FUNCTIONAL AAV CAPSIDS FOR SYSTEMIC ADMINISTRATION

SEQUENCE LISTING

The instant application contains a Sequence Listing which has been submitted electronically in eXtensible Markup Language (XML) format and is hereby incorporated by reference in its entirety. Said XML copy, created on Nov. 28, 2022, is named “421688-705021_SL.xml” and is 5 megabytes in size.

BACKGROUND

Recombinant adeno-associated viruses (rAAV) provide the leading platform for in vivo delivery of gene therapies. Current clinical trials employ a limited number of AAV capsids, primarily from naturally occurring human or primate serotypes such as AAV1, AAV2, AAV5, AAV6, AAV8, AAV9, AAVrh.10, AAV4rh.74, and AAVhu.67. These capsids often provide suboptimal targeting to tissues of interest, both due to poor infectivity of the tissue of interest and competing liver tropism. Increasing the dose to ensure infection of desired tissues can lead to dose-dependent liver toxicity. In addition, use of naturally-occurring capsids presents an immunological memory challenge—pre-immune patient populations are excluded from treatment and repeat dosing in a previously immune naïve patient is often not possible. Thus, there is a need for additional AAV capsids for use in gene therapy, in particular capsids that confer upon the rAAV high infectivity for specific tissues, such as muscle tissue and tissues in the central nervous system, and low liver tropism

SUMMARY OF THE INVENTION

Described herein is a system for high throughput engineering of functional AAV capsids with altered tropism for various tissues and capsid variants that have increased tropism for target tissues, such as muscle or central nervous system (CNS) tissues.

In various aspects, the present disclosure provides a viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 a VP1 polypeptide, wherein the 581-589 region comprises at least one amino acid substitution as compared to SEQ ID NO: 9, and wherein a recombinant viral capsid comprising the VP capsid polypeptide is capable of preferentially targeting a central nervous system (CNS) tissue at a DNA enrichment of at least 100-fold greater than a wild type AAV9.

In some aspects, the 581-589 region has a sequence selected from the group consisting of SEQ ID NO: 18, SEQ ID NO: 3472, SEQ ID NO: 262, SEQ ID NO: 3306, SEQ ID NO: 2028, SEQ ID NO: 2791, SEQ ID NO: 424, SEQ ID NO: 2536, SEQ ID NO: 1971, SEQ ID NO: 415, SEQ ID NO: 3846, SEQ ID NO: 3283, SEQ ID NO: 1956, SEQ ID NO: 3297, SEQ ID NO: 4545, SEQ ID NO: 2661, SEQ ID NO: 1576, SEQ ID NO: 425, SEQ ID NO: 709, SEQ ID NO: 2168, SEQ ID NO: 54, SEQ ID NO: 429, SEQ ID NO: 708, SEQ ID NO: 428, SEQ ID NO: 4119, SEQ ID NO: 3906, SEQ ID NO: 2456, SEQ ID NO: 2278, SEQ ID NO: 5006, SEQ ID NO: 426, SEQ ID NO: 307, SEQ ID NO: 5155, SEQ ID NO: 2640, SEQ ID NO: 4317, SEQ ID NO: 1145, SEQ ID NO: 430, and SEQ ID NO: 885. In some aspects, the recombinant viral capsid is capable of preferentially targeting the CNS tissue at a DNA enrichment of at least 200-fold greater than the wild type AAV9. In some aspects, the 581-589 region has a sequence selected from the group consisting of SEQ ID NO: 18, SEQ ID NO: 3472, SEQ ID NO: 262, SEQ ID NO: 3306, SEQ ID NO: 2028, SEQ ID NO: 2791, SEQ ID NO: 424, SEQ ID NO: 2536, SEQ ID NO: 1971, SEQ ID NO: 415, SEQ ID NO: 3846, SEQ ID NO: 3283, SEQ ID NO: 1956, and SEQ ID NO: 3297. In some aspects, the recombinant viral capsid is capable of preferentially targeting the CNS tissue at a DNA enrichment of at least 1000-fold greater than the wild type AAV9. In some aspects, the 581-589 region has a sequence of SEQ ID NO: 18.

In various aspects, the present disclosure provides a viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 a VP1 polypeptide, wherein the 581-589 region comprises at least one amino acid substitution as compared to SEQ ID NO: 9, and wherein a recombinant viral capsid comprising the VP capsid polypeptide is capable of preferentially targeting a central nervous system (CNS) tissue at an RNA enrichment of at least 100-fold greater than a wild type AAV9.

In some aspects, the 581-589 region has a sequence selected from the group consisting of SEQ ID NO: 18, SEQ ID NO: 3472, SEQ ID NO: 262, SEQ ID NO: 3306, SEQ ID NO: 2028, SEQ ID NO: 2791, SEQ ID NO: 424, SEQ ID NO: 2536, SEQ ID NO: 1971, SEQ ID NO: 415, SEQ ID NO: 3283, SEQ ID NO: 1956, SEQ ID NO: 3297, SEQ ID NO: 4545, SEQ ID NO: 2661, SEQ ID NO: 1576, SEQ ID NO: 425, SEQ ID NO: 709, SEQ ID NO: 54, SEQ ID NO: 429, SEQ ID NO: 428, SEQ ID NO: 426, and SEQ ID NO: 430. In some aspects, the recombinant viral capsid is capable of preferentially targeting the CNS tissue at an RNA enrichment of at least 200-fold or greater than the wild type AAV9. In some aspects, the 581-589 region has a sequence selected from the group consisting of SEQ ID NO: 18, SEQ ID NO: 3472, SEQ ID NO: 262, SEQ ID NO: 3306, SEQ ID NO: 2028, SEQ ID NO: 2791, SEQ ID NO: 424, SEQ ID NO: 2536, SEQ ID NO: 1971, SEQ ID NO: 415, SEQ ID NO: 3283, SEQ ID NO: 1956, SEQ ID NO: 2661, SEQ ID NO: 1576, SEQ ID NO: 425, SEQ ID NO: 709, SEQ ID NO: 54, SEQ ID NO: 430, SEQ ID NO: 569, and SEQ ID NO: 719. In some aspects, the recombinant viral capsid is capable of preferentially targeting the CNS tissue at an RNA enrichment of at least 500-fold or greater than the wild type AAV9. In some aspects, the 581-589 region has a sequence selected from the group consisting of SEQ ID NO: 18, SEQ ID NO: 3472, SEQ ID NO: 262, and SEQ ID NO: 2028.

In various aspects, the present disclosure provides a viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 a VP1 polypeptide, wherein the 581-589 region comprises at least one amino acid substitution as compared to SEQ ID NO: 9, and wherein a recombinant viral capsid comprising the VP capsid polypeptide is capable of preferentially targeting a central nervous system (CNS) tissue at a DNA enrichment of at least 5-fold greater than a wild type AAV5.

In various aspects, the present disclosure provides a viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 a VP1 polypeptide, wherein the 581-589 region comprises at least one amino acid substitution as compared to SEQ ID NO: 9, and wherein a recombinant viral capsid comprising the VP capsid polypeptide is capable of preferentially targeting a central nervous system (CNS) tissue at an RNA enrichment of at least 5-fold greater than a wild type AAV5.

In some aspects, the 581-589 region has a sequence selected from the group consisting of SEQ ID NO: 18, SEQ ID NO: 3472, SEQ ID NO: 262, SEQ ID NO: 3306, SEQ ID NO: 2028, SEQ ID NO: 2791, SEQ ID NO: 424, SEQ ID NO: 2536, SEQ ID NO: 1971, SEQ ID NO: 415, SEQ ID NO: 3283, SEQ ID NO: 1956, SEQ ID NO: 3297, SEQ ID NO: 4545, SEQ ID NO: 2661, SEQ ID NO: 1576, SEQ ID NO: 425, SEQ ID NO: 709, SEQ ID NO: 54, SEQ ID NO: 429, SEQ ID NO: 708, SEQ ID NO: 428, SEQ ID NO: 426, SEQ ID NO: 307, SEQ ID NO: 4317, SEQ ID NO: 430, and SEQ ID NO: 885. In some aspects, the recombinant viral capsid is capable of preferentially targeting the CNS tissue at an RNA enrichment of at least 20-fold greater than the wild type AAV5. In some aspects, the 581-589 region has a sequence selected from the group consisting of SEQ ID NO: 18, SEQ ID NO: 3472, SEQ ID NO: 262, SEQ ID NO: 3306, SEQ ID NO: 2028, SEQ ID NO: 2791, SEQ ID NO: 424, SEQ ID NO: 2536, SEQ ID NO: 1971, SEQ ID NO: 415, SEQ ID NO: 3283, SEQ ID NO: 1956, SEQ ID NO: 2661, SEQ ID NO: 1576, SEQ ID NO: 425, SEQ ID NO: 709, SEQ ID NO: 54, SEQ ID NO: 430, SEQ ID NO: 569, and SEQ ID NO: 719.

In various aspects, the present disclosure provides a viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence that is at least 85% identical to: (a) SEQ ID NO: 18, (b) SEQ ID NO: 3472, (c) SEQ ID NO: 262, (d) SEQ ID NO: 3306, (e) SEQ ID NO: 2028, (f) SEQ ID NO: 2791, (g) SEQ ID NO: 424, (h) SEQ ID NO: 2536, (i) SEQ ID NO: 1971, (j) SEQ ID NO: 415, (k) SEQ ID NO: 3846, (l) SEQ ID NO: 3283, (m) SEQ ID NO: 1956, (n) SEQ ID NO: 3297, (o) SEQ ID NO: 4545, (p) SEQ ID NO: 2661, (q) SEQ ID NO: 1576, (r) SEQ ID NO: 425, (s) SEQ ID NO: 709, (t) SEQ ID NO: 2168, (u) SEQ ID NO: 54, (v) SEQ ID NO: 429, (w) SEQ ID NO: 708, (x) SEQ ID NO: 428, (y) SEQ ID NO: 4119, (z) SEQ ID NO: 3906, (aa) SEQ ID NO: 2456, (bb) SEQ ID NO: 2278, (cc) SEQ ID NO: 5006, or (dd) SEQ ID NO: 426.

In some aspects, the 581-589 region has a sequence of: (a) SEQ ID NO: 18, (b) SEQ ID NO: 3472, (c) SEQ ID NO: 262, (d) SEQ ID NO: 3306, (e) SEQ ID NO: 2028, (f) SEQ ID NO: 2791, (g) SEQ ID NO: 424, (h) SEQ ID NO: 2536, (i) SEQ ID NO: 1971, (j) SEQ ID NO: 415, (k) SEQ ID NO: 3846, (l) SEQ ID NO: 3283, (m) SEQ ID NO: 1956, (n) SEQ ID NO: 3297, (o) SEQ ID NO: 4545, (p) SEQ ID NO: 2661, (q) SEQ ID NO: 1576, (r) SEQ ID NO: 425, (s) SEQ ID NO: 709, (t) SEQ ID NO: 2168, (u) SEQ ID NO: 54, (v) SEQ ID NO: 429, (w) SEQ ID NO: 708, (x) SEQ ID NO: 428, (y) SEQ ID NO: 4119, (z) SEQ ID NO: 3906, (aa) SEQ ID NO: 2456, (bb) SEQ ID NO: 2278, (cc) SEQ ID NO: 5006, or (dd) SEQ ID NO: 426.

In various aspects, the present disclosure provides a viral protein (VP) capsid polypeptide, 581-589 wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence that is at least 85% identical to any one of any one of SEQ ID NO: 12-SEQ ID NO: 3937 or SEQ ID NO: 3938-SEQ ID NO: 4237.

In some aspects, the 581-589 region has a sequence of any one of any one of SEQ ID NO: 12-SEQ ID NO: 3937 or SEQ ID NO: 3938-SEQ ID NO: 4237. In some aspects, the VP capsid polypeptide is capable of assembling into a recombinant viral capsid.

In various aspects, the present disclosure provides a viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide is capable of assembling into a recombinant viral capsid, and wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region: comprises at least one amino acid substitution as compared to SEQ ID NO: 9, and confers on the recombinant viral capsid an infection rate for central nervous system (CNS) tissue with at least 3-fold higher CNS tissue tropism than a wild type VP capsid polypeptide of SEQ ID NO: 1; wherein the VP capsid polypeptide comprises at least one amino acid substitution as compared to each of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 8.

