Muscle-targeted capsid

Abstract

The present invention provides novel capsid variants for viral vectors that target muscle tissue.

Description

[Technical Field]

[0001] The present disclosure relates to viral capsids. [Background technology]

[0002] Recombinant AAV (rAAV) is the most commonly used delivery vehicle for gene therapy and gene editing. Nevertheless, rAAV containing natural capsid variants have limited cell tropism. In fact, currently used rAAV mainly affects the liver after systemic delivery. Furthermore, the transduction efficiency of conventional rAAV in other cell types, tissues, and organs by these conventional rAAV with natural capsid variants is insufficient. Therefore, AAV-mediated polynucleotide delivery to affected areas affecting cells, tissues, and organs other than the liver, such as skeletal muscle, usually requires a large dose of virus (usually about 2 × 10 14 rAAV requires the injection of 1000 mg / kg of rAAV, which often results in liver toxicity. Furthermore, the large doses required when using conventional rAAV make it extremely difficult to produce sufficient quantities of therapeutic rAAV for administration to adult patients. Furthermore, due to differences in gene expression and physiology, mouse and primate models respond differently to viral capsids. The transduction efficiency of different viral particles varies between different species, and as a result, preclinical studies in mice often do not accurately reflect the results in primates, including humans. Therefore, improved rAAVs for use in the treatment of various genetic diseases are needed. Summary of the Invention

[0003] The present invention provides novel capsid protein variants for viral vectors that target muscle tissue. The capsid protein variants contain amino acid sequences that confer muscle tropism. For example, the capsid protein variants of the present invention may contain the sequences listed in Tables 1 to 4.

[0004] In particular, the capsid protein variants of the present invention comprise deletions, substitutions and / or insertions relative to the wild-type viral vector capsid.

[0005] In embodiments of the invention, the capsid protein comprises an amino acid sequence selected from the first column of Table 2a or 4a, and the amino acid sequence is in hypervariable region VIII (HVR VIII) relative to wild-type AAV9. The capsid protein variant may comprise substitutions at amino acids 586, 587, and 588 relative to the wild-type AAV9 vector capsid. For example, the substitutions at amino acids 586, 587, and 588 relative to the wild-type AAV9 vector capsid may be amino acid sequences selected from the first column of one of Tables 2b or 4b. In embodiments of the invention, the capsid protein variant may further comprise an insertion. For example, the capsid protein may comprise a 7-mer insertion selected from the second column of one of Tables 2b or 4b. The insertion may be at a position after amino acid 588 relative to the wild-type AAV9 vector.

[0006] In embodiments of the invention, the capsid protein comprises an amino acid sequence selected from the first column of Table 1a or 3a, and the amino acid sequence is in hypervariable region IV (HVR IV) relative to wild-type AAV9. The capsid protein variant may comprise substitutions at amino acids 453, 454, and 455 relative to the wild-type AAV9 vector capsid. For example, the substitutions at amino acids 453, 454, and 455 relative to the wild-type AAV9 vector capsid may be amino acid sequences selected from the first column of one of Tables 1b or 3b. In embodiments of the invention, the capsid protein variant may further comprise an insertion. For example, the capsid protein may comprise a 7-mer insertion selected from the second column of one of Tables 1b or 3b. The insertion may be at a position after amino acid 455 relative to the wild-type AAV9 vector.

[0007] Advantageously, viral vectors having a capsid protein variant comprising an amino acid sequence as set forth in Table 1a, 2a, 3a, or 4a exhibit increased muscle tropism compared to AAV vectors that do not comprise an amino acid sequence selected from the first column of any one of Tables 1a, 2a, 3a, or 4a.

[0008] Each of Tables 1-4 is explained below: Tables 1a and 1b below provide a selection of muscle-targeting myotropic variants that first arose in mice, where the 10-mer variant sequence is in hypervariable region IV (HVR IV) relative to wild-type AAV9. Tables 2a and 2b below provide a selection of muscle-targeting myotropic variants that first arose in mice, where the 10-mer variant sequence is in hypervariable region VIII (HVR VIII) relative to wild-type AAV9. Tables 3a and 3b below provide a selection of muscle-targeting myotropic variants that first arose in NHPs, where the 10-mer variant sequence is in hypervariable region IV (HVR IV) relative to wild-type AAV9. Tables 4a and 4b below provide a selection of muscle-targeting myotropic variants that arose initially in NHPs, where the 10-mer variant sequence is in hypervariable region VIII (HVR VIII) relative to wild-type AAV9.

