AAV capsid variants for gene therapy

Abstract

Adeno-associated virus (AAV) particles have emerged as a useful vehicle for gene delivery to various organs and tissues. Provided herein are variant AAV capsid proteins and variant capsid protein-containing particles. These variant AAV particle compositions can be used to modify tissue tropism and transduction efficiency. In some embodiments, the compositions described herein are useful for producing rAAV particles and / or delivering one or more genes of interest to target tissues.

Description

[Technical Field]

[0001] Related Applications This application claims the benefit under 35 U.S.C. § 119(e) of the filing date of U.S. Provisional Application Serial No. 62 / 886,915, entitled "AAV CAPSID VARIANTS FOR GENE THERAPY," filed August 14, 2019, which is incorporated herein by reference. [Background technology]

[0002] background Recombinant adeno-associated viruses (rAAVs) have entered the clinic as gene transfer vectors for a number of different diseases. In some cases, vectors are delivered directly into the tissue of interest, but there are an increasing number of diseases for which delivery is systemic. Therefore, the development of new AAV variants with different tissue tropisms and higher transduction efficiencies is highly desirable. Summary of the Invention

[0003] Abstract Disclosed herein are variant recombinant adeno-associated virus (rAAV) capsid proteins, nucleic acids encoding such proteins, and rAAV particles comprising such proteins (e.g., encapsidating an rAAV genome encoding a gene of interest). In some embodiments, the compositions described herein are useful for producing rAAV particles and / or delivering one or more genes of interest to a target tissue (e.g., muscle tissue, such as cardiac or skeletal muscle).

[0004] When used for the treatment of muscle disease, a large amount of virus is required to transduce the skeletal muscle and / or cardiac muscle of the body.Unfortunately, a large amount of virus can cause many different types of undesirable immune responses, which can be life-threatening for patients.Therefore, it is necessary to optimize the tropism of virus for target tissue, while reducing the uptake of virus in non-target tissue.

[0005] In some embodiments, disclosed herein are variant recombinant adeno-associated virus (rAAV) serotype 8 (AAV8) capsid proteins having one or more amino acid substitutions that enhance tissue tropism (e.g., muscle tropism). In some embodiments, the rAAV capsid protein contains one or more additional amino acid substitutions that reduce tropism for the brain and / or liver.

[0006] In some embodiments, the rAAV8 capsid protein comprises one or more of any one or more of the alterations as shown in Figure 1. In some embodiments, the rAAV8 capsid protein comprises the amino acid sequence as set forth in SEQ ID NO:11.

[0007] In some embodiments, the rAAV8 capsid protein further comprises any one or more of the following alterations: N500I; N263S; G264A; T265S; S266T; deletion of G268; T270S; and T274H. In some embodiments, the rAAV8 capsid protein comprises the amino acid sequence as set forth in SEQ ID NO: 15. In some embodiments, the rAAV8 capsid protein comprises the amino acid sequence as set forth in SEQ ID NO: 21. In some embodiments, the rAAV8 capsid protein comprises the amino acid sequence as set forth in SEQ ID NO: 27.

[0008] In some embodiments, the rAAV8 capsid protein contains the following substitutions: A24D, D41N, Q84K, R92K, T158A, K163S, R169K, L189I, A195G, V199L, P201S, S225A, G264S, A269S, R313K, S315N, E350D, Q362E, Q413E, T415S, T417Q, Y447F, T453S, N459G, T462Q, G46 In some embodiments, the rAAV8 capsid protein comprises one or more of the following amino acid sequences: 4L, N471S, T472N, A474S, N475A, T494V, A507G, G508A, N517D, A520V, T528S, D531E, D532G, N540S, N549G, A551G, A555V, D559K, E578Q, I581Q, T591A, Q594I, I595V, N665S, S667A, N670A, S712N, V722T, and Y733F. ...

[0009] In some embodiments, the rAAV8 capsid protein further comprises any one or more of the following alterations: N500I; N263S; S264A; T265S; S266T; deletion of G268; S269A; T270S; and T274H. In some embodiments, the rAAV8 capsid protein comprises the amino acid sequence as set forth in SEQ ID NO: 17. In some embodiments, the rAAV8 capsid protein comprises the amino acid sequence as set forth in SEQ ID NO: 23. In some embodiments, the rAAV8 capsid protein comprises the amino acid sequence as set forth in SEQ ID NO: 29.

[0010] In some embodiments, the rAAV8 capsid protein contains the following substitutions: K31Q, D41N, G42A, Q84K, R92K, Q105K, T158A, K163S, S180T, P201S, S225A, G264S, A269S, R313K, S315N, E350D, Q362E, Q413E, T415S, T417Q, Y447F, T453S, N459G, T462Q, G464L, N465G, N466G, N467G, N468G, N469G, N470G, N471G, N472G, N473G, N474G, N475G, N476G, N477G, N478G, N479G, N480G, N481G, N482G, N483G, N484G, N485G, N486G, N487G, N488G, N489G, N490G, N491G, N492G, N493G, N494G, N495G, N496G, N497G, N498G, N499G, N500G, N501G, N502G, N503G, N504G, N505G, N506G, N510G, N511G, N512G, N513G, N514G, N515G, N516G, N517G, N518G, N520G, N521G, N522G, N523G, N524G, N525G, N526 In some embodiments, the rAAV8 capsid protein comprises one or more of the following amino acid sequences: 471S, T472N, A474S, N475A, T494V, A507G, G508A, N517D, A520V, T528S, D531E, D532G, N540S, N549G, A551G, A555V, D559K, I581Q, Q588S, Q589A, Q594I, I595V, N665S, S667A, N670A, S712N, V722T, and Y733F. In some embodiments, the rAAV8 capsid protein comprises the amino acid sequence as set forth in SEQ ID NO: 13.

[0011] In some embodiments, the rAAV8 capsid protein further comprises any one or more of the following alterations: N500I; N263S; S264A; T265S; S266T; deletion of G268; S269A; T270S; and T274H. In some embodiments, the rAAV8 capsid protein comprises the amino acid sequence as set forth in SEQ ID NO: 19. In some embodiments, the rAAV8 capsid protein comprises the amino acid sequence as set forth in SEQ ID NO: 25. In some embodiments, the rAAV8 capsid protein comprises the amino acid sequence as set forth in SEQ ID NO: 31.

[0012] Further disclosed are variant rAAV capsid proteins of serotypes other than serotype 8 (e.g., AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV9, AAV10, AAVrhlO, AAVrhl74, AAVanc80L65, etc.) comprising any one or more of the disclosed amino acid alterations. In some embodiments, the amino acid substitution is at a position in the capsid protein of the serotype other than serotype 8 that corresponds to the position of the substitution in AAV8. Thus, in some embodiments, one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) of the amino acid substitutions set forth in Figure 1, Figure 4, and / or SEQ ID NOs: 2, 4, 6, 11-13, 15, 17, 19, 21, 23, 25, 27, 29, and / or 31 may be included in an rAAV8-based capsid protein, or in an rAAV capsid protein based on any of the different serotypes (e.g., capsid proteins set forth in SEQ ID NOs: 7-10), and / or in related rAAV particles (e.g., those comprising a nucleic acid encoding a gene of interest). In some embodiments, the variant capsid protein may be a variant VP1, VP2, or VP3 capsid protein. In some embodiments, the variant capsid protein comprises a subset of the amino acid substitutions shown in Figures 1 and 4, e.g., a subset having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 fewer amino acid substitutions than those shown in Figures 1 and 4.

[0013] Also disclosed are variant recombinant AAV (e.g., rAAV8) particles comprising the disclosed recombinant AAV (e.g., rAAV8) capsid proteins. In some embodiments, the AAV (e.g., rAAV8) particles comprise a nucleic acid comprising a gene of interest. In some embodiments, the nucleic acid is single-stranded. In some embodiments, the nucleic acid is double-stranded.

[0014] Also disclosed are compositions comprising a plurality of variant recombinant AAV (e.g., rAAV8) particles disclosed herein. These compositions may include a pharmaceutically acceptable carrier.

[0015] Also disclosed is a method of transducing a cell with a gene of interest, the method comprising providing a cell with a composition disclosed herein, wherein the AAV (e.g., rAAV8) particles in the composition comprise the gene of interest. Also provided is a method of transducing a cell with a gene of interest, the method comprising providing a cell with a composition comprising a plurality of recombinant AAV (e.g., rAAV8) particles comprising a variant recombinant AAV (e.g., rAAV8) capsid protein disclosed herein, wherein the AAV (e.g., rAAV8) particles in the composition comprise the gene of interest. In some embodiments, the gene of interest encodes a therapeutic protein. In some embodiments, the therapeutic protein is an antibody or antibody fragment, a peptibody, a growth factor, a hormone, a membrane protein, a cytokine, a chemokine, an activating or inhibitory peptide acting on a cell surface receptor or ion channel, a cell-penetrating peptide targeting an intracellular process, an enzyme, a nuclease, or other protein used for gene editing. In some embodiments, the subject is a mammal. In some embodiments, the subject is a human.

[0016] The following drawings form part of this specification and are included to further demonstrate certain aspects of the present disclosure. Certain aspects of the present disclosure may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein. It should be understood that the data illustrated in the drawings in no way limit the scope of the present disclosure. [Brief explanation of the drawings]

[0017] [Figure 1A]Figures 1A-1E show alignments of AAV variants SL1.2, SL2, and SL3 with other commonly used AAV vectors, AAV8, AAV9, AAVrh10, AAVrh74, and AAVanc80L65. Red indicates identical amino acids across all compared capsids; orange indicates one of the compared sequences substitutes a different amino acid; green indicates two of the compared sequences substitute different amino acids; and blue indicates three or more of the compared sequences substitute different amino acids. [Figure 1B] Figures 1A-1E show alignments of AAV variants SL1.2, SL2, and SL3 with other commonly used AAV vectors, AAV8, AAV9, AAVrh10, AAVrh74, and AAVanc80L65. Red indicates identical amino acids across all compared capsids; orange indicates one of the compared sequences substitutes a different amino acid; green indicates two of the compared sequences substitute different amino acids; and blue indicates three or more of the compared sequences substitute different amino acids. [Figure 1C] Figures 1A-1E show alignments of AAV variants SL1.2, SL2, and SL3 with other commonly used AAV vectors, AAV8, AAV9, AAVrh10, AAVrh74, and AAVanc80L65. Red indicates identical amino acids across all compared capsids; orange indicates one of the compared sequences substitutes a different amino acid; green indicates two of the compared sequences substitute different amino acids; and blue indicates three or more of the compared sequences substitute different amino acids. [Figure 1D]Figures 1A-1E show alignments of AAV variants SL1.2, SL2, and SL3 with other commonly used AAV vectors, AAV8, AAV9, AAVrh10, AAVrh74, and AAVanc80L65. Red indicates identical amino acids across all compared capsids; orange indicates one of the compared sequences substitutes a different amino acid; green indicates two of the compared sequences substitute different amino acids; and blue indicates three or more of the compared sequences substitute different amino acids. [Figure 1E] Figures 1A-1E show alignments of AAV variants SL1.2, SL2, and SL3 with other commonly used AAV vectors, AAV8, AAV9, AAVrh10, AAVrh74, and AAVanc80L65. Red indicates identical amino acids across all compared capsids; orange indicates one of the compared sequences substitutes a different amino acid; green indicates two of the compared sequences substitute different amino acids; and blue indicates three or more of the compared sequences substitute different amino acids.

[0018] [Figure 2] FIG. 2 depicts a "tree diagram" of AAV variants SL1.2, SL2, SL3, AAV8, AAV9, AAVrhlO, AAVrh74 and AAVanc80L65 based on the sequences of their capsid proteins. [Figure 3] Figure 3 shows the % sequence homology / divergence of AAV variants SL1.2, SL2, SL3, AAV8, AAV9, AAVrhlO, AAVrh74 and AAVanc80L65.

