Recombinant adeno-associated virus virion (raav), its use, pharmaceutical composition and recombinan

专利摘要:
VARIANT ADENO-ASSOCIATED VIRUS (AAV) CAPSID PROTEIN, RECOMBINANT ADENO-ASSOCIATED VIRUS (RAAV), ITS USE, PHARMACEUTICAL COMPOSITION, ISOLATED NUCLEIC ACID AND HOST CELL. The present disclosure provides adeno-associated virus (AAV) virions with altered capsid protein, where AAV virions exhibit greater infectivity of retinal cells when administered via intravitreal injection compared to wild-type AAV. The present disclosure further provides methods for delivering a gene product to a retinal cell in a subject and methods for treating eye disease.
公开号:BR112013027120B1
申请号:R112013027120-5
申请日:2012-04-20
公开日:2022-02-01
发明作者:David V. Schaffer；Ryan R. Klimczak；James T. Koerber；John G. Flannery；Deniz Dalkara Mourot；Meike Visel；Leah C.T. Byrne
申请人:The Regents Of The University Of California；
IPC主号:

专利说明:

CROSS REFERENCE
[001] This application claims the benefit of Provisional Patent Application US 61/478,355, filed April 22, 2011, the disclosure of which is incorporated herein by reference in its entirety. DECLARATION RELATED TO FEDERAL SPONSORED RESEARCH
[002] This invention was made with government support under Grant Nos. EY016994-02 and EY1018241 granted by the National Eye Institute of the National Institutes of Health. The government has certain rights in the invention. FUNDAMENTALS
[003] Photoreceptors are the first neurons in the retina to receive and process visual information, converting electromagnetic radiation into hyperpolarized responses through phototransduction. The overwhelming majority of inherited retinal diseases result in the loss of these cells, either directly, as in dominant mutations that affect the folding of the rhodopsin protein, or indirectly, as recessive mutations that affect the retinal recycling pathways in the retinal pigment epithelium (RPE). ).
[004] AAV belongs to the family Parvoviridae and genus Dependovirus, whose members require co-infection with a helper virus such as adenovirus to promote replication, and AAV stabilizes a latent infection in the absence of a helper. Virions are composed of a 25 nm icosahedral capsid encompassing a 4.9 kb single-stranded DNA genome with two reading regions: rep and cap. The nonstructural rep gene encodes four regulatory proteins essential for viral replication, while cap encodes three structural proteins (VP1-3) that assemble a 60-mer capsid shell. This viral capsid mediates the ability of AAV vectors to overcome many of the biological barriers of viral transduction, including cell surface receptor binding, endocytosis, intracellular trafficking, and unpacking in the nucleus.
[005] US Patent Publication 2005/0053922 ; US Patent Publication 2009/0202490; Allocca et al. (2007) J. Virol. 81: 11372; Boucas et al. (2009) J. Gene Med. 11: 1103. SUMMARY OF THE INVENTION
[006] The present disclosure provides adeno-associated virus (AAV) virions with altered capsid protein, wherein AAV virions exhibit greater infectivity of a retinal cell when administered via intravitreal injection compared to wild type AAV. The present disclosure further provides methods for delivering a further gene product to a retinal cell in a subject and methods for treating eye disease. BRIEF DESCRIPTION OF THE DRAWINGS
[007] Figure 1 provides a representative three-dimensional model of AAV2 containing a random heptamer after amino acid 587.
[008] Figure 2 depicts higher levels of intravitreal transduction by AAV2 variant 7M8 (right), compared to AAV2 (left).
[009] Figure 3 provides representative fluorescence images of retinal cryopathies showing the expression of green fluorescent protein (GFP) resulting from 7M8 containing the GFP gene under the control of ubiquitous CAG promoter (left) or a Rho promoter specific for the photoreceptor (right).
[0010] Figure 4 represents GFP+ photoreceptor cells per million retinal cells as counted by flow cytometry, followed by transduction by 7M8 or by 7M8 containing 4 tyrosine mutations (7m8.4YF).
[0011] Figure 5 provides an amino acid sequence of AAV2VP1 (SEQ ID NO: 1).
[0012] Figure 6 provides amino acid sequences corresponding to AAV2 amino acids 570-610 (Figure 5) of an AAV capsid VP1 protein from various AAV serotypes.
[0013] Figures 7A-I depict structural improvements in Rs1h-/- mouse retina following gene transfer.
[0014] Figures 8A-D represent functional rescue of the A and B waves of electroretinogram following the release of the RS1 gene.
[0015] Figures 9A-E depict sustained improvements in retinal thickness measured at 10 months after 7m8-rho-RS1 treatment.
[0016] Figure 10 provides an amino acid sequence of retinoschisin.
[0017] Figure 11 provides an amino acid sequence of brain-derived neurotrophic factor.
[0018] Figure 12 provides an amino acid sequence of RPE65.
[0019] Figures 13A-C provide the nucleotide sequence of the 7m8-rho-RS1 construct.
[0020] Figure 14 provides an amino acid sequence of peripherin 2.
[0021] Figure 15 provides an amino acid sequence of peripherin.
[0022] Figure 16 provides an amino acid sequence of GTPase interacting protein 1 from retinitis pigmentosa.
[0023] Figures 17A-C provide an amino acid sequence alignment of loop IV (GH loop) regions of the AAV capsid protein. Insertion sites are shown in bold and underlined.
[0024] Figures 18A-C provide an amino acid sequence alignment of the GH loop regions of the AAV capsid protein, with heterologous peptide insertions.
[0025] Figure 19 provides a fundus fluorescence image showing GFP expression in primate central retina 9 weeks after administration of 7m8 containing GFP under the control of connexin36 promoter. DEFINITIONS
[0026] The term "retinal cell" may refer herein to any type of cell comprising the retina, such as retinal ganglion cells, amacrym cells, horizontal cells, bipolar cells, and photoreceptor cells including rods and cones, Müller glial cells , and retinal pigment epithelium.
[0027] "AAV" is an abbreviation for adeno-associated virus and may be used to refer to the virus itself or derivatives thereof. The term covers all subtypes and both naturally occurring and recombinant forms, except where required otherwise. The abbreviation "rAAV" refers to recombinant adeno-associated virus, also referred to as a recombinant AAV vector (or "rA-AV vector"). The term "AAV" includes AAV type 1 (AAV-1), AAV type 2 (AAV-2), AAV type 3 (AAV-3), AAV type 4 (AAV-4), AAV type 5 (AAV-5) , AAV type 6 (AAV-6), AAV type 7 (AAV-7), AAV type 8 (AAV-8), avian AAV, bovine AAV, canine AAV, equine AAV, primate AAV, non-primate AAV, and oviN AAV "Primate AAV" refers to AAV that infects primates, "non-primate AAV" refers to an AAV that infects non-primate mammals, "bovine AAV" refers to AAV that infects bovine mammals, and so on.
[0028] The genomic sequences of various AAV serotypes, as well as the sequences of native terminal repeats (TRs), Rep proteins, and capsid subunits are known in the art. Said sequences can be found in the literature or public databases such as GenBank. See, for example, GenBank Accession Numbers NC_002077 (AAV-1), AF063497 (AAV-1), NC_001401 (AAV-2), AF043303 (AAV-2), NC_001729 (AAV-3), NC_001829 (AAV-4) ,U89790 (AAV-4), NC_006152 (AAV-5), AF513851 (AAV-7), AF513852 (AAV-8), and NC_006261 (AAV-8); the disclosures of which are incorporated by reference herein for teaching AAV nucleic acid and amino acid sequences. See also, for example, Srivistava et al. (1983) J. Virology 45:555; Chiorini et al. (1998) J. Virology 71:6823; Chiorini et al. (1999) J. Virology 73: 1309; Bantel-Schaal et al. (1999) J. Virology 73:939; Xiao et al. (1999) J. Virology 73:3994; Muramatsu et al. (1996) Virology 221:208; Shade et al., (1986) J. Virol. 58:921; Gao et al. (2002) Proc. nat. academy Sci. USA 99: 11854; Morris et al. (2004) Virology 33:375-383; international patent publications WO 00/28061, WO 99/61601, WO 98/11244; and US patent 6,156,303.
[0029] An "rAAV vector" as used herein refers to an AAV vector comprising a polynucleotide sequence not of AAV origin (i.e., a polynucleotide heterologous to AAV), typically a sequence of interest for the genetic transformation of a cell. In general, the heterologous polynucleotide is flanked by at least one, and usually two, AAV inverted terminal repeats (ITRs). The term rAAV vector includes both rAAV vector particles and rAAV vector plasmids. An rAAV vector can be single-stranded (ssAAV) or self-complementary (scAAV).
[0030] An "AAV virus" or "AAV viral particle" or "rAAV vector particle" refers to a viral particle composed of at least one AAV capsid protein (typically all capsid proteins of a wild-type AAV) and an encapsidated polynucleotide rAAV vector. If the particle comprises a heterologous polynucleotide (i.e., a polynucleotide in addition to the wild-type AAV genome as a transgene to be delivered to a mammalian cell), it is typically referred to as an "rAAV vector particle" or simply a "rAAV vector". Thus, rAAV particle production necessarily includes rAAV vector production, as a vector is contained within an rAAV particle.
[0031] "Packaging" refers to a series of intracellular events that result in the assembly and encapsidation of an AAV particle.
[0032] AAV "rep" and "cap" genes refer to polynucleotide sequences encoding adeno-associated virus replication and encapsidation proteins. AAV rep and cap are referred to herein as AAV "packaging genes".
[0033] A "helper virus" for AAV refers to a virus that allows AAV (e.g., wild-type AAV) to be replicated and packaged by a mammalian cell. A variety of said helper viruses for AAV are known in the art, including adenoviruses, herpes viruses and pox viruses such as vaccinia. Adenoviruses include a number of different subgroups, although Adenovirus type 5 subgroup C is most commonly used. Various adenoviruses of human, non-human mammal and avian origin are known and available from depositories such as the ATCC. Viruses in the herpes family include, for example, herpes simplex virus (HSV) and Epstein-Barr virus (EBV), as well as cytomegalovirus (CMV) and pseudorabies virus (PRV); which are still available from depositories such as ATCC.
[0034] "Helper virus functions" refers to the functions encoded in a helper virus genome that enable AAV replication and packaging (in conjunction with other requirements for replication and packaging described here). As described herein, "helper virus function" can be provided in a variety of ways, including providing helper virus or providing, for example, polynucleotide sequences encoding the functions required for a trans-producing cell. For example, a plasmid or other expression vector comprising nucleotide sequences encoding one or more adenoviral proteins in a producer cell together with an rAAV vector.
[0035] An "infectious" virus or viral particle is one that comprises a competently assembled viral capsid and is capable of releasing a polynucleotide component into a cell for which the viral species is tropic. The term does not necessarily imply any ability to replicate the virus. Assays for counting viral particles are described elsewhere in this disclosure and in the art. Viral infectivity can be expressed as the ratio of infectious viral particles to total viral particles. Methods for determining the ratio of infectious viral particle to total viral particles are known in the art. See, for example, Grainger et al. (2005) Mol. The R. 11:S337 (which describes a TCID50 infectious titer assay); and Zolotukhin et al. (1999) Gene Ther. 6:973. See also Examples.
[0036] A "replication competent" virus (e.g., a replication competent AAV) refers to a wild-type virus that is phenotypically infectious and is still capable of being replicated in an infected cell (i.e., in the presence of a helper virus or helper virus functions). In the case of AAV, replication competence generally requires the presence of functional AAV packaging genes. In general, rAAV vectors as described herein are replication-incompetent in mammalian cells (especially human cells) by virtue of the lack of one or more AAV packaging genes. Typically, said rAAV vectors are devoid of any AAV packaging gene to minimize the possibility that replication competent AAV is generated by recombination between AAV packaging genes and an input rAAV vector. In many embodiments, the rAAV vector preparations as described herein are those that contain some if any replication-competent AAV (rcAAV, still referred to as RCA) (e.g., less than about 1 rcAAV per 10 2 rAAV particles, less than than about 1 rcAAV per 104 rAAV particles, less than about 1 rcAAV per 108 rAAV particles, less than about 1 rcA-AV per 1012 rAAV particles, or no rcAAV).
[0037] The term "polynucleotide" refers to a polymeric form of nucleotides of any length, including deoxyribonucleotides or ribonucleotides, or analogs thereof. A polynucleotide may comprise modified nucleotides, such as methylated nucleotides and nucleotide analogues, and may be interrupted by non-nucleotide components. If present, modifications to the nucleotide structure can be conferred before or after assembly of the polymer. The term polynucleotide, as used herein, interchangeably refers to single-stranded and double-stranded molecules. Unless otherwise specified or required, any embodiment of the invention described herein that is a polynucleotide includes both double-stranded forms and each of the complementary single-stranded or predicted forms to prepare the double-stranded form.
[0038] Nucleic acid hybridization reactions can be carried out under different "stringency" conditions. Conditions that increase the stringency of a hybridization reaction are widely known and published in the art. See, for example, Sambrook et al. Molecular Cloning, A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989, incorporated herein by reference. For example, see page 7.52 of Sambrook et al. Examples of relevant conditions include (in order of increasing stringency): incubation temperatures of 25°C, 37°C, 50°C and 68°C; buffer concentrations of 10 x SSC, 6 x SSC, 1 x SSC, 0.1 x SSC (where 1 x SSC is 0.15 M NaCl and 15 mM citrate buffer) and their equivalents using other buffer systems; formamide concentrations of 0%, 25%, 50%, and 75%; incubation times from 5 minutes to 24 hours; 1, 2, or more washing steps; wash incubation times of 1, 2, or 15 minutes; and wash solutions of 6 x SSC, 1 x SSC, 0.1 x SSC, or deionized water. An example of stringent hybridization conditions at 50°C or greater and 0.1 x SSC (15 mM sodium chloride/1.5 mM sodium citrate). Another example of stringent hybridization conditions is overnight incubation at 42°C in a solution: 50% formamide, 1 x SSC (150 mM NaCl, 15 mM sodium citrate), 50 mM sodium phosphate (pH 7.6) , 5 x Denhardt's solution, 10% dextran sulfate, and 20 µg/ml denatured cut salmon sperm DNA, followed by washing the filters in 0.1 x SSC at about 65°C. As another example, stringent hybridization conditions comprise: prehybridization for 8 hours overnight at 65°C in a solution comprising 6X single-strength citrate (SSC) (1X SSC is 0.15M NaCl, 0.015M sodium citrate). Na, pH 7.0), 5X Denhardt's solution, 0.05% sodium pyrophosphate and 100 μg/ml herring sperm DNA; hybridization for 18-20 hours at 65°C in a solution containing 6X SSC, 1X Denhardt's solution, 100 μg/ml yeast tRNA and 0.05% sodium pyrophosphate; and washing filters at 65°C for 1 h in a solution containing 0.2X SSC and 0.1% SDS (sodium dodecyl sulfate).
[0039] Stringent hybridization conditions are hybridization conditions that are at least stringent than the above representative conditions. Other stringent hybridization conditions are known in the art and may further be employed to identify nucleic acids of this particular embodiment of the invention.
[0040] "Tm" is the temperature in degrees Celsius at which 50% of a polynucleotide duplex made of complementary strands of hydrogen bonded in an antiparallel direction by Watson-Crick base pairing dissociates into single strands under the conditions of the experiment . Tm can be predicted according to a standard formula, such as: Tm= 81.5 + 16.6 log[X+] + 0.41 (%G/C) - 0.61 (%F) - 600/L
[0041] where [X+] is the concentration of cation (generally sodium ion,Na+) in mol/L; (%G/C) is the number of G and C residues as a percentage of total residues in the duplex; (%F) is the percent formamide in solution (weight/vol); and L is the number of nucleotides in each strand of the duplex.
[0042] A polynucleotide or polypeptide has a certain percentage of "sequence identity" to another polynucleotide or peptide, meaning that, when aligned, that percentage of bases or amino acids are the same when comparing the two sequences. Sequence similarity can be determined in a number of different ways. To determine sequence identity, sequences can be aligned using methods and computer programs, including BLAST, available on the internet at ncbi.nlm.nih.gov/BLAST/. Another alignment algorithm is FASTA, available in the Genetics Computing Group (GCG) package, of Madison, Wisconsin, USA, a wholly owned subsidiary of Oxford Molecular Group, Inc. Other techniques for alignment are described in Methods in Enzymology, vol. 266: Computer Methods for Macromolecular Sequence Analysis (1996), ed. Doolittle, Academic Press, Inc., a division of Harcourt Brace & Co., San Diego, California, USA. Of particular interest are alignment programs that allow gaps in the sequence. Smith-Waterman is a type of algorithm that allows gaps in sequence alignments. See Meth. Mol. Biol. 70: 173-187 (1997). Furthermore, the GAP program using the Needleman and Wunsch alignment method can be used to align the sequences. See J. Mol. Biol. 48: 443-453 (1970).
[0043] Of interest is the BestFit program using Smith and Waterman's local homology algorithm (Advances in Applied Mathematics 2: 482-489 (1981) to determine sequence identity. The gap generation penalty will generally range from 1 to 5, usually 2 to 4 and in many modalities it will be 3. The gap extension penalty will generally range from about 0.01 to 0.20 and in many cases will be 0.10. The program has default parameters determined by the inserted sequences to be compared. Preferably, sequence identity is determined using the default parameters determined by the program. This program is also available from the Genetics Computing Group (GCG) package, of Madison, Wisconsin, USA.
[0044] Another program of interest is the FastDB algorithm. FastDB is described in Current Methods in Sequence Comparison and Analysis, Macromolecule Sequencing and Synthesis, Selected Methods and Applications, pp. 127-149, 1988, Alan R. Liss, Inc. The percent of sequence identity is calculated by FastDB based on the following parameters: Mismatch Penalty: 1.00;Gap Penalty: 1.00;Gap Size Penalty: 0.33; and Join Penalty: 30.0.
[0045] A "gene" refers to a polynucleotide containing at least one reading frame that is capable of encoding a particular protein after being transcribed and translated.
[0046] A "gene product" is a molecule that results from the expression of a particular gene. Gene products include, for example, a polypeptide, an aptamer, an interfering RNA, an aptamer, an interfering RNA, an mRNA, and the like.
