyltransferase (ChAT), luciferase and others well known in this field of technology.

[00437] The sequences present in the expression cassette, a cccDNA vector expression construct for controlled transgene expression described herein, can be codon-optimized for the target host cell. As used herein, the term “codon-optimized” or “codon optimization” refers to the process of modifying a nucleic acid sequence to enhance expression in cells of a vertebrate animal of interest, such as a mouse or human, by replacing at least one, more than one , or a significant number of codons from a native sequence (e.g., a prokaryotic sequence) to codons that are more frequently or most frequently used in the genes of that vertebrate. Different species exhibit a particular affinity for certain codons of a particular amino acid. Typically, codon optimization does not change the amino acid sequence of the original translated protein. Optimized codons can be determined using, for example, the Gene Forge® custom gene synthesis and codon optimization platform from Aptagen (Aptagen, Inc., 2190 Fox Mill Rd. Suite 300, Herndon, Va. 20171) or other publicly available database.

[00438] Many organisms exhibit a tendency to use certain codons when encoding the insertion of a particular amino acid into a growing peptide chain. Codon preference or codon ambiguity, differences in codon usage between organisms, is due to the degeneracy of the genetic code and is well documented for many organisms. Codon bias often correlates with the translation efficiency of messenger RNA (mRNA), which in turn is thought to depend, in part, on the properties of the codons being translated and the availability of certain transfer RNA (tRNA) molecules. The predominance of selected tRNAs in a cell typically reflects the codons most frequently used in peptide synthesis. Accordingly, genes can be tailored for optimal gene expression in a given organism based on codon optimization.

[00439] Due to the large number of gene sequences available for a wide range of animal, plant and microbial species, relative frequencies of codon usage can be calculated (Nakamura Y., et al. “Codon usage tabulated from the international DNA sequence databases: status for the year 2000” Nucl Acids Res 28:292 (2000)).

[00440] As noted herein, the cccDNA vector for controlled transgene expression disclosed herein may encode a protein or peptide, or a therapeutic nucleic acid sequence, or a therapeutic agent, including, without limitation, one or more agonists, antagonists, anti-apoptotic factors, inhibitors, receptors, cytokines, cytotoxins, erythropoietic agents, glycoproteins, growth factors, growth factor receptors, hormones, hormone receptors, interferons, interleukins, interleukin receptors, nerve growth factors, neuroactive peptides, neuroactive peptide receptors, proteases, protease inhibitors , protein decarboxylases, protein kinases, protein kinase inhibitors, enzymes, receptor binding proteins, transport proteins or one or more of their inhibitors, serotonin receptors or one or more of their uptake inhibitors, serpins, serpin receptors, tumor suppressors, diagnostic molecules, chemotherapeutic agents, cytotoxins , or any combination thereof.

[00441] cccDNA vectors are also useful for stopping gene expression. For example, in one embodiment, a cccDNA vector for controlled transgene expression can be used to express an antisense nucleic acid or functional RNA to induce knockdown of a target gene. As a non-limiting example, expression of the HIV receptors CXCR4 and CCR5 was successfully ablated in primary human T cells, see Schumann et al. (2015), PNAS 112(33): 10437–10442, which is incorporated herein by reference in its entirety. Another target gene for inhibition is PD-1, wherein the cDNA vector may express an inhibitory nucleic acid or RNAi or functional RNA to inhibit PD-1 expression. PD-1 expresses the cell surface immune checkpoint receptor on chronically active T cells seen in malignancy. See Schumann et al. higher.

[00442] In some embodiments, cccDNA vectors are useful for correcting a defective gene by expressing a transgene that targets the diseased gene. Non-limiting examples of diseases or disorders amenable to treatment with the cccDNA vector as described herein are listed, along with their related and associated genes, in Tables A-C of US Patent Publication 2014/0170753, which is incorporated herein by reference in full.

[00443] In alternative embodiments, cccDNA vectors are used to insert an expression cassette for expressing a therapeutic protein or reporter protein into a safe harbor gene, such as an inactive intron. In certain embodiments, a promoterless cassette is inserted into a safe harbor gene. In such embodiments, the promoterless cassette may utilize safe harbor gene regulatory elements (promoters, enhancers, and signal peptides), with a non-limiting example of an insertion at a safe harbor locus being the insertion at the albumin locus described in Blood (2015) 126(15):1777- 1784, which is incorporated herein by reference in its entirety. Insertion into albumin has the advantage of allowing the transgene to be secreted into the blood (see, for example, Example 22). In addition, the genomic safe harbor site can be determined using methods known in the art and described, for example, in Papapetrou, ER & Schambach, A. Molecular Therapy 24(4):678-684 (2016), or Sadelain et al. Nature Reviews Cancer 12:51–58 (2012), the contents of which are incorporated herein by reference in their entirety. This document specifically provides that safe harbor sites in the adeno-associated virus (AAV) genome (eg, the AAVS1 safe harbor site) can be used with the methods and compositions described herein (see, for example, Oceguera-Yanez et al. Methods 101 :43-55 (2016), or Tiyaboonchai, A et al. Stem Cell Res 12(3):630-7 (2014), the contents of which are incorporated by reference in their entirety). For example, the AAVS1 genomic safe harbor site can be used with vectors and cccDNA compositions as described herein for the purposes of hematopoiesis-specific transgene expression and gene silencing in embryonic stem cells (eg, human embryonic stem cells) or induced pluripotent stem cells (iPS -cells). In addition, this document provides that synthetic or commercially available homology-directed repair donor templates for insertion into the AAVS1 safe harbor site (AASV1) on chromosome 19 can be used with vectors or cccDNA compositions as described herein. For example, homology-directed repair templates and guide RNA can be purchased from commercial sources, such as System Biosciences (Palo Alto, Calif.), and cloned into a cccDNA vector.

[00444] In some embodiments, cccDNA vectors are used to express a transgene, or knock out or reduce expression of a target gene in a T cell, for example, to modify the T cell to improve adoptive cell transfer and/or CAR-T therapies (see eg Example 24). In some embodiments, a cccDNA vector for controlled transgene expression, as described herein, can express transgenes that knock out genes. Non-limiting examples of therapeutically relevant T cell knockouts are described in PNAS (2015) 112(33):10437-10442, which is incorporated herein by reference in its entirety.

C. Additional diseases for gene therapy:

[00445] In general, the cccDNA vector for controlled transgene expression described herein can be used to deliver any transgene as described above to treat, prevent, or alleviate symptoms associated with any gene expression disorder. Illustrative examples of disease conditions include, but are not limited to: cystic fibrosis (and other lung diseases), hemophilia A, hemophilia B, thalassemia, anemia and other blood disorders, AIDS, Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, epilepsy and other neurological disorders, cancer, diabetes mellitus, muscular dystrophies (eg, Duchenne, Becker), Hurler's disease, adenosine deaminase deficiency, metabolic defects, degenerative retinal diseases (and other eye diseases), mitochondria (eg, Leber's hereditary optic neuropathy (LEON), syndrome Leia and subacute sclerosing encephalopathy), myopathies (eg, scapulohumeral-facial myopathy (SSFM) and cardiomyopathies), diseases of solid organs (eg, brain, liver, kidneys, heart); and the like. In some embodiments, the cccDNA vectors described herein may be advantageously used in the treatment of individuals with metabolic disorders (eg, ornithine transcarbamylase deficiency).

[00446] In some embodiments, the cccDNA vector for controlled transgene expression described herein can be used to treat, alleviate, and/or prevent a disease or disorder caused by a mutation in a gene or gene product. Typical examples of diseases or disorders that can be treated using cccDNA vectors include, but are not limited to, metabolic diseases or disorders (eg, Fabry disease, Gaucher disease, phenylketonuria (PKU), glycogen storage disease); urea cycle diseases or disorders (eg, ornithine transcarbamylase (OTC) deficiency); lysosomal storage diseases or disorders (eg, metachromatic leukodystrophy (MLD), mucopolysaccharidosis type II (MPSII; Gunther syndrome)); liver diseases or disorders (eg, progressive familial intrahepatic cholestasis (PFIC); blood diseases or disorders (eg, hemophilia (A and B), thalassemia, and anemia); cancers and tumors, and genetic diseases or disorders (eg, cystic fibrosis) .

[00447] In yet another aspect, a cccDNA vector for controlled transgene expression, as described herein, can be used to deliver a heterologous nucleotide sequence in situations where regulation of the level of expression of a transgene is desired (for example, transgenes encoding hormones or growth factors, as described in this document).

[00448] Accordingly, in some embodiments, the cccDNA vector for controlled transgene expression described herein can be used to correct abnormal levels and/or function of a gene product (eg, absence or defect of a protein) that lead to a disease or disorder. Said cccDNA vector may produce a functional protein and/or modify levels of said protein to alleviate or reduce symptoms resulting from a particular disease or disorder, or to provide benefit to a particular disease or disorder caused by the absence or defect of the protein. For example, treatment of TK deficiency can be achieved by producing a functional TK enzyme; treatment of hemophilia A and B can be achieved by modifying the levels of factor VIII, factor IX and factor X; treatment of phenylketonuria can be achieved by modifying levels of the enzyme phenylalanine hydroxylase; treatment of Fabry disease or Gaucher disease can be achieved by producing functional alpha-galactosidase or beta-glucocerebrosidase, respectively; treatment of MLD or MPSII can be achieved by producing functional arylsulfatase A or iduronate-2-sulfatase, respectively; treatment of cystic fibrosis can be carried out by producing a functional regulator of transmembrane conductance in cystic fibrosis; treatment of glycogen disease can be carried out by restoring the function of the functional enzyme glucose-6-phosphatase; and treatment of PFIC can be achieved by producing functional genes ATP8B1, ABCB11, ABCB4 or TJP2.

[00449] In alternative embodiments, the cccDNA vectors described herein can be used to provide antisense nucleic acid to a cell in vitro or in vivo. For example, if the transgene is an RNA interference (RNAi) molecule, expression of an antisense nucleic acid or RNAi in a target cell reduces the level of expression of a particular protein by that cell. Accordingly, transgenes, which are RNAi molecules or antisense nucleic acids, can be introduced to reduce the expression of a particular protein in a subject in need thereof. Antisense nucleic acids can also be introduced into cells in vitro to regulate cell physiology, for example to optimize cell or tissue culture systems.

[00450] In some embodiments, representative examples of transgenes encoded by a cccDNA vector for controlled transgene expression include, but are not limited to: X, lysosomal enzymes (e.g., hexosaminidase A associated with Tay-Sachs disease or iduronate sulfatase associated with Gunther syndrome). MPS II), erythropoietin, angiostatin, endostatin, superoxide dismutase, globin, leptin, catalase, tyrosine hydroxylase, and cytokines (for example, interferon-α, interferon-β, interferon-γ, interleukin-2, interleukin-4, interleukin-12, granulocyte-macrophage colony-stimulating factor, lymphotoxin, etc.), peptide growth factors and hormones (for example, somatotropin, insulin, insulin-like growth factors 1 and 2, platelet-derived growth factor (TRF), epidermal growth factor (EGF), fibroblast growth factor (NGF), nerve growth factor (NGF), neurotrophic factor-3 and 4, brain-derived neurotrophic factor (BDNF), glial growth factor (GGF), transforming growth factors α and β; etc.), receptors (for example, tumor necrosis factor receptor).

[00451] In some exemplary embodiments, the transgene encodes a monoclonal antibody specific for one or more desired targets. Exemplary cccDNA vectors for the controlled expression of antibodies and fusion proteins in the methods described herein are disclosed in international application PCT/US19/18016, filed February 14, 2019, which is incorporated herein by reference in its entirety.

[00452] In some example embodiments, the cDNA vector encodes more than one transgene. In some exemplary embodiments, the transgene encodes a fusion protein comprising two different polypeptides of interest. In some embodiments, the transgene encodes an antibody, including a full-length antibody or antibody fragment, as defined herein. In some embodiments, said antibody is an antigen binding domain or immunoglobulin variable domain sequence as defined herein. Other illustrative examples of transgene sequences encode suicide gene products (thymidine kinase, cytosine deaminase, diphtheria toxin, cytochrome P450, deoxycytidine kinase, and tumor necrosis factor), proteins that confer resistance to drugs used in anticancer therapy, and tumor suppressor gene products.

[00453] In an exemplary embodiment, a transgene expressed by a cccDNA vector for controlled transgene expression may be used to treat muscular dystrophy in a subject in need thereof, the method comprising: administering an effective amount of the cccDNA vector described for treating, ameliorating, or preventing herein, wherein said cccDNA vector contains a heterologous nucleic acid encoding dystrophin, minidystrophin, microdystrophin, myostatin propeptide, follistatin, soluble activin type II receptor, IGF-1 (insulin-like growth factor), anti-inflammatory polypeptides such as dominant mutant I- kappa-B, sarcospan, utrophin, microdystrophin, laminin-α2, α-sarcoglycan, β-sarcoglycan, γ-sarcoglycan, δ-sarcoglycan, IGF-1, antibody or antibody fragment against myostatin or myostatin propeptide, and/or RNAi against myostatin . In specific embodiments, the cccDNA vector can be introduced into skeletal, diaphragmatic and/or cardiac muscle, as described elsewhere in this document.

[00454] In some embodiments, said cccDNA vector for controlled transgene expression may be used to deliver the transgene to skeletal, cardiac, or diaphragmatic muscle to produce a polypeptide (e.g., enzyme) or functional RNA (e.g., RNAi, microRNA, antisense RNA), that normally circulate in the blood, or for systemic delivery to other tissues for the purpose of treating, ameliorating and/or preventing a disorder (eg, metabolic disorder such as diabetes (eg, insulin), hemophilia (eg, VIII), mucopolysaccharidosis disorder (eg , Sly's syndrome, Hurler's syndrome, Scheie's syndrome, Hurler-Scheie's syndrome, Gunther's syndrome, Sanfilippo's syndrome A, B, C, D, Morquio's syndrome, Maroteaux-Lamy syndrome, etc.) or a lysosomal storage disorder (such as Gaucher's disease [glucocerebrosidase], Pompe disease [lysosomal acid alpha-glucosidase] or Fabry disease [alpha-galactosidase A]) or glycogen storage disorders (such as Pompe disease [lysosomal acid alpha-glucosidase]). Other suitable proteins for the treatment, amelioration and/or prevention of metabolic disorders are described above.

[00455] In other embodiments, a cccDNA vector for controlled transgene expression, as described herein, can be used to deliver a transgene in a method of treating, alleviating, and/or preventing a metabolic disorder in a subject in need thereof. Illustrative examples of metabolic disorders and transgenes encoding polypeptides are described herein. Optionally, the polypeptide is secreted (eg, a polypeptide that is a secreted polypeptide in its natural state, or modified to provide secretion, for example, by forming a functional link with a secretory signal sequence, as known to those skilled in the art).

[00456] Another aspect of the present invention relates to a method of treating, ameliorating and/or preventing congenital heart failure or PAD in a subject in need thereof, the method comprising administering a cccDNA vector for controlled expression of a transgene, as described herein, to the subject - a mammal, wherein said cDNA vector contains a transgene encoding, for example, Ca ²⁺ -ATPase of the sarcoplasmic endoreticulum (SERCA2a), an angiogenic factor, an inhibitor of phosphatase I (I-1), RNAi to phospholamban; phospholamban inhibitory or dominant negative molecule such as phospholamban S16E, zinc finger protein that regulates the phospholamban gene, β2-adrenergic receptor, beta-2-adrenergic receptor kinase (BARK), PI3-kinase, calsarcan, beta inhibitor β-adrenergic receptor kinase (βARKct), protein phosphatase 1 inhibitor 1, S100A1, parvalbumin, adenylyl cyclase type 6, a molecule that knocks down G protein-coupled receptor kinase type 2, such as truncated constitutively active βARKct, Pim-1, PGC-1α , SOD-1, SOD-2, EC-SOD, kallikrein, HIF, thymosin-β4, mir-1, mir-133, mir-206 and/or mir-208.

[00457] The cccDNA vectors described herein can be administered to the lungs of a subject by any suitable means, optionally by administering an aerosol suspension of inhalable particles containing the cccDNA vectors that the subject inhales. These inhalable particles can be liquid or solid. Aerosols of liquid particles containing cccDNA vectors can be produced by any suitable means, for example, using a pneumatic aerosol nebulizer or an ultrasonic nebulizer, as known to those skilled in the art. See, for example, US Pat. No. 4,501,729. Particulate aerosols containing cccDNA vectors can similarly be produced using any particulate drug aerosol generator using techniques known in the pharmaceutical field.

[00458] In some embodiments, these cDNA vectors can be introduced into CNS tissue (eg, brain, eye). In specific embodiments, cccDNA vectors, as described herein, can be administered to treat, alleviate, or prevent CNS diseases, including genetic disorders, neurodegenerative disorders, psychiatric disorders, and tumors. Illustrative examples of CNS diseases include, but are not limited to, Alzheimer's disease, Parkinson's disease, Huntington's disease, Canavan's disease, Leigh's disease, Refsum's disease, Tourette's syndrome, primary lateral sclerosis, amyotrophic lateral sclerosis, progressive muscular atrophy, Pick's disease, muscular dystrophy, multiple sclerosis , myasthenia gravis, Binswanger's disease, injury due to spinal cord or head injury, Tay-Sachs disease, Lesch-Nyhan disease, epilepsy, cerebral infarction, mental disorders, including mood disorders (eg, depression, bipolar affective disorder, persistent affective disorder disorder, secondary mood disorder), schizophrenia, drug or drug dependence (e.g., alcoholism and other substance abuse disorders), neuroses (e.g., anxiety, obsessive disorder, somatoform disorder, dissociative disorder, grief, postpartum depression), psychosis (e.g. hallucinations and delusions), dementia, paranoia, attention deficit disorder, psychosexual disorders, sleep disorders, pain-related disorders, eating or weight-related disorders (eg, obesity, cachexia, anorexia nervosa and bulimia), and cancers and tumors of the central nervous system (for example, pituitary tumors).

[00459] Ocular disorders that may be treated, ameliorated, or prevented by the cccDNA vectors of the invention include ophthalmic disorders affecting the retina, posterior tract, and optic nerve (e.g., retinitis pigmentosa, diabetic retinopathy and other retinal degenerative diseases, uveitis, age-related macular degeneration, glaucoma). Many ophthalmic diseases and disorders are associated with one or more of three types of features: (1) angiogenesis, (2) inflammation, and (3) degeneration. In some embodiments, a cccDNA vector for controlled transgene expression, as described herein, can be used to deliver antiangiogenic factors; anti-inflammatory factors; factors that slow down cell degeneration, promote cell preservation or promote cell growth, and combinations of the above. For example, diabetic retinopathy is characterized by angiogenesis. Diabetic retinopathy can be treated by delivering one or more antiangiogenic factors either intraocularly (eg, into the vitreous) or periocularly (eg, into the sub-Tenon's space). One or more neurotrophic factors may be delivered together, either intraocularly (eg, intravitreally) or periocularly. Additional ocular diseases that may be treated, ameliorated, or prevented by the cccDNA vectors of this invention include geographic atrophy, vascular or wet macular degeneration, Stargardt disease, Leber congenital amaurosis (LCA), Usher syndrome, pseudoxanthoma elasticum (PEC), X-linked retinitis pigmentosa (XLRP), X-linked retinal dissection (XLRS), choroideremia, Leber congenital optic neuropathy (LCON), achromatopsia, rod-cone dystrophy, Fuchs' corneal endothelial dystrophy, diabetic macular edema; eye cancer and tumors.

[00460] In some embodiments, inflammatory ocular diseases or disorders (eg, uveitis) can be treated, alleviated, or prevented using the cccDNA vectors of this invention. One or more anti-inflammatory factors can be expressed by intraocular (eg, vitreous or anterior chamber) administration of a cccDNA vector for controlled transgene expression, as described herein. In other embodiments, eye diseases or disorders characterized by retinal degeneration (eg, retinitis pigmentosa) can be treated, alleviated, or prevented using the cccDNA vectors of the present invention. To treat such diseases associated with retinal degeneration, intraocular (eg, vitreal) administration of a cccDNA vector described herein encoding one or more neurotrophic factors can be used. In some embodiments, diseases or disorders that include both angiogenesis and retinal degeneration (eg, age-related macular degeneration) can be treated using the cccDNA vectors of the invention. Treatment of age-related macular degeneration can be achieved by introducing a cccDNA vector, as described herein, encoding one or more neurotrophic factors intraocularly (eg, into the vitreous), and/or encoding one or more antiangiogenic factors, intraocularly or periocularly (eg, into the region of sub-Tenon space). Glaucoma is characterized by increased eye pressure and loss of retinal ganglion cells. Treatment of glaucoma involves the administration of one or more neuroprotective agents that protect cells from excitotoxic damage using a cccDNA vector as described herein. Accordingly, such agents include N-methyl-D-aspartate (NMDA) antagonists, cytokines and neurotrophic factors, which can be delivered intraocularly, optionally intravitreally, using a cccDNA vector as described herein.

[00461] In other embodiments, a cccDNA vector for controlled transgene expression, as described herein, can be used to treat seizures, for example, to reduce the occurrence, frequency, or severity of seizures. The effectiveness of a therapeutic treatment for seizures can be assessed based on behavioral signs ( eg, trembling, eye or mouth tics) and/or electrographic means (most seizures are characterized by distinctive electrographic abnormalities). Thus, the cccDNA vector for controlled transgene expression described herein can also be used to treat epilepsy characterized by multiple seizures over time. In one exemplary embodiment, somatostatin (or an active fragment thereof) is administered to the brain using a cccDNA vector as described herein to treat a pituitary tumor. In this embodiment, a cccDNA vector, as described herein, encoding somatostatin (or an active fragment thereof) is introduced into the pituitary gland by microinfusion. Likewise, this treatment can be used to treat acromegaly (abnormal secretion of growth hormone by the pituitary gland). The nucleic acid sequence (eg, GenBank Accession No. J00306) and amino acid sequence (eg, GenBank Accession No. P01166; contains the processed active peptides somatostatin-28 and somatostatin-14) of somatostatins are known to those skilled in the art. In specific embodiments, the cDNA vector may encode a transgene that contains a secretory signal, as described in US Pat. No. 7,071,172.

[00462] Another aspect of the invention relates to the use of a cccDNA vector for controlled transgene expression, as described herein, to produce antisense RNA, RNAi, or other functional RNA (eg, a ribozyme) for systemic delivery to a subject in vivo. Accordingly, in some embodiments, the cccDNA vector may contain a transgene that encodes an antisense nucleic acid, a ribozyme (e.g., as described in US Pat. No. 5,877,022), RNAs that affect spliceosome-mediated trans-splicing (see Puttaraju et al. , (1999) Nature Biotech. 17:246; US Patent No. 6013487; US Patent No. 6083702), interfering RNAs (RNAi) mediating gene silencing (see Sharp et al., (2000) Science 287:2431), or others untranslated RNAs such as guide RNAs (Gorman et al., (1998) Proc. Nat. Acad. Sci. USA 95:4929; US Pat. No. 5,869,248 to Yuan et al.), and the like.

[00463] In some embodiments, the cccDNA vector for controlled transgene expression may also further comprise a transgene encoding a reporter polypeptide (e.g., an enzyme such as green fluorescent protein or alkaline phosphatase). In some embodiments, the transgene that encodes a reporter protein useful for experimental or diagnostic purposes is selected from any of: β-lactamase, β-galactosidase (LacZ), alkaline phosphatase, thymidine kinase, green fluorescent protein (GFP), chloramphenicol acetyltransferase (ChAT) , luciferase and others well known in the art. In some aspects, cccDNA vectors containing a transgene encoding a reporter polypeptide can be used for diagnostic purposes or as markers of cccDNA vector activity in a subject to which they are administered.

[00464] In some embodiments, the cccDNA vector for controlled expression of a transgene may contain a transgene or heterologous nucleotide sequence that has homology to and recombines with a locus on the host chromosome. This approach can be used to correct a genetic defect in a host cell.

[00465] In some embodiments, the cDNA vector for controlled transgene expression may contain a transgene that can be used to express an immunogenic polypeptide in a subject, for example, for vaccination. The transgene may encode any immunogen of interest known in the art, including, without limitation, human immunodeficiency virus immunogens, influenza virus immunogens, gag proteins, tumor antigens, cancer antigens, bacterial antigens, viral antigens, and the like.

[00466] D. Testing for Successful Gene Expression Using a cccDNA Vector

[00467] Assays well known in the art can be used to test the efficiency of gene delivery using a cccDNA vector, and can be performed in both in vitro and in vivo models. One skilled in the art can evaluate knockin or knockout of a desired transgene using cccDNA by measuring the mRNA and protein levels of the desired transgene (eg, reverse transcription-PCR, Western blotting, and enzyme-linked immunosorbent assay (ELISA) techniques). Nucleic acid changes by cccDNA (eg, point mutations or deletions of DNA regions) can be assessed by deep sequencing of the target genomic DNA. In one embodiment, the cccDNA contains a reporter protein that can be used to assess the expression of a desired transgene, for example, by examining the expression of the reporter protein by fluorescence microscopy or using a fluorescent plate reader. For in vivo applications, protein function assays can be used to test the functionality of a given gene and/or gene product to determine the success of gene expression. For example, it is envisaged that a point mutation in the CFTR gene of the cystic fibrosis transmembrane conductance regulator (CFTR), which inhibits the ability of CFTR to move anions (eg, Cl ^- ) through the anion channel, can be corrected by delivering to the subject a functional (i.e., unmutated) CFTR gene with using a cccDNA vector. Once the cDNA vector is introduced, one skilled in the art can assess the ability of anions to move through the anion channel to determine the success of delivery and expression of the CFTR gene. One skilled in the art will be able to determine the best test to measure protein functionality in vitro or in vivo.

[00468] It is provided herein that the effects of gene expression of a transgene from a cccDNA vector in a cell or subject may last for at least 1 month, at least 2 months, at least 3 months, at least four months, at least 5 months, at least six months, at least 10 months, at least 12 months, at least 18 months, at least 2 years, at least 5 years, at least 10 years, at least at least 20 years, or may be permanent.

[00469] In some embodiments, a transgene in an expression cassette, expression construct, or cDNA vector described herein may be codon-optimized for a host cell. As used herein, the term “codon-optimized” or “codon optimization” refers to the process of modifying a nucleic acid sequence to enhance expression in vertebrate cells of interest, e.g., mouse or human (e.g., humanized), by replacing at least one more than one or a significant number of codons from a native sequence (eg, a prokaryotic sequence) to the codons that are most or most frequently used in the genes of a given vertebrate. Different species exhibit a particular affinity for certain codons of a particular amino acid. Typically, codon optimization does not change the amino acid sequence of the original translated protein. Optimized codons can be determined using, for example, the Gene Forge® custom gene synthesis and codon optimization platform from Aptagen (Aptagen, Inc.) or other publicly available database.

XII. Introduction

[00470] Examples of methods of administering a cccDNA vector for controlled expression of a transgene disclosed herein include oral, rectal, transmucosal, intranasal, inhalation (e.g., aerosol), buccal (e.g., sublingual), vaginal, intrathecal, intraocular, transdermal , intraendothelial, in utero (or in ovo), parenteral (e.g., intravenous, subcutaneous, intradermal, intracranial, intramuscular [including skeletal, diaphragmatic, and/or cardiac], intrapleural, intracerebral, and intraarticular), topical (for example, on the surface of the skin and mucous membranes, including the surface of the respiratory tract, and percutaneous administration), intralymphatic administration, etc., as well as direct injection into a tissue or organ (for example, into the liver, eye, skeletal muscle, cardiac muscle, diaphragm muscle or brain).

[00471] The cccDNA vector for controlled expression of the transgene may be administered to any site in the body of a subject, including, without limitation, a site selected from the group consisting of brain, skeletal muscle, smooth muscle, heart, diaphragm, respiratory tract epithelium , liver, kidney, spleen, pancreas, skin and eyes. The introduction of the specified cDNA vector for controlled expression of the transgene into the tumor (for example, into or near the tumor or lymph node) can also be carried out. The most appropriate route in any particular case will depend on the nature and severity of the condition being treated, ameliorated and/or prevented, and the nature of the particular cDNA vector used. In addition, cccDNA allows the introduction of more than one transgene in a single vector or in multiple cccDNA vectors (eg, cccDNA cocktail).

[00472] Administration of a cccDNA vector for controlled expression of a transgene described herein into skeletal muscle in accordance with the present invention includes, without limitation, administration into skeletal muscle of the extremities (e.g., upper arm, forearm, thigh, and/or lower leg), back, neck, head (eg tongue), chest, abdomen, pelvis/perineum and/or fingers. The cccDNA vector, as described herein, can be delivered to skeletal muscle by intravenous injection, intra-arterial injection, intraperitoneal injection, limb perfusion, (optionally, isolated limb perfusion - leg and/or arm; see, for example, Arruda et al ., (2005) Blood 105: 3458-3464), and/or direct intramuscular injection. In specific embodiments, the cccDNA vector described herein is introduced into a limb (arm and/or leg) of a subject (e.g., a subject with a muscular dystrophy, such as Duchenne muscular dystrophy (DMD)) by perfusing the limb, optionally perfusing an isolated limb (for example, by intravenous or intra-articular administration). In certain embodiments, a cccDNA vector for controlled transgene expression, as described herein, can be introduced without the use of “hydrodynamic” methods.

[00473] Administration of a cccDNA vector for controlled transgene expression as described herein into cardiac muscle includes administration into the left atrium, right atrium, left ventricle, right ventricle, and/or septum. The cccDNA vector as described herein can be delivered to the cardiac muscle by intravenous administration, intra-arterial administration such as intra-aortic administration, direct injection into the heart (eg, left atrium, right atrium, left ventricle, right ventricle), and/ or coronary artery perfusion. Administration into the diaphragmatic muscle may be accomplished by any suitable route, including intravenous administration, intra-arterial administration, and/or intraperitoneal administration. Administration into smooth muscle may be by any suitable route, including intravenous administration, intra-arterial administration, and/or intraperitoneal administration. In one embodiment, administration may be performed into endothelial cells present in, near and/or on smooth muscle.