In some aspects, the 581-589 region comprises a sequence of X¹X²X³X⁴X⁵X⁶X⁷X⁸X⁹, wherein X¹, X², X³, X⁴, X⁵, X⁶, X⁷, X⁸, and X⁹are each individually selected from A, R, N, D, C, E, Q, G, H, I, L, K, M, F, P, S, T, W, Y, and V. In some aspects, the VP capsid polypeptide has a sequence of SEQ ID NO: 2, wherein residues 581 to 589 of SEQ ID NO: 2 (X¹X²X³X⁴X⁵X⁶X⁷X⁸X⁹) correspond to the 581-589 region. In some aspects, the 581-589 region has a sequence that is at least 70%, at least 77%, at least 80%, at least 88%, at least 90%, or at least 95% identical to any one of SEQ ID NO: 12-SEQ ID NO: 3937. In some aspects, the 581-589 region has a sequence of any one of SEQ ID NO: 12-SEQ ID NO: 3937. In some aspects, the 581-589 region has a sequence that is at least 70%, at least 77%, at least 80%, at least 88%, at least 90%, or at least 95% identical to any one of SEQ ID NO: 3938-SEQ ID NO: 4237. In some aspects, the 581-589 region has a sequence of any one of SEQ ID NO: 3938-SEQ ID NO: 4237.

In some aspects, the VP capsid polypeptide comprises an amino acid sequence at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 98.5%, at least 99%, or at least 99.5% identical to SEQ ID NO: 1. In some aspects, the infection rate for CNS tissue is at least 2.5-fold, at least 3-fold, at least 3.5-fold, at least 4-fold, at least 5-fold, at least 10-fold, at least 20-fold, at least 50-fold, at least 75-fold, at least 100-fold, at least 200-fold, at least 500-fold, or at least 1000-fold higher than an infection rate of the wild type VP capsid polypeptide for CNS tissue. In some aspects, the 581-589 region confers on a recombinant viral capsid assembled from the VP capsid polypeptide improved stability compared to a wild type AAV5 capsid comprising a peptide of SEQ ID NO: 1. In some aspects, the 581-589 region confers lower toxicity upon administration to a subject compared to a wild type VP capsid polypeptide of SEQ ID NO: 1. In some aspects, the CNS tissue is selected from forebrain cortex, occipital cortex, temporal cortex, thalamus, hypothalamus, substantia nigra, hippocampus DG, hippocampus CA1, hippocampus CA3, cerebellum, and any combination thereof.

In various aspects, the present disclosure provides a pharmaceutical composition comprising a VP capsid polypeptide as described herein.

In some aspects, the VP capsid polypeptide is assembled into a recombinant viral capsid. In some aspects, the pharmaceutical composition further comprises a payload encapsidated by the recombinant viral capsid. In some aspects, the payload encodes a therapeutic polynucleotide or a therapeutic peptide. In some aspects, the payload encodes a guide RNA, a tRNA, a suppressor tRNA, a siRNA, a miRNA, an mRNA, a shRNA, a circular RNA, or an antisense oligonucleotide (ASO), a ribozyme, a DNAzyme, an aptamer, or any combination thereof. In some aspects, the payload encodes a component of a CRISPR/Cas system, an adenosine deaminase acting on RNA (ADAR) enzyme, a transcriptional activator, or a transcriptional repressor.

In various aspects, the present disclosure provides a recombinant adeno-associated virus (rAAV), comprising a VP capsid polypeptide as described herein assembled into a recombinant viral capsid and a payload encapsidated by the recombinant viral capsid.

In some aspects, the rAAV further comprises a VP2 polypeptide comprising the 581-589 region and a VP3 polypeptide comprising the 581-589 region. In some aspects, the payload encodes a therapeutic polynucleotide or a therapeutic peptide. In some aspects, the payload encodes a guide RNA, a tRNA, a suppressor tRNA, a siRNA, a miRNA, an mRNA, a shRNA, a circular RNA, or an antisense oligonucleotide (ASO), a ribozyme, a DNAzyme, an aptamer, or any combination thereof. In some aspects, the payload encodes a component of a CRISPR/Cas system, an adenosine deaminase acting on RNA (ADAR) enzyme, a transcriptional activator, or a transcriptional repressor.

In some aspects, the DNA enrichment is at least 200-fold greater than the wild type AAV9. In some aspects, the DNA enrichment is at least 1000-fold greater than the wild type AAV9.

In various aspects, the present disclosure provides a method of transcribing a payload in a central nervous system (CNS) tissue of a subject, the method comprising: administering a recombinant adeno-associated virus (rAAV) to the subject, wherein the rAAV encapsidates the payload, and wherein the rAAV comprises a VP capsid polypeptide comprising a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide, wherein the 581-589 region comprises at least one amino acid substitution as compared to SEQ ID NO: 9; infecting the CNS tissue with the rAAV; and preferentially transcribing the payload to the CNS tissue infected by the rAAV at an RNA enrichment of at least 100-fold greater than a wild type AAV9.

In some aspects, the DNA enrichment is at least 200-fold greater than the wild type AAV9. In some aspects, the DNA enrichment is at least 500-fold greater than the wild type AAV9.

In some aspects, the DNA enrichment is at least 10-fold greater than the wild type AAV5.

In some aspects, the DNA enrichment is at least 20-fold greater than the wild type AAV5.

In various aspects, the present disclosure provides a method of delivering a payload to a central nervous system (CNS) tissue of a subject, the method comprising: administering a recombinant adeno-associated virus (rAAV) to the subject, wherein the rAAV encapsidates the payload, and wherein the rAAV comprises a VP capsid polypeptide comprising a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide, wherein the 581-589 region has a sequence that is at least 85% identical to: (a) SEQ ID NO: 18, (b) SEQ ID NO: 3472, (c) SEQ ID NO: 262, (d) SEQ ID NO: 3306, (e) SEQ ID NO: 2028, (f) SEQ ID NO: 2791, (g) SEQ ID NO: 424, (h) SEQ ID NO: 2536, (i) SEQ ID NO: 1971, (j) SEQ ID NO: 415, (k) SEQ ID NO: 3846, (l) SEQ ID NO: 3283, (m) SEQ ID NO: 1956, (n) SEQ ID NO: 3297, (o) SEQ ID NO: 4545, (p) SEQ ID NO: 2661, (q) SEQ ID NO: 1576, (r) SEQ ID NO: 425, (s) SEQ ID NO: 709, (t) SEQ ID NO: 2168, (u) SEQ ID NO: 54, (v) SEQ ID NO: 429, (w) SEQ ID NO: 708, (x) SEQ ID NO: 428, (y) SEQ ID NO: 4119, (z) SEQ ID NO: 3906, (aa) SEQ ID NO: 2456, (bb) SEQ ID NO: 2278, (cc) SEQ ID NO: 5006, or (dd) SEQ ID NO: 426; infecting the CNS tissue with the rAAV with at least 3-fold higher CNS tissue tropism as compared to a wild type AAV5 capsid comprising a peptide of SEQ ID NO: 1; and delivering the payload to the CNS tissue infected by the rAAV.

In various aspects, the present disclosure provides a method of delivering a payload to a central nervous system (CNS) tissue of a subject, the method comprising: administering a recombinant adeno-associated virus (rAAV) to the subject, wherein the rAAV encapsidates the payload, and wherein the rAAV comprises a VP capsid polypeptide comprising a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide, wherein the 581-589 region comprises at least one amino acid substitution as compared to residues 581 to 589 of SEQ ID NO: 1, and wherein the VP capsid polypeptide comprises at least one amino acid substitution as compared to each of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 8; infecting the CNS tissue with the rAAV with at least 3-fold higher CNS tissue tropism as compared to a wild type AAV5 capsid comprising a peptide of SEQ ID NO: 1; and delivering the payload to the CNS tissue infected by the rAAV.

In some aspects, the method further comprises expressing a therapeutic polypeptide or a therapeutic polynucleotide encoded by the payload in the CNS tissue infected by the rAAV. In some aspects, the method further comprises producing a therapeutic effect in the CNS tissue of the subject. In some aspects, the therapeutic effect is produced upon administration of from 1×10⁵to 5×10¹⁴rAAVs per kg subject weight. In some aspects, the method comprises administering an amount of the rAAV sufficient to produce the therapeutic effect without producing a toxicity in the subject. In some aspects, the CNS tissue comprises forebrain cortex, occipital cortex, temporal cortex, thalamus, hypothalamus, substantia nigra, hippocampus DG, hippocampus CA1, hippocampus CA3, cerebellum, or a combination thereof.

In some aspects, the DNA enrichment is at least 200-fold greater than the wild type AAV9. In some aspects, the DNA enrichment is at least 1000-fold greater than the wild type AAV9.

In some aspects, the DNA enrichment is at least 200-fold greater than the wild type AAV9. In some aspects, the DNA enrichment is at least 500-fold greater than the wild type AAV9.

In some aspects, the DNA enrichment is at least 10-fold greater than the wild type AAV5.

In some aspects, the DNA enrichment is at least 20-fold greater than the wild type AAV5.

In various aspects, the present disclosure provides a method of treating a condition in a subject, the method comprising: administering a recombinant adeno-associated virus (rAAV) to the subject, wherein the rAAV encapsidates a payload, and wherein the rAAV comprises a VP capsid polypeptide comprising a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide, wherein the 581-589 region has a sequence that is at least 85% identical to: (a) SEQ ID NO: 18, (b) SEQ ID NO: 3472, (c) SEQ ID NO: 262, (d) SEQ ID NO: 3306, (e) SEQ ID NO: 2028, (f) SEQ ID NO: 2791, (g) SEQ ID NO: 424, (h) SEQ ID NO: 2536, (i) SEQ ID NO: 1971, (j) SEQ ID NO: 415, (k) SEQ ID NO: 3846, (l) SEQ ID NO: 3283, (m) SEQ ID NO: 1956, (n) SEQ ID NO: 3297, (o) SEQ ID NO: 4545, (p) SEQ ID NO: 2661, (q) SEQ ID NO: 1576, (r) SEQ ID NO: 425, (s) SEQ ID NO: 709, (t) SEQ ID NO: 2168, (u) SEQ ID NO: 54, (v) SEQ ID NO: 429, (w) SEQ ID NO: 708, (x) SEQ ID NO: 428, (y) SEQ ID NO: 4119, (z) SEQ ID NO: 3906, (aa) SEQ ID NO: 2456, (bb) SEQ ID NO: 2278, (cc) SEQ ID NO: 5006, or (dd) SEQ ID NO: 426; infecting a CNS tissue of the subject with the rAAV; delivering the payload to the CNS tissue infected by the rAAV; and producing a therapeutic effect in the CNS tissue, thereby treating the condition.

In various aspects, the present disclosure provides a method of treating a condition in a subject, the method comprising: administering a recombinant adeno-associated virus (rAAV) to the subject, wherein the rAAV encapsidates a payload, and wherein the rAAV comprises a VP capsid polypeptide a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide, wherein the 581-589 region comprises at least one amino acid substitution as compared to residues 581 to 589 of SEQ ID NO: 1, and wherein the VP capsid polypeptide does not have the sequence of any of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, or SEQ ID NO: 8; infecting a CNS tissue of the subject with the rAAV with at least 3-fold higher CNS tissue tropism as compared to a wild type AAV5 capsid comprising a peptide of SEQ ID NO: 1; delivering the payload to the CNS tissue infected by the rAAV; and producing a therapeutic effect in the CNS tissue, thereby treating the condition.

In some aspects, the condition is a neurological condition. In some aspects, the neurological condition is an aromatic l-amino acid decarboxylase (AADC) deficiency, Alzheimer's disease, a tauopathy, a synucleinopathy, Batten disease, mucopolysaccharidosis type III, frontotemporal dementia, Parkinson's disease, corticobasal degeneration, progressive supranuclear palsy, chronic traumatic encephalopathy, Gaucher disease, Canavan disease, Tay-Sachs disease, Huntington's disease, Protocki-Lupski syndrome, amyotrophic lateral sclerosis, Down syndrome, Sanfilippo disease type A, Sanfilippo disease type B, or Rett syndrome. In some aspects, the condition is Rett syndrome. In some aspects, the payload encodes a guide RNA that targets an mRNA encoding by MECP2 or a tRNA that targets a premature stope codon in MECP2. In some aspects, the condition is Parkinson's disease. In some aspects, the payload encodes a guide RNA targeting an mRNA encoding LRRK2, GBA, α-synuclein, AADC, GDNF, Neurturin, GAD, FOXG1, K_v7.2, fragile X mental retardation protein, tau, or laforin. In some aspects, the condition is Alzheimer's disease. In some aspects, the payload encodes a guide RNA targeting an mRNA encoding amyloid precursor protein (APP), alpha-synuclein (SNCA), Tau protein (MAPT), or Apolipoprotein E (ApoE). In some aspects, the payload encodes a transgene encoding amyloid precursor protein (APP), alpha-synuclein (SNCA), Tau protein (MAPT), or Apolipoprotein E (ApoE).