[0047] A "short interferen" or "short interfering RNA" or siRNA is a nucleotide duplex RNA that is targeted to a gene of interest (a "target gene"). An "RNA duplex" refers to the structure formed by the complementary pairing between two regions of an RNA molecule. siRNA is "targeted" to a gene in which the nucleotide sequence of the portion of the duplex portion of the siRNA is complementary to a nucleotide sequence of the target gene. In some embodiments, the duplex length of siRNAs is less than 30 nucleotides. In some embodiments, the duplex may be 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, or 10 nucleotides in length. length. In some embodiments, the length of the duplex is 19-25 nucleotides in length. The duplex RNA portion of the siRNA may be part of the hairpin structure. In addition to the duplex portion, the hairpin structure may contain a loop portion positioned between the two sequences that form the duplex. Loop handle can vary in length. In some embodiments the loop is 5, 6, 7, 8, 9, 10, 11, 12, or 13 nucleotides in length. The hairpin structure may also contain 3' or 5' overlapping positions. In some embodiments, the overlap is a 3' or 5' overlap of 0, 1, 2, 3, 4, or 5 nucleotides in length.
[0048] A "small hairpin RNA," or shRNA, is a polynucleotide construct that can be prepared to express an interfering RNA such as siRNA.
[0049] "Recombinant," as applied to a polynucleotide means that the polynucleotide is the product of various combinations of cloning, restriction or ligation steps, and other procedures that result in a construct that is distinct from a polynucleotide found in nature. A recombinant virus is a viral particle comprising a recombinant polynucleotide. The terms respectively include replicas of the original polynucleotide construct and progeny of the original virus construct.
[0050] A "control element" or "control sequence" is a nucleotide sequence involved in an interaction of molecules that contribute to the functional regulation of a polynucleotide, including replication, duplication, transcription, processing, translation or degradation of the polynucleotide. Regulation can affect the frequency, speed or specificity of the process, and it can be ameliorating or inhibitory in nature. Control elements known in the art include, for example, transcriptional regulatory sequences such as promoters and enhancers. A promoter is a region of DNA capable under certain conditions of binding RNA polymerase and initiating transcription of a coding region usually located downstream (in the 3' direction) of the promoter.
[0051] "Operably linked" or "operationally linked" refers to a juxtaposition of genetic elements, where the elements are in a relationship that allows them to operate in an expected way. For example, a promoter is operably linked to a coding region if the promoter helps initiate transcription of the coding sequence. There may be intervening residues between the promoter and the coding region as long as this functional relationship is maintained.
[0052] An "expression vector" is a vector comprising a region that encodes a polypeptide of interest, and is used to effect expression of the protein in an intended target cell. An expression vector further comprises control elements operably linked to the coding region to facilitate expression of the protein in the target. The combination of control elements and a gene or genes to which they are operatively linked for expression is sometimes referred to as an "expression cassette," a large number of which are known and available in the art or can be readily constructed from the components that are available in the art.
[0053] "Heterologous" means derived from an entity genotypically distinct from that of the rest of the entity to which it is being compared. For example, a polynucleotide introduced by genetic engineering techniques into a plasmid or vector derived from a different species is a heterologous polynucleotide. A promoter removed from its native coding sequence and operably linked to a coding sequence with which it is not naturally found linked is a heterologous promoter. Thus, for example, an rAAV that includes a heterologous nucleic acid that encodes a heterologous gene product is an rAAV that includes a nucleic acid not normally included in a naturally occurring wild-type AAV, and the encoded heterologous gene product is a product of gene not normally encoded by a naturally occurring wild-type AAV.
[0054] The terms "genetic alteration" and "genetic modification" (grammatical variants thereof) are used interchangeably here to refer to a process in which a genetic element (e.g., a polynucleotide) is introduced into a cell other than mitosis or meiosis. The element may be heterologous to the cell, or it may be an additional copy or improved version of an element already present in the cell. Genetic alteration can be effected, for example, by transferring a cell with a recombinant plasmid through any process known in the art, such as electroporation, calcium phosphate precipitation, or contact with a polynucleotide-liposome complex. Genetic alteration may also be effected, for example, by transduction or infection with a DNA or RNA virus or viral vector. Generally, the genetic element is introduced on a chromosome or minichromosome in the cell; but any alteration that modifies the phenotype and/or genotype and its progeny is included in this term.
[0055] A cell is said to be "stably" altered, transduced, genetically modified, or transformed with a genetic sequence if the sequence is available to perform its function during extended cell culture in vitro. Generally speaking, a cell is said to be "hereditarily" altered (genetically modified) in which a genetic change is introduced that is still heritably by descent from the altered cell.
[0056] The terms "polypeptide," "peptide" and "protein" are used interchangeably herein to refer to amino acid polymers of any length. The terms also include an amino acid polymer that has been modified, for example, disulfide bond formation, glycosylation, lipidation, phosphorylation, or conjugation to a tag component. Polypeptides such as anti-angiogenic polypeptides, neuroprotective polypeptides, and the like, when discussed in the context of delivering a gene product to a mammalian subject and compositions thereof, refer to the respective intact polypeptide, or any genetically modified fragment or derivative thereof, which retain the biochemical function of the intact protein. Similarly, nucleic acids encoding anti-angiogenic polypeptides, nucleic acids encoding neuroprotective polypeptides, and other so-called nucleic acids for use in delivering a gene product to a mammalian subject (which may be referred to as " transgenes" to be delivered to a recipient cell), include polynucleotides that encode the intact polypeptide or any genetically modified fragment derived therefrom that has the desired biochemical function.
[0057] An "isolated" plasmid, nucleic acid, vector, virus, virion, host cell, or other substance refers to a preparation despite at least some of the other components that may still be present where the substance or a substance similar occurs or is initially prepared from. Thus, for example, an isolated substance can be prepared using a purification technique to enrich it from a source mixture. Enrichment can be measured on an absolute basis, as weight per volume of solution, or it can be measured in relation to the second, potentially interfering present in the source mixture. Enhanced enrichments of the modalities of this disclosure are increasingly isolated. An isolated plasmid, nucleic acid, vector, host cell virus or other substance is in some embodiments purified, e.g. from about 80% to about 90% pure, at least about 90% pure, at least about 95% pure pure, at least about 98% pure, or at least about 99%, or more pure.
[0058] As used herein, the terms "treatment," "to treat," and the like, refer to obtaining a pharmacological and/or physiological effect. The effect may be prophylactic in terms of completely or partially preventing a disease or symptom thereof and/or in terms of partially or completely curing a disease and/or adverse effect attributable to the disease. "Treatment," as used herein, covers any treatment of a disease in a mammal, particularly a human, includes: (a) preventing the disease from occurring in a subject who may be predisposed to the disease or at risk of acquiring the disease, but has not yet been diagnosed as having this; (b) inhibit the disease, that is, stop its development; and (c) alleviating the disease, that is, causing the disease to regress.
[0059] The terms "individual," "host," "subject" and "patient" are used interchangeably herein, and refer to a mammal, including but not limited to humans and non-human primates; mammalian sport animals (eg horses); mammalian farm animals (eg sheep, goats, etc.); mammalian pets (dogs, cats, etc.); and rodents (eg mice, rats, etc.).
[0060] Before the present invention is described in more detail, it should be understood that this invention is not limited to the particular embodiments described, which may, of course, vary accordingly. It is further understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, as the scope of the present invention will be limited only by the appended claims.
[0061] If a range of values is provided, it is understood that each value intermediate, to the tenth of a unit of the lower limit unless the context clearly indicates otherwise, between the upper and lower limit of that range and any other stated value or intermediate in this stated range, is included in the invention. The upper and lower limits of such minor ranges may independently be included in the minor ranges and are also included in the invention, subject to any specifically excluded limit in the indicated range. When the stated range includes one or both of these limits, ranges excluding one or both of those included are also included in the invention.
[0062] Unless otherwise stated, all scientific and technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. While all methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention, preferred methods and materials are now described. All publications mentioned herein are incorporated herein by reference to disclose and describe methods and/or materials in relation to the cited publications.
[0063] It should be noted that as used herein and in the appended claims, the singular forms "a", "an", "a/o" include referent plurals unless the context clearly indicates otherwise. Thus, for example, reference to a "recombinant AAV virion" includes a plurality of said virions and references to "photoreceptor cell" includes reference to one or more photoreceptor cells and equivalents thereof known to those skilled in the art, and so on. . It should be further noted that the claims will be worded to exclude any optional elements. As such, this statement is intended to serve as a background to the use of said proprietary terminology such as "solely," "only" and the like in connection with mention of claimed elements, or use of a "negative" limitation.
[0064] It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may still be provided in combination in a single embodiment. On the other hand, various features of the invention, which are, for the sake of brevity, described in the context of a single embodiment, may also be provided separately or in any appropriate subcombinations. All combinations of embodiments pertaining to the invention are specifically included by the present invention and are disclosed herein just as if each and every combination were individually and explicitly disclosed. Furthermore, all subcombinations of the various embodiments and elements thereof are also specifically included by the present invention and are disclosed herein just as if each subcombination were individually and explicitly disclosed herein.
[0065] The publications discussed here are provided solely for your disclosures prior to the filing date of the present application. Nothing herein should be construed as an admission that the present invention is not entitled to advance such publication by virtue of the prior invention. In addition, the publication dates provided may differ from the actual publication dates which may need to be independently confirmed. DETAILED DESCRIPTION
[0066] The present disclosure provides adeno-associated virus (AAV) virions with altered capsid protein, wherein AAV virions exhibit greater infectivity of a retinal cell when administered via intravitreal injection compared to wild-type AAV. The present disclosure further provides methods for delivering a gene product to a retinal cell in an individual and methods for treating eye disease.
[0067] The retinal cell can be a photoreceptor (eg, rods; cones), a retinal ganglion cell (RGC), a Müller cell (a Müller glial cell), a bipolar cell, an amacryma cell, a horizontal cell, or a retinal pigment epithelium (RPE) cell.CAPSID POLYPEPTIDES, AAV VARIANTS
[0068] The present disclosure provides a variant AAV capsid protein, wherein the variant AAV capsid protein comprises an insertion of about 5 amino acids to about 11 amino acids at an insertion site in the GH loop of the capsid protein or loop IV, relative to the corresponding parental AAV capsid protein, and wherein the variant capsid protein, when present on an AAV virion, confers increased infectivity of a retinal cell compared to the infectivity of the retinal cell by an AAV virion comprising the capsid protein corresponding parental AAV. In some cases, the retinal cell is a photoreceptor cell (eg, rods; cones). In other cases, the retinal cell is an RGC. In other cases, the retina cell is an RPE cell. In other cases, the retinal cell is a Müller cell. Other retinal cells include amacryma cells, bipolar cells, and horizontal cells. An "insertion of about 5 amino acids to about 11 amino acids" is further referred to herein as a "peptide insert" (e.g., a heterologous peptide insert). A "matching parental AAV capsid protein" refers to an AAV capsid protein of the same AAV serotype, without the peptide insert.
[0069] The insertion site is in the GH loop, or IV loop, of the AAV capsid protein, for example, in a solvent accessible portion of the GH loop, or IV loop, of the AAV capsid protein. For the GH loop/IV loop of AAV capsid, see, for example, van Vliet et al. (2006) Mol. The R. 14:809; Padron et al. (2005) J. Virol. 79:5047; and Shen et al. (2007) Mol. The R. 15:1955. For example, the insertion site may be within amino acids 411-650 of an AAV capsid protein, as described in Figures 17A and 17B. For example, the insertion site may be within amino acids 570-611 of AAV2, amino acids 571-612 of AAV1, amino acids 560-601 of AAV5, amino acids 571 to 612 of AAV6, amino acids 572 to 613 of AAV7, at amino acids 573 to 614 of AAV8, amino acids 571 to 612 of AAV9, or amino acids 573 to 614 of AAV10, as depicted in Figure 6.
[0070] In some cases, from about 5 amino acids to about 11 amino acids are inserted into an insertion site in the GH loop or IV loop of the capsid protein relative to the corresponding parental AAV capsid protein. For example, the insertion site may be between amino acids 587 and 588 of AAV2, or the corresponding capsid subunit positions of another AAV serotype. It should be noted that the 587/588 insertion site is based on an AAV2 capsid protein. From about 5 amino acids to about 11 amino acids can be inserted into a corresponding site in an AAV serotype other than AAV2 (eg, AAV8, AAV9, etc.). Those skilled in the art would know, based on a comparison of the amino acid sequences of capsid proteins from various AAV serotypes, where an insertion site "corresponding to amino acids 587-588 of AAV2" could be in a capsid protein of any serotype. AAV provided. The sequences corresponding to amino acids 570-611 of AAV2 VP1 capsid protein (see Figure 5) in various AAV serotypes are shown in Figure 6. See, for example, GenBank Accession No. NP_049542 for AAV1; GenBank Accession No. AAD13756 for AAV5; GenBank Accession No. AAB95459 for AAV6; GenBank Accession No. YP_077178 for AAV7; GenBank Accession No. YP_077180 for AAV8; GenBank Accession No. AAS99264 for AAV9 and GenBank Accession No. AAT46337 for AAV10.
[0071] In some embodiments, the insertion site is a single insertion site between two adjacent amino acids located between amino acids 570-614 of VP1 of any AAV serotype, for example, the insertion site is between two adjacent amino acids located at amino acids 570-614 of VP1 of any AAV serotype. 610, amino acids 580-600, amino acids 570575, amino acids 575-580, amino acids 580-585, amino acids 585590, amino acids 590-600, or amino acids 600-614, of VP1 of any AAV serotype or variant. For example, the insertion site may be between amino acids 580 and 581, amino acids 581 and 582, amino acids 583 and 584, amino acids 584 and 585, amino acids 585 and 586, amino acids 586 and 587, amino acids 587 and 588, amino acids 588 and 589, or amino acids 589 and 590. The insertion site may be between amino acids 575 and 576, amino acids 576 and 577, amino acids 577 and 578, amino acids 578 and 579, or amino acids 579 and 580. The insertion site may be between amino acids 590 and 591, amino acids 591 and 592, amino acids 592 and 593, amino acids 593 and 594, amino acids 594 and 595, amino acids 595 and 596, amino acids 596 and 597, amino acids 597 and 598, amino acids 598 and 599, or amino acids 599 and 600.
[0072] For example, the insertion site can be between amino acids 587 and 588 of AAV2, between amino acids 590 and 591 of AAV1, between amino acids 575 and 576 of AAV5, between amino acids 590 and 591 of AAV6, between amino acids 589 and 590 of AAV7, between amino acids 590 and 591 of AAV8, between amino acids 588 and 589 of AAV9, or between amino acids 588 and 589 of AAV10.
[0073] As another example, the insertion site may be between amino acids 450 and 460 of an AAV capsid protein, as shown in Figure 17A. For example, the insertion site may be at (e.g., immediately N-terminal to) amino acid 453 of AAV2, amino acid 454 of AAV1, amino acid 454 of AAV6, amino acid 456 of AAV7, amino acid 456 of AAV8, AAV9 amino acid 454, or AAV10 amino acid 456, as shown in Figure 17A.
[0074] In some embodiments, an object capsid protein includes a GH loop comprising an amino acid sequence containing at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 98% minus about 99%, or 100%, amino acid sequence identity to an amino acid sequence set forth in Figure 18A-C. insertion peptides
[0075] As noted above, a peptide of about 5 amino acids to about 11 amino acids in length is inserted into the GH loop of an AAV capsid. The insertion peptide is 5 amino acids, 6 amino acids, 7 amino acids, 8 amino acids, 9 amino acids, 10 amino acids, or 11 amino acids in length.
[0076] The insertion peptide may comprise an amino acid sequence of any of the formulas set out below.
[0077] For example, an insertion peptide may be a peptide 5 to 11 amino acids in length, where the insertion peptide is of Formula I:Y1Y2X1X2X3X4X5X6X7Y3Y4
[0078] where:
[0079] each of Y1-Y4, if present, is independently selected from Ala, Leu, Gly, Ser, and Thr;
[0080] X1; if present, is selected from Leu, Asn, and Lys;
[0081] X2 is selected from Gly, Glu, Ala, and Asp;
[0082] X3 is selected from Glu, Thr, Gly, and Pro;
[0083] X4 is selected from Thr, He, Gln, and Lys;
[0084] X5 is selected from Thr and Wing;
[0085] X6 is selected from Arg, Asn, and Thr;
[0086] X7, if present, is selected from Pro and Asn.
[0087] As another example, an insertion peptide may be a peptide 5 to 11 amino acids in length, where the insertion peptide is of Formula IIa:Y1Y2X1X2X3X4X5X6X7Y3Y4
[0088] where:
[0089] each of Y1-Y4, if present, is independently selected from Ala, Leu, Gly, Ser, and Thr; each of X1-X4 is any amino acid;
[0090] X5 is Thr;