[0009] As described above for the HVR IV variant, the three upstream amino acids are at positions 453, 454, and 455. The 7-mer insert for the HVR IV variant begins with "RGD" and is inserted after amino acid 455. For the HVR VIII variant, the three upstream amino acids are at positions 586, 587, and 588. The 7-mer insert for the HVR VIII variant begins with "RGD" and is inserted after amino acid 588.

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[0161] [Table 152] DETAILED DESCRIPTION OF THE INVENTION

[0162] The present invention provides novel capsid protein variants for viral vectors that target muscle tissue. The capsid protein variants contain amino acid sequences that confer muscle tropism. For example, the capsid protein variants of the present invention may contain the sequences listed in Tables 1 to 4.

[0163] Specifically, each of Tables 1 to 4 is explained below: Tables 1a and 1b below provide a selection of muscle-targeting myotropic variants that first arose in mice, where the 10-mer variant sequence is in hypervariable region IV (HVR IV) relative to wild-type AAV9. Tables 2a and 2b below provide a selection of muscle-targeting myotropic variants that first arose in mice, where the 10-mer variant sequence is in hypervariable region VIII (HVR VIII) relative to wild-type AAV9. Tables 3a and 3b below provide a selection of muscle-targeting myotropic variants that first arose in NHPs, where the 10-mer variant sequence is in hypervariable region IV (HVR IV) relative to wild-type AAV9. Tables 4a and 4b below provide a selection of muscle-targeting myotropic variants that arose initially in NHPs, where the 10-mer variant sequence is in hypervariable region VIII (HVR VIII) relative to wild-type AAV9.

[0164] Adeno-associated virus vector AAV is a viral vector that is particularly suitable for delivering genetic material to mammalian cells.AAV is not known to cause disease in mammals and induces very mild immune responses.In addition, AAV can infect cells at multiple stages, whether they are in a resting state or in the phase of the cell replication cycle.Advantageously, AAV DNA is not systematically inserted into the host genome at random sites, which reduces the oncogenicity of this vector.

[0165] AAVs have been engineered to deliver a variety of treatments for genetic diseases, particularly those caused by single nucleotide polymorphisms ("SNPs"). Genetic diseases that have been studied with AAV vectors include cystic fibrosis, hemophilia, arthritis, macular degeneration, muscular dystrophy, Parkinson's disease, congestive heart failure, and Alzheimer's disease. AAVs can be used as vectors to deliver engineered nucleic acids to a host and transcribe the nucleic acid into a protein of interest using the host's own ribosomes. See, e.g., Western., Virology 160:38-47 (1987); U.S. Pat. No. 4,797,368; WO 93 / 24641; Kotin, Human Gene Therapy 5:793-801 (1994); and Muzyczka, J. Clin. Invest. 94:1351 (1994). AAV has some defects in its replication and / or pathogenicity and may therefore be safer than adenovirus. In some embodiments, AAV can integrate into a specific site on chromosome 19 in human cells without any discernible side effects. In some embodiments, the capacity of an AAV vector, system thereof, and / or AAV particle can be up to about 4.7 kb. An AAV vector or system thereof can comprise one or more engineered capsid polynucleotides described herein.

[0166] AAV is a small, non-enveloped, replication-defective virus that infects humans and other primates and has a linear, single-stranded DNA genome. Natural AAV serotypes exhibit liver tropism. As a result, transfection of non-liver tissues with conventional AAV vectors is hindered by the virus's natural liver tropism. Furthermore, because the liver acts to degrade materials delivered to a subject, transfection of non-liver tissues with unmodified AAV vectors requires a large dose to provide a sufficient viral load to overcome the liver and reach the non-liver tissues. More than 30 natural serotypes of AAV are available. Many natural variants of AAV capsids exist. AAV serotypes include, but are not limited to, AAV serotypes AAV1, AAV2, AAV3, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, and AAV13. AAVs can be engineered using conventional molecular biology techniques, allowing these particles to be optimized for, for example, cell-specific delivery, minimized immunogenicity, tuned stability and particle lifetime, efficient degradation, and precise delivery to the nucleus. AAV vectors can be specifically targeted to one or more types of cells by selecting the appropriate combination of AAV serotype, promoter, and delivery method.