In some aspects, the method comprises administering from 1×10⁵to 5×10¹⁴rAAVs per kg subject weight. In some aspects, the method comprises infecting the CNS tissue with at least 2.5-fold, at least 3-fold, at least 3.5-fold, at least 4-fold, at least 5-fold, at least 10-fold, at least 20-fold, at least 50-fold, at least 75-fold, at least 100-fold, at least 200-fold, at least 500-fold, or at least 1000-fold higher tissue tropism than a wild type AAV5 capsid comprising a peptide of SEQ ID NO: 1.

In some aspects, the payload encodes a therapeutic protein, therapeutic polynucleotide, a guide RNA, a tRNA, a suppressor tRNA, a siRNA, a miRNA, an mRNA, a shRNA, a circular RNA, an antisense oligonucleotide (ASO), a ribozyme, a DNAzyme, an aptamer, or any combination thereof. In some aspects, the guide RNA is a CRISPR/Cas guide RNA or an ADAR guide RNA. In some aspects, the therapeutic protein is selected from the group consisting of aromatic l-amino acid decarboxylase (AADC), amyloid precursor protein (APP), α-synuclein, microtubule associated protein tau (MAPT), ApoE, nerve growth factor (NGF), telomerase reverse transcriptase (TERT), CLN2, CLN3, CLN6, N-acetyl-α-glucosaminidase (NAGLU), granulin, glucosylceramidase 8 (GBA), aspartoacylase, GDNF, neurturin (NTN), glutamate decarboxylase (GAD), FOXG1, K_v7.2, laforin, leucine rich repeat kinase 2 (LRRK2), hexosaminidase A (HEXA), hexosaminidase B (HEXB), huntingtin, cholesterol 24-hydroxylase, C9orf72, superoxide dismutase 1 (SOD1), dual specificity tyrosine-phosphorylation-regulated kinase 1A (DYRKA1), N-sulfoglucosamine sulfohydrolase (SGSH), surfeit locus protein 1 (SURF1), GABA transporter 1 (GAT1), and methyl-CpG binding protein 2 (MECP2). In some aspects, the therapeutic polynucleotide targets an mRNA encoding a protein selected from the group consisting of aromatic l-amino acid decarboxylase (AADC), amyloid precursor protein (APP), α-synuclein, microtubule associated protein tau (MAPT), ApoE, nerve growth factor (NGF), telomerase reverse transcriptase (TERT), CLN2, CLN3, CLN6, N-acetyl-α-glucosaminidase (NAGLU), granulin, glucosylceramidase 8 (GBA), aspartoacylase, GDNF, neurturin (NTN), glutamate decarboxylase (GAD), FOXG1, K_v7.2, laforin, leucine rich repeat kinase 2 (LRRK2), hexosaminidase A (HEXA), hexosaminidase B (HEXB), huntingtin, cholesterol 24-hydroxylase, C9orf72, superoxide dismutase 1 (SOD1), dual specificity tyrosine-phosphorylation-regulated kinase 1A (DYRKA1), N-sulfoglucosamine sulfohydrolase (SGSH), surfeit locus protein 1 (SURF1), GABA transporter 1 (GAT1), and methyl-CpG binding protein 2 (MECP2). In some aspects, the payload encodes a component of a CRISPR/Cas system, an adenosine deaminase acting on RNA (ADAR) enzyme, a transcriptional activator, or a transcriptional repressor. In some aspects, the component of the CRISPR/Cas system comprises a Cas3, a Cas8, a Cas10, a Cas9, a Cas4, a Cas12, a Cas13, a guide RNA, or a combination thereof. In some aspects, the ADAR enzyme is ADAR1 or ADAR2. In some aspects, the transcriptional activator is VP64. In some aspects, the transcriptional repressor is KRAB.

In some aspects, the method comprises systemically administering the rAAV to the subject. In some aspects, the method comprises administering the rAAV via intravenous administration, intramuscular administration, intraperitoneal administration, or oral administration. In some aspects, the method further comprises expressing a therapeutic polypeptide or a therapeutic polynucleotide encoded by the payload in the CNS tissue infected by the rAAV. In some aspects, the method comprises expressing the therapeutic polypeptide or the therapeutic polynucleotide with higher CNS tissue tropism compared to a wild type AAV5 capsid comprising a peptide of SEQ ID NO: 1. In some aspects, the method comprises expressing the therapeutic polypeptide or the therapeutic polynucleotide with at least 3-fold, at least 3.5-fold, at least 4-fold, at least 5-fold, at least 10-fold, at least 20-fold, at least 50-fold, at least 75-fold, at least 100-fold, at least 200-fold, at least 500-fold higher, or at least 1000-fold higher tropism compared to the wild type AAV5 capsid.

In some aspects, the method comprises producing a toxicity in the subject that is lower than a toxicity produced upon systemic administration of a comparable number of wild type AAV5 capsids comprising a VP capsid polypeptide of SEQ ID NO: 1. In some aspects, the method comprises producing a toxicity in the subject that is lower than a toxicity produced upon systemic administration of a number of wild type AAV5 capsids comprising a VP capsid polypeptide of SEQ ID NO: 1 sufficient to deliver a comparable number of payloads to the CNS tissue. In some aspects, the method comprises producing a concentration of neutralizing antibodies in the subject that is lower than a concentration of neutralizing antibodies produced upon systemic administration of a comparable number of wild type AAV5 capsids comprising a VP capsid polypeptide of SEQ ID NO: 1. In some aspects, the method comprises producing a concentration of neutralizing antibodies in the subject that is lower than a concentration of neutralizing antibodies produced upon intravitreal administration of a comparable number of wild type AAV2 capsids. In some aspects, the method comprises producing a concentration of neutralizing antibodies in the subject that is lower than a concentration of neutralizing antibodies produced upon intravitreal administration of a comparable number of wild type AAV8 capsids. In some aspects, the method comprises producing a concentration of neutralizing antibodies in the subject that is lower than a concentration of neutralizing antibodies produced upon intravitreal administration of a comparable number of wild type AAV9 capsids. In some aspects, the VP capsid polypeptide further comprises one or more mutations outside of the 581-589 region that contributes to reduced production of neutralizing antibodies relative to a wild type AAV capsid. In some aspects, the method comprises producing a concentration of neutralizing antibodies in the subject that is lower than a concentration of neutralizing antibodies produced upon systemic administration of a number of wild type AAV5 capsids comprising a VP capsid polypeptide of SEQ ID NO: 1 sufficient to deliver a comparable number of payloads to the CNS tissue.

In various aspects, the present disclosure provides a viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 a VP1 polypeptide, wherein the 581-589 region comprises at least one amino acid substitution as compared to SEQ ID NO: 9, and wherein a recombinant viral capsid comprising the VP capsid polypeptide is capable of preferentially targeting central nervous system tissue and a cardiac muscle tissue.

In some aspects, the 581-589 region has a sequence having at least 85% sequence identity to a sequence selected from the group consisting of SEQ ID NO: 18, SEQ ID NO: 2536, SEQ ID NO: 3846, SEQ ID NO: 4545, SEQ ID NO: 2661, SEQ ID NO: 1576, SEQ ID NO: 4317, SEQ ID NO: 23, SEQ ID NO: 22, SEQ ID NO: 964, SEQ ID NO: 4288, SEQ ID NO: 4338, SEQ ID NO: 5037, and SEQ ID NO: 4936. In some aspects, the 581-589 region has a sequence selected from the group consisting of SEQ ID NO: 18, SEQ ID NO: 2536, SEQ ID NO: 3846, SEQ ID NO: 4545, SEQ ID NO: 2661, SEQ ID NO: 1576, SEQ ID NO: 4317, SEQ ID NO: 23, SEQ ID NO: 22, SEQ ID NO: 964, SEQ ID NO: 4288, SEQ ID NO: 4338, SEQ ID NO: 5037, and SEQ ID NO: 4936.

In various aspects, the present disclosure provides a viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 a VP1 polypeptide, wherein the 581-589 region comprises at least one amino acid substitution as compared to SEQ ID NO: 9, and wherein a recombinant viral capsid comprising the VP capsid polypeptide is capable of preferentially targeting central nervous system tissue and a skeletal muscle tissue.

In some aspects, the 581-589 region has a sequence having at least 85% sequence identity to a sequence selected from the group consisting of SEQ ID NO: 18, SEQ ID NO: 3472, SEQ ID NO: 262, SEQ ID NO: 3306, SEQ ID NO: 2536, SEQ ID NO: 1971, SEQ ID NO: 3283, SEQ ID NO: 3297, SEQ ID NO: 4545, SEQ ID NO: 2661, SEQ ID NO: 307, SEQ ID NO: 4317, SEQ ID NO: 1268, SEQ ID NO: 4288, SEQ ID NO: 724, and SEQ ID NO: 5037. In some aspects, the 581-589 region has a sequence selected from the group consisting of SEQ ID NO: 18, SEQ ID NO: 3472, SEQ ID NO: 262, SEQ ID NO: 3306, SEQ ID NO: 2536, SEQ ID NO: 1971, SEQ ID NO: 3283, SEQ ID NO: 3297, SEQ ID NO: 4545, SEQ ID NO: 2661, SEQ ID NO: 307, SEQ ID NO: 4317, SEQ ID NO: 1268, SEQ ID NO: 4288, SEQ ID NO: 724, and SEQ ID NO: 5037.

In various aspects, the present disclosure provides a viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 a VP1 polypeptide, wherein the 581-589 region comprises at least one amino acid substitution as compared to SEQ ID NO: 9, and wherein a recombinant viral capsid comprising the VP capsid polypeptide is capable of preferentially targeting central nervous system tissue and a muscle tissue.

In some aspects, the 581-589 region has a sequence having at least 85% sequence identity to a sequence selected from the group consisting of SEQ ID NO: 18, SEQ ID NO: 3472, SEQ ID NO: 262, SEQ ID NO: 2536, SEQ ID NO: 3846, SEQ ID NO: 3297, SEQ ID NO: 4545, SEQ ID NO: 2661, SEQ ID NO: 1576, SEQ ID NO: 4317, SEQ ID NO: 23, SEQ ID NO: 22, SEQ ID NO: 964, SEQ ID NO: 4288, SEQ ID NO: 4290, SEQ ID NO: 4338, and SEQ ID NO: 5037. In some aspects, the 581-589 region has a sequence selected from the group consisting of SEQ ID NO: 18, SEQ ID NO: 3472, SEQ ID NO: 262, SEQ ID NO: 2536, SEQ ID NO: 3846, SEQ ID NO: 3297, SEQ ID NO: 4545, SEQ ID NO: 2661, SEQ ID NO: 1576, SEQ ID NO: 4317, SEQ ID NO: 23, SEQ ID NO: 22, SEQ ID NO: 964, SEQ ID NO: 4288, SEQ ID NO: 4290, SEQ ID NO: 4338, and SEQ ID NO: 5037.

In various aspects, the present disclosure provides a recombinant adeno-associated virus (rAAV) for use in a method of treating a condition in a subject, the method comprising: administering an rAAV to the subject, wherein the rAAV encapsidates a payload, and wherein the rAAV comprises a VP capsid polypeptide comprising a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide, wherein the 581-589 region has a sequence that is at least 85% identical to: (a) SEQ ID NO: 18, (b) SEQ ID NO: 3472, (c) SEQ ID NO: 262, (d) SEQ ID NO: 3306, (e) SEQ ID NO: 2028, (f) SEQ ID NO: 2791, (g) SEQ ID NO: 424, (h) SEQ ID NO: 2536, (i) SEQ ID NO: 1971, (j) SEQ ID NO: 415, (k) SEQ ID NO: 3846, (l) SEQ ID NO: 3283, (m) SEQ ID NO: 1956, (n) SEQ ID NO: 3297, (o) SEQ ID NO: 4545, (p) SEQ ID NO: 2661, (q) SEQ ID NO: 1576, (r) SEQ ID NO: 425, (s) SEQ ID NO: 709, (t) SEQ ID NO: 2168, (u) SEQ ID NO: 54, (v) SEQ ID NO: 429, (w) SEQ ID NO: 708, (x) SEQ ID NO: 428, (y) SEQ ID NO: 4119, (z) SEQ ID NO: 3906, (aa) SEQ ID NO: 2456, (bb) SEQ ID NO: 2278, (cc) SEQ ID NO: 5006, or (dd) SEQ ID NO: 426; infecting a CNS tissue of the subject with the rAAV; and delivering the payload to the CNS tissue infected by the rAAV.