[0091] X6 is Arg; and
[0092] X7 is Pro.
[0093] As another example, an insertion peptide may be a peptide 5 to 11 amino acids in length, where the insertion peptide is of Formula IIb:Y1Y2X1X2X3X4X5X6X7Y3Y4
[0094] where:
[0095] each of Y1-Y4, if present, is independently selected from Ala, Leu, Gly, Ser, and Thr;
[0096] X1; if present, is selected from Leu and Asn;
[0097] X2, if present, is selected from Gly and Glu;
[0098] X3 is selected from Glu and Thr;
[0099] X4 is selected from Thr and Ile;
[00100] X5 is Thr;
[00101] X6 is Arg; and
[00102] X7 is Pro.
[00103] As another example, an insertion peptide may be a peptide 5 to 11 amino acids in length, where the insertion peptide is of Formula III:Y1Y2X1X2X3X4X5X6X7Y3Y4
[00104] where:
[00105] each of Y1-Y4, if present, is independently selected from Ala, Leu, Gly, Ser, and Thr;
[00106] X1; if present, it is Lys;
[00107] X2 is selected from Ala and Asp;
[00108] X3 is selected from Gly and Pro;
[00109] X4 is selected from Gln and Lys;
[00110] X5 is selected from Thr and Wing;
[00111] X6 is selected from Asn and Thr;
[00112] X7, if present, is Asn.
[00113] As another example, an insertion peptide may be a peptide 5 to 11 amino acids in length, where the insertion peptide is of Formula IV:Y1Y2X1X2X3X4X5X6X7Y3Y4
[00114] where:
[00115] each of Y1-Y4, if present, is independently selected from Ala, Leu, Gly, Ser, and Thr;
[00116] X1; if present, it is a positively charged amino acid; or is selected from Leu, Asn, Arg, Ala, Ser, and Lys;
[00117] X2 is a negatively charged amino acid or an uncharged amino acid; or is selected from Gly, Glu, Ala, Val, Thr, and Asp;
[00118] X3 is a negatively charged amino acid or an uncharged amino acid; or is selected from Glu, Thr, Gly, Asp, or Pro;
[00119] X4 is selected from Thr, Le, Gly, Lys, Asp, and Gln;
[00120] X5 is a polar amino acid, an alcohol (an amino acid containing a free hydroxyl group), or a hydrophobic amino acid; or is selected from Thr, Ser, Val, and Ala;
[00121] X6 is a positively charged amino acid or an uncharged amino acid; or is selected from Arg, Val, Lys, Pro, Thr, and Asn; and
[00122] X7, if present, is a positively charged or uncharged amino acid; or is selected from Pro, Gly, Phe, Asn, and Arg.
[00123] As non-limiting examples, the insertion peptide may comprise an amino acid sequence selected from LGETTRP (SEQ ID NO:13), NETITRP (SEQ ID NO:14), KAGQANN (SEQ ID NO:15), KDPKTTN (SEQ ID NO:16), KDTDTTR (SEQ ID NO:57), RAGGSVG (SEQ ID NO:58), AVDTTKF (SEQ ID NO:59), and STGKVPN (SEQ ID NO:60).
[00124] In some cases, the insertion peptide has 1 to 4 spacer amino acids (Y1——Y4) at the amino terminus and/or the carboxyl terminus of any of LGETTRP (SEQ ID NO:13), NE - TITRP (SEQ ID NO:14), KAGQANN (SEQ ID NO:15), KDPKTTN (SEQ ID NO:16), KDTDTTR (SEQ ID NO:57), RAGGSVG (SEQ ID NO:58), AVDTTKF (SEQ ID NO:59), and STGKVPN (SEQ ID NO:60).
[00125] Suitable spacer amino acids include, but are not limited to, leucine, alanine, glycine and serine.
[00126] For example, in some cases, an insertion peptide has one of the following amino acid sequences:
[00127] LALGETTRPA (SEQ ID NO:45); LANETITRPA (SEQ ID NO:46), LAKAGQANNA (SEQ ID NO:47), LAKDPKTTNA (SEQ ID NO:48), LAKDTDTTRA (SEQ ID NO:61), LARAGGSVGA (SEQ ID NO:62), LAAVDTTKFA (SEQ ID NO: 63), and LASTGKVPNA (SEQ ID NO:64).
[00128] As another example, in some cases an insertion peptide has one of the following amino acid sequences: AAL-GETTRPA (SEQ ID NO:49); AANETITRPA (SEQ ID NO:50), AA-KAGQANNA (SEQ ID NO:51), and AAKDPKTTNA (SEQ ID NO:52). As yet another example, in some cases, an insertion peptide has one of the following amino acid sequences: GLGETTRPA (SEQ ID NO:53); GNETITRPA (SEQ ID NO:54), GKAGQANNA (SEQ ID NO:55), and GKDPKTTNA (SEQ ID NO:56). As another example, in some cases, an insert peptide comprises one of KDTDTTR (SEQ ID NO:57), RAGGSVG (SEQ ID NO:58), AVDTTKF (SEQ ID NO:59), and STGKVPN (SEQ ID NO:60 ), flanked at the C-terminus in AA and at the N-terminus in A; or comprises one of KDTDTTR (SEQ ID NO:57), RAGGSVG (SEQ ID NO:58), AVDTTKF (SEQ ID NO:59), and STGKVPN (SEQ ID NO:60) flanked at the C-terminus by G and at N - terminal by A.
[00129] In some embodiments, an object AAV variant capsid does not include any other amino acid substitutions, insertions, or deletions, other than the insertion of about 5 amino acids to about 11 amino acids in the GH loop or IV loop relative to a protein of corresponding parental AAV capsid. In other embodiments, an object variant AAV capsid includes from 1 to about 25 amino acid insertions, deletions, or substitutions, compared to the parental AAV capsid protein, plus an insertion of about 5 amino acids to about 11 amino acids in the parent AAV capsid protein. GH loop or IV loop relative to the corresponding parental AAV capsid protein. For example, in some embodiments, an object variant AAV capsid includes from 1 to about 5, from about 5 to about 10, from about 10 to about 15, from about 15 to about 20, or from about 10 to about 10. from 20 to about 25 amino acid insertions, deletions, or substitutions, compared to the parental AAV capsid protein, plus an insertion of about 5 amino acids to about 11 amino acids in the GH loop or IV loop relative to the AAV protein. corresponding parental AAV capsid.
[00130] In some embodiments, an object variant capsid polypeptide does not include one, two, three, or four of the following amino acid substitutions: Y273F, Y444F, Y500F, and Y730F.
[00131] In some embodiments, an object variant capsid polypeptide comprises, in addition to an insertion peptide as described above, one, two, three or four of the following amino acid substitutions: Y273F, Y444F, Y500F and Y730F.
[00132] In some embodiments, an AAV variant capsid polypeptide is a chimeric capsid, for example, the capsid comprises a part of an AAV capsid from a first AAV serotype and a part of an AAV capsid from a second serotype; and comprises an insertion of about 5 amino acids to about 11 amino acids into the GH loop or IV loop relative to the corresponding parental AAV capsid protein.
[00133] In some embodiments, an object variant capsid protein comprises an amino acid sequence containing at least about 85%, at least about 90%, at least about 95%, at least about 98%, or at least about 99% amino acid sequence identity to the amino acid sequence given in Figure 5; and an insertion of about 5 amino acids to about 11 amino acids into the GH loop or IV loop relative to the corresponding parental AAV capsid protein.
[00134] In some embodiments, an object variant capsid protein is isolated, eg purified. In some cases, an object variant capsid protein is included in an AAV vector, which is still provided. As described in detail below, an object variant capsid protein can be included in a recombinant AAV virion. RECOMBINANT AAV VIRION
[00135] The present disclosure provides an adeno-associated virus (rAAV) virion comprising: a) a variant AAV capsid protein, wherein the variant AAV capsid protein comprises an insertion of about 5 amino acids to about 11 amino acids at a site insertion into the GH loop of the capsid protein or loop IV, relative to the corresponding parental AAV capsid protein, and wherein the variant capsid protein confers increased infectivity of a retinal cell compared to the infectivity of the retinal cell by an AAV virion comprising the corresponding parental AAV capsid protein; and b) a heterologous nucleic acid comprising a nucleotide sequence that encodes a gene product. In some cases, the retinal cell is a photoreceptor cell (eg, rods and/or cones). In other cases, the retinal cell is an RGC cell. In other cases, the retina cell is an RPE cell. In other cases, the retinal cell is a Müller cell. In other cases, retinal cells may include amacryma cells, bipolar cells, and horizontal cells. An "insert of about 5 amino acids to about 11 amino acids" is further referred to herein as a "peptide insert" (e.g., a heterologous peptide insert). A "matching parental AAV capsid protein" refers to an AAV capsid protein of the same AAV serotype, without the peptide insert.
[00136] The insertion site is in the GH loop, or IV loop, of the AAV capsid protein, for example, in a solvent accessible portion of the GH loop, or IV loop, of the AAV capsid protein. For the GH loop, see, for example, van Vliet et al. (2006) Mol. The R. 14:809; Padron et al. (2005) J. Virol. 79:5047; and Shen et al. (2007) Mol. The R. 15:1955. For example, the insertion site is at amino acids 570-611 of AAV2, amino acids 571-612 of AAV1, amino acids 560-601 of AAV5, amino acids 571 to 612 of AAV6, amino acids 572 to 613 of AAV7, at amino acids 573 to 614 of AAV8, at amino acids 571 to 612 of AAV9, or at amino acids 573 to 614 of AAV10.
[00137] From about 5 amino acids to about 11 amino acids are inserted into an insertion site in the GH loop or IV loop of the capsid protein relative to the corresponding parental AAV capsid protein. For example, the insertion site may be between amino acids 587 and 588 of AAV2, or the corresponding capsid subunit positions of another AAV serotype. It should be noted that the 587/588 insertion site is based on an AAV2 capsid protein. From about 5 amino acids to about 11 amino acids can be inserted into a corresponding site in an AAV serotype other than AAV2 (eg, AAV8, AAV9, etc.). Those skilled in the art would know, based on a comparison of the amino acid sequences of capsid proteins from various AAV serotypes, where an insertion site "corresponding to amino acids 587-588 of AAV2" could be on a capsid protein of any serotype. AAV provided. The sequences corresponding to amino acids 570-611 of AAV2 VP1 capsid protein (see Figure 5) in various AAV serotypes are shown in Figure 6.
[00138] In some embodiments, the insertion site is a single insertion site between two adjacent amino acids located between amino acids 570-614 of VP1 of any AAV serotype, for example, the insertion site is between two adjacent amino acids located at amino acids 570 -614, amino acids 580-600, amino acids 570575, amino acids 575-580, amino acids 580-585, amino acids 585590, amino acids 590-600, or amino acids 600-610, of VP1 of any AAV serotype or variant. For example, the insertion site may be between amino acids 580 and 581, amino acids 581 and 582, amino acids 583 and 584, amino acids 584 and 585, amino acids 585 and 586, amino acids 586 and 587, amino acids 587 and 588, amino acids 588 and 589, or amino acids 589 and 590. The insertion site may be between amino acids 575 and 576, amino acids 576 and 577, amino acids 577 and 578, amino acids 578 and 579, or amino acids 579 and 580. The insertion site may be between amino acids 590 and 591, amino acids 591 and 592, amino acids 592 and 593, amino acids 593 and 594, amino acids 594 and 595, amino acids 595 and 596, amino acids 596 and 597, amino acids 597 and 598, amino acids 598 and 599, or amino acids 599 and 600.
[00139] For example, the insertion site can be between amino acids 587 and 588 of AAV2, between amino acids 590 and 591 of AAV1, between amino acids 575 and 576 of AAV5, between amino acids 590 and 591 of AAV6, between amino acids 589 and 590 of AAV7, between amino acids 590 and 591 of AAV8, between amino acids 588 and 589 of AAV9, or between amino acids 589 and 590 of AAV10. insertion peptides
[00140] As noted above, an object rAAV virion comprises a peptide of about 5 amino acids to about 11 amino acids in length inserted into the GH loop of the AAV capsid. The insertion peptide has a length of 5 amino acids, 6 amino acids, 7 amino acids, 8 amino acids, 9 amino acids, 10 amino acids, or 11 amino acids.
[00141] The insertion peptide may comprise an amino acid sequence of any of the formulas set out below.
[00142] For example, an insertion peptide may be a peptide 5 to 11 amino acids in length, where the insertion peptide is of Formula I:Y1Y2X1X2X3X4X5X6X7Y3Y4
[00143] where:
[00144] each of Y1-Y4, if present, is independently selected from Ala, Leu, Gly, Ser, and Thr;
[00145] X1; if present, is selected from Leu, Asn, and Lys;
[00146] X2 is selected from Gly, Glu, Ala, and Asp;
[00147] X3 is selected from Glu, Thr, Gly, and Pro;
[00148] X4 is selected from Thr, He, Gln, and Lys;
[00149] X5 is selected from Thr and Wing;
[00150] X6 is selected from Arg, Asn, and Thr;
[00151] X7, if present, is selected from Pro and Asn.
[00152] As another example, an insertion peptide may be a peptide 5 to 11 amino acids in length, where the insertion peptide is of Formula IIa:Y1Y2X1X2X3X4X5X6X7Y3Y4
[00153] where:
[00154] each of Y1-Y4, if present, is independently selected from Ala, Leu, Gly, Ser, and Thr; each of X1-X4 is any amino acid;
[00155] X5 is Thr;
[00156] X6 is Arg; and
[00157] X7 is Pro.
[00158] As another example, an insertion peptide may be a peptide 5 to 11 amino acids in length, where the insertion peptide is of Formula IIb:Y1Y2X1X2X3X4X5X6X7Y3Y4
[00159] where:
[00160] each of Y1-Y4, if present, is independently selected from Ala, Leu, Gly, Ser, and Thr;
[00161] X1, if present, is selected from Leu and Asn;
[00162] X2, if present, is selected from Gly and Glu;
[00163] X3 is selected from Glu and Thr;
[00164] X4 is selected from Thr eLe;
[00165] X5 is Thr;
[00166] X6 is Arg; and
[00167] X7 is Pro.
[00168] As another example, an insertion peptide may be a peptide 5 to 11 amino acids in length, where the insertion peptide is of Formula III:Y1Y2X1X2X3X4X5X6X7Y3Y4
[00169] where:
[00170] each of Y1-Y4, if present, is independently selected from Ala, Leu, Gly, Ser, and Thr;
[00171] X1; if present, it is Lys;
[00172] X2 is selected from Ala and Asp;
[00173] X3 is selected from Gly and Pro;
[00174] X4 is selected from Gln and Lys;
[00175] X5 is selected from Thr and Wing;
[00176] X6 is selected from Asn and Thr;
[00177] X7, if present, is Asn.
[00178] As another example, an insertion peptide may be a peptide 5 to 11 amino acids in length, where the insertion peptide is of Formula IV:Y1Y2X1X2X3X4X5X6X7Y3Y4
[00179] where:
[00180] each of Y1-Y4, if present, is independently selected from Ala, Leu, Gly, Ser, and Thr;
[00181] X1; if present, it is a positively charged or uncharged amino acid; or is selected from Leu, Asn, Arg, Ala, Ser, and Lys;
[00182] X2 is a negatively charged amino acid or an uncharged amino acid; or is selected from Gly, Glu, Ala, Val, Thr, and Asp;
[00183] X3 is a negatively charged amino acid or an uncharged amino acid; or is selected from Glu, Thr, Gly, Asp, or Pro;
[00184] X4 is selected from Thr, Le, Gly, Lys, Asp, and Gln;
[00185] X5 is a polar amino acid, an alcohol (an amino acid containing a free hydroxyl group), or a hydrophobic amino acid; or is selected from Thr, Ser, Val, and Ala;
[00186] X6 is a positively charged amino acid or an uncharged amino acid; or is selected from Arg, Val, Lys, Pro, Thr, and Asn; and
[00187] X7, if present, is a positively charged or uncharged amino acid; or is selected from Pro, Gly, Phe, Asn, and Arg.
[00188] As non-limiting examples, the insertion peptide may comprise an amino acid sequence selected from LGETTRP (SEQ ID NO:13), NETITRP (SEQ ID NO:14), KAGQANN (SEQ ID NO:15), KDPKTTN (SEQ ID NO:16), KDTDTTR (SEQ ID NO:57), RAGGSVG (SEQ ID NO:58), AVDTTKF (SEQ ID NO:59), and STGKVPN (SEQ ID NO:60).
[00189] In some cases, the insertion peptide has 1 to 4 amino-terminal (Y1-Y4) amino-terminal and/or carboxyl-terminal amino acids of any of LGETTRP (SEQ ID NO:13), NETITRP (SEQ ID NO:14), KAGQANN (SEQ ID NO:15), KDPKTTN (SEQ ID NO:16), KDTDTTR (SEQ ID NO:57), RAGGSVG (SEQ ID NO:58), AVDTTKF (SEQ ID NO:59) , and STGKVPN (SEQ ID NO:60).
[00190] Suitable spacer amino acids include, but are not limited to, leucine, alanine, glycine and serine.
[00191] For example, in some cases, an insertion peptide has one of the following amino acid sequences:
[00192] LALGETTRPA (SEQ ID NO:45); LANETITRPA (SEQ ID NO:46), LAKAGQANNA (SEQ ID NO:47), LAKDPKTTNA (SEQ ID NO:48), LAKDTDTTRA (SEQ ID NO:61), LARAGGSVGA (SEQ ID NO:62), LAAVDTTKFA (SEQ ID NO: 63), and LASTGKVPNA (SEQ ID NO:64).
[00193] As another example, in some cases, an insertion peptide has one of the following amino acid sequences: AAL-GETTRPA (SEQ ID NO:49); AANETITRPA (SEQ ID NO:50), AA-KAGQANNA (SEQ ID NO:51), and AAKDPKTTNA (SEQ ID NO:52). As yet another example, in some cases, an insertion peptide has one of the following amino acid sequences: GLGETTRPA (SEQ ID NO:53); GNETITRPA (SEQ ID NO:54), GKAGQANNA (SEQ ID NO:55), and GKDPKTTNA (SEQ ID NO:56). As another example, in some cases, an insert peptide comprises one of KDTDTTR (SEQ ID NO:57), RAGGSVG (SEQ ID NO:58), AVDTTKF (SEQ ID NO:59), and STGKVPN (SEQ ID NO:60 ), flanked at the C-terminus by AA and at the N-terminus by A; or comprises one of KDTDTTR (SEQ ID NO:57), RAGGSVG (SEQ ID NO:58), AVDTTKF (SEQ ID NO:59), and STGKVPN (SEQ ID NO:60) flanked at the C-terminus by G and at N - terminal by A.
[00194] In some embodiments, an object rAAV virion capsid does not include any other amino acid substitutions, insertions, or deletions, other than an insertion of about 7 amino acids to about 10 amino acids into the GH loop or IV loop relative to the protein of corresponding parental AAV capsid. In other embodiments, an object rAAV virion capsid includes from 1 to about 25 amino acid insertions, deletions, or substitutions, compared to the parental AAV capsid protein, plus an insertion of about 7 amino acids at about of 10 amino acids in the GH loop or IV loop relative to the corresponding parental AAV capsid protein. For example, in some embodiments, an object rAAV virion capsid includes from 1 to about 5, from about 5 to about 10, from about 10 to about 15, from about 15 to about 20, or from about 10 to about 20. about 20 to about 25 amino acid insertions, deletions, or substitutions compared to the parental AAV capsid protein, plus an insertion of about 7 amino acids to about 10 amino acids in the GH loop or IV loop relative to the AAV protein. corresponding parental AAV capsid.
[00195] In some embodiments, an object rAAV virion capsid does not include one, two, three, or four of the following amino acid substitutions: Y273F, Y444F, Y500F, and Y730F.
[00196] In some embodiments, an object variant capsid polypeptide comprises, in addition to an insertion peptide as described above, one, two, three, or four of the following amino acid substitutions: Y273F, Y444F, Y500F, and Y730F.
[00197] In some embodiments, an object rAAV virion capsid is a chimeric capsid, for example, the capsid comprises a part of an AAV capsid of a first AAV serotype and a part of an AAV capsid of a second serotype; and comprises an insertion of about 5 amino acids to about 11 amino acids in the GH loop or IV loop relative to the corresponding parental AAV capsid protein.
[00198] In some embodiments, an object rAAV virion comprises a capsid protein comprising an amino acid sequence containing at least about 85%, at least about 90%, at least about 95%, at least about 98%, or at least about 99% amino acid sequence identity to the amino acid sequence given in Figure 5; and an insertion of about 5 amino acids to about 11 amino acids into the GH loop or IV loop relative to the corresponding parental AAV capsid protein.
[00199] In some embodiments, an object rAAV virion comprises a capsid protein that includes a GH loop comprising an amino acid sequence containing at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% amino acid sequence identity to an amino acid sequence set forth in Figure 18A-C.