[0167] Previous approaches to identifying AAV sequences that correlate with tropism have relied on comparison of highly related existing serotypes and their distinct characteristics, random domain swaps between unrelated serotypes, or conformational considerations to identify motifs that define liver tropism. For example, mapping determinants of AAV tropism has been performed by comparing closely related serotypes. One such example is a single amino acid change (E531K) between AAV1 and AAV6, which improves mouse liver transduction in AAV1. See Wu et al. (2006) J. Virol., 80(22):11393-7, incorporated herein by reference. Another example is the reciprocal domain swap between AAV2 and AAV8, which alters tropism but fails to identify a strong, specific tissue-targeting motif. See Rauppet et al. (201) J. Virol., 86(17):9396-408, incorporated herein by reference. Furthermore, global considerations of the structures only highlight gross differences between better or worse liver transducers that are more observational than practically useful. Nam et al (2007) J. Virol., 81(22):12260-71.

[0168] AAVs with modified tissue tropism that can be used with the present invention are described in U.S. Pat. Nos. 9,695,220, 9,719,070; 10,119,125; 10,526,584; U.S. Patent Application Publication No. 2018-0369414; U.S. Patent Application Publication No. 2020-0123504; U.S. Patent Application Publication No. 2020-0318082; WO 2015 / 054653; WO 2016 / 179496; WO 2017 / 100791; and WO 2019 / 217911, the contents of each of which are incorporated herein by reference in their entirety.

[0169] The AAV vector or system thereof may include one or more regulatory molecules, such as a promoter, enhancer, or repressor. In some embodiments, the AAV vector or system thereof may include one or more polynucleotides that can encode one or more regulatory proteins. In some embodiments, the one or more regulatory proteins may be selected from Rep78, Rep68, Rep52, Rep40, variants thereof, and combinations thereof. In some embodiments, a muscle-specific promoter can drive expression of the engineered AAV capsid polynucleotide.

[0170] An AAV vector or system thereof can include one or more polynucleotides that can encode one or more capsid proteins, such as the engineered AAV capsid proteins described elsewhere herein, that are capable of assembling to construct the protein shell (engineered capsid) of an AAV viral particle. The engineered capsid can have cell-, tissue-, and / or organ-specific tropism.

[0171] AAV vectors or systems thereof can be configured to produce AAV particles with specific serotypes. In some embodiments, the serotypes can be AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, AAV-8, AAV-9, or any combination thereof. In some embodiments, the AAV can be AAV1, AAV-2, AAV-5, AAV-9, or any combination thereof. The AAV of the AAV can be selected based on the cells to be targeted; for example, AAV serotypes 1, 2, 5, 9, or hybrid capsids AAV-1, AAV-2, AAV-5, AAV-9, or any combination thereof can be selected for targeting brain and / or neural cells; AAV-4 can be selected for targeting cardiac tissue; and AAV-8 can be selected for delivery to the liver. Thus, in some embodiments, an AAV vector or system thereof capable of producing AAV particles capable of targeting the brain and / or neural cells may be configured to produce AAV particles having serotypes 1, 2, 5, or hybrid capsids AAV-1, AAV-2, AAV-5, or any combination thereof. In some embodiments, an AAV vector or system thereof capable of producing AAV particles capable of targeting cardiac tissue may be configured to produce AAV particles having the AAV-4 serotype. In some embodiments, an AAV vector or system thereof capable of producing AAV particles capable of targeting the liver may be configured to produce AAV particles having the AAV-8 serotype. See also Srivastava. 2017. Curr. Opin. Virol. 21:75-80.

