JP2008523093A - Regulated expression of transgenes in the mammalian central nervous system - Google Patents

Abstract

  Recombinant adeno-associated virus (rAAV) vectors are provided for delivering regulatable transgenes to the mammalian central nervous system (CNS). Also provided are methods for treating patients with neurodegenerative diseases using vectors, and kits for constructing or using vectors, or for performing the methods of the invention. The transgene sequence is expressed from a promoter / enhancer region that contains one or more binding sites for transcription factors that respond to small molecule derivatives. Both the transgene construct and the construct containing the transcription factor are delivered to the target cells. Regulatable transgenes are delivered as transcription factors on the same rAAV vector or on separate vectors. A transcription factor may comprise two polypeptide chains, eg, a DNA binding domain and a transcriptional activation domain, that are active in the presence of a dimerization product such as rapamycin or a non-immunogenic analog thereof. A dimer is formed. The vectors, methods and kits of the invention may be used to deliver genes such as AADC or GDNF to the brain of patients with neurodegenerative diseases such as Parkinson's disease, where expression of AADC or GDNF in the brain is later Modulation is possible by treating the patient with rapamycin or a rapamycin analog.

Description

  The present invention relates to the regulation of genes introduced by gene therapy, in particular to the regulation of transgene expression transduced in the mammalian central nervous system (CNS).

  Many human diseases are caused by abnormal expression of genes. If the gene is underexpressed, or if the gene product itself is deleted, the absence of the corresponding functional gene product may be addressed by delivering the lost gene product to the patient. However, protein delivery is often difficult and only provides benefits for a limited period of time, meaning that protein must be re-administered regularly or indefinitely in the case of chronic disease To do. Repeated administration is costly, inconvenient and may be painful for poor patients. In addition, the level of the gene product can change rapidly between immediately after administration of one dose and immediately before administration of the next dose. This level of variability is particularly problematic in therapeutic agents with low pharmacokinetics (eg, short half-life) or a low therapeutic index.

  Gene therapy can be used by delivering a gene to cells that exhibit suboptimal expression of the gene rather than the gene product. In addition to providing low or incomplete expression of endogenous genes, gene therapy also has a beneficial effect when expressed in target cells such as cytokines, hormones, antibodies or genetically modified proteins Can also be used to deliver other genes that may have A gene delivered by gene therapy refers herein to a transgene. Gene therapy has the ability to deliver a constant level of gene product indefinitely when the transgene is stably expressed in the target cell. Gene therapy only needs to be performed once or at least infrequently compared to repeated delivery of the gene product itself. Proteins administered systemically are difficult to enter the brain due to the blood-brain barrier, and direct stereotactic injection into the brain is impractical for repeated administration, so gene therapy is a particularly useful method for delivering therapeutic proteins to the brain. preferable.

(Adeno-associated virus mediated gene therapy)
Viral vectors have been developed to assist in the efficient delivery of transgenes to target cells, a process implying transduction herein. One viral system that has been used for gene delivery is adeno-associated virus (AAV). AAV is a parvovirus belonging to the genus Dependvirus. AAV has several attractive features not found in other viruses. First, AAV can infect a wide range of host cells, including non-dividing cells. The ability to infect non-dividing cells makes AAV a particularly suitable option for transducing CNS tissue, eg, the brain. Secondly, AAV can infect cells from different species. Third, AAV is not associated with human or animal disease and does not appear to alter the biological properties of the host cell through integration. In fact, it is estimated that 80-85% of the human population is infected with the virus. Finally, AAV is stable in a wide range of physical and chemical conditions and is easy to produce, store and transport.

  The AAV genome is a straight single-stranded DNA molecule about 4681 nucleotides (nt) long. AAV genomes generally include internal non-repeating genomes with terminal inverted sequences (ITRs) located at both ends. ITRs are approximately 145 nt long. ITRs act as an origin of replication for DNA replication and a packaging signal for the viral genome.

  The internal non-repetitive portion of the genome contains two major open reading frames due to AAV replication (rep) and capsid (cap) genes. The rep and cap genes encode proteins that the virus replicates and packages the viral genome into virions. In particular, one family containing at least four viral proteins is expressed from the rep region of AAV; Rep78, Rep68, Rep52, and Rep40 have been named for their apparent molecular weight. The cap region of AAV encodes at least three proteins; VP1, VP2, and VP3.

  AAV is a helper-dependent virus. That is, co-infection with a helper virus (eg, adenovirus, herpes virus or vaccinia) is generally required to form AAV virions. In the absence of co-infection with helper virus, AAV establishes a latent state where the viral genome persists as an episome or inserts into the host cell chromosome, but no infectious virions are produced. Subsequent infection with helper virus “rescue” the integrated genome, ie, allow it to replicate and package the genome into infectious AAV virions. While AAV can infect cells of different origin, the helper virus must be the same species as the host cell. Thus, for example, human AAV will replicate in canine cells co-infected with canine adenovirus.

  AAV vectors deliver the gene of interest by deleting the internal non-repeating portion of the AAV genome (ie, rep and cap) and inserting a heterologous gene (“transgene”) between the ITRs. Designed. The transgene may be linked to a heterologous promoter. Terminal signals such as polyadenylation sites may also be included.

  To obtain a stock of infectious recombinant AAV (rAAV) containing the transgene, a suitable producer cell line is transfected with an AAV expression vector containing the transgene sandwiched between AAV ITRs. AAV helper functions and auxiliary functions are also expressed in the producer cells. Helper functions are generally functions provided by AAV-encoding genes (eg, rep and cap), and auxiliary functions are generally wild type AAV (wtAAV) helper virus (eg, adenovirus) Supplied by helper virus when replicating in co-infected cells. The gene encoding the helper function in WtAAV must be supplied separately because it is removed during the construction of the rAAV vector to make space for the transgene sequence. In the presence of helper and auxiliary functions, vector constructs containing transgenes and AAV ITRs are replicated and packaged to form recombinant AAV virions (rAAV). The production of rAAV stock is described in US Pat. Nos. 5,622,856, 6,001,650, 6,027,931, 6,365,403, which are incorporated herein by reference. No. 6,376,237, 5,945,335, 6,004,797, and 6,482,633.

  The rAAV stock thus prepared can then be used to introduce transgenes into target cells in vitro by infecting the target cells with rAAV in the absence of helper virus, or into an in vivo patient. . Since patient cells lack the rep and cap genes as well as auxiliary functional genes, the rAAV vector lacks replication in the target cells. That is, they cannot further replicate and package their own genome. Similarly, without a source of rep and cap genes, wtAAV cannot be formed in a patient's cells.

(Regulation of transgene expression)
One disadvantage of AAV-mediated gene therapy and general gene therapy is that once administered, it is not possible to reverse the treatment or to regulate its effects. Unlike drug delivery, which can be interrupted whenever necessary, successful gene therapy provides sustained long-term gene expression after vector administration. The ability to block transgene expression is an important safety consideration in the treatment of human patients. Furthermore, the ability to down regulate transgene expression would be useful to ensure proper pharmacological control of the transgene product within the patient.

  Various systems have been developed to regulate transgene expression using small molecule derivatives. Rivera et al. (1996) Nat. Med. 2: 1028-32, No et al. (1996) Proc. Natl. Acad. Sci. USA 93: 3346-51, Gossen and Bujard (1992) Proc. Natl. Acad. Sci. USA 89: 5547-51, the GeneSwitch® system (Valentis, Burlingame, Calif.). These systems are based on the use of improved transcription factor activity controlled by small molecule drugs and transgene expression promoted by regulated transcription factors. One such system is the formation of functional transcription factors derived from two synthetic fusion proteins by the addition of rapamycin, based on induction by rapamycin (referred to herein as the “dimerizer system”). About. Rivera et al. (1996) Nat. Med. 2: 1028-32. Rapamycin is an orally bioavailable small molecule drug closely related to the natural product immunosuppressant FK506, which binds to the cellular protein FKBP12 with high affinity (200 pM) and the complex binds to FRAP. Rapamycin has an intrinsic immunosuppressive effect, but non-immunosuppressive analogs can be used in conjunction with a modified FRAP gene sequence to facilitate transcription in dimeric systems without unwanted immunosuppression. Has been developed. Pollock et al. (2000) Proc. Natl. Acad. Sci. USA 97: 13221-26.

  A system based on dimerization of in vivo genetic regulation is described by Rivera et al. (1996) Nat. Med. 2: 1028-32 and Pollock et al. (2000) Proc. Natl. Acad. Sci. USA 97: 13221-26. Dimerization systems are available for muscle (Ye et al. (1999) Science 283: 88-91, Rivera et al. (1999) Proc. Natl. Acad. Sci. USA 96: 8657-62, Johnston et al. (2003) Mol. Ther. 7: 493-7), viral vectors that deliver genes to the liver (Aurichio et al. (2002) Gene Ther. 9: 963-71), and the eye (Aurichio et al. (2002) Mol. Ther. 6: 238-42). Has been adopted. The dimerization system is also an element of the ARGENT transcription technology platform of ARIAD Pharmaceuticals, Inc. (Cambridge, Mass.). This technique further describes Crabtree et al. US Pat. No. 6,043,082, which generally describes systems based on ARGENT dimerization, and synthetic DNA binding domains, p65 transcriptional activation domains and reduced immunosuppression Clackson et al., US Pat. No. 6,649,595, describes a dimer-based system using rapamycin or a rapamycin analog, including rapalogs having US Pat. No. 6,043,082 and US Pat. No. 6,649,595 are hereby incorporated by reference in their entirety.

  The dimerization system is sufficient for humans in that the functional elements of the transcriptionally active fusion protein are all human-derived proteins and therefore reduce the potential for adverse immune responses in humans. Has advantages for adaptation. Rivera et al. (1999) Proc. Natl. Acad. Sci. USA 96: 8657-62.

(Parkinson's disease)
Parkinson's disease (PD) is an example of a disease that can be adapted for gene therapy, particularly AAV gene therapy. PD is the second most common neurodegenerative disease in the United States and affects over 1 million people. PD is characterized by reduced locomotor activity, difficulty walking, posture instability, stiffness and tremor. These clinical signs are a direct result of degeneration of colored nerves (ie dopaminergic nerves) within the substantia nigra of the basal ganglia region of the brain. Progressive degeneration of the substantia nigra causes a reduction in available dopamine, since the substantia nigra is the synthesis site for important catecholamine neurotransmitters.

Dopamine is synthesized at the terminal nerve endings of the substantia nigra, especially the dopaminergic nerves of the substantia nigra. Dopaminergic nerves project to the striatum, particularly to the innervated putamen and caudate nucleus. Three enzymes are required for effective dopamine biosynthesis; they are tyrosine hydroxylase (TH), guanosine triphosphate cyclohydrolase I (GCH), and aromatic L-amino acid decarboxylase (AADC). It is. Tyrosine hydroxylase adds a hydroxyl group to the amino acid tyrosine to synthesize L-dihydroxyphenylalanine (L-dopa). GCH catalyzes the first step and the biosynthesis rate-limiting step of BH ₄ cofactor necessary for TH activity. Finally, AADC removes the terminal carboxyl group of L-dopa to produce dopamine.