In various aspects, the present disclosure provides a recombinant adeno-associated virus (rAAV) for use in a method of treating a condition in a subject, the method comprising: administering an rAAV to the subject, wherein the rAAV encapsidates a payload, and herein the rAAV comprises a VP capsid polypeptide a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide, wherein the 581-589 region comprises at least one amino acid substitution as compared to residues 581 to 589 of SEQ ID NO: 1, and wherein the VP capsid polypeptide does not have the sequence of any of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, or SEQ ID NO: 8; infecting a CNS tissue of the subject with the rAAV with at least 3-fold higher CNS tissue tropism as compared to a wild type AAV5 capsid comprising a peptide of SEQ ID NO: 1; and delivering the payload to the CNS tissue infected by the rAAV.

In various aspects, the present disclosure provides a viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 a VP1 polypeptide, wherein the 581-589 region comprises at least one amino acid substitution as compared to SEQ ID NO: 9, and wherein a recombinant viral capsid comprising the VP capsid polypeptide is capable of preferentially targeting a cardiac muscle tissue at a DNA enrichment of at least 25-fold greater than a wild type AAV9.

In some aspects, the 581-589 region has a sequence selected from the group consisting of SEQ ID NO: 348, SEQ ID NO: 2536, SEQ ID NO: 1576, SEQ ID NO: 4955, SEQ ID NO: 5017, SEQ ID NO: 4349, SEQ ID NO: 4964, SEQ ID NO: 293, SEQ ID NO: 4314, SEQ ID NO: 4995, SEQ ID NO: 4366, SEQ ID NO: 4961, SEQ ID NO: 4952, SEQ ID NO: 5075, SEQ ID NO: 4354, SEQ ID NO: 5060, SEQ ID NO: 5138, SEQ ID NO: 5206, SEQ ID NO: 4288, SEQ ID NO: 5092, SEQ ID NO: 4059, SEQ ID NO: 4295, SEQ ID NO: 5030, SEQ ID NO: 4938, SEQ ID NO: 2661, SEQ ID NO: 5215, SEQ ID NO: 4960, SEQ ID NO: 4969, SEQ ID NO: 5023, SEQ ID NO: 4934, SEQ ID NO: 4545, SEQ ID NO: 4963, SEQ ID NO: 4363, SEQ ID NO: 5159, and SEQ ID NO: 4973.

In various aspects, the present disclosure provides a viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence that is at least 85% identical to: (a) SEQ ID NO: 348, (b) SEQ ID NO: 2536, (c) SEQ ID NO: 1576, (d) SEQ ID NO: 4955, (e) SEQ ID NO: 5017, (f) SEQ ID NO: 4349, (g) SEQ ID NO: 4964, (h) SEQ ID NO: 293, (i) SEQ ID NO: 4314, (j) SEQ ID NO: 4995, (k) SEQ ID NO: 4366, (l) SEQ ID NO: 4961, (m) SEQ ID NO: 4952, (n) SEQ ID NO: 5075, (o) SEQ ID NO: 4354, (p) SEQ ID NO: 5060, (q) SEQ ID NO: 5138, (r) SEQ ID NO: 5206, (s) SEQ ID NO: 4288, (t) SEQ ID NO: 5092, (u) SEQ ID NO: 4059, (v) SEQ ID NO: 4295, (w) SEQ ID NO: 5030, (x) SEQ ID NO: 4938, (y) SEQ ID NO: 2661, (z) SEQ ID NO: 5215, (aa) SEQ ID NO: 4960, (bb) SEQ ID NO: 4969, (cc) SEQ ID NO: 5023, or (dd) SEQ ID NO: 4934.

In various aspects, the present disclosure provides a viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 a VP1 polypeptide, wherein the 581-589 region comprises at least one amino acid substitution as compared to SEQ ID NO: 9, and wherein a recombinant viral capsid comprising the VP capsid polypeptide is capable of preferentially targeting a skeletal muscle tissue at a DNA enrichment of at least 15-fold greater than a wild type AAV9.

In some aspects, the 581-589 region has a sequence selected from the group consisting of SEQ ID NO: 3472, SEQ ID NO: 3297, SEQ ID NO: 2661, SEQ ID NO: 4545, SEQ ID NO: 348, SEQ ID NO: 18, SEQ ID NO: 4349, SEQ ID NO: 293, SEQ ID NO: 5017, SEQ ID NO: 4955, SEQ ID NO: 4366, SEQ ID NO: 5092, SEQ ID NO: 210, SEQ ID NO: 4059, SEQ ID NO: 2536, SEQ ID NO: 5206, and SEQ ID NO: 1971.

In various aspects, the present disclosure provides a viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence that is at least 85% identical to: (a) SEQ ID NO: 3472, (b) SEQ ID NO: 3297, (c) SEQ ID NO: 2661, (d) SEQ ID NO: 4545, (e) SEQ ID NO: 348, (f) SEQ ID NO: 18, (g) SEQ ID NO: 4349, (h) SEQ ID NO: 293, (i) SEQ ID NO: 5017, (j) SEQ ID NO: 4955, (k) SEQ ID NO: 4366, (l) SEQ ID NO: 5092, (m) SEQ ID NO: 210, (n) SEQ ID NO: 4059, (o) SEQ ID NO: 2536, (p) SEQ ID NO: 5206, (q) SEQ ID NO: 1971, (r) SEQ ID NO: 262, (s) SEQ ID NO: 4343, (t) SEQ ID NO: 4995, (u) SEQ ID NO: 4009, (v) SEQ ID NO: 5190, (w) SEQ ID NO: 724, (x) SEQ ID NO: 4288, (y) SEQ ID NO: 4973, (z) SEQ ID NO: 4314, (aa) SEQ ID NO: 1561, (bb) SEQ ID NO: 5147, (cc) SEQ ID NO: 4238, or (dd) SEQ ID NO: 4961.

In various aspects, the present disclosure provides a viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 a VP1 polypeptide, wherein the 581-589 region comprises at least one amino acid substitution as compared to SEQ ID NO: 9, and wherein a recombinant viral capsid comprising the VP capsid polypeptide is capable of preferentially targeting a muscle tissue at a DNA enrichment of at least 20-fold greater than a wild type AAV9.

In some aspects, the 581-589 region has a sequence selected from the group consisting of SEQ ID NO: 348, SEQ ID NO: 2536, SEQ ID NO: 4955, SEQ ID NO: 5017, SEQ ID NO: 4349, SEQ ID NO: 293, SEQ ID NO: 1576, SEQ ID NO: 2661, SEQ ID NO: 4964, SEQ ID NO: 4314, SEQ ID NO: 4995, SEQ ID NO: 4366, SEQ ID NO: 4545, SEQ ID NO: 4961, SEQ ID NO: 5206, SEQ ID NO: 5092, SEQ ID NO: 3472, SEQ ID NO: 5075, SEQ ID NO: 4059, SEQ ID NO: 4354, SEQ ID NO: 5060, SEQ ID NO: 4288, SEQ ID NO: 4952, SEQ ID NO: 5138, SEQ ID NO: 18, SEQ ID NO: 5030, SEQ ID NO: 3297, SEQ ID NO: 4295, SEQ ID NO: 4363, SEQ ID NO: 4963, SEQ ID NO: 4973, SEQ ID NO: 5159, SEQ ID NO: 4960, SEQ ID NO: 4938, SEQ ID NO: 5157, SEQ ID NO: 5023, and SEQ ID NO: 4969.

In various aspects, the present disclosure provides a viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence that is at least 85% identical to: (a) SEQ ID NO: 348, (b) SEQ ID NO: 2536, (c) SEQ ID NO: 4955, (d) SEQ ID NO: 5017, (e) SEQ ID NO: 4349, (f) SEQ ID NO: 293, (g) SEQ ID NO: 1576, (h) SEQ ID NO: 2661, (i) SEQ ID NO: 4964, (j) SEQ ID NO: 4314, (k) SEQ ID NO: 4995, (l) SEQ ID NO: 4366, (m) SEQ ID NO: 4545, (n) SEQ ID NO: 4961, (o) SEQ ID NO: 5206, (p) SEQ ID NO: 5092, (q) SEQ ID NO: 3472, (r) SEQ ID NO: 5075, (s) SEQ ID NO: 4059, (t) SEQ ID NO: 4354, (u) SEQ ID NO: 5060, (v) SEQ ID NO: 4288, (w) SEQ ID NO: 4952, (x) SEQ ID NO: 5138, (y) SEQ ID NO: 18, (z) SEQ ID NO: 5030, (aa) SEQ ID NO: 3297, (bb) SEQ ID NO: 4295, (cc) SEQ ID NO: 4363, or (dd) SEQ ID NO: 4963.

In some aspects, the 581-589 region has a sequence of any one of any one of SEQ ID NO: 4238-SEQ ID NO: 4933 or SEQ ID NO: 4934-SEQ ID NO: 5233.

INCORPORATION BY REFERENCE

All references cited herein are incorporated by reference to the same extent as if each individual publication, database entry (e.g., Genbank sequences or GeneID entries), patent application, or patent, was specifically and individually indicated incorporated by reference in its entirety, for all purposes. This statement of incorporation by reference is intended by Applicants, pursuant to 37 C.F.R. § 1.57(b)(1), to relate to each and every individual publication, database entry (e.g., Genbank sequences or GeneID entries), patent application, or patent, each of which is clearly identified in compliance with 37 C.F.R. § 1.57(b)(2), even if such citation is not immediately adjacent to a dedicated statement of incorporation by reference. The inclusion of dedicated statements of incorporation by reference, if any, within the specification does not in any way weaken this general statement of incorporation by reference. Citation of the references herein is not intended as an admission that the reference is pertinent prior art, nor does it constitute any admission as to the contents or date of these publications or documents.

BRIEF DESCRIPTION OF THE DRAWINGS

These and other features, aspects, and advantages of the present invention will become better understood with regard to the following description, and accompanying drawings where:

FIG. 1 is a schematic of the high throughput AAV capsid engineering system.

FIG. 2A provides a side view (top panel) and top view (bottom panel) of key residues of known AAV capsids that have been shown to interact with target cells.

FIG. 2B illustrates salient features of the AAV capsid library described in EXAMPLE 1, showing the region of introduced diversity, with residue numbering corresponding to the numbering of amino acids in AAV5 VP1.

FIG. 3B is a schematic highlighting identification of AAV spike-in control DNA in CNS, liver, heart, and skeletal muscle by NGS analysis of NHP tissues post-selection.

FIG. 3C demonstrates that DNA from 94% of capsid variants predicted to target the CNS were found in the CNS.

FIG. 3D shows a comparison of DNA abundance in the CNS for three engineered AAV5 variants, SEQ ID NO: 18 (AAGRFNYFG), SEQ ID NO: 54 (AEDYWDLGA), and SEQ ID NO: 2028 (MDDICEFYA), of the present disclosure compared with wild type AAV5 and AAV9 spike-ins shows much greater infection than the highest (100×) controls.

FIG. 3E shows that engineered AAV5 variants of the present disclosure exhibit decreased liver infection compared to wild type (parental) AAV5 control.