[0172] It will be recognized that while different serotypes can provide some level of cell-, tissue-, and / or organ-specificity, each serotype remains polytropic and, therefore, tissue toxicity may occur when that serotype is used to target tissues in which it is less efficient at transduction. Thus, in addition to achieving some tissue-targeting capability by selecting an AAV of a particular serotype, it will be recognized that the tropism of an AAV serotype can be modified by the genetically engineered AAV capsids described herein. As described elsewhere herein, variants of wild-type AAV of any serotype can be produced by the methods described herein and determined to have a particular cell-specific tropism that may be the same as or different from the reference wild-type AAV serotype. In some embodiments, the cell-, tissue-, and / or specificity of the wild-type serotype can be enhanced (e.g., it can become more selective or specific for a particular cell type for which the serotype is already biased). For example, wild-type AAV-9 is biased toward human muscle and brain (see, e.g., Srivastava. 2017. Curr. Opin. Virol. 21:75-80). The inclusion of engineered AAV capsid and / or capsid protein variants of wild-type AAV-9 described herein can reduce or eliminate neuronal tropism and / or increase muscle specificity, likely resulting in a relative decrease in neuronal specificity, thus improving muscle specificity compared to wild-type AAV-9. As previously mentioned, the inclusion of engineered capsid and / or capsid protein variants of a wild-type AAV serotype can have a different tropism than the wild-type reference AAV serotype. For example, engineered AAV capsid and / or capsid protein variants of AAV-9 can have properties for tissues other than muscle or brain in humans.

[0173] In some embodiments, the AAV vector is a hybrid AAV vector or system thereof. Hybrid AAV is an AAV containing a genome with elements from one serotype packaged in a capsid derived from at least one different serotype. For example, if rAAV2 / 5 is produced and the production method is based on the helper-free transient transfection method described above, the first and third plasmids (adenosyl helper plasmids) will be the same as those discussed for rAAV2 production. However, the second plasmid, pRepCap, will be different. In this plasmid, called pRep2 / Cap5, the Rep gene is still derived from AAV2, and the Cap gene is derived from AAV5. The production scheme is the same as the above-mentioned approach for AAV2 production. The resulting rAAV is called rAAV2 / 5, and its genome is based on recombinant AAV2 and its capsid is based on AAV5. It is assumed that the cell or tissue tropism exhibited by this AAV2 / 5 hybrid virus is the same as that of AAV5. It will be appreciated that wild-type hybrid AAV particles have the same specificity as the non-hybrid wild-type serotypes described above.

[0174] The advantages achieved by the wild-type-based hybrid AAV system can be combined with the increased and customizable cellular properties that can be achieved with engineered AAV capsids, and can be combined by producing hybrid AAVs that may contain engineered AAV capsids as described elsewhere herein. It will be recognized that hybrid AAVs may contain engineered AAV capsids that contain genomes with elements from a serotype different from the reference wild-type serotype of which the engineered AAV capsid is a variant. For example, hybrid AAVs may be produced that contain engineered AAV capsids that are variants of the AAV-9 serotype, used to package genomes containing components from the AAV-2 serotype (e.g., rep elements). As with the wild-type-based hybrid AAVs described above, the tropism of the resulting AAV particles will be that of the engineered AAV capsid.

[0175] In some embodiments, the AAV vector or system thereof is configured as a "gutless" vector, similar to those vectors described in connection with retroviral vectors. In some embodiments, a "gutless" AAV vector or system thereof may have cis-acting viral DNA elements involved in genome amplification and packaging associated with the heterologous sequence of interest (e.g., a genetically engineered AAV capsid polynucleotide).

[0176] The vectors described herein can be constructed using any suitable process or technique. In some embodiments, one or more suitable recombination and / or cloning methods or techniques can be used with the vectors described herein. Suitable recombination and / or cloning techniques and / or methods include, but are not limited to, those described in U.S. Patent Application Publication No. 2004-0171156A1. Other suitable methods and techniques are described elsewhere herein.

[0177] Numerous publications describe the construction of recombinant AAV vectors, including U.S. Patent No. 5,173,414; Tratschin et al., Mol. Cell. Biol. 5:3251-3260 (1985); Tratschin, et al., Mol. Cell. Biol. 4:2072-2081 (1984); Hermonat & Muzyczka, PNAS 81:6466-6470 (1984); and Samulski et al., J. Virol. 63:03822-3828 (1989). Any of the techniques and / or methods may be used and / or applied to the construction of AAV or other vectors described herein. AAV vectors are discussed elsewhere herein.

[0178] In some embodiments, a vector may have one or more insertion sites, such as restriction endonuclease recognition sequences (also called "cloning sites"). In some embodiments, one or more insertion sites (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more insertion sites) are located upstream and / or downstream of one or more sequence elements of one or more vectors.