[00200] An object rAAV virion exhibits at least 10 times, at least 15 times, at least 20 times, at least 25 times, at least 50 times, or more than 50 times, increased infectivity of a retinal cell compared to infectivity of retinal cell by an AAV virion comprising the corresponding parental AAV capsid protein.
[00201] In some cases, an object rAAV virion exhibits at least 10 times, at least 15 times, at least 20 times, at least 25 times, at least 50 times, or more than 50 times, increased infectivity of a cell of retina, when administered via intravitreal injection, compared to retinal cell infectivity by an AAV virion comprising the corresponding parental AAV capsid protein, when administered via intravitreal injection.
[00202] In some embodiments, an object rAAV virion exhibits at least 10 times, at least 15 times, at least 20 times, at least 25 times, at least 50 times, or more than 50 times, increased infectivity of a photoreceptor cell (rod or cone), compared to the infectivity of the photoreceptor cell by an AAV virion comprising the corresponding parental AAV capsid protein.
[00203] In some embodiments, an object rAAV virion exhibits at least 10 times, at least 15 times, at least 20 times, at least 25 times, at least 50 times, or more than 50 times, increased infectivity of a photoreceptor cell (rod or cone), when administered via intravitreal injection, compared to the infectivity of the photoreceptor cell by an AAV virion comprising the corresponding parental AAV capsid protein, when administered via intravitreal injection.
[00204] In some embodiments, an object rAAV virion exhibits at least 10 times, at least 15 times, at least 20 times, at least 25 times, at least 50 times, or more than 50 times, increased infectivity of a RGC, compared to the infectivity of RGC by an AAV virion comprising the corresponding parental AAV capsid protein.
[00205] In some embodiments, an object rAAV virion exhibits at least 10 times, at least 15 times, at least 20 times, at least 25 times, at least 50 times, or more than 50 times, increased infectivity of a RGC, when administered via intravitreal injection, compared to the infectivity of RGC by an AAV virion comprising the corresponding parental AAV capsid protein, when administered via intravitreal injection.
[00206] In some embodiments, an object rAAV virion exhibits at least 10 times, at least 15 times, at least 20 times, at least 25 times, at least 50 times, or more than 50 times, increased infectivity of a RPE cell, compared to the infectivity of the RPE cell by an AAV virion comprising the corresponding parental AAV capsid protein.
[00207] In some embodiments, an object rAAV virion exhibits at least 10 times, at least 15 times, at least 20 times, at least 25 times, at least 50 times, or more than 50 times, increased infectivity of a RPE cell, when administered via intravitreal injection, compared to the infectivity of the RPE cell by an AAV virion comprising the corresponding parental AAV capsid protein, when administered via intravitreal injection.
[00208] In some embodiments, an object rAAV virion exhibits at least 10 times, at least 15 times, at least 20 times, at least 25 times, at least 50 times, or more than 50 times, increased infectivity of a cell Müller, compared to Müller cell infectivity by an AAV virion comprising the corresponding parental AAV capsid protein.
[00209] In some embodiments, an object rAAV virion exhibits at least 10 times, at least 15 times, at least 20 times, at least 25 times, at least 50 times, or more than 50 times, increased infectivity of a cell Müller, when administered via intravitreal injection, compared to Müller cell infectivity by an AAV virion comprising the corresponding parental AAV capsid protein, when administered via intravitreal injection.
[00210] In some embodiments, an object rAAV virion exhibits at least 10 times, at least 15 times, at least 20 times, at least 25 times, at least 50 times, or more than 50 times, increased infectivity of a cell bipolar, compared to bipolar cell infectivity by an AAV virion comprising the corresponding parental AAV capsid protein.
[00211] In some embodiments, an object rAAV virion exhibits at least 10 times, at least 15 times, at least 20 times, at least 25 times, at least 50 times, or more than 50 times, increased infectivity of a cell bipolar, when administered via intravitreal injection, compared to bipolar cell infectivity by an AAV virion comprising the corresponding parental AAV capsid protein, when administered via intravitreal injection.
[00212] In some embodiments, an object rAAV virion exhibits at least 10 times, at least 15 times, at least 20 times, at least 25 times, at least 50 times, or more than 50 times, increased infectivity of a cell amacryma, compared to the infectivity of the amacryma cell by an AAV virion comprising the corresponding parental AAV capsid protein.
[00213] In some embodiments, an object rAAV virion exhibits at least 10 times, at least 15 times, at least 20 times, at least 25 times, at least 50 times, or more than 50 times, increased infectivity of a cell amacryma, when administered via intravitreal injection, compared to the infectivity of the amacryma cell by an AAV virion comprising the corresponding parental AAV capsid protein, when administered via intravitreal injection.
[00214] In some embodiments, an object rAAV virion exhibits at least 10 times, at least 15 times, at least 20 times, at least 25 times, at least 50 times, or more than 50 times, increased infectivity of a cell horizontal, compared to the infectivity of the horizontal cell by an AAV virion comprising the corresponding parental AAV capsid protein.
[00215] In some embodiments, an object rAAV virion exhibits at least 10 times, at least 15 times, at least 20 times, at least 25 times, at least 50 times, or more than 50 times, increased infectivity of a cell horizontal cell when administered via intravitreal injection compared to horizontal cell infectivity by an AAV virion comprising the corresponding parental AAV capsid protein when administered via intravitreal injection.
[00216] In some cases, an object rAAV virion exhibits at least 10 times, at least 15 times, at least 20 times, at least 25 times, at least 50 times, or more than 50 times, increased ability to cross the membrane limiting factor (ILM), compared to the ability of an AAV virion comprising the corresponding parental AAV capsid protein to cross the ILM.
[00217] In some cases, an object rAAV virion exhibits at least 10 times, at least 15 times, at least 20 times, at least 25 times, at least 50 times, or more than 50 times, increased capacity when administered via intravitreal injection, to cross the inner limiting membrane (ILM), compared to the ability of an AAV virion comprising the corresponding parental AAV capsid protein to cross the ILM when administered via intravitreal injection.
[00218] An object rAAV virion can traverse the ILM, and can even traverse cell layers, including Müller cells, amacrym cells, etc., in reaching photoreceptor cells and or RPE cells. For example, an object rAAV virion, when administered via intravitreal injection, may cross the ILM, and may even pass through cell layers, including Müller cells, amacrym cells, etc., to reach photoreceptor cells and/or RPE cells.
[00219] In some embodiments, an object rAAV virion selectively infects a retinal cell, for example, an object rAAV virion infects a retinal cell 10 times, 15 times, 20 times, 25 times, 50 times, or more than 50 times more specificity than a non-retina cell, for example, a cell outside the eye. For example, in some embodiments, an object rAAV virion selectively infects a retinal cell, for example, an object rAAV virion infects a photoreceptor cell 10 times, 15 times, 20 times, 25 times, 50 times, or more than 50 times. times more specific than a non-retina cell, for example, a cell outside the eye.
[00220] In some embodiments, an object rAAV virion selectively infects a photoreceptor cell, for example, an object rAAV virion infects a photoreceptor cell 10 times, 15 times, 20 times, 25 times, 50 times, or more than 50 times , specificity than a non-photoreceptor cell present in the eye, e.g. a retinal ganglion cell, a Müller cell, etc.
[00221] In some embodiments, an object rAAV virion exhibits at least 10 times, at least 15 times, at least 20 times, at least 25 times, at least 50 times, or more than 50 times, increased infectivity of a cell photoreceptor cell when administered via intravitreal injection compared to the infectivity of the photoreceptor cell by an AAV virion comprising the corresponding parental AAV capsid protein when administered via intravitreal injection. gene products
[00222] An object rAAV virion comprises a heterologous nucleic acid comprising a nucleotide sequence encoding a gene product. In some embodiments, the gene product is an interfering RNA. In some embodiments, the gene product is an aptamer. In some embodiments, the gene product is a polypeptide. In some embodiments, the gene product is a site-specific nuclease that provides site-specific knock-down of gene function.Interfering RNA
[00223] Where the gene product is an interfering RNA (RNAi), appropriate RNAi includes RNAi that reduces the level of an apoptotic or angiogenic factor in a cell. For example, an RNAi can be a shRNA or siRNA that reduces the level of a gene product that induces or promotes apoptosis in a cell. Genes whose gene products induce or promote apoptosis are referred to herein as "pro-apoptotic genes" and the products of these genes (mRNA; protein) are referred to as "pro-apoptotic gene products." Pro-apoptotic gene products include, for example, Bax, Bid, Bak, and Bad gene products. See, for example, US patent 7,846,730.
[00224] Interfering RNAs could still be against an angiogenic product, e.g. VEGF (e.g. Cand5; see e.g. US Patent Publication No. 2011/0143400; US Patent Publication 2008/0188437; and Reich et al. ( 2003) Mol. Vis. 9:210), VEGFR1 (e.g., Sirna-027; see, e.g., Kaiser et al. (2010) Am. J. Ophthalmol. 150:33; and Shen et al. (2006) Gene Ther. 13:225), or VEGFR2 ( Kou et al. (2005) Biochem. 44: 15064 ). See further, US Patents 6,649,596, 6,399,586, 5,661,135, 5,639,872, and 5,639,736; and US patents 7,947,659 and 7,919,473. aptamers
[00225] Where the gene product is an aptamer, exemplary aptamers of interest include an aptamer against vascular endothelial growth factor (VEGF). See, for example, Ng et al. (2006) Nat. Rev. Drug Discovery 5: 123; and Lee et al. (2005) Proc. natl. academy Sci. USA 102:18902. For example, a VEGF aptamer may comprise the nucleotide sequence 5'-cgcaaucagugaaugcuuauacauccg-3' (SEQ ID NO: 17). Still suitable for use is a PDGF-specific aptamer, eg E10030; see, for example, Ni and Hui (2009) Ophthalmologica 223:401; and Akiyama et al. (2006) J. Cell Physiol. 207:407). polypeptides
[00226] Where the gene product is a polypeptide, the polypeptide is generally a polypeptide that enhances the function of a retinal cell, for example, the function of a rod or cone photoreceptor cell, a retinal ganglion cell, a retinal Müller, a bipolar cell, an amacryma cell, a horizontal cell, or a pigmented epithelial cell. Exemplary polypeptides include neuroprotective polypeptides (e.g., GDNF, CNTF, NT4, NGF, and NTN); anti-angiogenic polypeptides (eg, a soluble vascular endothelial growth factor (VEGF) receptor); a VEGF-binding antibody; a VEGF-binding fragment (eg, a single-chain anti-VEGF antibody); endostatin; tumstatin; angiostatin, a soluble Flt polypeptide (Lai et al. (2005) Mol. Ther. 12:659), an Fc fusion protein comprising a soluble Flt polypeptide (see, for example, Pechan et al. (2009) Gene Ther. 16 : 10), pigment epithelium-derived factor (PEDF), a soluble Tie-2 receptor, etc.); tissue inhibitor of metalloproteinases-3 (TIMP-3); a light-responsive opsin, eg, a rhodopsin; anti-apoptotic polypeptides (e.g., Bcl-2, Bcl-Xl); and the like. Suitable polypeptides include, but are not limited to, glial-derived neurotrophic factor (GDNF); fibroblast growth factor 2; neurturin (NTN); ciliary neurotrophic factor (CNTF); nerve growth factor (NGF); neurotrophin-4 (NT4); brain-derived neurotrophic factor (BDNF; for example, a polypeptide comprising an amino acid sequence containing at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100%, amino acid sequence identity to a contiguous stretch of about 200 amino acids to 247 amino acids of the amino acid sequence depicted in Figure 11 (SEQ ID NO: 11)); epidermal growth factor; rhodopsin; X-linked inhibitor of apoptosis; and Sonic hedgehog.
[00227] Suitable light responsive opsins include, for example, a light responsive opsin as described in US Patent Publication 2007/0261127 (e.g. ChR2; Chop2); US Patent Publication 2001/0086421; US Patent Publication 2010/0015095 ; and Diester et al. (2011) Nat. Neuroscience 14:387.
[00228] Suitable polypeptides include retinoschisin (for example, a polypeptide comprising an amino acid sequence containing at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100%, amino acid sequence identity to a contiguous stretch of about 200 amino acids to 224 amino acids of the amino acid sequence depicted in Figure 10 (SEQ ID NO: 10) Suitable polypeptides include, for example, protein 1 interacting with Retinitis pigmentosa GTPase (RGPR) (see, for example, GenBank Accession Nos. Q96KN7, Q9EPQ2, and Q9GLM3) (for example, a polypeptide comprising an amino acid sequence containing at least about 90%, at least about 95% , at least about 98%, at least about 99%, or 100% amino acid sequence identity to a contiguous stretch of about 1150 amino acids to about 1200 amino acids, or from about 1200 amino acids to 1286 amino acids - acids, of the amino acid sequence described as in Figure 16 (SEQ ID NO:21); peripherin-2 (Prph2) (see, for example, GenBank Accession No. NP_000313 (for example, a polypeptide comprising an amino acid sequence containing at least about 90%, at least about 95%, at least about 98 %, at least about 99%, or 100% amino acid sequence identity to a contiguous stretch of about 300 amino acids to 346 amino acids of the amino acid sequence depicted in Figure 14 (SEQ ID NO: 19); and Travis et al (1991) Genomics 10:733); peripherin (e.g., a polypeptide comprising an amino acid sequence containing at least about 90%, at least about 95%, at least about 98%, at least about 99% , or 100%, amino acid sequence identity to a contiguous stretch of about 400 amino acids to about 470 amino acids of the amino acid sequence depicted in Figure 15 (SEQ ID NO:20); a pigment epithelium-specific protein retina (RPE65), (e.g., a polypeptide comprising an amino acid sequence containing at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% amino acid sequence identity to a contiguous stretch of about 200 amino acids to 247 amino acids from the amino acid sequence depicted in Figure 12 (SEQ ID NO: 12)) (see, for example, GenBank AAC39660; and Morimura et al. (1998) Proc. natl. academy Sci. USA 95:3088); and the like.
[00229] Appropriate polypeptides further include: CHM (choroidermia (Rab escort 1 protein)), a polypeptide which, when defective or missing, causes choroideremia (see, for example, Donnelly et al. (1994) Hum. Mol. Genet 3:1017 and van Bokhoven et al (1994) Hum.Mol.Genet.3:1041); and Crumbs homolog 1 (CRB1), a polypeptide that, when defective or missing, causes Leber congenital amaurosis and retinitis pigmentosa (see, for example, den Hollander et al. (1999) Nat. Genet. 23:217; and GenBank Accession No. CAM23328).
[00230] Appropriate polypeptides further include polypeptides which, when defective or missing, lead to acromotopsia, where said polypeptides include, for example, cGMP-triggered channel alpha subunit of cone photoreceptor (CNGA3) (see, for example, GenBank N Accession No. NP_001289, and Booij et al (2011) Ophthalmology 118: 160-167 ); Cone photoreceptor cGMP-triggered cation channel beta subunit (CNGB3) (see, for example, Kohl et al. (2005) Eur J Hum Genet. 13(3):302 ); guanine nucleotide binding protein (G protein), alpha transducer activity polypeptide 2 (GNAT2) (ACHM4); and ACHM5; and polypeptides that, when defective or missing, lead to various forms of color blindness (eg, L-opsin, M-opsin, and S-opsin). See Mancuso et al. (2009) Nature 461(7265):784-787.Site-Specific Endonucleases
[00231] In some cases, a gene product of interest is a site-specific endonuclease that provides gene function-specific knock-down, for example, where the endonuclease knocks out an allele associated with a retinal disease. For example, where a dominant allele encodes a defective copy of a gene that, when wild-type, is a retinal framework protein and/or provides normal retinal function, a site-specific endonuclease can be targeted to the defective allele and knockout of the defective allele.
[00232] In addition to knocking out a defective allele, a site-specific nuclease can also be used to stimulate homologous recombination with a donor DNA that encodes a functional copy of the protein encoded by the defective allele. So, for example, an object rAAV virion can be used to release both a site-specific endonuclease that knocks out a defective allele, and it can be used to release a functional copy of the defective allele, resulting in repair of the defective allele, thus, providing for the production of a functional retinal protein (e.g., functional retinoschisin, functional RPE65, functional peripherin, etc.). See, for example, Li et al. (2011) Nature 475:217. In some embodiments, an object rAAV virion comprises a heterologous nucleotide sequence that encodes a site-specific endonuclease; and a heterologous nucleotide sequence that encodes a functional copy of a defective allele, where the functional copy encodes the functional retinal protein. Functional retinal proteins include, for example, retinoschisin, RPE65, retinitis pigmentosa regulatory GTPase interaction protein 1 (RGPR), peripherin, peripherin-2, and the like.
[00233] Site-specific endonucleases that are suitable for use include, for example, zinc finger nucleases (ZFNs); and transcriptional activator-type effector nucleases (TALENs), where said site-specific endonucleases are non-naturally occurring and are modified to target a specific gene. Such site-specific nucleases can be designed to cut off sites within a genome, and non-homologous splicing ends then repair the break while inserting or deleting various nucleotides. Said site-specific endonucleases (also referred to as "INDELs") then throw the protein out and effectively knock out the gene. See, for example, US Patent Publication 2011/0301073. regulatory sequences
[00234] In some embodiments, a nucleotide sequence encoding a gene product of interest is operably linked to a constitutive promoter. In other embodiments, a nucleotide sequence encoding a gene product of interest is operably linked to an inducible promoter. In some cases, a nucleotide sequence encoding a gene product of interest is operably linked to a tissue-specific, or cell-type-specific, regulatory element. For example, in some cases, a nucleotide sequence encoding a gene product of interest is operably linked to a photoreceptor-specific regulatory element (e.g., a photoreceptor-specific promoter), e.g., a regulatory element that confers ex- selective pressure of the operably linked gene in a photoreceptor cell. Photoreceptor-specific regulatory elements include, for example, a rhodopsin promoter; a rhodopsin kinase promoter ( Young et al. (2003) Ophthalmol. Vis. Sci. 44:4076 ); a beta phosphodiesterase promoter ( Nicoud et al. (2007) J. Gene Med. 9: 1015 ); a retinitis pigmentosa gene promoter (Nicoud et al. (2007) supra); a retinoid interphotoreceptor binding protein (IRBP) gene enhancer (Nicoud et al. (2007) supra); an IRBP gene promoter ( Yokoyama et al. (1992) Exp Eye Res. 55:225 ). PHARMACEUTICAL COMPOSITIONS