[0019] [Figure 4A]Figures 4A-4J show the alignment of AAV variants SL1.2, SL1.2L, SL1.2B, SL1.2LB, SL2, SL2L, SL2B, SL2LB, SL3, SL3L, SL3B, and SL3LB with other commonly used AAV vectors, AAV8, AAV9, AAVrh10, AAVrh74, and AAVanc80L65. The "L" suffix refers to a single amino acid change reported to result in less efficient liver targeting. The "B" suffix refers to eight amino acid substitutions / deletions reported to impair blood-brain barrier crossing. The "LB" suffix refers to a single amino acid change reported to result in less efficient liver targeting (L) combined with eight amino acid substitutions / deletions reported to impair blood-brain barrier crossing. Red indicates identical amino acids among all compared capsids; orange indicates that one of the compared sequences substitutes a different amino acid; green indicates that two of the compared sequences substitute a different amino acid; and blue indicates that three or more of the compared sequences substitute a different amino acid. [Figure 4B]Figures 4A-4J show the alignment of AAV variants SL1.2, SL1.2L, SL1.2B, SL1.2LB, SL2, SL2L, SL2B, SL2LB, SL3, SL3L, SL3B, and SL3LB with other commonly used AAV vectors, AAV8, AAV9, AAVrh10, AAVrh74, and AAVanc80L65. The "L" suffix refers to a single amino acid change reported to result in less efficient liver targeting. The "B" suffix refers to eight amino acid substitutions / deletions reported to impair blood-brain barrier crossing. The "LB" suffix refers to a single amino acid change reported to result in less efficient liver targeting (L) combined with eight amino acid substitutions / deletions reported to impair blood-brain barrier crossing. Red indicates identical amino acids among all compared capsids; orange indicates that one of the compared sequences substitutes a different amino acid; green indicates that two of the compared sequences substitute a different amino acid; and blue indicates that three or more of the compared sequences substitute a different amino acid. [Figure 4C]Figures 4A-4J show the alignment of AAV variants SL1.2, SL1.2L, SL1.2B, SL1.2LB, SL2, SL2L, SL2B, SL2LB, SL3, SL3L, SL3B, and SL3LB with other commonly used AAV vectors, AAV8, AAV9, AAVrh10, AAVrh74, and AAVanc80L65. The "L" suffix refers to a single amino acid change reported to result in less efficient liver targeting. The "B" suffix refers to eight amino acid substitutions / deletions reported to impair blood-brain barrier crossing. The "LB" suffix refers to a single amino acid change reported to result in less efficient liver targeting (L) combined with eight amino acid substitutions / deletions reported to impair blood-brain barrier crossing. Red indicates identical amino acids among all compared capsids; orange indicates that one of the compared sequences substitutes a different amino acid; green indicates that two of the compared sequences substitute a different amino acid; and blue indicates that three or more of the compared sequences substitute a different amino acid. [Figure 4D]Figures 4A-4J show the alignment of AAV variants SL1.2, SL1.2L, SL1.2B, SL1.2LB, SL2, SL2L, SL2B, SL2LB, SL3, SL3L, SL3B, and SL3LB with other commonly used AAV vectors, AAV8, AAV9, AAVrh10, AAVrh74, and AAVanc80L65. The "L" suffix refers to a single amino acid change reported to result in less efficient liver targeting. The "B" suffix refers to eight amino acid substitutions / deletions reported to impair blood-brain barrier crossing. The "LB" suffix refers to a single amino acid change reported to result in less efficient liver targeting (L) combined with eight amino acid substitutions / deletions reported to impair blood-brain barrier crossing. Red indicates identical amino acids among all compared capsids; orange indicates that one of the compared sequences substitutes a different amino acid; green indicates that two of the compared sequences substitute a different amino acid; and blue indicates that three or more of the compared sequences substitute a different amino acid. [Figure 4E]Figures 4A-4J show the alignment of AAV variants SL1.2, SL1.2L, SL1.2B, SL1.2LB, SL2, SL2L, SL2B, SL2LB, SL3, SL3L, SL3B, and SL3LB with other commonly used AAV vectors, AAV8, AAV9, AAVrh10, AAVrh74, and AAVanc80L65. The "L" suffix refers to a single amino acid change reported to result in less efficient liver targeting. The "B" suffix refers to eight amino acid substitutions / deletions reported to impair blood-brain barrier crossing. The "LB" suffix refers to a single amino acid change reported to result in less efficient liver targeting (L) combined with eight amino acid substitutions / deletions reported to impair blood-brain barrier crossing. Red indicates identical amino acids among all compared capsids; orange indicates that one of the compared sequences substitutes a different amino acid; green indicates that two of the compared sequences substitute a different amino acid; and blue indicates that three or more of the compared sequences substitute a different amino acid. [Figure 4F]Figures 4A-4J show the alignment of AAV variants SL1.2, SL1.2L, SL1.2B, SL1.2LB, SL2, SL2L, SL2B, SL2LB, SL3, SL3L, SL3B, and SL3LB with other commonly used AAV vectors, AAV8, AAV9, AAVrh10, AAVrh74, and AAVanc80L65. The "L" suffix refers to a single amino acid change reported to result in less efficient liver targeting. The "B" suffix refers to eight amino acid substitutions / deletions reported to impair blood-brain barrier crossing. The "LB" suffix refers to a single amino acid change reported to result in less efficient liver targeting (L) combined with eight amino acid substitutions / deletions reported to impair blood-brain barrier crossing. Red indicates identical amino acids among all compared capsids; orange indicates that one of the compared sequences substitutes a different amino acid; green indicates that two of the compared sequences substitute a different amino acid; and blue indicates that three or more of the compared sequences substitute a different amino acid. [Figure 4G]Figures 4A-4J show the alignment of AAV variants SL1.2, SL1.2L, SL1.2B, SL1.2LB, SL2, SL2L, SL2B, SL2LB, SL3, SL3L, SL3B, and SL3LB with other commonly used AAV vectors, AAV8, AAV9, AAVrh10, AAVrh74, and AAVanc80L65. The "L" suffix refers to a single amino acid change reported to result in less efficient liver targeting. The "B" suffix refers to eight amino acid substitutions / deletions reported to impair blood-brain barrier crossing. The "LB" suffix refers to a single amino acid change reported to result in less efficient liver targeting (L) combined with eight amino acid substitutions / deletions reported to impair blood-brain barrier crossing. Red indicates identical amino acids among all compared capsids; orange indicates that one of the compared sequences substitutes a different amino acid; green indicates that two of the compared sequences substitute a different amino acid; and blue indicates that three or more of the compared sequences substitute a different amino acid. [Figure 4H]Figures 4A-4J show the alignment of AAV variants SL1.2, SL1.2L, SL1.2B, SL1.2LB, SL2, SL2L, SL2B, SL2LB, SL3, SL3L, SL3B, and SL3LB with other commonly used AAV vectors, AAV8, AAV9, AAVrh10, AAVrh74, and AAVanc80L65. The "L" suffix refers to a single amino acid change reported to result in less efficient liver targeting. The "B" suffix refers to eight amino acid substitutions / deletions reported to impair blood-brain barrier crossing. The "LB" suffix refers to a single amino acid change reported to result in less efficient liver targeting (L) combined with eight amino acid substitutions / deletions reported to impair blood-brain barrier crossing. Red indicates identical amino acids among all compared capsids; orange indicates that one of the compared sequences substitutes a different amino acid; green indicates that two of the compared sequences substitute a different amino acid; and blue indicates that three or more of the compared sequences substitute a different amino acid. [Figure 4I]Figures 4A-4J show the alignment of AAV variants SL1.2, SL1.2L, SL1.2B, SL1.2LB, SL2, SL2L, SL2B, SL2LB, SL3, SL3L, SL3B, and SL3LB with other commonly used AAV vectors, AAV8, AAV9, AAVrh10, AAVrh74, and AAVanc80L65. The "L" suffix refers to a single amino acid change reported to result in less efficient liver targeting. The "B" suffix refers to eight amino acid substitutions / deletions reported to impair blood-brain barrier crossing. The "LB" suffix refers to a single amino acid change reported to result in less efficient liver targeting (L) combined with eight amino acid substitutions / deletions reported to impair blood-brain barrier crossing. Red indicates identical amino acids among all compared capsids; orange indicates that one of the compared sequences substitutes a different amino acid; green indicates that two of the compared sequences substitute a different amino acid; and blue indicates that three or more of the compared sequences substitute a different amino acid. [Figure 4J]Figures 4A-4J show the alignment of AAV variants SL1.2, SL1.2L, SL1.2B, SL1.2LB, SL2, SL2L, SL2B, SL2LB, SL3, SL3L, SL3B, and SL3LB with other commonly used AAV vectors, AAV8, AAV9, AAVrh10, AAVrh74, and AAVanc80L65. The "L" suffix refers to a single amino acid change reported to result in less efficient liver targeting. The "B" suffix refers to eight amino acid substitutions / deletions reported to impair blood-brain barrier crossing. The "LB" suffix refers to a single amino acid change reported to result in less efficient liver targeting (L) combined with eight amino acid substitutions / deletions reported to impair blood-brain barrier crossing. Red indicates identical amino acids among all compared capsids; orange indicates that one of the compared sequences substitutes a different amino acid; green indicates that two of the compared sequences substitute a different amino acid; and blue indicates that three or more of the compared sequences substitute a different amino acid.

[0020] [Figure 5] Figure 5 depicts a "tree diagram" of AAV variants SL1.2, SL1.2L, SL1.2B, SL1.2LB, SL2, SL2L, SL2B, SL2LB, SL3, SL3L, SL3B, SL3LB, AAV8, AAV9, AAVrhlO, AAVrh74 and AAVanc80L65 based on the sequences of their capsid proteins. [Figure 6] Figure 6 shows the % sequence homology / divergence of AAV variants SL1.2, SL1.2L, SL1.2B, SL1.2LB, SL2, SL2L, SL2B, SL2LB, SL3, SL3L, SL3B, SL3LB, AAV8, AAV9, AAVrhlO, AAVrh74 and AAVanc80L65.

[0021] [Figure 7]Figure 7 shows Western blot quantification of the blot shown in Figure 8. Delivery of three variants (SL1.2, SL2, and SL3) is compared with uptake of the native serotypes AAV9 and AAVrh10 and the engineered variant AAVanc80L65. Experiments were performed using a known subsaturating dose (1 x 10 vg / kg) administered via tail vein systemic delivery to 6-month-old DBA / 2J male mice. Green fluorescent protein (GFP) was used as a reporter protein under the control of the beta-actin promoter for expression in all tissues. Variant SL2 has improved cardiac tissue uptake compared to the control serotype, while SL3 has uptake more similar to AAV9. SL3 has the highest uptake in skeletal muscle and brain of any of the AAV serotypes tested.

[0022] [Figure 8] FIG. 8 shows the Western blot data corresponding to the quantification shown in FIG. [Figure 9] Figure 9 shows the superiority of SL3 in uptake in both skeletal and cardiac muscle. Western blots and quantification of heart and diaphragm are shown. Data were obtained by injecting 1 x 10 vg / kg of AAV9, SL1.2, SL2, or SL3 via the tail vein (systemic delivery) into 6-month-old DBA / 2J male mice. Green fluorescent protein (GFP) was used as a reporter protein under the control of the beta-actin promoter for expression in all tissues.

[0023] [Figure 10] Figure 10 shows the superiority of SL3 in uptake in both skeletal and cardiac muscle. Western blot and quantification of quadriceps muscle are shown. Data were obtained by injecting 1 x 10 vg / kg of AAV9, SL1.2, SL2, or SL3 via the tail vein (systemic delivery) into 6-month-old DBA / 2J male mice. Green fluorescent protein (GFP) was used as a reporter protein under the control of the beta-actin promoter for expression in all tissues.

[0024] [Figure 11] Figure 11 shows the distribution of green fluorescent protein (GFP; brightness) in skeletal muscle and heart after systemic injection of AAV9, SL1.2, SL2, or SL3 at 5 x 10 vg / kg or 1 x 10 vg / kg. The pattern of expression supports the conclusions of the Western blot data and indicates that SL3 is a superior vector for transduction of heart and skeletal muscle. DETAILED DESCRIPTION OF THE INVENTION

[0025] Detailed Description AAV-derived vectors are promising tools for human gene therapy applications due to their low pathogenicity, episomal localization, and stable transgene expression compared to other vectors. AAV particles show great potential for the delivery of therapeutic genes. The organ or tissue tropism of AAV particles is highly, if not completely, dependent on the structure of the particle surface or capsid. Disclosed herein are AAV variants that modify the tropism and / or efficiency of AAV transduction compared to AAV8. These vectors can be used in clinical settings for either veterinary or human use. Tissue tropism can vary depending on mammalian species. The basis for differences in tissue tropism and efficiency is mainly due to variations in the sequence of surface loops.

[0026] AAV serotype 8 (AAV8) has tropism for skeletal muscle, retinal pigment epithelium, photoreceptor cells, cardiac tissue, and hepatocytes and can be used to deliver genes to these. Typical for AAVs, the AAV8 capsid is composed of three proteins, VP1, VP2, and VP3, which are the products of three distinct but overlapping transcripts of a single AAV cap gene. Provided herein are compositions and methods for variant recombinant AAV8-like capsid proteins and particles that alter the tropism and / or efficiency of AAV transduction compared to AAV8. The present disclosure is based, at least in part, on the identification of recombinant AAV (e.g., rAAV8) variant proteins and particles that have altered tissue tropism and altered transduction efficiency compared to wild-type rAAV (e.g., rAAV8) proteins and particles.

[0027] As used herein, the term "variant" refers to a nucleic acid or protein that has characteristics that deviate from those found in nature. For example, a "variant" is at least about 70% identical, at least about 80% identical, at least about 90% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, at least about 99% identical, at least about 99.5% identical, or at least about 99.9% identical to a wild-type nucleic acid or protein. For example, a transgene variant is a nucleic acid that contains one or more substitutions in the nucleotides of a transgene compared to its wild-type sequence. These substitutions may be silent, i.e., they do not modify the amino acid sequence of any encoded protein (or do not otherwise result in a variant amino acid sequence). Alternatively, these substitutions can result in modifications of the amino acid sequence of the encoded protein, resulting in the encoded protein having one or more amino acid substitutions (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 10-15, or 15-20 amino acid substitutions) compared to the wild-type protein sequence. These substitutions include chemical modifications as well as truncations. The term further encompasses functional fragments of wild-type nucleic acid or amino acid sequences. These modifications of the reference sequence can occur at the 5' or 3' end of the reference sequence, or elsewhere in between, individually among nucleotides or peptides in the reference sequence, or in one or more contiguous groups within the reference sequence.