FIG. 4 is a bar graph showing relative accumulation, expressed as log 10 fold change in brain accumulation relative to other tissues, of variant AAV capsids comprising 581-589 regions of SEQ ID NO: 415 (DARVYRALD), SEQ ID NO: 569 (DSASPIMGM), SEQ ID NO: 428 (DAWQMLSGN), SEQ ID NO: 430 (DAWSYQCYH), SEQ ID NO: 708 (EAWMYHQFH), SEQ ID NO: 3906 (YSGVRVTGY), SEQ ID NO: 709 (EAWSKLEQP), SEQ ID NO: 3297 (TAGRFNYFD), SEQ ID NO: 1956 (LVGWTLQHV), SEQ ID NO: 885 (ESWMKLEWQ), SEQ ID NO: 3283 (TAAFAYKYE), SEQ ID NO: 3472 (TSHYITFTP), SEQ ID NO: 426 (DAWMMMWGS), SEQ ID NO: 3306 (TANVYRSGQ), SEQ ID NO: 1971 (LWGWTLQHQ), SEQ ID NO: 425 (DAWLQLKDN), SEQ ID NO: 262 (CATRFNIGG), SEQ ID NO: 424 (DAWCFISSY), SEQ ID NO: 18 (AAGRFNYFG), SEQ ID NO: 54 (AEDYWDLGA), or SEQ ID NO: 2028 (MDDICEFYA). Accumulation was compared to wild type AAV capsids of AAV9, AAV1, AAV PHP.B, AAV2, and AAV5.

FIG. 5 is a bar graph showing relative accumulation, expressed as log 2 fold change in muscle accumulation relative to other tissues, of variant AAV capsids comprising 581-589 regions of SEQ ID NO: 4318 (CKEWYVRDR), SEQ ID NO: 4960 (CWREFSFQN), SEQ ID NO: 4371 (CVSLHSISM), SEQ ID NO: 3975 (QAHHYNILN), SEQ ID NO: 5215 (CVRTLQSPD), SEQ ID NO: 4598 (LAWSQLLTP), SEQ ID NO: 4359 (CVATKSRML), SEQ ID NO: 5665 (RQTTYDCLD), SEQ ID NO: 257 (CAHYAQWGK), SEQ ID NO: 344 (CSSGFHLFH), SEQ ID NO: 5607 (QCGFIRLNE), SEQ ID NO: 3528 (TVTHMSQHC), SEQ ID NO: 5155 (CVIWYHKYE), SEQ ID NO: 5363 (GCMCIRHAY), SEQ ID NO: 4633 (MACWKNATE), SEQ ID NO: 2525 (QAGSSFHQA), SEQ ID NO: 386 (CYQFWSQYS), SEQ ID NO: 4970 (CINFEIKLG), SEQ ID NO: 5143 (CWRHNIISP), SEQ ID NO: 5178 (CIKWVDIFP), SEQ ID NO: 2505 (PTQFYGPAF), SEQ ID NO: 3709 (VSASFQQHV), SEQ ID NO: 5056 (CVLNYFQFG), SEQ ID NO: 5017 (CFYKIQHKG), SEQ ID NO: 5040 (CVLRMGTFC), SEQ ID NO: 5161 (CTHMMGKSP), SEQ ID NO: 1128 (GFANFMMNF), SEQ ID NO: 2204 (MTMTATTFG), SEQ ID NO: 4316 (CIRHAQDCY), SEQ ID NO: 5463 (KISPWDGFY), SEQ ID NO: 4944 (CWKWQMMFG), SEQ ID NO: 4955 (CVWSQILGG), SEQ ID NO: 4952 (CVILSTRDK), SEQ ID NO: 3260 (SVSPCFCNS), SEQ ID NO: 384 (CYMPFKMQH), SEQ ID NO: 5038 (CVVWPVLGQ), SEQ ID NO: 2371 (NMTMAHQAS), SEQ ID NO: 289 (CIHSVHFAA), SEQ ID NO: 2985 (RVDAFNRGT), SEQ ID NO: 4508 (GSCLKIAQE), SEQ ID NO: 4292 (CAMLLQAVQ), SEQ ID NO: 4995 (CVGYRVHSP), SEQ ID NO: 4949 (CYHQVFSQG), SEQ ID NO: 5077 (CVGSQLHAM), SEQ ID NO: 2370 (NMNTFGKMN), SEQ ID NO: 5193 (CWSHLYFQT), SEQ ID NO: 4568 (KANEDLKQF), SEQ ID NO: 5206 (CFFYPMSMS), SEQ ID NO: 4215 (LYAFYMSSE), SEQ ID NO: 4841 (SSDQYEEMN), SEQ ID NO: 5092 (CISNYLKQG), SEQ ID NO: 4335 (CPMKHIQDR), SEQ ID NO: 5029 (CSFNIHKHT), SEQ ID NO: 3258 (SVRMFDAQY), SEQ ID NO: 4349 (CSSYLVTAN), SEQ ID NO: 348 (CTASWMSWD), or SEQ ID NO: 2729 (QQQQTNFQN). AAV variants that were generated using machine learning (ML) are denoted with arrows. Accumulation was compared to wild type AAV capsids of AAV9, AAV PHP.B, AAV1, AAV2, and AAV5.

FIG. 6 is a Circos plot depicting variant AAV5 capsids identified in a primary screen performed as illustrated in FIG. 1. Venn diagrams in the figure are not to scale. Over 1 million variant capsids were identified as unique to cardiac tissue, over 100,000 variants were identified as unique to skeletal muscle tissue, and over 1,000 variants were identified as shared between cardiac tissue and skeletal muscle tissue. The variants identified in cardiac tissue, skeletal muscle tissue, or both cardiac and skeletal muscle tissue exhibited low shared identity with liver (about 0.7% overlap) and other tissues (e.g., non-muscle tissues).

FIG. 7A illustrates the normalized counts of plasmid library DNA compared to assembled virus DNA for wild type AAV controls (textured points) and for variant AAV candidates (gray circles). Levels of assembled virus for variant AAV candidates were comparable to wild type AAV controls at 1× spike-in frequency.

FIG. 7B shows DNA levels in liver versus CNS for variant AAV candidates (gray circles), wild type controls (textured points), and select AAV candidates with CNS tropism (open circles). Circle size for the select AAV candidates corresponds to amount of functional transduction in CNS tissue, with larger circles representing higher RNA expression in CNS tissue. CNS-tropic AAV candidates were enriched in CNS tissue relative to liver tissue.

FIG. 7C shows DNA levels in muscle versus CNS for variant AAV candidates (gray circles), wild type controls (textured points), and select AAV candidates with CNS tropism (open circles). Circle size for the select AAV candidates corresponds to amount of functional transduction in CNS tissue, with larger circles representing higher RNA expression in CNS tissue. CNS-tropic AAV candidates were enriched in CNS tissue relative to muscle tissue.

FIG. 8A shows a bar graph of the proportion of candidates from different library sub-sets that were identified as muscle-specific in a secondary screen. Muscle candidates generated using machine learning (“Muscle ML Synthetic”) contained a higher proportion of muscle-specific sequences than candidates identified in muscle in a primary screen (“Muscle Observed”), CNS targeted sequences, or negative control sequences. Muscle candidate sequences generated using machine learning were five times more likely to be heart- and/or skeletal muscle-specific than muscle candidates identified in the primary screen and were 20 times more likely to be heart- and/or skeletal muscle-specific than CNS targeted variants. Candidates were classified as muscle-specific if they were >2-fold enriched in heart and/or skeletal muscle relative to all other tissues and viral input (FDR <0.1, permutations with α-RRA to account for consistency across multiple barcodes for each capsid).

FIG. 8B shows violin and box and whisker plots comparing the predicted probability of muscle specificity from primary screen for candidates that were (“yes”) or were not (“no”) identified as muscle-specific in the secondary screen. Candidates were classified as muscle-specific if they were >2-fold enriched in heart and/or skeletal muscle relative to all other tissues and viral input (FDR <0.1, permutations with α-RRA to account for consistency across multiple barcodes for each capsid).

FIG. 9 shows the CNS prediction score for CNS targeted candidates identified from multiple non-human primates (NHPs), multiple samples, observational enrichment, frequency enrichment, or sequence similarity, or generated by machine learning. CNS targeted candidates generated using machine learning had, on average, higher CNS prediction scores than candidates identified from other sources.

FIG. 10 is a plot showing relative accumulation and functional transduction of wild type AAV9 capsids at varying concentrations (top) and three identified CNS tissue-tropic AAV variants (SEQ ID NO: 18, SEQ ID NO: 424, and SEQ ID NO: 2028, bottom) in different tissues following systemic administration. RNA expression, representing functional transduction in each tissue, is shown on the x-axis. Circle size represents relative accumulation of AAV virions in each tissue, as measured by DNA. The AAV variants are highly selective for CNS tissue.

FIG. 11 is a plot showing relative accumulation and functional transduction of three identified CNS tissue-tropic AAV variants (SEQ ID NO: 18, SEQ ID NO: 424, and SEQ ID NO: 2028) in different CNS tissues following systemic administration. DNA quantification of a given AAV variant is shown on the x-axis. Circle size corresponds to amount of functional transduction of a given AAV variant in each tissue, with larger circles representing higher RNA expression in CNS tissue. The AAV variants show broad functional transduction in CNS tissues.

FIG. 12 illustrates the relative functional transduction of RNA expression in different regions of the brain of wild type AAV9 (left) and a CNS tissue-tropic AAV variant of SEQ ID NO: 2028 (right). Brain regions sampled include cortex (forebrain), cortex (occipital), cortex (temporal), thalamus, hypothalamus, hippocampus, cerebellum, caudate, putamen, pons, medulla, midbrain, and substantia nigra. Darker shading indicates higher transduction, as measured by RNA expression. The AAV variant shows broad functional transduction in CNS tissues.

FIG. 13 shows that three AAV variants of the present disclosure (SEQ ID NO: 18, SEQ ID NO: 424, and SEQ ID NO: 2028, also shown in FIG. 11) functionally transduce neurons. CAG promoter-driven RNA expression highlights functional transduction of any cell type while SYN promoter-driven RNA expression highlights functional transduction of neurons.

FIG. 14 shows that some AAV variants of the present disclosure that target CNS also demonstrate dorsal root ganglion (DRG) depletion.

FIG. 15 shows that promoter usage differentiates CNS AAV variants and heart muscle AAV variants. For example, certain AAV CNS variants of the present disclosure show CAG and SYN promoter-driven RNA expression, while certain AAV heart muscle variants show only CAG promoter-driven RNA expression.

FIG. 16A is bar plot of group intersection size for capsids enriched in cardiac muscle tissue in non-human primates (NHPs), mice, or both NHPs and mice.

FIG. 16B is a scatter plot showing log 2-fold change in enrichment in cardiac tissue in NHPs versus mice for capsid variants or wild type AAV capsids. Seventeen variants enriched in cardiac tissue in both NHPs and mice are shown in dark circles.

FIG. 17A is bar plot of group intersection size for capsids enriched in CNS tissue in non-human primates (NHPs) or mice.

FIG. 17B is a scatter plot showing log 2-fold change in enrichment in CNS tissue in NHPs versus mice for capsid variants or wild type AAV capsids.

FIG. 18 is a scatter plot of the average Z-score of CNS tissue enrichment for screened variant AAV capsids, ordered by average variant capsid rank for twelve methods of differential expression analysis. The high scoring variant capsid, corresponding to the outlined points in the upper left quadrant, were ranked consistently highly across multiple analysis strategies and ranked consistently higher than wild type AAV5 (wtAAV5) and wild type AAV9 (wtAAV9).

DETAILED DESCRIPTION OF THE INVENTION

Unless described otherwise, all technical and scientific terms used herein have the meaning commonly understood by one of ordinary skill in the art to which the invention pertains.

Unless otherwise stated, whenever a range is recited, the range is inclusive of the recited endpoints. For example, the region from amino acid residue 581 to amino acid residue 589 of SEQ ID NO: 1 includes amino acid residues 581 and 589.

