CN119101132A

CN119101132A - AAV capsid protein variants targeting retina and uses thereof

Info

Publication number: CN119101132A
Application number: CN202310673303.9A
Authority: CN
Inventors: 吴小兵; 马文豪; 赵天意; 武志杰
Original assignee: Beijing Jinlan Gene Technology Co ltd
Current assignee: Beijing Jinlan Gene Technology Co ltd
Priority date: 2023-06-07
Filing date: 2023-06-07
Publication date: 2024-12-10

Abstract

本发明涉及靶向视网膜的AAV衣壳蛋白变体、编码其的分离核酸、包含所述核酸的载体和宿主细胞、和包含所述AAV衣壳蛋白变体的重组AAV载体及其治疗用途。The present invention relates to an AAV capsid protein variant targeting the retina, an isolated nucleic acid encoding the same, a vector and a host cell comprising the nucleic acid, and a recombinant AAV vector comprising the AAV capsid protein variant and therapeutic uses thereof.

Description

Retina-targeting AAV capsid protein variants and uses thereof

Technical Field

The present invention relates to the field of adeno-associated viruses (AAV), and in particular to AAV capsid protein variants, isolated nucleic acids encoding the same, vectors and host cells comprising the nucleic acids, and recombinant AAV vectors comprising the AAV capsid protein variants, and therapeutic uses thereof.

Background

Recombinant adeno-associated virus (rAAV) is the most commonly used vector for gene replacement therapy and gene editing in vivo, but selective transduction of specific tissues after in vivo delivery remains a challenge. AAV transduction has a number of steps, including binding to cell surface receptors, intracellular trafficking, transgene expression, etc., which may limit the efficacy of the vector. It has been proposed to improve or extend capsid targeting of AAV by utilizing an AAV capsid protein evolution strategy to allow selective delivery of genes to specific target tissues or cell types, thereby increasing viral transduction rates.

CN 112011571A discloses an AAV9 capsid protein variant AAV-php.eb obtained by AAV capsid protein evolution. The amino acid sequence of this variant replaces the original amino acid AQ with a DGTLAVPFK peptide between positions 586 and 589 of the amino acid sequence corresponding to the wild-type AAV9 capsid protein. The authors suggested that this replacement could increase the efficiency of infection of the nervous system by capsid proteins, and that the use of AAV viral vectors comprising the capsid variants for the treatment of spinal muscular atrophy by intrathecal administration was examined by model mice. However, this document does not relate to the study of targeting of retinal tissue.

In view of the application prospects of AAV in the treatment of inherited retinal diseases, there remains a need in the art to develop new capsid protein variants to improve the specific targeting and transduction efficiency of recombinant adeno-associated viruses and their carried transgenes to retinal cells.

Summary of The Invention

In an effort to develop new capsid protein variants, the present inventors have obtained the capsid protein variants of the present invention by capsid protein variant screening. The AAV capsid variants of the invention have an amino acid sequence substitution of SEQ ID NO.5 between amino acid positions 586 and 589 (amino acid positions numbered according to SEQ ID NO: 6) as compared to the wild-type AAV9 capsid protein. The variant of a capsid protein according to the invention resulting from said substitution is herein designated CapX-120. Animal experiments prove that the capsid protein variants CapX-120 can enhance the transduction efficiency of adeno-associated viral vectors on retinal tissues through comparison of the expression of the reporter gene and the fluorescence intensity. Thus, the use of the capsid protein variants according to the present invention allows the construction of highly efficient and suitable AAV viral vectors for specific tissue targeting required for the treatment of ophthalmic diseases, improving the therapeutic effects on the relevant diseases while minimizing toxicity due to off-target effects.

Thus, in a first aspect, the invention provides an adeno-associated virus (AAV) capsid protein variant having an amino acid sequence of SEQ ID No.5 inserted between amino acid 586 and 589 relative to a parent AAV capsid protein, in place of residues 587-588 of the parent AAV capsid protein, wherein the amino acid positions are numbered according to SEQ ID No. 6.

In some embodiments, a capsid protein variant according to the invention confers increased transduction of retinal cells, preferably selected from the group consisting of photoreceptor cells, RPE layer cells, outer nuclear layer cells, inner nuclear layer cells, and ganglion layer cells, by AAV virions comprising the same, as compared to a parent AAV capsid protein. In some embodiments, the retinal cells are cone cells and/or rod cells. In some embodiments, the capsid protein variant confers a higher transduction rate on cone and rod cells of the retina relative to other cell types of the retina.

In some embodiments, the parental AAV capsid protein is selected from the group consisting of AAV serotypes of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, and AAV 13. In some embodiments, the parent AAV capsid protein has a surface exposed hypervariable loop VIII corresponding in structure to the AAV9 capsid protein. In some preferred embodiments, the parent AAV capsid protein is an AAV9 capsid protein.

In some more preferred embodiments, the parent AAV capsid protein is an AAV9 capsid protein comprising or consisting of the amino acid sequence:

(a) Amino acid sequence of amino acids 203 to 736 of SEQ ID NO. 6;

(b) Amino acid sequence of amino acids 137 to 736 of SEQ ID NO. 6, or

(C) The amino acid sequence of SEQ ID NO. 6.

In some embodiments, the parent AAV capsid protein is a VP1 capsid protein and comprises the amino acid sequence of SEQ ID No. 6 or an amino acid sequence having at least 95%, 96%, 97%, 98%, 99%, 99.5% or 100% identity thereto. In some embodiments, a capsid protein variant according to the invention is a variant of the parent AAV capsid protein, and preferably is a VP1 capsid protein comprising the amino acid sequence of SEQ ID NO 4 or an amino acid sequence having at least 95%, 96%, 97%, 98%, 99%, 99.5% or 100% identity thereto.

In some embodiments, the parent AAV capsid protein is a VP2 capsid protein and comprises the amino acid sequence of amino acids 137 to 736 of SEQ ID No. 6 or an amino acid sequence having at least 95%, 96%, 97%, 98%, 99%, 99.5% or 100% identity thereto. In some embodiments, a capsid protein variant according to the invention is a variant of the parent AAV capsid protein, and preferably is a VP2 capsid protein comprising the N-terminal truncated amino acid sequence of SEQ ID No. 4 starting at amino acid 137, or an amino acid sequence having at least 95%, 96%, 97%, 98%, 99%, 99.5% or 100% identity thereto.

In some embodiments, the parent AAV capsid protein is a VP3 capsid protein and comprises the amino acid sequence of amino acids 203 to 736 of SEQ ID No. 6 or an amino acid sequence having at least 95%, 96%, 97%, 98%, 99%, 99.5% or 100% identity thereto. In some embodiments, a capsid protein variant according to the invention is a variant of the parent AAV capsid protein, and preferably is a VP3 capsid protein comprising the N-terminal truncated amino acid sequence of SEQ ID No. 4 starting at amino acid 203, or an amino acid sequence having at least 95%, 96%, 97%, 98%, 99%, 99.5% or 100% identity thereto.

In some embodiments, the capsid protein variant according to the invention is a VP1, VP2 or VP3 capsid protein comprising the amino acid sequence of SEQ ID NO. 4 or an N-terminal truncated sequence thereof. In other embodiments, the capsid protein variant according to the invention is a VP1, VP2 or VP3 capsid protein encoded by a nucleic acid comprising the nucleotide sequence of SEQ ID NO. 3.

In some embodiments, the capsid protein variant according to the invention is a VP1, VP2 or VP3 capsid protein derived from a transcript encoding the amino acid sequence of SEQ ID No. 4 or comprising the nucleotide sequence of SEQ ID No. 3.

In a second aspect, the invention provides an isolated nucleic acid, wherein the nucleic acid comprises a polynucleotide encoding an AAV capsid protein variant according to the invention.

In some embodiments, a nucleic acid according to the invention comprises:

(a) A polynucleotide encoding SEQ ID NO. 4 or having an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 100% identical thereto, or

(B) The nucleotide sequence of SEQ ID NO. 3, or a nucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 100% identity thereto.

In some embodiments, a nucleic acid according to the invention is capable of transcribing and expressing at least one or any two or all three AAV capsid proteins selected from VP1, VP2 and VP3. The expression "capable of transcription and expression" in the context of a nucleic acid means that the nucleic acid, when placed under the control of a suitable promoter, can be transcribed and expressed in a suitable cell into which the nucleic acid is introduced to produce the gene product in question.

In a third aspect, the invention provides a nucleic acid vector comprising a nucleic acid according to the invention encoding an AAV capsid protein variant according to the invention. In some embodiments, the nucleic acid vector further comprises a nucleic acid encoding an AAV REP protein. In some embodiments, the nucleic acid vector is a plasmid.

In a fourth aspect, the invention provides an isolated host cell comprising a nucleic acid according to the invention encoding an AAV capsid protein variant according to the invention. In some embodiments, the host cell is a recombinant adeno-associated viral vector (rAAV) producer cell. In some embodiments, the nucleic acid stably transfects the producer cell. In other embodiments, the producer cell further comprises a nucleic acid encoding an AAV REP protein (particularly a REP protein of AAV9 or AAV 2). In other embodiments, the producer cell further comprises a nucleic acid capable of trans-providing helper viral functions required for AAV replication and assembly (e.g., E1a and E1b helper genes from adenovirus, and/or E4, E2a, and VA helper genes). In other embodiments, the producer cell further comprises a rAAV genome carrying the heterologous nucleic acid.

In a fifth aspect, the invention provides the use and method of using a nucleic acid according to the invention encoding an AAV capsid protein variant according to the invention, a nucleic acid vector according to the invention, or a host cell or production cell according to the invention for the preparation of a recombinant adeno-associated virus (rAAV) vector. In some embodiments, the use or method comprises:

(i) Providing a producer cell according to the invention;

(ii) Culturing the cell under conditions that allow packaging to produce rAAV virions comprising a recombinant AAV genome, and

(Iii) The cultured host cells or medium are harvested to collect the recombinant AAV viral vectors produced.

In a sixth aspect, the invention provides a recombinant adeno-associated virus (rAAV) vector comprising a capsid protein variant according to the invention or produced using a producer cell of the invention comprising a nucleic acid according to the invention.