[00474] In some embodiments, the cccDNA vector for controlled expression of a transgene of the present invention is administered to skeletal muscle, diaphragm muscle, and/or cardiac muscle (e.g., to treat, alleviate, and/or prevent muscular dystrophy or heart disease (e.g., PAD or congestive heart failure).

A. Ex vivo treatment

[00475] In some embodiments, the cells are removed from the subject, the cDNA vector is introduced into them, and the cells are then returned to the subject. Methods for extracting cells from a subject for ex vivo treatment and then reintroducing them back into the subject are known in the art (see, for example, US Pat. No. 5,399,346; the disclosure of which is incorporated herein in its entirety). Alternatively, the cccDNA vector is introduced into cells from another subject, into cultured cells, or into cells from any other suitable source, and the cells are administered to a subject in need thereof.

[00476] The cells transduced with the cccDNA vector are preferably administered to a subject in a "therapeutically effective amount" in combination with a pharmaceutical carrier. Those skilled in the art will appreciate that therapeutic effects do not have to be complete or provide a cure, as long as the subject receives some benefit.

[00477] In some embodiments, the cccDNA vector for controlled transgene expression may encode a transgene (sometimes referred to as a heterologous nucleotide sequence) that is any polypeptide that is desired to be produced in a cell in vitro, ex vivo, or in vivo. For example, in contrast to the use of cccDNA vectors in a method of treatment as described herein, in some embodiments, the cccDNA vectors can be introduced into cultured cells and the expressed gene product isolated therefrom, for example, to produce antigens or vaccines.

[00478] cccDNA vectors can be used in both veterinary and human medicine. Suitable subjects for ex vivo gene delivery methods as described above include both birds (e.g., chickens, ducks, geese, quail, turkeys, and pheasants) and mammals (e.g., humans, bovine, sheep, goat, equine, feline, canids and lagomorphs), with mammals being preferred. People are the most preferred subjects. People include newborns, infants, adolescents and adults.

[00479] One aspect of the technology described herein relates to a method for delivering a transgene into a cell. Typically, in in vitro methods, the cccDNA vector for controlled transgene expression can be introduced into a cell using the methods disclosed herein as well as other methods known in the art. The cDNA vectors disclosed herein are preferably introduced into the cell in a biologically effective amount. If a cccDNA vector is introduced into a cell in vivo (eg, a subject), a biologically effective amount of the cccDNA vector is an amount sufficient to ensure transduction and expression of the transgene in the target cell.

C. Unit dosage forms

[00480] In some embodiments, the pharmaceutical compositions may be conveniently presented in a unit dosage form. The unit dosage form will typically be adapted to one or more specific routes of administration of the pharmaceutical composition. In some embodiments, the unit dosage form is adapted for administration by inhalation. In some embodiments, the unit dosage form is adapted for administration via a vaporizer. In some embodiments, the unit dosage form is adapted for administration via a nebulizer. In some embodiments, the unit dosage form is adapted for administration using an aerosol generator. In some embodiments, the unit dosage form is adapted for oral administration, for buccal administration, or for sublingual administration. In some embodiments, the unit dosage form is adapted for intravenous, intramuscular, or subcutaneous administration. In some embodiments, the unit dosage form is adapted for intrathecal or intracerebroventricular administration. In some embodiments, the pharmaceutical composition is formulated for topical use. The amount of active ingredient that can be combined with the carrier material to form a unit dosage form is usually that amount of said compound that provides a therapeutic effect.

XIII. Various Applications

[00481] The compositions and cccDNA vectors provided herein can be used to deliver a transgene for various purposes, as described above. In some embodiments, the transgene may encode a protein or be a functional RNA, and in some embodiments may be a protein or functional RNA that is modified for research purposes, for example, to create a somatic transgenic animal model carrying one or more mutations, or a corrected gene sequence, for example to study the function of a target gene. In another example, a transgene encodes a protein or functional RNA to create an animal model of a disease.

[00482] In some embodiments, the transgene encodes one or more peptides, polypeptides, or proteins that are useful for treating, ameliorating, or preventing disease states in a mammalian subject. The transgene expressed by the cccDNA vector for controlled expression of the transgene is administered to a patient in an amount sufficient to treat a disease associated with an abnormal gene sequence that may result in any one or more of the following: decreased expression, absence of expression, or impairment of function of the target gene .

[00483] Some embodiments provide for the use of cccDNA vectors in diagnostic and screening methods in which a transgene is transiently or stably expressed in a cell culture system or, alternatively, in a transgenic animal model.

[00484] Another aspect of the technology described herein provides a method for transducing a population of mammalian cells. In general, the method includes at least the step of introducing into one or more cells of the population a composition containing an effective amount of one or more cccDNAs disclosed herein.

[00485] The invention further provides compositions, as well as therapeutic and/or diagnostic kits, that include one or more disclosed cccDNA vectors or cccDNA compositions formulated using one or more additional ingredients, or prepared together with one or more instructions on their application.

[00486] The cell for introducing the cccDNA vector for controlled transgene expression as disclosed herein can be of any type, including, but not limited to, neural cells (including cells of the peripheral and central nervous systems, in particular brain cells), cells lung, retinal cells, epithelial cells (eg, intestinal and respiratory epithelial cells), muscle cells, dendritic cells, pancreatic cells (including islet cells), liver cells, myocardial cells, bone cells (eg, bone marrow stem cells), hematopoietic stem cells, spleen cells, keratinocytes, fibroblasts, endothelial cells, prostate cells, germ cells, etc. Alternatively, the cell may be any progenitor cell. As a further alternative, the cell may be a stem cell (eg, neural stem cell, liver stem cell). As yet another alternative, the cell may be a cancer or tumor cell. In addition, said cells may come from any species as stated above.

[00487] Certain embodiments of the technology described herein may be defined according to any of the following numbered paragraphs:

1. A composition containing a cccDNA vector that can be administered in repeated doses to increase the level of expression of the transgene from the previous level of expression, wherein said cccDNA vector contains asymmetric or symmetric ICP sequences flanking the polynucleotide sequence of the transgene operably linked to the promoter, with at least at least one of the ICPs is replication competent ICPs.

2. The composition of claim 1, wherein the cDNA vector expresses the transgene for a period of time selected from at least 42 days, at least 84 days, and at least 132 days.

3. The composition of claim 1, wherein the transgene is a genetic drug selected from any of: a nucleic acid, an inhibitor, a peptide or polypeptide, an antibody or antibody fragment, a fusion protein, an antigen, an antagonist, an agonist, or an RNAi molecule.

4. A method for increasing the level of transgene expression in a cell, including:

a. introducing into the cell at the first time point a priming dose of the composition to achieve expression of a heterologous nucleic acid sequence, and

b. introducing into the cell at a second time point a dose of the composition to increase the level of expression of the heterologous nucleic acid sequence compared to the level of expression of the heterologous nucleic acid achieved after administration of the composition at the first time point, or to increase the level of expression of the heterologous nucleic acid sequence to achieve the desired level of expression,

wherein the composition administered at the first and second time points contains a non-viral capsid-free DNA vector with covalently closed ends (ccDNA), wherein the cccDNA vector contains a heterologous nucleic acid sequence encoding a transgene functionally located between two asymmetric or symmetric sequences of inverted terminal repeats (ICP) AAV, one of the ICP contains a functional AAV end resolution site and a Rep binding site, and one of the ICP contains a deletion, insertion or substitution relative to the other ICP,

Moreover, cDNA, when digested with a restriction enzyme having a single recognition site on the cDNA vector, demonstrates the presence of characteristic bands of linear and continuous DNA, different from linear and discontinuous DNA controls when analyzed on a non-denaturing gel.

5. The method of claim 4, wherein the two asymmetric or symmetric inverted terminal repeat (ITR) sequences are AAB ICRs.

6. The method according to claims 4-5, wherein the ICP containing a functional end resolution site and a Rep binding site is a wild-type AAV ICP.

7. The method according to any one of claims 4-6, in which the AAV ICPs are AAV-2 ICPs.

8. The method according to any one of claims 4 to 7, wherein the two asymmetric or symmetric ICPs are a pair of ICPs selected from the group consisting of: (i) SEQ ID NO: 1 and SEQ ID NO: 4; and (ii) SEQ ID NO: 3 and SEQ ID NO: 2.

9. The method according to any one of claims 4 to 8, wherein the cDNA vector is administered in combination with a pharmaceutically acceptable carrier and/or excipient.

10. The method according to any one of claims 4-9, in which the second time point occurs after at least 30 days, or at least 60 days, or 60-90 days, or 90-120 days, or about 3-6 months after the first moment in time.

11. The method according to any one of claims 4 to 10, wherein the heterologous nucleic acid sequence encodes a therapeutic transgene and the desired level of expression of the transgene is a therapeutically effective amount.

12. The method according to any one of claims 4 to 11, wherein the cccDNA vector is derived from a vector polynucleotide, wherein the vector polynucleotide encodes a heterologous nucleic acid operably located between two asymmetric or symmetric inverted terminal repeat (ITR) sequences, at least one of The ICP contains a functional end resolution site and a Rep binding site, and one of the ICPs contains a deletion, insertion, or substitution relative to the other ICP; the presence of the Rep protein induces replication of the vector polynucleotide and production of a DNA vector in the insect cell, wherein the DNA vector can be produced by a process comprising the steps of:

a. incubating a population of insect cells bearing a vector polynucleotide that lacks viral capsid coding sequences in the presence of a Rep protein under conditions effective to, and for a period of time sufficient to induce, the production of a capsid-free non-viral DNA vector in the insect cells, wherein the insect cells do not capable of producing capsid-free non-viral DNA inside insect cells; And

b. collection and isolation of capsid-free non-viral DNA from insect cells;

wherein the presence of a capsid-free non-viral DNA vector isolated from insect cells can be confirmed by digesting DNA isolated from insect cells with a restriction enzyme having a single recognition site on the DNA vector and analyzing said digested DNA material on a non-denaturing gel for confirmation the presence of characteristic bands of linear and continuous DNA, different from bands of linear and discontinuous DNA.

13. The method according to any one of claims 4 to 11, further comprising introducing into the cell, at one or more time points after the second time point, a dose of a composition to increase the level of expression of the heterologous nucleic acid sequence compared to the level of expression of the heterologous nucleic acid achieved after administering the composition at a second time point or at a previous time point, or to increase the expression level of a heterologous nucleic acid sequence to achieve a desired expression level,

wherein the composition administered at one or more time points after the second time point contains a non-viral capsid-free DNA vector with covalently closed ends (ccDNA), wherein the cccDNA vector contains a heterologous nucleic acid sequence encoding a transgene functionally located between two asymmetric or symmetric AAV inverted terminal repeat (IRT) sequences, one of the IRTs contains a functional AAV end resolution site and a Rep binding site, and one of the IRTs contains a deletion, insertion or substitution relative to the other IRT,

in this case, ccDNA, when digested with a restriction enzyme that has a single recognition site on the ccDNA vector, demonstrates the presence of characteristic bands of linear and continuous DNA, different from the linear and discontinuous DNA controls when analyzed on a non-denaturing gel.

14. The method according to any one of claims 4 to 13, wherein the cccDNA vector, administered at any of the first, second or any subsequent time point, is administered in combination with a pharmaceutically acceptable carrier and/or excipient.

15. The method according to any one of claims 1 to 14, wherein the ccDNA vector introduced at the first, second or any subsequent time point is the same ccDNA vector containing the same transgene or a modified transgene.

16. The method according to any one of claims 1 to 15, wherein the ccDNA vector introduced at the first, second or any subsequent time point is another ccDNA vector containing the same transgene or a modified transgene.

17. The method of claim 16, wherein the other cDNA vector has a different promoter operably linked to the same transgene or a modified transgene.

18. The method according to any one of claims 1 to 17, wherein the transgene is a genetic drug.

19. A method of treating a disease in a subject, comprising:

a. administering to the subject at a first time point a priming dose of a composition comprising a non-viral, capsid-free, covalently closed-end DNA (ccDNA) vector to achieve expression of a heterologous nucleic acid sequence, and

b. administering to the subject, at a second time point, a dose of a composition containing a non-viral capsid-free, covalently closed-ended DNA (ccDNA) vector to increase the level of expression of the heterologous nucleic acid sequence compared to the level of expression of the heterologous nucleic acid achieved after administration of the composition at the first time point, or to increase the expression level of a heterologous nucleic acid sequence to achieve a desired level of expression, thereby treating a disease in a subject,

wherein the cccDNA vector introduced at the first and second time points contains a heterologous nucleic acid sequence encoding a transgene functionally located between two asymmetric or symmetric AAV inverted terminal repeat (ITR) sequences, wherein one of the ITRs contains a functional AAV terminal resolution site and a Rep binding site, and one of the ICPs contains a deletion, insertion or substitution relative to the other ICP,

wherein the cDNA vector, when digested with a restriction enzyme having a single recognition site on the cDNA vector, demonstrates the presence of characteristic bands of linear and continuous DNA, different from the linear and discontinuous DNA controls when analyzed on a non-denaturing gel.

20. The method of claim 19, wherein the two asymmetric or symmetric inverted terminal repeat (ITR) sequences are AAV ICRs.

21. The method according to claim 19-20, wherein the ICP containing a functional end resolution site and a Rep binding site is a wild-type AAV ICP.

22. The method according to any one of claims 19-21, in which the AAV ICPs are AAV-2 ICPs.

23. The method according to any one of claims 19 to 22, wherein the two asymmetric ICPs are a pair of ICPs selected from the group consisting of: (i) SEQ ID NO: 1 and SEQ ID NO: 4; and (ii) SEQ ID NO: 3 and SEQ ID NO: 2.

24. The method according to any one of claims 19 to 23, wherein the cDNA vector is administered in combination with a pharmaceutically acceptable carrier and/or excipient.

25. The method according to any one of claims 19-24, in which the second time point occurs after at least 30 days, or at least 60 days, or 60-90 days, or 90-120 days, or about 3-6 months after the first moment in time.

26. The method according to any one of claims 19 to 25, wherein the desired level of transgene expression achieved after administration of the composition at the second time point is a therapeutically effective amount of the transgene.

27. The method according to any one of claims 19 to 26, further comprising administering to the subject, at one or more time points after the second time point, a dose of a composition comprising an cccDNA vector to increase the level of expression of the heterologous nucleic acid sequence compared to the level of expression of the heterologous nucleic acid achieved after administration of the composition at a second time point or at a previous time point, or to increase the expression level of a heterologous nucleic acid sequence to a desired expression level,

wherein the composition administered at one or more time points after the second time point contains a non-viral capsid-free DNA vector with covalently closed ends (ccDNA), wherein the cccDNA vector contains a heterologous nucleic acid sequence encoding a transgene functionally located between two asymmetric or symmetric AAV inverted terminal repeat (IRT) sequences, one of the IRTs contains a functional AAV end resolution site and a Rep binding site, and one of the IRTs contains a deletion, insertion or substitution relative to the other IRT,

wherein cDNA, when digested with a restriction enzyme having a single recognition site on the cDNA vector, demonstrates the presence of characteristic bands of linear and continuous DNA, different from linear and discontinuous DNA controls when analyzed on a non-denaturing gel.

28. The method according to any one of claims 19 to 27, wherein the desired level of transgene expression achieved after administration of the composition at one or more time points after the second time point is a therapeutically effective amount of the transgene.

29. The method according to any one of claims 19 to 28, wherein the cccDNA vector administered at the first, second or any subsequent time point is administered with a pharmaceutically acceptable carrier.

30. The method according to any one of claims 19-29, in which one or more time points after the second time point occur after at least 30 days, or at least 60 days, or 60-90 days, or 90-120 days, or approximately 3-6 months after the previous point in time.

31. The method according to any one of claims 19 to 30, wherein the ccDNA vector introduced at the first, second or any subsequent time point is the same ccDNA vector containing the same transgene or a modified transgene.

32. The method according to any one of claims 19 to 30, wherein the ccDNA vector introduced at the first, second or any subsequent time point is another ccDNA vector containing the same transgene or a modified transgene.

33. The method of claim 31, wherein the other cDNA vector has a different promoter operably linked to the same transgene or a modified transgene.

34. The method according to any one of claims 19 or 33, in which the ccDNA vector is derived from a vector polynucleotide, wherein the vector polynucleotide encodes a heterologous nucleic acid operably located between two asymmetric or symmetric inverted terminal repeat (ITR) sequences, at least one of The ICP contains a functional end resolution site and a Rep binding site, and one of the ICPs contains a deletion, insertion, or substitution relative to the other ICP; wherein the presence of the Rep protein induces replication of the vector polynucleotide and production of a DNA vector in the insect cell, wherein the DNA vector can be produced by a process comprising the steps of:

a. incubating a population of insect cells bearing a vector polynucleotide that lacks viral capsid coding sequences in the presence of a Rep protein under conditions effective to, and for a period of time sufficient to induce, the production of a capsidless non-viral DNA vector in the insect cells, wherein the insect cells do not capable of producing capsid-free non-viral DNA inside insect cells; And

b. collection and isolation of capsid-free non-viral DNA from insect cells;

wherein the presence of a capsid-free non-viral DNA vector isolated from insect cells can be confirmed by digesting DNA isolated from insect cells with a restriction enzyme having a single recognition site on the DNA vector and analyzing said digested DNA material on a non-denaturing gel for confirmation the presence of characteristic bands of linear and continuous DNA, different from bands of linear and discontinuous DNA.

35. The method according to any one of claims 19 to 34, wherein the transgene is a genetic drug.

36. A composition containing a cccDNA vector that can be administered in repeated doses to maintain the level of stable expression of the transgene, wherein the ccDNA vector contains asymmetric or symmetric ICP sequences flanking the polynucleotide sequence of the transgene operably linked to the promoter, wherein at least one of the ICPs is competent for ICP replication.

37. The composition of claim 36, wherein the cDNA vector expresses the transgene for a period of time selected from at least 42 days, at least 84 days, and at least 132 days.

38. The composition of claim 36, wherein the transgene is a genetic drug selected from any of: a nucleic acid, an inhibitor, a peptide or polypeptide, an antibody or antibody fragment, an antigen, an antagonist, an agonist, or an RNAi molecule.

39. A method for maintaining the level of transgene expression in a cell, including:

a. introducing into the cell at the first time point a priming dose of the composition to achieve expression of a heterologous nucleic acid sequence, and

b. introducing into the cell at a second time point a dose of the composition to compensate for any decrease in the level of expression of the heterologous nucleic acid sequence after administration of the composition at the first time point,

wherein the composition administered at the first and second time points contains a non-viral capsid-free DNA vector with covalently closed ends (ccDNA), wherein the cccDNA vector contains a heterologous nucleic acid sequence encoding a transgene functionally located between two asymmetric or symmetric sequences of inverted terminal repeats (ICP) AAV, one of the ICP contains a functional AAV end resolution site and a Rep binding site, and one of the ICP contains a deletion, insertion or substitution relative to the other ICP,

wherein cDNA, when digested with a restriction enzyme having a single recognition site on the cDNA vector, demonstrates the presence of characteristic bands of linear and continuous DNA, different from linear and discontinuous DNA controls when analyzed on a non-denaturing gel.

40. The method of claim 39, wherein the two asymmetric or symmetric inverted terminal repeat (ITR) sequences are AAV ICRs.

41. The method according to claims 39-40, wherein the ICP containing a functional end resolution site and a Rep binding site is a wild-type AAV ICP.

42. The method according to any one of claims 39-41, in which the AAV ICPs are AAV-2 ICPs.

43. The method according to any one of claims 39 to 42, wherein the two asymmetric ICPs are a pair of ICPs selected from the group consisting of: (i) SEQ ID NO: 1 and SEQ ID NO: 4; and (ii) SEQ ID NO: 3 and SEQ ID NO: 2.

44. The method according to any one of claims 39 to 43, wherein the cDNA vector is administered in combination with a pharmaceutically acceptable carrier and/or excipient.

45. The method according to any one of claims 39-44, in which the second time point occurs after at least 30 days, or at least 60 days, or 60-90 days, or 90-120 days, or about 3-6 months after the first moment in time.

46. The method of any one of claims 39 to 45, wherein the heterologous nucleic acid sequence encodes a therapeutic transgene and the sustained level of expression of the transgene is a therapeutically effective amount.

47. The method according to any one of claims 39-46, in which the ccDNA vector is derived from a vector polynucleotide, wherein the vector polynucleotide encodes a heterologous nucleic acid operably located between two asymmetric inverted terminal repeat (ITR) sequences, at least one of the IRTs contains a functional end resolution site and a Rep binding site, and one of the ICPs contains a deletion, insertion, or substitution relative to the other ICP; wherein the presence of the Rep protein induces replication of the vector polynucleotide and production of a DNA vector in the insect cell, and the DNA vector can be produced by a process comprising the steps of:

a. incubating a population of insect cells bearing a vector polynucleotide that lacks viral capsid coding sequences in the presence of a Rep protein under conditions effective to, and for a period of time sufficient to induce, the production of a capsidless non-viral DNA vector in the insect cells, wherein the insect cells do not capable of producing capsid-free non-viral DNA inside insect cells; And

b.collection and isolation of capsid-free non-viral DNA from insect cells;

wherein the presence of a capsid-free non-viral DNA vector isolated from insect cells can be confirmed by digesting DNA isolated from insect cells with a restriction enzyme having a single recognition site on the DNA vector and analyzing said digested DNA material on a non-denaturing gel for confirmation the presence of characteristic bands of linear and continuous DNA, different from bands of linear and discontinuous DNA.

48. The method according to any one of claims 39-47, further comprising introducing into the cell, at one or more time points after the second time point, an additional dose of the composition to increase the level of expression of the heterologous nucleic acid sequence compared to the level of expression of the heterologous nucleic acid achieved after administration of the composition at a second time point or at a previous time point, or to increase the expression level of a heterologous nucleic acid sequence to maintain a desired level of sustained expression,

wherein the composition administered at one or more time points after the second time point contains a non-viral capsid-free DNA vector with covalently closed ends (ccDNA), wherein the cccDNA vector contains a heterologous nucleic acid sequence encoding a transgene functionally located between two asymmetric or symmetric AAV inverted terminal repeat (IRT) sequences, one of the IRTs contains a functional AAV end resolution site and a Rep binding site, and one of the IRTs contains a deletion, insertion or substitution relative to the other IRT,

in this case, ccDNA, when digested with a restriction enzyme that has a single recognition site on the ccDNA vector, demonstrates the presence of characteristic bands of linear and continuous DNA, different from the linear and discontinuous DNA controls when analyzed on a non-denaturing gel.

49. The method according to any one of claims 39 to 48, wherein the cccDNA vector administered at any of the first, second or any subsequent time point is administered in combination with a pharmaceutically acceptable carrier and/or excipient.

50. The method according to any one of claims 39 to 49, wherein the ccDNA vector introduced at the first, second or any subsequent time point is the same ccDNA vector containing the same transgene or a modified transgene.

51. The method according to any one of claims 39 to 50, wherein the ccDNA vector introduced at the first, second or any subsequent time point is another ccDNA vector containing the same transgene or a modified transgene.

52. The method of claim 51, wherein the other cDNA vector has a different promoter operably linked to the same transgene or to a modified transgene.

53. The method according to any one of claims 1 to 52, wherein the transgene is a genetic drug.

54. A method of treating a disease in a subject, comprising:

a. administering to the subject at a first time point a priming dose of a composition comprising a non-viral, capsid-free, covalently closed-end DNA (ccDNA) vector to achieve expression of a heterologous nucleic acid sequence, and

b. administering to the subject at a second time point a dose of a composition containing a non-viral capsid-free covalently closed-ended DNA (ccDNA) vector to maintain the level of expression of the heterologous nucleic acid sequence at a desired steady-state level compared to the level of expression of the heterologous nucleic acid achieved after administration of the composition at the first point in time, thereby providing treatment for the disease in the subject,

wherein the cccDNA vector introduced at the first and second time points contains a heterologous nucleic acid sequence encoding a transgene functionally located between two asymmetric or symmetric AAV inverted terminal repeat (ITR) sequences, wherein one of the ITRs contains a functional AAV terminal resolution site and a Rep binding site, and one of the ICPs contains a deletion, insertion or substitution relative to the other ICP,

wherein the cDNA vector, when digested with a restriction enzyme having a single recognition site on the cDNA vector, demonstrates the presence of characteristic bands of linear and continuous DNA, different from the linear and discontinuous DNA controls when analyzed on a non-denaturing gel.

55. The method of claim 54, wherein the two asymmetric or symmetric inverted terminal repeat (ITR) sequences are AAV ICRs.

56. The method of claim 54 or claim 55, wherein the ICP comprising the functional end resolution site and the Rep binding site is a wild-type AAV ICP.

57. The method according to any one of claims 54-56, in which the AAV ICPs are AAV-2 ICPs.

58. The method according to any one of claims 54 to 57, wherein the two asymmetric ICPs are a pair of ICPs selected from the group consisting of: (i) SEQ ID NO: 1 and SEQ ID NO: 4; and (ii) SEQ ID NO: 3 and SEQ ID NO: 2.

59. The method according to any one of claims 54 to 58, wherein the cDNA vector is administered in combination with a pharmaceutically acceptable carrier and/or excipient.

60. The method according to any one of claims 54-59, in which the second time point occurs after at least 30 days, or at least 60 days, or 60-90 days, or 90-120 days, or about 3-6 months after the first moment in time.

61. The method of any one of claims 54 to 60, wherein the desired level of transgene expression achieved after administration of the composition at the second time point is a therapeutically effective amount of the transgene.

62. The method of any one of claims 54 to 61, further comprising administering to the subject at one or more time points after the second time point a dose of a composition comprising a cccDNA vector to increase the level of expression of the heterologous nucleic acid sequence compared to the level of expression of the heterologous nucleic acid achieved after administration of the composition at the second time point, in order to maintain the required stable level of expression of the heterologous nucleic acid,

wherein the composition administered at one or more time points after the second time point contains a non-viral capsid-free DNA vector with covalently closed ends (ccDNA), wherein the cccDNA vector contains a heterologous nucleic acid sequence encoding a transgene functionally located between two asymmetric or symmetric AAV inverted terminal repeat (IRT) sequences, one of the IRTs contains a functional AAV end resolution site and a Rep binding site, and one of the IRTs contains a deletion, insertion or substitution relative to the other IRT,

wherein cDNA, when digested with a restriction enzyme having a single recognition site on the cDNA vector, demonstrates the presence of characteristic bands of linear and continuous DNA, different from linear and discontinuous DNA controls when analyzed on a non-denaturing gel.

63. The method of any one of claims 54 to 62, wherein the desired level of transgene expression achieved after administration of the composition at one or more time points after the second time point is a therapeutically effective amount of the transgene.

64. The method according to any one of claims 54 to 63, wherein the cccDNA vector administered at the first, second or any subsequent time point is administered with a pharmaceutically acceptable carrier.

65. The method according to any one of claims 54-64, in which one or more time points after the second time point occurs after at least 30 days, or at least 60 days, or 60-90 days, or 90-120 days, or approximately 3-6 months after the previous point in time.

66. The method according to any one of claims 54 to 65, wherein the ccDNA vector introduced at the first, second or any subsequent time point is the same ccDNA vector containing the same transgene or a modified transgene.

67. The method according to any one of claims 54 to 65, wherein the ccDNA vector introduced at the first, second or any subsequent time point is another ccDNA vector containing the same transgene or a modified transgene.

68. The method of claim 67, wherein the other cDNA vector has a different promoter operably linked to the same transgene or to a modified transgene.

69. The method according to any one of claims 54 to 68, wherein the cccDNA vector is derived from a vector polynucleotide, wherein the vector polynucleotide encodes a heterologous nucleic acid operably located between two asymmetric or symmetric inverted terminal repeat (ITR) sequences, at least one of The ICP contains a functional end resolution site and a Rep binding site, and one of the ICPs contains a deletion, insertion, or substitution relative to the other ICP; wherein the presence of the Rep protein induces replication of the vector polynucleotide and production of a DNA vector in the insect cell, wherein the DNA vector can be produced by a process comprising the steps of:

c. incubating a population of insect cells bearing a vector polynucleotide that lacks viral capsid coding sequences in the presence of a Rep protein under conditions effective to, and for a period of time sufficient to induce, the production of a capsid-free non-viral DNA vector in the insect cells, wherein the insect cells do not capable of producing capsid-free non-viral DNA inside insect cells; And

d. collection and isolation of capsid-free non-viral DNA from insect cells;

wherein the presence of a capsid-free non-viral DNA vector isolated from insect cells can be confirmed by digesting DNA isolated from insect cells with a restriction enzyme having a single recognition site on the DNA vector and analyzing said digested DNA material on a non-denaturing gel for confirmation the presence of characteristic bands of linear and continuous DNA, different from bands of linear and discontinuous DNA.

70. The method according to any one of claims 54 to 69, wherein the transgene is a genetic drug.

EXAMPLES

[00488] The following examples are provided by way of illustration and not limitation. One skilled in the art will appreciate that cccDNA vectors can be constructed from any of the wild-type or modified CPIs described herein, and that the following exemplary methods can be used to construct and evaluate the activity of such cccDNA vectors. Although the methods are illustrated with specific cDNA vectors, they are applicable to any cDNA vector as described.