  Treatment of PD currently involves oral administration of L-dopa, often in combination with a related inhibitor of AADC. Concurrent with the progression of PD, the majority of patients experience a decrease in AADC content in the affected area of the brain (ie, substantia nigra). Since AADC is required to convert L-dopa to dopamine, increasing the L-dopa dose is necessary for therapeutic effects, but this often leads to increased side effects. Furthermore, as the substantia nigra deteriorates, AADC continues to decline, often reaching a level where the benefits of treatment with L-dopa administration are no longer recognized.

(AADC)
One approach based on gene therapy for PD therapy is to supply genes encoding one or more enzymes related to dopamine biosynthesis, such as AADC. Supplying AADC restores the effectiveness of L-dopa treatment. AAV-derived vectors and methods used for PD treatment by delivery and expression of AADC in the brain of a mammalian patient are described in US Patent Publication No. 2002/0172664, which is hereby incorporated by reference in its entirety. Is done.

  Parkinson's disease (PD) is characterized by progressive loss of dopaminergic nerves in the substantia nigra and severe reduction of dopamine in the striatum. Hornykiewicz (1975) Natl. Inst. Drug Abuse Res. Monogr. Ser. 13-21. The 6-OHDA model is created by chemically damaging the medial forebrain bundle that projects from the substantia nigra to the striatum, and is histopathologically similar to PD. Ungerstedt (1971) Acta Physiol. Scand. Suppl. 367: 69-93. Rats unilaterally hemispherically 6-OHDA damaged develop rapid contralateral rotational activity in response to therapeutic doses of L-dopa or dopamine receptor agonists. Ungerstedt (1971). In previous studies, transferring a gene encoding human AADC (hAADC) into the rat or non-human primate striatum and exogenously delivering L-dopa has been associated with Parkinson's disease (PD). Have been shown to be able to restore dopamine to an effective level and reduce the need for L-dopa. Bankiewicz et al. (2000) Exp. Neurol. 164: 2-14, Sanchez-Pernaute et al. (2001) Mol. Ther. 4: 324-30.

(GDNF)
Another gene therapy based approach to the treatment of PD is to deliver genes that inhibit or delay the progression of the degenerative process. Glial cell-derived neurotrophic factor (GDNF) is a potent neurotrophic factor that has been shown to enhance DA neuronal survival in both in vitro and in vivo PD animal models (Bjorklund et al., Brain Res. (2000) 886: 82-98, Bohn, MC, Mol.Ther. (2000) 1: 494-496, et al., J. Neural Trans.Suppl. (2000) 58: 181-191), It was integrated into the present specification by explicit reference. AAV-derived vectors and methods of PD treatment by delivery and expression of GDNF in the brain of a mammalian patient are described in US Patent Application Publication No. 2003/0050273, incorporated herein by reference.

  While expression of AADC or GDNF in the brains of Parkinson patients is beneficial, overexpression of these genes can cause dangerous side effects. Typical vectors used in gene therapy are designed to incorporate a strong constitutive promoter that is intended to maximize overall transgene expression to ensure therapeutic efficacy Has been. In many past studies, attempts were made to maximize expression from the few transduced cells, when transduction efficiency is expected to be relatively low, and when the transgene product is transduced. It may be a reasonable goal if it is secreted from the cells into the systemic circulation. Several previous studies of transgene expression regulated by dimerization relate to such secreted proteins. Rivera et al. (1996) Nat. Med. 2: 1028-32, Rivera et al. (1999) Proc. Natl. Acad. Sci. USA 96: 8657-62, Ye et al. (1999) Science 283: 88-91, Pollock et al. (2000) Proc. Natl. Acad. Sci. USA 97: 13221-26, Auricchio et al. (2002) Gene Ther. 9: 963-71, Johnston et al. (2003) Mol. Ther. 7: 493-7, Auricchio et al. (2002) Mol. Ther. 6: 238-42.

  The therapeutic benefit when the non-secreted transgene product is overexpressed in some cells and totally deficient in other cells, as opposed to the product of secreted proteins accumulating in the circulatory system Cannot be achieved. Gene therapy utilizing non-secretory proteins requires transgene expression that is regulated at the desired level within each transduced cell, and it is particularly important that the expression of such proteins can be regulated after transduction. Can be.

  Gene therapy for neurodegenerative diseases and other CNS disorders is an expanding technology field (Tinsley and Eriksson (2004) Acta Neurol. Scand. 109: 1-8), and transgene regulation is both dosage and safety. It may be necessary for both sexual purposes. Regulatory gene expression may not be necessary in the case of AADC therapy because dopamine production is in principle controllable by exogenous delivery of the precursor L-dopa, but regulation is the amount of enzyme delivered It can be beneficial to precisely control or to terminate treatment if necessary. Regulation is necessary for gene delivery of neurotrophic factors (such as GDNF) where overexpression is a detrimental effect, and the degree of control required is determined by the particular application.

  There is a need for methods of regulating gene expression introduced into a patient's CNS (eg, brain) by gene therapy, and vectors that allow such regulation. In particular, there is a need for methods of regulating transgene expression in the brain of patients suffering from neurodegenerative diseases such as PD. Preferably, the regulatory system not only has a low background level of transgene expression under repressed conditions, but also exhibits a high induction rate. Optimal systems will also minimize the chance of an adverse immune response during human gene therapy by including functional elements all derived from human proteins.

(Summary of the Invention)
These as well as other needs in the art are compatible with the present invention. That is, the present invention provides vectors, methods and kits for AAV-mediated gene therapy, in which transgene expression in target cells of the nervous system can be regulated using derivatives.

  In one aspect, the present invention relates to a recombinant AAV (rAAV) vector in which transgene expression can be regulated after transgene transduction into the CNS (eg, brain) of a patient.

  In one embodiment, modulation is achieved using a transcription factor comprising two polypeptide elements that are only activated when the two elements bind to each other, and these elements are present in the presence of the derivative. Bond only to each other. In one embodiment, the two polypeptides include a DNA binding domain fusion and an activation domain fusion. A typical DNA binding domain fusion includes two DNA binding domains derived from the human transcription factor Zif268, a homeodomain derived from Oct-1, and three drug binding domains derived from the cellular receptor of FK506. A typical active domain fusion includes the rapamycin binding domain of human FRAP fused to a transcriptional activation domain derived from the p65 subunit of NFκB.

  In one embodiment, the first rAAV vector contains a transgene and the second rAAV vector contains a sequence of transcription factor elements. In another embodiment, a single rAAV vector comprises a transcription factor element and a transgene sequence.

  In one embodiment, the modulation is made by administration of a derivative. In another embodiment, the derivative is a dimerization such as rapamycin or a non-immunosuppressive analog thereof, such as AP21967.

  In another aspect, the invention relates to a method of treating a patient with an rAAV vector that can modulate transgene expression after the transgene has been transduced into the patient's CNS (eg, brain). In one embodiment, the method of treatment involves administration of a derivative, eg, a dimerization such as rapamycin or a non-immunosuppressive analog thereof, eg, AP21967.

  In yet another aspect, the present invention relates to a kit for constructing the vector of the present invention or for performing the method of the present invention. In one embodiment, the kit includes a first rAAV vector having a polylinker region for cloning the transgene of interest of the user. In another embodiment, the kit further comprises a second rAAV vector encoding a transcription factor capable of regulating expression from a transgene cloned into the first rAAV vector. In yet another embodiment, the kit includes a derivative, for example, a dimer such as rapamycin or a non-immunosuppressive analog thereof, such as AP21967.

  In one embodiment, the present invention relates to the treatment of human neurodegenerative diseases such as Parkinson's disease (PD).

  In some embodiments, the regulated transgene is AADC or GDNF, but in general, the regulated transgene can be any gene that is desired for expression in the target tissue.

  These and other embodiments of the invention will be readily made by those of ordinary skill in the art for the disclosure herein.

(Explanation of the figure)
FIG. 1A is a schematic of an rAAV vector (AAV-CMV-TF) encoding a transcription factor activation and DNA binding domain for transgene expression regulation.

  FIG. 1B is a schematic of a recombinant AAV vector encoding hAADC (AAV-Z12-hAADC) whose expression can be regulated by the addition of rapamycin or a derivative thereof.

  FIG. 1C is a schematic of a recombinant AAV vector (AAV-CMV-hAADC2) encoding hAADC whose expression is driven by a constitutive CMV promoter.

FIG. 2 shows the results of experiments in which D7-4 cells were transduced with various rAAV constructs and treated with 0, 5 or 25 nM rapamycin analog AP21967. Untreated controls are also shown. “OD” value refers to relative AADC expression (measured at OD ₄₀₅ in AADC expression ELISA) calculated using a standard curve obtained from cells transduced with AAV-CMV-hAADC2 in a reference lot. To do.

  FIG. 3 is a time series of experiments in which the rotational response to L-dopa was measured as a function of rapamycin treatment in 6-OHDA-damaged rats.

  FIG. 4 shows the rotational response to 5 mg / kg L-dopa in 6-OHDA-damaged rats, expressed as contralateral rotations per hour as a function of time along the time series shown in FIG. Rats are injected with vehicle alone (control) or a vector encoding regulatable hAADC and the corresponding transcription factor. Data do not show results for week 1 and week 2. As shown in FIG. 3, injection is performed on day 0, and that day is set as the day on which the number of weeks is started (for example, “3 weeks” means 21 days after injection). For simplicity, the four rapamycin injections shown in FIG. 3 (“Rapamycin”) are represented in FIG. 4 as a single arrow.

  FIG. 5A shows the results of AADC immunohistochemistry in rat striatum 7 weeks after injection treated with rapamycin together with an AAV vector having the sequence required for rapamycin-regulated expression of hAADC. The scale bar represents 75 μm.

  FIG. 5B shows experimental results similar to FIG. 5A, except that the rats are untreated with rapamycin.

  FIG. 6 shows AADC immunohistochemistry results of typical rat-derived whole-mount brain sections in three treatment groups obtained 7 weeks after injection: A: vector injection (+) rapamycin; B: vector injection (−) Rapamycin; C: vehicle injection (+) rapamycin. As shown in FIG. 4, rats were injected with vehicle alone (FIG. 6C) or a vector encoding regulatable hAADC and corresponding transcription factors (FIGS. 6A and 6B). The rats of FIGS. 6A and 6C were then treated with rapamycin (as shown in FIG. 3), but the rat of FIG. 6B was not treated. The left hemisphere is the site of 6-OHDA injury and intrastriatal vector (or vehicle) injection, and the right hemisphere shows endogenous staining of normal rat AADC positive fibers.

  FIG. 7 shows AADC expression 7 weeks after injection measured by Western blot analysis in the three treatment groups considered according to FIG. The upper figure is the AADC and β-actin bands observed by electrophoresis of typical rat brain derived proteins from each of the three treatment groups. β-actin was used as a control. The bar graph shows the average integrated image density and standard deviation of the protein bands in the AADC band of each of the three treatment groups.

  FIG. 8A shows a rAAV comprising transcription factors activating transcription and DNA binding domain fusions, and regulatable transcription encoding the transgene hGDNF, wherein expression of hGDNF can be regulated by rapamycin or the like. 1 is a diagram of a vector (AAV-TF-Z8-hGDNF).

  FIG. 8B shows that expression of the transcription factor DNA binding domain is promoted by the SV40 promoter on a transcription separate from that of the activation domain, and that the activation domain is expressed from the opposite strand (ie, in the opposite direction). FIG. 8B is a schematic of a vector similar to that shown in FIG.