In some embodiments, a rAAV vector comprising a capsid protein variant according to the invention exhibits increased transduction of retinal cells relative to a rAAV vector comprising a wild-type AAV capsid protein or relative to a reference rAAV vector comprising a parent capsid protein, preferably, the retinal cells are selected from the group consisting of rods, RPE, outer nuclear, inner nuclear, and ganglion. In some embodiments, the retinal cells are photoreceptor cells, cone cells and/or rod cells.

The rAAV vector according to the invention comprises in its genome:

a.5 'and 3' AAV Inverted Terminal Repeat (ITR) sequences, and

B. An expression construct comprising a heterologous nucleic acid positioned between the 5 'and 3' ITRs, wherein the heterologous nucleic acid encodes a gene product of interest,

Preferably, wherein the expression construct comprises the following elements functionally linked to each other in the direction of transcription:

The presence of a promoter which,

The presence of a heterologous nucleic acid,

-A transcription terminator.

In some embodiments, the heterologous nucleic acid encodes a gene product of interest for gene replacement, gene suppression, or gene editing, preferably the gene product of interest is a protein or RNA.

An important and controversial problem in retinal gene therapy is that subretinal injection can result in transient separation of the RPE from the photoreceptors, adversely affecting the diseased retina. Intravitreal delivery is, relatively speaking, a minimally invasive retinal delivery modality and can therefore more safely transduce the retina. However, most viral vectors, including most AAV serotypes, do not transduce photoreceptors and RPEs upon intravitreal injection. See Gene therapy of inherited retinal degenerations:prospects and challenges,Hum.Gene Ther.2015;26:193-200; also https:// doi.org/10.1016/j.molmed.2018.06.006. Therefore, there is a need in the art to develop AAV serotypes that are capable of efficiently transducing photoreceptors by intravitreal injection.

As shown in the examples, the capsid protein variants of the present invention exhibit higher transduction properties for retinal cells, particularly near-saturated transduction levels for the primary target of hereditary retinal degeneration (IRD), photoreceptors (rod cone), compared to the parent capsid protein by vitreous injection. Thus, the capsid protein variants of the invention may be advantageously applied in the treatment of hereditary retinal related diseases, including IRDs.

In some preferred embodiments, accordingly, the invention provides rAAV vectors according to the invention comprising in the genome a heterologous nucleic acid for the treatment of a hereditary retinal related disease. In some embodiments, the heterologous nucleic acid encodes a gene product of interest that can be used for gene replacement, gene suppression, or gene editing of a gene associated with a hereditary retinal related disease. Examples of such genes include, for example, but are not limited to, congenital amaurosis-related genes such as RPE65, total color blindness (Achromatopsia) -related genes such as CNGB3, choroidal-free disease (Choroideremia) -related genes such as CHM and REP1, leber hereditary optic neuropathy (Leber hereditary optic neuropathy) -related genes such as ND4, macular degeneration-related genes such as VEGF, sFLt1 and CD59, retinitis pigmentosa (RETINITIS PIGMENTOSA) -related genes such as ChR2, MERTK, RLBP1 and PDE6B, X-linked retinitis pigmentosa (X-LINKED RETINITIS pigmentosa) -related genes such as RPGR, and X-linked retinal split (X-linked retinoschisis) -related genes such as RS1.

In some embodiments, preferably, in a rAAV vector according to the invention, the heterologous gene is combined with a retinal tissue or cell type specific promoter, such as a rod and/or cone specific promoter. In some cases, the combination will be beneficial in reducing the dose and/or toxicity of rAAV administration.

In a seventh aspect, the invention provides a pharmaceutical composition comprising a recombinant AAV viral vector according to the invention and a pharmaceutically acceptable carrier.

In an eighth aspect, the invention provides a method of delivering a heterologous nucleic acid to a subject in need thereof, wherein the method comprises administering to the subject a recombinant AAV viral vector according to the invention or a pharmaceutical composition according to the invention. Accordingly, the present invention also provides the use of a recombinant AAV viral vector according to the invention or a pharmaceutical composition according to the invention in the manufacture of a medicament for delivering a heterologous nucleic acid to a subject in need thereof.

In some embodiments according to this aspect of the invention, the rAAV vector comprises an AAV9 capsid protein variant having the amino acid sequence of SEQ ID No. 11 between amino acid 586 and 589. In some embodiments, methods according to the invention are used to treat or prevent a hereditary retinal-related disease, e.g., congenital amaurosis, total color blindness, retinitis (X-linked retinitis pigmentosa), choroidal-free disease, leber's hereditary optic neuropathy, macular degeneration (e.g., age-related macular degeneration), X-linked retinal split, diabetic retinopathy, diabetic macular edema, or choroidal neovascularization.

In some embodiments, the delivery comprises a vitreous injection or subretinal injection. More preferably, the delivery is by way of a less invasive intravitreal injection.

In a ninth aspect, the invention provides a method of delivering a heterologous nucleic acid to an isolated or in vitro cultured cell, comprising contacting the cell with a rAAV viral vector according to the invention, preferably the cell is a retinal cell, such as a rod cell and/or cone cell.

Drawings

FIG. 1 schematically shows pRDAV-CMV-EGFP-Cap9 backbone plasmid (construct 1). ITR is AAV Inverted Terminal Repeat (ITR), CMV promoter is cytomegalovirus promoter, EGFP is coding gene of enhanced green fluorescent protein, short pA is short polyadenylation signal, p40 is p40 promoter from AAV9, CAP9 is coding gene of wild AAV9 serum capsid protein.

FIG. 2 schematically shows the pRDAV-CMV-EGFP-Cap9Δ -588 backbone plasmid (construct 2) produced on the basis of construct 1.

FIG. 3 shows a schematic representation of the results of microscopic examination (×40) of retinas of mice infected with CapX-120 capsid variant virus.

FIG. 4 shows a schematic representation of the results of microscopic examination (×200) of retinas of mice infected with CapX-120 capsid variant virus.

FIG. 5 shows the average fluorescence intensity and fold change in fluorescence intensity detected in the cone rod layer, RPE layer, outer nuclear layer, inner nuclear layer and visual ganglion layer of mouse retinal tissue via vitrectomy CapX-120 capsid variant virus relative to control rAAV9 virus.

FIG. 6 shows analysis of changes in transduction rates (i.e., EGFP-positive cell numbers) of the outer and inner layers of the retina caused by CapX-120 capsid variant virus infection relative to control rAAV9 virus.

FIG. 7 shows the sequence alignment of the capsid protein variant of SEQ ID NO. 4 with SEQ ID NO. 6. This sequence alignment shows that the amino acid residue of SEQ ID NO. 4 corresponds to the position number of SEQ ID NO. 6. As can be seen, between amino acid residues 586-589 corresponding to SEQ ID NO. 6, SEQ ID NO. 4 has an inserted amino acid sequence YGNPSQKL.

Detailed Description

Unless defined otherwise hereinafter, all technical and scientific terms used in this specification have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In addition, the materials, methods, and examples described herein are illustrative only and are not intended to be limiting. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims.

Definition of the definition

In this context, the term "about" when used in conjunction with a numerical value is intended to encompass numerical values within a range having a lower limit of 5% less than the specified numerical value and an upper limit of 5% greater than the specified numerical value. The term is also intended to encompass values within the specified number ± 1%, ±0.5%, or ± 0.1%.

In this document, the expression "and/or" is used to denote any one of the listed related items, or any and all possible combinations of a plurality of the listed related items.

The terms "comprises" or "comprising" when used herein are intended to include the recited element, integer or step, or group of elements, integers or steps, but not to exclude any other element, integer or step, or group of other elements, integers or steps. In this document, the terms "comprises" or "comprising" when used herein, unless otherwise indicated, also encompass the circumstance of consisting of the recited elements, integers or steps. For example, when referring to a polypeptide/protein "comprising" a particular sequence, it is also intended to encompass polypeptides/proteins consisting of that particular sequence.

Herein, the term "AAV capsid protein variant" or "AAV variant capsid protein" refers to an AAV capsid protein having at least one modification (including deletion, insertion, and/or substitution) in the amino acid sequence relative to a parent AAV capsid protein. The variant AAV capsid protein may have at least 90%, 91%, or 95% identity to the amino acid sequence of the parent capsid protein.

In this context, "parent AAV capsid protein" or "parent capsid protein" refers to an AAV capsid protein that is the template for introducing the capsid protein mutation at positions 586-589 according to the invention. The parent of the variant AAV capsid protein may be a naturally occurring or "wild-type" AAV capsid protein, but may be other than a wild-type capsid protein, such as a capsid protein that incorporates one or more (e.g., no more than 10 or 5, 4,3, 2, 1) amino acid modifications in the native AAV capsid protein. It will be appreciated that where the parent is a non-natural capsid protein, it does not comprise the capsid protein mutation at positions 586-589 according to the invention. In some embodiments, preferably, the parent capsid protein is a native AAV9 serotype capsid protein, e.g., VP1 protein having the amino acid sequence of SEQ ID No. 6, or the corresponding VP2 or VP3 protein having an N-terminal truncation.

In this context, when referring to amino acid positions in an AAV capsid protein or a segment thereof, reference is made to amino acid positions numbered according to SEQ ID NO. 6. The amino acid position in the sequence of the capsid protein corresponding to the amino acid position of SEQ ID NO. 6 can be determined by amino acid sequence alignment of the AAV capsid protein in question with SEQ ID NO. 6. For example, when referring to position 586 of a capsid protein, reference is made to amino acid residue 586 of SEQ ID NO. 6, or to amino acid residues which occur in corresponding positions of other capsid proteins by alignment. It will be appreciated that in the present disclosure, when an AAV capsid protein variant involves an amino acid insertion or deletion at a segment of a particular amino acid position, the alignment of the AAV capsid protein variant with SEQ ID No. 6 at that segment can be visually detected, and if necessary, gaps introduced in one or both sequences, to ensure that the segment of the AAV capsid protein is in the corresponding region of position in the alignment with the corresponding segment of SEQ ID No. 6 after the introduction of the gaps, and has the corresponding amino acid residue number. For example, for an AAV capsid protein variant (e.g., SEQ ID NO: 4) having the sequence of SEQ ID NO:5 (YGNPSQKL) inserted between positions 586-589, the alignment of the variant with SEQ ID NO:6 at that segment can be visually inspected and, if necessary, a gap introduced before position 587 of SEQ ID NO:6, thereby allowing the insertion region of the variant to be in the region of the corresponding position in the alignment with positions 586-589 of SEQ ID NO:6 after the gap is introduced and to have the insertion sequence YGNPSQKL between the amino acid residues corresponding to positions 586-589 of SEQ ID NO: 6. See fig. 7. Sequence alignment for amino acid position determination may be performed using Basic Local ALIGNMENT SEARCH Tool available from https:// blast.ncbi.lm.nih.gov/blast.cgi, using default parameters.