[00235] The present disclosure provides a pharmaceutical composition comprising: a) an object rAAV virion, as described above; and b) a pharmaceutically acceptable carrier, diluent, or buffer. In some embodiments, the pharmaceutically acceptable carrier, diluent, excipient, or buffer is suitable for use in a human.
[00236] Said excipients, carriers, diluents and buffers include any pharmaceutical agent that can be administered without undue toxicity. Pharmaceutically acceptable excipients include, but are not limited to, liquids such as water, saline, glycerol and ethanol. Pharmaceutically acceptable salts may be included herein, for example, mineral acid salts such as hydrochlorides, hydrobromides, phosphates, sulfates, and the like; and salts of organic acids such as acetates, propionates, malonates, benzoates, and the like. In addition, auxiliary substances, such as wetting or emulsifying agents, pH buffering substances, and the like, may be present in said vehicles. A wide variety of pharmaceutically acceptable excipients are known in the art and need not be discussed in detail here. Pharmaceutically acceptable excipients have been extensively described in a variety of publications, including, for example, A. Gennaro (2000) "Remington: The Science and Practice of Pharmacy," 20th edition, Lippincott, Williams, &Wilkins; Pharmaceutical Dosage Forms and Drug Delivery Systems (1999) H.C. Ansel et al., eds., 7th ed., Lippincott, Williams, &Wilkins; and Handbook of Pharmaceutical Excipients (2000) A.H. Kibbe et al., eds., 3rd ed. Amer. Pharmaceutical Assoc. METHODS FOR DELIVERY OF A GENE PRODUCT TO A RETINA CELL AND METHODS OF TREATMENT
[00237] The present disclosure provides a method for delivering a gene product to a retinal cell in a subject, the method comprising administering to the subject an object rAAV virion as described above. The gene product can be a polypeptide or an interfering RNA (e.g., a shRNA, a siRNA, and the like), an aptamer, or a site-specific endonuclease, as described above. Delivering a gene product to a retinal cell may provide treatment for a retinal disease. The retinal cell can be a photoreceptor, a retinal ganglion cell, a Müller cell, a bipolar cell, an amacryma cell, a horizontal cell, or a retinal pigmented epithelial cell. In some cases, the retinal cell is a photoreceptor cell, for example, a rod or cone cell.
[00238] The present disclosure provides a method of treating a disease of the retina, the method comprising administering to a subject in need thereof an effective amount of an object rAAV virion as described above. An object rAAV virion may be administered via intraocular injection, by intravitreal injection, or by any other convenient mode or route of administration. Other convenient modes or routes of administration include, for example, intravenous, intranasal, etc.
[00239] A "therapeutically effective amount" will be in a relatively wide range that can be determined through experimentation and/or clinical studies. For example, for in vivo injection, i.e. injection directly into the eye, a therapeutically effective dose will be on the order of about 106 to about 1015 rAAV virions, for example from about 108 to 1012 rAAV virions. For in vitro transduction, an effective amount of rAAV virions to be delivered to cells will be on the order of about 108 to about 1013 rAAV virions. Other effective dosages can be readily established by one of skill in the art through routine studies that establish dose-response curves.
[00240] In some embodiments, more than one administration (e.g. two, three, four or more administrations) may be employed to obtain the desired level of gene expression over a period of various intervals, e.g. daily, weekly , monthly, yearly, etc.
[00241] Eye diseases that can be treated using an object method include, but are not limited to, acute macular neuroretinopathy; Behcet's disease; choroidal neovascularization; diabetic uveitis; histoplasmosis; macular degeneration, such as macular degeneration, non-exudative age-related macular degeneration, and exudative age-related macular degeneration; edema, such as macular edema, cystoid macular edema, and diabetic macular edema; multifocal choroiditis; ocular trauma affecting a posterior ocular site or site; eye tumors; retinal disorders such as central retinal vein occlusion, diabetic retinopathy (including diabetic proliferative retinopathy), proliferative vitreoretinopathy (PVR), retinal arterial occlusive disease, retinal detachment, uveitic retinal disease; sympathetic ophthalmia; Vogt Koyanagi-Harada syndrome (VKH); uveal diffusion; a posterior eye condition caused by or influenced by laser eye treatment; posterior eye conditions caused by or influenced by photodynamic therapy; photocoagulation, radiation retinopathy; epiretinal membrane disorders; retinal branch vein occlusion; anterior ischemic optic neuropathy; diabetic retina dysfunction not retinopathy; retinoschisis; retinitis pigmentosa; glaucoma; Usher syndrome, rod-cone dystrophy; Stargardt's disease (background flavimaculate); inherited macular degeneration; chorioretinal degeneration; Leber congenital amaurosis; congenital stationary night blindness; choroideremia; Bardet-Biedl syndrome; macular telan-giectasia; Leber's hereditary optic neuropathy; retinopathy of prematurity; and color vision disorders, including achromatopsia, protanopia, deuteranopia, and tritanopia.NUCLEIC ACIDS AND HOST CELLS
[00242] The present disclosure provides an isolated nucleic acid comprising a nucleotide sequence encoding an object variant adeno-associated virus (AAV) capsid protein as described above, wherein the variant AAV capsid protein comprises an insert of about 5 amino acids to about 11 amino acids in the GH loop or loop IV relative to the corresponding parental AAV capsid protein, and where the variant capsid protein, when present on an AAV virion, provides increased infectivity of a retinal cell compared to retinal cell infectivity by an AAV virion comprising the corresponding parental AAV capsid protein. An isolated object nucleic acid may be an AAV vector, for example a recombinant AAV vector.Insertion peptides
[00243] A variant AAV capsid protein encoded by an object nucleic acid has an insertion peptide of about 5 amino acids to about 11 amino acids in length is inserted into the GH loop of an AAV capsid. The insertion peptide is 5 amino acids, 6 amino acids, 7 amino acids, 8 amino acids, 9 amino acids, 10 amino acids, or 11 amino acids in length.
[00244] The insertion peptide may comprise an amino acid sequence of any of the formulas set out below.
[00245] For example, an insertion peptide may be a peptide of 5 to 11 amino acids in length, where the insertion peptide is of Formula I:Y1Y2X1X2X3X4X5X6X7Y3Y4
[00246] where:
[00247] each of Y1-Y4, if present, is independently selected from Ala, Leu, Gly, Ser, and Thr;
[00248] X1; if present, is selected from Leu, Asn, and Lys;
[00249] X2 is selected from Gly, Glu, Ala, and Asp;
[00250] X3 is selected from Glu, Thr, Gly, and Pro;
[00251] X4 is selected from Thr, He, Gln, and Lys;
[00252] X5 is selected from Thr and Wing;
[00253] X6 is selected from Arg, Asn, and Thr;
[00254] X7, if present, is selected from Pro and Asn.
[00255] As another example, an insertion peptide may be a peptide 5 to 11 amino acids in length, where the insertion peptide is of Formula IIa:Y1Y2X1X2X3X4X5X6X7Y3Y4
[00256] where:
[00257] each of Y1 -Y4, if present, is independently selected from Ala, Leu, Gly, Ser, and Thr; each of X1 - X4 is any amino acid;
[00258] X5 is Thr;
[00259] X6 is Arg; and
[00260] X7 is Pro.
[00261] As another example, an insertion peptide may be a peptide 5 to 11 amino acids in length, where the insertion peptide is of Formula IIb:Y1Y2X1X2X3X4X5X6X7Y3Y4
[00262] where:
[00263] each of Y1 -Y4, if present, is independently selected from Ala, Leu, Gly, Ser, and Thr; X1; if present, is selected from Leu and Asn;
[00264] X2, if present, is selected from Gly and Glu;
[00265] X3 is selected from Glu and Thr;
[00266] X4 is selected from Thr and Le;
[00267] X5 is Thr;
[00268] X6 is Arg; and
[00269] X7 is Pro.
[00270] As another example, an insertion peptide may be a peptide 5 to 11 amino acids in length, where the insertion peptide is of Formula III:Y1Y2X1X2X3X4X5X6X7Y3Y4
[00271] where:
[00272] each of Y1-Y4, if present, is independently selected from Ala, Leu, Gly, Ser, and Thr;
[00273] X1, if present, is Lys;
[00274] X2 is selected from Ala and Asp;
[00275] X3 is selected from Gly and Pro;
[00276] X4 is selected from Gln and Lys;
[00277] X5 is selected from Thr and Wing;
[00278] X6 is selected from Asn and Thr;
[00279] X7, if present, is Asn.
[00280] As another example, an insertion peptide may be a peptide 5 to 11 amino acids in length, where the insertion peptide is of Formula IV:Y1Y2X1X2X3X4X5X6X7Y3Y4
[00281] where:
[00282] each of Yi -Y4, if present, is independently selected from Ala, Leu, Gly, Ser, and Thr;
[00283] X1 ; if present, it is a positively charged or uncharged amino acid; or is selected from Leu, Asn, Arg, Ala, Ser, and Lys;
[00284] X2 is a negatively charged amino acid or an uncharged amino acid; or is selected from Gly, Glu, Ala, Val, Thr, and Asp;
[00285] X3 is a negatively charged amino acid or an uncharged amino acid; or is selected from Glu, Thr, Gly, Asp, or Pro;
[00286] X4 is selected from Thr, Le, Gly, Lys, Asp, and Gln;
[00287] X5 is a polar amino acid, an alcohol (an amino acid containing a free hydroxyl group), or a hydrophobic amino acid; or is selected from Thr, Ser, Val, and Ala;
[00288] X6 is a positively charged amino acid or an uncharged amino acid; or is selected from Arg, Val, Lys, Pro, Thr, and Asn; and
[00289] X7, if present, is a positively charged or uncharged amino acid; or is selected from Pro, Gly, Phe, Asn, and Arg.
[00290] As non-limiting examples, the insertion peptide may comprise an amino acid sequence selected from LGETTRP (SEQ ID NO:13), NETITRP (SEQ ID NO:14), KAGQANN (SEQ ID NO:15), KDPKTTN (SEQ ID NO:16), KDTDTTR (SEQ ID NO:57), RAGGSVG (SEQ ID NO:58), AVDTTKF (SEQ ID NO:59), and STGKVPN (SEQ ID NO:60).
[00291] In some cases, the insertion peptide has 1 to 4 amino-terminal (Yi-Y4) amino-terminal and/or carboxyl-terminal amino acids of any of LGETTRP (SEQ ID NO:13), NETITRP (SEQ ID NO:14), KAGQANN (SEQ ID NO:15), KDPKTTN (SEQ ID NO:16), KDTDTTR (SEQ ID NO:57), RAGGSVG (SEQ ID NO:58), AVDTTKF (SEQ ID NO:59) , and STGKVPN (SEQ ID NO:60).
[00292] Suitable spacer amino acids include, among others, leucine, alanine, glycine, and serine.
[00293] For example, in some cases, an insertion peptide has one of the following amino acid sequences: LALGETTRPA (SEQ ID NO:45); LANETITRPA (SEQ ID NO:46), LAKAGQANNA(SEQ ID NO:47), LAKDPKTTNA (SEQ ID NO:48), LAKDTDTTRA(SEQ ID NO:61), LARAGGSVGA (SEQ ID NO:62), LAAVDTTKFA(SEQ ID NO: :63), and LASTGKVPNA (SEQ ID NO:64). As another example, in some cases an insertion peptide has one of the following amino acid sequences: AALGETTRPA (SEQ ID NO:49); AANETITRPA (SEQ ID NO:50), AAKAGQANNA (SEQ ID NO:51), and AAKDPKTTNA (SEQ ID NO:52). As yet another example, in some cases, an insertion peptide has one of the following amino acid sequences: GLGETTRPA (SEQ ID NO:53); GNETITRPA (SEQ ID NO:54), GKAGQANNA (SEQ ID NO:55), and GKDPKTTNA (SEQ ID NO:56). As another example, in some cases, an insert peptide comprises one of KDTDTTR (SEQ ID NO:57), RAGGSVG (SEQ ID NO:58), AVDTTKF (SEQ ID NO:59), and STGKVPN (SEQ ID NO:60 ), flanked at the C-terminus by AA and at the N-terminus by A; or comprises one of KDTDTTR (SEQ ID NO:57), RAGGSVG (SEQ ID NO:58), AVDTTKF (SEQ ID NO:59), and STGKVPN (SEQ ID NO:60) flanked at C-terminus by G and at N -terminal by A.
[00294] An object recombinant AAV vector can be used to generate an object recombinant AAV virion as described above. Thus, the present disclosure provides a recombinant AAV vector which, when introduced into an appropriate cell, can provide for production of a recombinant AAV virion.
[00295] The present invention further provides host cells, for example, isolated (genetically modified) host cells, comprising an object nucleic acid. An object host cell may be an isolated cell, for example a cell in in vitro culture. A host cell object is useful for producing an object rAAV virion, as described below. Where an object host cell produces an object rAAV virion, this is referred to as a "packaging cell." In some embodiments, an object host cell is stably genetically modified as an object nucleic acid. In other embodiments, an object host cell is transiently genetically modified with an object nucleic acid.
[00296] An object nucleic acid is introduced stably or transiently into a host cell using established techniques, among others, electroporation, calcium phosphate precipitation, liposome-mediated transfection, and the like. For stable transformation, an object nucleic acid will generally still include a selectable marker, for example, any of the well-known selectable markers such as neomycin resistance, and the like.
[00297] An object host cell is generated by introducing an object nucleic acid into any of a variety of cells, e.g. mammalian cells, including, e.g., murine cells, and primate cells (e.g., human cells ). Suitable mammalian cells include, but are not limited to, primary cells and cell lines, where appropriate cell lines include, but are not limited to, 293 cells, COS cells, HeLa cells, Vero cells, 3T3 mouse fibroblasts, C3H10T1/2 fibroblasts, CHO, and the like. Non-limiting examples of suitable host cells include, for example, HeLa cells (e.g., American Type Culture Collection (ATCC) No. CCL-2), CHO cells (e.g., ATCC Nos. CRL9618, CCL61, CRL9096 ), 293 cells (e.g. ATCC No. CRL-1573), Vero cells, NIH 3T3 cells (e.g. ATCC No. CRL-1658), Huh-7 cells, BHK cells (e.g. ATCC No. CCL10), PC12 cells (ATCC No. CRL1721), COS cells, COS-7 cells (ATCC No. CRL1651), RATI cells, mouse L cells (ATCC No. CCLI.3), human embryonic kidney cells (HEK) (ATCC No. CRL1573), HLHepG2 cells, and the like. An object host cell can further be made using a baculovirus to infect insect cells such as Sf9 cells, which produce AAV (see, for example, US patent 7,271,002; US patent application 12/297,958)
[00298] In some embodiments, a genetically modified host cell subject includes, in addition to a nucleic acid comprising a nucleotide sequence that encodes a variant AAV capsid protein, as described above, a nucleic acid that comprises a nucleotide sequence that encodes one or more AAV rep proteins. In other embodiments, an object host cell further comprises an rAAV vector. An rAAV virion can be generated using an object host cell. Methods for generating an rAAV virion are described in, for example, US Patent Publication 2005/0053922 and US Patent Publication 2009/0202490. EXAMPLES
[00299] The following examples are presented in order to provide those skilled in the art with a complete disclosure and description of how to prepare and use the present described invention, and are not intended to limit the scope of what the inventors consider their invention to be, nor are they intended to represent that the experiments below are all or only the experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (eg amounts, temperature, etc.), but some experimental errors and deviations must be accounted for. Unless otherwise indicated, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Celsius, and pressure is at or near atmospheric. Standard abbreviations can be used, eg bp, base pairs; kb, kilobases; pi, picoliters; s or sec, seconds; min, minutes; h or hr, hours; aa, amino acids; kb, kilobases; bp, base pairs; nt, nucleotides; i.m., intramuscular; i.p., intraperitoneally; s.c., subcutaneous; and the like.Example 1: AAV variant with enhanced retinal cell transduction
[00300] The approach used was to create a peptide display library by introducing a single AvrII site into the wild-type AAV2 genome between amino acid 587 and 588 by polymerase chain reaction (PCR) mutagenesis. A random insertion of 21 nucleotides, 7mer For, was used to synthesize dsDNA inserts, along with the antisense primer 7mer Rev. The resulting dsDNA inserts were cloned into the AvrII site of the genome after digestion with NheI, producing a diverse 7mer display library that was then packaged (Perabo et al., 2003; Müller et al., 2003). The virus was generated so that each viral genome was packaged or encapsulated with the capsid protein variant that genome encoded. In this regard, functional enhancements identified through selection can be linked to the genome sequence encoding the improved function contained within the viral capsid.
[00301] This library was subjected to positive selection in rho-GFP mice (Wensel et al. (2005) Vision Res. 45:3445). Briefly, in one round of selection, adult rho-GFP mice were injected intravitrally with 2 μL of dialyzed phosphate-buffered saline (PBS), iodixanol-purified library with a genomic titer of approximately 1x1012 viral genomes (vg)/mL. A disposable 30 1/2 gauge ultrafine needle was passed through the sclera of the animal's eye, at the equator and close to the limbus, in the vitreous cavity. The injection of 2μl of virus was performed with direct observation of the needle in the center of the vitreous cavity. One week after injection, the eyes were enucleated and the retinas dissociated using a papain protease light treatment, followed by fluorescence activated cell sorter (FACS) isolation of the photoreceptor populations. The successful virions were then amplified in PCR from genomic extractions and further cloned and repackaged for injection.
[00302] Further iterations of this selection were performed, narrowing the group of variants to a subgroup with the most permissive mutations. After three iterations, a round of error prone PCR was performed to create another generation of variants for selection. In total, this process was repeated for two generations. In this regard, this process of directed evolution created photoreceptor-permissive AAV variants through the application of positive selection and induced mutagenesis, similar to the process of natural evolution.
[00303] Subsequently, the cap genes of fifty variants were sequenced to determine the most prominent and successful variants to have permissive mutations for intravitreal photoreceptor transduction. Of the 50 clones, 46 generated readable sequences from a 7mer insert. Notably, nearly two-thirds of the clones contained the same distinct 7mer motif (~588LGETTRP~; SEQ ID NO: 13). Interestingly, the next most prominent variant (~588 NETITRP-; SEQ ID NO: 14) also contained a flanking motif consisting of a positively charged arginine residue sandwiched between polar threonine and a non-polar proline (TRP) residue. .
Table 1. Sequencing of isolated variants of directed evolution reveals a high degree of convergence in virtual libraries. All variants derived from the 7mer AAV2 library, with approximately 64% of variants containing the same 7mer motif (~588LGETTRP~ (SEQ ID NO: 13)).
[00304] Among the 7mer insert sequences, there were moderate preferences at particular positions, for example, a positively charged amino acid at position 1; a negatively charged amino acid at position 2; an alcohol (e.g., an amino acid containing an alcohol group (a free hydroxyl group), such as Thr or Ser) at position 5.
[00305] The 7mer inserts were flanked by spacers, as shown in Table 2:

[00306] Figure 1. Representative three-dimensional capsid model of AAV2 containing a random heptamer (shown in orange) after amino acid 587. This area of AAV2 capsid possibly participates in cell surface receptor binding.
[00307] In light of the high degree of library convergence from the selection described above, a recombinant form of AAV2 ~588LGETTRP~ (SEQ ID NO: 13; called 7M8) was cloned and packaged the vector with a scCAG-GFP transgene to view your transduction profile. Three weeks after intravitreal injection in adult mice, robust expression in several cell types, including retinal ganglion cells (RGCs) and Müller cells, was observed. Importantly, photoreceptor transduction in retinas injected with 7M8, as seen by GFP expression in nuclei of the outer nuclear layer (ONL) (red arrows) and in outer segments (Figure 2, blue arrow), was observed, whereas AAV2 showed no discernible photoreceptor expression.
[00308] Figure 2 Variant AAV2 7M8 (right) demonstrates higher levels of intravitreal photoreceptor transduction relative to AAV2 (left). Confocal microscopy of transverse retinal sections three weeks after intravitreal injection of 2 μL of 1x1012 vg/mL AAV2 7M8 and AAV2 scCAG GFP in adult mice. Red arrows (upper) denote photoreceptor nuclei and blue arrow (upper) denotes outer photoreceptor segments.
[00309] In light of these gains in retinal cell transduction, an attempt was made to increase specificity in expression through the use of an ssRho-eGFP transgene containing a photoreceptor-specific rhodopsin promoter to better address transduction efficiencies. specifically in photoreceptors (Figure 3). Indeed the use of a photoreceptor-specific Rho promoter limited GFP expression to the photoreceptors. An attempt was made to improve the transduction efficiency of 7M8 by combining a rational design approach to the previous directed evolution approach. Therefore, four exposed tyrosine residues were mutagenized to phenylalanines in the 7M8 capsid (Y273F, Y444F, Y500F, and Y730F) which has previously been shown to increase photoreceptor infectivity (Petrs-Silva et al., 2009). Interestingly, the addition of mutations reduced the number of transduced photoreceptors compared to the original virus as shown by FAC classification of GFP(+) photoreceptors from 7m8 or 7m8-4YF infected retinas (Figure 4).
[00310] Figure 3. Representative fluorescence images of retinal cryo-slices showing the expression of 7m8 GFP containing the GFP gene under the control of ubiquitous CAG promoter (left) or a photoreceptor-specific Rho promoter (right).
[00311] Figure 4. GFP(+) photoreceptor cells per million retinal cells as counted by flow cytometry. 7m8 transduces more than 2x the amount of photoreceptors compared to 7m8 containing 4 tyrosine mutations (top).Example 2: Treatment of retinoschisis
[00312] Using the 7m8-rho-RS 1 expression construct, a functional retinoschisin (RS1) protein was released to retinoschisin-deficient mice (Rs1h-deficient mice; Rs1h is the human RS1 mouse homologue). The vector comprises a nucleotide sequence that encodes a functional retinoschisin protein in transcriptional control of a rhodopsin promoter. See Figures 13A-C , where the bolded and underlined nucleotide sequence (nucleotides 4013-4851) is the rhodopsin promoter; and nucleotides 4866-5540 (with the atg start and tga stop sequences shown in bold) encode a human RS1 protein.
[00313] The 7m8-rho-RS l construct was administered intravitreally to Rs1h-/- mice at P15. Rs1h -/- mice were generated by disrupting exon 3 of the Rs1h gene as described (Weber et al. (2002) Proc. Natl. Acad. Sci. USA 99:6222). Rs1h-/- mice are deficient in the Rs1h protein product, have an electronegative ERG (eg, a reduced b wave with relative preservation of an a wave), and retinal layering similar to that seen in human retinoschisis patients. Injection of 7m8-rho-RS l vector at Rs1h-/- led to high levels of panretinal RS1 expression of photoreceptors in the retina. RS1 expression led to improved retinal morphology with a reduction in the number and size of retinal cavities as seen in spectral domain optical coherence tomography (SD-OCT) imaging (Figures 7A-I), a b-wave rescue. ERG (Figures 8A-D), and long-term structural preservation of the retina (Figures 9A-E).
[00314] Figures 7A-I. Representative high resolution SD-OCT images of retinas injected with 7m8-rho-GFP (left column), 7m8-rho-RS1 (middle column), or uninjected WT animals (right column). Background images were taken in the inner nuclear layer of the upper retina and exclude other layers (a-c). Transverse images of the superior (d-f) and inferior (g-i) retina were taken using the optic nerve head as a landmark.
[00315] The untreated RS1 retina increases in overall thickness when measured from the Inner Boundary Membrane (ILM) to the photoreceptors, as the pathology progresses due to the fissures that divide the inner retina. This process is distinct from that seen in more degenerative retinal diseases (RDD) which do not form fissures but have progressive photoreceptor cell death in INL and concomitant retinal thinning and loss of ERG amplitude. In RS1, the ONL thins as the photoreceptors die from the disease, but the disease is distinct from the general change in retinal thickening. It is generally thought that successful therapy for RS1 could return retinal thickness to wild type and ameliorate photoreceptor loss in ONL. In most RDD beyond Rs1, the loss of photoreceptors, marked by ONL thinning, parallels a reduction in physiological retinal output as measured by ERG amplitude. RS1 is one of the very few examples of a retinal disease in which the pathology increases the thickness of the retina with concomitant loss of erg amplitude. In summary, restoring the RS1 gene product, an "extracellular retinal glue" thins the retinal background to wild-type thickness, and the erg amplitude returns to normal levels as the fissure resolves.
[00316] Figure 8a shows a comparison of functional rescue from non-Rs1-/- untreated eyes to eyes injected with AAV2-rho-RS1, 7m8-rho-GFP, and 7m8-rho-RS1 both at 1 month (left) and 4 months (right) after injection. One month after injection, 7m8-rho-RS1 led to considerable rescue of ERG b-wave amplitude, whereas AAV2-rho.RS1 was statistically indistinguishable from untreated eyes.
[00317] After 4 months, 7m8-rho-RS 1 amplitude still increases in the direction of wild-type amplitude (right). Figure 8b shows representative ERG traces of eyes injected with 7m8-rho-RS1 that show increased amplitude of an a wave and wave b and a shorter waveform to wild-type eyes compared to eyes injected with 7m8-rho-GFP. Figure 8c shows the full-field b-wave scotopeak amplitude resulting from a high-intensity stimulus (1 log cd x s/m2) was recorded on a monthly basis starting one month after injection at P15 for each condition. Three responses were recorded and weighted for each eye at each time point.
[00318] Mean amplitudes of wave b ERG were plotted as a function of time after injection. n=7 was used for both conditions. Figure 8d shows an analysis of ERG responses under scotopic (upper traces, stimulus ranges from -3 to 1 log cd xs/m2) and photopic (lower traces, ranges from -0.9 to 1.4 log cd xs/m2) conditions ) indicates improved function of rods and cones over a range of stimulus intensities.
[00319] Figures 9A-E. Sustained improvements in retinal thickness measured at 10 months after 7m8-rho-RS1 treatment. Representative cross-sectional SD-OCT images of retinas treated with a) 7m8-rho-RS1 or b) or 7m8-rho-GFP 10 months after optic nerve head centered injection. Measurements of c) retinal thickness, d) ONL thickness, and e) inner and outer segment thickness are plotted as a function of optic nerve head.
[00320] Example 3: AAV variant used to deliver a protein to monkey retina cells
[00321] A recombinant AAV2 virion (7m8 carrying GFP under the control of a connexin36 promoter) was generated. The recombinant AAV2 virion included an AAV2 capsid variant with a LALGETTRPA peptide insert between amino acids 587 and 588 of the AAV2 capsid, and GFP in transcriptional control from a connexin36 promoter, which is expressed in interneurons. The rAAV2 virion was injected intravitreal into the eye of a monkey. The data are shown in Figure 18.
[00322] Figure 18 provides a fundus fluorescence image showing GFP expression in the fundus of the central primate retina 9 weeks after administration of 7m8 containing GFP under the control of the connexin36 promoter. Compared to the parental AAV2 serotype (Yin et al, IOVS 52(5); 2775), a higher level of expression was observed in the foveal ring, and visible fluorescence was observed in the central retina outside the fovea.REFERENCE LISTINGDaiger SP, Bowne SJ, Sullivan LS (2007) Perspective on genes and mutations causing retinitis pigmentosa. Arch Ophthalmol 125: 151-158. Dalkara D, Kolstad KD, Caporale N, Visel M, Klimczak RR, et al. (2009) Inner Limiting Membrane Barriers to AAV Mediated Retinal Transduction from the Vitreous. Mol Ther.den Hollander AI, Roepman R, Koenekoop RK, Cremers FP (2008) Leber congenital amaurosis: genes, proteins and disease mechanisms. Prog Retin Eye Res 27: 391-419. Gruter O, Kostic C, Crippa SV, Perez MT, Zografos L, et al. (2005) Lentiviral vector-mediated gene transfer in adult mouse photoreceptors is impaired by the presence of a physical barrier. Gene Ther 12: 942-947. Maguire AM, Simonelli F, Pierce EA, Pugh EN, Jr., Mingozzi F, et al. (2008) Safety and efficacy of gene transfer for Leber's congenital amaurosis. N Engl J Med 358: 2240-2248. Mancuso K, Hauswirth WW, Li Q, Connor TB, Kuchenbecker JA, et al. (2009) Gene therapy for red-green color blindness in adult primates. Nature 461: 784-787. McGee Sanftner LH, Abel H, Hauswirth WW, Flannery JG (2001) Glial cell line derived neurotrophic factor delays photoreceptor degeneration in a transgenic rat model of retinitis pigmentosa. Mol Ther 4: 622-629. Muller OJ, Kaul F, Weitzman MD, Pasqualini R, Arap W, et al. (2003) Random peptide libraries displayed on adeno-associated viruses to select for targeted gene therapy vectors. Nat Biotechnol 21: 1040-1046. Nakazawa T. et al. (2007) Attenuated glial reactions and photoreceptor degeneration after retinal detachment in mice deficient in glial fibrillary acidic protein and vimentin. Invest Ophthamol Vis Sci 48: 2760-8.Nakazawa T. et al. (2006) Characterization of cytokine responses to retinal detachment in rats. Mol Vis 12: 867-78. Perabo L, Buning H, Kofler DM, Ried MU, Girod A, et al. (2003) In vitro selection of viral vectors with modified tropism: the adeno-associated virus display. Mol Ther 8: 151-157.Petrs-Silva H, Dinculescu A, Li Q, Min SH, Chiodo V, et al. (2009) High-efficiency transduction of the mouse retina by tyrosinemutant AAV serotype vectors. Mol Ther 17: 463-471. Reme CE, Grimm C, Hafezi F, Wenzel A, Williams TP (2000) Apoptosis in the Retina: The Silent Death of Vision. News Physiol Sci 15: 120-124.Rolling F (2004) Recombinant AAV-mediated gene transfer to the retina: gene therapy perspectives. Gene Ther 11 Suppl 1: S26-32. Wensel TG, Gross AK, Chan F, Sykoudis K, Wilson JH (2005) Rhodopsin-EGFP knock-ins for imaging quantal gene alterations. Vision Res 45: 3445-3453. Zhong L, Li B, Mah CS, Govindoamy L, Agbandje-McKenna M, et al. (2008) Next generation of adeno-associated virus 2 vectors: point mutations in tyrosines lead to high-efficiency transduction at lower doses. Proc Natl Acad Sci US A 105: 7827-7832.
[00323] While the present invention has been described with reference to specific embodiments thereof, it should be understood by those skilled in the art that various changes may be made and equivalents may be substituted without departing from the true spirit and scope of the invention. Furthermore, many modifications can be made to adapt a situation, material, composition, object, process, step or steps of the process, to the object, spirit and particular scope of the present invention. All said modifications are intended to be within the scope of the claims appended hereto.