“Homology” or “identity” or “similarity” can refer to sequence similarity between two peptides or between two nucleic acid molecules. Homology can be determined by comparing a position in each sequence which can be aligned for purposes of comparison. When a position in the compared sequence can be occupied by the same base or amino acid, then the molecules can be homologous at that position. A degree of homology between sequences can be a function of the number of matching or homologous positions shared by the sequences. An “unrelated” or “non-homologous” sequence shares less than 40% identity, or alternatively less than 25% identity, with one of the sequences of the disclosure. Sequence homology can refer to a % identity of a sequence to a reference sequence. As a practical matter, whether any particular sequence can be at least 50%, 60%, 70%, 77.7%, 80%, 85%, 88.8%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to any sequence described herein (which can correspond with a particular nucleic acid or amino acid sequence described herein), such particular polypeptide sequence can be determined conventionally using known computer programs such the Bestfit program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, 575 Science Drive, Madison, Wis. 53711). When using Bestfit or any other sequence alignment program to determine whether a particular sequence is, for instance, 95% identical to a reference sequence, the parameters can be set such that the percentage of identity can be calculated over the full length of the reference sequence and that gaps in sequence homology of up to 5% of the total reference sequence can be allowed. The term percent “identity” or percent “homology,” in the context of two or more nucleic acid or polypeptide sequences, refer to two or more sequences or subsequences that have a specified percentage of nucleotides or amino acid residues that are the same, when compared and aligned for maximum correspondence, as measured using one of the sequence comparison algorithms described below (e.g., BLASTP and BLASTN or other algorithms available to persons of skill) or by visual inspection. Depending on the application, the percent “identity” can exist over a region of the sequence being compared, e.g., over a functional domain, or, alternatively, exist over the full length of the two sequences to be compared. For sequence comparison, typically one sequence acts as a reference sequence to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are input into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. The sequence comparison algorithm then calculates the percent sequence identity for the test sequence(s) relative to the reference sequence, based on the designated program parameters. For purposes herein, determination of percent identity and sequence similarity is performed using the BLAST algorithm, which is described in Altschul et al., J. Mol. Biol. 215:403-410 (1990). Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (www.ncbi.nlm.nih.gov/). For example, where an amino acid sequence consisting of 9 residues is compared with another 9 residue amino acid sequence, if 8 residues match then the sequences have 88.8% identity, if 7 residues match then the sequences have 77.7% identity.

In some cases, the identity between a reference sequence (query sequence, e.g., a sequence of the disclosure) and a subject sequence, also referred to as a global sequence alignment, can be determined using the FASTDB computer program. In some embodiments, parameters for a particular embodiment in which identity can be narrowly construed, used in a FASTDB amino acid alignment, can include: Scoring Scheme=PAM (Percent Accepted Mutations) 0, k-tuple=2, Mismatch Penalty=1, Joining Penalty=20, Randomization Group Length=0, Cutoff Score=1, Window Size=sequence length, Gap Penalty=5, Gap Size Penalty=0.05, Window Size=500 or the length of the subject sequence, whichever can be shorter. According to this embodiment, if the subject sequence can be shorter than the query sequence due to N- or C-terminal deletions, not because of internal deletions, a manual correction can be made to the results to take into consideration the fact that the FASTDB program does not account for N- and C-terminal truncations of the subject sequence when calculating global percent identity. For subject sequences truncated at the N- and C-termini, relative to the query sequence, the percent identity can be corrected by calculating the number of residues of the query sequence that can be lateral to the N- and C-terminal of the subject sequence, which can be not matched/aligned with a corresponding subject residue, as a percent of the total bases of the query sequence. A determination of whether a residue can be matched/aligned can be determined by results of the FASTDB sequence alignment. This percentage can be then subtracted from the percent identity, calculated by the FASTDB program using the specified parameters, to arrive at a final percent identity score. This final percent identity score can be used for the purposes of this embodiment. In some cases, only residues to the N- and C-termini of the subject sequence, which can be not matched/aligned with the query sequence, can be considered for the purposes of manually adjusting the percent identity score. That is, only query residue positions outside the farthest N- and C-terminal residues of the subject sequence can be considered for this manual correction. For example, a 90-residue subject sequence can be aligned with a 100-residue query sequence to determine percent identity. The deletion occurs at the N-terminus of the subject sequence, and therefore, the FASTDB alignment does not show a matching/alignment of the first 10 residues at the N-terminus. The 10 unpaired residues represent 10% of the sequence (number of residues at the N- and C-termini not matched/total number of residues in the query sequence) so 10% can be subtracted from the percent identity score calculated by the FASTDB program. If the remaining 90 residues were perfectly matched, the final percent identity can be 90%. In another example, a 90-residue subject sequence can be compared with a 100-residue query sequence. This time the deletions can be internal deletions, so there can be no residues at the N- or C-termini of the subject sequence which can be not matched/aligned with the query. In this case, the percent identity calculated by FASTDB can be not manually corrected. Once again, only residue positions outside the N- and C-terminal ends of the subject sequence, as displayed in the FASTDB alignment, which can be not matched/aligned with the query sequence can be manually corrected for.

Peptide sequences for use in the present invention may include one or more conservative amino acid substitutions, such that the resulting sequence has a similar amino acid sequence and/or retains the same function. The skilled person is aware that various amino acids have similar biochemical properties and thus are “conservative”. One or more such amino acids of a protein, polypeptide or peptide can often be substituted by one or more other such amino acids without eliminating a desired activity of that protein, polypeptide or peptide. Thus, the amino acids glycine, alanine, valine, leucine, and isoleucine can often be substituted for one another (amino acids having aliphatic side chains). Of these possible substitutions it is preferred that glycine and alanine are used to substitute for one another (since they have relatively short side chains) and that valine, leucine and isoleucine are used to substitute for one another (since they have larger aliphatic side chains which are hydrophobic). Other amino acids which can often be substituted for one another include: phenylalanine, tyrosine and tryptophan (amino acids having aromatic side chains); lysine, arginine and histidine (amino acids having basic side chains); aspartate and glutamate (amino acids having acidic side chains); asparagine and glutamine (amino acids having amide side chains); and cysteine and methionine (amino acids having sulfur containing side chains). It should be appreciated that amino acid substitutions within the scope of the present invention can be made using naturally occurring or non-naturally occurring amino acids. For example, the methyl group on an alanine may be replaced with an ethyl group, and/or minor changes may be made to the peptide backbone. Whether or not natural or synthetic amino acids are used, it is preferred that only L-amino acids are present. Substitutions of this nature are often referred to as “conservative substitutions”.

As used herein, “tropism” of a rAAV for a tissue may refer to the ability of a given rAAV to preferentially infect a given cell type or tissue. A degree of tropism may be determined by a ratio of an infection rate in a targeted tissue to an infection rate in a different, non-targeted tissue. As used herein, increased tropism for a given cell type or tissue, such as increased tropism conferred by a 581-589 region, is determined relative to a wild type AAV5 capsid. As used herein, “detargeting” of a rAAV to a tissue may refer to the ability of a given rAAV to avoid infecting a detargeted tissue or cell type while infecting one or more other tissues or cell types. A degree of detargeting may be determined by a ratio of an infection rate in a detargeted tissue to an infection rate of a different, non-detargeted tissue. As used herein, increased detargeting for a given cell type or tissue, such as increased detargeting conferred by a 581-589 region, is determined relative to a wild type AAV5 capsid.

As used herein, “tissue tropism” refers to a preference of a virus having an engineered VP capsid polypeptide of the present disclosure to infect a given tissue or be enriched in or accumulate in a given tissue. A “tissue-tropic” rAAV may specifically target or infect a first tissue or set of tissues and may not target or infect a second tissue or set of tissues. For example, a “CNS-tropic” rAAV may specifically target or infect CNS tissue and may not target or infect muscle, skin, bone, or other tissue or tissues. A “tissue-detargeted” rAAV may specifically avoid targeting or avoid infection of the detargeted tissue or set of tissues while infecting a second tissue or set of tissues. For example, a “liver-detargeted” rAAV may not target or infect liver tissue but may infect one or more other tissues, such as nervous, muscle, skin, bone, and/or other tissue. Tissue tropism or tissue detargeting, when used as a relative term and depending on the context in which it is described herein, refers to an increase or decrease in tissue tropism of a given rAAV virion having a first capsid polypeptide in a first tissue as compared to a second tissue and/or refers to an increase or decrease in tissue tropism of a given rAAV virion having a first capsid polypeptide to an rAAV virion having a second capsid polypeptide. In some embodiments, the first tissue can be a group of tissues. In some embodiments, the second tissue can be a group of tissues. For example, the first tissue may be CNS tissues, which comprise cortex forebrain, cortex occipital, cortex temporal, thalamus, hypothalamus, substantia nigra, hippocampus DG, hippocampus CA1, hippocampus CA3, and cerebellum and the second tissue may be a non-CNS tissue consisting collectively of liver, skeletal muscle, heart, lung, spleen, lymph node, bone marrow, mammary gland, skin, adrenal gland, thyroid, colon, sciatic nerve, and spinal cord tissues. As another example, the first tissue may be liver tissue and the second tissue may be non-liver tissue consisting collectively of CNS tissues, skeletal muscle, heart, lung, spleen, lymph node, bone marrow, mammary gland, skin, adrenal gland, thyroid, colon, sciatic nerve, and spinal cord tissues.

In some embodiments, the rAAV virions of the present disclosure may also be referred to as preferentially targeting a given tissue or having tissue selectivity for a given tissue. For example, an rAAV virion that preferentially targets CNS tissue may specifically target or infect CNS tissue and may not target or infect skeletal muscle, cardiac muscle, or other tissues or may target other tissues to a lesser degree than CNS tissue. As another example, an rAAV virion that has retinal tissue selectivity may specifically target or infect CNS tissue and may not target or infect skeletal muscle, cardiac muscle, or other tissues or may target other tissues to a lesser degree than CNS tissue.

For simplicity throughout this disclosure, viral capsid protein is generally referred to as “VP.” Viral capsid protein is referred to as VP1 when referencing AAV5 VP1 positional notation. In all cases, viral capsid sequences and mutations disclosed herein should be understood as pertaining to all isoforms of the capsid protein (VP1, VP2, and VP3), as a mixture of these isoforms assemble to form virions. The positional amino acid residue designations “581 to 589” are relative to the translational start of the VP1 polypeptide and should be adjusted accordingly to the relative start sites of VP2 and VP3. It should be understood that the present disclosure, when describing any particular VP1 sequence with mutations at particular amino acid residue positions, necessarily also encompasses corresponding mutations in VP2 and VP3. For example, any consensus sequence or specific sequence of a VP1 capsid protein having one or more mutations in the 581-589 region, corresponding to amino acid residues 581 to 589 of VP1, also encompasses VP2 and VP3 capsid proteins having said one or more mutations in an amino acid residue region in VP2 and VP3 corresponding to the amino acid residues of the VP1 581 to 589 region. For example, the amino acid residues of the 581 to 589 region of VP1 (SEQ ID NO: 1; MSFVDHPPDWLEEVGEGLREFLGLEAGPPKPKPNQQHQDQARGLVLPGYNYLGPGNG LDRGEPVNRADEVAREHDISYNEQLEAGDNPYLKYNHADAEFQEKLADDTSFGGNLG KAVFQAKKRVLEPFGLVEEGAKTAPTGKRIDDHFPKRKKARTEEDSKPST SSDAEAGPS GSQQLQIPAQPASSLGADTMSAGGGGPLGDNNQGADGVGNASGDWHCDSTWMGDRV VTKSTRTWVLPSYNNHQYREIKSGSVDGSNANAYFGYSTPWGYFDFNRFHSHWSPRD WQRLINNYWGFRPRSLRVKIFNIQVKEVTVQDSTTTIANNLTSTVQVFTDDDYQLPYVV GNGTEGCLPAFPPQVFTLPQYGYATLNRDNTENPTERSSFFCLEYFPSKMLRTGNNFEFT YNFEEVPFHSSFAPSQNLFKLANPLVDQYLYRFVSTNNTGGVQFNKNLAGRYANTYKN WFPGPMGRTQGWNLGSGVNRASVSAFATTNRMELEGASYQVPPQPNGMTNNLQGSN TYALENTMIFNSQPANPGTTATYLEGNMLITSESETQPVNRVAYNVGGQMATNNQSST TAPATGTYNLQEIVPGSVWMERDVYLQGPIWAKIPETGAHFHPSPAMGGFGLKHPPPM MLIKNTPVPGNIT SFSDVPVSSFITQYSTGQVTVEMEWELKKENSKRWNPEIQYTNNYN DPQFVDFAPDSTGEYRTTRPIGTRYLTRPL) correspond to the amino acid residues of the 445 to 453 region of VP2 (SEQ ID NO: 10; TAPTGKRIDDHFPKRKKARTEEDSKPSTSSDAEAGPSGSQQLQIPAQPASSLGADTMSAG GGGPLGDNNQGADGVGNASGDWHCDSTWMGDRVVTKSTRTWVLPSYNNHQYREIKS GSVDGSNANAYFGYSTPWGYFDFNRFHSHWSPRDWQRLINNYWGFRPRSLRVKIFNIQ VKEVTVQDSTTTIANNLTSTVQVFTDDDYQLPYVVGNGTEGCLPAFPPQVFTLPQYGY ATLNRDNTENPTERSSFFCLEYFPSKMLRTGNNFEFTYNFEEVPFHSSFAPSQNLFKLAN PLVDQYLYRFVSTNNTGGVQFNKNLAGRYANTYKNWFPGPMGRTQGWNLGSGVNRA SVSAFATTNRMELEGASYQVPPQPNGMTNNLQGSNTYALENTMIFNSQPANPGTTATY LEGNMLITSESETQPVNRVAYNVGGQMATNNQSSTTAPATGTYNLQEIVPGSVWMERD VYLQGPIWAKIPETGAHFHPSPAMGGFGLKHPPPMMLIKNTPVPGNITSFSDVPVSSFITQ YSTGQVTVEMEWELKKENSKRWNPEIQYTNNYNDPQFVDFAPDSTGEYRTTRPIGTRY LTRPL) and to the amino acid residues of 389 to 397 region of VP3 (SEQ ID NO: 11; MSAGGGGPLGDNNQGADGVGNASGDWHCDSTWMGDRVVTKSTRTWVLPSYNNHQY REIKSGSVDGSNANAYFGYSTPWGYFDFNRFHSHWSPRDWQRLINNYWGFRPRSLRVK IFNIQVKEVTVQDSTTTIANNLTSTVQVFTDDDYQLPYVVGNGTEGCLPAFPPQVFTLPQ YGYATLNRDNTENPTERSSFFCLEYFPSKMLRTGNNFEFTYNFEEVPFHSSFAPSQNLFK LANPLVDQYLYRFVSTNNTGGVQFNKNLAGRYANTYKNWFPGPMGRTQGWNLGSGV NRASVSAFATTNRMELEGASYQVPPQPNGMTNNLQGSNTYALENTMIFNSQPANPGTT ATYLEGNMLITSESETQPVNRVAYNVGGQMATNNQSSTTAPATGTYNLQEIVPGSVWM ERDVYLQGPIWAKIPETGAHFHPPSPAMGGFGLKHPPPMMLIKNTPVPGNITSFSDVPVSS FITQYSTGQVTVEMEWELKKENSKRWNPEIQYTNNYNDPQFVDFAPDSTGEYRTTRPIG TRYLTRPL). As used herein, “wild type AAV5” or “wild type AAV5 capsid polypeptide” refers to a VP1 capsid polypeptide of SEQ ID NO: 1, a VP2 capsid polypeptide of SEQ ID NO: 10, a VP3 capsid polypeptide of SEQ ID NO: 11, or a combination thereof. Also as used herein, a “wild type 581-589 region” refers to a 581 to 589 region of VP1 having a sequence of