"AAV virions" and "AAV virions" are used interchangeably herein to refer to whole virions comprising an AAV capsid and an AAV nucleic acid genome (including wild-type AAV genome and recombinant AAV genome) packaged in the capsid. In this regard, the strand of an AAV nucleic acid molecule packaged into any one of the AAV virions can be either the sense (e.g., "sense") strand or the "antisense" strand, and both strands have equal infectivity.

The term "recombinant AAV vector" is used interchangeably herein with "recombinant AAV virion", "recombinant AAV virion" and refers to a non-wild-type recombinant AAV virion capable of functioning as a vehicle for a nucleic acid of interest. Typically, the viral vector comprises a capsid and a viral genome packaged therein, and preferably the viral genome comprises a nucleic acid of interest inserted therein to be delivered to a target cell or tissue. Herein, "recombinant" may be abbreviated as "r", e.g., recombinant AAV may be referred to as rAAV. Thus, herein, the terms "recombinant AAV virions" and "recombinant AAV vectors" may also be used interchangeably with "rAAV virions", "rAAV virions" or "rAAV vectors". Typically, recombinant AAV vectors are infectious, but replication defective.

Herein, for purposes of this disclosure, rAAV capsids may be from or derived from various adeno-associated viral serotypes including, but not limited to, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, and AAV13. For example, the rAAV can have a viral protein capsid comprised of AAV capsid proteins from the serotype and/or variants thereof.

Herein, for purposes of this disclosure, the viral genome packaged in a rAAV vector is recombinant, i.e., AAV genomic DNA having a genetic modification relative to wild-type AAV genomic DNA. To produce recombinant AAV virions that can deliver a nucleic acid of interest to a tissue or cell, it is often only necessary to retain the Inverted Terminal Repeat (ITR) cis element in the genome, while the remaining sequences required for viral packaging can be provided in trans. Thus, typically, a rAAV can have one or more AAV wild-type genes, such as rep and/or cap genes, deleted in whole or in part, and thus be replication defective, but retain the functional flanking ITR sequences necessary for rescue, replication, and packaging of the AAV virion. More typically, the recombinant AAV viral genome packaged in a rAAV virion may retain only functional ITR sequences and preferably comprises or consists of one or more exogenous nucleotide sequences located between two AAV ITR sequences. It will be appreciated that in the case of a rAAV, the functional ITR sequence may be, but is not necessarily, a wild-type nucleotide sequence, which may be altered, for example, by insertion, deletion or substitution of nucleotides, so long as it still provides the functions required for rescue, replication and packaging. Thus, herein, a rAAV vector is a viral vector comprising at least the functional ITRs required for cis-providing replication and packaging of the virus. Moreover, it is understood that the recombinant AAV viral genome may comprise two or more (e.g., three) ITR sequences, and that these ITR sequences may be the same or different.

The term AAV "inverted terminal repeat" (INVERTED TERMINAL REPEAT) or "ITR" is used interchangeably herein to refer to a functional inverted terminal repeat cis-acting element from the AAV viral genome and encompasses wild-type ITR sequences and variant ITR sequences.

The wild-type ITR of a native AAV virus comprises a Rep protein binding site (RBS) and a terminal melting site trs (terminal resolution site) in the sequence, which can be recognized by the Rep protein binding and create a notch at the trs, and can form a unique "T" letter-like secondary structure that plays an important role in the life cycle of the AAV virus. The earliest AAV virus, AAV2, had an "inverted terminal repeat" (ITR) located at both ends of the genome and having a length of 145bp in a palindromic-hairpin structure. Thereafter, different ITR sequences were found in AAV viruses of various serotypes, but both formed a hairpin structure and had a Rep binding site. Recombinant AAV viral vectors based on these wild-type ITR sequences are typically single-stranded AAV vectors (ssAAV).

A variant ITR can be, for example, a non-natural ITR sequence comprising one or more nucleotide deletions, substitutions, and/or additions, and/or truncations relative to a wild-type ITR sequence, but still functional, i.e., capable of being used to produce a rAAV viral vector. It has been found that, unlike ssAAV described above, by engineering the ITRs, deletion of the trs and optionally D sequences in one side ITR sequence of the AAV virus, the packaged recombinant AAV viral vector can be made to carry self-complementary genomic DNA, thereby producing a virus known as scaAAV (self-complementary AAV). The packaging capacity of scAAV viral vectors is half that of ssAAV viral vectors, approximately 2.2kb-2.5kb, but transduction efficiency is higher after infection of cells. See, for example, self-complementary AAV Vectors, ADVANCES AND Applications, https:// doi.org/10.1038/mt.2008.171. Such variant ITR sequences, also referred to herein as Δitrs, are useful for producing scAAV viruses. The present disclosure contemplates not only ssAAV vectors generated by combining two wild-type ITRs, but also scAAV vectors generated by combining a Δitr sequence with a wild-type ITR.

As will be appreciated by those skilled in the art, the viral capsid proteins and viral genome ITR sequences of the recombinant adeno-associated virus may be from the same or different AAV viral serotypes. In this context, therefore, recombinant adeno-associated virus may be expressed in terms of the AAV viral serotype from which the viral capsid is derived, alone, or in terms of the AAV viral serotype from which the viral capsid and viral genomic ITR sequences are derived. In the latter case, the separation is herein carried out using an identifier "/", followed by the source serotype of the capsid, followed by the source serotype of the ITR. For example, the numeral 9 in the expression recombinant AAV9 indicates that the recombinant adeno-associated virus has a capsid from AAV9 serotype, while the numeral preceding the identifier "/" in the expression recombinant AAV2/9 indicates that the recombinant adeno-associated virus has a wild-type or variant ITR sequence from AAV2, and the numeral following the identifier "/" indicates that the recombinant adeno-associated virus has capsid protein from AAV 9. In some embodiments, for example, a rAAV virus according to the invention can be an AAV9 virus having variant capsid proteins from an AAV9 serotype, or an AAV2/9 virus further comprising ITR sequences from AAV 2.

As used herein, the term "host cell" refers to a cell into which an exogenous polynucleotide has been introduced, including the progeny of such a cell. Examples of host cells include, but are not limited to, microorganisms, yeast cells, insect cells, and mammalian cells. In some embodiments, the host cell is a mammalian cell. In some embodiments, the host cell is a retinal cell. The host cell may be an in vitro, ex vivo or in vivo cell, depending on the context in which the term is used. In other embodiments, the host cell is a producer cell, e.g., "HEK293 cell" or "293 cell", or a cell line derived from the cell, for producing a rAAV vector according to the invention.

In this context, in some embodiments, the term "producer cell" or "producer cell line" refers to a population of cells capable of continuous or long-term growth and division in vitro. It is known in the art that during storage or passaging of such clonal cell populations, spontaneous or induced changes in karyotype can occur and thus may not be exactly the same as the ancestor cell or culture, but still retain the desired characteristics of the original producer cell. Such producer cells or cell lines are encompassed within the scope of the invention.

The terms "exogenous" or "heterologous" as used herein in describing a nucleic acid or protein are used interchangeably to refer to the nucleic acid or protein being exogenous or heterologous with respect to a virus, host cell or subject or other organism comprising the nucleic acid or protein, or with respect to a flanking nucleic acid or polypeptide to which it is linked, i.e., the nucleic acid or protein is present in the virus, host cell or subject or organism in a non-native state, or is non-naturally linked to the flanking nucleic acid or polypeptide. For example, a nucleic acid introduced by recombinant techniques into a particular viral genome or host cell or subject so as to be linked to a sequence to which it is not natively linked or in a non-native chromosomal or cellular location or state, is heterologous with respect to the viral genome or host cell or subject. Thus, nucleic acids that are inserted into the same host cell as the cell from which they were derived or into the same organism from which they were derived, but which are present in a non-natural state, e.g., are present in different copy numbers or under the control of different regulatory elements, are exogenous or heterologous.

As used herein, "isolated" nucleic acid refers to a nucleic acid molecule that is synthesized synthetically or isolated from at least some components of the natural environment in which it is contained. For example, an isolated nucleic acid may be part of a larger nucleic acid, or part of a vector or composition of matter, or may be contained within a cell, and still be "isolated" provided that the larger nucleic acid, vector, composition of matter, or particular cell is not the natural environment of the nucleic acid. In some embodiments, an "isolated" nucleic acid is enriched by at least about 10-fold, 100-fold, 1000-fold, 10,000-fold, or more relative to the starting material.

The term "functionally linked," also referred to herein as "operatively linked," means that the specified components are in a relationship that allows them to function in the intended manner. For example, a promoter or enhancer sequence is operably linked to a coding sequence if it stimulates or modulates transcription of the coding sequence in a suitable host cell or other expression system. In general, promoters operably linked to a transcribable sequence are contiguous with the transcribable sequence, i.e., they are cis-acting (cis-acting). However, some transcriptional regulatory sequences (e.g., enhancers) need not be physically adjacent or in close proximity to the coding sequence that they enhance transcription.

In this context, the term sequence "identity" is used to describe the sequence structural similarity between two amino acid sequences or polynucleotide sequences. To determine the percent identity of two amino acid sequences or two nucleic acid sequences, the sequences may be aligned for optimal comparison purposes (e.g., gaps may be introduced in one or both of the first and second amino acid sequences or nucleic acid sequences for optimal alignment or non-homologous sequences may be discarded for comparison purposes). In a preferred embodiment, the length of the reference sequences aligned for comparison purposes is at least 30%, preferably at least 40%, more preferably at least 50%, 60%, and even more preferably at least 70%, 80%, 90%, or 100% of the length of the reference sequences. Amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position.