[0028] In some embodiments, a variant recombinant adeno-associated virus (rAAV) capsid protein disclosed herein has a Y to F substitution at position 447 and a T to V substitution at position 494 made in a background containing serotype 8 (AAV8) VP1, VP2, or VP3. In some embodiments, the variant recombinant AAV (e.g., rAAV8) capsid protein comprises the amino acid sequence set forth in SEQ ID NO: 11. In some embodiments, the variant recombinant AAV (e.g., rAAV8) capsid protein has the amino acid sequence identified as the SL1.2 capsid protein in Figures 1A-1E. In some embodiments, the variant recombinant AAV (e.g., rAAV8) capsid protein comprises one or more of the following substitutions: K163S, R169K, S180T, L189I, T417Q, Y447F, T462Q, G464L, T494V, D559K, T591A, and Y733F. In one embodiment, the variant recombinant AAV (e.g., rAAV8) capsid protein comprises the substitutions K163S, R169K, S180T, L189I, T417Q, Y447F, T462Q, G464L, T494V, D559K, T591A, and Y733F.

[0029] In some embodiments, the variant recombinant AAV (e.g., rAAV8) capsid protein contains the following substitutions: A24D, D41N, Q84K, R92K, T158A, K163S, R169K, L189I, A195G, V199L, P201S, S225A, G264S, A269S, R313K, S315N, E350D, Q362E, Q413E, T415S, T417Q, Y447F, T453S, N459G, T and one or more of 462Q, G464L, N471S, T472N, A474S, N475A, T494V, A507G, G508A, N517D, A520V, T528S, D531E, D532G, N540S, N549G, A551G, A555V, D559K, E578Q, I581Q, T591A, Q594I, I595V, N665S, S667A, N670A, S712N, V722T, and Y733F. In one embodiment, the variant recombinant AAV (e.g., rAAV8) capsid protein comprises the substitutions A24D, D41N, Q84K, R92K, T158A, K163S, R169K, L189I, A195G, V199L, P201S, S225A, G264S, A269S, R313K, S315N, E350D, Q362E, Q413E, T415S, T417Q, Y447F, T453S, N459G, T In some embodiments, the variant recombinant AAV (e.g., rAAV8) comprises the amino acid sequence set forth in SEQ ID NO: 12. In some embodiments, the variant recombinant AAV (e.g., rAAV8) capsid protein has the amino acid sequence identified as the SL2 capsid protein in Figures 1A-1E.

[0030] In some embodiments, the variant rAAV capsid protein has a Y to F substitution at position 447 and a T to V substitution at position 494 made in a background comprising serotype 8 (AAV8) VP1, VP2, or VP3, and contains any one or more of the following substitutions: K31Q, D41N, G42A, Q84K, R92K, Q105K, T158A, K163S, S180T, P201S, S225A, G264S, A269S, R313K, S315N, E35 0D, Q362E, Q413E, T415S, T417Q, Y447F, T453S, N459G, T462Q, G464L, N471S, T472N, A474S, N475A, T494V, A507G, G508A, N517D, A520V, T528S, D531E, D532G, N540S, N549G, A551G, A555V, D559K, I581Q, Q588S, Q589A, Q594I, I595V, N665S, S667A, N670A, S712N, V722T and Y733F. In one embodiment, the variant recombinant AAV (e.g., rAAV8) capsid protein contains the substitutions K31Q, D41N, G42A, Q84K, R92K, Q105K, T158A, K163S, S180T, P201S, S225A, G264S, A269S, R313K, S315N, E350D, Q362E, Q413E, T415S, T417Q, Y447F, T453S, N459G, T462 In some embodiments, the variant recombinant AAV (e.g., rAAV8) comprises the amino acid sequence set forth in SEQ ID NO: 13. In some embodiments, the variant recombinant AAV (e.g., rAAV8) capsid protein has the amino acid sequence identified as the SL3 capsid protein in Figures 1A-1E.

[0031] AAV8 structure and capsid proteins The wild-type AAV genome is composed of single-stranded deoxyribonucleic acid (ssDNA) of either positive or negative sense. At each end of the DNA strand is an inverted terminal repeat (ITR). Between the ITRs are two open reading frames (ORFs): rep and cap. The rep ORF comprises four overlapping genes encoding the Rep proteins required for the AAV life cycle. The cap ORF contains overlapping nucleotide sequences of the capsid proteins VP1, VP2, and VP3, which interact together to form the icosahedral symmetric capsid.

[0032] Wild-type AAV8 amino acid sequence: (SEQ ID NO: 7)

[0033] Wild-type AAV9 amino acid sequence: (SEQ ID NO: 8)

[0034] AAVrh10 amino acid sequence: (SEQ ID NO: 9)

[0035] AAVrh74 amino acid sequence: (SEQ ID NO: 10)

[0036] AAVanc80L65 amino acid sequence: (SEQ ID NO: 32)

[0037] Variant recombinant AAV8 capsid proteins Provided herein is a nucleic acid encoding any one of the variant rAAV capsid proteins disclosed herein. In some embodiments, a plasmid comprises a nucleic acid encoding a variant rAAV capsid protein.

[0038] Some non-limiting examples of nucleic acids encoding variant AAV capsid proteins are provided in Table 1. These variants are referred to as SL1.2 (encoded by SEQ ID NO:1), SL1.2L (encoded by SEQ ID NO:14), SL1.2B (encoded by SEQ ID NO:20), SL1.2LB (encoded by SEQ ID NO:26), SL2 (encoded by SEQ ID NO:3), SL2L (encoded by SEQ ID NO:16), SL2B (encoded by SEQ ID NO:22), SL2LB (encoded by SEQ ID NO:28), SL3 (encoded by SEQ ID NO:5), SL3L (encoded by SEQ ID NO:18), SL3B (encoded by SEQ ID NO:24), and SL2LB (encoded by SEQ ID NO:28).

[0039] The tissue tropism and transduction efficiency of AAV particles are determined by the nature of the amino acid residues exposed on the surface of the capsid (Wu et al., J Virol. 2006, 80(22):11393-7). Therefore, manipulating the amino acids of the capsid protein provides an opportunity to fine-tune the tissue tropism of the particle and improve the transduction efficiency. However, certain manipulations of the capsid protein, such as amino acid substitutions, can cause the capsid to misfold or not form at all.

[0040] Disclosed herein is an rAAV variant that has increased transduction efficiency compared with wild-type AAV.In some embodiments, transduction efficiency is increased by removing the phosphorylation site that reduces the efficiency of AAV8.Disclosed herein is an rAAV8 variant that has sequence modification that prevents phosphorylation.Also disclosed herein is an rAAV variant that has altered tissue tropism compared with wild-type AAV.

[0041] In some embodiments, the rAAV variants disclosed herein have increased transduction efficiency and / or tropism in skeletal muscle compared to wild-type AAV. In some embodiments, the rAAV variants disclosed herein have increased transduction efficiency and / or tropism in cardiac tissue compared to wild-type AAV. In some embodiments, the rAAV variants disclosed herein have increased transduction efficiency and / or tropism in central nervous system compared to wild-type AAV. In some embodiments, the rAAV variants disclosed herein have increased transduction efficiency and / or tropism in cardiac tissue, but do not have enhanced transduction or tropism in CNS tissue. In some embodiments, the rAAV variants disclosed herein have increased transduction efficiency and / or tropism in CNS tissue, but do not have enhanced transduction or tropism in cardiac tissue.

[0042] Thus, it is useful to use the rAAV variants disclosed herein to deliver genes to the CNS, cardiac muscle and / or skeletal muscle tissue, for example, to treat a person with that tissue disease or condition.

[0043] Thus, provided herein are rAAV capsid proteins that contain alterations (e.g., substitutions) compared to the wild-type AAV8 sequence (e.g., as set forth in SEQ ID NO:7). In some embodiments, the amino acid substitutions in any one of the variant AAV capsid proteins disclosed herein are located in the variable region. In some embodiments, one or more amino acid substitutions belong to a recognized variable region or an exposed loop in the AAV capsid sequence. In some embodiments, all of the amino acid substitutions belong to a recognized variable region or an exposed loop in the AAV capsid sequence. It should be understood that any positioning of an amino acid as described herein is with respect to the sequence of the wild-type AAV8 sequence as set forth in SEQ ID NO:7.

[0044] Some non-limiting examples of variant AAV capsid proteins are provided in Table 1. These variants are referred to as SL1.2 (SEQ ID NO:11), SL1.2L (SEQ ID NO:15), SL1.2B (SEQ ID NO:21), SL1.2LB (SEQ ID NO:26), SL2 (SEQ ID NO:12), SL2L (SEQ ID NO:17), SL2B (SEQ ID NO:23), SL2LB (SEQ ID NO:28), SL3 (SEQ ID NO:13), SL3L (SEQ ID NO:19), SL3B (SEQ ID NO:25), and SL2LB (SEQ ID NO:31). [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5] [Table 1-6] [Table 1-7] [Table 1-8] [Table 1-9] [Table 1-10] [Table 1-11] [Table 1-12] [Table 1-13] [Table 1-14] [Table 1-15] [Table 1-16]

[0045] It should be understood that any one of the variant recombinant AAV (e.g., rAAV8) capsid proteins disclosed herein may include any single amino acid substitution described herein or any combination of the amino acid substitutions described herein.

[0046] For example, in some embodiments, SL1.2, SL2 and SL3 are further mutated. In some embodiments, SL1.2, SL2 and SL3 are further mutated to modify the tropism of the variant to specific tissues (e.g., heart, nerve or other muscle tissues). In some embodiments, further mutation of SL1.2, SL2 and SL3 improves the uptake of SL1.2, SL2 and / or SL3 in target tissues (e.g., heart, nerve or other muscle tissues). In some embodiments, further mutation of SL1.2, SL2 and SL3 reduces the uptake of SL1.2, SL2 and / or SL3 in non-target tissues (e.g., nerve or liver tissues).

[0047] In some embodiments, the capsid region spanning from amino acid Glu578 to Gly596 is further mutated in SL1.2, SL2, and / or SL3. In some embodiments, one amino acid is further mutated from asparagine 500 to isoleucine in SL1.2, SL2, and / or SL3. In some embodiments, the additional mutation introduced into any of SL1.2, SL2, and SL3 is as described in Pulicherla, et al., Mol. Ther. 19:6, 1070-78 (2011). In some embodiments, the mutation of asparagine 500 to isoleucine in L1.2, SL2, and / or SL3 results in reduced liver tissue tropism.

[0048] In some embodiments, a mutation of asparagine 500 to isoleucine in SL1.2 results in an AAV variant protein having the amino acid sequence of SEQ ID NO: 15 ("SL1.2L"). In some embodiments, a mutation of asparagine 500 to isoleucine in SL1.2 results in an AAV variant protein encoded by a nucleic acid having the sequence of SEQ ID NO: 14. In some embodiments, a mutation of asparagine 500 to isoleucine in SL2 results in an AAV variant protein having the amino acid sequence of SEQ ID NO: 17 ("SL2L"). In some embodiments, a mutation of asparagine 500 to isoleucine in SL2 results in an AAV variant protein encoded by a nucleic acid having the sequence of SEQ ID NO: 16. In some embodiments, a mutation of asparagine 500 to isoleucine in SL3 results in an AAV variant protein having the amino acid sequence of SEQ ID NO: 19 ("SL3L"). In some embodiments, the mutation of asparagine 500 to isoleucine in SL3 results in an AAV variant protein encoded by a nucleic acid having a sequence encoded by a nucleic acid having the sequence of SEQ ID NO:18.

[0049] In some embodiments, seven amino acids are further mutated in SL1.2: asparagine 263 to serine; glycine 264 to alanine; threonine 265 to serine; serine 266 to threonine; deletion of glycine 268; threonine 270 to serine; and threonine 274 to histidine ("seven amino acid mutations in SL1.2"). In some embodiments, eight amino acids are further mutated in SL2: asparagine 263 to serine; serine 264 to alanine; threonine 265 to serine; serine 266 to threonine; deletion of glycine 268; serine 269 to alanine; threonine 270 to serine; and threonine 274 to histidine ("eight amino acid mutations in SL2"). In some embodiments, eight amino acids are further mutated in SL3: asparagine 263 to serine; serine 264 to alanine; threonine 265 to serine; serine 266 to threonine; deletion of glycine 268; serine 269 to alanine; threonine 270 to serine; and threonine 274 to histidine ("eight amino acid mutations in SL3"). In some embodiments, the additional mutations introduced into any of SL1.2, SL2, and SL3 are those described in Albright, et al., Mol. Ther. 26:2, 510-23 (2018). In some embodiments, seven amino acid mutations in SL1.2 or eight amino acid mutations in SL2 and / or SL3 result in reduced brain tissue tropism.