  FIG. 8C is a schematic of a recombinant AAV vector encoding hGDNF (AAV-CMV-hGDNF) whose expression is driven by a constitutive CMV promoter.

  FIG. 9 shows a transient response with either a regulatory TF-GDNF plasmid (AAV-TF-Z8-hGDNF) or a constitutive CMV-GDNF plasmid (AAV-CMV-hGDNF) depending on 0, 5 or 25 nM rapamycin treatment. Shows GDNF expression in picogram units (pg) in HelaD7-4 cells transfected with.

(Detailed description of the invention)
The practice of the present invention is carried out according to conventional methods of protein chemistry, biochemistry, recombinant DNA technology and pharmacology within the art, unless otherwise specified. Such techniques are explained fully in the literature. T.A. E. Creighton, Proteins: Structures and Molecular Properties (W. H. Freeman and Company, 1993), A.C. L. Lehninger, Biochemistry (Worth Publishers, Inc., current addition), Sambrook et al., Molecular Cloning: A Laboratory Manual (2nd Edition, 1989), Methodology. , Remington's Pharmaceutical Science, 18th Edition (Easton, Pa .: Mack Publishing Company, 1990).

(I, definition)
All scientific and technical terms used in this application have meanings commonly used in the art unless otherwise specified. As used in this application, the following words or phrases have specific meanings.

  As used in the specification and claims, the singular forms “a”, “an”, and “the” include plural references unless the content clearly dictates otherwise. There must be. Thus, for example, reference to “a vector” includes a mixture of two or more such vectors, and the like.

  The term “transgene”, as used herein, in some embodiments, regardless of whether it is derived from a transgene, including additional copies or sequence changes of an endogenous gene already present in the target cell. It refers to a gene that is delivered to a target cell. A transgene can be a gene sequence or partial gene sequence obtained from an organism, a genetically engineered sequence change of such a gene, or a synthetic (non-naturally occurring) DNA sequence. The transgene may direct the production of a messenger RNA encoding the protein or may encode a biologically active RNA molecule such as antisense, ribozyme, triplex forming, RNAi or other RNA sequence.

  A “regulatable transgene” as used herein is a gene whose expression can be altered by administration of a derivative after being transduced into a target cell. Regulation of therapeutic transgene expression provides pharmacological control of the level of the transgene product.

  “Expression cassette” means a construct capable of directing the expression of a sequence or gene of interest. An expression cassette includes a promoter or promoter / enhancer operably linked to a sequence or gene of interest (to effect direct transcription thereof), and often also includes a polyadenylation sequence. Within certain embodiments of the invention, the expression cassette described herein may be included in an adenovirus-associated virus construct.

  “Recombinant” as used herein with respect to nucleic acids relates to all or some of the polynucleotides with which the genomic, cDNA, viral, semi-synthetic, or synthetically derived polynucleotide is originally associated, depending on its origin or manipulation. Means not. The term “recombinant” as used with respect to a protein or polypeptide means a polypeptide produced by expression of a recombinant polynucleotide. In general, the gene of interest is cloned and then expressed in the transformed animal, as further described below. A host organism expresses an external gene under expression conditions to produce a protein.

  A “coding sequence” or a sequence that “encodes” a selected polypeptide is transcribed (in the case of DNA) into a polypeptide in vivo when placed under the control of suitable regulatory sequences (or “control elements”) and A nucleic acid molecule that is translated (in the case of mRNA). The boundaries of the coding sequence can be determined by a start codon at the 5 '(amino) terminus and a translation stop codon at the 3' (carboxy) terminus. A coding sequence can include, but is not limited to, viral-derived cDNA, prokaryotic or eukaryotic mRNA, genomic DNA sequences from viral or prokaryotic DNA, and even synthetic DNA sequences. A transcription termination sequence may be located 3 'to the coding sequence.

  Typical “control elements” are: transcription promoter, transcription enhancer element, transcription termination signal, polyadenylation sequence (localized at the 3 ′ translation stop codon), translation initiation optimized sequence (localized at the 5 ′ coding sequence). Including, but not limited to, translation termination sequences.

  The term “nucleic acid” also includes DNA and RNA, and analogs thereof such as modified backbones (eg, phosphorothioates), as well as peptide nucleic acids (PNA) and the like. The invention includes nucleic acids (eg, for antisense or probes) that include sequences complementary to those described above.

  The term “transfection” is used to mean the uptake of external DNA into a cell. A cell is “transfected” when exogenous DNA is introduced inside the cell membrane. Many transfection techniques are generally known in the art. Graham et al. (1973) Virology, 52: 456, Sambrook et al. (1989) Molecular Cloning, a laboratory manual, Cold Spring Harbor Laboratories, New York, Ms. Bio, et al. (1986) See Gene 13: 197, 1981. Such techniques can be used to introduce one or more exogenous DNA moieties, such as plasmid vectors and other nucleic acid molecules, into suitable host cells. The term refers to both stable and transient uptake of genetic material.

  The term “transduction” means delivery of a DNA molecule to a recipient cell in vivo or in vitro via a vector, such as a recombinant adeno-associated virus vector.

  The term “heterologous”, related to nucleic acid sequences, such as gene sequences and regulatory sequences, usually refers to sequences that do not bind together and / or are not generally associated with a particular cell. Thus, a “heterologous” region of a nucleic acid construct or vector is a fragment of a nucleic acid that is naturally associated with or bound to another nucleic acid molecule that is not found in association with the other molecule. For example, a heterologous region of a nucleic acid construct can include a coding sequence that is flanked by sequences that are not found in relation to the original coding sequence. Another example of a heterologous coding sequence is a construct where the coding sequence itself is not found in nature (eg, a synthetic sequence having a different codon from the wild type gene). Similarly, cells transformed with constructs that are generally not present in the cell are considered heterogeneous for the purposes of the present invention. Allelic variation or spontaneous mutation phenomenon does not result in heterologous DNA as used herein.

  The term “control element” refers to a promoter region, polyadenylation signal, transcription termination sequence, upstream regulatory domain, origin of replication, in-sequence ribosome entry site (“ IRES "), enhancer, and the like. All of these control elements are not always necessary if the chosen coding sequence is capable of replication, transcription and translation in a suitable host cell.

  As used herein, the term “promoter region” refers in a general sense to a nucleic acid region comprising a DNA regulatory sequence, which is bound by an RNA polymerase and downstream (3 ′ direction) coding sequence. Derived from a gene that can initiate transcription of.

  “Operably linked” means an arrangement of elements that are set up so that the elements so described perform their normal functions. Thus, a control element operably linked to a coding sequence can result in the expression of the coding sequence. A control element need not be adjacent to a coding sequence as long as it serves to direct the expression of the coding sequence. Thus, for example, even if an intervening untranslated transcription sequence is placed between the promoter sequence and the coding sequence, the promoter sequence is still considered “operably linked” to the coding sequence.

  By “isolated” with respect to a nucleotide sequence is meant that the particular molecule is present in the substantial absence of other biological macromolecules of the same type. Thus, an “isolated nucleic acid molecule that encodes a specific polypeptide” means a nucleic acid molecule that is substantially free of other nucleic acid molecules that do not encode a polypeptide of interest, but is fundamental to the nature of the compound. Additional bases or molecules that do not deleteriously affect may be included.

  A “vector” is capable of transferring nucleic acid sequences into target cells (eg, viral vectors, non-viral vectors, particulate carriers, and liposomes). Typically, “vector construct”, “expression vector”, and “gene transfer vector” refer to a nucleic acid construct that directs the expression of a nucleic acid of interest and is capable of transferring a nucleic acid sequence into a target cell. The term therefore includes cloning and expression carriers, as well as viral vectors.

  “Patient” refers to humans and other primates, including chimpanzees and other apes and non-human apes such as monkey species, domestic animals such as cattle, sheep, pigs, goats and horses, and domestic mammals such as dogs and cats Chordates including birds, including laboratory animals including rodents, mice, rats, guinea pigs, domestic birds, wild birds and game birds such as chickens, turkeys and other poultry birds, ducks, geese, and the like It means Amon, but it is not limited to them. The term does not denote a particular age. Therefore, each of adults and newborns is covered.

  By “therapeutically effective dose or amount” of an AAV vector encoding a regulatable transcription factor and / or transgene, a positive therapeutic response when the AAV vector is administered as described herein, eg, An amount that ameliorates the symptoms of neurological disease or inhibits progression is contemplated. For example, administration of a therapeutically effective amount of an AAV vector results in expression of a therapeutic factor (eg, AADC, GDNF) in a patient treating Parkinson's disease, for example, improving motor function or shaking at rest May improve symptoms such as relief. The appropriate amount of AAV vector required will vary from patient to patient depending on species, year, and patient's overall condition, the degree of condition being treated, and the particular compound used, the dosage form, and the like. It will change.

(II. Embodiment of the present invention)
Before describing the details of the present invention, it is to be understood that the present invention is not limited to the particular exemplified molecule or method parameters, and may naturally be varied as such. is there. It will also be appreciated that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting. In addition, the practice of the present invention will employ conventional methods within the ordinary technical scope of virology, microbiology, molecular biology, recombinant DNA techniques and immunology, unless otherwise specified. Such techniques are explained fully in the literature. Sambrook et al., Molecular Cloning: A Laboratory Manual (2nd edition, 1989), DNA Cloning: A Practical Approach, vol. I & II (Edited by D. Glover), Oligonucleotide Synthesis (Edited by N. Gait, 1984), A Practical Guide to Molecular Cloning (1984), and Fundamental Virology, 2nd Edition. See I & II (B.N. Fields and D.M.Knipe). Although many of the materials and methods are similar or equivalent to those described herein and can be used in the practice of the present invention, the preferred materials and methods are described herein.

  The present invention provides vector constructions, methods and kits for regulating transgene expression in target cells. In one embodiment, the sequences necessary for transduction of a transgene regulatable in a cell are provided on multiple separate recombinant AAV vector molecules. Examples 1 and 2 show the construction and use of specific examples of the double vector of the invention. In another embodiment, a single recombinant AAV vector carries all the sequences necessary for transgene transduction that can be regulated in the target cell. Example 3 shows the construction and use of one embodiment of such a single vector of the invention.

  In one embodiment, a regulatable form of the AADC gene is delivered to the target cell. Examples 1 and 2 show the delivery of a regulatable form of the AADC gene. In another embodiment, a regulatable form of the GDNF gene is delivered to the target cell. Example 3 shows the delivery of a regulatable form of the GDNF gene.

  As used herein, the term transgene, in certain embodiments, refers to the target cell regardless of the origin of the transgene, including additional copies of endogenous genes already present in the target cell, or sequence changes. Means the gene to be delivered. A transgene is a gene sequence or partial gene sequence obtained from several organisms, a genetically modified sequence change of such a gene, or a synthetic (non-naturally occurring) DNA sequence obtain. The transgene can direct the production of a messenger RNA encoding the protein or can encode a biologically active RNA molecule such as an antisense, ribozyme, triplex forming, RNAi or other RNA sequence.

  As used herein, a regulatable transgene is a gene whose expression can be altered by administration of a derivative after the gene has been transduced into a target cell. Regulation of therapeutic transgene expression provides pharmacological control at the transgene product level. One such small molecule drug is rapamycin.