Example 1: Construction of cDNA Vectors Using an Insect Cell Based Method

[00489] The production of cccDNA vectors using a polynucleotide construct template is described in Example 1 of PCT/US18/49996, which is incorporated herein by reference in its entirety. For example, the polynucleotide construct template used to produce the cDNA vectors of the present invention may be a cDNA plasmid, a cDNA bacmid, and/or a cDNA baculovirus. Without being limited by theory, in a permissive host cell, in the presence of e.g. , Rep, a polynucleotide construct template having two symmetrical ICPs and an expression construct, wherein at least one of the ICPs is modified relative to the wild-type ICP sequence, is replicated to form cccDNA vectors. Production of a cccDNA vector occurs in two stages: firstly, excision (“release”) of the template from the matrix backbone (for example, ccDNA plasmid, ccDNA bacmid, ccDNA baculovirus genome, etc.) through Rep proteins and, secondly, -second, Rep-mediated replication of the excised cDNA vector.

[00490] An exemplary method for producing cDNA vectors uses a cDNA plasmid as described herein. In Figures 1A and 1B, the matrix of the polynucleotide construct of each cDNA plasmid includes both a left modified ICP and a right modified ICP, with the following sequences located between the indicated ICPs: (i) enhancer/promoter; (ii) transgene cloning site; (iii) a post-transcriptional response element (eg, woodchuck hepatitis virus post-transcriptional regulatory element (WPRE)); and (iv) a polyadenylation signal (eg, from the bovine growth hormone gene (BGHpA). Unique restriction endonuclease recognition sites (R1-R6) (depicted in Figure 1A and Figure 1B) were also introduced between all components to facilitate the introduction of new genetic components into specific sites of the construct. The enzyme sites R3 (PmeI) GTTTAAAC (SEQ ID NO: 123) and R4 (PacI) TTAATTAA (SEQ ID NO: 124) were inserted into the cloning site by genetic engineering methods to introduce the open reading frame of the transgene. These sequences were cloned into the pFastBac HT B plasmid obtained from ThermoFisher Scientific.

[00491] Production of cccDNA-bacmid

[00492] DH10Bac competent cells (MAX EFFICIENCY® DH10Bac™ competent cells, Thermo Fisher) were transformed with either test or control plasmids according to the protocol according to the manufacturer's instructions. To obtain recombinant cccDNA bacmids, recombination between the plasmid and the baculovirus shuttle vector was induced in DH10Bac cells. Recombinant bacteria were selected by positive selection screening based on blue-white screening in E. coli (marker Φ80dlacZΔM15 provides α-complementation of the β-galactosidase gene from the bacmid vector) on a bacterial agar plate containing X-gal and IPTG with antibiotics for selection transformants, and maintenance of bacmids and transposase plasmids. White colonies resulting from transposition that disrupts the β-galactoside indicator gene were collected and cultured in 10 ml of medium.

[00493] Recombinant cccDNA bacmids were isolated from E. coli and transfected into Sf9 or Sf21 insect cells using FugeneHD to produce infectious baculovirus. Adherent Sf9 or Sf21 insect cells were cultured in 50 ml of medium in T25 flasks at 25°C. After 4 days, the culture medium (containing the P0 virus) was separated from the cells and filtered through a 0.45-μm pore size filter, separating infectious baculovirus particles from cells or cellular debris.

[00494] Optionally, the first generation of baculovirus (P0) was amplified by infecting naive Sf9 or Sf21 insect cells in 50-500 ml of medium. Cells were maintained in suspension cultures in an orbital shaker incubator at 130 rpm at 25°C, monitoring cell diameter and cell viability until the cells reached a diameter of 18-19 nm (from a naïve cell diameter of 14-15 nm) and density of about 4.0E+6 cells/ml. 3-8 days after infection, baculovirus P1 particles in the medium were collected after centrifugation to remove cells and debris, followed by filtration through a 0.45-μm pore size filter.

[00495] The cDNA baculovirus containing the test constructs was collected and the infectivity or titer of the baculovirus was determined. Specifically, 4 × 20 ml Sf9 cell cultures at a density of 2.5E+6 cells/ml were treated with baculovirus P1 at the following dilutions: 1/1000, 1/10000, 1/50000, 1/100000, and incubated at 25-27° C. Infectivity was determined daily for 4-5 days by the increase in cell diameter and cell cycle arrest, as well as changes in cell viability.

[00496] The "Rep plasmid" was produced in the expression vector "pFASTBAC ^TM -Dual" (ThermoFisher) containing either Rep78 (SEQ ID NO: 131 or 133) and Rep52 (SEQ ID NO: 132) or Rep68 ( SEQ ID NO: 130) and Rep40 (SEQ ID NO: 129). The indicated Rep plasmid was transformed into competent DH10Bac cells (“MAX EFFICIENCY® DH10Bac™ Competent Cells” (Thermo Fisher)) according to the protocol provided by the manufacturer. Recombination between the Rep plasmid and the baculovirus shuttle vector in DH10Bac cells was induced to produce recombinant bacmids (“Rep bacmids”). Recombinant bacmids were selected using positive selection, which included blue-white screening in E. coli (marker Φ80dlacZΔM15 provides α-complementation of the β-galactosidase gene from the bacmid vector) on a bacterial agar plate containing X-gal and IPTG. Isolated white colonies were collected and inoculated into 10 ml of selective medium (kanamycin, gentamicin, tetracycline in LB broth). Recombinant bacmids (Rep bacmids) were isolated from E. coli, and Sf9 or Sf21 insect cells were transfected with these Rep bacmids to produce infectious baculovirus.

[00497] Sf9 or Sf21 insect cells were cultured in 50 ml of medium for 4 days and an infectious recombinant baculovirus (“Rep-baculovirus”) was isolated from the culture. Optionally, first generation Rep baculovirus (P0) was amplified by infecting naive insect cells with Sf9 or Sf21 and culturing in 50-500 ml of medium. 3-8 days after infection, baculovirus P1 particles in the medium were collected by separating cells by centrifugation or filtration, or using another fractionation method. Rep-baculovirus was collected and the infectivity of the baculovirus was determined. Specifically, 4 x 20 ml Sf9 cell cultures at a density of 2.5 x 10 ⁶ cells/ml were treated with baculovirus P1 at the following dilutions: 1/1000, 1/10000, 1/50000, 1/100000, and incubated. Infectivity was determined daily for 4-5 days by the increase in cell diameter and cell cycle arrest, as well as changes in cell viability.

[00498] Preparation and characterization of cccDNA vector

[00499] As shown in Figure 4B, Sf9 insect cell culture medium containing either (1) a sample containing ccDNA bacmid or ccDNA baculovirus or (2) Rep baculovirus described above was then added to the fresh cell culture Sf9 (2.5E+6 cells/ml, 20 ml) at a ratio of 1:1000 and 1:10000, respectively. Then the cells were cultured at 130 rpm at 25°C. 4-5 days after co-infection, the diameter and viability of the cells were determined. When the cell diameter reached 18-20 nm with a viability of about 70-80%, the cell cultures were centrifuged, the medium was removed and the cell pellets were collected. Cell pellets were first resuspended in a sufficient volume of aqueous medium - water or buffer. The cccDNA vector was isolated and purified from cells using the Qiagen MIDI PLUS™ purification protocol (Qiagen, treating 0.2 mg cell pellet weight per column).

[00500] Yield values for cccDNA vectors produced and purified from Sf9 insect cells were initially determined based on UV absorbance at 260 nm.

[00501] Evaluation of cccDNA vectors can be accomplished by identification using agarose gel electrophoresis under native or denaturing conditions, as illustrated in Figure 4D, where (a) the presence of characteristic bands migrating at twice the size on denaturing gels is observed compared with native gels after restriction endonuclease digestion and gel electrophoretic analysis, and (b) the presence of monomer and dimer bands (2×) on denaturing gels for undigested material is characteristic of the presence of cccDNA vector.

[00502] The structures of the isolated cccDNA vectors were further analyzed by digesting DNA obtained from coinfected Sf9 cells (as described herein) with restriction endonucleases selected based on a) the presence of only one cut site in the ccDNA vectors, and b) obtaining the resulting fragments large enough to be clearly visible when fractionated on a 0.8% denaturing agarose gel (>800 bp). As demonstrated in Figures 4D and 4E, linear discontinuous DNA vectors and linear and continuous cccDNA vectors can be distinguished by the size of their reaction products—for example, a discontinuous DNA vector is expected to produce fragments of 1 t. .By. and 2 kb, while for a cDNA vector with a continuous structure the formation of fragments of 2 kb is expected. and 4 kb.

[00503] Thus, in order to qualitatively demonstrate that the isolated cccDNA vectors have covalently closed ends, as required by definition, samples were digested with a restriction endonuclease identified in the context of the specific DNA vector sequence as having a single restriction site, preferably , leading to the formation of two cleavage products of unequal size (for example, 1000 bp and 2000 bp). After digestion and electrophoresis on a denaturing gel (which separates two complementary DNA strands), linear DNA that does not have covalently closed ends will be separated into 1000 bp fragments. and 2000 bp, while covalently closed DNA (i.e. cDNA vector) will be divided into fragments 2 times larger (2000 bp and 4000 bp), since two chains The DNA is linked and, when unrolled, will be twice as long (even though it is single-stranded). In addition, when the monomeric, dimeric and n-mer forms of DNA vectors are digested, they will all be separated into fragments of the same size due to the binding of the ends of the multimeric DNA vectors (see Fig. 4D).

[00504] As used herein, the phrase “assay for identification of DNA vectors by agarose gel electrophoresis under native and denaturing gel conditions” refers to an assay for assessing the presence of closed ends of ccDNA by performing restriction endonuclease digestion followed by electrophoretic assessment of the digestion products. One example of such an analysis is given below, although one skilled in the art will appreciate that many variations of this example known in the art are possible. The restriction endonuclease is selected to be an enzyme that makes a single cut of the cccDNA vector of interest to produce products of approximately 1/3× and 2/3× the length of the DNA vector. This ensures separation of bands on both the native and denaturing gels. It is important to remove the buffer from the sample before denaturation. A Qiagen PCR product purification kit or desalting “spun columns” such as GE Healthcare ILUSTRA(MicroSpin(G-25) columns) are some of the options known in the art for endonuclease digestion. for example, i) DNA digestion with suitable restriction endonuclease(s), 2) application, for example, to a Qiagen PCR product purification kit, eluting with distilled water, iii) addition of a 10x denaturing solution (10×=0.5 M NaOH, 10 mM EDTA), adding 10X unbuffered dye solution and running the assay together with DNA molecular weight markers obtained by adding 10X denaturing solution in a 4 replicate experiment at 0.8-1.0% gel, pre-incubated with 1 mM EDTA and 200 mM NaOH to ensure uniformity of NaOH concentration in the gel and gel chamber, and running the gel in the presence of 1× denaturing solution (50 mM NaOH, 1 mM EDTA). One skilled in the art will understand what voltage to use to perform electrophoresis based on the size and desired time to obtain results. After electrophoresis, the gels are dried and neutralized in 1× TBE (Tris-borate-EDTA buffer) or TAE (Tris-acetate-EDTA buffer) and transferred to distilled water or 1× TBE/TAE with 1× SYBR Gold. The bands can then be visualized, for example, using Thermo Fisher's SYBR® Gold Nucleic Acid Gel Stain (10,000X concentrate in DMSO) and epifluorescent light (blue) or UV (312 nm).

[00505] The purity of the resulting cccDNA vector can be assessed using any method known in the art. As one non-limiting example of a method, the contribution of the cDNA plasmid to the total UV absorbance of the sample can be assessed by comparing the fluorescence intensity of the cDNA vector with a standard. For example, in the case of UV absorbance determination, if 4 μg of ccDNA vector was loaded on the gel, and the fluorescence intensity of the ccDNA vector is equivalent to a 2 kb band. with a known mass of 1 μg, then this corresponds to the presence of 1 μg of cDNA vector, and the cDNA vector constitutes 25% of the total amount of UV absorbing material. The intensity of the band on the gel is then plotted against the calculated input amount to which the band corresponds - for example, if the entire ccDNA vector corresponds to 8 kb, and the excised comparative band corresponds to 2 kb, then the intensity of the band is graph will be 25% of the total amount administered, which in this case will be 0.25 μg when 1.0 μg is administered. Using titration of the cDNA vector plasmid to construct a calibration curve, the amount of the cDNA vector band is then calculated from the regression line equation, which this can then be used to determine the percentage of total input represented by the cccDNA vector, or the percentage of purity.

[00506] For purposes of comparison, Example 1 describes the preparation of cccDNA vectors using an insect cell-based method and a polynucleotide construct template, which is also described in Example 1 of PCT/US18/49996, incorporated herein by reference in its entirety. For example, the polynucleotide construct template used to produce the cDNA vectors of the present invention in accordance with Example 1 may be a cDNA plasmid, a cDNA bacmid, and/or a cDNA baculovirus. Without being limited by theory, in a permissive host cell , in the presence of, for example, Rep, a polynucleotide construct template having two symmetrical ICPs and an expression construct, at least one of the ICPs being modified relative to the wild-type ICP sequence, is replicated to form cDNA vectors. Production of a cccDNA vector occurs in two stages: firstly, excision (“release”) of the template from the matrix backbone (for example, ccDNA plasmid, ccDNA bacmid, ccDNA baculovirus genome, etc.) through Rep proteins and, secondly, -second, Rep-mediated replication of the excised cDNA vector.

[00507] An example of a method for producing cccDNA vectors using an insect cell is its production from a ccDNA plasmid, as described herein. In Figures 1A and 1B, the matrix of the polynucleotide construct of each cDNA plasmid includes both a left modified ICP and a right modified ICP, with the following sequences located between the indicated ICPs: (i) enhancer/promoter; (ii) transgene cloning site; (iii) a post-transcriptional response element (eg, woodchuck hepatitis virus post-transcriptional regulatory element (WPRE)); and (iv) a polyadenylation signal (eg, from the bovine growth hormone gene (BGHpA). Unique restriction endonuclease recognition sites (R1-R6) were also introduced between all components (depicted in Figure 1A and Figure 1B) to facilitate the introduction of new genetic components into specific sites of the construct. The enzyme sites R3 (PmeI) GTTTAAAC (SEQ ID NO: 123) and R4 (PacI) TTAATTAA (SEQ ID NO: 124) were inserted into the cloning site by genetic engineering methods to introduce the open reading frame of the transgene. These sequences were cloned into the pFastBac HT B plasmid obtained from ThermoFisher Scientific.

[00508] Production of cccDNA-bacmid

[00509] DH10Bac competent cells (MAX EFFICIENCY® DH10Bac™ competent cells, Thermo Fisher) were transformed with either test or control plasmids according to the protocol according to the manufacturer's instructions. To obtain recombinant cccDNA bacmids, recombination between the plasmid and the baculovirus shuttle vector was induced in DH10Bac cells. Recombinant bacmids were selected by a positive selection screen based on the blue-white screen in E. coli (marker Φ80dlacZΔM15 provides α-complementation of the β-galactosidase gene from the bacmid vector) on a bacterial agar plate containing X-gal (5-bromo-4 -chloro-3-indolyl-beta-D-galactopyranoside) and IPTG (isopropylthiogalactoside) with antibiotics to select transformants, and maintain bacmids and transposase plasmids. White colonies resulting from transposition that disrupts the β-galactoside indicator gene were collected and cultured in 10 ml of medium.

[00510] Recombinant cccDNA bacmids were isolated from E. coli and transfected into Sf9 or Sf21 insect cells using FugeneHD to produce infectious baculovirus. Adherent Sf9 or Sf21 insect cells were cultured in 50 ml of medium in T25 flasks at 25°C. After 4 days, the culture medium (containing the P0 virus) was separated from the cells and filtered through a filter with a pore size of 0.45 μm, separating infectious baculovirus particles from the cells or cellular debris.

[00511] Optionally, the first generation of baculovirus (P0) was amplified by infecting naive Sf9 or Sf21 insect cells in 50-500 ml of medium. Cells were maintained in suspension cultures in an orbital shaker incubator at 130 rpm at 25°C, monitoring cell diameter and cell viability until the cells reached a diameter of 18-19 nm (from a naïve cell diameter of 14-15 nm) and density of about 4.0E+6 cells/ml. 3-8 days after infection, baculovirus P1 particles in the medium were collected after centrifugation to remove cells and debris, followed by filtration through a 0.45-μm pore size filter.

[00512] The cDNA baculovirus containing the test constructs was collected and the infectivity or titer of the baculovirus was determined. Specifically, 4 × 20 ml of Sf9 cell culture with a density of 2.5E+6 cells/ml were treated with baculovirus P1 at the following dilutions: 1/1000, 1/10000, 1/50000, 1/100000, and incubated at 25-27° C. Infectivity was determined daily for 4-5 days by the increase in cell diameter and cell cycle arrest, as well as changes in cell viability.

[00513] The "Rep plasmid" was produced in the expression vector "pFASTBAC ^TM -Dual" (ThermoFisher) containing either Rep78 (SEQ ID NO: 131 or 133) and Rep52 (SEQ ID NO: 132) or Rep68 ( SEQ ID NO: 130) and Rep40 (SEQ ID NO: 129). The indicated Rep plasmid was transformed into competent DH10Bac cells (“MAX EFFICIENCY® DH10Bac™ Competent Cells” (Thermo Fisher)) according to the protocol provided by the manufacturer. Recombination was induced between the Rep plasmid and the baculovirus shuttle vector in DH10Bac cells to produce recombinant bacmids (“Rep bacmids”). Recombinant bacmids were selected using positive selection, which included blue-white screening in E. coli (marker Φ80dlacZΔM15 provides α-complementation of the β-galactosidase gene from the bacmid vector) on a bacterial agar plate containing X-gal and IPTG. Isolated white colonies were collected and inoculated into 10 ml of selective medium (kanamycin, gentamicin, tetracycline in LB broth). Recombinant bacmids (Rep bacmids) were isolated from E. coli, and Sf9 or Sf21 insect cells were transfected with these Rep bacmids to produce infectious baculovirus.

[00514] Sf9 or Sf21 insect cells were cultured in 50 ml of medium for 4 days and an infectious recombinant baculovirus (“Rep-baculovirus”) was isolated from the culture. Optionally, first generation Rep baculovirus (P0) was amplified by infecting naive insect cells with Sf9 or Sf21 and culturing in 50-500 ml of medium. 3-8 days after infection, baculovirus P1 particles in the medium were collected by separating cells by centrifugation or filtration, or using another fractionation method. Rep-baculovirus was collected and the infectivity of the baculovirus was determined. Specifically, 4 × 20 ml of Sf9 cell culture with a density of 2.5 × 10 ⁶ cells/ml were treated with baculovirus P1 at the following dilutions: 1/1000, 1/10000, 1/50000, 1/100000, and incubated. Infectivity was determined daily for 4-5 days by the increase in cell diameter and cell cycle arrest, as well as changes in cell viability.

[00515] Preparation and characterization of cccDNA vector

[00516] Sf9 insect cell culture medium containing either (1) a sample containing cccDNA bacmid or cccDNA baculovirus and (2) Rep baculovirus described above was then added to a fresh Sf9 cell culture (2.5E+6 cells/ml, 20 ml) in a ratio of 1:1000 and 1:10000, respectively. Then the cells were cultured at 130 rpm at 25°C. 4-5 days after co-infection, the diameter and viability of the cells were determined. When the cell diameter reached 18-20 nm with a viability of about 70-80%, the cell cultures were centrifuged, the medium was removed and the cell pellets were collected. Cell pellets were first resuspended in a sufficient volume of aqueous medium - water or buffer. The cccDNA vector was isolated from cells and purified using the Qiagen MIDI PLUS™ purification protocol (Qiagen, treating 0.2 mg cell pellet weight per column).

[00517] Yield values for cccDNA vectors produced and purified from Sf9 insect cells were initially determined based on UV absorbance at 260 nm. The correct configuration of purified closed-end cccDNA vectors can be assessed using the electrophoretic methodology described in Example 5.

EXAMPLE 2: Preparation of synthetic ccDNA by cutting double-stranded DNA from a molecule

[00518] A synthetic method for producing cccDNA vectors is described in Examples 2-6 of International Application PCT/US19/14122, filed January 18, 2019, which is incorporated herein by reference in its entirety. One typical example of a method for producing a cDNA vector using a synthetic method involves cutting out a double-stranded DNA molecule. Briefly, a cDNA vector can be produced using a double-stranded DNA construct, for example, see FIGS. 7A-8E of application PCT/US19/14122. In some embodiments, the double-stranded DNA construct is a cccDNA plasmid, see, for example, Figure 6 of international patent application PCT/US2018/064242, filed December 6, 2018).

[00519] In some embodiments, the cDNA vector construct comprises a regulatory switch, as described herein.

[00520] For illustrative purposes, Example 2 describes the preparation of cccDNA vectors as examples of closed-end DNA vectors produced using this method. However, although cccDNA vectors are described in this Example as an example illustrating in vitro synthetic production methods for producing a closed-end DNA vector by excision of a double-stranded polynucleotide containing an ICP and an expression cassette (e.g., a heterologous nucleic acid sequence), followed by ligation free 3' and 5' ends as described herein, one skilled in the art will know that, as stated above, a double-stranded DNA polynucleotide molecule can be modified to produce any desired closed-end DNA vector. including, without limitation, doggybone DNA, dumbbell DNA, and the like.

[00521] The method includes (i) excision of a sequence encoding an expression cassette from a double-stranded DNA construct, and (ii) formation of hairpin structures in one or more ICPs, and (iii) joining the free 5' and 3' ends by ligation, for example, using T4 DNA ligase.

[00522] The double-stranded DNA construct contains, in order from 5' to 3': a first restriction endonuclease site; left ICP; expression cassette; right ICP; and a second restriction endonuclease site. The double-stranded DNA construct is then contacted with one or more restriction endonucleases to generate double-strand breaks at both restriction endonuclease sites. Both sites may be targets of the same endonuclease, or each site may be the target of different endonucleases, provided that the restriction sites are not included in the ccDNA vector template. As a result, the sequence between the restriction endonuclease sites is excised from the rest of the double-stranded DNA construct. After ligation, a DNA vector with closed ends is formed.

[00523] One or both of the ICPs used in the method may be wild-type ICPs. Modified ICPs may also be used, and the modification may include the deletion, insertion or substitution of one or more nucleotides from the wild-type ICP in the sequences forming arms B and B' and/or arms C and C' (see, for example, Fig. 3B and 3D), and may have two or more hairpin loops or one hairpin loop. A modified hairpin loop ICP can be produced by genetic modification of an existing oligonucleotide or by biological and/or chemical de novo synthesis.

[00524] In a non-limiting example, the left and right ICP-6 (SEQ ID NOs: 111 and 112) include deletions of 40 nucleotides in arms B-B' and C-C' compared to wild-type AAB2 ICP. The nucleotides remaining in the modified ICP are predicted to form a single hairpin structure. The Gibbs free energy of structure expansion is about -54.4 kcal/mol. Other ICP modifications can also be made, including optional deletion of a functional Rep binding site or Trs site.

EXAMPLE 3: Production of ccDNA by Design of Oligonucleotides

[00525] Another example of a method for producing a cccDNA vector using a synthetic method involving the assembly of various oligonucleotides is presented in Example 3 of PCT/US19/14122, in which a ccDNA vector is prepared by synthesizing 5' and 3' ICP oligonucleotides and ligation ICP oligonucleotides with a double-stranded polynucleotide containing an expression cassette. FIG. 11B of PCT/US19/14122 shows an example of a method for ligating a 5'-ICP oligonucleotide and a 3'-ICP oligonucleotide to a double-stranded polynucleotide containing an expression cassette.

[00526] As disclosed herein, ICP oligonucleotides may comprise ICP-DT or modified ICPs (eg, see FIGS. 6A, 6B, 7A and 7B of application PCT/US19/14122, which is incorporated herein in its entirety). Exemplary ICP oligonucleotides include, but are not limited to, SEQ ID NO: 134-145 (eg, see Table 7 in PCT/US19/14122). Modified ICPs may include the deletion, insertion or substitution of one or more nucleotides relative to the wild-type ICP in the sequences forming arms B and B' and/or arms C and C'. ICP oligonucleotides containing ICP-DT or mod-ICP as described herein, intended for use in cell-free synthesis, can be prepared by genetic modification or biological and/or chemical synthesis. As described herein, the ICP oligonucleotides in Examples 2 and 3 may contain ICP-DT or modified ICPs (mod-ICPs) in symmetric or asymmetric configurations, as discussed herein.

EXAMPLE 4: Production of ccDNA using a single-stranded DNA molecule

[00527] Another example of a method for producing a cccDNA vector using a synthetic method, presented in Example 4 of PCT/US19/14122, uses single-stranded linear DNA containing two sense ICPs that flank the sense sequence of the expression cassette and are covalently attached to two antisense ICPs , which flank the antisense expression cassette, and the ends of the single-stranded linear DNA are then ligated to form a single-stranded molecule with closed ends. One non-limiting example involves synthesizing and/or producing a single-stranded DNA molecule, annealing the portions of the molecule to form a single linear DNA molecule that has one or more base-paired secondary structure regions, and then ligating the free 5' and 3' ends to each other to form a closed single-chain molecule.

[00528] A typical single-stranded DNA molecule for producing a cDNA vector contains, in the 5' to 3' direction:

first semantic ICP;

the semantic sequence of the expression cassette;

second semantic ICP;

second antisense ICP;

antisense sequence of the expression cassette; And

the first antisense ICP.

[00529] A single-stranded DNA molecule for use in the typical method of Example 4 can be prepared using any DNA synthesis methodology described herein, for example, in vitro DNA synthesis, or by digesting a DNA construct (e.g., a plasmid) with nucleases and melting the resulting dsDNA fragments to form single-stranded DNA (ssDNA) fragments.

[00530] Annealing can be accomplished by reducing the temperature to below the calculated melting temperatures of sense and antisense sequence pairs. The melting point depends on the content of the specific nucleotide bases and the characteristics of the solution used, such as salt concentration. One skilled in the art can readily calculate melting temperatures for any given combination of sequence and solution.

[00531] The free 5' and 3' ends of the annealed molecule can be ligated to each other or ligated to a hairpin molecule to form a cccDNA vector. Typical examples of suitable ligation techniques and hairpin molecules are described in Examples 2 and 3.

EXAMPLE 5: Purification and/or confirmation of cccDNA production

[00532] Any of the DNA vector products produced by the methods described herein, for example, including the insect cell-based production methods described in Example 1, or the synthetic production methods described in Examples 2-4, can be purified, for example , to remove impurities, unused components, or by-products, using methods generally known to qualified persons; and/or can be analyzed to confirm that the produced DNA vector (in this case, the ccDNA vector) is the desired molecule. An example of a method for purifying a DNA vector, such as cccDNA, is using the Qiagen Midi Plus purification protocol (Qiagen) and/or gel purification.

[00533] The following is a typical method for confirming the identity of cDNA vectors.

[00534] Evaluation of cccDNA vectors can be done by identifying, using agarose gel electrophoresis under native or denaturing conditions, as illustrated in Figures 4C and 4D, (a) the presence of characteristic bands migrating at twice the size on denaturing gels according to compared to native gels after restriction endonuclease digestion and gel electrophoretic analysis, and (b) the presence of monomer and dimer bands (2×) on denaturing gels for undigested material indicative of the presence of cccDNA vector.

[00535] The structures of the isolated cccDNA vectors were further analyzed by digesting the purified DNA with restriction endonucleases selected to ensure a) the presence of only one cut site in the ccDNA vectors, and b) the resulting fragments are large enough to be clearly visible when fractionated into 0.8% denaturing agarose gel (>800 bp). As demonstrated in Figures 4C and 4D, linear discontinuous DNA vectors and linear and continuous cccDNA vectors can be distinguished by the size of their reaction products—for example, discontinuous DNA vectors are expected to produce 1-ton fragments .By. and 2 kb, while for a cDNA vector with a continuous structure one would expect the formation of fragments of 2 kb in size. and 4 kb.

[00536] Thus, in order to qualitatively demonstrate that the isolated cccDNA vectors have covalently closed ends, as required by definition, samples were digested with a restriction endonuclease identified in the context of the specific DNA vector sequence as having a single restriction site, preferably , leading to the formation of two cleavage products of unequal size (for example, 1000 bp and 2000 bp). After digestion and electrophoresis on a denaturing gel (which separates two complementary DNA strands), linear DNA that does not have covalently closed ends will be separated into 1000 bp fragments. and 2000 bp, while covalently closed DNA (i.e. cDNA vector) will be divided into fragments 2 times larger (2000 bp and 4000 bp), since two chains The DNA is linked and, when unrolled, will be twice as long (even though it is single-stranded). In addition, when the monomeric, dimeric and n-mer forms of DNA vectors are digested, they will all be separated into fragments of the same size due to the binding of the ends of the multimeric DNA vectors (see Figures 4D and 4E).

[00537] As used herein, the phrase “assay for the identification of DNA vectors by agarose gel electrophoresis under native and denaturing conditions” refers to an assay for assessing the presence of closed ends of cccDNA by performing restriction endonuclease digestion followed by electrophoretic evaluation of the digestion products. One example of such an analysis is given below, although one skilled in the art will appreciate that many variations of this example known in the art are possible. The restriction endonuclease is selected to be an enzyme that makes a single cut of the cDNA vector of interest to produce products of approximately 1/3× and 2/3× the length of the DNA vector. This ensures band separation on both native and denaturing gels. It is important to remove the buffer from the sample before denaturation. A Qiagen PCR product purification kit or desalting “spun columns,” such as the GE Healthcare ILUSTRA™ MicroSpin™ G-25 columns, are some of the options known in the art for endonuclease digestion. The analysis involves, for example, i) digestion of DNA with suitable restriction endonuclease(s), 2) application, for example, to a Qiagen PCR product purification kit, eluting with distilled water, iii) addition of a 10× denaturing solution (10×=0 .5 M NaOH, 10 mM EDTA), adding 10× unbuffered dye solution and running the assay together with DNA molecular weight markers obtained by adding 10× denaturing solution in a 4-replicate experiment, at 0.8–1 .0% gel, pre-incubated with 1 mM EDTA and 200 mM NaOH to ensure uniformity of NaOH concentration in the gel and gel chamber, and running the gel in the presence of 1× denaturing solution (50 mM NaOH, 1 mM EDTA). One skilled in the art will understand what voltage to use to perform electrophoresis depending on the size and desired time to obtain results. After electrophoresis, the gels are dried and neutralized in 1× TBE (Tris-borate-EDTA buffer) or TAE (Tris-acetate-EDTA buffer) and transferred to distilled water or 1× TBE/TAE with 1× SYBR Gold. The bands can then be visualized, for example, using Thermo Fisher's SYBR® Gold Nucleic Acid Gel Stain (10,000X concentrate in DMSO) and epifluorescent light (blue) or UV (312 nm). The above gel-based method can be adapted for purification purposes by isolating the cDNA vector from the gel band and allowing it to be renatured.