[0050] In some embodiments, seven amino acid mutations in SL1.2 result in an AAV variant protein having the amino acid sequence of SEQ ID NO:21 ("SL1.2B"). In some embodiments, seven amino acid mutations in SL1.2 result in an AAV variant protein encoded by a nucleic acid having the sequence of SEQ ID NO:20. In some embodiments, eight amino acid mutations in SL2 result in an AAV variant protein having the amino acid sequence of SEQ ID NO:23 ("SL2B"). In some embodiments, eight amino acid mutations in SL2 result in an AAV variant protein encoded by a nucleic acid having the sequence of SEQ ID NO:22. In some embodiments, eight amino acid mutations in SL3 result in an AAV variant protein having the amino acid sequence of SEQ ID NO:25 ("SL3B"). In some embodiments, eight amino acid mutations in SL3 result in an AAV variant protein encoded by a nucleic acid having the sequence of SEQ ID NO:24.

[0051] In some embodiments, eight amino acids are further mutated in SL1.2: asparagine 500 to isoleucine; asparagine 263 to serine; glycine 264 to alanine; threonine 265 to serine; serine 266 to threonine; deletion of glycine 268; threonine 270 to serine; and threonine 274 to histidine ("seven amino acid mutations in SL1.2"). In some embodiments, the eight amino acid mutations in SL1.2 result in a reduced tropism for liver and brain tissue. In some embodiments, the eight amino acid mutations in SL1.2 result in an AAV variant protein having the amino acid sequence of SEQ ID NO: 27 ("SL1.2LB"). In some embodiments, the eight amino acid mutations in SL1.2 result in an AAV variant protein encoded by a nucleic acid having the sequence of SEQ ID NO: 26.

[0052] In some embodiments, nine amino acids are further mutated in SL2: asparagine 500 to isoleucine; asparagine 263 to serine; serine 264 to alanine; threonine 265 to serine; serine 266 to threonine; deletion of glycine 268; serine 269 to alanine; threonine 270 to serine; and threonine 274 to histidine ("nine amino acid mutations in SL2"). In some embodiments, the nine amino acid mutations in SL2 result in a reduced tropism for liver and brain tissue. In some embodiments, the nine amino acid mutations in SL2 result in an AAV variant protein having the amino acid sequence of SEQ ID NO: 29 ("SL2LB"). In some embodiments, the nine amino acid mutations in SL2 result in an AAV variant protein encoded by a nucleic acid having the sequence of SEQ ID NO: 28.

[0053] In some embodiments, nine amino acids are further mutated in SL3: asparagine 500 to isoleucine; asparagine 263 to serine; serine 264 to alanine; threonine 265 to serine; serine 266 to threonine; deletion of glycine 268; serine 269 to alanine; threonine 270 to serine; and threonine 274 to histidine ("nine amino acid mutations in SL3"). In some embodiments, the nine amino acid mutations in SL3 result in a reduced tropism for liver and brain tissue. In some embodiments, the nine amino acid mutations in SL3 result in an AAV variant protein having the amino acid sequence of SEQ ID NO: 31 ("SL3LB"). In some embodiments, the nine amino acid mutations in SL3 result in an AAV variant protein encoded by a nucleic acid having the sequence of SEQ ID NO: 30.

[0054] Also contemplated herein are variant rAAV capsid proteins of serotypes other than serotype 8. In some embodiments, any one of the amino acid changes described herein is in a variable region of a capsid protein of a serotype other than serotype 8 that is homologous to a variable region of AAV8 (e.g., 1, 2, 3, 3B, 4, 5, 6, 7, 9, 10, 11, 12, or 13). In some embodiments, variant rAAV capsids may be made in the context of a cap gene of one serotype being delivered together with a rep gene of a different serotype.

[0055] Recombinant AAV vectors As used herein, the term "vector" can refer to a nucleic acid vector (e.g., a plasmid or a recombinant viral genome), a wild-type AAV genome, or a virus containing a viral genome. In some embodiments, the term "vector" can refer to a viral particle, such as an AAV viral particle.

[0056] The wild-type AAV genome is a single-stranded deoxyribonucleic acid (ssDNA) of either positive or negative sense. The genome contains two inverted terminal repeats (ITRs), one at each end of the DNA strand, and two open reading frames (ORFs): rep and cap, located between the ITRs. The rep ORF contains four overlapping genes encoding the Rep proteins required for the AAV life cycle. The cap ORF contains overlapping genes encoding capsid proteins: VP1, VP2, and VP3, which interact together to form the viral capsid. VP1, VP2, and VP3 are translated from a single mRNA transcript that can be spliced ​​in two different ways. Longer or shorter introns are excised, resulting in the formation of two mRNA isoforms: approximately 2.3 kb and approximately 2.6 kb long. The capsid is a supramolecular assembly of approximately 60 individual capsid protein subunits into a non-enveloped T-1 icosahedral lattice that can protect the AAV genome. The mature AAV capsid comprises VP1, VP2, and VP3 (molecular masses of approximately 87, 73, and 62 kDa, respectively) in a ratio of approximately 1:1:10.

[0057] Recombinant AAV (rAAV) particles may comprise a recombinant nucleic acid vector (hereinafter referred to as an "rAAV vector"), which may, at a minimum, comprise: (a) one or more heterologous nucleic acid regions comprising a sequence encoding a transgene; and (b) one or more regions comprising a sequence that facilitates integration of the heterologous nucleic acid region into the genome of a subject (optionally, together with one or more nucleic acid regions comprising sequences that facilitate expression). In some embodiments, the sequence that facilitates integration of the heterologous nucleic acid region into the genome of a subject (optionally, together with one or more nucleic acid regions comprising sequences that facilitate expression) is an inverted terminal repeat (ITR) sequence (e.g., a wild-type ITR sequence or an engineered ITR sequence) that flanks the one or more nucleic acid regions (e.g., the heterologous nucleic acid region).

[0058] In some embodiments, the rAAV nucleic acid vector comprises one or more transgenes operably linked to a promoter, each of which comprises a sequence encoding a protein or polypeptide of interest, wherein the one or more transgenes are flanked on each side by ITR sequences. In some embodiments, the nucleic acid vector further comprises a region encoding a Rep protein as described herein, operably linked to a promoter (either in the region adjacent to the ITR or outside the region or nucleic acid). The ITR sequences may be derived from any AAV serotype (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10), or from more than one serotype. In some embodiments, the ITR sequences are derived from AAV2 or AAV6 serotypes. In some embodiments, the first serotype provided herein is not AAV2 or AAV8 serotype. In some embodiments, the ITR sequences of the first serotype are derived from AAV3, AAV5, or AAV6. In some embodiments, the ITR sequences are derived from AAV2, AAV3, AAV5, or AAV6. In some embodiments, the ITR sequences are of the same serotype as the capsid (e.g., AAV6 ITR sequences and an AAV6 capsid, etc.). In some embodiments, the ITR sequences are derived from the AAVrh.10 serotype.

[0059] In some embodiments, recombinant AAV (e.g., rAAV8) particles containing any one of the variant rAAV capsid proteins disclosed herein comprise the ITRs and / or rep ORF of serotype 8. In some embodiments, the rAAV particles are pseudotyped rAAV particles, which include (a) a capsid comprising a capsid protein containing a modification described herein made in the context of serotype 8, and (b) a nucleic acid vector comprising ITRs from another serotype (e.g., AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV9, or AAV10).

[0060] The particles in the rAAV particles or rAAV preparations disclosed herein can be of any AAV serotype, including any derivative or pseudotype (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 2 / 1, 2 / 5, 2 / 8, 2 / 9, 3 / 1, 3 / 5, 3 / 8, or 3 / 9). As used herein, the serotype of rAAV rAAV particles refers to the serotype of the capsid protein of the recombinant virus. In some embodiments, the rAAV particles are rAAV6 or rAAV9. Non-limiting examples of derivatives or pseudotypes include AAVrh.10, AAVrh.74, AAV2 / 1, AAV2 / 5, AAV2 / 6, AAV2 / 8, AAV2 / 9, AAV2-AAV3 hybrid, AAVhu.14, AAV3a / 3b, AAVrh32.33, AAV-HSC15, AAV-HSC17, AAVhu.37, AAVrh.8, CHt-P6, AA Examples include V2.5, AAV6.2, AAV2i8, AAV-HSC15 / 17, AAVM41, AAV9.45, AAV6(Y445F / Y731F), AAV2.5T, AAV-HAE1 / 2, AAV clone 32 / 83, AAVShH10, AAV2(Y->F), AAV8(Y733F), AAV2.15, AAV2.4, AAVM41, and AAVr3.45. Such AAV serotypes and derivatives / pseudotypes, as well as methods for producing such derivatives / pseudotypes, are known in the art (see, e.g., Mol Ther. 2012 Apr;20(4):699-708. doi: 10.1038 / mt.2011.287. Epub 2012 Jan 24. The AAV vector toolkit: poised at the clinical crossroads. Asokan A1, Schaffer DV, Samulski RJ.). In certain embodiments, the capsid of any of the rAAV particles disclosed herein is of the AAVrh.10 serotype. In some embodiments, the capsid is of the AAV2 / 6 serotype.In some embodiments, the rAAV particle is a pseudotyped rAAV particle, which includes (a) an rAAV vector that includes ITRs from one serotype (e.g., AAV2, AAV3), and (b) a capsid that comprises capsid proteins from another serotype (e.g., AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, or AAV10). Methods for generating and using pseudotyped rAAV vectors are known in the art (see, e.g., Duan et al., J. Virol., 75:7662-7671, 2001; Halbert et al., J. Virol., 74:1524-1532, 2000; Zolotukhin et al., Methods, 28:158-167, 2002; and Auricchio et al., Hum. Molec. Genet., 10:3075-3081, 2001).

[0061] ITR sequences and plasmids containing ITR sequences are known in the art and commercially available (e.g., products and services available from Vector Biolabs, Philadelphia, PA; Cellbiolabs, San Diego, CA; Agilent Technologies, Santa Clara, CA; and Addgene, Cambridge, MA; and Gene delivery to skeletal muscle results in sustained expression and systemic delivery of a therapeutic protein. Kessler PD, et al. Proc Natl Acad Sci U S A. 1996 Nov 26;93(24):14082-7; and Curtis A. Machida. Methods in Molecular Medicine™. Viral Vectors for Gene Therapy Methods and Protocols. 10.1385 / 1-59259-304-6:201 (Copyright) Humana Press Inc. 2003. Chapter 10. Targeted Integration by Adeno-Associated Virus. Matthew D. Weitzman, Samuel M. (See Young Jr., Toni Cathomen and Richard Jude Samulski; U.S. Patent Nos. 5,139,941 and 5,962,313; all of which are incorporated herein by reference.) In some embodiments, the rAAV comprises a pTR-UF-11 plasmid backbone, a plasmid containing the AAV2 ITRs. This plasmid is commercially available from the American Type Culture Collection (ATCC MBA-331).

[0062] Provided herein are variant recombinant AAV (e.g., rAAV8) particles. In some embodiments, the particles are empty particles (e.g., those that do not contain a nucleic acid vector containing a gene of interest). In some embodiments, the AAV8 particles contain a nucleic acid vector containing a gene of interest. As used herein, a "gene of interest" refers to a gene that encodes a target RNA or protein.

[0063] Thus, in some embodiments, the rAAV vector includes one or more regions containing sequences that promote expression of a gene of interest, e.g., an expression control sequence operably linked to the nucleic acid. Many such sequences are known in the art. Non-limiting examples of expression control sequences include promoters, insulators, silencers, response elements, introns, enhancers, initiation sites, internal ribosome entry site (IRES) termination signals, and poly(A) signals. Any combination of such control sequences is contemplated herein (e.g., promoters and enhancers). In some embodiments, the rAAV vector includes a promoter operably linked to the coding sequence of a gene of interest to promote expression of the gene of interest. As used herein, "promoter" refers to a nucleic acid control region in which the initiation and rate of transcription of the remainder of the nucleic acid sequence are controlled. A promoter drives transcription of the nucleic acid sequence it controls; therefore, it is typically located at or near the transcription start site of the gene. A promoter may have a length of, for example, 100 to 1,000 nucleotides. In some embodiments, a promoter is operably linked to a nucleic acid, or sequence of a nucleic acid (nucleotide sequence). In some embodiments, one or more promoters may be operably linked to a coding nucleotide sequence in a heterologous nucleic acid. A promoter is considered to be "operably linked" to a nucleic acid sequence that it controls when it is in the correct functional location and orientation relative to the sequence that it controls and / or regulates (e.g., controls ("drives") transcription initiation and / or expression of that sequence). A promoter may be a constitutive promoter, a tissue-specific promoter, an inducible promoter, or a synthetic promoter.

[0064] For example, constitutive promoters of different strengths can be used. The nucleic acid vectors described herein may contain one or more constitutive promoters, such as viral promoters or promoters from mammalian genes, that are generally active in promoting transcription. Non-limiting examples of constitutive viral promoters include herpes simplex virus (HSV), thymidine kinase (TK), Rous sarcoma virus (RSV), simian virus 40 (SV40), mouse mammary tumor virus (MMTV), and Ad E1A cytomegalovirus (CMV) promoters. Non-limiting examples of constitutive mammalian promoters include various housekeeping gene promoters, such as those exemplified by the β-actin promoter (e.g., chicken β-actin promoter) and the human elongation factor-1α (EF-1α) promoter. In some embodiments, a chimeric viral / mammalian promoter may include a chimeric CMV / chicken beta-actin (CBA, CB, or CAG) promoter. In some embodiments, a shortened or truncated promoter is used.