(SEQ ID NO: 9)

ATGTYNLQE.

As used herein, “581-589 region” refers to a region or fragment of VP1 corresponding to amino acid residues 581 to 589 relative to the translational start of the VP1 polypeptide. The 581-589 region may also be referred to as a “variant region.” The 581-589 region corresponds to amino acid residues 445 to 453 of VP2 and to amino acid residues 389 to 397 of VP3. The 581-589 region may confer tissue tropism to an AAV, and defined variants (e.g., SEQ ID NO: 12-SEQ ID NO: 3937, SEQ ID NO: 3938-SEQ ID NO: 4237, SEQ ID NO: 4238-SEQ ID NO: 4933, SEQ ID NO: 4934-SEQ ID NO: 5233, SEQ ID NO: 5234-SEQ ID NO: 5807, or SEQ ID NO: 5808-SEQ ID NO: 5811) may be engineered to confer tissue tropism to an rAAV formed from viral capsid polypeptides (VP1, VP2, and VP3) comprising the 581-589 region. The VP1 with a generalized 581-589 region is provided in SEQ ID NO: 2 (MSFVDHPPDWLEEVGEGLREFLGLEAGPPKPKPNQQHQDQARGLVLPGYNYLGPGNG LDRGEPVNRADEVAREHDISYNEQLEAGDNPYLKYNHADAEFQEKLADDTSFGGNLG KAVFQAKKRVLEPFGLVEEGAKTAPTGKRIDDHFPKRKKARTEEDSKPSTSSDAEAGPS GSQQLQIPAQPASSLGADTMSAGGGGPLGDNNQGADGVGNASGDWHCDSTWMGDRV VTKSTRTWVLPSYNNHQYREIKSGSVDGSNANAYFGYSTPWGYFDFNRFHSHWSPRD WQRLINNYWGFRPRSLRVKIFNIQVKEVTVQDSTTTIANNLTSTVQVFTDDDYQLPYVV GNGTEGCLPAFPPQVFTLPQYGYATLNRDNTENPTERSSFFCLEYFPSKMLRTGNNFEFT YNFEEVPFHSSFAPSQNLFKLANPLVDQYLYRFVSTNNTGGVQFNKNLAGRYANTYKN WFPGPMGRTQGWNLGSGVNRASVSAFATTNRMELEGASYQVPPQPNGMTNNLQGSN TYALENTMIFNSQPANPGTTATYLEGNMLITSESETQPVNRVAYNVGGQMATNNQSST TAPX¹X²X³X⁴X⁵X⁶X⁷X⁸X⁹IVPGSVWMERDVYLQGPIWAKIPETGAHFHPSPAMGGFGLK HPPPMMLIKNTPVPGNITSFSDVPVSSFITQYSTGQVTVEMEWELKKENSKRWNPEIQYT NNYNDPQFVDFAPDSTGEYRTTRPIGTRYLTRPL), in which the 581-589 region has a sequence of X¹X²X³X⁴X⁵X⁶X⁷X⁸X⁹; wherein X¹, X², X³, X⁴, X⁵, X⁶, X⁷, X⁸, and X⁹are each independently selected from A, R, N, D, C, E, Q, G, H, I, L, K, M, F, P, S, T, W, Y, and V.

It should be understood that the present disclosure includes polynucleotide sequences encoding for any sequence disclosed herein. For example, if an amino acid sequence is provided, the present disclosure also encompasses a polynucleotide sequence encoding for said amino acid sequence.

It should be understood that further embodiments include mutations in VP1, VP2, VP3, or any combination thereof that do not alter the desired properties (e.g., a particular tissue tropism) or affect viral assembly, as described herein.

An rAAV virion is made of a capsid that may include the engineered AAV5 VP capsid polypeptides disclosed herein (e.g., VP1, VP2, and VP3 capsid polypeptides).

Engineered Capsids and Capsid Polypeptides for Tissue Tropism

Described herein are engineered capsids, engineered capsid polypeptides, and 581-589 regions of capsid polypeptides that confer tissue tropism (e.g., tissue-specific accumulation, tissue-specific infection, or both) to a viral capsid. In some embodiments, an engineered capsid may comprise one or more engineered capsid polypeptides assembled into a recombinant adeno-associated virus (rAAV) viral capsid. In some embodiments, an engineered capsid polypeptide may comprise a variation in the 581-589 region. The 581-589 region of viral capsid polypeptides, corresponding to amino acid residues 581 to 589 of the VP1 polypeptide, amino acid residues 445 to 453 of the VP2 polypeptide, and amino acid residues 389 to 397 of the VP3 polypeptide, is located at the AAV interface that interacts with host cells and tissues.

Also described herein are methods of using engineered capsids comprising engineered capsid polypeptides with 581-589 regions for tissue-specific delivery of a payload (e.g., a polynucleotide, such as a transgene) encapsidated by the engineered capsid. Recombinant AAVs comprising VP capsid polypeptides with 581-589 regions engineered for tissue specificity may be used to specifically infect a target tissue. Using tissue-tropic rAAV viral capsids for payload delivery provides numerous advantages over using adeno-associated virus (AAV) viral capsids that lack tissue tropism including reduced toxicity, lower dose needed to produce a therapeutic effect, wider therapeutic window, and reduced immune response. Furthermore, tissue-specific payload delivery may enable targeted therapies even when administering systemically. For example, a central nervous (CNS) tissue-tropic rAAV viral capsid may be systemically administered to specifically deliver a payload to the CNS for treatment of a neurological disease. In another example, a muscle-tropic rAAV viral capsid may be systemically administered to specifically deliver a payload to muscle for treatment of a muscular disease, including cardiac muscle and vascular diseases.

In some embodiments, a tissue-tropic capsid of the present disclosure may be CNS tissue-tropic. For example, a CNS tissue-tropic capsid polypeptide may confer tropism for one or more CNS tissues (e.g., hippocampus (dentate gyrus, CA1, or CA3), cerebellum, hypothalamus, cortex (occipital, temporal, or forebrain), substantia nigra, thalamus, or combinations thereof). In some embodiments, a tissue-tropic capsid, engineered capsid polypeptide, or 581-589 region of a capsid polypeptide may be muscle-tropic. For example, a muscle-tropic capsid polypeptide may confer tropism for one or more muscle tissues (e.g., aorta, esophagus, heart (including atrium, ventricle, valves), skeletal muscle (including biceps, brachii, femoris, diaphragm, gastrocnemius, tibialis anterior, triceps, quadriceps, including vastus lateralis)), or muscle fibers (e.g., type I (slow oxidative) fibers, type IC fibers, type II fibers, type IIC fibers, type IIA (fast oxidative/glycolytic) fibers, type IIAX fibers, type IIXA fibers, type IIX (fast glycolytic) fibers, or combinations thereof).

An engineered capsid polypeptide of the present disclosure may comprise one or more amino acid substitutions relative to an AAV5 viral protein (VP) polypeptide (e.g., a VP1 polypeptide of SEQ ID NO: 1, a VP2 polypeptide of SEQ ID NO: 10, or a VP3 polypeptide of SEQ ID NO: 11). The engineered capsid polypeptide may comprise one or more amino acid substitutions relative to a VP1 polypeptide of any or all of SEQ ID NO: 3-SEQ ID NO: 8. In some embodiments, the engineered capsid polypeptide may comprise a 581-589 region comprising one or more amino acid substitutions in a region of a VP polypeptide (e.g., a 581-589 region of VP1, VP2, VP3, or a combination thereof) corresponding to amino acid residues 581 to 589 of VP1 (e.g., SEQ ID NO: 1), amino acid residues 445 to 453 of VP2 (e.g., SEQ ID NO: 10), or amino acid residues 389 to 397 of VP3 (e.g., SEQ ID NO: 11). In some embodiments, the 581-589 region may be present in VP1, VP2, and VP3. An engineered viral capsid may be assembled from VP1, VP2, and VP3 polypeptides comprising a 581-589 region with one or more amino acid substitutions. In some embodiments, the 581-589 region may confer or increase the likelihood of conferring CNS tissue-tropism. For example, a 581-589 region with increased likelihood of conferring CNS tissue-tropism may comprise a sequence of any one of SEQ ID NO: 12-SEQ ID NO: 3937, SEQ ID NO: 3938-SEQ ID NO: 4237, or SEQ ID NO: 5234-SEQ ID NO: 5807. In some embodiments, the 581-589 region may confer or increase the likelihood of conferring muscle-tropism. For example, a 581-589 region with increased likelihood of conferring muscle-tropism may comprise a sequence of any one of SEQ ID NO: 4238-SEQ ID NO: 4933, SEQ ID NO: 4934-SEQ ID NO: 5233, or SEQ ID NO: 5808-SEQ ID NO: 5811.

In some embodiments, the 581-589 region may confer or increase the likelihood of conferring CNS tissue-tropism. For example, a 581-589 region with increased likelihood of conferring CNS tissue-tropism may comprise a sequence of any one of SEQ ID NO: 12-SEQ ID NO: 3937 or SEQ ID NO: 3938-SEQ ID NO: 4237. In some embodiments, the 581-589 region may confer or increase the likelihood of conferring muscle-tropism. For example, a 581-589 region with increased likelihood of conferring muscle-tropism may comprise a sequence of any one of SEQ ID NO: 4238-SEQ ID NO: 4933 or SEQ ID NO: 4934-SEQ ID NO: 5233.

Capsid Engineering Methods

Disclosed herein is a system for high-throughput engineering of engineered AAV capsids with modified function, including increased or decreased infectivity of desired tissues, such as increased targeting of the central nervous system (CNS), increased targeting of muscle, decreased targeting of non-CNS tissue, or decreased targeting of non-muscle tissue relative to a wild type AAV5 capsid (e.g., comprising a VP capsid polypeptide of SEQ ID NO: 1). A general schematic of the process is shown in FIG. 1. However, it should be understood that the present disclosure also encompasses reasonable variations or extensions to the method that are understood to those of ordinary skill in the art. As shown in FIG. 1, the method may begin with production of a capsid library with theoretical diversity of 5×10¹¹(5e11) unique sequence variants. Higher or lower theoretical diversities are also encompassed herein. For example, a capsid library may have a theoretical diversity of from about 1×10³(1e3) to about 1×10²⁰(1e20). The library may then be cloned into plasmids, transformed into bacteria, and subsequently, library plasmids are screened for productive virion assembly in a production cell line. The assembled virions may then be administered intravenously into non-human primates (NHP). After a period of time sufficient for distribution, infection, and stable transduction, the NHP may be sacrificed, organs harvested, and sequences of AAV capsids in each tissue may be determined by deep sequencing.