Sequence comparison and calculation of percent identity between two sequences can be accomplished using mathematical algorithms. In a preferred embodiment, the percentage identity between two amino acid sequences is determined using the Needlema and Wunsch ((1970) J.mol.biol.48:444-453) algorithms (available at http:// www.gcg.com) that have been integrated into the GAP program of the GCG software package, using the Blossum 62 matrix or the PAM250 matrix and the GAP weights 16, 14, 12, 10, 8, 6 or 4 and the length weights 1,2, 3, 4, 5 or 6. In yet another preferred embodiment, the percentage of identity between two nucleotide sequences is determined using the GAP program in the GCG software package (available at http:// www.gcg.com) using the NWS gapdna.CMP matrix and the GAP weights 40, 50, 60, 70 or 80 and the length weights 1,2, 3, 4, 5 or 6. A particularly preferred set of parameters (and one that should be used unless otherwise indicated) is the Blossum 62 scoring matrix employing gap penalty 12, gap extension penalty 4, and frameshift gap penalty 5.

The percent identity between two amino acid sequences or nucleotide sequences can also be determined using PAM120 weighted remainder table, gap length penalty 12, gap penalty 4) using the e.meyers and w.miller algorithm that has been incorporated into the ALIGN program (version 2.0) ((1989) CABIOS, 4:11-17).

As used herein, the term "conservative" amino acid or nucleotide change refers to a neutral or near neutral amino acid or nucleotide change that results in a protein or nucleic acid molecule that contains the amino acid or nucleotide change that substantially retains its original function. For example, conservative amino acid substitutions are those in which an amino acid is substituted or substituted for a different amino acid whose side chain has similar biochemical properties (e.g., charge, hydrophobicity, and size). Such conservatively modified variants are additive to and do not exclude polymorphic variants, inter-species homologs and alleles. The following group 8 contains amino acids which are conservative substitutions for each other, 1) alanine (A), glycine (G), 2) aspartic acid (D), glutamic acid (E), 3) asparagine (N), glutamine (Q), 4) arginine (R), lysine (K), 5) isoleucine (I), leucine (L), methionine (M), valine (V), 6) phenylalanine (F), tyrosine (Y), tryptophan (W), 7) serine (S), threonine (T), and 8) cysteine (C), methionine (M) (see, e.g., creaton, proteins (1984)). The conservation of amino acids or nucleotide changes in a particular polypeptide sequence or nucleotide sequence can be readily detected by one of ordinary skill in the art by conventional means, such as functional assay assays.

As used herein, "individual" or "subject" refers to a mammal. Examples of mammals include, but are not limited to, humans, non-human primates (e.g., cynomolgus, rhesus), rodents, and other mammals, e.g., cows, pigs, horses, dogs. Herein, mammals include individuals at all developmental stages (including embryonic and fetal stages).

As used herein, the term "treatment" refers to a clinical intervention intended to alter the natural course of a disease in an individual undergoing treatment. Desirable therapeutic effects include, but are not limited to, preventing occurrence or recurrence of a disease, alleviating symptoms, reducing any direct or indirect pathological consequences of a disease, preventing metastasis, reducing the rate of disease progression, improving or moderating the disease state, and alleviating or improving prognosis. The term "treating" also encompasses modifying or improving at least one physical parameter, including physical parameters that may not be discernable by the patient.

As used herein, the term "preventing" refers to preventing or delaying the onset or progression or course of a disease or disorder. Herein, "prevention" generally refers to a hospital intervention performed before at least one symptom of a disease occurs.

Aspects of the invention are described below.

Capsid protein variants

Adeno-associated virus (AAV) is a non-pathogenic parvovirus consisting of a non-enveloped capsid and a 4.7kb single-stranded DNA genome within it. The genome comprises three Open Reading Frames (ORFs), flanked by Inverted Terminal Repeats (ITRs) that are the origins of viral replication and packaging signals. The rep ORF encodes four nonstructural proteins that play a role in viral replication, transcriptional regulation, site-specific integration, and viral particle assembly. The cap ORF encodes three structural proteins (VP 1-3) that assemble to form the viral capsid.

AAV viral capsids consist of a total of 60 VP monomers from three VP capsid proteins arranged in an icosahedral structure, with the molar ratio VP1:vp2:vp3 of the three capsid proteins "optimal" values of about 1:1:10. The three capsid proteins VP1, VP2, VP3 are all encoded by the cap gene of AAV and are produced from the same transcript, regulated by the p40 promoter. The three VP proteins have a common C-terminal sequence, but different N-terminal start sites, and VP2 and VP3 produced by alternative splicing are two truncated forms of VP 1. As an example, the capsid protein sequence of AAV9 is shown below, wherein VP1 specific amino acid sequences are shown in black and bold (VP 1 unique region). The amino acid sequence common to VP1 and VP2 ("VP 1/VP2 consensus region") is underlined and italicized, and the entire VP3 is comprised in VP1 and VP2, being the amino acid common to all three capsid proteins ("consensus VP3 region"), is shown in bold and italicized below.

VP1 and VP2 are mainly localized in the nucleus, whereas monomeric VP3 is distributed both in the nucleus and in the cytoplasm. The assembled AAV capsid outer surface consists of VP3 sequences (including VP3 and the C-terminus of VP1 and VP 2), with the N-termini of VP1 and VP2 being located within the capsid.

There are nine protruding loops of VP called Variable Regions (VR). VR varies from AAV serotype and is responsible for serotype specific receptor binding differences. Because of their exposed positions and their function in receptor binding, VR forming a protruding loop is an ideal location for capsid modification to redirect or extend AAV tropism (i.e., cell surface targeting).

To develop new cell/tissue targeted AAV capsid protein variants, the inventors performed AAV capsid protein variant screening. A plasmid library was generated by inserting a random sequence into the cap ORF at the amino acid position corresponding to the top of the VR-VIII loop (positions 586-588) and constructing an AAV transfer plasmid containing the reporter gene and the cap ORF to couple the genotype and phenotype of the AAV capsid variant with the reporter gene expression. AAV capsid protein variants that are capable of more efficiently and/or more specifically transducing retinal cells (particularly photoreceptor cells) in a vitreoadministrable manner are then identified by capsid variant screening and in vivo tissue distribution detection in animals. Thus, the inventors have established novel capsid protein variants of the invention.

Thus, in one aspect, the invention provides AAV capsid protein variants. In one embodiment, an AAV capsid protein variant according to the invention is derived from a parent AAV capsid protein having inserted between amino acid positions 586 and 589 of the parent capsid protein the peptide of SEQ ID NO. 11 (YGNPSQKL) to replace the original residues 587-588 (numbering according to AAV9 VP1 of SEQ ID NO. 6). The capsid protein modifications according to the invention do not interfere with capsid assembly and genome packaging and allow rAAV to achieve modified retinal cell targeting.

In other embodiments, the invention provides an AAV capsid protein variant comprising an intervening peptide between amino acids 586 and 589 of a parent AAV capsid protein, in place of amino acid residues at positions 567-568 of the original, said peptide comprising the amino acid sequence of SEQ ID NO. 5. In some embodiments, the parental AAV capsid protein is a capsid protein of an AAV serotype selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, and AAV 13. In some preferred embodiments, the parent AAV capsid protein is an AAV9 serotype capsid protein. In some embodiments, the parent AAV capsid protein comprises or consists of an AAV9 capsid protein comprising the amino acid sequence:

(a) Amino acid sequence of amino acids 203 to 736 of SEQ ID NO. 6;

(b) Amino acid sequence of amino acids 137 to 736 of SEQ ID NO. 6;

(c) The amino acid sequence of SEQ ID NO. 6;

(d) An amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 100% identity to the amino acid sequence of any one of (a) - (c), or

(E) Having one or more (e.g., 1, 2, 3, 4, 5, 7, 9, or 10) amino acid residue substitutions, deletions, and/or additions (preferably conservative amino acid substitutions) as compared to the amino acid sequence of any one of (a) - (c).

In other embodiments, AAV capsid protein variants according to the invention are derived from transcripts encoding the amino acid sequence shown in SEQ ID No. 6 with the intervening peptide sequence of SEQ ID No.11 between amino acid positions 586 and 589. The expression "derived from" a transcript in relation to a capsid protein variant herein means that said transcript is capable of producing said capsid protein variant when subjected to conditions suitable for its expression to produce a protein, including different forms of capsid protein variants, such as VP1, VP2 or VP3 capsid protein variants, produced from the transcript by use of alternative translation initiation sites and/or alternative splicing. In some embodiments, therefore, AAV capsid proteins of the invention derived from transcripts may be capsid protein variants selected from VP1, VP2, and VP 3. In some preferred embodiments, AAV capsid protein variants according to the invention are derived from transcripts encoding the amino acid sequences depicted in SEQ ID NO. 4. In some preferred embodiments, AAV capsid protein variants according to the invention are derived from transcripts comprising the nucleotide sequence depicted in SEQ ID NO. 3.

In some embodiments, in addition to the mutation at positions 586-589 according to the invention, a capsid protein variant according to the invention may or may not comprise amino acid substitutions at one or more other amino acid positions, e.g., 2 to 5, 5 to 10, or 10 to 15 amino acid substitutions.

In some embodiments, AAV capsid protein variants according to the invention can be further modified, for example, to enhance or expand delivery of the recombinant vector. Methods of constructing and screening libraries of recombinant AAV capsid proteins are well known in the art. See, for example, US 2005/0053922 and US 2009/0202490, the disclosures of which are incorporated herein by reference in their entirety.

AAV capsid protein variants disclosed herein are identified by intravitreal injection into animals and retinal tissue detection. In some embodiments, the variant capsid proteins disclosed herein confer increased retinal cell transduction of a rAAV virion comprising the same as compared to transduction of a retinal cell with a rAAV virion comprising the corresponding parent AAV capsid protein or wild-type AAV. For example, a variant capsid protein of the invention results in greater uptake of AAV virions by retinal cells (particularly in vivo retinal cells) relative to AAV virions comprising a parent AAV capsid protein or wild type AAV. In some embodiments, AAV virions comprising a variant capsid protein of the invention preferentially transduce one or more retinal cells (e.g., rod cells; cone cells), e.g., 2-fold or 5-fold or more efficiently than other retinal cells. In some embodiments, the retinal cells are selected from the group consisting of photoreceptor cells (e.g., cone and rod cells), retinal Pigment Epithelium (RPE) layer cells, outer nuclear layer cells, inner nuclear layer cells (e.g., bipolar cells), and optic nerve layer cells. In some embodiments, the retinal cell is a photoreceptor cell (e.g., rod; cone). In some embodiments, the retinal cell is a Retinal Ganglion Cell (RGC). In some embodiments, the retinal cell is a retinal pigment epithelial cell (RPE cell). In some embodiments, the retinal cell is a bipolar cell. The transduction efficiency of AAV virions to retinal cells, e.g., increased transduction efficiency, transduced cell type selection, transduced priority, etc., can be assessed in vitro or in vivo using a variety of methods known in the art for measuring gene expression. For example, AAV capsid proteins can be used to package a rAAV genome comprising a reporter gene (e.g., a fluorescent protein under the control of a constitutive promoter) and to assess transduction efficiency and/or transduced cell type selectivity/preference by detecting expression of the reporter gene (e.g., fluorescent microscopy) in an in vitro cell-based assay (e.g., after transfection of a target cell or set of target cells) or in an animal model-based assay.