[00538] Evaluation of the purity of the resulting cccDNA vector can be carried out using any method known in the art. As one non-limiting example of a method, the contribution of the cDNA plasmid to the total UV absorbance of the sample can be assessed by comparing the fluorescence intensity of the cDNA vector with a standard. For example, in the case of UV absorbance determination, if 4 μg of ccDNA vector was loaded onto the gel, and the fluorescence intensity of the ccDNA vector is equivalent to a 2 kb band. with a known mass of 1 μg, then this corresponds to the presence of 1 μg of cDNA vector, and the cDNA vector constitutes 25% of the total amount of UV absorbing material. The intensity of the band on the gel is then plotted against the calculated input amount to which the band corresponds - for example, if the entire ccDNA vector corresponds to 8 kb, and the excised comparative band corresponds to 2 kb, then the intensity of the band is graph will be 25% of the total amount administered, which in this case will be 0.25 μg when 1.0 μg is administered. Using titration of the cDNA vector plasmid to construct a calibration curve, the amount of the cDNA vector band is then calculated from the regression line equation, which this can then be used to determine the percentage of total input represented by the cccDNA vector, or the percentage of purity.

EXAMPLE 6: Controlled expression of a cccDNA transgene: expression of a cccDNA vector transgene in vivo can be maintained and/or increased by repeated administration

[00539] An cccDNA vector was prepared according to the methods described in Example 1 above using a cccDNA plasmid containing a CAG promoter (SEQ ID NO: 72) and a luciferase transgene (SEQ ID NO: 56) flanked by asymmetric ICPs (eg , 5'-IKP-DT (SEQ ID NO: 2) and 3' mod-IKP (SEQ ID NO: 3)), and were evaluated in various in vivo treatment programs. This cDNA vector was used in all subsequent experiments described in Examples 6-10. In Example 6, the cccDNA vector was purified, formulated with lipid nanoparticles (LNP-ccDNA) and injected into the tail vein of each CD-1® IGS mouse. Liposomes were prepared with a suitable lipid mixture containing four components to form lipid nanoparticle (LNP) liposomes, including cationic lipids, auxiliary lipids, cholesterol, and PEG lipids.

[00540] To assess sustained in vivo transgene expression from a cccDNA vector over an extended period of time, cccDNA LNPs were administered in sterile phosphate-buffered saline (PBS) by intravenous tail vein injection into CDG-1® IGS mice approximately 5-5 years of age. 7 weeks. Three groups were evaluated at different dosages: 0.1 mg/kg, 0.5 mg/kg and 1.0 mg/kg, ten mice per group (except for the 1.0 mg/kg group, which had 15 mice). Injections were given on day 0. Five mice from each group received an injection of an additional identical dose on day 28. Luciferase expression was measured by IVIS imaging following intravenous administration of CD- ^1® IGS mice (Charles River Laboratories; wild-type (WT) mice) . Luciferase expression was assessed by IVIS imaging after intraperitoneal administration of 150 mg/kg luciferin substrate on days 3, 4, 7, 14, 21, 28, 31, 35, and 42 and regularly (eg, weekly, biweekly, or every 10 days, or every 2 weeks), in the period between days 42-110. The results are presented in Figure 6. This figure is a graph showing luciferase transgene expression measured by IVIS imaging for at least 132 days, according to 3 different administration protocols.

[00541] Studies were continued to examine the effect of repeated dosing, for example, repeated administration of LNP-ccDNA expressing luciferase to subjects receiving LNP-ccDNA. In particular, the possibility of increasing expression levels by one or more additional injections of the cccDNA vector was assessed.

[00542] This study assessed the biodistribution of cccDNA vector luciferase expression by IVIS in CD-1® IGS mice after an initial intravenous administration of 1.0 mg/kg (i.e., priming dose) on days 0 and 28 (group A ). The second administration of cccDNA vector was done by tail vein injection of 3 mg/kg (group B) or 10 mg/kg (group C) in 1.2 ml tail vein injection on day 84. Five (5) mice were used in this study. CD-1® were in each of Groups A, B, and C. IVIS imaging of luciferase expression in mice was performed before additional dosing on days 49, 56, 63, and 70 as described above, and after repeat dosing on day 84 and on days 91, 98, 105, 112 and 132. Luciferase expression was assessed and detected in all three groups A, B and C until at least day 110 (the longest period of assessment).

[00543] It has been demonstrated that the level of luciferase expression is increased by repeated dosing (ie, repeated administration of a cccDNA composition) of LNP-ccDNA-Luc, as determined by assessing luciferase activity in the presence of luciferin. The results are presented in Figure 6, which is a graph showing luciferase transgene expression measured by IVIS imaging for at least 110 days according to 3 different administration protocols (groups A, B and C). Mice that did not receive any additional boost dose (priming dose of 1 mg/kg (i.e., group A)) exhibited stable luciferase expression observed throughout the study. Mice in group B that received a repeat dose of 3 mg/kg cccDNA vector showed approximately a sevenfold increase in observed luminosity compared to mice in group C. Surprisingly, mice that received a repeat dose of 10 mg/kg cccDNA vector showed a 17-fold increase in observed luminosity. increase in observed luciferase luminosity compared to mice not receiving any repeat dose (group A).

[00544] Group A demonstrates luciferase expression in CD-1 ^® IGS mice following intravenous tail vein administration of 1 mg/kg cccDNA vector on days 0 and 28. Groups B and C demonstrate luciferase expression in CD-1 ^® IGS mice. administered 1 mg/kg of cccDNA vector at the first time point (day 0) and a repeat dose of cccDNA vector at the second time point on day 84. Surprisingly, the second administration (i.e., repeat dose) of cccDNA vector increased expression by less than at least 7 times, and even up to 17 times.

[00545] Surprisingly, a 3-fold increase in dose (i.e., amount) of cccDNA vector upon repeated dosing in Group B (i.e., upon repeated dosing at 3 mg/kg) resulted in a 7-fold increase in luciferase expression. Also unexpectedly, a 10-fold increase in the amount of cccDNA vector upon repeated dosing (i.e., 10 mg/kg repeated dosing) in group C resulted in a 17-fold increase in luciferase expression. Thus, a second administration (i.e., repeat dose) of cccDNA increased expression by at least 7-fold, and even up to 17-fold. This shows that the increase in transgene expression from the repeat dose is greater than expected and is dependent on the dose or amount of cccDNA vector at the repeat dose and appears to be synergistic with the initial transgene expression after the initial priming on day 0. Thus, the dose-dependent increase in transgene expression is not additive; rather, the level of transgene expression is dose-dependent and super-proportional to the total amount of cccDNA vector introduced at each time point.

[00546] Both Groups B and C showed a significant dose-dependent increase in luciferase expression compared to control mice (Group A) that did not receive a repeat dose of the cccDNA vector at the second time point. Taken together, these data indicate that transgene expression from a cccDNA vector can be increased in a dose-dependent manner by re-dosing (ie, reintroducing) the cccDNA vector at at least a second time point.

[00547] Taken together, the data in Figure 6 demonstrate that the level of transgene expression from cccDNA vectors can be maintained at a constant level for at least 84 days and can be increased in vivo after a repeated dose of cccDNA vector administered at least at least at the second point in time.

EXAMPLE 7: Stable expression of a transgene in vivo with cDNA vectors prepared as compositions in LNPs

[00548] The reproducibility of the results obtained in Example 6 with another lipid nanoparticle was assessed in vivo in mice. Mice were dosed on day 0 with either a cccDNA vector containing a luciferase transgene driven by the CAG promoter encapsulated in LNPs different from those used in Example 6, or the same LNPs containing poly(C) but without the ccDNA or luciferase gene. Specifically, male CD-1® mice, approximately 4 weeks of age, received a single injection of 0.5 mg/kg LNP-TTX (tetrodotoxin)-luciferase or control LNP-poly(C) administered intravenously via the lateral tail vein on day 0. B day 14, animals were administered systemic luciferin at a dose of 150 mg/kg via intraperitoneal injection at a dose of 2.5 ml/kg. Approximately 15 minutes after luciferin administration, each animal was imaged using an in vivo imaging system (“IVIS”).

[00549] As shown in Figure 7, significant fluorescence in the liver was observed in all four mice that received cccDNA, and very little fluorescence was observed in the animals beyond the injection site, indicating that LNPs mediate liver-specific delivery of the cccDNA construct, and that the delivered cccDNA vector was capable of controlled, sustained expression of its transgene for at least two weeks after administration.

EXAMPLE 8: Sustained expression of a transgene in the liver in vivo after administration of a cccDNA vector

[00550] In a separate experiment, the localization of LNP-delivered cccDNA in the liver of experimental animals was assessed. The cccDNA vector containing the functional transgene of interest was encapsulated in the same LNPs as used in Example 7 and administered to mice in vivo at a dose of 0.5 mg/kg by intravenous injection. After 6 hours, mice were sacrificed and liver samples were collected, formalin fixed, and paraffin embedded using standard protocols. RNAscope® in situ hybridization experiments were performed to visualize cccDNA vectors in tissue using a probe specific for the ccDNA transgene and detection using a chromogenic reaction and hematoxylin staining (Advanced Cell Diagnostics). Figure 8 presents results indicating the presence of cctDNA in hepatocytes.

EXAMPLE 9: Sustained expression of cDNA transgene in eyes in vivo

[00551] The persistence of cccDNA vector transgene expression in tissues other than the liver was assessed to determine the tolerability and expression of the cccDNA vector following ocular administration in vivo. On day 0, male Sprague Dawley rats, approximately 9 weeks old, were injected subretinal with 5 μl of either cDNA vector containing the luciferase transgene, formulated with jetPEI® transfection reagent (Polyplus), or plasmid DNA encoding luciferase, formulated with "jetPEI®", in both cases at a concentration of 0.25 µg/µl. Four rats were tested in each group. Animals were sedated and the test drug was injected subretinal into the right eye using a 33-gauge needle. The left eye of each animal was left intact. Immediately after injection, eyes were examined by optical coherence tomography or fundus imaging to confirm the presence of a subretinal bleb. Rats received buprenorphine and topical antibiotic ointment according to standard procedures.

[00552] On days 7, 14, 21, 28 and 35, animals in both groups were systemically administered freshly prepared luciferin at a dose of 150 mg/kg via intraperitoneal injection in an amount of 2.5 ml/kg. 5-15 minutes after luciferin administration, all animals were imaged using IVIS under isoflurane anesthesia. Total flux [photon/s] and average flux (photon/s/sr/cm ² ) in the region of interest covering the eye were obtained with an exposure of 5 minutes. The results are presented graphically as the average luminosity for each experimental group in the treated eye ( "injected") relative to the luminosity increments for each experimental group in the untreated eye ("uninjected") (Fig. 9B). Significant fluorescence was easily detected in eyes with the cDNA vector but was much weaker in eyes with the plasmid (Figure 9A). After 35 days, plasmid-injected rats were sacrificed, while studies continued in cccDNA-injected rats with luciferin injection and IVIS imaging on days 42, 49, 56, 63, 70, and 99. The results demonstrate that the cccDNA vector administered by a single injection into rat eye, mediates transgene expression in vivo, and such expression was maintained at a high level for at least 99 days after injection.

EXAMPLE 10: Long-term dosing and repeated dosing of cccDNA vector in Rag2 mice

[00553] In situations where one or more transgenes encoded in a cccDNA vector gene expression cassette are expressed in a host environment (e.g., a cell or subject), and the expressed protein is recognized as foreign, there is a possibility that the host will develop adaptive immune response, which can lead to unwanted depletion of the expression product, which may be mistaken for lack of expression. In some cases, this may occur with a reporter molecule that is heterologous to the normal host environment. Accordingly, cccDNA vector transgene expression was assessed in vivo in the Rag2 mouse model, which lacks B and T cells and therefore does not mount an adaptive immune response to non-mouse-native proteins such as luciferase. Briefly, c57bl/6 and Rag2 knockout mice were treated on day 0 with an intravenous tail vein injection of 0.5 mg/kg LNP-encapsulated ccDNA vector expressing luciferase or poly(C) control, and on day 21 some mice were given a repeat dose of either the same LNP-encapsulated cccDNA vector at the same dose level. All experimental groups consisted of 4 mice each. IVIS imaging was performed after luciferin injection as described in Example 9 at weekly intervals.

[00554] When comparing the total flux observed in IVIS assays, the fluorescence observed in wild-type mice (an indirect measure of the presence of expressed luciferase) injected with LNP-ccDNA-vector-Luc gradually decreased after day 21, while Rag2 mice , which were administered the same drug, showed relatively constant, stable luciferase expression throughout the 42 days of the experiment (Fig. 10A). The observed decline in wild-type mice around day 21 corresponds to the time window in which an adaptive immune response can be expected to emerge. Repeated administration of the LNP-ccDNA vector to Rag2 mice resulted in a marked increase in expression that was robustly maintained for at least the 21 days observed in this study (Figure 10B). The results suggest that adaptive immunity may play a role in expression of a non-native protein from a cccDNA vector in the host, and that the observed decrease in expression at 20+ days from initial administration may indicate an interfering adaptive immune response to the expressed molecule rather than to decrease expression (or in addition to it). It should be noted that this response is expected to be low when native proteins are expressed in the host, where presumably the host will correctly recognize the expressed molecules as self and will not mount such an immune response.

EXAMPLE 11: Effect of liver-specific expression and CpG modulation on sustained expression

[00555] As described in Example 10, an undesirable host immune response may in some cases artificially dampen what would otherwise be robust expression of one or more desired transgenes from the introduced cccDNA vector. Two approaches were used to evaluate the effect of preventing and/or attenuating a potential host immune response on sustained expression from a cccDNA vector. First, since the cDNA-Luc vector used in the previous examples was under the control of the constitutive CAG promoter, a similar construct was generated using the liver-specific promoter (hAAT) or another constitutive promoter (hEF-1) to determine whether eliminating long-term exposure to myeloid cells or non-liver tissues will reduce any observed immune effects. Second, some cDNA-luciferase constructs have been engineered to have reduced CpG content, a known trigger of the host immune response. The expression of the cccDNA-encoded luciferase gene was measured when these modified cccDNA vectors with promoter switching were administered to mice.

[00556] Three different cDNA vectors were used, each encoding a luciferase transgene. The first ccDNA vector had a large number of unmethylated CpGs (about 350) and contained a constitutive CAG promoter (“CAG ccDNA”); the second had a moderate amount of unmethylated CpG (about 60) and contained the liver-specific hAAT promoter (“low-CpG hAAT ccDNA”); and the third was a methylated form of the second, lacking the unmethylated CpG, and also containing the hAAT promoter (“hAAT ccDNA without CpG”). Otherwise, the cccDNA vectors were identical. Vectors were prepared as described above.

[00557] Four groups of four male CD-1® mice, approximately 4 weeks old, received one of the LNP-encapsulated cccDNA vectors or a poly(C) control. On day 0, each mouse received one intravenous tail vein injection of 0.5 mg/kg cccDNA vector in a volume of 5 ml/kg. Body weight was recorded on days -1, -, 1, 2, 3, 7 and weekly thereafter until killing mice. Whole blood and serum samples were collected on days 0, 1, and 35. Intravital imaging was performed on days 7, 14, 21, 28, and 35, and weekly thereafter using an in vivo imaging system (IVIS). For imaging, each mouse was administered luciferin at a dose of 150 mg/kg via intraperitoneal injection in a volume of 2.5 ml/kg. After 15 minutes, each mouse was anesthetized and imaged. Mice were sacrificed on day 93 and terminal tissues, including liver and spleen, were collected. Cytokine measurements were performed 6 hours post-dose on day 0.

[00558] Although all cccDNA-treated mice showed significant fluorescence on days 7 and 14, in the cccDNA CAG mice fluorescence decreased rapidly after day 14 and more gradually decreased throughout the remainder of the study. In contrast, in hAAT cDNA mice with low CpG and no CpG, total flux remained at a consistently high level (Figure 11). This suggests that targeting the cccDNA vector specifically to the liver resulted in sustained and long-lasting expression of the transgene from the vector for at least 77 days after a single injection. Constructs with minimal or no CpG content had similar long-term sustained expression profiles, whereas the constitutive promoter construct with high CpG content showed a decrease in expression over time, suggesting that host immune activation upon introduction of the cccDNA vector may play a role. in any decrease in expression observed when such a vector is administered to a subject. These results provide alternative methods for tailoring the duration of the response to the desired level by selecting a tissue-restricted promoter and/or changing the CpG content of the cDNA vector in the event of an observed host immune response, a potentially transgene-specific response.

LINKS TO SOURCES

[00559] All publications and references, including, without limitation, patents and patent applications referred to in this specification and the examples provided herein, are incorporated by reference in their entirety as if specifically and individually for each individual publication or reference is indicated to be incorporated by reference herein. Any patent application for which this application claims priority is also incorporated herein by reference in the manner described above for publications and references.

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LIST OF SEQUENCES

<110>GENERATION BIO CO.

<120>CONTROLLED EXPRESSION OF TRANSGENES USING

DNA VECTORS

CLOSED END (ccDNA)

<130>080170-091190-WOPT

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<400>20

aggaacccct agtgatggag ttggccactc cctctctgcg cgctcgctcg ctcactgagg 60

ccgggcgaaa atcgcccgac gcccgggctt tgcccgggcg gcctcagtga gcgagcgagc

120

gcgcagctgc ctgcagg 137

<210>21

<211>135

<212>DNA

<213>Artificial sequence

<220>

<223>Description of artificial sequence: Synthetic

polynucleotide

<400>21

aggaacccct agtgatggag ttggccactc cctctctgcg cgctcgctcg ctcactgagg 60

ccgggcgaaa cgcccgacgc ccgggctttg cccgggcggc ctcagtgagc gagcgagcgc

120

gcagctgcct gcagg 135

<210>22

<211>133

<212>DNA

<213>Artificial sequence

<220>

<223>Description of artificial sequence: Synthetic

polynucleotide

<400>22

aggaacccct agtgatggag ttggccactc cctctctgcg cgctcgctcg ctcactgagg 60

ccgggcaaag cccgacgccc gggctttgcc cgggcggcct cagtgagcga gcgagcgcgc

120

agctgcctgc agg 133

<210>23

<211>139

<212>DNA

<213>Artificial sequence

<220>

<223>Description of artificial sequence: Synthetic

polynucleotide

<400>23

aggaacccct agtgatggag ttggccactc cctctctgcg cgctcgctcg ctcactgagg 60

ccgggcgacc aaaggtcgcc cgacgcccgg gtttcccggg cggcctcagt gagcgagcga

120

gcgcgcagct gcctgcagg 139

<210>24

<211>137

<212>DNA

<213>Artificial sequence

<220>

<223>Description of artificial sequence: Synthetic

polynucleotide

<400>24

aggaacccct agtgatggag ttggccactc cctctctgcg cgctcgctcg ctcactgagg 60

ccgggcgacc aaaggtcgcc cgacgcccgg tttccgggcg gcctcagtga gcgagcgagc

120

gcgcagctgc ctgcagg 137

<210>25

<211>135

<212>DNA

<213>Artificial sequence

<220>

<223>Description of artificial sequence: Synthetic

polynucleotide

<400>25

aggaacccct agtgatggag ttggccactc cctctctgcg cgctcgctcg ctcactgagg 60

ccgggcgacc aaaggtcgcc cgacgcccgt ttcgggcggc ctcagtgagc gagcgagcgc

120

gcagctgcct gcagg 135

<210>26

<211>133

<212>DNA

<213>Artificial sequence

<220>

<223>Description of artificial sequence: Synthetic

polynucleotide

<400>26

aggaacccct agtgatggag ttggccactc cctctctgcg cgctcgctcg ctcactgagg 60

ccgggcgacc aaaggtcgcc cgacgccctt tgggcggcct cagtgagcga gcgagcgcgc

120

agctgcctgc agg 133

<210>27

<211>131

<212>DNA

<213>Artificial sequence

<220>

<223>Description of artificial sequence: Synthetic

polynucleotide

<400>27

aggaacccct agtgatggag ttggccactc cctctctgcg cgctcgctcg ctcactgagg 60

ccgggcgacc aaaggtcgcc cgacgccttt ggcggcctca gtgagcgagc gagcgcgcag

120

ctgcctgcag g 131

<210>28

<211>129

<212>DNA

<213>Artificial sequence

<220>

<223>Description of artificial sequence: Synthetic

polynucleotide

<400>28

aggaacccct agtgatggag ttggccactc cctctctgcg cgctcgctcg ctcactgagg 60

ccgggcgacc aaaggtcgcc cgacgctttg cggcctcagt gagcgagcga gcgcgcagct

120

gcctgcagg 129

<210>29

<211>127

<212>DNA

<213>Artificial sequence

<220>

<223>Description of artificial sequence: Synthetic

polynucleotide

<400>29

aggaacccct agtgatggag ttggccactc cctctctgcg cgctcgctcg ctcactgagg 60

ccgggcgacc aaaggtcgcc cgacgtttcg gcctcagtga gcgagcgagc gcgcagctgc

120

ctgcagg 127

<210>30

<211>122

<212>DNA

<213>Artificial sequence

<220>

<223>Description of artificial sequence: Synthetic

polynucleotide

<400>30

aggaacccct agtgatggag ttggccactc cctctctgcg cgctcgctcg ctcactgagg 60

ccgggcgacc aaaggtcgcc cgacggcctc agtgagcgag cgagcgcgca gctgcctgca

120

gg 122

<210>31

<211>130

<212>DNA

<213>Artificial sequence

<220>

<223>Description of artificial sequence: Synthetic

polynucleotide

<400>31

aggaacccct agtgatggag ttggccactc cctctctgcg cgctcgctcg ctcactgagg 60

ccgggcgacc aaaggtcgcc cgacgcccgg gcggcctcag tgagcgagcg agcgcgcagc

120

tgcctgcagg 130

<210>32

<211>120

<212>DNA

<213>Artificial sequence

<220>

<223>Description of artificial sequence: Synthetic

polynucleotide

<400>32

cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggaaacc cgggcgtgcg 60

cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact aggggttcct

120

<210>33

<211>122

<212>DNA

<213>Artificial sequence

<220>

<223>Description of artificial sequence: Synthetic

polynucleotide

<400>33

cctgcaggca gctgcgcgct cgctcgctca ctgaggccgt cgggcgacct ttggtcgccc 60

ggcctcagtg agcgagcgag cgcgcagaga gggagtggcc aactccatca ctaggggttc

120

ct 122

<210>34

<211>122

<212>DNA

<213>Artificial sequence

<220>

<223>Description of artificial sequence: Synthetic

polynucleotide

<400>34

cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcaaag cccgggcgtc 60

ggcctcagtg agcgagcgag cgcgcagaga gggagtggcc aactccatca ctaggggttc

120

ct 122

<210>35

<211>129

<212>DNA

<213>Artificial sequence

<220>

<223>Description of artificial sequence: Synthetic

polynucleotide

<400>35

cctgcaggca gctgcgcgct cgctcgctca ctgaggcgcc cgggcgtcgg gcgacctttg 60

gtcgcccggc ctcagtgagc gagcgagcgc gcagagaggg agtggccaac tccatcacta

120

ggggttcct 129

<210>36

<211>101

<212>DNA

<213>Artificial sequence

<220>

<223>Description of artificial sequence: Synthetic

polynucleotide

<400>36

cctgcaggca gctgcgcgct cgctcgctca ctgaggcaaa gcctcagtga gcgagcgagc 60

gcgcagagag ggagtggcca actccatcac taggggttcc t 101

<210>37

<211>139

<212>DNA

<213>Artificial sequence

<220>

<223>Description of artificial sequence: Synthetic

polynucleotide

<400>37

cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcaaag cccgggcgtc 60

gggcgacttt gtcgcccggc ctcagtgagc gagcgagcgc gcagagaggg agtggccaac

120

tccatcacta ggggttcct 139

<210>38

<211>137

<212>DNA

<213>Artificial sequence

<220>

<223>Description of artificial sequence: Synthetic

polynucleotide

<400>38

cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcaaag cccgggcgtc 60

gggcgatttt cgcccggcct cagtgagcga gcgagcgcgc agagagggag tggccaactc

120

catcactagg ggttcct 137

<210>39

<211>135

<212>DNA

<213>Artificial sequence

<220>

<223>Description of artificial sequence: Synthetic

polynucleotide

<400>39

cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcaaag cccgggcgtc 60

gggcgtttcg cccggcctca gtgagcgagc gagcgcgcag agagggagtg gccaactcca

120

tcactagggg ttcct 135

<210>40

<211>133

<212>DNA

<213>Artificial sequence

<220>

<223>Description of artificial sequence: Synthetic

polynucleotide

<400>40

cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcaaag cccgggcgtc 60

gggctttgcc cggcctcagt gagcgagcga gcgcgcagag agggagtggc caactccatc

120

actaggggtt cct 133

<210>41

<211>139

<212>DNA

<213>Artificial sequence

<220>

<223>Description of artificial sequence: Synthetic

polynucleotide

<400>41

cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggaaacc cgggcgtcgg 60

gcgacctttg gtcgcccggc ctcagtgagc gagcgagcgc gcagagaggg agtggccaac

120

tccatcacta ggggttcct 139

<210>42

<211>137

<212>DNA

<213>Artificial sequence

<220>

<223>Description of artificial sequence: Synthetic

polynucleotide

<400>42

cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccggaaaccg ggcgtcgggc 60

gacctttggt cgcccggcct cagtgagcga gcgagcgcgc agagagggag tggccaactc

120

catcactagg ggttcct 137

<210>43

<211>135

<212>DNA

<213>Artificial sequence

<220>

<223>Description of artificial sequence: Synthetic

polynucleotide

<400>43

cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgaaacggg cgtcgggcga 60

cctttggtcg cccggcctca gtgagcgagc gagcgcgcag agagggagtg gccaactcca

120

tcactagggg ttcct 135

<210>44

<211>133

<212>DNA

<213>Artificial sequence

<220>

<223>Description of artificial sequence: Synthetic

polynucleotide

<400>44

cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccaaagggcg tcgggcgacc 60

tttggtcgcc cggcctcagt gagcgagcga gcgcgcagag agggagtggc caactccatc

120

actaggggtt cct 133

<210>45

<211>131

<212>DNA

<213>Artificial sequence

<220>

<223>Description of artificial sequence: Synthetic

polynucleotide

<400>45

cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc caaaggcgtc gggcgacctt 60

tggtcgcccg gcctcagtga gcgagcgagc gcgcagagag ggagtggcca actccatcac

120

taggggttcct 131

<210>46

<211>129

<212>DNA

<213>Artificial sequence

<220>

<223>Description of artificial sequence: Synthetic

polynucleotide

<400>46

cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc aaagcgtcgg gcgacctttg 60

gtcgcccggc ctcagtgagc gagcgagcgc gcagagaggg agtggccaac tccatcacta

120

ggggttcct 129

<210>47

<211>127

<212>DNA

<213>Artificial sequence

<220>

<223>Description of artificial sequence: Synthetic

polynucleotide

<400>47

cctgcaggca gctgcgcgct cgctcgctca ctgaggccga aacgtcgggc gacctttggt 60

cgcccggcct cagtgagcga gcgagcgcgc agagagggag tggccaactc catcactagg

120

ggttcct 127

<210>48

<211>122

<212>DNA

<213>Artificial sequence

<220>

<223>Description of artificial sequence: Synthetic

polynucleotide

<400>48

aggaacccct agtgatggag ttggccactc cctctctgcg cgctcgctcg ctcactgagg 60

ccgggcgacc aaaggtcgcc cgacggcctc agtgagcgag cgagcgcgca gctgcctgca

120

gg 122

<210>49

<211>12

<212>DNA

<213>Artificial sequence

<220>

<223>Description of artificial sequence: Synthetic

oligonucleotide

<400>49

cgatcgttcg at 12

<210>50

<211>12

<212>DNA

<213>Artificial sequence

<220>

<223>Description of artificial sequence: Synthetic

oligonucleotide

<400>50

atcgaaccat cg 12

<210>51

<211>12

<212>DNA

<213>Artificial sequence

<220>

<223>Description of artificial sequence: Synthetic

oligonucleotide

<400>51

atcgaacgat cg 12

<210>52

<211>165

<212>DNA

<213>Artificial sequence

<220>

<223>Description of artificial sequence: Synthetic

polynucleotide

<400>52

aggaacccct agtgatggag ttggccactc cctctctgcg cgctcgctcg ctcactgagg 60

ccgcccgggc aaagcccggg cgtcgggcga cctttggtcg cccggcctca gtgagcgagc

120

gagcgcgcag agagggagtg gccaactcca tcactagggg ttcct 165

<210>53

<211>140

<212>DNA

<213>Artificial sequence

<220>

<223>Description of artificial sequence: Synthetic

polynucleotide

<400>53

cccctagtga tggagttggc cactccctct ctgcgcgctc gctcgctcac tgaggccgcc 60

cggggcaaagc ccgggcgtcg ggcgaccttt ggtcgcccgg cctcagtgag cgagcgagcg

120

cgcagagaga tcactagggg 140

<210>54

<211>91

<212>DNA

<213>Artificial sequence

<220>

<223>Description of artificial sequence: Synthetic

oligonucleotide

<400>54

gcgcgctcgc tcgctcactg aggccgcccg ggcaaagccc gggcgtcggg cgacctttgg 60

tcgcccggcc tcagtgagcg agcgagcgcg c 91

<210>55

<211>91

<212>DNA

<213>Artificial sequence

<220>

<223>Description of artificial sequence: Synthetic

oligonucleotide

<400>55

gcgcgctcgc tcgctcactg aggccgggcg accaaaggtc gcccgacgcc cgggctttgc 60

ccgggcggcc tcagtgagcg agcgagcgcg c 91

<210>56

<211>1662

<212>DNA

<213>Artificial sequence

<220>

<223>Description of artificial sequence: Synthetic

polynucleotide

<400>56

gccgccacca tggaagacgc caaaaacata aagaaaggcc cggcgccatt ctatccgctg 60

gaagatggaa ccgctggaga gcaactgcat aaggctatga agagatacgc cctggttcct

120

ggaacaattg cttttacaga tgcacatatc gaggtggaca tcacttacgc tgagtacttc

180

gaaatgtccg ttcggttggc agaagctatg aaacgatatg ggctgaatac aaatcacaga

240

atcgtcgtat gcagtgaaaa ctctcttcaa ttctttatgc cggtgttggg cgcgttattt

300

atcggagttg cagttgcgcc cgcgaacgac atttataatg aacgtgaatt gctcaacagt

360

atgggcattt cgcagcctac cgtggtgttc gtttccaaaa aggggttgca aaaaattttg

420

aacgtgcaaa aaaagctccc aatcatccaa aaaattatta tcatggattc taaaacggat

480

taccagggat ttcagtcgat gtacacgttc gtcacatctc atctacctcc cggttttaat

540

gaatacgatt ttgtgccaga gtccttcgat agggacaaga caattgcact gatcatgaac

600

tcctctggat ctactggtct gcctaaaggt gtcgctctgc ctcatagaac tgcctgcgtg

660

agattctcgc atgccagaga tcctattttt ggcaatcaaa tcattccgga tactgcgatt

720

ttaagtgttg ttccattcca tcacggtttt ggaatgttta ctacactcgg atatttgata

780

tgtggatttc gagtcgtctt aatgtataga tttgaagaag agctgtttct gaggagcctt

840

caggattaca agattcaaag tgcgctgctg gtgccaaccc tattctcctt cttcgccaaa

900

agcactctga ttgacaaata cgatttatct aatttacacg aaattgcttc tggtggcgct

960

cccctctcta aggaagtcgg ggaagcggtt gccaagaggt tccatctgcc aggtatcagg

1020

caaggatatg ggctcactga gactacatca gctattctga ttacacccga gggggatgat

1080

aaaccgggcg cggtcggtaa agttgttcca ttttttgaag cgaaggttgt ggatctggat

1140

accgggaaaa cgctgggcgt taatcaaaga ggcgaactgt gtgtgagagg tcctatgatt

1200

atgtccggtt atgtaaacaa tccggaagcg accaacgcct tgattgacaa ggatggatgg

1260

ctacattctg gagacatagc ttactgggac gaagacgaac acttcttcat cgttgaccgc

1320

ctgaagtctc tgattaagta caaaggctat caggtggctc ccgctgaatt ggaatccatc

1380

ttgctccaac accccaacat cttcgacgca ggtgtcgcag gtcttcccga cgatgacgcc

1440

ggtgaacttc ccgccgccgt tgttgttttg gagcacggaa agacgatgac ggaaaaagag

1500

atcgtggatt acgtcgccag tcaagtaaca accgcgaaaa agttgcgcgg aggagttgtg

1560

tttgtggacg aagtaccgaa aggtcttacc ggaaaactcg acgcaagaaa aatcagagag

1620

atcctcataa aggccaagaa gggcggaaag atcgccgtgt aa 1662

<210>57

<211>453

<212>PROTEIN

<213>Artificial sequence

<220>

<223>Description of artificial sequence: Synthetic

polypeptide

<220>

<221>MOD_RES

<222>(1)..(1)