In one embodiment, the regulatable gene expression system of the present invention provides specific dimerization of immunophilin FKBP and lipid kinase homologue FRAP in the presence of rapamycin or AP21967, a non-immunosuppressive rapamycin analog. Utilizing it produces an active transcription factor complex that activates a specifically inducible promoter. Rivera et al. (1996) Nat. Med. 2: 1028-32. The structure of AP21967 is provided below.

AP21967 (molecular weight 1017.4 Da) is a 7- methylindolyl analog represented by compound 69 of US Pat. No. 6,649,595 (the '595 patent), as described in Example 5 therein. , 7-methylindole can be prepared by substituting dimethoxybenzene. AP21967 corresponds to compound 71 of the '595 patent except that it has a 7-methyl group on the indole ring.

  Other rapamycin analogs can also be used without departing from the scope of the present invention. The rapamycin analog will not interact with the patient's endogenous FRAP and will not potentially have the unwanted immunosuppressive and cell cycle inhibitory effects of rapamycin. Pollock et al. (2000) Proc. Natl. Acad. Sci. USA 97: 13221-26. FRAP variants can be made to maintain the ability to bind to non-immunosuppressive rapamycin analogs through the use of the FRB portion of a dimerized activation domain fusion protein. See, for example, Pollock et al. (2000).

(Regulated expression of AADC)
Experiments are performed using AAV vector-mediated modulation of rapamycin-dependent AADC transgene expression in vitro in HeLaD7-4 cells and in an in vivo rodent model of Parkinson's disease (PD). Expression of the human AADC (hAADC) transgene is made dependent on functional transcription factor (TF) reconstitution with dimerized rapamycin using the vectors further described in Example 1 and FIGS. 1A and 1B. The Transcription factor AAV vectors are used to deliver the transcription factor DNA binding and activation domains, and expression AAV vectors are used to deliver hAADC transgenes under the control of a regulatable promoter.

(In vitro AADC regulation)
An experiment performing dimerization-dependent AADC expression in human cells in vitro is described in Example 1. The dimerization product, AP21967 used in Example 1, is a non-immunosuppressive rapamycin analog that has approximately 3 times less activity than rapamycin. The results are shown in FIG. 2, and the rightmost data show rapamycin dose-dependent hAADC expression (AAV− in cells cotransduced with a vector encoding a dimerization-dependent transcription factor (AAV-CMV-TF). Z12-hAADC). The ELISA data shows that AADC expression is 9 times higher in the presence of 25 nM AP21967 than in the absence of AP21967. 25 nM is the maximally effective concentration of AP21967 and provides a stable level for transgene expression in vitro. HAADC expression from a regulatable promoter in 25 nM AP21967 is about half the level of hAADC expression under the control of the constitutive CMV promoter (AAV-CMV-hAADC2) (FIG. 1C, and Sanftner et al. (2004) Mol. Ther.9: 403-9).

  Cells with both vectors (AAV-Z12-hAADC and AAV-CMV-TF) show only low levels of AADC production in the absence of AP21967, ie the system is not “leaky”. The absence of leakage confirms that the transgene can be totally blocked as needed and can be critical to gene therapy to provide a wide range of pharmacological regulation.

(AADC regulation in vivo)
The same vector used to perform AAV-mediated transduction of dimerized regulatory hAADC in vitro (FIGS. 1A and 1B) is a surgical model of striatal denervation as described in Example 2. In vivo in a 6-hydroxydopamine (6-OHDA) rat model of Parkinson's disease. The treatment schedule is represented schematically in FIG. As shown in FIG. 4, Parkinson's disease rats are transduced with both AAV-CMV-TF and AAV-Z12-hAADC, and then their rotational behavior is measured after a series of rapamycin treatments.

  Rapamycin is used in in vivo experiments rather than AP21967 used in vitro, for the simple reason that the appropriate concentration of rapamycin has been previously investigated. Although the dimerized rapamycin has many advantageous properties for use as a derivative in human gene therapy, its unique immunosuppressive properties can limit its utility. Non-immunosuppressive analogs of rapamycin, such as AP21967, may be excellent derivatives of transgene expression, particularly in the treatment of human patients. The appropriate dose of AP21967 in animals and human patients who are in vivo may be determined by common laboratory methods, including clinical trials.

  Transduction of a regulated hAADC vector with a combination of L-dopa and rapamycin administration produces behavioral effects in 6-OHDA-damaged rats consistent with production of significant levels of dopamine. As shown in FIG. 4, rapamycin treatment reversibly increases hAADC expression in the damaged striatum, a change to 5 mg / kg that is reversible by cessation of rapamycin use in the vector-injected (+) rap group Indicated by the rotation response. The rotational response to L-dopa of the vector-injected (+) rapamycin group was significantly greater than both the vector-injected (−) rapamycin group and the vehicle-injected control group immediately after rapamycin treatment at 3, 5 and 7 weeks. Increase (P <0.001) but return to near control levels at 4 and 6 weeks.

  In quantitative real-time PCR, rats in the vector-injected (+) rapamycin group and vector-injected (-) rapamycin group were used to eliminate the possibility of different transduction efficiencies as an explanation for the different results observed in the two groups. Confirm transduction with an equivalent copy number of hAADC gene.

  Immunohistochemistry and protein expression assays performed at the end of the study (Table 1 and FIGS. 5, 6 and 7) support behavioral results as discussed in detail below.

  FIG. 5A shows intense immunohistochemical staining of AADC and therefore effective transgene expression in the spinous nerve in the middle of the rat striatum from the vector-injected (+) rapamycin group, while FIG. Only very low levels of AADC expression are shown in rats from the injected (-) rapamycin group.

  6A-6C represent low magnification views of whole-mount brain sections stained with AADC immunohistochemistry. The results show that in the left (treated) hemisphere of the vector-injected (+) rapamycin group (FIG. 6A) compared to the left (treated) hemisphere of the rat from the vector-injected (−) rapamycin group (FIG. 6A). Provides a visual confirmation that is a significantly higher level of expression. The results for the rats from the vector-injected (−) rapamycin group are similar to those for the control vehicle-injected rats (FIG. 6C).

  A three-dimensional analysis of immunohistochemistry confirms that the expression level, measured by positive cell count, is significantly increased when the vector is administered in combination with rapamycin. Table 1 presents the results of transgene-derived immunostaining and estimated positive cell counts by quantitative steric analysis in rapamycin-induced and rapamycin-uninduced rats. Rapamycin-induced rats show approximately twice as much anteroposterior diffusion of AADC immunostaining, diffusion volume of AADC immunostaining, and number of AADC positive cells compared to uninduced rats.

  Overall protein analysis shows even greater differences at the protein level. As shown in FIG. 7, Western blot analysis of hAADC enzyme levels after striatal protein sample electrophoresis shows significantly higher hAADC expression in the (+) rapamycin group compared to the (−) rapamycin group. When endogenous AADC expression is subtracted from the plotted data in FIG. 7, hAADC levels are 88% lower in the (−) rapamycin group compared to the (+) rapamycin group.

  The low level expression observed in uninduced rats suggests “leakage” in the in vivo regulatory system, but the experimental results described in Example 2 indicate that hAADC gene expression is induced by the dimerized rapamycin Indicates. The reason for the leakage of the system is not clear and may be caused by unregulated expression from the minimal IL-2 promoter in the absence of bound transcription factors, but is not intended to be limited by theory. However, the low level of hAADC observed without induction is not sufficient to elicit behavior in response to sub-therapeutic doses of L-dopa, and the small amount produced by a promoter inducible in the absence of an inducing agent. It suggests that gene expression may be acceptable for this particular application.

  The appropriate dosage of rapamycin (or the like) to produce the desired level of transgene induction in vivo may be determined experimentally on a case-by-case basis, as is common in treatment protocols. Dosage may be adjusted by phenotypic measurement or trial and error based on surrogate markers. The dosage may also be adjusted to achieve a predetermined target concentration of rapamycin in the patient's target tissue or blood. When introduced by intravenous injection, typical dosages range from 0.01 to 50 mg / kg, preferably from 0.1 to 10 mg / kg, for example 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.8, 0.9, 1, 1.5, 2, 3, 4, 5, 6, 7, 8, 9 or 10 mg / kg. In treating human patients, dosages can be determined with reference to the results of clinical trials or can be inferred from animal data.

  Recombinant AAV vectors can be surgically introduced into various regions of the brain, depending on the transgene to be delivered, its mechanism of action, and the desired target cells. For example, basal ganglia are groups of nerves located under the cortex. They include the caudate nucleus, the putamen, and the pallidal bulb. Together, the caudate nucleus and the putamen form the basal ganglia striatum (or simply the striatum). The caudate and putamen are interconnected with the substantia nigra consisting of the substantia nigra (SNpc) and the substantia nigra-like part. SNpc is a normal site of dopamine biosynthesis, and SNpc degeneration is a feature of PD. Transduction of dopamine synthesis by transducing genes in the basal ganglia striatal putamen or caudate nucleus into genes encoding enzymes of the dopamine biosynthetic pathway, such as AADC, thereby functionally reducing it The SNpc network can be overcome.

  Striatal cells of the basal ganglia can be transduced using a variety of techniques known in the art. For example, stereotaxic injection is a common surgical technique used by neurosurgeons to administer various compounds to the CNS. Direct injection can also be used. When using this technique, anatomical maps from CT, PET, or MRI scans can be used by the surgeon to assist in selecting the injection site. Other techniques, including convection-enhanced delivery (described in detail in US Pat. No. 6,309,634, incorporated herein by reference), have been employed in the methods of the invention to deliver rAAV virions to the CNS. obtain.

  The experiments described in Examples 1 and 2 relate to an embodiment of the dual vector of the present invention where separate vectors are used to deliver the transcription factor fusion protein and the transgene. In other embodiments, a different gene is used in place of hAADC in the expression vector. Up to about 5000-5200 nucleotides (nt) can be packaged in an AAV virion, optimally about 4500 nt. Dong et al. (1996) Hum. Gene Ther. 7: 2101-12. Given other required sequence elements (eg, ITR region, promoter, transcription factor binding sequence, etc.) such as the one represented in FIG. 1B, the transgene sequence in the expression vector is approximately 2500. There may also be no nucleotides (nt). Within this size limitation, the desired transgene can be delivered using the rAAV expression vector construct shown in the dual vector embodiment of the invention. Improved versions of optimized expression vectors with shorter ITRs, promoters or transcription factor binding regions can be constructed to allow delivery of longer transgene sequences.

  The results of Examples 1 and 2 show that dimerization-dependent hAADC transgene expression is regulated in human cultured cells and in vivo rat nerves using the dual vector rAAV-mediated embodiment of the present invention. Show you get.

(GDNF regulation in a single vector system)
As described in Example 3, experiments were performed using the single vector embodiment of the present invention, and a single rAAV vector was required for dimerization-dependent regulation of GDNF transgene expression. Were designed to express the protein. A schematic of a regulated rAAV hGDNF expression vector is provided in FIG. 8A, and a schematic of a control vector with constitutive (unregulated) hGDNF expression is provided in FIG. 8C. (FIG. 8B shows an alternative design of a regulatory GDNF expression vector not used in Example 3.) Plasmid vectors are transiently transfected into HEK-293 cells in vitro. Cells are then treated with media containing 0, 5 or 25 nM rapamycin.