RAAV production

In some aspects, the invention provides the use of nucleic acids encoding the capsid protein variants of the invention, vectors and host cells comprising the same in the preparation of rAAV vectors.

General principles of rAAV production are reviewed in, for example, carter,1992,Current Opinions in Biotechnology,1533-539; and Muzyczka,1992,CUM Topics in Microbial.and Immunol., 158:97-129). Various methods are described in ：WO 95/13365;WO 95/13392;WO 96/17947;PCT/US98/18600;WO 97/09441;WO 97/08298;WO 97/21825;WO 97/06243;WO 99/11764;US 5,786,211;US 5,871,982; and US 6,258,595. These documents, particularly the portions thereof related to rAAV production, are hereby incorporated by reference in their entirety.

Two terminal inverted repeats (ITRs) in the AAV genome have been shown to be cis-acting elements necessary for AAV integration, replication, rescue and packaging. Two open reading frames, called Rep and Cap, located between ITR sequences in the wild-type AAV genome encode 4 Rep proteins (Rep 78, rep68, rep52 and Rep 40) and 3 Cap proteins (VP 1, VP2 and VP 3), respectively, and 1 Assembly Activator Protein (AAP) involved in AAV replication and packaging. Transcription of Rep78 and Rep68 is controlled by the p5 promoter, and Rep52 and Rep40 initiate transcription by the p19 promoter, with the p40 promoter regulating transcription of the Cap proteins VP1, VP2, and VP 3.

Typically, to produce rAAV virions, the cis-acting element ITR needs to be maintained in the rAAV genome, but the REP and CAP proteins required for replication and packaging can be provided in trans. Thus, typically, an infectious rAAV virion can be produced by transfecting a suitable host cell (e.g., a suitable packaging cell) capable of trans-providing the deleted AAV function (e.g., rep and/or cap protein) with a vector (e.g., a plasmid) carrying a rAAV genome that lacks all or a portion of the rep and/or cap genes.

Herein, a nucleic acid vector, also referred to as an "AAV transfer vector," carrying the rAAV genome to be transferred into a target cell, may be in the form of a variety of nucleic acid vectors suitable for gene transfer, e.g., a plasmid. In this context, AAV functions deleted in the rAAV genome that need to be provided in trans to assist packaging of the rAAV genome into productive AAV virions, also referred to as "AAV helper functions," e.g., rep and/or cap genes deleted from the rAAV genome, or nucleic acids encoding AAV rep and/or cap expression products or functional variants thereof. In some embodiments, AAV helper functions may be provided by vectors encoding the helper functions, e.g., in the form of plasmids, phages, transposons, cosmids, viruses, or virions. Typically, such vectors provide for transient expression of AAV rep and/or cap nucleic acids, but lack AAV ITRs and are therefore neither capable of replication nor packaging themselves. In other embodiments, AAV helper functions may be provided by cells or cell lines that stably express the helper functions.

Adeno-associated virus (AAV) is a non-enveloped virus of the parvoviridae family, belonging to the genus dependent parvovirus (dependoparvovirus), and requires the presence of helper virus for replication and assembly, yielding infectious virions. Viruses that allow AAV (e.g., wild-type AAV) to be replicated and packaged by mammalian cells are also referred to herein as "AAV helper viruses. A wide variety of helper viruses for AAV are known in the art, including adenoviruses, herpesviruses, and poxviruses. The functions required for AAV replication and packaging encoded by the helper virus genome are herein also referred to as "helper virus functions", e.g., E1a and E1b helper genes, and E4, E2a and VA helper genes from adenoviruses. The helper virus function may be provided in the form of a plasmid or other vector, and/or in the form of a stable cell line expressing the helper gene. Furthermore, as will be appreciated by those skilled in the art, AAV helper functions (rep/cap genes) and helper virus functions (e.g., E4, E2a and VA helper genes) may be provided on a single or separate vector. In some aspects, helper virus function may also be provided by wild-type adenovirus. For example, wild-type adenovirus and AAV transfer vectors can be used to transduce cell lines that contain and stably express AAV Rep and Cap proteins, thereby producing infectious rAAV viral particles.

In some aspects, techniques for producing rAAV particles involve providing cells with rAAV genome, rep and cap genes to be packaged, and helper virus functions. Such techniques are well known in the art. For example, rAAV producer cells (or packaging cells) can be produced that comprise a rAAV genome, AAV rep and cap genes separate from (i.e., not in) the rAAV genome, and helper virus functions. Thereafter, the producer cells can be used to produce the infectious rAAV particle of interest.

In some aspects, the invention thus provides a producer cell (or packaging cell) useful for preparing a recombinant adeno-associated virus (rAAV) vector comprising a cap gene encoding a capsid protein variant of the invention, and optionally further comprising a nucleic acid encoding an AAV rep gene and/or a nucleic acid encoding a helper virus function. In some embodiments, the producer cell further comprises a rAAV genome. In some embodiments, the rAAV genome comprises the cap gene and/or the rep gene. In other embodiments, the rAAV genome does not comprise the cap gene and rep gene.

In other aspects, the invention also provides methods for preparing recombinant adeno-associated virus (rAAV) vectors. In some embodiments, the method comprises:

(i) Providing a producer cell according to the invention;

(ii) Culturing the cell under conditions that allow packaging to produce a rAAV virion comprising the recombinant AAV genome, and

In some embodiments, the producer cell comprises a nucleic acid according to the invention encoding an AAV capsid protein variant according to the invention. In some embodiments, a nucleic acid according to the invention comprises:

In some embodiments, a nucleic acid according to the invention is capable of transcribing and expressing at least one or any two or all three AAV capsid proteins selected from VP1, VP2 and VP3.

In some embodiments, the nucleic acid according to the invention is introduced into the producer cell in the form of a vector. In some embodiments, the vector further comprises a nucleic acid encoding an AAV REP protein. In some embodiments, the vector is a plasmid.

In some embodiments, the nucleic acid according to the invention is stably transfected and integrated in the genome of the producer cell.

In other embodiments, the producer cell further comprises (e.g., is stably integrated with) a nucleic acid encoding an AAV REP protein (particularly a REP protein of AAV9 or AAV 2). In other embodiments, the producer cell further comprises (e.g., stably integrated or introduced via a vector) a nucleic acid capable of providing helper viral functions (e.g., E1a and E1b helper genes, and/or E4, E2a, and VA helper genes, from adenovirus) in trans.

In some embodiments, step (i) of the methods according to the invention comprises introducing a recombinant AAV genome comprising a heterologous nucleic acid into a producer cell according to any of the embodiments described above. In some embodiments, the rAAV genome to be introduced into the host cell lacks AAV rep and cap DNA. In some embodiments, the rAAV genome to be introduced into the host cell comprises cap DNA encoding a capsid protein variant of the invention, but lacks AAV REP DNA. In some embodiments, the rAAV genome is introduced into the host cell by an AAV transfer vector.

In some embodiments, the rAAV production methods according to the invention further comprise the step of generating a producer cell (or cell line). In some embodiments, the producer cell (or cell line) expresses one or more components necessary for rAAV genome packaging, the rAAV genome lacking AAV rep and cap genes, the AAV rep gene separate from the rAAV genome, and the cap gene encoding a capsid protein variant according to the invention. Thereafter, the producer cells can be infected with a helper virus (e.g., adenovirus) to produce infectious rAAV virions.

In some preferred embodiments, a rAAV production method according to the invention comprises co-transfecting three plasmids to generate a producer cell (or cell line), wherein the three plasmids are a plasmid providing a rAAV genome comprising a heterologous nucleic acid and flanking ITRs (a transfer plasmid), a plasmid providing AAV helper functions (cap and/or rep proteins), a plasmid providing helper virus (e.g., adenovirus or herpes simplex virus) functions. In other embodiments, the invention also contemplates the use of adenovirus or baculovirus in place of the plasmids described above to introduce AAV helper functions and helper virus functions.

rAAV

In some aspects, the invention provides rAAV vectors comprising a variant capsid protein according to the invention or produced using a rAAV production method according to the invention. In some embodiments, a rAAV vector according to the invention has a rAAV genome packaged in a viral capsid consisting of a capsid protein variant of the invention. In some embodiments, the rAAV genome according to the invention is ssAAV genome or scAAV genome.

In some embodiments, a recombinant AAV vector according to the invention comprises in its genome:

aav Inverted Terminal Repeat (ITR) sequences, and

B. an expression construct comprising a heterologous nucleic acid, wherein the heterologous nucleic acid encodes a gene product of interest,

The presence of a promoter which,

A heterologous nucleic acid encoding a gene product of interest,

-A transcription terminator.

In some embodiments, the genome lacks AAV rep and cap DNA, i.e., no AAV rep or cap DNA is present in the genome.

In some embodiments, a rAAV genome disclosed herein comprises two AAV ITRs flanking the expression construct. In some embodiments, the ITR sequences flanking the expression construct in the rAAV genome are native, variant, or modified AAV ITR sequences. In some embodiments, at least one ITR sequence is a native, variant, or modified AAV ITR sequence. In some embodiments, both ITR sequences are native, variant, or modified AAV ITR sequences. In some embodiments, one of the flanking ITRs is a modified AAV ITR sequence that allows production from a complementary genome, while the other ITR is a native AAV ITR sequence.

In a rAAV genome according to the invention, an AAV ITR can be derived or derivable from any AAV serotype, including, but not limited to, AAV serotypes AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, AAV-7, AAV-8, AAV-9, AAV-10, AAV-11, AAV-12, and AAV-13. The nucleotide sequence of the genome of AAV serotypes is known. For example, the complete genome of AAV-1 is provided in GenBank accession NC-002077, the complete genome of AAV-2 is provided in GenBank accession NC 001401 and Srivastava et al, virol [ virology ],45:555-564{ 1983), and the AAV-9 genome is provided in Gao et al, J.virol [ J virology ],78:6381-6388 (2004).