<223>Any amino acid

<400>57

Xaa Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ala Phe Ser Ser Tyr

20 25 30

Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Val Ile Trp Phe Asp Gly Thr Lys Lys Tyr Tyr Thr Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Thr Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Asp Arg Gly Ile Gly Ala Arg Arg Gly Pro Tyr Tyr Met Asp

100 105 110

Val Trp Gly Lys Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys

115 120 125

Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly

130 135 140

Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro

145 150 155 160

Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr

165 170 175

Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val

180 185 190

Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn

195 200 205

Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro

210 215 220

Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu

225 230 235 240

Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp

245 250 255

Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp

260 265 270

Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly

275 280 285

Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn

290 295 300

Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp

305 310 315 320

Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro

325 330 335

Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu

340 345 350

Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn

355 360 365

Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile

370 375 380

Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr

385 390 395 400

Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys

405 410 415

Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys

420 425 430

Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu

435 440 445

Ser Leu Ser Pro Gly

450

<210>58

<211>214

<212>PROTEIN

<213>Artificial sequence

<220>

<223>Description of artificial sequence: Synthetic

polypeptide

<400>58

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser Thr Pro Leu

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210>59

<211>1310

<212>DNA

<213>Artificial sequence

<220>

<223>Description of artificial sequence: Synthetic

polynucleotide

<400>59

ggagccgaga gtaattcata caaaaggagg gatcgccttc gcaaggggag agcccaggga 60

ccgtccctaa attctcacag acccaaatcc ctgtagccgc cccacgacag cgcgaggagc

120

atgcgctcag ggctgagcgc ggggagagca gagcacacaa gctcatagac cctggtcgtg

180

ggggggagga ccggggagct ggcgcggggc aaactgggaa agcggtgtcg tgtgctggct

240

ccgccctctt cccgagggtg ggggagaacg gtatataagt gcggcagtcg ccttggacgt

300

tctttttcgc aacgggtttg ccgtcagaac gcaggtgagg ggcgggtgtg gcttccgcgg

360

gccgccgagc tggaggtcct gctccgagcg ggccggggccc cgctgtcgtc ggcggggatt

420

agctgcgagc attcccgctt cgagttgcgg gcggcgcggg aggcagagtg cgaggcctag

480

cggcaacccc gtagcctcgc ctcgtgtccg gcttgaggcc tagcgtggtg tccgcgccgc

540

cgccgcgtgc tactccggcc gcactctggt cttttttttt tttgttgttg ttgccctgct

600

gccttcgatt gccgttcagc aataggggct aacaaaggga gggtgcgggg cttgctcgcc

660

cggagcccgg agaggtcatg gttggggagg aatggaggga caggagtggc ggctggggcc

720

cgcccgcctt cggagcacat gtccgacgcc acctggatgg ggcgaggcct ggggtttttc

780

ccgaagcaac caggctgggg ttagcgtgcc gaggccatgt ggccccagca cccggcacga

840

tctggcttgg cggcgccgcg ttgccctgcc tccctaacta gggtgaggcc atcccgtccg

900

gcaccagttg cgtgcgtgga aagatggccg ctcccggggcc ctgttgcaag gagctcaaaa

960

tggaggacgc ggcagcccgg tggagcgggc gggtgagtca cccacacaaa ggaagagggc

1020

ctggtccctc accggctgct gcttcctgtg accccgtggt cctatcggcc gcaatagtca

1080

cctcgggctt ttgagcacgg ctagtcgcgg cggggggagg ggatgtaatg gcgttggagt

1140

ttgttcacat ttggtgggtg gagactagtc aggccagcct ggcgctggaa gtcatttttg

1200

gaatttgtcc ccttgagttt tgagcggagc taattctcgg gcttcttagc ggttcaaagg

1260

tatcttttaa accctttttt aggtgttgtg aaaaccaccg ctaattcaaa 1310

<210>60

<211>16

<212>DNA

<213>Artificial sequence

<220>

<223>Description of artificial sequence: Synthetic

oligonucleotide

<400>60

gcgcgctcgc tcgctc 16

<210>61

<211>6

<212>DNA

<213>Artificial sequence

<220>

<223>Description of artificial sequence: Synthetic

oligonucleotide

<400>61

ggttga 6

<210>62

<211>4

<212>DNA

<213>Artificial sequence

<220>

<223>Description of artificial sequence: Synthetic

oligonucleotide

<400>62

agtt 4

<210>63

<211>6

<212>DNA

<213>Artificial sequence

<220>

<223>Description of artificial sequence: Synthetic

oligonucleotide

<400>63

ggttgg 6

<210>64

<211>6

<212>DNA

<213>Artificial sequence

<220>

<223>Description of artificial sequence: Synthetic

oligonucleotide

<400>64

agttgg 6

<210>65

<211>6

<212>DNA

<213>Artificial sequence

<220>

<223>Description of artificial sequence: Synthetic

oligonucleotide

<400>65

agttga 6

<210>66

<211>6

<212>DNA

<213>Artificial sequence

<220>

<223>Description of artificial sequence: Synthetic

oligonucleotide

<400>66

rrttrr 6

<210>67

<211>581

<212>DNA

<213>Artificial sequence

<220>

<223>Description of artificial sequence: Synthetic

polynucleotide

<400>67

gagcatctta ccgccattta ttcccatatt tgttctgttt ttcttgattt gggtatacat 60

ttaaatgtta ataaaacaaa atggtggggc aatcatttac atttttaggg atatgtaatt

120

actagttcag gtgtattgcc acaagacaaa catgttaaga aactttcccg ttatttacgc

180

tctgttcctg ttaatcaacc tctggattac aaaatttgtg aaagattgac tgatattctt

240

aactatgttg ctccttttac gctgtgtgga tatgctgctt tatagcctct gtatctagct

300

attgcttccc gtacggcttt cgttttctcc tccttgtata aatcctggtt gctgtctctt

360

ttagaggagt tgtggcccgt tgtccgtcaa cgtggcgtgg tgtgctctgt gtttgctgac

420

gcaaccccca ctggctgggg cattgccacc acctgtcaac tcctttctgg gactttcgct

480

ttccccctcc cgatcgccac ggcagaactc atcgccgcct gccttgcccg ctgctggaca

540

ggggctaggt tgctgggcac tgataattcc gtggtgttgt c 581

<210>68

<211>225

<212>DNA

<213>Artificial sequence

<220>

<223>Description of artificial sequence: Synthetic

polynucleotide

<400>68

tgtgccttct agttgccagc catctgttgt ttgcccctcc cccgtgcctt ccttgaccct 60

ggaaggtgcc actcccactg tcctttccta ataaaatgag gaaattgcat cgcattgtct

120

gagtaggtgt cattctattc tggggggtgg ggtggggcag gacagcaagg gggaggattg

180

ggaagacaat agcaggcatg ctggggatgc ggtgggctct atggc 225

<210>69

<211>8

<212>DNA

<213>Artificial sequence

<220>

<223>Description of artificial sequence: Synthetic

oligonucleotide

<400>69

actgaggc 8

<210>70

<211>8

<212>DNA

<213>Artificial sequence

<220>

<223>Description of artificial sequence: Synthetic

oligonucleotide

<400>70

gcctcagt 8

<210>71

<211>16

<212>DNA

<213>Artificial sequence

<220>

<223>Description of artificial sequence: Synthetic

oligonucleotide

<400>71

gagcgagcga gcgcgc 16

<210>72

<211>1923

<212>DNA

<213>Artificial sequence

<220>

<223>Description of artificial sequence: Synthetic

polynucleotide

<400>72

tcaatattgg ccattagcca tattattcat tggttatata gcataaatca atattggcta 60

ttggccattg catacgttgt atctatatca taatatgtac atttatattg gctcatgtcc

120

aatatgaccg ccatgttggc attgattatt gactagttat taatagtaat caattacggg

180

gtcattagtt catagcccat atatggagtt ccgcgttaca taacttacgg taaatggccc

240

gcctggctga ccgcccaacg acccccgccc attgacgtca ataatgacgt atgttcccat

300

agtaacgcca atagggactt tccattgacg tcaatgggtg gagtatttac ggtaaactgc

360

ccacttggca gtacatcaag tgtatcatat gccaagtccg ccccctattg acgtcaatga

420

cggtaaatgg cccgcctggc attatgccca gtacatgacc ttacgggact ttcctacttg

480

gcagtacatc tacgtattag tcatcgctat taccatggtc gaggtgagcc ccacgttctg

540

cttcactctc cccatctccc ccccctcccc acccccaatt ttgtatttat ttatttttta

600

attattttgt gcagcgatgg gggcgggggg gggggggggg cgcgcgccag gcggggcggg

660

gcggggcgag gggcggggcg gggcgaggcg gagaggtgcg gcggcagcca atcagagcgg

720

cgcgctccga aagtttcctt ttatggcgag gcggcggcgg cggcggccct ataaaaagcg

780

aagcgcgcgg cgggcgggag tcgctgcgac gctgccttcg ccccgtgccc cgctccgccg

840

ccgcctcgcg ccgcccgccc cggctctgac tgaccgcgtt actcccacag gtgagcgggc

900

gggacggccc ttctcctccg ggctgtaatt agcgcttggt ttaatgacgg cttgtttctt

960

ttctgtggct gcgtgaaagc cttgaggggc tccgggaggg ccctttgtgc gggggggagc

1020

ggctcggggg gtgcgtgcgt gtgtgtgtgc gtggggagcg ccgcgtgcgg cccgcgctgc

1080

ccggcggctg tgagcgctgc gggcgcggcg cggggctttg tgcgctccgc agtgtgcgcg

1140

aggggagcgc ggccgggggc ggtgccccgc ggtgcggggg gggctgcgag gggaacaaag

1200

gctgcgtgcg gggtgtgtgc gtgggggggt gagcaggggg tgtgggcgcg gcggtcgggc

1260

tgtaaccccc ccctgcaccc ccctccccga gttgctgagc acggcccggc ttcgggtgcg

1320

gggctccgta cggggcgtgg cgcggggctc gccgtgccgg gcggggggtg gcggcaggtg

1380

ggggtgccgg gcggggcggg gccgcctcgg gccggggagg gctcggggga ggggcgcggc

1440

ggcccccgga gcgccggcgg ctgtcgaggc gcggcgagcc gcagccattg ccttttatgg

1500

taatcgtgcg agagggcgca gggacttcct ttgtcccaaa tctgtgcgga gccgaaatct

1560

gggaggcgcc gccgcacccc ctctagcggg cgcggggcga agcggtgcgg cgccggcagg

1620

aaggaaatgg gcggggaggg ccttcgtgcg tcgccgcgcc gccgtcccct tctccctctc

1680

cagcctcggg gctgtccgcg gggggacggc tgccttcggg ggggacgggg cagggcgggg

1740

ttcggcttct ggcgtgtgac cggcggctct agagcctctg ctaaccatgt tttagccttc

1800

ttctttttcc tacagctcct gggcaacgtg ctggttattg tgctgtctca tcatttgtcg

1860

acagaattcc tcgaagatcc gaaggggttc aagcttggca ttccggtact gttggtaaag

1920

cca 1923

<210>73

<211>1272

<212>DNA

<213>Artificial sequence

<220>

<223>Description of artificial sequence: Synthetic

polynucleotide

<400>73

aggctcagag gcacacagga gtttctgggc tcaccctgcc cccttccaac ccctcagttc 60

ccatcctcca gcagctgttt gtgtgctgcc tctgaagtcc acactgaaca aacttcagcc

120

tactcatgtc cctaaaatgg gcaaacattg caagcagcaa acagcaaaca cacagccctc

180

cctgcctgct gaccttggag ctggggcaga ggtcagagac ctctctgggc ccatgccacc

240

tccaacatcc actcgacccc ttggaatttc ggtggagagg agcagaggtt gtcctggcgt

300

ggtttaggta gtgtgagagg gtccgggttc aaaaccactt gctgggtggg gagtcgtcag

360

taagtggcta tgccccgacc ccgaagcctg tttccccatc tgtacaatgg aaatgataaa

420

gacgcccatc tgatagggtt tttgtggcaa ataaacattt ggtttttttg ttttgttttg

480

ttttgttttt tgagatggag gtttgctctg tcgcccaggc tggagtgcag tgacacaatc

540

tcatctcacc acaaccttcc cctgcctcag cctcccaagt agctgggatt acaagcatgt

600

gccaccacac ctggctaatt ttctattttt agtagagacg ggtttctcca tgttggtcag

660

cctcagcctc ccaagtaact gggattacag gcctgtgcca ccacacccgg ctaatttttt

720

ctatttttga cagggacggg gtttcaccat gttggtcagg ctggtctaga ggtaccggat

780

cttgctacca gtggaacagc cactaaggat tctgcagtga gagcagaggg ccagctaagt

840

ggtactctcc cagagactgt ctgactcacg ccaccccctc caccttggac acaggacgct

900

gtggtttctg agccaggtac aatgactcct ttcggtaagt gcagtggaag ctgtacactg

960

cccaggcaaa gcgtccgggc agcgtaggcg ggcgactcag atcccagcca gtggacttag

1020

cccctgtttg ctcctccgat aactggggtg accttggtta atattcacca gcagcctccc

1080

ccgttgcccc tctggatcca ctgcttaaat acggacgagg acagggccct gtctcctcag

1140

cttcaggcac caccactgac ctgggacagt gaatccggac tctaaggtaa atataaaatt

1200

tttaagtgta taatgtgtta aactactgat tctaattgtt tctctctttt agattccaac

1260

ctttggaact ga 1272

<210>74

<211>1177

<212>DNA

<213>Artificial sequence

<220>

<223>Description of artificial sequence: Synthetic

polynucleotide

<400>74

ggctcagagg ctcagaggca cacaggagtt tctgggctca ccctgccccc ttccaacccc 60

tcagttccca tcctccagca gctgtttgtg tgctgcctct gaagtccaca ctgaacaaac

120

ttcagcctac tcatgtccct aaaatgggca aacattgcaa gcagcaaaca gcaaacacac

180

agccctccct gcctgctgac cttggagctg gggcagaggt cagagacctc tctgggccca

240

tgccacctcc aacatccact cgaccccttg gaatttcggt ggagaggagc agaggttgtc

300

ctggcgtggt ttaggtagtg tgagagggtc cgggttcaaa accacttgct gggtggggag

360

tcgtcagtaa gtggctatgc cccgaccccg aagcctgttt ccccatctgt acaatggaaa

420

tgataaagac gcccatctga tagggttttt gtggcaaata aacatttggt ttttttgttt

480

tgttttgttt tgttttttga gatggaggtt tgctctgtcg cccaggctgg agtgcagtga

540

cacaatctca tctcaccaca accttcccct gcctcagcct cccaagtagc tgggattaca

600

agcatgtgcc accacacctg gctaattttc tatttttagt agagacgggt ttctccatgt

660

tggtcagcct cagcctccca agtaactggg attacaggcc tgtgccacca cacccggcta

720

attttttcta tttttgacag ggacggggtt tcaccatgtt ggtcaggctg gtctagaggt

780

accggatctt gctaccagtg gaacagccac taaggattct gcagtgagag cagaggcca

840

gctaagtggt actctcccag agactgtctg actcacgcca ccccctccac cttggacaca

900

ggacgctgtg gtttctgagc caggtacaat gactcctttc ggtaagtgca gtggaagctg

960

tacactgccc aggcaaagcg tccgggcagc gtaggcgggc gactcagatc ccagccagtg

1020

gacttagccc ctgtttgctc ctccgataac tggggtgacc ttggttaata ttcaccagca

1080

gcctcccccg ttgcccctct ggatccactg cttaaatacg gacgaggaca gggccctgtc

1140

tcctcagctt caggcaccac cactgacctg ggacagt 1177

<210>75

<211>547

<212>DNA

<213>Artificial sequence

<220>

<223>Description of artificial sequence: Synthetic

polynucleotide

<400>75

ccctaaaatg ggcaaacatt gcaagcagca aacagcaaac acacagccct ccctgcctgc 60

tgaccttgga gctggggcag aggtcagaga cctctctggg cccatgccac ctccaacatc

120

cactcgaccc cttggaattt ttcggtggag aggagcagag gttgtcctgg cgtggtttag

180

gtagtgtgag aggggaatga ctcctttcgg taagtgcagt ggaagctgta cactgcccag

240

gcaaagcgtc cgggcagcgt aggcgggcga ctcagatccc agccagtgga cttagcccct

300

gtttgctcct ccgataactg gggtgacctt ggttaatatt caccagcagc ctcccccgtt

360

gcccctctgg atccactgct taaatacgga cgaggacagg gccctgtctc ctcagcttca

420

ggcaccacca ctgacctggg acagtgaatc cggactctaa ggtaaatata aaatttttaa

480

gtgtataatg tgttaaacta ctgattctaa ttgtttctct cttttagatt ccaacctttg

540

gaactga 547

<210>76

<211>556

<212>DNA

<213>Artificial sequence

<220>

<223>Description of artificial sequence: Synthetic

polynucleotide

<400>76

ccctaaaatg ggcaaacatt gcaagcagca aacagcaaac acacagccct ccctgcctgc 60

tgaccttgga gctggggcag aggtcagaga cctctctggg cccatgccac ctccaacatc

120

cactcgaccc cttggaattt cggtggagag gagcagaggt tgtcctggcg tggtttaggt

180

agtgtgagag gggaatgact cctttcggta agtgcagtgg aagctgtaca ctgcccaggc

240

aaagcgtccg ggcagcgtag gcgggcgact cagatcccag ccagtggact tagcccctgt

300

ttgctcctcc gataactggg gtgaccttgg ttaatattca ccagcagcct cccccgttgc

360

ccctctggat ccactgctta aatacggacg aggacactcg agggccctgt ctcctcagct

420

tcaggcacca ccactgacct gggacagtga atccggacat cgattctaag gtaaatataa

480

aatttttaag tgtataattt gttaaactac tgattctaat tgtttctctc ttttagattc

540

caacctttgg aactga 556

<210>77

<211>1179

<212>DNA

<213>Artificial sequence

<220>

<223>Description of artificial sequence: Synthetic

polynucleotide

<400>77

ggctccggtg cccgtcagtg ggcagagcgc acatcgccca cagtccccga gaagttgggg 60

ggaggggtcg gcaattgaac cggtgcctag agaaggtggc gcggggtaaa ctgggaaagt

120

gatgtcgtgt actggctccg cctttttccc gagggtgggg gagaaccgta tataagtgca

180

gtagtcgccg tgaacgttct ttttcgcaac gggtttgccg ccagaacaca ggtaagtgcc

240

gtgtgtggtt cccgcgggcc tggcctcttt acgggttatg gcccttgcgt gccttgaatt

300

acttccacct ggctgcagta cgtgattctt gatcccgagc ttcgggttgg aagtgggtgg

360

gagagttcga ggccttgcgc ttaaggagcc ccttcgcctc gtgcttgagt tgaggcctgg

420

cctgggcgct ggggccgccg cgtgcgaatc tggtggcacc ttcgcgcctg tctcgctgct

480

ttcgataagt ctctagccat ttaaaatttt tgatgacctg ctgcgacgct ttttttctgg

540

caagatagtc ttgtaaatgc gggccaagat ctgcacactg gtatttcggt ttttggggcc

600

gcgggcggcg acggggcccg tgcgtcccag cgcacatgtt cggcgaggcg gggcctgcga

660

gcgcggccac cgagaatcgg acggggggtag tctcaagctg gccggcctgc tctggtgcct

720

ggtctcgcgc cgccgtgtat cgccccgccc tgggcggcaa ggctggcccg gtcggcacca

780

gttgcgtgag cggaaagatg gccgcttccc ggccctgctg cagggagctc aaaatggagg

840

acgcggcgct cgggagagcg ggcgggtgag tcacccacac aaaggaaaag ggcctttccg

900

tcctcagccg tcgcttcatg tgactccacg gagtaccggg cgccgtccag gcacctcgat

960

tagttctcga gcttttggag tacgtcgtct ttaggttggg gggaggggtt ttatgcgatg

1020

gagtttcccc acactgagtg ggtggagact gaagttaggc cagcttggca cttgatgtaa

1080

ttctccttgg aatttgccct ttttgagttt ggatcttggt tcattctcaa gcctcagaca

1140

gtggttcaaa gtttttttct tccatttcag gtgtcgtga 1179

<210>78

<211>141

<212>DNA

<213>Artificial sequence

<220>

<223>Description of artificial sequence: Synthetic

polynucleotide

<400>78

aataaacgat aacgccgttg gtggcgtgag gcatgtaaaa ggttacatca ttatcttgtt 60

cgccatccgg ttggtataaa tagacgttca tgttggtttt tgtttcagtt gcaagttggc

120

tgcggcgcgc gcagcacctt t 141

<210>79

<211>317

<212>DNA

<213>Artificial sequence

<220>

<223>Description of artificial sequence: Synthetic

polynucleotide

<400>79

ggtgtggaaa gtccccaggc tccccagcag gcagaagtat gcaaagcatg catctcaatt 60

agtcagcaac caggtgtgga aagtccccag gctccccagc aggcagaagt atgcaaagca

120

tgcatctcaa ttagtcagca accatagtcc cgcccctaac tccgcccatc ccgcccctaa

180

ctccgcccag ttccgcccat tctccgcccc atggctgact aatttttttt atttatgcag

240

aggccgaggc cgcctcggcc tctgagctat tccagaagta gtgaggaggc ttttttggag

300

gcctaggctt ttgcaaa 317

<210>80

<211>241

<212>DNA

<213>Artificial sequence

<220>

<223>Description of artificial sequence: Synthetic

polynucleotide

<400>80

gagggcctat ttcccatgat tccttcatat ttgcatatac gatacaaggc tgttagagag 60

ataattggaa ttaatttgac tgtaaacaca aagatattag tacaaaatac gtgacgtaga

120

aagtaataat ttcttgggta gtttgcagtt ttaaaattat gttttaaaat ggactatcat

180

atgcttaccg taacttgaaa gtatttcgat ttcttggctt tatatatctt gtggaaagga

240

c 241

<210>81

<211>215

<212>DNA

<213>Artificial sequence

<220>

<223>Description of artificial sequence: Synthetic

polynucleotide

<400>81

gaacgctgac gtcatcaacc cgctccaagg aatcgcgggc ccagtgtcac taggcgggaa 60

cacccagcgc gcgtgcgccc tggcaggaag atggctgtga gggacagggg agtggcgccc

120

tgcaatattt gcatgtcgct atgtgttctg ggaaatcacc ataaacgtga aatgtctttg

180

gatttgggaa tcgtataaga actgtatgag accac 215

<210>82

<211>546

<212>DNA

<213>Artificial sequence

<220>

<223>Description of artificial sequence: Synthetic

polynucleotide

<400>82

ccctaaaatg ggcaaacatt gcaagcagca aacagcaaac acacagccct ccctgcctgc 60

tgaccttgga gctggggcag aggtcagaga cctctctggg cccatgccac ctccaacatc

120

cactcgaccc cttggaattt ttcggtggag aggagcagag gttgtcctgg cgtggtttag

180

gtagtgtgag aggggaatga ctcctttcgg taagtgcagt ggaagctgta cactgcccag

240

gcaaagcgtc cgggcagcgt aggcgggcga ctcagatccc agccagtgga cttagcccct

300

gtttgctcct ccgataactg gggtgacctt ggttaatatt caccagcagc ctcccccgtt

360

gcccctctgg atccactgct taaatacgga cgaggacagg gccctgtctc ctcagcttca

420

ggcaccacca ctgacctggg acagtgaatc cggactctaa ggtaaatata aaatttttaa

480

gtgtataatg tgttaaacta ctgattctaa ttgtttctct cttttagatt ccaacctttg

540

gaactg 546

<210>83

<211>576

<212>DNA

<213>Artificial sequence

<220>

<223>Description of artificial sequence: Synthetic

polynucleotide

<400>83

tagtaatcaa ttacggggtc attagttcat agcccatata tggagttccg cgttacataa 60

cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt gacgtcaata

120

atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca atgggtggag

180

tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc aagtacgccc

240

cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta catgacctta

300

tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac catggtgatg

360

cggttttggc agtacatcaa tgggcgtgga tagcggtttg actcacgggg atttccaagt

420

ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg ggactttcca

480

aaatgtcgta acaactccgc cccattgacg caaatgggcg gtaggcgtgt acggtgggag

540

gtctatataa gcagagctgg tttagtgaac cgtcag 576

<210>84

<211>150

<212>DNA

<213>Artificial sequence

<220>

<223>Description of artificial sequence: Synthetic

polynucleotide

<400>84

ataaacgata acgccgttgg tggcgtgagg catgtaaaag gttacatcat tatcttgttc 60

gccatccggt tggtataaat agacgttcat gttggttttt gtttcagttg caagttggct

120

gcggcgcgcg cagcaccttt gcggccatct 150

<210>85

<211>1313

<212>DNA

<213>Artificial sequence

<220>

<223>Description of artificial sequence: Synthetic

polynucleotide

<400>85

ggagccgaga gtaattcata caaaaggagg gatcgccttc gcaaggggag agcccaggga 60

ccgtccctaa attctcacag acccaaatcc ctgtagccgc cccacgacag cgcgaggagc

120

atgcgcccag ggctgagcgc gggtagatca gagcacacaa gctcacagtc cccggcggtg

180

gggggagggg cgcgctgagc gggggccagg gagctggcgc ggggcaaact gggaaagtgg

240

tgtcgtgtgc tggctccgcc ctcttcccga gggtggggga gaacggtata taagtgcggt

300

agtcgccttg gacgttcttt ttcgcaacgg gtttgccgtc agaacgcagg tgagtggcgg

360

gtgtggcttc cgcgggcccc ggagctggag ccctgctctg agcgggccgg gctgatatgc

420

gagtgtcgtc cgcagggttt agctgtgagc attcccactt cgagtggcgg gcggtgcggg

480

ggtgagagtg cgaggcctag cggcaacccc gtagcctcgc ctcgtgtccg gcttgaggcc

540

tagcgtggtg tccgccgccg cgtgccactc cggccgcact atgcgttttt tgtccttgct

600

gccctcgatt gccttccagc agcatgggct aacaaaggga gggtgtgggg ctcactctta

660

aggagcccat gaagcttacg ttggatagga atggaagggc aggaggggcg actggggccc

720

gcccgccttc ggagcacatg tccgacgcca cctggatggg gcgaggcctg tggctttccg

780

aagcaatcgg gcgtgagttt agcctacctg ggccatgtgg ccctagcact gggcacggtc

840

tggcctggcg gtgccgcgtt cccttgcctc ccaacaaggg tgaggccgtc ccgcccggca

900

ccagttgctt gcgcggaaag atggccgctc ccggggccct gttgcaagga gctcaaaatg

960

gaggacgcgg cagcccggtg gagcgggcgg gtgagtcacc cacacaaagg aagagggcct

1020

tgcccctcgc cggccgctgc ttcctgtgac cccgtggtct atcggccgca tagtcacctc

1080

gggcttctct tgagcaccgc tcgtcgcggc ggggggaggg gatctaatgg cgttggagtt

1140

tgttcacatt tggtgggtgg agactagtca ggccagcctg gcgctggaag tcattcttgg

1200

aatttgcccc tttgagtttg gagcgaggct aattctcaag cctcttagcg gttcaaaggt

1260

attttctaaa cccgtttcca ggtgttgtga aagccaccgc taattcaaag caa 1313

<210>86

<211>213

<212>DNA

<213>Artificial sequence

<220>

<223>Description of artificial sequence: Synthetic

polynucleotide

<400>86

taagatacat tgatgagttt ggacaaacca caactagaat gcagtgaaaa aaatgcttta 60

tttgtgaaat ttgtgatgct attgctttat ttgtaaccat tataagctgc aataaacaag

120

ttaacaacaa caattgcatt cattttatgt ttcaggttca gggggaggtg tgggaggttt

180

tttaaagcaa gtaaaacctc tacaaatgtg gta 213

<210>87

<211>7

<212>PROTEIN

<213>Artificial sequence

<220>

<223>Description of artificial sequence: Synthetic

peptide

<400>87

Pro Lys Lys Lys Arg Lys Val

15

<210>88

<211>19

<212>PROTEIN

<213>Artificial sequence

<220>

<223>Description of artificial sequence: Synthetic

peptide

<400>88

Met Asp Trp Thr Trp Arg Ile Leu Phe Leu Val Ala Ala Ala Thr Gly

1 5 10 15

Ala His Ser

<210>89

<211>19

<212>PROTEIN

<213>Artificial sequence

<220>

<223>Description of artificial sequence: Synthetic

peptide

<400>89

Met Leu Pro Ser Gln Leu Ile Gly Phe Leu Leu Leu Trp Val Pro Ala

1 5 10 15

Ser Arg Gly

<210>90

<211>7

<212>PROTEIN

<213>Monkey virus 40

<400>90

Pro Lys Lys Lys Arg Lys Val

15

<210>91

<211>21

<212>DNA

<213>Monkey virus 40

<400>91

cccaagaaga agaggaaggt g 21

<210>92

<211>16

<212>PROTEIN

<213>Unknown

<220>

<223>Description of the unknown:

Bipartite NLS sequence of nucleoplasmin

<400>92

Lys Arg Pro Ala Ala Thr Lys Lys Ala Gly Gln Ala Lys Lys Lys Lys

1 5 10 15

<210>93

<211>9

<212>PROTEIN

<213>Unknown

<220>

<223>Description of the unknown:

NLS sequence C-myc

<400>93

Pro Ala Ala Lys Arg Val Lys Leu Asp

15

<210>94

<211>11

<212>PROTEIN

<213>Unknown

<220>

<223>Description of the unknown:

NLS sequence C-myc

<400>94

Arg Gln Arg Arg Asn Glu Leu Lys Arg Ser Pro

1 5 10

<210>95

<211>38

<212>PROTEIN

<213>Homo sapiens

<400>95

Asn Gln Ser Ser Asn Phe Gly Pro Met Lys Gly Gly Asn Phe Gly Gly

1 5 10 15

Arg Ser Ser Gly Pro Tyr Gly Gly Gly Gly Gln Tyr Phe Ala Lys Pro

20 25 30

Arg Asn Gln Gly Gly Tyr

35

<210>96

<211>42

<212>PROTEIN

<213>Unknown

<220>

<223>Description of the unknown:

IBB domain from the importin-alpha sequence

<400>96

Arg Met Arg Ile Glx Phe Lys Asn Lys Gly Lys Asp Thr Ala Glu Leu

1 5 10 15

Arg Arg Arg Arg Val Glu Val Ser Val Glu Leu Arg Lys Ala Lys Lys

20 25 30

Asp Glu Gln Ile Leu Lys Arg Arg Asn Val

35 40

<210>97

<211>8

<212>PROTEIN

<213>Unknown

<220>

<223>Description of the unknown:

Fibroids T protein sequence

<400>97

Val Ser Arg Lys Arg Pro Arg Pro

15

<210>98

<211>8

<212>PROTEIN

<213>Unknown

<220>

<223>Description of the unknown:

Fibroids T protein sequence

<400>98

Pro Pro Lys Lys Ala Arg Glu Asp

15

<210>99

<211>8

<212>PROTEIN

<213>Homo sapiens

<400>99

Pro Gln Pro Lys Lys Lys Pro Leu

15

<210>100

<211>12

<212>PROTEIN

<213>Mus musculus

<400>100

Ser Ala Leu Ile Lys Lys Lys Lys Lys Met Ala Pro

1 5 10

<210>101

<211>70

<212>DNA

<213>Artificial sequence

<220>

<223>Description of artificial sequence: Synthetic

oligonucleotide

<400>101

gcgcgctcgc tcgctcactg aggccgcccg ggaaacccgg gcgtgcgcct cagtgagcga 60

gcgagcgcgc 70

<210>102

<211>70

<212>DNA

<213>Artificial sequence

<220>

<223>Description of artificial sequence: Synthetic

oligonucleotide

<400>102

gcgcgctcgc tcgctcactg aggcgcacgc ccgggtttcc cgggcggcct cagtgagcga 60

gcgagcgcgc 70

<210>103

<211>72

<212>DNA

<213>Artificial sequence

<220>

<223>Description of artificial sequence: Synthetic

oligonucleotide

<400>103

gcgcgctcgc tcgctcactg aggccgtcgg gcgacctttg gtcgcccggc ctcagtgagc 60

gagcgagcgc gc 72

<210>104

<211>72

<212>DNA

<213>Artificial sequence

<220>

<223>Description of artificial sequence: Synthetic

oligonucleotide

<400>104

gcgcgctcgc tcgctcactg aggccgggcg accaaaggtc gcccgacggc ctcagtgagc 60

gagcgagcgc gc 72

<210>105

<211>72

<212>DNA

<213>Artificial sequence

<220>

<223>Description of artificial sequence: Synthetic

oligonucleotide

<400>105

gcgcgctcgc tcgctcactg aggccgcccg ggcaaagccc gggcgtcggc ctcagtgagc 60

gagcgagcgc gc 72

<210>106

<211>72

<212>DNA

<213>Artificial sequence

<220>

<223>Description of artificial sequence: Synthetic

oligonucleotide

<400>106

gcgcgctcgc tcgctcactg aggccgacgc ccgggctttg cccgggcggc ctcagtgagc 60

gagcgagcgc gc 72

<210>107

<211>83

<212>DNA

<213>Artificial sequence

<220>

<223>Description of artificial sequence: Synthetic

oligonucleotide

<400>107

gcgcgctcgc tcgctcactg aggccgcccg ggcaaagccc gggcgtcggg ctttgcccgg 60

cctcagtgag cgagcgagcg cgc 83

<210>108

<211>83

<212>DNA

<213>Artificial sequence

<220>

<223>Description of artificial sequence: Synthetic

oligonucleotide

<400>108

gcgcgctcgc tcgctcactg aggccgggca aagcccgacg cccgggcttt gcccgggcgg 60

cctcagtgag cgagcgagcg cgc 83

<210>109

<211>77

<212>DNA

<213>Artificial sequence

<220>

<223>Description of artificial sequence: Synthetic

oligonucleotide

<400>109

gcgcgctcgc tcgctcactg aggccgaaac gtcgggcgac ctttggtcgc ccggcctcag 60

tgagcgagcg agcgcgc 77

<210>110

<211>77

<212>DNA

<213>Artificial sequence

<220>

<223>Description of artificial sequence: Synthetic

oligonucleotide

<400>110

gcgcgctcgc tcgctcactg aggccgggcg accaaaggtc gcccgacgtt tcggcctcag 60

tgagcgagcg agcgcgc 77

<210>111

<211>51

<212>DNA

<213>Artificial sequence

<220>

<223>Description of artificial sequence: Synthetic

oligonucleotide

<400>111

gcgcgctcgc tcgctcactg aggcaaagcc tcagtgagcg agcgagcgcg c 51

<210>112

<211>51

<212>DNA

<213>Artificial sequence

<220>

<223>Description of artificial sequence: Synthetic

oligonucleotide

<400>112

gcgcgctcgc tcgctcactg aggctttgcc tcagtgagcg agcgagcgcg c 51

<210>113

<211>80

<212>DNA

<213>Artificial sequence

<220>

<223>Description of artificial sequence: Synthetic

oligonucleotide

<400>113

gcgcgctcgc tcgctcactg aggccgcccg ggcgtcgggc gacctttggt cgcccggcct 60

cagtgagcga gcgagcgcgc 80

<210>114

<211>80

<212>DNA

<213>Artificial sequence

<220>

<223>Description of artificial sequence: Synthetic

oligonucleotide

<400>114

gcgcgctcgc tcgctcactg aggccgggcg accaaaggtc gcccgacgcc cgggcggcct 60

cagtgagcga gcgagcgcgc 80

<210>115

<211>79

<212>DNA

<213>Artificial sequence

<220>

<223>Description of artificial sequence: Synthetic

oligonucleotide

<400>115

gcgcgctcgc tcgctcactg aggcgcccgg gcgtcgggcg acctttggtc gcccggcctc 60

agtgagcgag cgagcgcgc 79

<210>116

<211>79

<212>DNA

<213>Artificial sequence

<220>

<223>Description of artificial sequence: Synthetic

oligonucleotide

<400>116

gcgcgctcgc tcgctcactg aggccgggcg accaaaggtc gcccgacgcc cgggcgcctc 60

agtgagcgag cgagcgcgc 79

<210>117

<211>5

<212>PROTEIN

<213>Influenza virus

<400>117

Asp Arg Leu Arg Arg

15

<210>118

<211>7

<212>PROTEIN

<213>Influenza virus

<400>118

Pro Lys Gln Lys Lys Arg Lys

15

<210>119

<211>10

<212>PROTEIN

<213>Hepatitis delta virus

<400>119

Arg Lys Leu Lys Lys Lys Ile Lys Lys Leu

1 5 10

<210>120

<211>10

<212>PROTEIN

<213>Mus musculus

<400>120

Arg Glu Lys Lys Lys Phe Leu Lys Arg Arg

1 5 10

<210>121

<211>20

<212>PROTEIN

<213>Homo sapiens

<400>121

Lys Arg Lys Gly Asp Glu Val Asp Gly Val Asp Glu Val Ala Lys Lys

1 5 10 15

Lys Ser Lys Lys

20

<210>122

<211>17

<212>PROTEIN

<213>Homo sapiens

<400>122

Arg Lys Cys Leu Gln Ala Gly Met Asn Leu Glu Ala Arg Lys Thr Lys

1 5 10 15

Lys

<210>123

<211>8

<212>DNA

<213>Artificial sequence

<220>

<223>Description of artificial sequence: Synthetic

oligonucleotide

<400>123

gtttaaac 8

<210>124

<211>8

<212>DNA

<213>Artificial sequence

<220>

<223>Description of artificial sequence: Synthetic

oligonucleotide

<400>124

ttaattaa 8

<210>125

<211>141

<212>DNA

<213>Artificial sequence

<220>

<223>Description of artificial sequence: Synthetic

polynucleotide

<400>125

aataaacgat aacgccgttg gtggcgtgag gcatgtaaaa ggttacatca ttatcttgtt 60

cgccatccgg ttggtataaa tagacgttca tgttggtttt tgtttcagtt gcaagttggc

120

tgcggcgcgc gcagcacctt t 141

<210>126

<211>317

<212>DNA

<213>Artificial sequence

<220>

<223>Description of artificial sequence: Synthetic

polynucleotide

<400>126

ggtgtggaaa gtccccaggc tccccagcag gcagaagtat gcaaagcatg catctcaatt 60

agtcagcaac caggtgtgga aagtccccag gctccccagc aggcagaagt atgcaaagca

120

tgcatctcaa ttagtcagca accatagtcc cgcccctaac tccgcccatc ccgcccctaa

180

ctccgcccag ttccgcccat tctccgcccc atggctgact aatttttttt atttatgcag

240

aggccgaggc cgcctcggcc tctgagctat tccagaagta gtgaggaggc ttttttggag

300

gcctaggctt ttgcaaa 317

<210>127

<211>72

<212>DNA

<213>Artificial sequence

<220>

<223>Description of artificial sequence: Synthetic

oligonucleotide

<400>127

gcgcgctcgc tcgctcactg aggccgggcg accaaaggtc gcccgacggc ctcagtgagc 60

gagcgagcgc gc 72

<210>128

<211>60

<212>DNA

<213>Artificial sequence

<220>

<223>Description of artificial sequence: Synthetic

oligonucleotide

<400>128

gagacagaca cactcctgct atgggtactg ctgctctggg ttccaggttc cactggtgac 60

<210>129

<211>1260

<212>DNA

<213>Adeno-associated virus-2

<400>129

atggagctgg tcgggtggct cgtggacaag gggattacct cggagaagca gtggatccag 60

gaggaccagg cctcatacat ctccttcaat gcggcctcca actcgcggtc ccaaatcaag

120

gctgccttgg acaatgcggg aaagattatg agcctgacta aaaccgcccc cgactacctg

180

gtgggccagc agcccgtgga ggacatttcc agcaatcgga tttataaaat tttggaacta

240

aacgggtacg atccccaata tgcggcttcc gtctttctgg gatgggccac gaaaaagttc

300

ggcaagagga acaccatctg gctgtttggg cctgcaacta ccgggaagac caacatcgcg

360

gaggccatag cccacactgt gcccttctac gggtgcgtaa actggaccaa tgagaacttt

420

cccttcaacg actgtgtcga caagatggtg atctggtggg aggaggggaa gatgaccgcc

480

aaggtcgtgg agtcggccaa agccattctc ggaggaagca aggtgcgcgt ggaccagaaa

540

tgcaagtcct cggcccagat agacccgact cccgtgatcg tcacctccaa caccaacatg

600

tgcgccgtga ttgacgggaa ctcaacgacc ttcgaacacc agcagccgtt gcaagaccgg

660

atgttcaaat ttgaactcac ccgccgtctg gatcatgact ttgggaaggt caccaagcag

720

gaagtcaaag actttttccg gtgggcaaag gatcacgtgg ttgaggtgga gcatgaattc

780

tacgtcaaaa agggtggagc caagaaaaga cccgccccca gtgacgcaga tataagtgag

840

cccaaacggg tgcgcgagtc agttgcgcag ccatcgacgt cagacgcgga agcttcgatc

900

aactacgcag acaggtacca aaacaaatgt tctcgtcacg tgggcatgaa tctgatgctg

960

tttccctgca gacaatgcga gagaatgaat cagaattcaa atatctgctt cactcacgga

1020

cagaaagact gtttagagtg ctttcccgtg tcagaatctc aacccgtttc tgtcgtcaaa

1080

aaggcgtatc agaaactgtg ctacattcat catatcatgg gaaaggtgcc agacgcttgc

1140

actgcctgcg atctggtcaa tgtggatttg gatgactgca tctttgaaca ataaatgatt

1200

taaatcaggt atggctgccg atggttatct tccagattgg ctcgaggaca ctctctctga

1260

<210>130

<211>1932

<212>DNA

<213>Adeno-associated virus-2

<400>130

atgccggggt tttacgagat tgtgattaag gtccccagcg accttgacga gcatctgccc 60

ggcatttctg acagctttgt gaactgggtg gccgagaagg aatgggagtt gccgccagat

120

tctgacatgg atctgaatct gattgagcag gcacccctga ccgtggccga gaagctgcag

180

cgcgactttc tgacggaatg gcgccgtgtg agtaaggccc cggaggccct tttctttgtg

240

caatttgaga agggagagag ctacttccac atgcacgtgc tcgtggaaac caccggggtg

300

aaatccatgg ttttgggacg tttcctgagt cagattcgcg aaaaactgat tcagagaatt

360

taccgcggga tcgagccgac tttgccaaac tggttcgcgg tcacaaagac cagaaatggc

420

gccggaggcg ggaacaaggt ggtggatgag tgctacatcc ccaattactt gctccccaaa

480

acccagcctg agctccagtg ggcgtggact aatatggaac agtatttaag cgcctgtttg

540

aatctcacgg agcgtaaacg gttggtggcg cagcatctga cgcacgtgtc gcagacgcag

600

gagcagaaca aagagaatca gaatcccaat tctgatgcgc cggtgatcag atcaaaaact

660

tcagccaggt acatggagct ggtcgggtgg ctcgtggaca aggggattac ctcggagaag

720

cagtggatcc aggaggacca ggcctcatac atctccttca atgcggcctc caactcgcgg

780

tcccaaatca aggctgcctt ggacaatgcg ggaaagatta tgagcctgac taaaaccgcc

840

cccgactacc tggtgggcca gcagcccgtg gaggacatt ccagcaatcg gatttataaa

900

attttggaac taaacgggta cgatccccaa tatgcggctt ccgtctttct gggatgggcc

960

acgaaaaagt tcggcaagag gaacaccatc tggctgtttg ggcctgcaac taccgggaag

1020

accaacatcg cggaggccat agcccacact gtgcccttct acgggtgcgt aaactggacc

1080

aatgagaact ttcccttcaa cgactgtgtc gacaagatgg tgatctggtg ggaggagggg

1140

aagatgaccg ccaaggtcgt ggagtcggcc aaagccattc tcggaggaag caaggtgcgc

1200

gtggaccaga aatgcaagtc ctcggcccag atagacccga ctcccgtgat cgtcacctcc

1260

aacaccaaca tgtgcgccgt gattgacggg aactcaacga ccttcgaaca ccagcagccg

1320

ttgcaagacc ggatgttcaa atttgaactc acccgccgtc tggatcatga ctttgggaag

1380

gtcaccaagc aggaagtcaa agactttttc cggtgggcaa aggatcacgt ggttgaggtg

1440

gagcatgaat tctacgtcaa aaagggtgga gccaagaaaa gacccgcccc cagtgacgca

1500

gatataagtg agcccaaacg ggtgcgcgag tcagttgcgc agccatcgac gtcagacgcg

1560

gaagcttcga tcaactacgc agacaggtac caaaacaaat gttctcgtca cgtgggcatg

1620

aatctgatgc tgtttccctg cagacaatgc gagagaatga atcagaattc aaatatctgc

1680

ttcactcacg gacagaaaga ctgtttagag tgctttcccg tgtcagaatc tcaacccgtt

1740

tctgtcgtca aaaaggcgta tcagaaactg tgctacattc atcatatcat gggaaaggtg

1800

cgacgctt gcactgcctg cgatctggtc aatgtggatt tggatgactg catctttgaa

1860

caataaatga tttaaatcag gtatggctgc cgatggttat cttccagatt ggctcgagga

1920

cactctctct ga 1932

<210>131

<211>1876

<212>DNA

<213>Artificial sequence

<220>

<223>Description of artificial sequence: Synthetic

polynucleotide

<400>131

cgcagccacc atggcggggt tttacgagat tgtgattaag gtccccagcg accttgacgg 60

gcatctgccc ggcatttctg acagctttgt gaactgggtg gccgagaagg aatgggagtt

120

gccgccagat tctgacatgg atctgaatct gattgagcag gcacccctga ccgtggccga

180

gaagctgcag cgcgactttc tgacggaatg gcgccgtgtg agtaaggccc cggaggccct

240

tttctttgtg caatttgaga agggagagag ctacttccac atgcacgtgc tcgtggaaac

300

caccggggtg aaatccatgg ttttgggacg tttcctgagt cagattcgcg aaaaactgat

360

tcagagaatt taccgcggga tcgagccgac tttgccaaac tggttcgcgg tcacaaagac

420

cagaaatggc gccggaggcg ggaacaaggt ggtggatgag tgctacatcc ccaattactt

480

gctccccaaa acccagcctg agctccagtg ggcgtggact aatatggaac agtatttaag

540

cgcctgtttg aatctcacgg agcgtaaacg gttggtggcg cagcatctga cgcacgtgtc

600

gcagacgcag gagcagaaca aagagaatca gaatcccaat tctgatgcgc cggtgatcag

660

atcaaaaact tcagccaggt acatggagct ggtcgggtgg ctcgtggaca aggggattac

720

ctcggagaag cagtggatcc aggaggacca ggcctcatac atctccttca atgcggcctc

780

caactcgcgg tcccaaatca aggctgcctt ggacaatgcg ggaaagatta tgagcctgac

840

taaaaccgcc cccgactacc tggtgggcca gcagcccgtg gaggacatt ccagcaatcg

900

gatttataaa attttggaac taaacgggta cgatccccaa tatgcggctt ccgtctttct

960

gggatgggcc acgaaaaagt tcggcaagag gaacaccatc tggctgtttg ggcctgcaac

1020

taccgggaag accaacatcg cggaggccat agcccacact gtgcccttct acgggtgcgt

1080

aaactggacc aatgagaact ttcccttcaa cgactgtgtc gacaagatgg tgatctggtg

1140

ggaggagggg aagatgaccg ccaaggtcgt ggagtcggcc aaagccattc tcggaggaag

1200

caaggtgcgc gtggaccaga aatgcaagtc ctcggcccag atagacccga ctcccgtgat

1260

cgtcacctcc aacaccaaca tgtgcgccgt gattgacggg aactcaacga ccttcgaaca

1320

ccagcagccg ttgcaagacc ggatgttcaa atttgaactc acccgccgtc tggatcatga

1380

ctttgggaag gtcaccaagc aggaagtcaa agactttttc cggtgggcaa aggatcacgt

1440

ggttgaggtg gagcatgaat tctacgtcaa aaagggtgga gccaagaaaa gacccgcccc

1500

cagtgacgca gatataagtg agcccaaacg ggtgcgcgag tcagttgcgc agccatcgac

1560

gtcagacgcg gaagcttcga tcaactacgc agacaggtac caaaacaaat gttctcgtca

1620

cgtgggcatg aatctgatgc tgtttccctg cagacaatgc gagagaatga atcagaattc

1680

aaatatctgc ttcactcacg gacagaaaga ctgtttagag tgctttcccg tgtcagaatc

1740

tcaacccgtt tctgtcgtca aaaaggcgta tcagaaactg tgctacattc atcatatcat

1800

gggaaaggtg cgacgctt gcactgcctg cgatctggtc aatgtggatt tggatgactg

1860

catctttgaa caataa 1876

<210>132

<211>1194

<212>DNA

<213>Artificial sequence

<220>

<223>Description of artificial sequence: Synthetic

polynucleotide

<400>132

atggagctgg tcgggtggct cgtggacaag gggattacct cggagaagca gtggatccag 60

gaggaccagg cctcatacat ctccttcaat gcggcctcca actcgcggtc ccaaatcaag

120

gctgccttgg acaatgcggg aaagattatg agcctgacta aaaccgcccc cgactacctg

180

gtgggccagc agcccgtgga ggacatttcc agcaatcgga tttataaaat tttggaacta

240

aacgggtacg atccccaata tgcggcttcc gtctttctgg gatgggccac gaaaaagttc

300

ggcaagagga acaccatctg gctgtttggg cctgcaacta ccgggaagac caacatcgcg

360

gaggccatag cccacactgt gcccttctac gggtgcgtaa actggaccaa tgagaacttt

420

cccttcaacg actgtgtcga caagatggtg atctggtggg aggaggggaa gatgaccgcc

480

aaggtcgtgg agtcggccaa agccattctc ggaggaagca aggtgcgcgt ggaccagaaa

540

tgcaagtcct cggcccagat agacccgact cccgtgatcg tcacctccaa caccaacatg

600

tgcgccgtga ttgacgggaa ctcaacgacc ttcgaacacc agcagccgtt gcaagaccgg

660

atgttcaaat ttgaactcac ccgccgtctg gatcatgact ttgggaaggt caccaagcag

720

gaagtcaaag actttttccg gtgggcaaag gatcacgtgg ttgaggtgga gcatgaattc

780

tacgtcaaaa agggtggagc caagaaaaga cccgccccca gtgacgcaga tataagtgag

840

cccaaacggg tgcgcgagtc agttgcgcag ccatcgacgt cagacgcgga agcttcgatc

900

aactacgcag accgctacca aaacaaatgt tctcgtcacg tgggcatgaa tctgatgctg

960

tttccctgca gacaatgcga gagaatgaat cagaattcaa atatctgctt cactcacgga

1020

cagaaagact gtttagagtg ctttcccgtg tcagaatctc aacccgtttc tgtcgtcaaa

1080

aaggcgtatc agaaactgtg ctacattcat catatcatgg gaaaggtgcc agacgcttgc

1140

actgcctgcg atctggtcaa tgtggatttg gatgactgca tctttgaaca ataa 1194

<210>133

<211>1876

<212>DNA

<213>Artificial sequence

<220>

<223>Description of artificial sequence: Synthetic

polynucleotide

<400>133

cgcagccacc atggcggggt tttacgagat tgtgattaag gtccccagcg accttgacgg 60

gcatctgccc ggcatttctg acagctttgt gaactgggtg gccgagaagg aatgggagtt

120

gccgccagat tctgacatgg atctgaatct gattgagcag gcacccctga ccgtggccga

180

gaagctgcag cgcgactttc tgacggaatg gcgccgtgtg agtaaggccc cggaggccct

240

tttctttgtg caatttgaga agggagagag ctacttccac atgcacgtgc tcgtggaaac

300

caccggggtg aaatccatgg ttttgggacg tttcctgagt cagattcgcg aaaaactgat

360

tcagagaatt taccgcggga tcgagccgac tttgccaaac tggttcgcgg tcacaaagac

420

cagaaatggc gccggaggcg ggaacaaggt ggtggatgag tgctacatcc ccaattactt

480

gctccccaaa acccagcctg agctccagtg ggcgtggact aatatggaac agtatttaag

540

cgcctgtttg aatctcacgg agcgtaaacg gttggtggcg cagcatctga cgcacgtgtc

600

gcagacgcag gagcagaaca aagagaatca gaatcccaat tctgatgcgc cggtgatcag

660

atcaaaaact tcagccaggt acatggagct ggtcgggtgg ctcgtggaca aggggattac

720

ctcggagaag cagtggatcc aggaggacca ggcctcatac atctccttca atgcggcctc

780

caactcgcgg tcccaaatca aggctgcctt ggacaatgcg ggaaagatta tgagcctgac

840

taaaaccgcc cccgactacc tggtgggcca gcagcccgtg gaggacatt ccagcaatcg

900

gatttataaa attttggaac taaacgggta cgatccccaa tatgcggctt ccgtctttct

960

gggatgggcc acgaaaaagt tcggcaagag gaacaccatc tggctgtttg ggcctgcaac

1020

taccgggaag accaacatcg cggaggccat agcccacact gtgcccttct acgggtgcgt

1080

aaactggacc aatgagaact ttcccttcaa cgactgtgtc gacaagatgg tgatctggtg

1140

ggaggagggg aagatgaccg ccaaggtcgt ggagtcggcc aaagccattc tcggaggaag

1200

caaggtgcgc gtggaccaga aatgcaagtc ctcggcccag atagacccga ctcccgtgat

1260

cgtcacctcc aacaccaaca tgtgcgccgt gattgacggg aactcaacga ccttcgaaca

1320

ccagcagccg ttgcaagacc ggatgttcaa atttgaactc acccgccgtc tggatcatga

1380

ctttgggaag gtcaccaagc aggaagtcaa agactttttc cggtgggcaa aggatcacgt

1440

ggttgaggtg gagcatgaat tctacgtcaa aaagggtgga gccaagaaaa gacccgcccc

1500

cagtgacgca gatataagtg agcccaaacg ggtgcgcgag tcagttgcgc agccatcgac

1560

gtcagacgcg gaagcttcga tcaactacgc agacaggtac caaaacaaat gttctcgtca

1620

cgtgggcatg aatctgatgc tgtttccctg cagacaatgc gagagaatga atcagaattc

1680

aaatatctgc ttcactcacg gacagaaaga ctgtttagag tgctttcccg tgtcagaatc

1740

tcaacccgtt tctgtcgtca aaaaggcgta tcagaaactg tgctacattc atcatatcat

1800

gggaaaggtg cgacgctt gcactgcctg cgatctggtc aatgtggatt tggatgactg

1860

catctttgaa caataa 1876

<210>134

<211>51

<212>DNA

<213>Artificial sequence

<220>

<223>Description of artificial sequence: Synthetic

oligonucleotide

<400>134

ctaggccgcc cgggcaaagc ccgggcgtcg ggcgaccttt ggtcgcccgg c 51

<210>135

<211>65

<212>DNA

<213>Artificial sequence

<220>

<223>Description of artificial sequence: Synthetic

oligonucleotide

<400>135

ctaggactga ggccgcccgg gcaaagcccg ggcgtcgggc gacctttggt cgcccggcct 60

cagtc 65

<210>136

<211>67

<212>DNA

<213>Artificial sequence

<220>

<223>Description of artificial sequence: Synthetic

oligonucleotide

<400>136

ggactgaggc cgcccgggca aagcccgggc gtcgggcgac ctttggtcgc ccggcctcag 60

tcctgca 67

<210>137

<211>41

<212>DNA

<213>Artificial sequence

<220>

<223>Description of artificial sequence: Synthetic

oligonucleotide

<400>137

gtgcgggcga ccaaaggtcg cccgacgccc gggcgcactc a 41

<210>138

<211>56

<212>DNA

<213>Artificial sequence

<220>

<223>Description of artificial sequence: Synthetic

oligonucleotide

<400>138

ggactgaggc cgggcgacca aaggtcgccc gacgcccggg cggcctcagt cctgca 56

<210>139

<211>54

<212>DNA

<213>Artificial sequence

<220>

<223>Description of artificial sequence: Synthetic

oligonucleotide

<400>139

ctaggactga ggccgcccgg gcgtcgggcg acctttggtc gcccggcctc agtc 54

<210>140

<211>48

<212>DNA

<213>Artificial sequence

<220>

<223>Description of artificial sequence: Synthetic

oligonucleotide

<400>140

ggactgaggc cgggcgacca aaggtcgccc gacggcctca gtcctgca 48

<210>141

<211>46

<212>DNA

<213>Artificial sequence

<220>

<223>Description of artificial sequence: Synthetic

oligonucleotide

<400>141

ctaggactga ggccgtcggg cgacctttgg tcgcccggcc tcagtc 46

<210>142

<211>67

<212>DNA

<213>Artificial sequence

<220>

<223>Description of artificial sequence: Synthetic

oligonucleotide

<400>142

ggactgaggc ccgggcgacc aaaggtcgcc cgacgcccgg gctttgcccg ggcgcctcag 60

tcctgca 67

<210>143

<211>47

<212>DNA

<213>Artificial sequence

<220>

<223>Description of artificial sequence: Synthetic

oligonucleotide

<400>143

atacctaggc acgcgtgtta ctagttatta atagtaatca attacgg 47

<210>144

<211>29

<212>DNA

<213>Artificial sequence

<220>

<223>Description of artificial sequence: Synthetic

oligonucleotide

<400>144

atacctaggg gccgcacgcg tgttactag 29

<210>145

<211>42

<212>DNA

<213>Artificial sequence

<220>

<223>Description of artificial sequence: Synthetic

oligonucleotide

<400>145

atacactcag tgcctgcagg cacgtggtcc ggagatccag ac 42

<210>146

<211>3754

<212>DNA

<213>Artificial sequence

<220>

<223>Description of artificial sequence: Synthetic

polynucleotide

<400>146

cctaggtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact aggggttcct 60

tgtagttaat gattaacccg ccatgctact tatcgcggcc gctcaatatt ggccattagc

120

catattattc attggttata tagcataaat caatattggc tattggccat tgcatacgtt

180

gtatctatat cataatatgt acatttatat tggctcatgt ccaatatgac cgccatgttg

240

gcattgatta ttgactagtt attaatagta atcaattacg gggtcattag ttcatagccc

300

atatatggag ttccgcgtta cataacttac ggtaaatggc ccgcctggct gaccgcccaa

360

cgacccccgc ccattgacgt caataatgac gtatgttccc atagtaacgc caatagggac

420

tttccattga cgtcaatggg tggagtattt acggtaaact gcccacttgg cagtacatca

480

agtgtatcat atgccaagtc cgccccctat tgacgtcaat gacggtaaat ggcccgcctg

540

gcattatgcc cagtacatga ccttacggga ctttcctact tggcagtaca tctacgtatt

600

agtcatcgct attaccatgg tcgaggtgag ccccacgttc tgcttcactc tccccatctc

660

ccccccctcc ccacccccaa ttttgtattt atttattttt taattatttt gtgcagcgat

720

gggggcgggg gggggggggg ggcgcgcgcc aggcggggcg gggcggggcg aggggcgggg

780

cggggcgagg cggagaggtg cggcggcagc caatcagagc ggcgcgctcc gaaagtttcc

840

ttttatggcg aggcggcggc ggcggcggcc ctataaaaag cgaagcgcgc ggcgggcggg

900

agtcgctgcg acgctgcctt cgccccgtgc cccgctccgc cgccgcctcg cgccgcccgc

960

cccggctctg actgaccgcg ttactcccac aggtgagcgg gcgggacggc ccttctcctc

1020

cgggctgtaa ttagcgcttg gtttaatgac ggcttgtttc ttttctgtgg ctgcgtgaaa

1080

gccttgaggg gctccgggag ggccctttgt gcggggggga gcggctcggg gggtgcgtgc

1140

gtgtgtgtgt gcgtggggag cgccgcgtgc ggcccgcgct gcccggcggc tgtgagcgct

1200

gcgggcgcgg cgcggggctt tgtgcgctcc gcagtgtgcg cgaggggagc gcggccgggg

1260

gcggtgcccc gcggtgcggg gggggctgcg aggggaacaa aggctgcgtg cggggtgtgt

1320

gcgtgggggg gtgagcaggg ggtgtgggcg cggcggtcgg gctgtaaccc ccccctgcac

1380

ccccctcccc gagttgctga gcacggcccg gcttcgggtg cggggctccg tacggggcgt

1440

ggcgcggggc tcgccgtgcc gggcgggggg tggcggcagg tgggggtgcc gggcggggcg

1500

gggccgcctc gggccgggga gggctcgggg gaggggcgcg gcggcccccg gagcgccggc

1560

ggctgtcgag gcgcggcgag ccgcagccat tgccttttat ggtaatcgtg cgagaggcg

1620

cagggacttc ctttgtccca aatctgtgcg gagccgaaat ctgggaggcg ccgccgcacc

1680

ccctctagcg ggcgcggggc gaagcggtgc ggcgccggca ggaaggaaat gggcggggag

1740

ggccttcgtg cgtcgccgcg ccgccgtccc cttctccctc tccagcctcg gggctgtccg

1800

cggggggacg gctgccttcg ggggggacgg ggcagggcgg ggttcggctt ctggcgtgtg

1860

accggcggct ctagagcctc tgctaaccat gttttagcct tcttcttttt cctacagctc

1920

ctgggcaacg tgctggttat tgtgctgtct catcatttgt cgacagaatt cctcgaagat

1980

ccgaaggggt tcaagcttgg cattccggta ctgttggtaa agccagttta aacgccgcca

2040

ccatggtgag caagggcgag gagctgttca ccggggtggt gcccatcctg gtcgagctgg

2100

acggcgacgt aaacggccac aagttcagcg tgtccggcga gggcgagggc gatgccacct

2160

acggcaagct gaccctgaag ttcatctgca ccaccggcaa gctgcccgtg ccctggccca

2220

ccctcgtgac caccctgacc tacggcgtgc agtgcttcag ccgctacccc gaccacatga

2280

agcagcacga cttcttcaag tccgccatgc ccgaaggcta cgtccaggag cgcaccatct

2340

tcttcaagga cgacggcaac tacaagaccc gcgccgaggt gaagttcgag ggcgacaccc

2400

tggtgaaccg catcgagctg aagggcatcg acttcaagga ggacggcaac atcctggggc

2460

acaagctgga gtacaactac aacagccaca acgtctatat catggccgac aagcagaaga

2520

acggcatcaa ggtgaacttc aagatccgcc acaacatcga ggacggcagc gtgcagctcg

2580

ccgaccacta ccagcagaac acccccatcg gcgacggccc cgtgctgctg cccgacaacc

2640

actacctgag cacccagtcc gccctgagca aagaccccaa cgagaagcgc gatcacatgg

2700

tcctgctgga gttcgtgacc gccgccggga tcactctcgg catggacgag ctgtacaagt

2760

aattaattaa gagcatctta ccgccatta ttcccatatt tgttctgttt ttcttgattt

2820

gggtatacat ttaaatgtta ataaaacaaa atggtggggc aatcatttac atttttaggg

2880

atatgtaatt actagttcag gtgtattgcc acaagacaaa catgttaaga aactttcccg

2940

ttatttacgc tctgttcctg ttaatcaacc tctggattac aaaatttgtg aaagattgac

3000

tgatattctt aactatgttg ctccttttac gctgtgtgga tatgctgctt tatagcctct

3060

gtatctagct attgcttccc gtacggcttt cgttttctcc tccttgtata aatcctggtt

3120

gctgtctctt ttagaggagt tgtggcccgt tgtccgtcaa cgtggcgtgg tgtgctctgt

3180

gtttgctgac gcaaccccca ctggctgggg cattgccacc acctgtcaac tcctttctgg

3240

gactttcgct ttccccctcc cgatcgccac ggcagaactc atcgccgcct gccttgcccg

3300

ctgctggaca ggggctaggt tgctgggcac tgataattcc gtggtgttgt ctgtgccttc

3360

tagttgccag ccatctgttg tttgcccctc ccccgtgcct tccttgaccc tggaaggtgc

3420

cactcccact gtcctttcct aataaaatga ggaaattgca tcgcattgtc tgagtaggtg

3480

tcattctatt ctggggggtg gggtggggca ggacagcaag ggggaggatt gggaagacaa

3540

tagcaggcat gctggggatg cggtgggctc tatggctcta gagcatggct acgtagataa

3600

gtagcatggc gggttaatca ttaactacac ctgcagcagg aacccctagt gatggagttg

3660

gccactccct ctctgcgcgc tcgctcgctc cctgcaggac tgaggccggg cgaccaaagg

3720

tcgcccgacg cccgggcggc ctcagtcctg cagg 3754

<210>147

<211>8418

<212>DNA

<213>Artificial sequence

<220>

<223>Description of artificial sequence: Synthetic

polynucleotide

<400>147

ggcagctgcg cgctcgctcg ctcacctagg ccgcccgggc aaagcccggg cgtcgggcga 60

cctttggtcg cccggcctag gtgagcgagc gagcgcgcag agagggagtg gccaactcca

120

tcactagggg ttccttgtag ttaatgatta acccgccatg ctacttatcg cggccgctca

180

atattggcca ttagccatat tattcattgg ttatatagca taaatcaata ttggctattg

240

gccattgcat acgttgtatc tatatcataa tatgtacatt tatattggct catgtccaat

300

atgaccgcca tgttggcatt gattattgac tagttattaa tagtaatcaa ttacggggtc

360

attagttcat agcccatata tggagttccg cgttacataa cttacggtaa atggcccgcc

420

tggctgaccg cccaacgacc cccgcccatt gacgtcaata atgacgtatg ttcccatagt

480

aacgccaata gggactttcc attgacgtca atgggtggag tatttacggt aaactgccca

540

cttggcagta catcaagtgt atcatatgcc aagtccgccc cctattgacg tcaatgacgg

600

taaatggccc gcctggcatt atgcccagta catgacctta cgggactttc ctacttggca

660

gtacatctac gtattagtca tcgctattac catggtcgag gtgagcccca cgttctgctt

720

cactctcccc atctcccccc cctccccacc cccaattttg tatttattta ttttttaatt

780

attttgtgca gcgatggggg cggggggggg gggggggcgc gcgccaggcg gggcggggcg

840

gggcgagggg cggggcgggg cgaggcggag aggtgcggcg gcagccaatc agagcggcgc

900

gctccgaaag tttcctttta tggcgaggcg gcggcggcgg cggccctata aaaagcgaag

960

cgcgcggcgg gcgggagtcg ctgcgacgct gccttcgccc cgtgccccgc tccgccgccg

1020

cctcgcgccg cccgccccgg ctctgactga ccgcgttact cccacaggtg agcgggcggg

1080

acggcccttc tcctccgggc tgtaattagc gcttggttta atgacggctt gtttcttttc

1140

tgtggctgcg tgaaagcctt gaggggctcc gggagggccc tttgtgcggg ggggagcggc

1200

tcggggggtg cgtgcgtgtg tgtgtgcgtg gggagcgccg cgtgcggccc gcgctgcccg

1260

gcggctgtga gcgctgcggg cgcggcgcgg ggctttgtgc gctccgcagt gtgcgcgagg

1320

ggagcgcggc cgggggcggt gccccgcggt gcgggggggg ctgcgagggg aacaaaggct

1380

gcgtgcgggg tgtgtgcgtg ggggggtgag cagggggtgt gggcgcggcg gtcgggctgt

1440

aacccccccc tgcacccccc tccccgagtt gctgagcacg gcccggcttc gggtgcgggg

1500

ctccgtacgg ggcgtggcgc ggggctcgcc gtgccgggcg gggggtggcg gcaggtgggg

1560

gtgccgggcg gggcggggcc gcctcggggcc ggggagggct cgggggaggg gcgcggcggc

1620

ccccggagcg ccggcggctg tcgaggcgcg gcgagccgca gccattgcct tttatggtaa

1680

tcgtgcgaga gggcgcaggg acttcctttg tcccaaatct gtgcggagcc gaaatctggg

1740

aggcgccgcc gcaccccctc tagcgggcgc ggggcgaagc ggtgcggcgc cggcaggaag

1800

gaaatgggcg gggagggcct tcgtgcgtcg ccgcgccgcc gtccccttct ccctctccag

1860

cctcggggct gtccgcgggg ggacggctgc cttcgggggg gacggggcag ggcggggttc

1920

ggcttctggc gtgtgaccgg cggctctaga gcctctgcta accatgtttt agccttcttc

1980

tttttcctac agctcctggg caacgtgctg gttattgtgc tgtctcatca tttgtcgaca

2040

gaattcctcg aagatccgaa ggggttcaag cttggcattc cggtactgtt ggtaaagcca

2100

gtttaaacgc cgccaccatg gtgagcaagg gcgaggagct gttcaccggg gtggtgccca

2160

tcctggtcga gctggacggc gacgtaaacg gccacaagtt cagcgtgtcc ggcgaggcg

2220

agggcgatgc cacctacggc aagctgaccc tgaagttcat ctgcaccacc ggcaagctgc

2280

ccgtgccctg gcccaccctc gtgaccaccc tgacctacgg cgtgcagtgc ttcagccgct

2340

accccgacca catgaagcag cacgacttct tcaagtccgc catgcccgaa ggctacgtcc

2400

aggagcgcac catcttcttc aaggacgacg gcaactacaa gacccgcgcc gaggtgaagt

2460

tcgagggcga caccctggtg aaccgcatcg agctgaaggg catcgacttc aaggaggacg

2520

gcaacatcct ggggcacaag ctggagtaca actacaacag ccacaacgtc tatatcatgg

2580

ccgacaagca gaagaacggc atcaaggtga acttcaagat ccgccacaac atcgaggacg

2640

gcagcgtgca gctcgccgac cactaccagc agaacacccc catcggcgac ggccccgtgc

2700

tgctgcccga caaccactac ctgagcaccc agtccgccct gagcaaagac cccaacgaga

2760

agcgcgatca catggtcctg ctggagttcg tgaccgccgc cgggatcact ctcggcatgg

2820

acgagctgta caagtaatta attaagagca tcttaccgcc atttattccc atatttgttc

2880

tgtttttctt gatttgggta tacatttaaa tgttaataaa acaaaatggt ggggcaatca

2940

tttacatttt tagggatatg taattactag ttcaggtgta ttgccacaag acaaacatgt

3000

taagaaactt tcccgttatt tacgctctgt tcctgttaat caacctctgg attacaaaat

3060

ttgtgaaaga ttgactgata ttcttaacta tgttgctcct tttacgctgt gtggatatgc

3120

tgctttatag cctctgtatc tagctattgc ttcccgtacg gctttcgttt tctcctcctt

3180

gtataaatcc tggttgctgt ctcttttaga ggagttgtgg cccgttgtcc gtcaacgtgg

3240

cgtggtgtgc tctgtgtttg ctgacgcaac ccccactggc tggggcattg ccaccacctg

3300

tcaactcctt tctgggactt tcgctttccc cctcccgatc gccacggcag aactcatcgc

3360

cgcctgcctt gcccgctgct ggacaggggc taggttgctg ggcactgata attccgtggt

3420

gttgtctgtg ccttctagtt gccagccatc tgttgtttgc ccctcccccg tgccttcctt

3480

gaccctggaa ggtgccactc ccactgtcct ttcctaataa aatgaggaaa ttgcatcgca

3540

ttgtctgagt aggtgtcatt ctattctggg gggtggggtg gggcaggaca gcaaggggga

3600

ggattgggaa gacaatagca ggcatgctgg ggatgcggtg ggctctatgg ctctagagca

3660

tggctacgta gataagtagc atggcgggtt aatcattaac tacacctgca gcaggaaccc

3720

ctagtgatgg agttggccac tccctctctg cgcgctcgct cgctccctgc aggactgagg

3780

ccgggcgacc aaaggtcgcc cgacgcccgg gcggcctcag tcctgcaggg agcgagcgag

3840

cgcgcagctg cctgcacggg cgcgccggta ccgggagatg ggggaggcta actgaaacac

3900

ggaagggagac aataccggaa ggaacccgcg ctatgacggc aataaaaaga cagaataaaa

3960

cgcacgggtg ttgggtcgtt tgttcataaa cgcggggttc ggtcccaggg ctggcactct

4020

gtcgataccc caccgagacc ccattgggac caatacgccc gcgtttcttc cttttcccca

4080

ccccaacccc caagttcggg tgaaggccca gggctcgcag ccaacgtcgg ggcggcaagc

4140

cctgccatag ccactacggg tacgtaggcc aaccactaga actatagcta gagtcctggg

4200

cgaacaaacg atgctcgcct tccagaaaac cgaggatgcg aaccacttca tccggggtca

4260

gcaccaccgg caagcgccgc gacggccgag gtctaccgat ctcctgaagc cagggcagat

4320

ccgtgcacag caccttgccg tagaagaaca gcaaggccgc caatgcctga cgatgcgtgg

4380

agaccgaaac cttgcgctcg ttcgccagcc aggacagaaa tgcctcgact tcgctgctgc

4440

ccaaggttgc cgggtgacgc acaccgtgga aacggatgaa ggcacgaacc cagttgacat

4500

aagcctgttc ggttcgtaaa ctgtaatgca agtagcgtat gcgctcacgc aactggtcca

4560

gaaccttgac cgaacgcagc ggtggtaacg gcgcagtggc ggttttcatg gcttgttatg

4620

actgtttttt tgtacagtct atgcctcggg catccaagca gcaagcgcgt tacgccgtgg

4680

gtcgatgttt gatgttatgg agcagcaacg atgttacgca gcagcaacga tgttacgcag

4740

cagggcagtc gccctaaaac aaagttaggt ggctcaagta tgggcatcat tcgcacatgt

4800

aggctcggcc ctgaccaagt caaatccatg cgggctgctc ttgatctttt cggtcgtgag

4860

ttcggagacg tagccaccta ctcccaacat cagccggact ccgattacct cgggaacttg

4920

ctccgtagta agacattcat cgcgcttgct gccttcgacc aagaagcggt tgttggcgct

4980

ctcgcggctt acgttctgcc caggtttgag cagccgcgta gtgagatcta tatctatgat

5040

ctcgcagtct ccggcgagca ccggaggcag ggcattgcca ccgcgctcat caatctcctc

5100

aagcatgagg ccaacgcgct tggtgcttat gtgatctacg tgcaagcaga ttacggtgac

5160

gatcccgcag tggctctcta tacaaagttg ggcatacggg aagaagtgat gcactttgat

5220

atcgacccaa gtaccgccac ctaacaattc gttcaagccg agatcggctt cccggccgcg

5280

gagttgttcg gtaaattgtc acaacgccgc gaatatagtc tttaccatgc ccttggccac

5340

gcccctcttt aatacgacgg gcaatttgca cttcagaaaa tgaagagttt gctttagcca

5400

taacaaaagt ccagtatgct ttttcacagc ataactggac tgatttcagt ttacaactat

5460

tctgtctagt ttaagacttt attgtcatag tttagatcta ttttgttcag tttaagactt

5520

tattgtccgc ccacacccgc ttacgcaggg catccattta ttactcaacc gtaaccgatt

5580

ttgccaggtt acgcggctgg tctgcggtgt gaaataccgc acagatgcgt aaggagaaaa

5640

taccgcatca ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg

5700

ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg

5760

gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag

5820

gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga

5880

cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct

5940

ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc

6000

tttctccctt cgggaagcgt ggcgctttct caatgctcac gctgtaggta tctcagttcg

6060

gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc

6120

tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca

6180

ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag

6240

ttcttgaagt ggtggcctaa ctacggctac actagaagga cagtatttgg tatctgcgct

6300

ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc

6360

accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga

6420

tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca

6480

cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat

6540

taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac

6600

caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt

6660

gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt

6720

gctgcaatga taccgcgaga cccacgctca ccggctccag atttatcagc aataaaccag

6780

ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct

6840

attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt

6900

gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc

6960

tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt

7020

agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg

7080

gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg

7140

actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct

7200

tgcccggcgt caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc

7260

attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt

7320

tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt

7380

tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg

7440

aaatgttgaa tactcatact cttccttttt caatattatt gaagcattta tcagggttat

7500

tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg

7560

cgcacatttc cccgaaaagt gccacctgaa attgtaaacg ttaatatttt gttaaaattc

7620

gcgttaaatt tttgttaaat cagctcattt tttaaccaat aggccgaaat cggcaaaatc

7680