[0179] The delivery vehicles, vectors, particles, nanoparticles, formulations and components thereof for expression of one or more elements of the genetically engineered AAV capsid system described herein are used in the above-mentioned documents, such as WO 2021 / 050974 and WO 2021 / 077000, and PCT International Application No. PCT / US2021 / 042812, the contents of which are incorporated herein by reference.

[0180] Additional AAV vectors are described in WO 2019 / 2071632, the contents of which are incorporated herein by reference.

[0181] Further AAV vectors are described in WO2020 / 086881 and WO2020 / 235543, the contents of each of which are incorporated herein by reference.

[0182] Additional AAV vectors are described in WO 2005 / 033321; WO 2006 / 110689; WO 2007 / 127264; WO 2008 / 027084; WO 2009 / 073103; WO 2009 / 073104; WO 2009 / 105084; WO 2009 / 134681; WO 2009 / 136977, the contents of each of which are incorporated herein by reference. Lett; WO 2010 / 051367; WO 2010 / 138675; WO 2001 / 038187; WO 2012 / 112832; WO 2015 / 054653; WO 2016 / 179496; WO 2017 / 100791; WO 2017 / 019994; WO 2018 / 209154; WO 2019 / 067982; WO No. 2019 / 195701; WO 2019 / 217911; WO 2020 / 041498; WO 2020 / 210839; U.S. Patent No. 7,906,111; U.S. Patent No. 9,737,618; U.S. Patent No. 10,265,417; U.S. Patent No. 10,485,883; U.S. Patent No. 10,695,441; U.S. Patent No. 10,722,598; U.S. Patent No. 8,999,678; U.S. Patent No. 10,301,648; U.S. Patent No. US Patent Nos. 10,626,415; 9,198,984; 10,155,931; 8,524,219; 9,206,238; 8,685,387; 9,359,618; 8,231,880; 8,470,310; 9,597,363; 8,940,290; 9,593,346; 10,501,757;U.S. Patent Nos. 10,786,568; 10,973,928; 10,519,198; 8,846,031; 9,617,561; 9,884,071; 10,406,173; 9,596,220; 9,719,010; 10,117,12 No. 5; U.S. Patent No. 10,526,584; U.S. Patent No. 10,881,548; U.S. Patent No. 10,738,087; U.S. Patent Application Publication No. 2011-023353; U.S. Patent Application Publication No. 2019-0015527; U.S. Patent Application Publication No. 2020-155704; U.S. Patent Application Publication No. 2017-0191079; U.S. Patent Application Publication No. 2019-0218574 No. 2020-0208176; U.S. Patent Application Publication No. 2020-0325491; U.S. Patent Application Publication No. 2019-0055523; U.S. Patent Application Publication No. 2020-0385689; U.S. Patent Application Publication No. 2009-0317417; U.S. Patent Application Publication No. 2016-0051603; U.S. Patent Application Publication No. 2016-00244783; U.S. US Patent Application Publication No. 2017-0183636; US Patent Application Publication No. 2020-0263201; US ​​Patent Application Publication No. 2020-0101099; US Patent Application Publication No. 2020-0318082; US Patent Application Publication No. 2018-0369414; US Patent Application Publication No. 2019-0330278; US Patent Application Publication No. 2020-0231986.

[0183] Capsid protein The capsid protein is the shell or coating of the virus that allows its delivery into the host. Without this protein, the nucleic acid would be destroyed by the host without entering the host cell and without initiating transcription and translation. The capsid protein can have the native conformation of a native AAV, or it can be modified.

[0184] In certain exemplary embodiments, the AAV capsid protein is an engineered AAV capsid protein that has reduced or eliminated uptake in non-muscle cells compared to the corresponding wild-type AAV capsid polypeptide.