In some embodiments, the heterologous nucleic acid comprised in the rAAV genome is operably linked to transcriptional control DNA, particularly promoter DNA, enhancer DNA, and polyadenylation signal sequence DNA that are functional in the target cell, to form an expression construct. The expression construct may also include intron sequences to facilitate processing of the RNA transcript when expressed in mammalian cells.

The heterologous nucleic acid comprised in the rAAV according to the invention is not particularly limited. In some embodiments, the heterologous nucleic acid encodes a gene product to be expressed in a target tissue/cell (e.g., one or more retinal cells). In some embodiments, expression of the gene product in a target cell/tissue will be beneficial in the treatment or prevention of a disease or disorder associated with a genetic defect in the tissue (e.g., retinopathy). In other embodiments, the heterologous nucleic acid comprises a polynucleotide encoding a marker or reporter protein.

Gene replacement (GENE REPLACEMENT) is one of the most "direct modes of gene therapy, which generally involves introducing and expressing a nucleic acid encoding a functional protein into a host cell to make up for the deletion of the functional protein in the host cell caused by a defective gene (e.g., a mutated retinopathy-related gene). As will be appreciated by those skilled in the art, depending on the size and nature of the functional protein, the full-length functional protein may be expressed by introducing one nucleic acid molecule encoding the protein into a host cell, or by introducing multiple nucleic acids each encoding a portion of the protein into the same host cell. In some embodiments, a rAAV according to the invention comprises an expression construct, wherein the expression construct comprises at least one heterologous nucleic acid for gene replacement operably linked to a promoter. In some embodiments, the at least one heterologous nucleic acid encodes a therapeutic protein.

Gene suppression may be by RNA interference (RNAi) means, such as antisense nucleic acids, sirnas or micrornas, to inhibit expression or translation of a defective gene in a host cell, thereby treating or preventing a functional disorder caused by the defective gene. In some embodiments, a rAAV according to the invention comprises an expression construct, wherein the expression construct comprises at least one heterologous nucleic acid operably linked to a promoter for gene suppression. In some embodiments, the at least one heterologous nucleic acid, upon introduction into host cell expression, will result in the production of siRNA or microRNA to inhibit expression or translation of a defective gene in the host cell.

Gene editing is another gene therapy approach to targeted repair of gene defects, including, but not limited to ZFNs, TALENs, and CRISP/Cas editing. In some embodiments, a rAAV according to the invention comprises an expression construct, wherein the expression construct comprises at least one heterologous nucleic acid operably linked to a promoter for gene editing (particularly CRISP/Cas9 editing). In some embodiments, expression of the heterologous nucleic acid in the cell/tissue of interest will target repair of a gene defect in said cell/tissue.

The promoter used for ligation with the heterologous nucleic acid in the expression construct is not particularly limited. In some embodiments of the rAAV vectors disclosed herein, the heterologous nucleic acid encoding the gene product is operably linked to a constitutive promoter. Suitable constitutive promoters include, for example, the cytomegalovirus promoter (CMV), the CMV early enhancer/chicken beta-actin (CBA) promoter/rabbit beta-globin intron (CAG) 7, the human elongation factor 1 alpha promoter (EF 1 alpha), the human phosphoglycerate kinase Promoter (PGK), the mitochondrial heavy chain promoter, the ubiquitin promoter. In other embodiments, the heterologous nucleic acid encoding a gene product is operably linked to an inducible promoter. In some cases, the heterologous nucleic acid encoding a gene product is operably linked to a tissue-specific or cell-type-specific regulatory element. For example, in some cases, a heterologous encoding a gene product is operably linked to a photoreceptor-specific regulatory element (e.g., a photoreceptor-specific promoter), such as a regulatory element that confers selective expression of an operably linked gene in a photoreceptor cell. Suitable photoreceptor-specific regulatory elements include, for example, the rhodopsin promoter, the rhodopsin kinase promoter, the beta phosphodiesterase gene promoter, the retinitis pigmentosa gene promoter, the opsin gene promoter, the retinal cleavage protein gene promoter.

In addition to the promoter and the heterologous nucleic acid, other regulatory sequences may be included in the expression construct as desired. The terms "regulatory sequence" and "expression control sequence" are used interchangeably herein to refer to a nucleic acid sequence that induces, inhibits or otherwise controls the transcription of a protein to which the coding nucleic acid sequence is operably linked. Regulatory sequences may be, for example, initiation sequences, enhancer sequences, intron sequences, transcription terminator sequences, and the like. In some embodiments, the expression construct further comprises a Kozak sequence located upstream (i.e., 5') of the heterologous nucleic acid.

Pharmaceutical composition

In one aspect, the invention provides a pharmaceutical composition comprising a rAAV vector of the invention. rAAV virions according to the present disclosure can be formulated after purification according to known methods to produce pharmaceutically useful compositions. The compositions of the present disclosure may be formulated for administration to a mammalian subject, such as a human, using techniques known in the art.

When the delivery system is formulated as a solution or suspension, the delivery system is in an acceptable carrier, such as an aqueous carrier. A variety of aqueous carriers can be used, for example, water, buffered water, 0.8% saline, 0.3% glycine, hyaluronic acid, and the like. These compositions may be sterilized by conventional well-known sterilization techniques or may be sterile filtered. The resulting aqueous solution may be packaged for use as is or lyophilized, the lyophilized formulation being combined with a sterile solution prior to administration.

The pharmaceutical composition according to the present invention may contain pharmaceutically acceptable auxiliary substances to approximate physiological conditions, such as pH adjusting and buffering agents, tonicity adjusting agents, wetting agents and the like, for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, triethanolamine oleate and the like. The pharmaceutical composition according to the invention may or may not comprise a preservative.

The genome titer of a viral vector, e.g., in the compositions and formulations disclosed herein, can be determined using a variety of standard methods, including, e.g., using PCR to determine the genome titer of the viral vector. Genome titers of viral vectors were determined using qPRC and ddPCR.

In a preferred embodiment, the pharmaceutical composition of the invention is in the form of a pharmaceutical formulation formulated for intravitreal delivery.

Therapeutic application

Hereditary retinal-related diseases encompass a large number of heterogeneous genetic diseases that affect 1 out of about 3000 (over 2 million people worldwide) and are the major sources of severe vision loss or blindness. AAV-based gene therapies have been proposed in which recombinant adeno-associated virus (rAAV) is used to deliver genes to one or more types of cells in the retina, for example, to replace a deleted gene or correct a defective gene.

The novel capsid protein variants of the present invention increase the targeting and transduction efficiency of AAV to a variety of retinal cells upon intravitreal delivery, thus providing a more effective means of gene delivery-based treatment of retinal-related eye diseases.

Thus, in one aspect, the invention provides methods of treating a disease using the recombinant AAV vectors of the invention or pharmaceutical compositions comprising the same. In one embodiment, the method comprises administering any recombinant AAV vector or pharmaceutical composition of the invention to a subject in need thereof. The recombinant AAV vector or pharmaceutical composition may be administered by any suitable route. In some embodiments, the treatment is therapeutic. In other embodiments, the treatment is prophylactic. In some embodiments, the subject is a mammal, wherein the mammal is especially a human, primate, dog, horse, cow, especially a human subject.

Ocular diseases that can be treated using the variant rAAV vectors or virions and/or methods disclosed herein include, but are not limited to, monogenic diseases, complex genetic diseases, acquired diseases, and traumatic injuries. Examples of such diseases include, but are not limited to, congenital amaurosis, total color blindness, retinitis (X-linked retinitis pigmentosa), choroidal-free disease, leber's hereditary optic neuropathy, macular degeneration (e.g., age-related macular degeneration), X-linked retinal splitting, diabetic retinopathy, diabetic macular edema, or choroidal neovascularization. Examples of such disease-associated genes include, for example, but are not limited to, congenital amaurosis-associated genes such as RPE65, total color blindness (Achromatopsia) -associated genes such as CNGB3, choroidal free disease (Choroideremia) -associated genes such as CHM and REP1, leber hereditary optic neuropathy (Leber hereditary optic neuropathy) -associated genes such as ND4, macular degeneration-associated genes such as VEGF, sFLt1 and CD59, retinitis pigmentosa (RETINITIS PIGMENTOSA) -associated genes such as ChR2, MERTK, RLBP1 and PDE6B, X-linked retinitis pigmentosa (X-LINKED RETINITIS pigmentosa) -associated genes such as RPGR, and X-linked retinal split (X-linked retinoschisis) -associated genes such as RS1. The gene products delivered by AAV variant capsids according to the invention may be used to alter the level of gene products or gene product activity directly or indirectly associated with retinal diseases and wounds.

One of ordinary skill in the art will be readily able to determine the amount of rAAV virions that are effective for a subject and monitor disease treatment by testing for changes in one or more functional or anatomical parameters, such as visual acuity, visual field, electrophysiological responses to light and darkness, color vision, contrast sensitivity, anatomy, retinal health and vasculature, ocular motility, fixation preferences, and stability. Some non-limiting methods for assessing retinal function and changes thereof include assessing vision (e.g., best corrected vision [ BCVA ], movement, navigation, object detection and discrimination), assessing vision (e.g., static and dynamic vision measurements), conducting clinical examinations (e.g., slit lamp examinations of the anterior and posterior segments of the eye), assessing electrophysiological responses to all bright and dark wavelengths (e.g., all forms of Electroretinogram (ERG) [ full view, multifocal and mode ], all forms of Visual Evoked Potential (VEP), electrooculography (EOG), color vision, dark adaptation, and/or contrast sensitivity). Non-limiting methods for assessing anatomical and retinal health and changes thereof include Optical Coherence Tomography (OCT), fundus photography, adaptive optical scanning laser ophthalmoscopy (AO-SLO), fluorescence and/or autofluorescence, measuring eyeball motility and eye movement (e.g., nystagmus, fixation preferences and stability), measuring reported results (changes in patient reported visual and non-visual guidance behaviors and activities, patient reported results [ PRO ], questionnaire-based quality of life assessment, daily activity and measurement of neurological function (e.g., functional Magnetic Resonance Imaging (MRI)).