[0065] Inducible promoters and / or regulatory elements can also be designed to achieve appropriate expression levels of the target protein or polypeptide.Non-limiting examples of suitable inducible promoters include those from genes such as cytochrome P450 genes, heat shock protein genes, metallothionein genes, and hormone-inducible genes such as estrogen gene promoters.Another example of an inducible promoter is the tetVP16 promoter, which is responsive to tetracycline.

[0066] Tissue-specific promoters and / or regulatory elements are also contemplated herein. In some embodiments, the promoter may be a tissue-specific promoter. As used herein, the term "tissue-specific promoter" refers to a promoter that can function only in a specific type of tissue, such as the heart. Thus, a "tissue-specific promoter" cannot drive the expression of a transgene in other types of tissue. In some embodiments, it may be beneficial to combine a variant rAAV particle as disclosed herein with a promoter that targets the same cells, tissues, or organs as the variant rAAV particle. For example, for a variant with improved cardiac tropism, it may be appropriate to use the cardiac troponin T promoter to achieve cardiac-specific expression.

[0067] In some embodiments, the promoter that can be used according to the present disclosure can include any promoter that can drive the expression of a transgene in the heart of a subject.In some embodiments, the promoter that can be used according to the present disclosure is a heart-restricted promoter.For example, the promoter is a heart-restricted promoter selected from cardiac troponin C, cardiac troponin I, and cardiac troponin T (cTnT).

[0068] Alternatively, the promoter may be a promoter from one of the following genes, without limitation: α-myosin heavy chain gene, 6-myosin heavy chain gene, myosin light chain 2v (MLC-2v) gene, myosin light chain 2a gene, CARP gene, cardiac α-actin gene, cardiac m2 muscarinic acetylcholine receptor gene, ANF, cardiac troponin C, cardiac troponin I, cardiac troponin T (cTnT), cardiac sarcoplasmic reticulum Ca-ATPase gene, skeletal α-actin; or an artificial cardiac promoter derived from the MLC-2v gene.

[0069] In some embodiments, the rAAV vector of the present disclosure further comprises a polyadenylation (pA) signal. Eukaryotic mRNA is typically transcribed as a precursor mRNA. The precursor mRNA is processed to produce a mature mRNA, which involves the polyadenylation process. The polyadenylation process begins with the termination of gene transcription. The 3'-most segment of the newly made precursor mRNA is first cleaved by a set of proteins. These proteins then synthesize a poly(A) tail at the 3' end of the RNA. The cleavage site typically contains a polyadenylation signal, such as AAUAAA. The poly(A) tail is important for the nuclear transport, translation, and stability of mRNA.

[0070] In some embodiments, the rAAV vectors of the present disclosure include at least, in 5' to 3' order, a first adeno-associated virus (AAV) inverted terminal repeat (ITR) sequence, a promoter operably linked to a first transgene, an IRES operably linked to a second transgene, a polyadenylation signal, and a second AAV inverted terminal repeat (ITR) sequence.

[0071] In some embodiments, the rAAV is circular. In some embodiments, the rAAV vector is linear. In some embodiments, the rAAV vector is single-stranded. In some embodiments, the rAAV vector is double-stranded. In some embodiments, the rAAV vector is a self-complementary rAAV vector. Any rAAV vector described herein may be encapsidated with a viral capsid such as an AAV6 capsid or any other serotype (e.g., a serotype of the same serotype as the ITR sequence).

[0072] The protein of interest may be a detectable marker or a therapeutic protein. The detectable marker may be a molecule that can be visualized (e.g., with the naked eye or under a microscope). In some embodiments, the detectable marker is a fluorescent molecule, a bioluminescent molecule, or a molecule that provides color (e.g., β-galactosidase, β-lactamase, β-glucuronidase, and spheroidenone). In some embodiments, the detectable marker is a fluorescent protein or a functional peptide or polypeptide thereof.

[0073] In some embodiments, the gene of interest encodes a therapeutic protein and is referred to as a "therapeutic gene." A therapeutic gene can provide a therapeutic effect in cells, tissues, or organs to which it is delivered. In some embodiments, the therapeutic gene encodes an antibody, a peptibody, a growth factor, a coagulation factor, a hormone, a membrane protein, a cytokine, a chemokine, an activating or inhibitory peptide acting on cell surface receptors or ion channels, a cell-penetrating peptide targeting intracellular processes, a thrombolytic agent, an enzyme, a bone morphogenetic protein, a nuclease or other protein used for gene editing, an Fc-fusion protein, an anticoagulant, a nuclease, a guide RNA, or other nucleic acid or protein for gene editing. In some embodiments, the gene of interest encodes a therapeutic RNA, for example, a small interfering RNA.

[0074] In some embodiments, the therapeutic gene (e.g., gene of interest) is a cardioprotective gene. Several cardioprotective genes are known, including heme oxygenase-1. This protein degrades the pro-oxidant heme to produce carbon monoxide and the antioxidant bilirubin, protecting the myocardium from ischemia / reperfusion injury (Franz et al., Circ. Res. 73:629-638, 1993). In inflammatory diseases, HO-1 is increased as a cytoprotective gene. However, it is usually in amounts insufficient to stop inflammation. A vigilant vector can provide amplified amounts of HO-1 when reduced oxygen indicates a need for HO-1. Another example of a cardioprotective gene is superoxide dismutase, which protects cardiac tissue from superoxide radicals generated during ischemia-reperfusion (Chen et al., Circulation 94:11412-11417, 1996; and Woo et al., Circulation 98:11255-11260, 1998). Genes that provide protective effects against other cardiac disease states, such as cardiac degeneration and heart failure, may also be used in the vectors of the present invention. An example of a gene that improves cardiac function is phospholamban (PLN). The PLN gene product regulates the strength of each heartbeat and is known to malfunction in heart failure (Zvaritch et al., J. Biol. Chem. 275:14985-14991, 2000). Any suitable cardioprotective gene that provides a therapeutic level of protection may be used in the vectors of the present invention.

[0075] In some embodiments, a cardioprotective gene is a gene important for a signaling pathway in cardioprotection. In some embodiments, the cardioprotective gene is any one of the following: protectomiRs (e.g., microRNA 125b*), ZAC1 transcription factor, pro-inflammatory genes such as phospholamban (PLN), cyclooxygenase (COX)-2 and inducible nitric oxide synthase (iNOS), antioxidant enzymes such as heme oxygenase (HO)-1, extracellular superoxidase dismutase and manganese superoxidase dismutase (ec-SOD and Mg-SOD), heat shock proteins (HSPs), growth factors such as insulin-like growth factor (IGF)-1 and hepatocyte growth factor (HGF), anti-apoptotic proteins such as Bcl-2 and Bcl-xL, pro-apoptotic proteins such as FasL, Bcl-2, Bax, caspase-3 and p53, and pro-angiogenic genes such as TGF-beta, sphingosine kinase 1 (SPK1), apoptosis repressor with a caspase recruitment domain caspase recruitment domain (ARC), wild-type cardiac troponin T, wild-type cardiac myosin-binding protein C, myosin light chain (either regulatory or essential), PI3K-Akt, and / or S100 variants.

[0076] S100 family proteins that may be used in accordance with the present disclosure include, without limitation, S100A1, S100A2, S100A3, S100A4, S100A5, S100A6, S100A7 (e.g., psoriasin), S100A8 (e.g., calgranulin A), S100A9 (e.g., calgranulin B), S100A10, S100A11, S100A12 (e.g., calgranulin C), S100A13, S100A14, S100A15 (e.g., koebnerisin), S100A16, S100B, S100P, and S100Z, or variants thereof.

[0077] In some embodiments, the S100 family protein may be S100 calcium-binding protein A1 (S100A1). In some embodiments, the S100A1 is cardiac S100A1 (cS100A1) or a variant thereof. The cS100A1 protein is a regulator of myocardial contractility. cS100A1 protein levels are decreased in right ventricular hypertrophy tissue in a model of pulmonary hypertension. Furthermore, S100A1 is a regulator of the genetic program underlying cardiac hypertrophy in that it inhibits alpha-1 adrenergic stimulation of hypertrophy genes, including MYH7, ACTA1, and S100B.

[0078] In cardiomyocytes, S100A1 binds to ryanodine receptor 2 (RYR2), the Ca2+ receptor in the sarco / endoplasmic reticulum. + S100A1 regulates the calcium-regulated network of SR, sarcomere, and mitochondrial function through regulation of the activity of SR Ca2+-ATPase (SERCA), titin, and mitochondrial F1-ATPase. Consequently, cardiomyocytes and hearts with increased S100A1 expression exhibit increased contractile and relaxation capacity, which is due to increased SR Ca2+ + Load and subsequent systolic Ca + In addition to enhanced release, SR Ca2 + Reduced leakage and Ca2 + Ca2 resulting from enhanced resequestration + This results in improved transient amplitude. At the same time, S100A1 increases mitochondrial high-energy phosphate production, thereby providing energy to cardiomyocytes via Ca2+ + This is coordinated with an increased adenosine 5'-triphosphate (ATP) requirement due to enhanced turnover. Reduced S100A1 expression in cardiomyocytes is associated with impaired contractile function, demonstrating the pathophysiological importance of this protein.

[0079] In some embodiments, the S100A1 cDNA (transgene) sequence of any of the polynucleotides of the disclosed rAAV vectors has 100% identity to the naturally occurring S100A1 sequence of human origin. In other embodiments, the S100A1 cDNA sequence has at least about 70% identity, at least about 80% identity, at least about 90% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, at least about 99% identity, at least about 99.5% identity, or at least about 99.9% identity to the naturally occurring S100A1 sequence.

[0080] In some embodiments, the S100A1 cDNA sequence is codon-optimized for expression in human cells, hi other embodiments, the S100A1 cDNA sequence is codon-optimized for expression in canine cells.

[0081] Aspects of the present disclosure provide compositions and methods that include the delivery of genes encoding apoptosis inhibitors (e.g., anti-apoptotic agents). Illustrative examples of apoptosis inhibitors include antagonists of fink, p35, crmA, Bcl-2, Bcl-XL, Mcl-1, E1B-19K, and pro-apoptotic agents (e.g., antisense, ribozyme, antibody, etc.) from adenovirus. In some embodiments, the apoptosis inhibitor is cardiac apoptosis repressor with caspase recruitment domain (ARC) or a variant thereof. In other embodiments, the apoptosis inhibitor is cardiac ARC or a variant thereof. In some embodiments, it may be desirable to deliver S100 family proteins and apoptosis inhibitors separately. In certain embodiments, genes encoding S100 family proteins are delivered simultaneously or sequentially with one or more small molecule apoptosis inhibitors. Other exemplary small molecule apoptosis inhibitors include c-Myc inhibitors, Bax inhibitors, p53 inhibitors, tBid inhibitors, caspase inhibitors, and inhibitors of pro-apoptotic BCL-2 family members.

[0082] cARC is an apoptosis-regulating protein expressed almost exclusively in myogenic cells. It contains a caspase recruitment domain (CARD), through which it blocks the activation of several initiator caspases. ARC also blocks caspase-independent events associated with apoptosis. Apoptosis caused by acute ischemia and subsequent ventricular remodeling has been implicated as a mediator of heart failure. Although postischemic heart failure can have multiple causes, recent attention has been directed toward understanding the contribution of apoptosis, or programmed cell death. Apoptosis is characterized by preservation of mitochondrial and sarcolemmal membranes, nuclear chromatin condensation, and phagocytosis by macrophages or nearby cells without eliciting an inflammatory response. Activation of apoptosis is known to occur through a mechanism involving caspases, a family of cysteine ​​proteases that are synthesized as inactive precursors and proteolytically cleaved to their active forms. ARC can block apoptosis activation by blocking caspases.

[0083] In certain embodiments, the cARC cDNA sequence is codon-optimized for expression in human cells. In other embodiments, the cARC cDNA sequence is codon-optimized for expression in canine cells.

[0084] In some embodiments, the gene of interest encodes an antisense molecule.Many antisense molecules that confer cardioprotective effects are known, including the antisense to angiotensin II type 1 receptor (Yang et al., Circulation 96:922-926, 1997; and Yang et al., Circ. Res. 83:552-559, 1998), the antisense to adrenergic beta-1 receptor (Chen et al., Pharmacol. Exp. Ther. 294:722-727, 2000), and the antisense to angiotensin converting enzyme (Chen et al., Pharmacol. Exp. Ther. 294:722-727, 2000), which has been shown to protect rat hearts from ischemia-reperfusion. In some embodiments, the antisense molecule is antisense to the angiotensin II type 1 receptor, antisense to the adrenergic beta-1 receptor, or antisense to angiotensin converting enzyme.

[0085] In some embodiments, the nucleic acid vector contained in the rAAV (e.g., rAAV8) particle comprises one or more of the following: (a) one or more heterologous nucleic acid regions comprising a gene of interest, and (b) one or more regions comprising inverted terminal repeat (ITR) sequences (e.g., wild-type ITR sequences or engineered ITR sequences) flanking the one or more nucleic acid regions (e.g., heterologous nucleic acid regions). In some embodiments, the nucleic acid vector in the rAAV particle comprises one or more nucleic acid regions comprising regulatory sequences that promote expression of the heterologous nucleic acid region (e.g., promoter). In some embodiments, the nucleic acid vector in the recombinant AAV (e.g., rAAV8) particle comprises one or more nucleic acid regions comprising sequences that promote integration of the heterologous nucleic acid region into the genome of a subject (optionally together with one or more nucleic acid regions comprising sequences that promote expression).