[0185] In some embodiments, a polynucleotide configured to be an AAV genome donor in an AAV vector system that can be used to produce the genetically engineered AAV particles described elsewhere herein can include a polynucleotide encoding a genetically engineered AAV capsid. In some embodiments, the polynucleotide encoding the genetically engineered AAV capsid can be operably linked to a polyadenylation tail. In some embodiments, the polyadenylation tail can be an SV40 polyadenylation tail. In some embodiments, the polynucleotide encoding the AAV capsid can be operably linked to a promoter. In some embodiments, the promoter can be a tissue-specific promoter. In some embodiments, the tissue-specific promoter is specific for muscle (e.g., cardiac, skeletal, and / or smooth muscle), neural and support cells (e.g., astrocytes, glial cells, Schwann cells, etc.), fat, spleen, liver, kidney, immune cells, cerebrospinal fluid cells, synovial cells, skin cells, cartilage, tendon, connective tissue, bone, pancreas, adrenal gland, blood cells, bone marrow cells, placenta, endothelial cells, and combinations thereof. In some embodiments, the promoter can be a constitutive promoter. Suitable tissue-specific and constitutive promoters are discussed elsewhere herein, are generally known in the art, and may be commercially available. Suitable muscle-specific promoters include, but are not limited to, CK8, MHCK7, myoglobin promoter (Mb), desmin promoter, muscle creatine kinase promoter (MCK) and variants thereof, and SPc5-12 synthetic promoter.

[0186] Described herein are various embodiments of engineered viral capsids, such as adeno-associated viral (AAV) capsids, that can be engineered to confer cell-specific tropism, such as muscle-specific tropism, to the engineered viral particles. The engineered viral capsids can be lentiviral, retroviral, adenoviral, or AAV capsids. The engineered capsids can be contained in engineered viral particles (e.g., engineered lentiviral, retroviral, adenoviral, or AAV viral particles) and can confer cell-specific tropism, reduced immunogenicity, or both to the engineered viral particles. The engineered viral capsids described herein can comprise one or more engineered viral capsid proteins described herein. The engineered viral capsids described herein can comprise one or more engineered viral capsid proteins described herein that can contain an n-mer motif or a muscle-specific targeting site composed of an n-mer motif as described elsewhere herein.

[0187] The genetically engineered viral capsid and / or capsid protein may be encoded by one or more genetically engineered viral capsid polynucleotides. In some embodiments, the genetically engineered viral capsid polynucleotide is a genetically engineered AAV capsid polynucleotide, a genetically engineered lentivirus capsid polynucleotide, a genetically engineered retrovirus capsid polynucleotide, or a genetically engineered adenovirus capsid polynucleotide. In some embodiments, the genetically engineered viral capsid polynucleotide (e.g., a genetically engineered AAV capsid polynucleotide, a genetically engineered lentivirus capsid polynucleotide, a genetically engineered retrovirus capsid polynucleotide, or a genetically engineered adenovirus capsid polynucleotide) may comprise a 3' polyadenylation signal. The polyadenylation signal may be an SV40 polyadenylation signal.

[0188] The engineered viral capsid may be a variant of a wild-type viral capsid. For example, in some embodiments, the engineered AAV capsid may be a variant of a wild-type AAV capsid. In some embodiments, the wild-type AAV capsid may be composed of VP1, VP2, VP3 capsid proteins, or a combination thereof. That is, the engineered AAV capsid may include one or more variants of wild-type VP1, wild-type VP2, and / or wild-type VP3 capsid proteins. In some embodiments, the serotype of the reference wild-type AAV capsid may be AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, AAV-8, AAV-9, or any combination thereof. In some embodiments, the serotype of the wild-type AAV capsid may be AAV-9. The engineered AAV capsid may have a tropism that differs from the tropism of the reference wild-type AAV capsid.

[0189] The genetically engineered viral capsid may contain between 1 and 60 genetically engineered capsid proteins. In some embodiments, the genetically engineered viral capsid may contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 genetically engineered capsid proteins. In some embodiments, the genetically engineered viral capsid may contain 0 to 59 wild-type viral capsid proteins. In some embodiments, the genetically engineered viral capsid may contain 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, or 59 wild-type viral capsid proteins.

[0190] In some embodiments, an engineered AAV capsid may contain between 1 and 60 engineered capsid proteins. In some embodiments, an engineered AAV capsid may contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 engineered capsid proteins. In some embodiments, the engineered AAV capsid may contain 0 to 59 wild-type AAV capsid proteins. In some embodiments, the engineered AAV capsid may contain 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, or 59 wild-type AAV capsid proteins.