In some embodiments, an effective amount of a rAAV virion results in a reduction in the rate of loss of retinal function, anatomical integrity, or retinal health, e.g., a 2-fold, 3-fold, 4-fold, or 5-fold or more reduction in the rate of loss, and thus the progression of the disease, e.g., a 10-fold or more reduction in the rate of loss, and thus the progression of the disease, in the subject.

In some embodiments, the rAAV virions are administered to the subject by intraocular injection, e.g., by intravitreal injection, by subretinal injection, by suprachoroidal injection, or by any other convenient mode or route of administration that will result in delivery of the rAAV virions to the eye. Other convenient modes or routes of administration include, but are not limited to, intravenous, intra-arterial, periocular, intracameral, subconjunctival and sub-tenon's injections and topical and intranasal administration. In some preferred embodiments, the rAAV virions are administered by intravitreal injection. In some embodiments, the rAAV virions are capable of traversing the vitreous of a subject and traversing the inner limiting membrane when administered by intravitreal injection and/or moving more efficiently through the retinal layer than the ability of AAV virions comprising the corresponding parental AAV capsid protein.

In one aspect, the invention also provides the use of a recombinant AAV viral vector of the invention or a pharmaceutical composition of the invention in the manufacture of a medicament for use in the above method. In some embodiments, the drug is used to deliver the heterologous nucleic acid into the retina of the subject. In other embodiments, the medicament is for treating or preventing a retinal related disease, e.g., congenital amaurosis, total color blindness, retinitis (X-linked retinitis), choroidal free disease, leber hereditary optic neuropathy, macular degeneration (e.g., age-related macular degeneration), X-linked retinal splitting, diabetic retinopathy, diabetic macular edema, or choroidal neovascularization. In another embodiment, the drug is in the form of a vitreous injection formulation.

Examples

The following description of the embodiments of the present invention will be made clearly and completely, and it is apparent that the described embodiments are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention. The various reagents involved in the examples are commercially available unless otherwise specified.

Example 1 construction of backbone plasmids for AAV9 capsid protein variant libraries

1. Construction of pRDAV-CMV-EGFP-Cap9 backbone plasmid (construct 1)

The original plasmid pRDAV-CMV-EGFP (brocade gene preservation) contained EcoRI and MluI cleavage sites after EGFP. Construct 1 (shown schematically in fig. 1) expressing AAV9 capsid protein (Cap 9) was generated as described below.

Cap9 complete ORF fragment amplification

The AAV9 complete genome is used as a template, a Cap9 complete ORF fragment is amplified by PCR and introduced into an enzyme digestion site, wherein the Cap9 complete ORF fragment is an ORF fragment with the length of 2637bp from a P40 promoter, a Cap9 stop codon TAA is stopped, a restriction enzyme MluI enzyme digestion site is introduced at the P40 end, and a restriction enzyme EcoRI enzyme digestion site is introduced at the Cap9 stop codon end.

The primer sequences used for PCR amplification are as follows:

Upstream primer (SEQ ID NO: 7): 5'-ACGCGTacgtcaaaaagggtggagc-3'

Downstream primer (SEQ ID NO: 8): 5'-GAATTCttacagattacgagtcaggtatctg-3'

The PCR amplification was performed by a procedure of 95℃pre-denaturation for 2min, 95℃denaturation for 15s, 56℃annealing for 15s, 72℃extension for 30s, repeated denaturation to 35 cycles of extension, 72℃finishing extension for 2min, and 4℃termination.

Production of construct 1

And (3) carrying out digestion on the PCR product by EcoRI and MluI, and recovering by gel electrophoresis to obtain the target fragment. The original plasmid pRDAV-CMV-EGFP was digested with EcoRI and MluI, and the resulting fragment of interest was ligated with the digested plasmid backbone, thereby obtaining plasmid backbone pRDAV-CMV-EGFP-Cap9, construct 1 (shown in FIG. 1), 10191bp in size, and the sequence shown in SEQ ID NO. 1.

2. Construction of pRDAV-CMV-EGFP-Cap 9. Delta. -588 backbone plasmid (construct 2)

A fragment of the wild-type Cap9 gene from the 881 st base position (c.881) to the 1985 th base position (c.1985) was selected, the alanine codon corresponding to amino acid 587 position was mutated to tyrosine codon (A587Y), and the sequence GGTAACCGTT AACTT (which contained BestEII and HpaI cleavage sites, bestEII cleavage produced sticky ends, hpaI cleavage produced blunt ends, whereby the cleavage products produced by both BestEII and HpaI cleavage were used, sticky ends at one end and blunt ends at the other end, so that self-ligation could be prevented, and the middle region of the cleavage site could be replaced by a randomized library fragment, as shown in FIG. 2), replacing the 3 bases encoding the amino acid residues of the original 588 site, thereby obtaining the Cap 9. Delta. -588 sequence encoding the 587 site mutation and the 588 site deletion. A DraIII restriction site was introduced at position c.881 of the Cap 9. Delta. -588 sequence, a BamHI restriction site was introduced at position c.1985, and gene synthesis was carried out by the company of Prinsepia Biotechnology. The synthetic product was constructed into construct 1 by means of cleavage ligation to replace the corresponding fragment of original Cap9 located between DraIII and BamHI cleavage sites, resulting in construct 2, pRDAV-CMV-EGFP-Cap 9. Delta. -588. The sequence of construct 2 is shown in SEQ ID NO. 2.

Example 2 construction of AAV capsid variant Virus library

Linearization framework carrier

Construct 2 of example 1 was used as a backbone vector and was double digested with BestEII and HpaI to linearize the backbone vector. The enzyme digestion system comprises a construct 2 (3 mug), cutSmart Buffer (5 mug), a restriction enzyme BestEII (0.5 mug) and a restriction enzyme HpaI (0.5 mug), and is incubated for 3-4 hours at 37 ℃ after water is added to 50 mug. And (3) after enzyme digestion, recovering the linearization vector through gel cutting.

Synthesis of Oligo library

The Oligo sequences used were as follows:

Oligo-F:5’-GTAACNNNNNNNNN-3’

Oligo-R:5’-NNNNNNNNN-3’

the Oligo is denatured at 95 ℃ and annealed slowly at room temperature to obtain an annealed product.

Library construction and screening

The Oligo annealing product was ligated to the linearized backbone (construct 2) using T4 DNA ligase to give a recombinant plasmid. The ligation system was annealed (2. Mu.L), linearized backbone vector (25 ng), T4 DNA ligase (0.5. Mu.L), ligation buffer (5. Mu.L), and water was added to 10. Mu.L at 16℃overnight to give recombinant plasmid with Oligo inserted.

The recombinant plasmid was transformed into E.coli HB101 competent cells as follows.

1) Adding 10 μl of recombinant plasmid into Ep tube containing 100 μl HB101 competent cells, slightly beating the tube wall for several times, mixing thoroughly, and ice-bathing for 30min;

2) The Ep tube was placed in a 42 ℃ water bath for 90s;

3) LB medium (0.5 mL,37 ℃) was slowly added to the Ep tube and shaken for 45min at 80 rpm;

4) The bacterial solution was spread on LB plates containing ampicillin (0.1 g/L), and cultured overnight at 37 ℃.

Single colonies were randomly picked and inoculated into a culture tube (LB medium containing 0.1g/L ampicillin), shaken at 37℃and 200rpm, and cultured overnight.

Plasmid extraction was performed as follows.

1) Centrifuging the bacterial liquid obtained by the method at 12000rpm for 1 minute, and pouring out a supernatant culture medium;

2) Adding 250 mu L of buffer P1/RNaseA mixed solution, and high-speed vortex to re-suspend bacteria;

3) Adding 250 μL buffer P2, and reversing upside down for 8-10 times;

4) Adding 350 mu L buffer P3, immediately reversing and uniformly mixing for 8-10 times to thoroughly neutralize the solution;

5) Centrifuging at 13000rpm for 10 min, and collecting supernatant;

6) Centrifuging 12000 for 1 minute, pouring out the waste liquid, adding 500 mu L of PW1,12000, centrifuging for 1 minute, and pouring out the waste liquid;

7) Adding 600 mu L of PW2,12000, centrifuging for 1 min, and pouring out the supernatant;

8) Repeating the step 7;

9) Idle at 12000rpm for 2 minutes;

10 30-50. Mu.L of the preheated eluent at 55 ℃ is added, and the mixture is kept stand for 2 minutes and centrifuged at 12000rpm for 1 minute.

The positive plasmids identified by concentration detection and cleavage were numbered and 10. Mu.L sequenced, and the positive plasmids were stored in a-20℃refrigerator to obtain AAV capsid libraries.

Sequencing to obtain AAV9 capsid protein (CAP 9) VP1 variant with number CapX-120, wherein the corresponding nucleotide sequence is shown in SEQ ID NO.3, and the corresponding amino acid sequence is shown in SEQ ID NO. 4. Sequence information obtained by sequencing is compared with the original capsid protein (CAP 9) sequence, and the substitution amino acid sequences of the variants at 587 and 588 sites are shown in SEQ ID NO.5 respectively.

EXAMPLE 3 packaging production of mutant AAV Virus and control AAV Virus

Reference (Xiao X,et al.Production of High-Titer Recombinant Adeno-Associated Virus Vectors in the Absence of Helper Adenovirus.J Virol.1998;72(3):2224-2232), uses a three plasmid packaging system to package and purify recombinant AAV viruses. Briefly, HEK293 cells were co-transfected with packaging plasmids expressing the Rep gene of AAV, helper plasmids providing the helper functions of AdV, and transfer plasmids carrying the ITRs of AAV and expressing variant CAP9 and green fluorescent protein eGFP (plasmid shown in FIG. 2 in which CAP9 variants were inserted) or transfer plasmids expressing wild-type CAP9 and green fluorescent protein eGFP (plasmid shown in FIG. 1) at a molar ratio of 1:1:1. After 72h transfection, cells and culture supernatants were collected and recombinant AAV virus was purified by PEG8000-NaCl & CH3Cl extraction. AAV viral products were solubilized with 0.01% Pluronic F68-PBS and rAAV titers were determined using the dot blot method. AAV capsid variant viruses numbered CapX-120, respectively, and control rAAV9 virus were obtained.