  The results of GDNF ELISA performed 3 days after transfection are shown in FIG. The data shows a regulated construct with an observed expression of up to about a quarter level of expression from constitutively expressed GDNF ("CMV-GDNF plasmid" meaning pAAV-CMV-hGDNF). Represents rapamycin dose-responsive expression of GDNF in cells transfected with “TF-GDNF plasmid” meaning pAAV-TF-Z8-hGDNF). GDNF expression from pCMV-GDNF is not increased by the addition of rapamycin.

  The experiment described in Example 3 demonstrates that a single vector regulatable AAV-GDNF construct can be constructed in human cultured cells such that GDNF expression can be regulated simply by the addition of the small molecule derivative rapamycin. To express. The results suggest that GDNF expression can be regulated in patients in vivo by a regulatable AAV-GDNF vector. The single vector of Example 3 serves as both a transcription factor vector and an expression vector in the dual vector embodiment discussed above. Compared to the two-vector method where two vectors need to be co-transfected into target cells, treatment with a single vector requires only one transduction event to introduce regulatable GDNF. Is advantageous. The advantages of this single vector embodiment are particularly significant for protocols that offer low transduction efficiencies such as gene therapy methods where a very small percentage of target cells are expected to be transduced simultaneously by both vectors. . For example, estimating the independent transduction efficiency, if the overall transduction efficiency is 5% for each vector, only 0.25% of the cells will be transduced with both vectors.

  Because vectors packaged in AAV virions cannot be longer than about 5000-5200 nucleotides (nt), single vector regulatable constructs are only useful for relatively short transgene delivery. For example, with the particular selection of transcription factor fusion proteins and regulatory elements used in the rAAV vector represented in FIG. 8A (as used in Example 3), the transgene (GDNF) sequence is approximately 600 nt. It is long. With optimized single vector regulatable AAV-constructs where unnecessary nucleotides have been removed from the fusion protein and regulatable elements with more efficient substitution of sequence elements, longer transgene sequences, such as Up to 850 or even 1000 nt can be delivered. Within the AAV-package size limits, the desired transgene can be delivered using the single vector construct of the present invention. The transgene can contain an active partial fragment of the desired gene rather than the full-length gene to facilitate delivery of a single vector.

The dose of rAAV virion required to achieve a particular therapeutic effect, eg, the unit dose of vector genome (vg / kg) per kilogram body weight, is required to obtain the rAAV virion route of administration, therapeutic effect It may depend on several factors including, but not limited to, the level of transgene expression, as well as the stability of the heterologous gene product. One technique in the art can determine the dose range of rAAV virions to treat a patient suffering from a particular disease or disorder based on the aforementioned factors, as well as other factors well known in the art. . In some embodiments of the present invention, the proper dose of rAAV used to generate transduction in ^mammals, from 1X10 8 vg / kg to ^1X10 15 vg / kg, preferably from the 4X10 ⁹ vg / kg 4X10 It can range up to ¹² vg / kg, but higher or lower doses may be obtained as determined by experimentation. Although AADC and GDNF are shown as exemplary transgenes in the examples herein, the particular transgene delivered is not limited to embodiments of the invention. If another gene or part of a gene is expected to provide a beneficial (eg, therapeutic) effect and its sequence is short enough to fit within an AAV vector construct that can be packaged into an AAV virion, It can be introduced into the brain using the vectors, methods and kits of the invention.

  As discussed above, AADC (OMIM 107930, EC 4.1.1.28, Genbank accession number M76180) is a transgene for dopamine biosynthesis. Other possible transgenes associated with dopamine biosynthesis are tyrosine hydroxylase (TH) (OMIM 191290, EC 1.14.16.2, Genbank accession number X05290) and guanosine triphosphate cyclohydrolase I (GCH). ) (OMIM600225, EC3.5.4.16, Genbank accession number NM_000161). Still other possible transgenes include GDNF (OMIM 6000083, Genbank accession number AX713049, L19063) and artemin (OMIM 603886, Genbank accession number AF109401), neuroturin (OMIM 602018, Genbank accession number). HSU78110), and neurotrophins including other members of the GDNF protein family, such as persephin (OMIM 602921, Genbank accession number AF040962). Saarma et al. (1999) Microscopy Res. Tech. 45: 292-302. Still other transgenes include IL-10 (OMIM 124092, Genbank accession number M57627).

  The OMIM number refers to the Online Mendelian Inheritance in Man database managed by John Hopkins University, the website of the National Library of Medicine www. ncbi. nlm. nih. gov / entrez. Is available on the internet through. The contents of all OMIM items described herein and the sequences described by accession numbers are incorporated herein by reference.

  Genbank accession numbers provide only representative complete cDNA sequences and are not intended to limit the scope of the invention. In particular, possible transgenes include other genes reported in Genbank, full-length and partial fragments of natural genes, homologous genes from other species, allelic variants of these sequences, and naturally occurring or artificial Includes genes with creation variants.

  The transgenes are also Alzheimer's disease, Huntington's disease, amyotrophic lateral sclerosis (ALS), multiple sclerosis, canavan disease, cerebral ischemia, progressive supranuclear palsy, Lewy body dementia, Shy-Dreger syndrome , AIDS cognitive syndrome, essential tremor, ataxia, basal ganglia degeneration, multiple system atrophy and retinal degeneration (eg, macular degeneration, diabetic retinopathy, retinitis pigmentosa, glaucoma) Also included are genes for treating other neurodegenerative diseases.

  The derivative is preferably selected from compounds that can be delivered to a human patient without significant toxicity or side effects. In preferred embodiments for treating the nervous system or other CNS disorders, the derivative is capable of crossing the blood brain barrier. In a more preferred embodiment, the derivative is a dimerization such as rapamycin or a non-immunosuppressive analog thereof.

  In some embodiments, the derivative is delivered locally, for example by subcutaneous, intramuscular, intraocular or intravenous injection. In preferred embodiments, the derivative is orally, topically by nasal delivery (spray nasal drops), by aerosol / pulmonary delivery (inhaler), by ocular delivery (eye drops) or by other convenient delivery modes. One that can be delivered relatively conveniently. The derivatives may also be delivered continuously or semi-continuously using transdermal patches, subcutaneous implants, implantable osmotic minipumps, mechanical infusion pumps or controlled release pharmaceutical compositions.

  The rAAV vector stock according to the present invention may be prepared using several methods known in the art for AAV virion production. Wild type AAV and helper virus can be used to provide the necessary replication functions for rAAV virion production, or plasmids can be used for helper function genes (eg, pHLP19), auxiliary function genes (eg, pladeno5). ), Or both in the case of triple transduction methods. See US Pat. Nos. 5,139,941, 5,622,856, 6,001,650 and 6,004,797, which are incorporated herein by reference.

  For in vivo delivery, rAAV virions are formulated into a pharmaceutical composition comprising an appropriate dose of one or more rAAV virions and a pharmaceutically acceptable excipient. Such excipients include pharmaceutical agents that do not induce themselves the production of antibodies that are detrimental to the particular recipient composition and can be administered without undue toxicity. Pharmaceutically acceptable excipients include, but are not limited to, liquids such as water, saline, glycerol and ethanol.

  Pharmaceutically acceptable salts include, for example, mineral salts such as hydrochloride, hydrobromide, phosphate, sulfate and the like, and acetate, propionate, malonate, benzoate , And similar salts thereof. In addition, auxiliary drugs such as wetting or emulsifying agents, pH buffering agents, and the like are present in such vehicles. Detailed discussion regarding pharmaceutically acceptable excipients is available at REMINGTON'S PHARMACEUTICAL SCIENCE (Mack Pub. Co., NJ 1991).

  In some embodiments, the rAAV virions of the present invention include excipients that increase the stability of the virus to long-term stock and repeated exposure to freeze-thaw cycles and reduce the attachment of the vector to the infusion device. Supplied as a pharmaceutical composition. In preferred embodiments, the excipient exhibits low toxicity in the target tissue, eg, the CNS. For example, virions can be stocked and supplied in a buffer containing 0.01% to 0.0001%, preferably 0.001% Pluronic F-68 (BASF, Mount Olive, NJ). Additional compounds and excipients are described in US Pat. Nos. 6,759,050 and 6,764,845, incorporated herein by reference.

  In another aspect, the present invention relates to a kit for the regulated expression of a transgene. In some embodiments, one or more vectors that are substantially identical to the vectors shown in FIGS. 1A and 1B, or FIG. 8A or 8B are included in such kits.

  In other embodiments, vectors similar to those depicted in FIGS. 1B, 8A, or 8B are such kits except that the AADC and GDNF coding sequences are replaced by convenient cloning sequences such as polylinkers. include. Expression vectors similar to the vector shown in FIG. 1B are useful in the dual vector method, while vectors similar to the vectors shown in FIGS. 8A and 8B are useful in the single vector method of the invention. The user of the kit of the present invention can clone the gene or gene fragment of interest into an expression vector and subsequently prepare rAAV virions for transduction.

  Kits that include expression vectors for use in the dual vector method may also include transcription factor vectors that are substantially similar to the vector shown in FIG. 1A.

  The kit may also optionally include rapamycin or the like.

  The kit also optionally includes plasmids for use in preparing rAAV virion stocks such as plasmids pHLP19 and pladeno5, as described more fully in US Pat. Nos. 6,001,650 and 6,004,797. But you can.

  The kit may include instructions for use.

  In some embodiments, the gene therapy vectors, methods and kits of the invention are administered to a patient suffering from a disease such as Parkinson's disease to provide a therapeutic effect. Generally speaking, a “therapeutic effect” is sufficient to change the desired outcome or the components of the disease (or disease) towards the end of clinical treatment, such that the patient's disease or disease shows an improvement in symptoms. Means the expression level of one or more transgenes, often reflected by an improvement in clinical signs or symptoms related to the disease or disorder. In the case of Parkinson's disease, the therapeutic effect can be, for example, an improvement in motor function (eg, delicate motor work) manifested by improved hand dexterity. Alternatively, reduced tremor at rest can also be a sign of PD improvement. There are several other art-known observation and measurable endpoints to examine the effectiveness of a therapy (ie, therapeutic effect) for a particular PD treatment.

  In human patients presenting with clinical signs and symptoms of PD, physicians use the well-known Parkinson's disease unity scale (to assess the extent of the disease and to measure the effectiveness of therapy for a particular treatment modality ( Often depends on UPDRS). Similar to the UPDRS system, scientists measure primate Parkinson's disease unified scale (PPRS, treatment evaluation) that measures other characteristics such as fine motor testing, tremors at rest, motor slowness, motor decline, and muscle stiffness. Score-CRS) is also used to evaluate PD characteristics in a PD primate model. The PPRS system is described in Langston et al. (2000) Ann. Neurol. 47: S79-89.

(III, experiment)
The following are specific examples for carrying out the present invention. In the examples, only exemplary purposes are proposed and are in no way intended to limit the scope of the invention. Efforts have been made to support accuracy with respect to quantities used (eg, amounts, temperature, etc.) but some experimental error and inaccuracies should be allowed for.

Example 1
(Regulated expression of AADC in vitro)
In vitro regulation of the AADC transgene is performed as follows.