[0086] In some embodiments, the AAV is a fully constructed AAV containing a modified cap gene according to the present invention, or a modified AAV capsid according to the present invention carrying a transgene with capsid proteins supplied in trans. In some embodiments, empty capsids containing the disclosed modified capsid proteins can be used to pretreat prior to use of filled AAV capsids to further enhance transduction.

[0087] Listed in Table 1 are exemplary rAAV capsid proteins or related gene sequences of the present disclosure. The rAAV capsid proteins shown in Table 1 include the amino acid sequences set forth as SEQ ID NOs: 11-13, 15, 17, 19, 21, 23, 25, 27, 29, and 31.

[0088] The rAAV capsid proteins of the present disclosure may comprise amino acid sequences having at least 70% identity, at least about 80% identity, at least about 90% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, at least about 99% identity, at least about 99.5% identity, or at least about 99.9% identity to the sequences set forth as SEQ ID NOs: 11-13, 15, 17, 19, 21, 23, 25, 27, 29, and 31.

[0089] The rAAV capsid proteins set forth in Table 1 are encoded by polynucleotides having the sequences set forth as SEQ ID NOs: 1, 3, 5, 14, 16, 18, 20, 22, 24, 26, 28, and 30. The rAAV capsid proteins of the present disclosure may be encoded by a nucleic acid sequence having at least 70% identity, at least about 80% identity, at least about 90% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, at least about 99% identity, at least about 99.5% identity, or at least about 99.9% identity to the sequences set forth as SEQ ID NOs: 1, 3, 5, 14, 16, 18, 20, 22, 24, 26, 28, and 30.

[0090] As a practical matter, whether any particular nucleic acid molecule is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to, for example, the nucleotide sequence of a transgene can be routinely determined using known computer programs. A preferred method for determining the best overall match between a query sequence (e.g., a sequence of the present disclosure) and a subject sequence, also referred to as a global sequence alignment, can be determined using the FASTDB or blastn computer program based on the algorithm of Brutlag et al. (Comp. App. Biosci. 6:237-245 (1990)). In sequence alignment, the query sequence and the subject sequence are both nucleotide sequences or both amino acid sequences. The result of the global sequence alignment is expressed as percent identity. Preferred parameters used in FASTDB amino acid alignment are as follows: Matrix=PAM 0, k-tuple=2, Mismatch Penalty=1, Joining Penalty=20, Randomization Group Length=0, Cutoff Score=1, Window Size=length of sequence, Gap Penalty=5, Gap Size Penalty=0.05, Window Size=500, or the length of the subject amino acid sequence, whichever is shorter. Whether nucleotides match / align is determined by the results of the FASTDB sequence alignment. This percentage is then subtracted from the percent identity calculated by the FASTDB program above using the specified parameters to arrive at a final percent identity score. This final percent identity score is the one used for purposes of the present disclosure. For subject sequences that are truncated at the 5' and / or 3' end compared to the query sequence, the percent identity is corrected by calculating the number of nucleotides of the query sequence that are located 5' or 3' to the query sequence that are not matched / aligned with the corresponding subject nucleotides as a percentage of the total bases in the query sequence.

[0091] In some embodiments, any of the disclosed rAAV amino acid vector sequences comprises a truncation at the 5' or 3' end compared to any one of SEQ ID NOs: 11-13, 15, 17, 19, 21, 23, 25, 27, 29, and 31. In some embodiments, any of the rAAV vectors comprises an amino acid sequence that differs by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or more than 18 amino acids from any one of SEQ ID NOs: 11-13, 15, 17, 19, 21, 23, 25, 27, 29, and 31. In some embodiments, any of the disclosed rAAV nucleic acid vector sequences comprises a truncation at the 5' or 3' end compared to any one of SEQ ID NOs: 1, 3, 5, 14, 16, 18, 20, 22, 24, 26, 28, and 30. In some embodiments, any of the rAAV vectors is encoded by a nucleic acid sequence that differs by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or more than 18 nucleic acids from any one of SEQ ID NOs: 1, 3, 5, 14, 16, 18, 20, 22, 24, 26, 28, and 30.

[0092] How to make rAAV particles Further provided herein are methods for producing rAAV particles. The rAAV particles comprise a viral capsid and an rAAV vector as described herein encapsidated by the viral capsid. Various methods for producing rAAV particles and nucleic acid vectors are known in the art and commercially available (see, for example, Zolotukhin et al. Production and purification of serotype 1, 2, and 5 recombinant adeno-associated viral vectors. Methods 28 (2002) 158-167; and US Patent Publication Numbers US20070015238 and US20120322861, which are incorporated herein by reference; and plasmids and kits available from ATCC and Cell Biolabs, Inc.). In some embodiments, a vector (e.g., a plasmid) containing a gene of interest may be combined with one or more helper plasmids containing, for example, rep genes (e.g., encoding Rep78, Rep68, Rep52, and Rep40) and cap genes (encoding VP1, VP2, and VP3, including modified VP regions as described herein) and introduced into recombinant cells, referred to as helper or producer cells, such that the nucleic acid vector is packaged or encapsidated within the capsid and subsequently purified.

[0093] Non-limiting examples of mammalian helper cells include HEK293 cells, COS cells, HeLa cells, BHK cells, or CHO cells (e.g., ATCC® CRL-1573™, ATCC® CRL-1651™, ATCC® CRL-1650™, ATCC® CCL-2, ATCC® CCL-10™, or ATCC® CCL-61™). A non-limiting example of an insect helper cell is Sf9 cell (see, e.g., ATCC® CRL-1711™). Helper cells may contain rep and / or cap genes encoding Rep and / or Cap proteins. In some embodiments, packaging is performed in vitro (e.g., outside the cell).

[0094] In some embodiments, a nucleic acid vector (e.g., a plasmid) containing a heterologous gene is combined with one or more helper plasmids, such as one containing a rep gene of a first serotype and a cap gene of the same or a different serotype, and transfected into helper cells to package rAAV particles. In some embodiments, the one or more helper plasmids include a first helper plasmid containing the rep gene and the cap gene, and a second helper plasmid containing one or more of the following helper genes: E1a gene, E1b gene, E4 gene, E2a gene, and VA gene. For clarity, helper genes are genes encoding the helper proteins E1a, E1b, E4, E2a, and VA. Helper plasmids and methods for producing such plasmids are known in the art and are commercially available (e.g., pDF6, pRep, pDM, pDG, pDP1rs, pDP2rs, pDP3rs, pDP4rs, pDP5rs, pDP6rs, pDG(R484E / R585E), and pDP8.ape plasmids from PlasmidFactory, Bielefeld, Germany; other products and services available from Vector Biolabs, Philadelphia, PA; Cellbiolabs, San Diego, CA; Agilent Technologies, Santa Clara, CA; and Addgene, Cambridge, MA; pxx6; Grimm et al. (1998), Novel Tools for Production and Purification of Recombinant Adeno-associated Virus Vectors, Human Gene Therapy, Vol. 9, 2745-2760; Kern, A. et al. (2003), Identification of a Heparin-Binding Motif on Adeno-Associated Virus Type 2 Capsids, Journal of Virology, Vol. 77, 11072-11081.;Grimm et al.(2003), "Helper Virus-Free, Optically Controllable, and Two-Plasmid-Based Production of Adeno-Associated Virus Vectors of Serotypes 1 to 6," Molecular Therapy, Vol. 7, 839-850; Kronenberg et al. (2005), "A Conformational Change in the Adeno-Associated Virus Type 2 Capside Leads to the Exposure of Hidden VP1 N Termini," Journal of Virology, Vol. 79, 5296-5303; and Moullier, P. and Snyder, RO (2008), "International efforts for recombinant adeno-associated viral vector reference standards," Molecular Therapy, Vol. 16, 1185-1188). Plasmids encoding wild-type AAV coding regions for specific serotypes are also known and available. For example, Addgene catalog #37825-AAV8.T includes a helper plasmid containing the rep and wild-type AAV8 cap genes (https: / / www.addgene.org / 37825 / #37825-AAV8).

[0095] ITR sequences and plasmids containing ITR sequences are known in the art and commercially available (e.g., products and services available from Vector Biolabs, Philadelphia, PA; Cellbiolabs, San Diego, CA; Agilent Technologies, Santa Clara, CA; and Addgene, Cambridge, MA; and Gene delivery to skeletal muscle results in sustained expression and systemic delivery of a therapeutic protein. Kessler PD, Podsakoff GM, Chen X, McQuiston SA, Colosi PC, Matelis LA, Kurtzman GJ, Byrne BJ. Proc Natl Acad Sci U S A. 1996 Nov 26;93(24):14082-7; and Curtis A. Machida. Methods in Molecular Medicine™. Viral Vectors for Gene Therapy Methods and Protocols. 10.1385 / 1-59259-304-6:201 (Copyright) Humana Press Inc. 2003. Chapter 10. Targeted Integration by Adeno-Associated Virus. Matthew D. Weitzman, Samuel M. Young Jr., Toni Cathomen and Richard Jude Samulski; see U.S. Patent Nos. 5,139,941 and 5,962,313; all of which are incorporated herein by reference).

[0096] Genebank reference numbers for the sequences of AAV serotypes 1, 2, 3, 3B, 4, 5, 6, 7, 8, 9, 10, 11, 12 and 13 are listed in patent publication WO2012064960, which is incorporated by reference herein in its entirety.

[0097] Non-limiting methods for rAAV particle production are described below. One or more helper plasmids are generated or obtained, containing the rep and cap ORFs for the desired AAV serotype, as well as the adenoviral VA, E2A (DBP), and E4 genes under the transcriptional control of their native promoters. In some embodiments, the one or more helper plasmids contain the rep gene, the cap gene, and optionally one or more of the adenoviral VA, E2A (DBP), and E4 genes under the transcriptional control of their native promoters. In some embodiments, the one or more helper plasmids contain the cap ORF (and optionally the rep ORF) for the desired AAV serotype, as well as the adenoviral VA, E2A (DBP), and E4 genes under the transcriptional control of their native promoters. The cap ORF may also contain one or more modifications to generate modified capsid proteins as described herein. For example, HEK293 cells (available from ATCC®) are transfected with a helper plasmid and a plasmid containing a nucleic acid vector via CaPO4-mediated transfection, lipids, or polymeric molecules such as polyethyleneimine (PEI). The HEK293 cells are then incubated for at least 60 hours to produce rAAV particles. Alternatively, HEK293 cells are transfected with a helper plasmid containing genes encoding AAV-ITRs, Rep and Cap proteins, containing one or more genes of interest, via the above-mentioned method, and co-transfected with a helper virus. A helper virus is a virus that allows AAV replication. Examples of helper viruses are adenovirus and herpesvirus.

[0098] Alternatively, in another example, an Sf9-based stable producer cell line is infected with a single recombinant baculovirus containing a nucleic acid vector.As a further alternative, in another example, an HEK293 or BHK cell line is infected with an HSV containing a nucleic acid vector, and optionally one or more helper HSVs containing the rep and cap ORFs and adenovirus VA, E2A (DBP) and E4 genes as described herein under the transcriptional control of their native promoters.The HEK293, BHK or Sf9 cells are then incubated for at least 60 hours to produce rAAV particles.The rAAV particles can then be purified using any method known in the art or described herein, for example, by iodixanol step gradient, CsCl gradient, chromatography, or polyethylene glycol (PEG) precipitation.

[0099] Methods for large-scale production of AAV using herpesvirus-based systems are also known. See, for example, Clement et al. (Hum Gene Ther. 2009, 20(8):796-806). Methods for generating exosome-associated AAV that may be more resistant to neutralizing anti-AAV antibodies are also known (Hudry et al., Gene Ther. 2016, 23(4):380-92; Macguire et al., Mol Ther. 2012, 20(5):960-71).

[0100] Methods for generating and using pseudotyped rAAV vectors are also known in the art (see, e.g., Duan et al., J. Virol., 75:7662-7671, 2001; Halbert et al., J. Virol., 74:1524-1532, 2000; Zolotukhin et al., Methods, 28:158-167, 2002; and Auricchio et al., Hum. Molec. Genet., 10:3075-3081, 2001).

[0101] composition The present disclosure is directed to a composition comprising one or more of the disclosed rAAV particles or preparations.In some embodiments, the rAAV preparation comprises an rAAV particle comprising an rAAV vector containing ITRs of a first serotype (e.g., AAV3, AAV5, AAV6 or AAV9) and a capsid protein that encapsidates the rAAV vector.In some embodiments, the capsid protein is of the first serotype (e.g., AAV3, AAV5, AAV6 or AAV9).In some embodiments, the preparation has at least 4-fold higher transduction efficiency (e.g., in human hepatocellular carcinoma cell lines such as Huh7) than the preparation prepared using an rAAV vector containing AAV2 ITRs.