[0191] In some embodiments, the engineered viral capsid protein may have an n-mer amino acid motif, where n is at least 3 amino acids. In some embodiments, n may be 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids. In some embodiments, the engineered AAV capsid may have a 6-mer or 7-mer amino acid motif. In some embodiments, the n-mer amino acid motif may be inserted between two amino acids in a wild-type viral protein (VP) (or capsid protein). In some embodiments, the n-mer motif may be inserted between two amino acids in a variable amino acid region in the viral capsid protein.

[0192] In some embodiments, an n-mer motif can be inserted between two amino acids in the variable amino acid region of an AAV capsid protein. The core of each wild-type AAV viral protein contains an eight-stranded beta-barrel motif (beta B to beta I) and an alpha helix (alpha A) that are conserved in autonomous parvovirus capsids (see, e.g., DiMattia et al. 2012. J. Virol. 86(12):6947-6958). Structural variable regions (VRs) occur in surface loops connecting the beta strands and cluster together, resulting in local variations on the capsid surface. AAV has 12 variable regions (also called hypervariable regions) (see, e.g., Weitzman and Linden. 2011. "Adeno-Associated Virus Biology." In Snyder, RO, Moullier, P. (eds.) Totowa, NJ: Humana Press). In some embodiments, one or more n-mer motifs may be inserted between two amino acids in one or more of the 12 variable regions of the wild-type AVV capsid protein. In some embodiments, the one or more n-mer motifs may be inserted between two amino acids in VR-I, VR-II, VR-III, VR-IV, VR-V, VR-VI, VR-VII, VR-III, VR-IX, VR-X, VR-XI, VR-XII, or a combination thereof, respectively. In some embodiments, the n-mer may be inserted between two amino acids in VR-III of the capsid protein. In some embodiments, the genetically engineered capsid may have an n-mer inserted between two adjacent amino acids between amino acids 262 and 269, between two adjacent amino acids between amino acids 327 and 332, between two adjacent amino acids between amino acids 382 and 386, between two adjacent amino acids between amino acids 452 and 460, between two adjacent amino acids between amino acids 488 and 505, between two adjacent amino acids between amino acids 545 and 558, between two adjacent amino acids between amino acids 581 and 593, or between two adjacent amino acids between amino acids 704 and 714 of the AAV9 viral protein. In some embodiments, the genetically engineered capsid may have an n-mer inserted between amino acids 588 and 589 of the AAV9 viral protein.In some embodiments, the engineered capsid may have a 7-mer motif inserted between amino acids 588 and 589 of the AAV9 viral protein. In other embodiments, the inserted motif is a 10-mer motif, with a substitution of amino acids 586-88 and an insertion before 589. SEQ ID NO: 1 is a reference AAV9 capsid sequence for at least the insertion sites described above. It will be recognized that n-mers may be inserted at analogous positions in AAV viral proteins of other serotypes. In some embodiments, as described above, n-mers may be inserted between any two adjacent amino acids within the AAV viral protein, and in some embodiments, the insertion is made in a variable region.

[0193] In some embodiments, the first 1, 2, 3, or 4 amino acids of an n-mer motif can be inserted, replacing 1, 2, 3, or 4 amino acids of the polypeptide preceding the insertion site. In some embodiments, the amino acids of the n-mer motif that replace one or more amino acids of the polypeptide into which the n-mer motif is inserted precede or immediately precede the "RGD" in the n-mer motif. For example, in one or more 10-mer insertions, the first three amino acids shown can replace 1 to 3 amino acids in the polypeptide into which they are inserted. Using AAV as another non-limiting example, one or more n-mer motifs can be inserted into an AAV9 capsid polypeptide between amino acids 588 and 589, replacing amino acids 586, 587, and 588, such that the amino acid immediately preceding the n-mer motif after insertion is residue 585. It will be appreciated that this principle may apply to other insertion situations and is not necessarily limited to insertions between residues 588 and 589 of the AAV9 capsid or at the equivalent position in other AAV capsids. Furthermore, it will be appreciated that in some embodiments, amino acids in the polypeptide into which the n-mer motif is inserted are not replaced by the n-mer motif.