(AAV vector)
Two vectors are constructed to practice dimerization-dependent expression of hAADC: AAV-CMV-TF and AAV-Z12-hAADC. FIG. 1A is a diagram of AAV-CMV-TF, in which a cytomegalovirus (CMV) enhancer / promoter promotes the expression of a bicistronic message of an activation domain and a DNA binding domain fusion protein. The activation domain fusion protein comprises a rapamycin binding domain (FRB ^* ) of human FRAP linked to a transcriptional activation domain from the p65 subunit (p65) of NFκB. The specific FRB domain (FRB _T2098L ) used in this example is represented in FIG. 1A (FRB ^* ) and contains a T to L mutation at position 2098 when compared to the wild type FRB sequence. Pollock et al. (2000) Proc. Natl. Acad. Sci. USA 97: 13221-26. FRB _T2098L can be dimerized with DNA binding domain fusions using either AP21967 or rapamycin.

  The in-sequence ribosome entry site (IRES) is derived from encephalomyocarditis virus. The DNA binding domain fusion protein comprises the human transcription factor Zif268, two DNA binding domains of the homeodomain from Oct-1 (ZFHD1), and three drug binding domains from the cytoplasmic receptor FK506 (3xFKBP). FIG. 1B is a schematic of AAV-Z12-hAADC, where human AADC (hAADC) expression is controlled by 12 binding sites of transcription factors linked to a minimal IL-2 promoter. AAV2 terminal inverted sequence (ITR), hAADC coding sequence (hAADC) and human growth hormone polyadenylation region (pA) are shown.

(In vitro expression analysis)
The ability to regulate hAADC gene expression was co-transduced into HeLaD7-4 cells with a 1: 1 ratio of AAV-CMV-TF and AAV-Z12-hAADC, followed by 0, 5 or 25 nM rapamycin analog AP21967. Measurements are made in vitro by treating the cells with (ARIAD Pharmaceuticals, Inc., Cambridge, Mass.). The results are represented in FIG. 2, which are non-transduced cells, cells transduced with transcription factor vector alone (AAV-CMV-TF), cells transduced with constitutive expression hAADC vector (AAV-CMV-hAADC2) ), As well as various control experiments involving cells transduced with a regulatory hAADC vector excluding the transcription factor vector (AAV-Z12-hAADC).

  The details of the experiment relating to the experimental method used in Example 1 are as follows.

(Construction of vector)
AAV-CMV-TF has been previously described (AAV-CMV-TF1Nc, Auricchio et al. (2002) Mol. Ther. 6: 238-42). The pAAV-hAADC CMV enhancer and promoter (Sanfiner et al. (2004) Mol. Ther. 9: 403-9) is a Z12 containing 12 binding sites in the DNA binding domain of ZFHD1 upstream of the minimal IL-2 promoter. -Make AAV-Z12-hAADC by substituting the region of IL2-SEAP (Rivera et al. (1996) Nat. Med. 2: 1028-32).

(Production of recombinant vectors)
Recombinant AAV vector (serotype 2) is generated by triple transfection in HEK-293 (ATCC Accession No. CRL 1573) cells and purified by cesium chloride density gradient centrifugation. Grimm et al. (2003) Blood 102: 2412-9. Briefly, recovery of cells containing rAAV is performed by microfluidizing and filtering through a 0.2-μm filter. The vector is purified from cell lysates extracted by cesium chloride density gradient centrifugation and contains 0.001% Pluronic F-68 (BASF, Mount Olive, NJ) supplemented with the vector loss in the delivery catheter. Concentrate by phosphate filtration and diafiltration with phosphate buffered saline (PBS-sorbitol) containing 5% sorbitol, pH 7.4. The purity of the vector is measured by SDS-PAGE. The purified rAAV vector used in this study only shows VP1, VP2, and VP3 by silver staining of SDS-PAGE gels. The titer is examined by Q-PCR analysis of the vector genome.

(In vitro AADC analysis by expression ELISA)
In the expression ELISA, an antibody against hAADC is used to measure the expression of hAADC protein in permeable HeLaD7-4 cells. HeLaD7-4 cells are transduced with rAAV vector and treated with three different concentrations (0, 5, 25 nM) of AP21967. Each group n = 3. Cells are seeded in 96-well plates 24 hours prior to transduction. Cells are transduced at an MOI of 10 ⁴ vg / cell (in each vector when using multiple vectors) in 100 μl complete medium.

An ELISA is performed on cells 48 hours after transduction. Briefly, the medium is aspirated and the cells are washed with PBS. Cells are fixed with 4% paraformaldehyde and incubated for 20 minutes at room temperature. Cells are washed with PBS, blocked with blocking buffer (3% goat serum in PBS, 0.5% Triton X-100) while shaking and then incubated at room temperature for 60 minutes with shaking. Primary antibody (AB136 rabbit anti-hAADC, Chemicon, 1: 1000) is diluted with wash buffer (1% goat serum in PBS, 0.5% Triton X-100) and allowed to react on a shaker for 60 minutes at room temperature. Plates are washed with wash buffer and reacted with secondary antibody (goat anti-rabbit IgG-AP (Vector Labs, Burlingame, Calif., 1: 1000 in wash buffer) in a shaker for 30 minutes at room temperature. Wash plate with wash buffer. Substrate solution [1Xp-nitrophenyl phosphate, 1X levamisole (quenching agent) in substrate buffer (100 mM NaHCO ₃ , pH 10.0) (Vector Labs, Burlingame, Calif.)] Is added and the plate is 30-60 Allow to react at room temperature for minutes, then read OD ₄₀₅ with a plate reader. The data from this hAADC expression ELISA is compared to the transduction dose response curve of the AAV-CMV-hAADC2 reference lot and reported as the relative optical density measured in cells transduced with the vector.

Example 2
(Regulated expression of AADC in vivo)
The same vector used for AAV-mediated transduction of dimerized regulatory hAADC in vitro is also tested in vivo in a 6-hydroxydopamine (6-OHDA) rat model of Parkinson's disease as follows. Due to the higher potential and known pharmacokinetics, Example 2 uses rapamycin instead of AP21667 as a dimerization. Rapamycin has a half-life of about 10 hours with rapid clearance in vivo. Gallant-Haidner et al. (2000) Ther. Drug Monitor. 22: 31-5.

  All rats used in this example are unilaterally injured on the left side with 6-OHDA. There are three experimental groups: vehicle injection control group (vehicle injection (+) rapamycin treatment), vector injection (−) rapamycin treatment control group, and vector injection (+) rapamycin treatment group. The vector-injected (-) rapamycin group provides a control for hAADC gene expression in the absence of rapamycin, i.e., a test to see if the system leaks.

A 1: 1 mixture of AAV-CMV-TF and AAV-Z12-hAADC (each 3 × ^{10 10} viral genomes (vg)) was injected into the ipsilateral side of unilateral 6-hydroxydopamine (6-OHDA) damaged rats. Only vehicle is injected into the vehicle injection control group. As discussed below, transgene expression is effected via intraperitoneal injection of rapamycin at a set point in time.

  FIG. 3 shows a time series of experiments described in this example. Before rAAV treatment, a reference rotation test with L-dopa (5 mg / kg) administration is performed in all groups. The vector or vehicle is injected into the striatum on day 0. Day 17 rats are induced with rapamycin or treated with diluent. Induction consists of 10 mg / kg / day of rapamycin intraperitoneal injection for 4 consecutive days. (Rapamycin is administered for 4 consecutive days to ensure maximum circulation level in the brain.) The arrows in FIG. 4 indicate the first 4 days of rapamycin induction. Test the rotational response to 5 mg / kg L-dopa in rats on day 21 again. Rats are allowed to recover from rapamycin for a week followed by a response test on day 28. Rats on day 31 are given a second induction and on day 35 the rotational response is tested. After recovering from rapamycin for one week, the rotation test was repeated on day 42. Rats on day 45 are given a third induction followed by a final rotation test on day 49 after the last induction. In this induced (+ rapamycin) state, rats are euthanized and immunohistochemistry is performed.

(Action rotation response to L-dopa)
Behavioral analysis using classical rotational response to dopamine agonists. In this case, the agonist is dopamine synthesized from exogenous L-dopa in the unilateral 6-OHDA rat model of PD. Ungerstedt (1971) Acta Physiol. Scand. Suppl. 367: 69-93. As shown in FIG. 4, vector-injected (+) rapamycin group rats 3 weeks after transduction were strongly contralaterally rotated to L-dopa (5 mg / kg) significantly higher than rats in the vector-injected (−) rapamycin group. Shows response (215.13 ± 73.86 for 16.33 ± 21.92 right turn in 60 minutes, P <0.001). Statistical differences are compared using one-way analysis of variance for multiple groups.

  In the vector-injected (+) rapamycin group, the contralateral rotational response to L-dopa at 3, 5, and 7 weeks showed a pre-injection score or time matched control (vector-injected (-) rapamycin group and vehicle infusion (+) Rapamycin group) significantly (P <0.001). In contrast, the vector-injected (+) rapamycin group is not significantly different from the two control groups (P> 0.05) at the time of pre-injection and the subsequent reduction of the rapamycin effect (weeks 4 and 6).

  The vector injection (−) rapamycin group and the vehicle injection control group are not significantly different at any time point (P> 0.05). While not intending to be bound by theory, the gradual increase after 7 weeks in both control groups may be due to susceptibility to repeated L-dopa treatment.

  The rotational response of the vector-injected (+) rapamycin group at 4 and 6 weeks, when “off” rapamycin, was not significantly different from the vector-injected (−) rapamycin group or vehicle-injected (+) rapamycin group at the same time , Showing the reversibility of inductive responses. Induction of hAADC expression, followed by decreased expression in untreated weeks, was observed in more than 3 consecutive rapamycin treatment cycles, strengthening the conclusion that induction of rapamycin-induced gene expression occurs in vivo.

(Quantification of hAADC transgene copy in injected striatum)
Quantitative real-time PCR is performed on all rats to confirm that an equal copy number of hAADC gene has been transduced into both vector injection groups. The copy number of hAADC gene in the vector-injected (+) rapamycin group (222 ± 92 genome copies / 20 ng DNA) (± SD) is no different from the vector-injected (−) rapamycin group (229 ± 48 genome copies / 20 ng DNA).

(Immunohistochemistry and quantification of expression)
The level of hAADC expression is assessed by immunohistochemical analysis 7 weeks after injection. A high-magnification enlarged view of a representative rat striatum injection site in the vector-injected (+) rapamycin group is shown in FIG. As represented in FIG. 5A, hAADC transgene expression is localized to the spinous nerve in the middle of the rat striatum. In contrast, rats injected with uninduced vectors (vector injected (−) rapamycin group) show very low levels of hAADC transgene expression (FIG. 5B).

FIG. 6 shows a low magnification view of AADC immunohistochemistry in whole-mount brain sections from representative rats from each group 7 weeks after striatal injection. AAV-CMV-TF + AAV-Z12-hAADC (each vector 3 × ^{10 10} vg) rats with rapamycin induction (A) or no rapamycin induction (B) are shown in comparison to the rapamycin-induced vehicle control (C). The left hemisphere is the site of 6-OHDA injury and intrastriatal vector (or vehicle) injection. The right hemisphere is intact and uninjected, and staining of the right hemisphere reflects endogenous staining from untreated AADC positive fibers.