[0102] As described herein, such compositions may further comprise pharmaceutical excipients, buffers, or diluents and may be formulated for administration to host cells ex vivo or to animals in situ, and particularly to humans. Such compositions may further optionally comprise liposomes, lipids, lipid complexes, microspheres, microparticles, nanospheres, or nanoparticles and may be otherwise formulated for administration to cells, tissues, organs, or the body of a human subject in need thereof. Such compositions may be formulated for use in a variety of therapies, such as amelioration, prevention, and / or treatment of conditions such as peptide deficiency, polypeptide deficiency, peptide overexpression, polypeptide overexpression, etc., including conditions that result in diseases or disorders such as those described herein.

[0103] A variety of formulations have been developed to facilitate the use of rAAV particles. For example, for administration of an injectable aqueous solution of rAAV particles, the solution may be suitably buffered if necessary, and the liquid diluent may first be made isotonic with sufficient saline or glucose. In some embodiments, a composition as provided herein comprises a plurality of any one of the variant rAAV particles disclosed herein. In some embodiments, a composition comprises a plurality of more than one of the variant rAAV particles disclosed herein. In some embodiments, "administering" or "administration" refers to providing a material to a subject in a pharmacologically useful manner.

[0104] Thus, in some embodiments, compositions of variant rAAV particles include a pharmaceutically acceptable carrier. The term "carrier" refers to a diluent, adjuvant, excipient, or vehicle with which the rAAV particles are administered. Such pharmaceutical carriers can be sterile liquids (e.g., water, oils, saline solutions, aqueous dextrose and glycerol solutions), suspending agents, preservatives (e.g., methyl-, ethyl-, and propyl-hydroxybenzoates), and pH adjusters (inorganic and organic acids and bases, etc.). In some embodiments, the carrier comprises a buffered saline solution (e.g., phosphate-buffered saline, HEPES-buffered saline). USP-grade carriers and excipients are particularly useful for delivery of rAAV particles to human subjects. Such compositions may further optionally include liposomes, lipids, lipid complexes, microspheres, microparticles, nanospheres, or nanoparticles, or may be otherwise formulated for administration to cells, tissues, organs, or the body of a subject in need thereof. Methods for making such compositions are well known and can be found, for example, in Remington: The Science and Practice of Pharmacy, 22nd Edition, Pharmaceutical Press (2012).

[0105] In some embodiments, a composition comprising any one of the rAAV particles disclosed herein comprises balanced salt solution (BSS) supplemented with 0.014% Tween 20 (polysorbate 20).

[0106] Typically, compositions will contain at least about 0.1% or more of a therapeutic agent (e.g., rAAV particles), although the percentage of active ingredient may vary and may conveniently be between about 1 or 2% to about 70% or 80% or more by weight or volume of the total formulation. Of course, the amount of therapeutic agent (e.g., rAAV particles) in each therapeutically useful composition may be prepared in a manner that will result in a suitable dosage in any given unit dose of compound. Factors such as solubility, bioavailability, biological half-life, route of administration, product shelf life, and other pharmacological considerations will be contemplated by those skilled in the art of preparing such pharmaceutical formulations, and accordingly, various dosages and treatment regimens may be desirable.

[0107] Pharmaceutical forms of rAAV particle compositions suitable for injectable use include sterile aqueous solutions or dispersions. In some embodiments, the forms are sterile and fluid to the extent that easy syringability exists. In some embodiments, the forms are stable under the conditions of manufacture and storage and are preserved against the contaminating action of microorganisms such as bacteria and fungi. In some embodiments, the forms are sterile. The carrier can be a solvent or dispersion medium containing, for example, water, saline, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, etc.), suitable mixtures thereof, and / or vegetable oils. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion, and by the use of surfactants.

[0108] The preparation of compositions for administration to a subject is known in the art. For example, one dose may be dissolved in 1 ml of isotonic NaCl solution and added to 1000 ml of subcutaneous infusion or injected at the proposed site of infusion (see, for example, Remington's Pharmaceutical Sciences, 15th Edition, pages 1035-1038 and 1570-1580). Some dosage variation will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for each individual subject. Furthermore, for human administration, preparations should meet sterility, pyrogenicity, and general safety and purity standards, such as those required by the FDA Office of Biologics standards.

[0109] rAAV gene therapy for heart disease The disclosed RAAV vectors, rAAV particles, or compositions comprising rAAV particles may be used for gene therapy of cardiac diseases in human subjects in need thereof. Examples of cardiac diseases that may be treated using the disclosed methods and compositions include, but are not limited to, cardiomyopathy and acute ischemia. In some embodiments, the cardiomyopathy is hypertrophic cardiomyopathy or dilated cardiomyopathy. Heart failure caused by cardiomyopathy or other cardiac diseases comprises two components: calcium regulatory dysfunction and apoptosis. When administered to a subject in need thereof via, for example, vascular delivery into the coronary artery and / or direct injection into the heart, compositions comprising RAAV vectors, particles, and rAAV particles may be used to treat such heart failure. Compositions comprising RAAV vectors, particles, and rAAV particles drive the co-expression of cS100A1 protein and ARC protein in the subject's cardiomyocytes. S100A1 improves calcium regulation, including normalizing the calcium transient in the sarcoplasmic reticulum, which leads to normal contractile function. ARC will not only block apoptosis initiated by mitochondrial and non-mitochondrial mechanisms (such as stretch-induced apoptosis), but will also improve mitochondrial function. Thus, the synergistic benefit of the two proteins expressed by the transgenes of the present disclosure may lead to better long-term outcomes by targeting both aspects of cardiomyopathy.

[0110] Methods for transducing cells Any one of the rAAV particles disclosed herein or the composition comprising any one of the rAAV particles can be used to transduce cells, tissues or organs.In some embodiments, the cells, tissues or organs transduced using any one of the variant rAAV particles disclosed herein are transduced with a gene of interest, which can be a therapeutic gene or something that is desired to be studied.In some embodiments, cells, tissues or organs are transduced in an in vitro setting, where cells, tissues or organs are incubated with or perfused with culture medium.Cells can be one of many cells cultured under specific conditions, or can be a part of a harvested organ, organoid, or part of an organism.

[0111] In some embodiments, cells, tissues, or organs are transduced in vivo, e.g., for the purpose of treating disease. In some embodiments, such rAAV particles contain a gene of interest that encodes a therapeutic protein or RNA.

[0112] In some embodiments, a composition comprising any one or more of the variant rAAV particles disclosed herein is provided to cells.In some embodiments, a composition comprising any one or more of the variant rAAV particles disclosed herein is provided to tissue in the CNS, to skeletal muscle, or to cardiac tissue.In some embodiments, a composition comprising rAAV particles is provided to cells in vivo, ex vivo, via intraperitoneal, intravenous, intramuscular, intracoronary, subcutaneous, intrathecal, intracranial, intravesicular, or oral delivery methods.In some embodiments, a composition comprising rAAV particles is provided to cells by intravenous, intramuscular, intracoronary, or intrathecal injection or administration.

[0113] In some embodiments, the method of transducing a cell with a gene of interest comprises providing to the cell any one of the compositions provided herein. In some embodiments, the variant rAAV particles used to transduce the cell with the gene of interest comprise SEQ ID NO: 11, 12, or 13.

[0114] Other aspects of the present disclosure relate to methods and preparations for use by a subject, such as a human or non-human subject, a host cell in situ in a subject, or a host cell derived from a subject. In some embodiments, the subject into which cells, tissues, or organs are transduced is a vertebrate (e.g., a mammal or reptile). In some embodiments, the mammalian subject is a human, a non-human primate, a dog, a cat, a hamster, a mouse, a rat, a pig, a horse, a cow, a mule, or a rabbit. Non-limiting examples of non-human primate subjects include macaques (e.g., cynomolgus monkeys or rhesus monkeys), marmosets, tamarins, spider monkeys, owl monkeys, plains monkeys, squirrel monkeys, baboons, gorillas, chimpanzees, and orangutans. In some embodiments, the subject is a model for a particular disease or used to study the pharmacokinetics and / or pharmacokinetics of a protein or siRNA encoded by a gene of interest.

[0115] In some embodiments, the subject has or is suspected of having a heart disease that may be treated by gene therapy. In some embodiments, the subject is in any stage of heart failure. In some embodiments, the heart failure is caused by cardiomyopathy. In some embodiments, the heart failure is hypertrophic cardiomyopathy or dilated cardiomyopathy.

[0116] "Treating" a disease, as that term is used herein, means reducing the frequency or severity of at least one sign or symptom of a disease or disorder experienced by a subject. The compositions described above or elsewhere herein are typically administered to a subject in an effective amount, which is an amount capable of providing a desired result. The desired result will depend on the active agent being administered. For example, an effective amount of rAAV particles may be the amount of particles capable of transferring an expression construct to a host cell, tissue, or organ. A therapeutically acceptable amount may be an amount capable of treating a disease. As is well known in the medical and veterinary fields, the dosage for any one subject depends on many factors, including the subject's size, body surface area, age, the particular composition to be administered, the active ingredients in the composition, the time and route of administration, general health, and other drugs being administered concomitantly.

[0117] The following examples are intended to be illustrative of certain aspects of the present disclosure and are intended to be non-limiting.

[0118] example Example 1. Three AAV variants ("SL1.2" (SEQ ID NO: 11), "SL2" (SEQ ID NO: 12), and "SL3" (SEQ ID NO: 13)) were designed and generated to alter the tropism and / or efficiency of AAV transduction compared to AAV8. Transduction efficiency was increased by removing phosphorylation sites that reduce the efficiency of AAV8. To alter tropism, a rational design approach was taken, comparing the known tropism of AAV8 with that of AAV5, AAV9, AAVrhlO, and AAV_ANC80L65, along with differences in the AAV capsid sequence (see Figure 1). Differences, corrected for by improved tropism to the heart and CNS, were then introduced into the AAV8 capsid sequence. SL1.2, SL2, and SL3 represent incremental additions of such sequences. AAV-SL1.2 is therefore closely related in sequence to AAV8, while AAV-SL2 and AAV-SL3 are more divergent but more closely related to AAVr10, AAVrh74, and AAV8 than to AAV9 or AAVanc80L65. The phylogeny and % divergence of SL1.2, SL2, and SL3 compared to other AAV serotypes are shown in Figures 2 and 3, respectively.

[0119] Example 2. To improve cardiac delivery of cardioprotective genes, mutant capsids were designed to modify regions believed to be important for myocardial uptake based on comparison with naturally occurring AAV serotypes. In particular, the capsid region spanning amino acids Glu578 to Gly596 differed in the three variants described herein (SL1.2, SL2, and SL3; see Example 1 and Figures 1-3).

[0120] Western blot quantification (Figure 7) of the blot shown in Figure 8 compared the delivery of three variants (SL1.2, SL2, and SL3) to the uptake of the native serotypes AAV9 and AAVrh10 and the engineered variant AAVanc80L65. AAV9 and AAVrh10 were chosen because they are two of the best described AAV serotypes for cardiac delivery. The experiment was conducted using a known subsaturating dose (1 x 10) of AAV administered to 6-month-old DBA / 2J male mice via tail vein systemic delivery. 13 The experiments were performed using a 1000-kDa (1000 mg / kg) immunization kit. Green fluorescent protein (GFP) was used as a reporter protein under the control of the beta-actin promoter for expression in all tissues. Figures 7 and 8 show that variant SL2 has improved uptake in cardiac tissue compared to the control serotype, while SL3 has uptake more similar to AAV9. SL3 was observed to have the highest uptake in skeletal muscle and brain of any of the AAV serotypes tested.

[0121] As shown in Table 2, it was also observed that the mutations introduced into SL2 and SL3 resulted in a substantial increase in production efficiency (e.g., yield for a given amount of DNA). The production efficiency for SL2 and SL3 increased such that the yield exceeded that of AAV9 and AAVrh10 by 1.5-4 fold, far exceeding that of the synthetic variant AAVanc80L65. AAVanc70L65 has been proposed to be more useful for cardiac delivery compared to AAV8, but based on the results described herein, it is actually inferior to AAV9, AAVrh10, and the inventive variants SL2 and SL3. The higher yields of SL2 and SL3 are important for the large-scale production required for systemic human gene therapy because these variants are easier and less expensive to produce.

[0122] The dominance of SL3 in uptake in both skeletal and cardiac muscle is shown in Figure 9 (Western blot and quantification of heart and diaphragm) and Figure 10 (Western blot and quantification of quadriceps). Data in Figures 9 and 10 are from 1 x 10 13 The reporter was GFP under the control of the beta-actin promoter for expression in all tissues. [Table 2]

[0123] Figure 11 shows the 5 x 10 13 vg / kg or 1 × 10 14 Figure 1 shows the distribution of green fluorescent protein (GFP; brightness) in skeletal muscle and heart after systemic injection of AAV9, SL1.2, SL2, or SL3 at 1000 mg / kg. The pattern of expression supports the conclusions of the Western blot data and indicates that SL3 is a superior vector for transduction of heart and skeletal muscle.