Example 4 rule of biological distribution of AAV capsid variant Virus in mice

AAV virus injection

Male SPF grade C57 mice, 18-20g, were selected from 6-8 weeks, and at least 3 mice per group were tested using rAAV9 virus encoding the fluorescent protein EFGP as a control and AAV capsid variant virus as described herein. The capsid variant virus CapX-120 was injected intravitreally at a dose of 2E10 gc/kg. After injection, the materials were dissected and harvested 28 days later, frozen sections of each tissue were prepared, and EGFP fluorescence was observed.

Mouse tissue section and immunofluorescence comparison

A. Preparation of frozen sections

1) Tissue fixation, namely fixing fresh tissue fixing liquid for more than 24 hours, taking out the tissue from the fixing liquid, and trimming and flattening the tissue of the target part by a surgical knife.

2) And (3) dewatering, namely placing the trimmed tissue into 15% sucrose solution, and transferring the trimmed tissue into 30% sucrose solution for dewatering and sinking in a 4 ℃ refrigerator.

3) OCT embedding, namely taking out the dehydrated tissue, slightly sucking the surface water by filter paper, placing the surface water on an embedding table upwards, dripping an OCT embedding agent around the tissue, placing the embedding table on a quick freezing table of a frozen microtome for quick freezing embedding, and slicing after OCT becomes white and hard. The direct frozen section of fresh tissue needs no fixed dehydration, and the OCT embedding agent is used for embedding and slicing after the tissue of the target part is leveled by a surgical knife.

4) And slicing, namely fixing the embedding table on a slicing machine, firstly roughly cutting the tissue surface to be flat, then starting slicing, wherein the slicing thickness is 8-10 mu m, putting a clean glass slide on the cut tissue piece, and pasting the tissue on the glass slide and writing a label.

B. Observing fluorescence

The frozen sections were protected with a fade-resistant capper (ProLong, thermo Fischer), and EGFP protein was observed under a fluorescence microscope using a blue excitation band module (488 nm) and images were collected. Statistical analysis of the images was performed using ImageJ software.

Results

Capsid protein mutants CapX-120

Experimental results show that, compared with the control group, the AAV capsid variants CapX-120 virus significantly enhance the intracellular fluorescence intensity after infection of the retina of the mice, increase the number of infected cells, and nearly saturate the infection rate of cone cells and rod cells (fig. 3 and 4). From the results of fluorescence analysis by ImageJ software, the average fluorescence intensities caused by CapX-120 virus infection in the cone rod, RPE, outer and inner layers were all 1 to 2 times higher than AAV9 virus (fig. 5), and the EGFP positive cell rates after CapX-120 virus infection were 2 to 3 times higher than AAV9 virus for the countable outer and inner layers than AAV9 virus (fig. 6). Taken together, the results indicate that AAV capsid protein mutants CapX-120 can significantly enhance the transduction efficiency of the retina and can be used as a better choice for AAV to treat ophthalmic diseases.

Some embodiments of the invention:

1. An adeno-associated virus (AAV) capsid protein variant having inserted between amino acid 586 and 589 of a parent AAV capsid protein the amino acid sequence of SEQ ID No.5, in place of residues 587-588 of the parent AAV capsid protein, wherein the amino acid positions are numbered according to SEQ ID No. 6.

2. The AAV capsid protein variant of embodiment 1, wherein said capsid protein variant confers increased transduction of an AAV virion comprising the same, as compared to a parent AAV capsid protein, to a retinal cell, preferably said retinal cell is selected from the group consisting of photoreceptor cells, retinal Pigment Epithelium (RPE) layer cells, outer nuclear layer cells, inner nuclear layer cells, and ganglion layer cells, more preferably said retinal cell is a rod cell and/or cone cell,

Still preferably, the capsid protein variant confers a higher transduction rate on cones and rod cells of the retina relative to other cell types of the retina.

3. The capsid protein variant of embodiment 1 or 2, wherein said parent AAV capsid protein is an AAV9 capsid protein.

4. The capsid protein variant of any one of embodiments 1-3, wherein said parent AAV capsid protein is an AAV9 capsid protein comprising or consisting of the amino acid sequence:

(a) Amino acid sequence of amino acids 203 to 736 of SEQ ID NO. 6;

(b) Amino acid sequence of amino acids 137 to 736 of SEQ ID NO. 6;

(c) The amino acid sequence of SEQ ID NO. 6.

5. The capsid protein variant according to any one of embodiments 1-4, wherein said capsid protein variant is a VP1, VP2 or VP3 capsid protein,

Preferably, the capsid protein variant:

(a) An amino acid sequence comprising SEQ ID NO. 4;

(b) Encoded by a nucleic acid comprising the nucleotide sequence of SEQ ID NO. 3, or

(C) Derived from an amino acid sequence encoding SEQ ID NO. 4 or a transcript comprising the nucleotide sequence of SEQ ID NO. 3.

6. An isolated nucleic acid, wherein the nucleic acid comprises a polynucleotide sequence encoding an AAV capsid protein variant of any one of embodiments 1-5.

7. The nucleic acid according to embodiment 6, comprising:

(a) Polynucleotides encoding the amino acid sequence of SEQ ID NO. 4, or

(B) The nucleotide sequence of SEQ ID NO. 3.

8. The nucleic acid according to embodiment 6 or 7, wherein the nucleic acid is capable of transcribing and expressing at least one or any two or all three AAV capsid proteins selected from the group consisting of VP1, VP2, and VP3.

9. A nucleic acid vector comprising the nucleic acid of any one of embodiments 6-8, optionally further comprising a polynucleotide encoding an AAV REP protein.

10. An isolated host cell comprising a nucleic acid according to any one of embodiments 6 to 8, optionally stably transfecting the host cell,

Preferably, the host cell further comprises a nucleic acid encoding an AAV REP protein and/or a nucleic acid encoding a helper virus function.

11. Use of the nucleic acid of any of embodiments 6-8 or the nucleic acid vector of embodiment 9 or the host cell of embodiment 10 for the preparation of a recombinant adeno-associated virus (rAAV) vector.

12. A recombinant adeno-associated virus (rAAV) vector comprising a capsid protein variant of any one of embodiments 1-5,

Preferably, the rAAV exhibits increased transduction of retinal cells relative to wild-type AAV, preferably, the retinal cells are selected from the group consisting of photoreceptor cells, RPE layer cells, outer nuclear layer cells, inner nuclear layer cells, and ganglion layer cells.

13. The rAAV vector according to embodiment 12, wherein the rAAV vector comprises in its genome:

a.5 'and 3' AAV Inverted Terminal Repeat (ITR) sequences, and

B. An expression construct comprising a heterologous nucleic acid located between the 5 'and 3' itrs, wherein the heterologous nucleic acid encodes a gene product of interest.

14. The rAAV vector according to embodiment 13, wherein the expression construct comprises the following elements functionally linked to each other in the direction of transcription:

promoters, preferably retina-specific promoters, such as rod-and/or cone-specific promoters,

The presence of a heterologous nucleic acid,

-A transcription terminator.

15. The AAV vector according to embodiment 13 or 14, wherein the heterologous nucleic acid encodes a gene product of interest for gene replacement, gene suppression or gene editing, preferably the gene product of interest is a protein or RNA.

16. A pharmaceutical composition comprising the recombinant AAV viral vector of any one of embodiments 12-15 and a pharmaceutically acceptable carrier.

17. A method of delivering a heterologous nucleic acid to a subject in need thereof, wherein the method comprises administering to the subject the recombinant AAV viral vector of any one of embodiments 12-15 or the pharmaceutical composition of embodiment 16.

18. The method of embodiment 17, wherein the method is for treating or preventing a retinal-related disorder, e.g., congenital amaurosis, total color blindness, retinitis (X-linked retinitis pigmentosa), choroidal-free disease, leber's hereditary optic neuropathy, macular degeneration (e.g., age-related macular degeneration), X-linked retinal splitting, diabetic retinopathy, diabetic macular edema, or choroidal neovascularization.

19. The method of embodiment 17 or 18, wherein said delivering comprises intravitreal injection.

20. A method of delivering a heterologous nucleic acid to an isolated or in vitro cultured cell comprising contacting the cell with the rAAV viral vector of embodiments 12-15, preferably the cell is a retinal cell, particularly a rod cell and/or cone cell.

21. Use of the recombinant AAV viral vector according to any of embodiments 12-15, for the preparation of a pharmaceutical composition for treating or preventing a retinal-related disease, e.g., selected from congenital amaurosis, achromatopsia, retinitis (X-linked retinitis pigmentosa), choroidemia, leber's hereditary optic neuropathy, macular degeneration (such as age-related macular degeneration), X-linked retinal split, diabetic retinopathy, diabetic macular edema, or choroidal neovascularization, preferably, the pharmaceutical composition is administered by ocular gene delivery (e.g., vitreous injection).

Claims

2. The AAV capsid protein variant of claim 1, wherein said capsid protein variant confers increased transduction of an AAV virion comprising the same, as compared to a parent AAV capsid protein, preferably said retinal cell is selected from the group consisting of photoreceptor cells, retinal Pigment Epithelium (RPE) layer cells, outer nuclear layer cells, inner nuclear layer cells, and ganglion layer cells, more preferably said retinal cell is a rod cell and/or cone cell,

3. The capsid protein variant of claim 1 or 2, wherein said parent AAV capsid protein is an AAV9 capsid protein.

4. An isolated nucleic acid, wherein the nucleic acid comprises a polynucleotide sequence encoding the AAV capsid protein variant of any one of claims 1-3.

5. A nucleic acid vector comprising the nucleic acid of claim 4, optionally further comprising a polynucleotide encoding an AAV REP protein.

6. An isolated host cell comprising the nucleic acid of claim 4, optionally stably transfecting said host cell,

7. Use of a nucleic acid according to claim 4 or a nucleic acid vector according to claim 5 or a host cell according to claim 6 for the preparation of a recombinant adeno-associated virus (rAAV) vector.

8. A recombinant adeno-associated virus (rAAV) vector comprising a capsid protein variant according to any one of claims 1-3,

9. A pharmaceutical composition comprising the recombinant AAV viral vector of claim 8 and a pharmaceutically acceptable carrier.

10. A method of delivering a heterologous nucleic acid to a subject in need thereof or to cells cultured ex vivo or in vitro, wherein the method comprises administering to the subject or the cells the recombinant AAV viral vector of claim 8 or the pharmaceutical composition of claim 9.