  6-OHDA damage causes a loss of endogenous AADC, resulting in low histochemical staining of the enzyme in the left hemisphere as well represented in the vehicle infusion control group of FIG. 6C. All rats in the vector-injected (+) rapamycin group show hAADC transgene staining on the left side injected (see, eg, FIG. 6A). Rats in the vector-injected (−) rapamycin group have low levels of hAADC transgene expression (see, eg, FIG. 6B). This hAADC expression is observed in 5 out of 6 of the vector-injected (−) rap rats. Compared to the vector-injected (+) rapamycin group, the vector-injected (-) rapamycin group had few positive cells, low staining per cell, and low levels from inducible promoters in the absence of rapamycin. Only hAADC expression is shown.

（表１）ラットの脳におけるＡＡＤＣ発現
「ｎ」は調べた半球の数である。
データ値を±標準偏差で表す。
ラパマイシン誘導群の平均の前後方向への拡張、拡張体積、およびＡＡＤＣ陽性細胞数は、Ｓｔｕｄｅｎｔのｔ−検定による「非誘導」群（Ｐ＜０．０２）の値と統計的に異なる。 Quantitative stereoanalysis is also performed on serial brain slices obtained from rats administered rapamycin immediately after intrastriatal injection and euthanized at 7 weeks. Sequential sections of brain tissue with fixed AADC immunostaining are evaluated and quantified by stereoanalysis. An optical fractionator stereoanalysis protocol is used, which is an efficient random counting method that combines a statistically optimized spatial sampling procedure with optical resolution. Gundersen et al. (1988) Apmis 96: 857-81 and Gundersen (1986) J. MoI. Microsc. 143 (Pt1): 3-45. The results are shown in Table 1. Table 1 shows that the vector-injected (+) rapamycin group has the broadest staining anteroposterior extension (3,720 ± 1,276 μm) (± SD), while the vector-injected (−) rapamycin group has lower levels It is clarified that it is a diffusion (1,920 ± 1,577 μm).

Table 1. AADC expression “n” in rat brain is the number of hemispheres examined.
Data values are expressed as ± standard deviation.
The average anteroposterior expansion, expansion volume, and number of AADC positive cells in the rapamycin-induced group are statistically different from those in the “non-induced” group (P <0.02) by Student's t-test.

Table 1 also represents the average distribution and expanded volume of transgene positive cells within the striatum. Vector injected (+) rapamycin group had the highest number of AADC positive cells (75,825 +/− 30,506 cells) followed by fewer positive in vector injected (−) rapamycin group (P <0.01) The number of cells (31,000 +/− 25,812 cells), and no detectable expression is seen in the vehicle injection control group (no data). Similarly, the vector-injected (+) rapamycin group shows a large number of hAADC striatal diffusion (15.75 ± 8.16 mm ³ ), while the vector-injected (−) rapamycin group has a striatal diffusion of 55 % Lower volume (7.09 ± 5.69 mm ³ ).

  Stereoanalysis is an accurate measurement of the number of neurons in the striatum transduced and the distribution of viral vector particles. However, the overall protein analysis is a more accurate measure of the amount of hAADC enzyme produced since the amount of hAADC made per cell can vary between groups. To confirm that the lower intensity hAADC expression in the non-induced group observed by immunostaining correlated with the lower total level of hAADC expression, total protein was extracted from serial tissue sections and analyzed by Western blot Find out by.

  Confirm lower hAADC protein concentration in uninduced group. FIG. 7 is a diagram of related gel bands from vector-injected (+/−) rapamycin and vehicle-injected (+) rapamycin unilateral 6-OHDA-injured rats showing changes in protein levels (50 kDa) of hAADC in the striatum. And a plot of the integrated band intensity. β-actin is included as a loading control. The band concentration of AADC was significantly higher in the vector-injected (+) rapamycin group compared to both control groups, P <0.001. Vector injected (+) rapamycin and (−) rapamycin western blot band densities from rats were 102.04 ± 7.02 megapixel (MP) and 30.63 ± 3.47 MP, respectively, 7 weeks after injection. is there. Average total AADC protein levels are reduced to 88% in the vector-injected (−) rapamycin group after correcting for endogenous rat AADC levels compared to the vector-injected (+) rapamycin group. These data show a greater difference in the hAADC enzyme levels between the two groups than the results estimated by stereoanalysis.

  Details regarding the experimental method used in Example 2 are as follows.

(Surgical procedure)
6-OHDA injured Sprague-Dawley rats (vehicle injection and vector injection (-) rat n = 6 for rapamycin group, and vector injection (+) rat n = 8 for rapamycin group) were used in Taconic Farms (Germantown, NY). ) Rats are housed 1 per cage under general conditions: controlled temperature and humidity, 12 hour photoperiod, and free provision of food and water. The vector is injected by convection-enhanced delivery (CED) to achieve optimal distribution into the striatum. Bankiewicz et al. (2000) Exp. Neurol. 164: 2-14, Lieberman et al. (1995) J. MoI. Neurosurg. 82: 1021-9. Briefly, polymer tubing (OD, 1/16 ″; ID, 0.030 ″; Oak Harbor, Washington) with the vector connected to a tube filled with olive oil delivered from a 1 ml airtight Hamilton syringe. To Upchurch Scientific). The vector is delivered by a programmable pump (Bioanalytical System, West Lafayette, IN). Connected to the tip of the polymer tubing is a cannula consisting of a fused silica capillary (OD, 164 μm; ID, 100 μm; Polymicro Technologies, Phoenix, Ariz.) Incorporated into a 27 gauge needle. Anesthesia is performed with 3% isoflurane (2 L / min) under O ₂ circulation and the rats are placed in a stereotaxic frame (Kopf, Tahanga, Calif.). Then maintain anesthesia with 1% isoflurane in O ₂ through a fixed mask in a stereotaxic frame. Drill wells (Burr holes) are opened above the target site and a cannula is inserted vertically from the caudate nucleus into the putamen at the following coordinates for bregma and dura mater. Set the incisor bar to 3.3mm, AP 0.0mm, ML-3.5mm, DV-5.0mm. Rats are injected unilaterally with 10 μl of two vectors in a 1: 1 ratio at a rate of 0.5 μl / min. At the end of the 20 minute infusion period, reduce the speed to 0 μl / min, rest for 5 minutes, and slowly remove the cannula.

(Behavior analysis)
Response to apomorphine (0.05 mg / kg, Sigma, St. Louis, MO) and L-dopa (methyl ester, Sigma) Rotomax rotational analysis software (Computer running AccuScan Instruments, Columbus, Ohio) Measure with an automated rotometer connected to. The total opposite rotation and ipsilateral rotation are calculated at 30 minutes or more (for apomorphine) and 60 minutes or more (for L-dopa). Only rats with an average rotation> 6 opposite rotations / min in 30 minutes in response to apomorphine (0.05 mg / kg) 3 weeks after injury are adequately damaged (Ungerstedt (1971) Acta Physiol. Scand. Suppl. 367: 69-93) and test the L-dopa response. These rats were co-administered with the following therapeutic range doses of 2.5 mg / kg benserazide (Sigma, St. Louis, MO) using an L-dopa response using 5 mg / kg L-dopa methyl ester. Measure. More than 95% of the rats tested do not rotate in response to 5 mg / kg L-dopa. Rats that eventually show contralateral rotation for this dose are excluded from the experiment. L-dopa response is assessed at different time points (3, 4, 5, 6 and 7 weeks) before surgery and after intrastriatal injection.

(Immunohistochemistry)
For histological studies, rats were infiltrated through the aorta with saline followed by 4% paraformaldehyde (vehicle injection and vector injection (−) rapamycin group, n = 6 rats and vector injection (+ ) Rapamycin group, n = 8 rats). Brains are perfused overnight with 4% paraformaldehyde, equilibrated with gradient sucrose solution, and frozen in isopentane. The brain is cut continuously with a claristat into 40 μm thick coronal pieces. Immunohistochemistry of AADC (Chemicon, Temecula, Calif., 1: 1500) is performed on free-floating sections. The section is treated with 3% hydrogen peroxide for 30 minutes to suppress endogenous peroxidase. After inhibiting nonspecific binding with 5% general goat serum, the sections are reacted overnight with primary antibody at room temperature. Biotinylated anti-rabbit IgG antibody (Vector Laboratories, Burlingame, Calif., 1: 300) followed by reaction with streptavidin-conjugated horseradish peroxidase (Vector Laboratories, 1: 300) for 1 hour at room temperature Is visualized with 3-3'-diaminobenzidine (DAB, Vector Laboratories) and hydrogen peroxide. The sections are mounted on gelatin-coated slides, dried, dehydrated with increasing amounts of ethanol, cleared with xylene, and mounted using Cytosal-60 (Richard-Allen Scientific, Kalamazoo, Michigan). The distribution of hAADC immunostaining in the front-rear direction is examined by the formula (nx12x40 μm). Here, n is the number of sections with hAADC positive cells, 40 μm is the thickness of the section, and is examined every 12 sections. Distribution volume and number of positive cells based on stereological methods at 63X magnification on a Zeiss microscope with stereo camera and stereology software (Microbrightfield, Williston, Vermont) Calculations are made in serial sections (every 12 sections) stained with AADC using a fractionator-optical dissection (Optical Fractorator). Each group CEE <5%. Results are reported as mean ± SD. Student's t-test is used to measure statistical significance.

(Quantitative real-time PCR)
The vector AAV-Z12-hAADC used in this study contains the human AADC target gene. Q-PCR primers and probes pair exons 2 and 3 of the AADC gene, thus extending introns not present in the vector sequence, and thus minimizing genomic DNA amplification. Real-time Q-PCR (Heid et al. (1996) Genome Res. 6: 986-94) is standardized with plasmid DNA containing the insert of the vector. The plasmid was linearized with restriction enzymes, purified, quantified by UV absorbance, Q-PCR dilution buffer (10 mM Tris-HCl, pH 8.0, 1 mM EDTA, 10 μg / ml yeast tRNA, and 0.1% Tween). 80) to provide 10 standards ranging from 3 to 10 ⁶ copies per reaction. Run each standard in three 50 μl reactions in a 96-well optical plate. Add 10 μl of sample containing 20 ng of DNA each to three Q-PCR wells containing 40 μl of reaction mixture. PCR is performed on an Applied Biosystems 7700 Sequence Detection System. Calculate the copy number of the hAADC gene by comparison with a calibration curve, multiply the copy number generated per well by 2, and estimate that one copy of double-stranded plasmid DNA is equivalent to two single-stranded vector genomes To do.

(Western blot analysis)
Ten serial sections (40 μm each) of the whole brain are homogenized separately with a manual homogenizer in a lysis buffer containing a mixture of phosphatase and protease inhibitors. Protein is quantified by the Bradford method. Protein samples (15 μg) were separated on SDS-PAGE gels (4-15% gradient gel; Bio-rad, Hercules, Calif.) And applied to polyvinylidene difluoride filters (Millipore, Bedford, Mass.). Transition.