权利要求:
Claims (22)
[0001]
1. Use of a recombinant adeno-associated virus (rAAV) virion, characterized in that it is for the preparation of a drug to treat an eye disease, wherein the drug comprises a pharmaceutically acceptable excipient, and wherein the adeno-associated virus virion recombinant (rAAV) comprises: a) a variant AAV capsid protein, wherein the variant AAV capsid protein comprises an insertion of a peptide into the GH loop of the capsid protein relative to a corresponding parental AAV capsid protein, in that the insert comprises an amino acid sequence selected from LALGETTRPA (SEQ ID NO: 45); LANETITRPA (SEQ ID NO: 46), LA-KAGQANNA (SEQ ID NO: 47), LAKDPKTTNA (SEQ ID NO: 48), KDTDTTR (SEQ ID NO: 57), RAGGSVG (SEQ ID NO: 58), AVDTTKF (SEQ ID NO:59), STGKVPN (SEQ ID NO:60), LAKDTDTTRA (SEQ ID NO:61), LARAGGSVGA (SEQ ID NO:62), LAAVDTTKFA (SEQ ID NO:63), and LASTGKVPNA (SEQ ID NO:64 ), and b) a heterologous nucleic acid comprising a nucleotide sequence encoding a gene product; wherein the rAAV virion infects a retinal cell, wherein the eye disease is glaucoma, retinitis pigmentosa, macular degeneration, retinoschisis, Leber, diabetic retinopathy, acromotopsia or color blindness.
[0002]
2. Use according to claim 1, characterized in that the insertion site is within amino acids 570 to 611 of the AAV2 capsid protein as set forth in SEQ ID NO: 1, or the corresponding positions in the capsid protein of another AAV serotype.
[0003]
3. Use according to claim 1 or 2, characterized in that the retinal cell is a photoreceptor, a retinal ganglion cell, a Müller cell, a bipolar cell, an amacrine cell, a horizontal or a retinal pigment epithelial cell.
[0004]
4. Use according to any one of claims 1 to 3, characterized in that said drug is suitable for intraocular injection.
[0005]
5. Use according to any one of claims 1 to 4, characterized in that said drug is suitable for intravitreal injection.
[0006]
6. Recombinant adeno-associated virus (rAAV) virion, characterized in that it comprises: a) a variant AAV capsid protein, wherein the variant AAV capsid protein comprises an insertion of a peptide into the GH loop of the protein the capsid relating to a corresponding parental AAV capsid protein, wherein the insert comprises an amino acid sequence selected from LALGETTRPA (SEQ ID NO: 45); LANETITRPA (SEQ ID NO: 46), LA-KAGQANNA (SEQ ID NO: 47), LAKDPKTTNA (SEQ ID NO: 48), LAK-DTDTTRA (SEQ ID NO: 61), LARAGGSVGA (SEQ ID NO: 62), LAAV - DTTKFA (SEQ ID NO: 63), and LASTGKVPNA (SEQ ID NO: 64), and wherein the variant capsid protein confers infectivity of a retinal cell, and b) a heterologous nucleic acid comprising a nucleotide sequence encoding a gene product.
[0007]
7. rAAV virion according to claim 6, characterized in that the insertion site is within amino acids 570 to 611 of the AAV2 capsid protein or the corresponding positions in the capsid protein of another AAV serotype.
[0008]
8. The rAAV virion according to claim 6 or 7, characterized in that the retinal cell is a photoreceptor, a retinal ganglion cell, a Müller cell, a bipolar cell, an amacrine cell, a cell horizontal or a retinal pigment epithelium cell.
[0009]
9. The rAAV virion according to claim 6, characterized in that the insertion site is between amino acids 587 and 588 of the AAV2 capsid protein as set out in SEQ ID NO: 1, or the corresponding positions in the capsid protein from another AAV serotype.
[0010]
10. The rAAV virion according to any one of claims 6 to 9, characterized in that the gene product is an interfering RNA, an aptamer or a polypeptide.
[0011]
11. rAAV virion according to claim 10, characterized in that the polypeptide is selected from the group consisting of: glia-derived neurotrophic factor, fibroblast growth factor 2, neurturin, ciliary neurotrophic factor, growth factor neurotrophic factor, brain-derived neurotrophic factor, epidermal growth factor, rhodopsin, X-linked apoptosis inhibitor, retinoschisin, retinal pigment epithelium-specific protein (RPE65), retinitis pigmentosa GTPase-interacting protein 1, peripherin , peripherin 2, a rhodopsin and Sonic hedgehog.
[0012]
12. Pharmaceutical composition, characterized in that it comprises: a) an rAAV virion as defined in any one of claims 6 to 11; and b) a pharmaceutically acceptable excipient.
[0013]
13. Recombinant adeno-associated virus virion 2 (rA-AV2), characterized in that it comprises: a) a variant AAV2 capsid protein, wherein the variant AAV capsid protein comprises an insertion of a peptide into the GH loop of the capsid protein relative to a corresponding parental AAV capsid protein, wherein the insertion comprises an amino acid sequence LALGETTRPA (SEQ ID NO: 45), wherein the insertion site is within amino acids 570611 of the established AAV2 capsid protein in SEQ ID NO: 1; and b) a heterologous nucleic acid comprising a nucleotide sequence that encodes a gene product.
[0014]
14. The rAAV2 virion, according to claim 13, characterized by the fact that the insertion site is between amino acids 587-588 of the AAV2 capsid protein set forth in SEQ ID NO: 1.
[0015]
15. rAAV2 virion, according to claim 13 or 14, characterized in that the gene product is an interference RNA or an aptamer.
[0016]
16. The rAAV2 virion according to any one of claims 13 to 15, characterized in that the gene product is a polypeptide.
[0017]
17. The rAAV2 virion according to claim 16, characterized in that the polypeptide is a polypeptide that improves the function of a retinal cell, a neuroprotective polypeptide or an antiangiogenic polypeptide.
[0018]
18. rAAV2 virion, according to claim 16, characterized by the fact that the polypeptide is selected from the group consisting of: glial-derived neurotrophic factor, fibroblast growth factor 2, neurturin, ciliary neurotrophic factor, growth hormone, brain-derived neurotrophic factor, epidermal growth factor, rhodopsin, apoptosis-linked inhibitor X, retinoschisin, RPE65, retinitis pigmentosa GTPase interactive protein-1, periphine, periphin-2, rhodopsin, and Sonic Hedgehog .
[0019]
19. The rAAV2 virion according to claim 16, characterized in that the polypeptide is selected from the group consisting of: a soluble vascular endothelial growth factor (VEGF) receptor, a VEGF binding antibody , an Fc fusion polypeptide comprising a soluble Flt polypeptide.
[0020]
20. The rAAV2 virion according to claim 18, characterized in that the polypeptide is an Fc fusion polypeptide comprising a soluble Flt polypeptide.
[0021]
21. rAAV2 virion, according to claim 16, characterized by the fact that the polypeptide is cone photoreceptor cGMP-activated cation channel alpha subunit (CNGA3) or cone photoreceptor-activated cation channel beta subunit. Cone Photoreceptor cGMP (CNGB3).
[0022]
22. rAAV2 virion, according to claim 16, characterized by the fact that the polypeptide is L-opsin, M-opsin or S-opsin.