[0124] When designing a new AAV vector that is intended to target tissues other than the liver, it is useful to minimize delivery to the liver, which typically takes up more vectors than any other tissue.In order to reduce the uptake of SL1.2, SL2 and SL3 by the liver, a single amino acid change (asparagine 500 to isoleucine) is introduced into each of SL1.2, SL2 and SL3 (see, for example, Pulicherla, et al., Mol. Ther. 19:6,1070-78 (2011)).The resulting vectors are herein designated as "SL1.2L" (SEQ ID NO: 15), "SL2L" (SEQ ID NO: 17) and "SL3L" (SEQ ID NO: 19).

[0125] In some applications, particularly when the central nervous system (CNS) is not the target, it is useful to prevent the virus from crossing the blood-brain barrier, thereby minimizing delivery to the brain. To reduce brain uptake of SL1.2, SL2, and SL3, seven amino acid changes (asparagine 263 to serine; glycine 264 to alanine; threonine 265 to serine; serine 266 to threonine; deletion of glycine 268; threonine 270 to serine; and threonine 274 to histidine) were introduced into SL1.2, and eight amino acid changes (asparagine 263 to serine; serine 264 to alanine; threonine 265 to serine; serine 266 to threonine; deletion of glycine 268; serine 269 to alanine; threonine 270 to serine; and threonine 274 to histidine) were introduced into each of SL2 and SL3 (e.g., Albright, et al., Mol. Ther. 26:2, 510-23). (2018)). The resulting vectors were designated herein as "SL1.2B" (SEQ ID NO: 21), "SL2B" (SEQ ID NO: 23), and "SL3B" (SEQ ID NO: 25).

[0126] Finally, in some cases, it may be useful to utilize AAV vectors that have reduced uptake in both liver and brain compared with naturally occurring AAV serotypes.Therefore, design SL1.2, SL2 and SL3 variants that incorporate the above-mentioned liver- and brain-related single amino acid alteration and eight amino acid alterations, respectively.The resulting vectors are herein designated as " SL1.2LB " (SEQ ID NO: 27), " SL2LB " (SEQ ID NO: 29) and " SL3LB " (SEQ ID NO: 31).

[0127] The phylogeny and % divergence of SL1.2, SL1.2L, SL1.2B, SL1.2LB, SL2, SL2L, SL2B, SL2LB, SL3, SL3L, SL3B, and SL3LB compared to other AAV serotypes are shown in Figures 5 and 6, respectively.

[0128] Other Aspects All of the features disclosed herein may be combined in any combination. Each feature disclosed herein may be replaced by an alternative feature serving the same, equivalent, or similar purpose. Thus, unless expressly stated otherwise, each feature disclosed is only an example of a generic series of equivalent or similar features.

[0129] From the above description, those skilled in the art can easily ascertain the essential features of the present disclosure, and can make various changes and modifications to the present disclosure to adapt it to various uses and conditions without departing from the spirit and scope thereof. Accordingly, other embodiments are also within the scope of the claims.

[0130] equivalent While several embodiments of the present invention have been described and illustrated herein, those skilled in the art will readily envision a variety of other means and / or structures for performing the functions and / or obtaining one or more of the results and / or advantages described herein, and each such variation and / or modification is deemed to be within the scope of the embodiments of the present invention described herein. More generally, those skilled in the art will readily appreciate that all parameters, dimensions, materials, and configurations described herein are intended to be exemplary, and that the actual parameters, dimensions, materials, and / or configurations will depend on the particular application for which the teachings of the present invention are used. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the present invention described herein. Accordingly, it should be understood that the foregoing embodiments are presented by way of example only, and that within the scope of the appended claims and equivalents thereto, embodiments of the present invention may be practiced otherwise than as specifically described or claimed. Inventive aspects of the present disclosure are directed to each individual feature, system, article, material, kit, and / or method described herein. In addition, any combination of two or more such features, systems, articles, materials, kits, and / or methods is within the inventive scope of the present disclosure, provided that such features, systems, articles, materials, kits, and / or methods are not mutually inconsistent.

[0131] All definitions, as defined and used herein, should be understood to take precedence over dictionary definitions, definitions in documents incorporated by reference, and / or ordinary meanings of the defined terms.

[0132] All references, patents, and patent applications disclosed herein are incorporated by reference with respect to the subject matter for which each is cited, which in some cases may include the entire document.

[0133] The indefinite articles "a" and "an," as used herein, in the specification, and in the claims, unless clearly indicated to the contrary, should be understood to mean "at least one."

[0134] The phrase "and / or," as used herein, in the specification, and in the claims, should be understood to mean "either or both" of the elements so conjoined, i.e., elements that are present conjunctively in some cases and disjunctively in other cases. Multiple elements listed with "and / or" should be construed in the same manner, i.e., "one or more" of the elements so conjoined. Other elements may optionally be present in addition to the elements specifically identified by the "and / or" clause, which may or may not be related to the elements specifically identified. Thus, as a non-limiting example, a reference to "A and / or B," when used in combination with open-ended language such as "comprising," may, in one embodiment, refer to A only (optionally including elements other than B); in another embodiment, it may refer to B only (optionally including elements other than A); in yet another embodiment, it may refer to both A and B (optionally including other elements), and so forth.

[0135] As used herein, in the specification and in the claims, "or" should be understood to have the same meaning as "and / or" as defined above. For example, when separating items in a list, "or" or "and / or" should be interpreted as inclusive, i.e., the inclusion of at least one, but also including more than one of a number or list of elements, and optionally additional unlisted items. Terms clearly indicated to the contrary, such as "only one of" or "exactly one of," or, when used in the claims, "consisting of," will refer to the inclusion of exactly one element of a number or list of elements. In general, the term "or," as used herein, should only be interpreted as indicating exclusive alternatives (i.e., "one or the other, but not both") when preceded by terms of exclusivity, such as "any of," "one of," "only one of," or "exactly one of." "Consisting essentially of," when used in the claims, shall have its ordinary meaning as used in the field of patent law.

[0136] As used herein in the specification and in the claims, the phrase "at least one," in reference to a list of one or more elements, should be understood to mean at least one element selected from any one or more of the elements in the list of elements, but not necessarily including at least one of each and every element specifically listed in the list of elements, and not excluding any combination of elements in the list of elements. This definition also allows for elements other than those specifically identified in the list of elements to which the phrase "at least one" refers, whether related or unrelated to the elements specifically identified, to be optionally present. Thus, as a non-limiting example, "at least one of A and B" (or, equivalently, "at least one of A or B" or, equivalently, "at least one of A and / or B") may refer, in one embodiment, to at least one (optionally more than one) A in the absence of B (and optionally including elements other than B); in another embodiment, to at least one (optionally more than one) B in the absence of A (and optionally including elements other than A); in yet another embodiment, to at least one (optionally more than one) A and at least one (optionally more than one) B (and optionally including other elements); etc.

[0137] Also, unless expressly indicated to the contrary, it should be understood that in any method claimed herein that includes more than one step or action, the order of the method steps or actions is not necessarily limited to the order in which the method steps or actions are described.

[0138] In the claims, as well as in the above specification, all transitional phrases such as "comprising," "including," "carrying," "having," "containing," "involving," "holding," "composed of," and the like, should be understood to be open-ended, i.e., meaning including, but not limited to. As set forth in the United States Patent Office Manual of Patent Examining Procedures, Section 2111.03, only the transitional phrases "consisting of" and "consisting essentially of" shall be closed or semi-closed transitional phrases, respectively. It should be understood that embodiments described herein using an open-ended transitional phrase (e.g., "comprising") are also contemplated, in alternative embodiments, as "consisting of" and "consisting essentially of" the feature recited by the open-ended transitional phrase. For example, if the disclosure describes "a composition comprising A and B," the disclosure also contemplates the alternative embodiments "a composition consisting of A and B" and "a composition consisting essentially of A and B."

[0139] References 1. Kay CN, Ryals RC, Aslanidi GV, Min SH, Ruan Q, Sun J, Dyka FM, Kasuga D, Ayala AE, Van Vliet K, Agbandje-McKenna M, Hauswirth WW, Boye SL, Boye SE. Targeting photoreceptors via intravitreal delivery using novel, capsid-mutated AAV vectors. PLoS One. 2013 Apr 26;8(4):e62097.

Claims

1. 1. A variant recombinant adeno-associated virus (rAAV) capsid protein comprising the amino acid sequence set forth in SEQ ID NO: 12, or an amino acid sequence that differs by 1, 2, 3, 4, 5, or 6 amino acids from the sequence of SEQ ID NO: 12, with the proviso that the capsid protein is selected from the group consisting of A24D, D41N, Q84K, R92K, T158A, K163S, R169K, L189I, A195G, V199L, P201S, S225A, G264S, A269S, R313K, S315N, E350D, Q362E, Q413E, T415S, T417Q, Y447F, T453S, N459 7. The variant capsid protein comprises G, T462Q, G464L, N471S, T472N, A474S, N475A, T494V, A507G, G508A, N517D, A520V, T528S, D531E, D532G, N540S, N549G, A551G, A555V, D559K, E578Q, I581Q, T591A, Q594I, I595V, N665S, S667A, N670A, S712N, V722T, and Y733F amino acid substitutions, wherein the positions of the substitutions are based on the wild-type AAV8 capsid protein amino acid sequence set forth in SEQ ID NO:

7.

2. 1. A variant recombinant adeno-associated virus (rAAV) capsid protein comprising the amino acid sequence set forth in SEQ ID NO: 13, or an amino acid sequence that differs by 1, 2, 3, 4, 5, or 6 amino acids from the sequence of SEQ ID NO: 13, provided that the capsid protein is selected from the group consisting of K31Q, D41N, G42A, Q84K, R92K, Q105K, T158A, K163S, S180T, P201S, S225A, G264S, A269S, R313K, S315N, E350D, Q362E, Q4 13E, T415S, T417Q, Y447F, T453S, N459G, T462Q, G464L, N471S, T472N, A474S, N475A, T494V, A507G, G508A, N517D, A520V, T528S, D531E, D532G, N540S, N549G, A551G, A555V, D559K, I581Q, Q588S, Q589A, Q594I, I595V, N665S, S667A, N670A, S712N, V722T, and Y733F amino acid substitutions wherein the positions of the substitutions are based on the wild-type AAV8 capsid protein amino acid sequence set forth in SEQ ID NO:

7.

3. A variant rAAV particle comprising the rAAV capsid protein of claim 1 or 2.

4. The variant rAAV particle of claim 3, further comprising a nucleic acid comprising a gene of interest.

5. The variant rAAV particle of claim 4, wherein the nucleic acid is single-stranded.

6. The variant rAAV particle of claim 4, wherein the nucleic acid is double-stranded.

7. A composition comprising a plurality of variant rAAV particles according to any one of claims 3 to 6.

8. 8. The composition of claim 7, further comprising a pharmaceutically acceptable carrier.

9. 9. The composition of claim 7 or 8 for use in a method for transducing cells with a gene of interest, the method comprising providing the composition to cells, wherein AAV particles in the composition comprise the gene of interest.

10. 10. The composition for use of claim 9, wherein the cell is in situ in a subject or derived from a subject, and the subject is a mammal.

11. The composition for use according to claim 10, wherein the mammal is a human.

12. The composition for use according to any one of claims 9 to 11, wherein the gene of interest encodes a therapeutic protein.

13. 13. The composition for use of claim 12, wherein the therapeutic protein is an antibody or antibody fragment, a peptibody, a growth factor, a hormone, a membrane protein, a cytokine, a chemokine, an activating or inhibitory peptide acting on a cell surface receptor or an ion channel, a cell-penetrating peptide that targets intracellular processes, an enzyme, a nuclease or other protein used for gene editing.

14. 1. A variant recombinant adeno-associated virus (rAAV) capsid protein comprising the amino acid sequence set forth in SEQ ID NO:11, or an amino acid sequence which differs by 1, 2, 3, 4, 5, or up to 6 amino acids from the sequence of SEQ ID NO:11, wherein said capsid protein comprises the following amino acid substitutions: K163S, R169K, S180T, L189I, T417Q, Y447F, T462Q, G464L, T494V, D559K, T591A, and Y733F, wherein the positions of the substitutions are based on the wild-type AAV8 capsid protein amino acid sequence set forth in SEQ ID NO:

7.

15. 15. The composition of claim 14, comprising a plurality of variant rAAV particles.

16. 16. The composition of claim 15, further comprising a pharmaceutically acceptable carrier.

17. 17. The composition of claim 15 or 16, for use in a method of transducing a cell with a gene of interest, the method comprising providing a composition of cells, wherein rAAV particles in the composition comprise the gene of interest.

18. 20. The composition for use in the method of claim 17, wherein the cell is in situ in a subject or derived from a subject, and the subject is a mammal.

19. 19. The composition for use according to claim 18, wherein the mammal is a human.

20. The composition for use according to any one of claims 17 to 19, wherein the gene of interest encodes a therapeutic protein.

21. 21. The composition for use of claim 20, wherein the therapeutic protein is an antibody or antibody fragment, a peptibody, a growth factor, a hormone, a membrane protein, a cytokine, a chemokine, an activating or inhibitory peptide acting on a cell surface receptor or an ion channel, a cell-penetrating peptide that targets intracellular processes, an enzyme, a nuclease, or other protein used for gene editing.

22. The composition for use according to any one of claims 11 and 17 to 19, wherein the gene of interest is a cardioprotective gene.

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