  The filter is blocked with 3% scheme milk and reacted with a polyclonal rabbit anti-AADC (1: 500, Chemicon, Temecula, Calif.) Primary antibody for 1 hour. The blots were then reacted with the corresponding HRP-conjugated secondary antibody (1: 3000; Amersham Biosciences, Arlington Heights, Ill.) For 1 hour at room temperature (RT) and ECL solution (PerkinElmer Life Sciences, Emeryville, Calif.). For 1 minute and then exposed to X-Omat film from Kodak (Rochester, NY) for 1-30 minutes. Finally, the blot was reacted in stripping buffer (67.5 mM Tris, pH 6.8, 2% SDS, and 0.7% β-mercaptoethanol) for 30 minutes at 50 ° C., and polyclonal rabbit anti-β-as a loading control. Re-pair with actin antibody (1: 1000; Alpha Diagnostics, San Antonio, Texas). Anti-AADC primary antibodies have been used extensively in previous studies, and the Western blot bands observed in this study show the same band size (~ 50 kDa) shown in the antibody information sheet.

  The density of each specific band is measured using a computer-assisted imaging analysis system (AlphaImager®, Alpha Innotech, San Leandro, Calif.). There is no significant difference in the band concentration of β-actin loading control between each group. By comparing the differences between vehicle-injected (+) rapamycin control group and vector-injected group, each specific band concentration was first normalized to the corresponding internal loading band concentration (each group n = 3). To do. The percent reduction in total protein is examined after subtracting the endogenous hAADC level seen in vehicle-injected rats from the vector-injected group.

Example 3
(Regulated expression of GDNF in vitro)
Mammalian cells are transduced with a GDNF transgene construct capable of dimerization control as follows.

(AAV-GDNF vector)
Figures 8A and 8B are schematics of recombinant AAV vector plasmid constructs for regulatable human GDNF (hGDNF) transgene delivery. A control vector for delivery of constitutively expressed hGDNF (pAAV-CMV-hGDNF) is shown in FIG. 8C.

  The regulatable construct (FIGS. 8A and 8B) relates to a single rAAV vector having a gene encoding hGDNF and elements of a transcription factor activation domain fusion protein and a DNA binding domain fusion protein. In both constructs, hGDNF expression is driven by a minimal IL-2 promoter adjacent to the eight binding sites for the dimerizable transcription factor described in further detail below.

  In the construct represented in FIG. 8A (pAAV-TF-Z8-hGDNF), the CMV enhancer / promoter is between the two with a single transcriptional expression encoding both the transcription factor activation and the DNA binding domain. Promotes an internal ribosome entry site (IRES). In contrast, in the construct depicted in FIG. 8B, the SV40 promoter promotes the expression of the DNA binding domain and the CMV enhancer / promoter promotes the expression of the activation domain of a different transcription (ie, in the opposite direction) from the opposite strand. To do.

  The DNA binding domain fusion protein comprises the human transcription factor Zif268, two DNA binding domains of the homeodomain from Oct-1 (ZFHD1), and three drug binding domains from the cytoplasmic receptor FK506 (3xFKBP).

The activation domain fusion protein comprises a rapamycin binding domain (FRB ^* ) of human FRAP linked to a transcriptional activation domain (p65) from the p65 subunit of NFκB. The FRB ^* represented in FIGS. 8A and 8B is described in Example 1.

  AAV2 terminal inverted sequence (ITR), minimal SV40 polyadenylation (Min.SV40 pA), minimal rabbit β-globin polyadenylation (Min.RBG pA) and minimal human growth hormone polyadenylation (Min.hGH pA) sequences are shown. .

  In the control vector pAAV-CMV-hGDNF (FIG. 8C), the CMV promoter / enhancer promotes hGDNF expression. AAV 2 terminal inverted sequence (ITR) and human growth hormone polyadenylation (pA) sequence are shown.

  The results of the rapamycin induction experiment are represented in FIG. 9 and GDNF expression is expressed as a function of vector construct and rapamycin concentration. pAAV-TF-Z8-hGDNF directs GDNF production in response to concentration when cells are treated with rapamycin, while pAAV-CMV-hGDNF is a constitutive (high) level of GDNF regardless of rapamycin treatment. Directs expression.

  Experimental details regarding the ELISA assay used in Example 3 are as follows.

(GDNF ELISA)
An ELISA assay to quantify GDNF expression is performed as follows.

HEK-293 cells (5 × 10 ⁵ cells / well) are grown overnight in two 6-well plates to a 60-70% population. Plates are transfected with 10 μg of either pAAV-TF-Z8-hGDNF or pAAV-CMV-hGDNF using 300 μM CaCl ₂ . After 6 hours, the medium is replaced with fresh medium containing rapamycin (both 0 nM, 5 nM, or 25 nM each) and cultured for 3 days. Both media and cells are collected separately, snap frozen and stored at -80 ° C until ELISA. All samples are treated with 1N HCl to acidify to pH 3.0 for 15 minutes and then neutralized back to about pH 7.6 with 1N NaOH.

Media samples are measured for the presence of GDNF by the Promega E _max® immunoassay system (Promega, Madison, Wis.). Coating buffer was added to a 96 well plate and incubated overnight at 4 ° C. Remove the coating buffer and dry the plate. Blocking buffer (200 μL) is added to the plate and incubated for 1 hour at room temperature without shaking. Remove blocking buffer and dry plate.

  Two 8-well stages of a 96 well plate are set as GDNF standard stages. Add 1 × Block & Sample Buffer (100 μL / well) to Standard Rows BH. Dilute GDNF standard solution (200 mL of 1000 pg / ml) is added to row A of the standard plate, making a 2-fold serial dilution of 100 μL / well up to row G. Column H is a buffer only control without GDNF.

  Experimental samples were diluted 1: 300 for pAAV-TF-Z8-hGDNF and 1: 1000 for pAAV-CMV-hGDNF, 100 μL of each was added to duplicate wells and incubated at room temperature for 6 hours with shaking (500 rpm) To do. Wash wells 5 times and use approximately 400 μl of the recommended wash buffer supplied with the kit each time. 100 μL of anti-hGDNF polyclonal antibody 1: 500 dilution (in 1X block & sample buffer) is added to each well and allowed to react overnight at 4 ° C. without shaking. Wash plate again as above. 100 μL of anti-chicken IgY, 1: 250 dilution of HRP conjugate (in 1X block & sample buffer) is added to each well and allowed to react for 2 hours at room temperature with shaking (500 rpm). Wash plate again as above. 100 μL of room temperature TMB One solution (including HRP substrate 3,3 ', 5,5'-tetramethylbenzidine) is added to each well and allowed to react for 15 minutes at room temperature without shaking. Color development is stopped by adding 100 μL of 1N hydrochloric acid to each well. Absorbance is measured at 450 nm in a plate reader within 30 minutes after addition of TMB. GDNF levels in pg / ml are examined by comparing signals obtained from experimental samples with GDNF standards on the same plate. Standard deviation is derived from the sum of four measurements of each sample (two samples on each of two plates).

  While specific examples of preferred embodiments of the present invention have been described, it will be understood by those skilled in the art that various changes and modifications can be made without departing from the spirit of the present invention. It will cover all such changes and modifications that are within the spirit and scope.

  All publications, patents, patent applications, sequences and database items referenced herein are hereby incorporated by reference.

FIG. 1A is a schematic of an rAAV vector (AAV-CMV-TF) encoding a transcription factor activation and DNA binding domain for transgene expression regulation. FIG. 1B is a schematic of a recombinant AAV vector encoding hAADC (AAV-Z12-hAADC) whose expression can be regulated by the addition of rapamycin or a derivative thereof. FIG. 1C is a schematic of a recombinant AAV vector (AAV-CMV-hAADC2) encoding hAADC whose expression is driven by a constitutive CMV promoter. FIG. 2 shows the results of experiments in which D7-4 cells were transduced with various rAAV constructs and treated with 0, 5 or 25 nM rapamycin analog AP21967. FIG. 3 is a time series of experiments in which the rotational response to L-dopa was measured as a function of rapamycin treatment in 6-OHDA-damaged rats. FIG. 4 shows the rotational response to 5 mg / kg L-dopa in 6-OHDA-damaged rats, expressed as contralateral rotations per hour as a function of time along the time series shown in FIG. FIG. 5A shows the results of AADC immunohistochemistry in rat striatum 7 weeks after injection treated with rapamycin together with an AAV vector having the sequence required for rapamycin-regulated expression of hAADC. The scale bar represents 75 μm. FIG. 5B shows experimental results similar to FIG. 5A, except that the rats are untreated with rapamycin. FIG. 6 shows AADC immunohistochemistry results of typical rat-derived whole-mount brain sections in three treatment groups obtained 7 weeks after injection: A: vector injection (+) rapamycin; B: vector injection (−) Rapamycin; C: vehicle injection (+) rapamycin. FIG. 7 shows AADC expression 7 weeks after injection measured by Western blot analysis in the three treatment groups considered according to FIG. FIG. 8A shows a rAAV comprising transcription factors activating transcription and DNA binding domain fusions, and regulatable transcription encoding the transgene hGDNF, wherein expression of hGDNF can be regulated by rapamycin or the like. 1 is a diagram of a vector (AAV-TF-Z8-hGDNF). FIG. 8B shows that expression of the transcription factor DNA binding domain is promoted by the SV40 promoter on a transcription separate from that of the activation domain, and that the activation domain is expressed from the opposite strand (ie, in the opposite direction). FIG. 8B is a schematic of a vector similar to that shown in FIG. FIG. 8C is a schematic of a recombinant AAV vector encoding hGDNF (AAV-CMV-hGDNF) whose expression is driven by a constitutive CMV promoter. FIG. 9 shows a transient response with either a regulatory TF-GDNF plasmid (AAV-TF-Z8-hGDNF) or a constitutive CMV-GDNF plasmid (AAV-CMV-hGDNF) depending on 0, 5 or 25 nM rapamycin treatment. Shows GDNF expression in picogram units (pg) in HelaD7-4 cells transfected with.

Claims (11)

A recombinant adeno-associated virus (AAV) vector encoding a regulatable transcription factor having transcription enhancing activity; and a recombinant AAV vector encoding a transgene;
A pharmaceutical composition for treating a patient suffering from a neurological disease, wherein the expression of the transgene is affected by the activity of a transcription factor.   2. The pharmaceutical composition of claim 1, wherein the regulatable transcription factor and the transgene are encoded on separate rAAV vectors.   The pharmaceutical composition of claim 1 or 2, wherein the activity of the regulatable transcription factor is increased in the presence of rapamycin or a rapamycin analog.   4. The pharmaceutical composition of claim 3, wherein the rapamycin analog is AP21967.   The pharmaceutical composition according to any one of claims 1 to 4, wherein the neurological disease is Parkinson's disease.   The pharmaceutical composition according to any one of claims 1 to 5, wherein the transgene is an aromatic L-amino acid decarboxylase (AADC).   The pharmaceutical composition according to any one of claims 1 to 5, wherein the transgene is glial cell-derived neurotrophic factor (GDNF).   8. A method of treating a patient suffering from a neurological disease comprising administering a pharmaceutical composition of any one of claims 1 to 7 in a pharmaceutically effective dose. A recombinant AAV vector encoding a regulatable transcription factor:
A kit for performing the method of claim 8, comprising a recombinant AAV vector encoding the transgene: and rapamycin or a rapamycin analog.   Use of a composition according to any of claims 1 to 7 in a method of treating a patient suffering from a neurological disorder.   Use of a recombinant AAV vector encoding a transgene and a recombinant AAV vector encoding a regulatable transcription factor having transcription enhancing activity in the manufacture of a composition for treating a patient suffering from a neurological disease, comprising: Use, wherein the expression of the transgene is affected by the activity of transcription factors.

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