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法律状态:
2018-01-23| B07D| Technical examination (opinion) related to article 229 of industrial property law [chapter 7.4 patent gazette]|

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2018-04-03| B06F| Objections, documents and/or translations needed after an examination request according [chapter 6.6 patent gazette]|

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2019-11-19| B07E| Notification of approval relating to section 229 industrial property law [chapter 7.5 patent gazette]|

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2020-03-31| B06U| Preliminary requirement: requests with searches performed by other patent offices: procedure suspended [chapter 6.21 patent gazette]|

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2021-08-10| B07A| Application suspended after technical examination (opinion) [chapter 7.1 patent gazette]|

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2021-11-23| B09A| Decision: intention to grant [chapter 9.1 patent gazette]|

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2022-02-01| B16A| Patent or certificate of addition of invention granted [chapter 16.1 patent gazette]|Free format text: PRAZO DE VALIDADE: 20 (VINTE) ANOS CONTADOS A PARTIR DE 20/04/2012, OBSERVADAS AS CONDICOES LEGAIS. |

优先权:

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申请号 | 申请日 | 专利标题

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US201161478355P| true| 2011-04-22|2011-04-22|

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US61/478,355|2011-04-22|

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PCT/US2012/034413|WO2012145601A2|2011-04-22|2012-04-20|Adeno-associated virus virions with variant capsid and methods of use thereof|

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