ccttataaat caaaagaata gaccgagata gggttgagtg ttgttccagt ttggaacaag

7740

agtccactat taaagaacgt ggactccaac gtcaaagggc gaaaaaccgt ctatcagggc

7800

gatggcccac tacgtgaacc atcaccctaa tcaagttttt tggggtcgag gtgccgtaaa

7860

gcactaaatc ggaaccctaa agggagcccc cgatttagag cttgacgggg aaagccggcg

7920

aacgtggcga gaaaggaagg gaagaaagcg aaaggagcgg gcgctagggc gctggcaagt

7980

gtagcggtca cgctgcgcgt aaccaccaca cccgccgcgc ttaatgcgcc gctacaggc

8040

gcgtcccatt cgccattcag gctgcaaata agcgttgata ttcagtcaat tacaaacatt

8100

aataacgaag agatgacaga aaaattttca ttctgtgaca gagaaaaagt agccgaagat

8160

gacggtttgt cacatggagt tggcaggatg tttgattaaa aacataacag gaagaaaaat

8220

gccccgctgt gggcggacaa aatagttggg aactgggagg ggtggaaatg gagtttttaa

8280

ggattattta gggaagagtg acaaaataga tgggaactgg gtgtagcgtc gtaagctaat

8340

acgaaaatta aaaatgacaa aatagtttgg aactagatt cacttatctg gttcggatct

8400

cctagtgagc tccctgca 8418

<210>148

<211>225

<212>DNA

<213>Artificial sequence

<220>

<223>Description of artificial sequence: Synthetic

polynucleotide

<400>148

tgtgccttct agttgccagc catctgttgt ttgcccctcc cccgtgcctt ccttgaccct 60

ggaaggtgcc actcccactg tcctttccta ataaaatgag gaaattgcat cgcattgtct

120

gagtaggtgt cattctattc tggggggtgg ggtggggcag gacagcaagg gggaggattg

180

ggaagacaat agcaggcatg ctggggatgc ggtgggctct atggc 225

<210>149

<211>1177

<212>DNA

<213>Artificial sequence

<220>

<223>Description of artificial sequence: Synthetic

polynucleotide

<400>149

ggctcagagg ctcagaggca cacaggagtt tctgggctca ccctgccccc ttccaacccc 60

tcagttccca tcctccagca gctgtttgtg tgctgcctct gaagtccaca ctgaacaaac

120

ttcagcctac tcatgtccct aaaatgggca aacattgcaa gcagcaaaca gcaaacacac

180

agccctccct gcctgctgac cttggagctg gggcagaggt cagagacctc tctgggccca

240

tgccacctcc aacatccact cgaccccttg gaatttcggt ggagaggagc agaggttgtc

300

ctggcgtggt ttaggtagtg tgagagggtc cgggttcaaa accacttgct gggtggggag

360

tcgtcagtaa gtggctatgc cccgaccccg aagcctgttt ccccatctgt acaatggaaa

420

tgataaagac gcccatctga tagggttttt gtggcaaata aacatttggt ttttttgttt

480

tgttttgttt tgttttttga gatggaggtt tgctctgtcg cccaggctgg agtgcagtga

540

cacaatctca tctcaccaca accttcccct gcctcagcct cccaagtagc tgggattaca

600

agcatgtgcc accacacctg gctaattttc tatttttagt agagacgggt ttctccatgt

660

tggtcagcct cagcctccca agtaactggg attacaggcc tgtgccacca cacccggcta

720

attttttcta tttttgacag ggacggggtt tcaccatgtt ggtcaggctg gtctagaggt

780

accggatctt gctaccagtg gaacagccac taaggattct gcagtgagag cagaggcca

840

gctaagtggt actctcccag agactgtctg actcacgcca ccccctccac cttggacaca

900

ggacgctgtg gtttctgagc caggtacaat gactcctttc ggtaagtgca gtggaagctg

960

tacactgccc aggcaaagcg tccgggcagc gtaggcgggc gactcagatc ccagccagtg

1020

gacttagccc ctgtttgctc ctccgataac tggggtgacc ttggttaata ttcaccagca

1080

gcctcccccg ttgcccctct ggatccactg cttaaatacg gacgaggaca gggccctgtc

1140

tcctcagctt caggcaccac cactgacctg ggacagt 1177

<210>150

<211>1326

<212>DNA

<213>Artificial sequence

<220>

<223>Description of artificial sequence: Synthetic

polynucleotide

<400>150

ctgcagggcc cactagtgga gccgagagta attcatacaa aaggagggat cgccttcgca 60

aggggagagc cccaggaccg tccctaaatt ctcacagacc caaatccctg tagccgcccc

120

acgacagcgc gaggagcatg cgcccagggc tgagcgcggg tagatcagag cacacaagct

180

cacagtcccc ggcggtgggg ggaggggcgc gctgagcggg ggccagggag ctggcgcggg

240

gcaaactggg aaagtggtgt cgtgtgctgg ctccgccctc ttcccgaggg tggggggagaa

300

cggtatataa gtgcggtagt cgccttggac gttctttttc gcaacgggtt tgccgtcaga

360

acgcaggtga gtggcgggtg tggcttccgc gggccccgga gctggagccc tgctctgagc

420

gggccgggct gatatgcgag tgtcgtccgc agggtttagc tgtgagcatt cccacttcga

480

gtggcgggcg gtgcgggggt gagagtgcga ggcctagcgg caaccccgta gcctcgcctc

540

gtgtccggct tgaggcctag cgtggtgtcc gccgccgcgt gccactccgg ccgcactatg

600

cgttttttgt ccttgctgcc ctcgattgcc ttccagcagc atgggctaac aaagggaggg

660

tgtggggctc actcttaagg agcccatgaa gcttacgttg gataggaatg gaagggcagg

720

aggggcgact ggggcccgcc cgccttcgga gcacatgtcc gacgccacct ggatggggcg

780

aggcctgtgg ctttccgaag caatcgggcg tgagtttagc ctacctgggc catgtggccc

840

tagcactggg cacggtctgg cctggcggtg ccgcgttccc ttgcctccca acaagggtga

900

ggccgtcccg cccggcacca gttgcttgcg cggaaagatg gccgctcccg gggccctgtt

960

gcaaggagct caaaatggag gacgcggcag cccggtggag cgggcgggtg agtcaccac

1020

acaaaggaag agggccttgc ccctcgccgg ccgctgcttc ctgtgacccc gtggtctatc

1080

ggccgcatag tcacctcggg cttctcttga gcaccgctcg tcgcggcggg gggaggggat

1140

ctaatggcgt tggagtttgt tcacatttgg tgggtggaga ctagtcaggc cagcctggcg

1200

ctggaagtca ttcttggaat ttgccccttt gagtttggag cgaggctaat tctcaagcct

1260

cttagcggtt caaaggtatt ttctaaaccc gtttccaggt gttgtgaaag ccaccgctaa

1320

ttcaaa 1326

<210>151

<211>573

<212>DNA

<213>Mus musculus

<400>151

gtaagagttt tatgtttttt catctctgct tgtatttttc tagtaatgga agcctggtat 60

tttaaaatag ttaaattttc ctttagtgct gatttctaga ttattattac tgttgttgtt

120

gttattattg tcattatttg catctgagaa cccttaggtg gttatattat tgatatattt

180

ttggtatctt tgatgacaat aatgggggat tttgaaagct tagctttaaa tttcttttaa

240

ttaaaaaaaa atgctaggca gaatgactca aattacgttg gatacagttg aatttattac

300

ggtctcatag ggcctgcctg ctcgaccatg ctatactaaa aattaaaagt gtgtgttact

360

aattttataa atggagtttc catttatatt tacctttatt tcttatttac cattgtctta

420

gtagatattt acaaacatga cagaaacact aaatcttgag tttgaatgca cagatataaa

480

cacttaacgg gttttaaaaa taataatgtt ggtgaaaaaa tataactttg agtgtagcag

540

agaggaacca ttgccacctt cagattttcc tgt 573

<210>152

<211>1993

<212>DNA

<213>Mus musculus

<400>152

acgatcggga actggcatct tcagggagta gcttaggtca gtgaagagaa gaacaaaaag 60

cagcatatta cagttagttg tcttcatcaa tctttaaata tgttgtgtgg tttttctctc

120

cctgtttcca cagacaagag tgagatcgcc catcggtata atgatttggg agaacaacat

180

ttcaaaggcc tgtaagttat aatgctgaaa gcccacttaa tatttctggt agtattagtt

240

aaagttttaa aacacctttt tccaccttga gtgtgagaat tgtagagcag tgctgtccag

300

tagaaatgtg tgcattgaca gaaagactgt ggatctgtgc tgagcaatgt ggcagccaga

360

gatcacaagg ctatcaagca ctttgcacat ggcaagtgta actgagaagc acacattcaa

420

ataatagtta attttaattg aatgtatcta gccatgtgtg gctagtagct cctttcctgg

480

agagagaatc tggagcccac atctaacttg ttaagtctgg aatcttattt tttatttctg

540

gaaaggtcta tgaactatag ttttgggggc agctcactta ctaactttta atgcaataag

600

atctcatggt atcttgagaa cattattttg tctctttgta gtactgaaac cttatacatg

660

tgaagtaagg ggtctatact taagtcacat ctccaacctt agtaatgttt taatgtagta

720

aaaaaatgag taattaattt atttttagaa ggtcaatagt atcatgtatt ccaaataaca

780

gaggtatatg gttagaaaag aaacaattca aaggacttat ataatatcta gccttgacaa

840

tgaataaatt tagagagtag tttgcctgtt tgcctcatgt tcataaatct attgacacat

900

atgtgcatct gcacttcagc atggtagaag tccatattcc tttgcttgga aaggcaggtg

960

ttcccattac gcctcagaga atagctgacg ggaagaggct ttctagatag ttgtatgaaa

1020

gatatacaaa atctcgcagg tatacacagg catgatttgc tggttgggag agccacttgc

1080

ctcatactga ggtttttgtg tctgcttttc agagtcctga ttgccttttc ccagtatctc

1140

cagaaatgct catacgatga gcatgccaaa ttagtgcagg aagtaacaga ctttgcaaag

1200

acgtgtgttg ccgatgagtc tgccgccaac tgtgacaaat cccttgtgag taccttctga

1260

ttttgtggat ctactttcct gctttctgga actctgtttc aaagccaatc atgactccat

1320

cacttaaggc cccgggaaca ctgtggcaga gggcagcaga gagattgata aagccaggt

1380

gatgggaatt ttctgtggga ctccatttca tagtaattgc agaagctaca atacactcaa

1440

aaagtctcac cacatgactg cccaaatggg agcttgacag tgacagtgac agtagatatg

1500

ccaaagtgga tgagggaaag accacaagag ctaaaccctg taaaaagaac tgtaggcaac

1560

taaggaatgc agagagaaga agttgccttg gaagagcata ccaactgcct ctccaatacc

1620

aatggtcatc cctaaaacat acgtatgaat aacatgcaga ctaagcaggc tacatttagg

1680

aatatacatg tatttacata aatgtatatg catgtaacaa caatgaatga aaactgaggt

1740

catggatctg aaagagagca agggggctta catgagagg tttggaggga ggggttggag

1800

ggagggaggt attattcttt agttttacag ggaacgtagt aaaaacatag gcttctccca

1860

aaggagcaga gcccatgagg agctgtgcaa ggttccccag cttgatttta cctgctcctc

1920

aaattccctt gatttgtttt tattataatg actttactcc tagcttttag tgtcagatag

1980

aaaacatgga agg 1993

<210>153

<211>1350

<212>DNA

<213>Homo sapiens

<400>153

taggaggctg aggcaggagg atcgcttgag cccaggagtt cgagaccagc ctgggcaaca 60

tagtgtgatc ttgtatctat aaaaataaac aaaattagct tggtgtggtg gcgcctgtag

120

tccccagcca cttggagggg tgaggtgaga ggattgcttg agcccgggat ggtccaggct

180

gcagtgagcc atgatcgtgc cactgcactc cagcctgggc gacagagtga gaccctgtct

240

cacaacaaca acaacaacaa caaaaaggct gagctgcacc atgcttgacc cagtttctta

300

aaattgttgt caaagcttca ttcactccat ggtgctatag agcacaagat tttatttggt

360

gagatggtgc tttcatgaat tcccccaaca gagccaagct ctccatctag tggacaggga

420

agctagcagc aaaccttccc ttcactacaa aacttcattg cttggccaaa aagagagtta

480

attcaatgta gacatctatg taggcaatta aaaacctatt gatgtataaa acagtttgca

540

ttcatggagg gcaactaaat acattctagg actttataaa agatcacttt ttatttatgc

600

acagggtgga acaagatgga ttatcaagtg tcaagtccaa tctatgacat caattattat

660

acatcggagc cctgccaaaa aatcaatgtg aagcaaatcg cagcccgcct cctgcctccg

720

ctctactcac tggtgttcat ctttggtttt gtgggcaaca tgctggtcat cctcatcctg

780

ataaactgca aaaggctgaa gagcatgact gacatctacc tgctcaacct ggccatctct

840

gacctgtttt tccttcttac tgtccccttc tgggctcact atgctgccgc ccagtgggac

900

tttggaaata caatgtgtca actcttgaca gggctctatt ttataggctt cttctctgga

960

atcttcttca tcatcctcct gacaatcgat aggtacctgg ctgtcgtcca tgctgtgttt

1020

gctttaaaag ccaggacggt cacctttggg gtggtgacaa gtgtgatcac ttgggtggtg

1080

gctgtgtttg cgtctctccc aggaatcatc tttaccagat ctcaaaaaga aggtcttcat

1140

tacacctgca gctctcattt tccatacagt cagtatcaat tctggaagaa tttccagaca

1200

ttaaagatag tcatcttggg gctggtcctg ccgctgcttg tcatggtcat ctgctactcg

1260

ggaatcctaa aaactctgct tcggtgtcga aatgagaaga agaggcacag ggctgtgagg

1320

cttatcttca ccatcatgat tgtttatttt 1350

<210>154

<211>1223

<212>DNA

<213>Homo sapiens

<400>154

tgacagagac tcttgggatg acgcactgct gcatcaaccc catcatctat gcctttgtcg 60

gggagaagtt cagaaactac ctcttagtct tcttccaaaa gcacattgcc aaacgcttct

120

gcaaatgctg ttctattttc cagcaagagg ctcccgagcg agcaagctca gtttacaccc

180

gatccactgg ggagcaggaa atatctgtgg gcttgtgaca cggactcaag tgggctggtg

240

acccagtcag agttgtgcac atggcttagt tttcatacac agcctgggct gggggtgggg

300

tggggagaggt cttttttaaa aggaagttac tgttatagag ggtctaagat tcatccatt

360

atttggcatc tgtttaaagt agattagatc ttttaagccc atcaattata gaaagccaaa

420

tcaaaatatg ttgatgaaaa atagcaacct ttttatctcc ccttcacatg catcaagtta

480

ttgacaaact ctcccttcac tccgaaagtt ccttatgtat atttaaaaga aagcctcaga

540

gaattgctga ttcttgagtt tagtgatctg aacagaaata ccaaaattat ttcagaaatg

600

tacaactttt tacctagtac aaggcaacat ataggttgta aatgtgttta aaacaggtct

660

ttgtcttgct atggggagaa aagacatgaa tatgattagt aaagaaatga cacttttcat

720

gtgtgatttc ccctccaagg tatggttaat aagtttcact gacttagaac caggcgagag

780

acttgtggcc tgggagagct ggggaagctt cttaaatgag aaggaatttg agttggatca

840

tctattgctg gcaaagacag aagcctcact gcaagcactg catgggcaag cttggctgta

900

gaaggagaca gagctggttg ggaagacatg gggaggaagg acaaggctag atcatgaaga

960

accttgacgg cattgctccg tctaagtcat gagctgagca ggggagatcct ggttggtgtt

1020

gcagaaggtt tactctgtgg ccaaaggagg gtcaggaagg atgagcattt agggcaagga

1080

gaccaccaac agccctcagg tcagggtgag gatggcctct gctaagctca aggcgtgagg

1140

atgggaagga gggaggtatt cgtaaggatg ggaaggaggg aggtattcgt gcagcatatg

1200

aggatgcaga gtcagcagaa ctg 1223

<210>155

<211>215

<212>DNA

<213>Artificial sequence

<220>

<223>Description of artificial sequence: Synthetic

polynucleotide

<400>155

gaacgctgac gtcatcaacc cgctccaagg aatcgcgggc ccagtgtcac taggcgggaa 60

cacccagcgc gcgtgcgccc tggcaggaag atggctgtga gggacagggg agtggcgccc

120

tgcaatattt gcatgtcgct atgtgttctg ggaaatcacc ataaacgtga aatgtctttg

180

gatttgggaa tcttataagt tctgtatgag accac 215

<210>156

<211>141

<212>DNA

<213>Artificial sequence

<220>

<223>Description of artificial sequence: Synthetic

polynucleotide

<400>156

cctgcaggca gctgcgcgct cgctcgctca cctaggccgc ccgggcaaag cccgggcgtc 60

gggcgacctt tggtcgcccg gcctaggtga gcgagcgagc gcgcagagag ggagtggcca

120

actccatcac taggggttcc t 141

<210>157

<211>19

<212>DNA

<213>Artificial sequence

<220>

<223>Description of artificial sequence: Synthetic

oligonucleotide

<400>157

gcgcgctcgc tcgctcacc 19

<210>158

<211>22

<212>DNA

<213>Artificial sequence

<220>

<223>Description of artificial sequence: Synthetic

oligonucleotide

<400>158

ctaggtgagc gagcgagcgc gc 22

<210>159

<211>75

<212>DNA

<213>Artificial sequence

<220>

<223>Description of artificial sequence: Synthetic

oligonucleotide

<400>159

cctgcaggac tgaggccgcc cgggcaaagc ccgggcgtcg ggcgaccttt ggtcgcccgg 60

cctcagtcct gcagg 75

<210>160

<211>130

<212>DNA

<213>Artificial sequence

<220>

<223>Description of artificial sequence: Synthetic

polynucleotide

<400>160

aggaacccct agtgatggag ttggccactc cctctctgcg cgctcgctcg ctcactgagt 60

gcgggcgacc aaaggtcgcc cgacgcccgg gcgcactcag tgagcgagcg agcgcgcagc

120

tgcctgcagg 130

<210>161

<211>142

<212>DNA

<213>Artificial sequence

<220>

<223>Description of artificial sequence: Synthetic

polynucleotide

<400>161

cctgcaggca gctgcgcgct cgctcgctcc ctaggactga ggccgcccgg gcgtcgggcg 60

acctttggtc gcccggcctc agtcctaggg agcgagcgag cgcgcagaga gggagtggcc

120

aactccatca ctaggggttc ct 142

<210>162

<211>80

<212>DNA

<213>Artificial sequence

<220>

<223>Description of artificial sequence: Synthetic

oligonucleotide

<400>162

gcgcgctcgc tcgctcactg agtgcgggcg accaaaggtc gcccgacgcc cgggcgcact 60

cagtgagcga gcgagcgcgc 80

<210>163

<211>21

<212>DNA

<213>Artificial sequence

<220>

<223>Description of artificial sequence: Synthetic

oligonucleotide

<400>163

gcgcgctcgc tcgctcactg a 21

<210>164

<211>18

<212>DNA

<213>Artificial sequence

<220>

<223>Description of artificial sequence: Synthetic

oligonucleotide

<400>164

gtgagcgagc gagcgcgc 18

<210>165

<211>89

<212>DNA

<213>Artificial sequence

<220>

<223>Description of artificial sequence: Synthetic

oligonucleotide

<400>165

gcgcgctcgc tcgctcactg aggccgcccg ggcaaagccc gggcgtcggg cgactttgtc 60

gcccggcctc agtgagcgag cgagcgcgc 89

<210>166

<211>89

<212>DNA

<213>Artificial sequence

<220>

<223>Description of artificial sequence: Synthetic

oligonucleotide

<400>166

gcgcgctcgc tcgctcactg aggccgggcg acaaagtcgc ccgacgcccg ggctttgccc 60

gggcggcctc agtgagcgag cgagcgcgc 89

<210>167

<211>87

<212>DNA

<213>Artificial sequence

<220>

<223>Description of artificial sequence: Synthetic

oligonucleotide

<400>167

gcgcgctcgc tcgctcactg aggccgcccg ggcaaagccc gggcgtcggg cgattttcgc 60

ccggcctcag tgagcgagcg agcgcgc 87

<210>168

<211>87

<212>DNA

<213>Artificial sequence

<220>

<223>Description of artificial sequence: Synthetic

oligonucleotide

<400>168

gcgcgctcgc tcgctcactg aggccgggcg aaaatcgccc gacgcccggg ctttgcccgg 60

gcggcctcag tgagcgagcg agcgcgc 87

<210>169

<211>85

<212>DNA

<213>Artificial sequence

<220>

<223>Description of artificial sequence: Synthetic

oligonucleotide

<400>169

gcgcgctcgc tcgctcactg aggccgcccg ggcaaagccc gggcgtcggg cgtttcgccc 60

ggcctcagtg agcgagcgag cgcgc 85

<210>170

<211>85

<212>DNA

<213>Artificial sequence

<220>

<223>Description of artificial sequence: Synthetic

oligonucleotide

<400>170

gcgcgctcgc tcgctcactg aggccgggcg aaacgcccga cgcccgggct ttgcccgggc 60

ggcctcagtg agcgagcgag cgcgc 85

<210>171

<211>89

<212>DNA

<213>Artificial sequence

<220>

<223>Description of artificial sequence: Synthetic

oligonucleotide

<400>171

gcgcgctcgc tcgctcactg aggccgcccg ggaaacccgg gcgtcgggcg acctttggtc 60

gcccggcctc agtgagcgag cgagcgcgc 89

<210>172

<211>89

<212>DNA

<213>Artificial sequence

<220>

<223>Description of artificial sequence: Synthetic

oligonucleotide

<400>172

gcgcgctcgc tcgctcactg aggccgggcg accaaaggtc gcccgacgcc cgggtttccc 60

gggcggcctc agtgagcgag cgagcgcgc 89

<210>173

<211>87

<212>DNA

<213>Artificial sequence

<220>

<223>Description of artificial sequence: Synthetic

oligonucleotide

<400>173

gcgcgctcgc tcgctcactg aggccgcccg gaaaccgggc gtcgggcgac ctttggtcgc 60

ccggcctcag tgagcgagcg agcgcgc 87

<210>174

<211>87

<212>DNA

<213>Artificial sequence

<220>

<223>Description of artificial sequence: Synthetic

oligonucleotide

<400>174

gcgcgctcgc tcgctcactg aggccgggcg accaaaggtc gcccgacgcc cggtttccgg 60

gcggcctcag tgagcgagcg agcgcgc 87

<210>175

<211>85

<212>DNA

<213>Artificial sequence

<220>

<223>Description of artificial sequence: Synthetic

oligonucleotide

<400>175

gcgcgctcgc tcgctcactg aggccgcccg aaacgggcgt cgggcgacct ttggtcgccc 60

ggcctcagtg agcgagcgag cgcgc 85

<210>176

<211>85

<212>DNA

<213>Artificial sequence

<220>

<223>Description of artificial sequence: Synthetic

oligonucleotide

<400>176

gcgcgctcgc tcgctcactg aggccggggcg accaaaggtc gcccgacgcc cgtttcgggc 60

ggcctcagtg agcgagcgag cgcgc 85

<210>177

<211>83

<212>DNA

<213>Artificial sequence

<220>

<223>Description of artificial sequence: Synthetic

oligonucleotide

<400>177

gcgcgctcgc tcgctcactg aggccgccca aagggcgtcg ggcgaccttt ggtcgcccgg 60

cctcagtgag cgagcgagcg cgc 83

<210>178

<211>83

<212>DNA

<213>Artificial sequence

<220>

<223>Description of artificial sequence: Synthetic

oligonucleotide

<400>178

gcgcgctcgc tcgctcactg aggccgggcg accaaaggtc gcccgacgcc ctttgggcgg 60

cctcagtgag cgagcgagcg cgc 83

<210>179

<211>81

<212>DNA

<213>Artificial sequence

<220>

<223>Description of artificial sequence: Synthetic

oligonucleotide

<400>179

gcgcgctcgc tcgctcactg aggccgccaa aggcgtcggg cgacctttgg tcgcccggcc 60

tcagtgagcg agcgagcgcg c 81

<210>180

<211>81

<212>DNA

<213>Artificial sequence

<220>

<223>Description of artificial sequence: Synthetic

oligonucleotide

<400>180

gcgcgctcgc tcgctcactg aggccggggcg accaaaggtc gcccgacgcc tttggcggcc 60

tcagtgagcg agcgagcgcg c 81

<210>181

<211>79

<212>DNA

<213>Artificial sequence

<220>

<223>Description of artificial sequence: Synthetic

oligonucleotide

<400>181

gcgcgctcgc tcgctcactg aggccgcaaa gcgtcgggcg acctttggtc gcccggcctc 60

agtgagcgag cgagcgcgc 79

<210>182

<211>79

<212>DNA

<213>Artificial sequence

<220>

<223>Description of artificial sequence: Synthetic

oligonucleotide

<400>182

gcgcgctcgc tcgctcactg aggccgggcg accaaaggtc gcccgacgct ttgcggcctc 60

agtgagcgag cgagcgcgc 79

<210>183

<211>81

<212>DNA

<213>Artificial sequence

<220>

<223>Description of artificial sequence: Synthetic

oligonucleotide

<400>183

ctgcgcgctc gctcgctcac tgaggccgaa acgtcgggcg acctttggtc gcccggcctc 60

agtgagcgag cgagcgcgca g 81

<210>184

<211>81

<212>DNA

<213>Artificial sequence

<220>

<223>Description of artificial sequence: Synthetic

oligonucleotide

<400>184

ctgcgcgctc gctcgctcac tgaggccggg cgaccaaagg tcgcccgacg tttcggcctc 60

agtgagcgag cgagcgcgca g 81

<210>185

<211>72

<212>DNA

<213>Artificial sequence

<220>

<223>Description of artificial sequence: Synthetic

oligonucleotide

<400>185

gcgcgctcgc tcgctcactg aggccgggcg accaaaggtc gcccgacggc ctcagtgagc 60

gagcgagcgc gc 72

<210>186

<211>80

<212>DNA

<213>Artificial sequence

<220>

<223>Description of artificial sequence: Synthetic

oligonucleotide

<400>186

gcgcgctcgc tcgctcactg aggccgggcg accaaaggtc gcccgacgcc cgggcggcct 60

cagtgagcga gcgagcgcgc 80

<210>187

<211>79

<212>DNA

<213>Artificial sequence

<220>

<223>Description of artificial sequence: Synthetic

oligonucleotide

<400>187

gcgcgctcgc tcgctcactg aggcgcccgg gcgtcgggcg acctttggtc gcccggcctc 60

agtgagcgag cgagcgcgc 79

<210>188

<211>48

<212>DNA

<213>Artificial sequence

<220>

<223>Description of artificial sequence: Synthetic

oligonucleotide

<400>188

ggagtcaaag ttctgtttgc cctgatctgc atcgctgtgg ccgaggcc 48

<210>189

<211>99

<212>DNA

<213>Artificial sequence

<220>

<223>Description of artificial sequence: Synthetic

oligonucleotide

<400>189

attcatacca acttgaagaa aaagttcagc ctcttcatcc tggtctttct cctgttcgca 60

gtcatctgtg tttggaagaa agggagcgac tatgaggcc 99

<210>190

<211>588

<212>DNA

<213>Artificial sequence

<220>

<223>Description of artificial sequence: Synthetic

polynucleotide

<400>190

gcccctctcc ctcccccccc cctaacgtta ctggccgaag ccgcttggaa taaggccggt 60

gtgcgtttgt ctatatgtta ttttccacca tattgccgtc ttttggcaat gtgagggccc

120

ggaaacctgg ccctgtcttc ttgacgagca ttcctagggg tctttcccct ctcgccaaag

180

gaatgcaagg tctgttgaat gtcgtgaagg aagcagttcc tctggaagct tcttgaagac

240

aaacaacgtc tgtagcgacc ctttgcaggc agcggaaccc cccacctggc gacaggtgcc

300

tctgcggcca aaagccacgt gtataagata cacctgcaaa ggcggcacaa ccccagtgcc

360

acgttgtgag ttggatagtt gtggaaagag tcaaatggct ctcctcaagc gtattcaaca

420

aggggctgaa ggatgcccag aaggtacccc attgtatggg atctgatctg gggcctcggt

480

gcacatgctt tacatgtgtt tagtcgaggt taaaaaaacg tctaggcccc ccgaaccacg

540

gggacgtggt tttcctttga aaaacacgat gataatatgg ccacaacc 588

<---

Claims (62)

1. A method for prolonging or increasing the expression level of a transgene in a subject, comprising: a) administering a first dose of a capsidless closed-end DNA (ccDNA) vector containing a nucleic acid cassette containing at least one promoter-linked transgene between flanking inverted terminal repeats (ITRs), the first dose containing an amount of cccDNA vector for expressing a transgene in a subject, wherein the transgene encodes a target protein; And b) administering a second dose of said cccDNA vector to continue expression of the target protein in the subject, wherein the second dose is in an amount that is the same as the first dose, or wherein the second dose is 2-10 times greater than the first dose of the ccDNA vector, wherein : the second dose increases the expression level of the target protein or the second dose prolongs the expression of the target protein; And the second dose does not produce an immune response sufficient to prevent expression of the target protein from being achieved, and wherein said method is not a process of modifying the genetic identity of the human germline. 2. A method for increasing transgene expression in a subject, comprising: a) administering a first dose of a capsidless closed-end DNA (ccDNA) vector containing a nucleic acid cassette containing at least one promoter-linked transgene between flanking inverted terminal repeats (ITRs), the first dose containing an amount of cccDNA vector for expressing a transgene in a subject, wherein the transgene encodes a target protein; And b) administering a second dose of said cccDNA vector to continue expression of the target protein in the subject, wherein the second dose is in an amount that is the same as the first dose, or wherein the second dose is 2-10 times greater than the first dose of the ccDNA vector, and: the second dose increases the expression level of the target protein; And the second dose does not produce an immune response sufficient to prevent expression of the target protein from being achieved, and wherein said method is not a process of modifying the genetic identity of the human germline. 3. A method for increasing transgene expression in a subject, comprising: a) administering a first dose of a capsidless closed-end DNA (ccDNA) vector containing a nucleic acid cassette containing at least one promoter-linked transgene between flanking inverted terminal repeats (ITRs), the first dose containing an amount of cccDNA vector for expressing a transgene in a subject, wherein the transgene encodes a target protein; And b) administering a second dose of said cccDNA vector to continue expression of the target protein in the subject, wherein the second dose is in an amount that is the same as the first dose, or wherein the second dose is 2-10 times greater than the first dose of the ccDNA vector, and: the second dose prolongs the expression of the target protein; And the second dose does not produce an immune response sufficient to prevent expression of the target protein from being achieved, and wherein said method is not a process of modifying the genetic identity of the human germline. 4. A method for increasing transgene expression in a subject, comprising: a) administering a first dose of a capsidless closed-end DNA (ccDNA) vector containing a nucleic acid cassette containing at least one promoter-linked transgene between flanking inverted terminal repeats (ITRs), the first dose containing an amount of cccDNA vector for expressing a transgene in a subject, wherein the transgene encodes a target protein; And b) administering a second dose of said cccDNA vector to continue expression of the target protein in the subject, wherein the second dose is in an amount that is the same as the first dose, or wherein the second dose is 2-10 times greater than the first dose of the ccDNA vector, and: the second dose increases the expression level of the target protein and the second dose prolongs the expression of the target protein; And the second dose does not produce an immune response sufficient to prevent expression of the target protein from being achieved, and wherein said method is not a process of modifying the genetic identity of the human germline. 5. The method according to claim 1, characterized in that said cDNA vector is administered in combination with a pharmaceutically acceptable carrier. 6. Method according to any one of paragraphs. 1-4, characterized in that said second dose is intended to be administered at a point in time when the level of transgene expression decreases. 7. Method according to any one of paragraphs. 1-4, characterized in that said second dose is intended to be administered at least 90 days after the first administration or wherein the administrations are carried out on a periodic schedule. 8. Method according to any one of paragraphs. 1-4, characterized in that said transgene encodes a therapeutic protein, and the target level of expression of the transgene is a therapeutically effective amount of the therapeutic protein. 9. Method according to any one of paragraphs. 1-4, characterized in that said target protein is an inhibitor protein or said target protein replaces a defective protein or a protein that is not expressed. 10. Method according to any one of paragraphs. 1-4, characterized in that said inhibitor protein is an antibody or fusion protein. 11. The method according to claim 1, characterized in that said promoter is an inducible or repressible promoter. 12. Method according to any one of paragraphs. 1-4, characterized in that said cDNA vector additionally includes a regulatory switch. 13. Method according to any one of paragraphs. 1-4, characterized in that the specified ccDNA vector, which is administered in the first, second or any subsequent dose, is a ccDNA vector of the same type containing the same transgene or a modified transgene. 14. Method according to any one of paragraphs. 1-4, characterized in that said second cDNA vector has a different promoter operably linked to the same transgene or a modified transgene. 15. Method according to any one of paragraphs. 1-4, characterized in that said two flanking inverted terminal repeats (ITRs) are AAB ITRs. 16. Method according to any one of paragraphs. 1-4, characterized in that at least one of the flanking ICPs contains a functional end resolution site and a Rep binding site. 17. The method according to claim 16, characterized in that said flanking AAV ICPs are AAV-2 ICPs. 18. Method according to any one of paragraphs. 1-4, characterized in that: said flanking ICPs are symmetrical or asymmetrical; said flanking ICPs are symmetrical or substantially symmetrical; said flanking ICPs are asymmetrical; or said flanking ICPs are derived from different viral serotypes. 19. Method according to any one of paragraphs. 1-4, characterized in that one or both of said flanking ICPs contain a sequence selected from the group consisting of sequences SEQ ID NO: 1, 2, 5-48. 20. Method according to any one of paragraphs. 1-4, characterized in that at least one of these flanking ICPs is changed relative to the wild-type AAV ICP sequence by deletion, addition or substitution, which affects the overall three-dimensional conformation of the specified ICP. 21. The method according to claim 1, characterized in that one or both of said flanking ICPs are obtained from an AAV serotype selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV 10 , AAV 11 and AAV 12. 22. The method according to claim 21, characterized in that: one or both of these flanking ICPs are synthetic; one or both of the flanking ICPs indicated are not wild type; both of these flanking ICPs are not wild type; one of said flanking ICP is a wild-type ICP; or both of the flanking ICPs are wild-type ICPs. 23. Method according to any one of paragraphs. 1-4, characterized in that one or both of said flanking ICPs are modified by deletion, insertion and/or substitution in at least one of the ICP regions selected from A, A', B, B', C, C', D and D'. 24. Method according to any one of paragraphs. 1-4, characterized in that said deletion, insertion and/or substitution results in the removal of all or part of the stem-loop structure, which, in wild-type ICP, contains regions A, A', B, B', C or C' . 25. Method according to any one of paragraphs. 1-4, characterized in that: one or both of these flanking ICPs is modified by deletion, insertion and/or substitution, which results in the removal of all or part of the stem-loop structure, which, in the wild type ICP, contains regions B and B' ; one or both of said flanking ICPs is modified by deletion, insertion and/or substitution that results in the removal of all or part of the stem-loop structure, which, in the wild type ICP, contains the C and C' regions; or one or both of said flanking ICPs is modified by deletion, insertion and/or substitution that results in the removal of part of the stem-loop structure which, in the wild-type ICP, contains regions B and B', and/or parts of the stem-loop structure which , in wild-type ICP, contains regions C and C'. 26. Method according to any one of paragraphs. 1-4, characterized in that one or both of said flanking ICPs contain a single stem-loop structure in a region which, in the wild-type ICP, contains a first stem-loop structure formed by regions B and B', and a second stem-loop structure formed by regions C and C', or one or both of these ICPs contain one stem and two loops in a region which, in the wild type ICP, contains a first stem-loop structure formed by regions B and B', and a second stem-loop structure , formed by areas C and C'. 27. Method according to any one of paragraphs. 1-4, characterized in that both flanking ICPs are changed in such a way that this leads to an overall three-dimensional symmetry when said ICPs are inverted relative to each other. 28. Method according to any one of paragraphs. 1-4, characterized in that said cDNA vector additionally includes at least one regulatory switch. 29. The method of claim 28, wherein the at least one regulatory switch is selected from the group consisting of a binary regulatory switch, a small molecule regulatory switch, a password-based regulatory switch, a nucleic acid-based regulatory switch, a post-transcriptional regulatory switch, radiation-controlled or ultrasound-controlled regulatory switch, hypoxia-mediated regulatory switch, inflammatory response regulatory switch, shift-activated regulatory switch and kill switch. 30. Method according to any one of paragraphs. 1-4, characterized in that the subject has a disease or disorder selected from the group consisting of cancer, autoimmune disease, neurodegenerative disorder, hypercholesterolemia, acute organ rejection, multiple sclerosis, postmenopausal osteoporosis, skin disease, asthma and hemophilia. 31. The method according to claim 30, characterized in that: said cancer is selected from solid tumor, soft tissue sarcoma, lymphoma and leukemia; said autoimmune disease selected from rheumatoid arthritis and Crohn's disease; said skin disease is selected from psoriasis and atopic dermatitis, or said neurodegenerative disorder is Alzheimer's disease. 32. The method of claim 30, further comprising administering to the subject a subsequent dose of said cccDNA vector after the second dose, wherein the subsequent dose is designed to increase the level of expression of the transgene compared to the level of expression of the transgene achieved after the second dose, or to increase the level of expression of the transgene for achieving the target expression level. 33. Method according to any one of paragraphs. 1-4, wherein said transgene is or encodes a genetic drug selected from the group consisting of a nucleic acid, an inhibitor, a peptide or polypeptide, an antibody or antibody fragment, a fusion protein, an antigen, an antagonist, an agonist and an RNAi molecule. 34. Method according to any one of paragraphs. 1-4, characterized in that said second or subsequent dose of the cDNA vector is selected from an amount that increases the expression of the transgene by at least 3-fold, or at least 5-fold, or at least 10-fold, or 2-15-fold, or 2-20-fold, compared to the expression of the transgene after administration the first or previous dose of the cDNA vector. 35. The method according to claim 34, characterized in that said second or subsequent dose of the cccDNA vector is determined by the dose relationship between the dose of the ccDNA vector and the level of expression of the transgene from the ccDNA vector to achieve the target level of transgene expression in the cell.