[0194] In some embodiments, the AAV capsid or other viral capsid or composition may be muscle-specific. In some embodiments, the muscle specificity of the engineered AAV or other viral capsid or other composition is conferred by a muscle-specific n-mer motif incorporated into the engineered AAV or other viral capsid or other composition described herein. Without intending to be bound by theory, it is believed that the n-mer motif confers a 3D structure to or within a domain or region of the engineered AAV or other viral capsid or other composition such that interaction of a viral particle or other composition containing the engineered AAV capsid or other viral capsid or other composition described herein has an increased or improved interaction (e.g., increased affinity) with cell surface receptors and / or other molecules on the surface of muscle cells. In some embodiments, the cell surface receptor is an AAV receptor (AAVR). In some embodiments, the cell surface receptor is a muscle cell-specific AAV receptor. In some embodiments, the cell surface receptor or other molecule is a cell surface receptor or other molecule selectively expressed on the surface of muscle cells. In some embodiments, the cell surface receptor or molecule is an integrin or dimer thereof, hi some embodiments, the cell surface receptor or molecule is a Vb6 integrin heterodimer.

[0195] In some embodiments, muscle-specific engineered viral particles or other compositions described herein containing a muscle-specific capsid, n-mer motif, or muscle-specific targeting moiety described herein may have increased uptake, delivery rate, transduction rate, efficiency, quantity, or a combination thereof in muscle cells compared to other cell types and / or other viral particles (such as, but not limited to, AAV) and other compositions that do not contain a muscle-specific n-mer motif of the invention.

[0196] As described elsewhere herein and / or in, for example, U.S. Provisional Patent Application Nos. 62 / 899,453, 62 / 916,207, 63 / 018,454, 63 / 242,008, and 63 / 345,14, first- and second-generation muscle-specific AAV capsids were developed using muscle-specific promoters, and the resulting capsid libraries were screened in mice and non-human primates.

[0197] Incorporation by Reference References and citations to other documents, such as patents, patent applications, patent publications, journals, books, articles, and web content, are made throughout this disclosure, and all such documents are incorporated herein by reference in their entirety for all purposes.

[0198] equivalent Various modifications of the invention and many further embodiments thereof, in addition to those shown and described herein, will become apparent to those skilled in the art from the entire contents of this specification, including the references to the scientific and patent literature set forth herein. The subject matter herein contains important information, exemplification and guidance that can be adapted to the practice of this invention in its various embodiments and equivalents thereof.

Claims

1. An adeno-associated virus (AAV) vector comprising a capsid protein comprising an amino acid sequence selected from the first column of any one of Tables 1a, 2a, 3a, or 4a.

2. 2. The AAV vector of claim 1, wherein the amino acid sequence is selected from the first column of Table 2a or 4a, and the amino acid sequence is in hypervariable region VIII (HVR VIII) relative to wild-type AAV9.

3. 3. The AAV vector of claim 2, wherein the capsid protein comprises substitutions at amino acids 586, 587, and 588 relative to a wild-type AAV9 vector capsid.

4. 4. The AAV vector of claim 3, wherein the substitutions at amino acids 586, 587, and 588 relative to the wild-type AAV9 vector capsid are substituted with an amino acid sequence selected from the first column of any one of Tables 2b or 4b.

5. 5. The AAV vector of claim 4, wherein the capsid protein comprises a 7-mer insertion selected from the second column of either Table 2b or 4b.

6. 6. The AAV vector of claim 5, wherein the 7-mer insertion is inserted after amino acid 588 relative to the wild-type AAV9 vector.

7. 2. The AAV vector of claim 1, wherein the amino acid sequence is selected from the first column of Table 1a or 3a, and the amino acid sequence is in hypervariable region IV (HVR IV) relative to wild-type AAV9.

8. 8. The AAV vector of claim 7, wherein the capsid protein comprises substitutions at amino acids 453, 454, and 455 relative to a wild-type AAV9 vector capsid.

9. 9. The AAV vector of claim 8, wherein the substitutions at amino acids 453, 454, and 465 relative to the wild-type AAV9 vector capsid are substituted with an amino acid sequence selected from the first column of any one of Tables 1b or 3b.

10. 10. The AAV vector of claim 9, wherein the capsid protein comprises a 7-mer insertion selected from the second column of either Table 1b or 3b.

11. 11. The AAV vector of claim 10, wherein the 7-mer insertion is inserted after amino acid 455 relative to the wild-type AAV9 vector.

12. 2. The AAV vector of claim 1, which exhibits increased muscle tropism compared to an AAV vector that does not comprise an amino acid sequence selected from the first column of any one of Tables 1a, 2a, 3a, or 4a.

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