FIG. 2A provides a side view (top panel) and top view (bottom panel) of the surface of a prototype AAV virion, identifying residues of known AAV capsids, including AAV2, AAV5, AAV6, and AAV9, that have been shown in the research literature to interact with target cells. These target-interacting residues correspond to amino acids 581 to 589 in the AAV5 VP1 capsid protein.

FIG. 2B shows the salient elements of the library plasmid, illustrating rep and cap coding sequences positioned between AAV ITRs. In the illustrated embodiment, further described in EXAMPLE 1, variation is introduced into each of residues 581 to 589 of the AAV5 cap protein (“Library variant region”) that is present in VP1, VP2, and VP3. Each of the 20 natural amino acids is introduced at each of the 9 positions of the 581-589 region, providing a theoretical library diversity of 20⁹(20{circumflex over ( )}9; approximately 5×10¹¹(5e11)) unique sequence variants.

The 581-589 region targeted for engineering is the most likely to interact with target cell receptors, and relatively tolerant to changes without disrupting virion assembly. Unlike earlier approaches that add unstructured peptides that protrude above the virion 3-fold axis of symmetry, the current approach introduces sequence diversity that alters the characteristics of the binding pocket. In addition, this approach may change the overall structure of the receptor-binding trimer, allowing for altered allosteric interactions outside the binding pocket (e.g., AAVR PKD1). Introduced diversity is non-random, thereby reducing missense and frameshifts of randomized libraries.

By cloning the polynucleotide encoding the capsid variants into the packaged viral genome (between the ITRs), the recombinant virions with variant capsids carry polynucleotides having their cognate mutation, so the unique variant providing the desired function can be identified by sequencing packaged virus or infected cells.

In some embodiments, the capsid is a capsid selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV 10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAV-DJ, AAV-DJ/8, AAV-DJ/9, AAV1/2, AAV.rh8, AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh43, AAV.Rh74, AAV.v66, AAV.Oligo001, AAV.SCH9, AAV.r3.45, AAV.RHM4-1, AAV.hu37, AAV.Anc80, AAV.Anc80L65, AAV.7m8, AAV.PhP.eB, AAV.PhP.V1, AAV.PHP.B, AAV.PhB.C1, AAV.PhB.C2, AAV.PhB.C3, AAV.PhB.C6, AAV.cy5, AAV2.5, AAV2tYF, AAV3B, AAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10, AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, AAV.HSC16, AAV.HSC17, or AAVhu68 (for example, as described in WO2020/033842, incorporated herein by reference in its entirety). For example, the capsid may be an AAV5 capsid. The hu68 capsid is described in WO 2018/160582, incorporated herein by reference in its entirety.

Such capsids may comprise a 581-589 region corresponding amino acid residues 581 to 589 of the AAV5 VP1, and as such analogous engineered VP capsids with desired tissue tropism or preference, ability to assemble, and exhibit various other desired traits are encompassed herein. Thus, any one of the engineered AAV5 VP capsid polypeptides disclosed herein having a mutation or substitution in the 581-589 region corresponding to the 581 to 589 region of AAV5 VP1 may be inserted into the corresponding region in any one of the other AAV capsids described herein and the present disclosure encompasses such variants.

In some embodiments, the capsid is a derivative, modification, or pseudotype of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV 10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAV-DJ, AAV-DJ/8, AAV-DJ/9, AAV1/2, AAV.rh8, AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh43, AAV.Rh74, AAV.v66, AAV.Oligo001, AAV.SCH9, AAV.r3.45, AAV.RHM4-1, AAV.hu37, AAV.Anc80, AAV.Anc80L65, AAV.7m8, AAV.PhP.eB, AAV.PhP.V1, AAV.PHP.B, AAV.PhB.C1, AAV.PhB.C2, AAV.PhB.C3, AAV.PhB.C6, AAV.cy5, AAV2.5, AAV2tYF, AAV3B, AAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10, AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, AAV.HSC16, AAV.HSC17, or AAVhu68. For example, the capsid may be a derivative of AAV5.

In some embodiments, capsid protein is a chimera of capsid proteins from two or more serotype selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV 10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAV-DJ, AAV-DJ/8, AAV-DJ/9, AAV1/2, AAV.rh8, AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh43, AAV.Rh74, AAV.v66, AAV.Oligo001, AAV.SCH9, AAV.r3.45, AAV.RHM4-1, AAV.hu37, AAV.Anc80, AAV.Anc80L65, AAV.7m8, AAV.PhP.eB, AAV.PhP.V1, AAV.PHP.B, AAV.PhB.C1, AAV.PhB.C2, AAV.PhB.C3, AAV.PhB.C6, AAV.cy5, AAV2.5, AAV2tYF, AAV3B, AAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10, AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, AAV.HSC16, AAV.HSC17, or AAVhu68 (for example, as described in WO2020/033842, incorporated herein by reference in its entirety). In certain embodiments, the capsid is an rh32.33 capsid, described in U.S. Pat. No. 8,999,678, incorporated herein by reference in its entirety.

Such capsids may comprise a 581-589 region corresponding to 581 to 589 of the AAV5 VP1, and as such analogous engineered VP capsids with desired tissue tropism or preference, ability to assemble, and exhibit various other desired traits are encompassed herein.

VP-Encoding Polynucleotides, Vectors, and Vector Libraries

Accordingly, in a first aspect, polynucleotides are provided. The polynucleotides encode an adeno-associated virus (AAV) VP1 capsid polypeptide having the amino acid sequence of SEQ ID NO: 2, wherein X¹, X², X³, X⁴, X⁵, X⁶, X⁷, X⁸, and X⁹are each independently selected from the 20 naturally occurring amino acids, using standard one letter codes, from A, R, N, D, C, E, Q, G, H, I, L, K, M, F, P, S, T, W, Y, and V. The sequence X¹X²X³X⁴X⁵X⁶X⁷X⁸X⁹of SEQ ID NO: 2 corresponds to the 581-589 region of VP1. The polynucleotide encodes a polypeptide that includes at least one mutation of the native AAV5 capsid and thus does not have the sequence of SEQ ID NO: 1. In addition, the polypeptide does not have the sequence of SEQ ID NO: 3 (MSFVDHPPDWLEEVGEGLREFLGLEAGPPKPKPNQQHQDQARGLVLPGYNYLGPGNG LDRGEPVNRADEVAREHDISYNEQLEAGDNPYLKYNHADAEFQEKLADDTSFGGNLG KAVFQAKKRVLEPFGLVEEGAKTAPTGKRIDDHFPKRKKARTEEDSKPSTSSDAEAGPS GSQQLQIPAQPASSLGADTMSAGGGGPLGDNNQGADGVGNASGDWHCDSTWMGDRV VTKSTRTWVLPSYNNHQYREIKSGSVDGSNANAYFGYSTPWGYFDFNRFHSHWSPRD WQRLINNYWGFRPRSLRVKIFNIQVKEVTVQDSTTTIANNLTSTVQVFTDDDYQLPYVV GNGTEGCLPAFPPQVFTLPQYGYATLNRDNTENPTERSSFFCLEYFPSKMLRTGNNFEFT YNFEEVPFHSSFAPSQNLFKLANPLVDQYLYRFVSTNNTGGVQFNKNLAGRYANTYKN WFPGPMGRTQGWNLGSGVNRASVSAFATTNRMELEGASYQVPPQPNGMTNNLQGSN TYALENTMIFNSQPANPGTTATYLEGNMLITSESETQPVNRVAYNVGGQMATNNQSST TAPATGTVNLQEIVPGSVWMERDVYLQGPIWAKIPETGAHFHPSPAMGGFGLKHPPPM MLIKNTPVPGNIT SFSDVPVSSFITQYSTGQVTVEMEWELKKENSKRWNPEIQYTNNYN DPQFVDFAPDSTGEYRTTRPIGTRYLTRPL), SEQ ID NO: 4 (MSFVDHPPDWLEEVGEGLREFLGLEAGPPKPKPNQQHQDQARGLVLPGYNYLGPGNG LDRGEPVNRADEVAREHDISYNEQLEAGDNPYLKYNHADAEFQEKLADDTSFGGNLG KAVFQAKKRVLEPFGLVEEGAKTAPTGKRIDDHFPKRKKARTEEDSKPSTSSDAEAGPS GSQQLQIPAQPASSLGADTMSAGGGGPLGDNNQGADGVGNASGDWHCDSTWMGDRV VTKSTRTWVLPSYNNHQYREIKSGSVDGSNANAYFGYSTPWGYFDFNRFHSHWSPRD WQRLINNYWGFRPRSLRVKIFNIQVKEVTVQDSTTTIANNLTSTVQVFTDDDYQLPYVV GNGTEGCLPAFPPQVFTLPQYGYATLNRDNTENPTERSSFFCLEYFPSKMLRTGNNFEFT YNFEEVPFHSSFAPSQNLFKLANPLVDQYLYRFVSTNNTGGVQFNKNLAGRYANTYKN WFPGPMGRTQGWNLGSGVNRASVSAFATTNRMELEGASYQVPPQPNGMTNNLQGSN TYALENTMIFNSQPANPGTTATYLEGNMLITSESETQPVNRVAYNVGGQMATNNQSST TAPATGTYNTQEIVPGSVWMERDVYLQGPIWAKIPETGAHFHPSPAMGGFGLKHPPPM MLIKNTPVPGNITSFSDVPVSSFITQYSTGQVTVEMEWELKKENSKRWNPEIQYTNNYN DPQFVDFAPDSTGEYRTTRPIGTRYLTRPL), SEQ ID NO: 5 (MSFVDHPPDWLEEVGEGLREFLGLEAGPPKPKPNQQHQDQARGLVLPGYNYLGPGNG LDRGEPVNRADEVAREHDISYNEQLEAGDNPYLKYNHADAEFQEKLADDTSFGGNLG KAVFQAKKRVLEPFGLVEEGAKTAPTGKRIDDHFPKRKKARTEEDSKPSTSSDAEAGPS GSQQLQIPAQPASSLGADTMSAGGGGPLGDNNQGADGVGNASGDWHCDSTWMGDRV VTKSTRTWVLPSYNNHQYREIKSGSVDGSNANAYFGYSTPWGYFDFNRFHSHWSPRD WQRLINNYWGFRPRSLRVKIFNIQVKEVTVQDSTTTIANNLTSTVQVFTDDDYQLPYVV GNGTEGCLPAFPPQVFTLPQYGYATLNRDNTENPTERSSFFCLEYFPSKMLRTGNNFEFT YNFEEVPFHSSFAPSQNLFKLANPLVDQYLYRFVSTNNTGGVQFNKNLAGRYANTYKN WFPGPMGRTQGWNLGSGVNRASVSAFATTNRMELEGASYQVPPQPNGMTNNLQGSN TYALENTMIFNSQPANPGTTATYLEGNMLITSESETQPVNRVAYNVGGQMATNNQSST TAPTTGTYNLQEIVPGSVWMERDVYLQGPIWAKIPETGAHFHPSPAMGGFGLKHPPPM MLIKNTPVPGNITSFSDVPVSSFITQYSTGQVTVEMEWELKKENSKRWNPEIQYTNNYN DPQFVDFAPDSTGEYRTTRPIGTRYLTRPL), SEQ ID NO: 6 (MSFVDHPPDWLEEVGEGLREFLGLEAGPPKPKPNQQHQDQARGLVLPGYNYLGPGNG LDRGEPVNRADEVAREHDISYNEQLEAGDNPYLKYNHADAEFQEKLADDTSFGGNLG KAVFQAKKRVLEPFGLVEEGAKTAPTGKRIDDHFPKRKKARTEEDSKPSTSSDAEAGPS GSQQLQIPAQPASSLGADTMSAGGGGPLGDNNQGADGVGNASGDWHCD STWMGDRV VTKSTRTWVLPSYNNHQYREIKSGSVDGSNANAYFGYSTPWGYFDFNRFHSHWSPRD WQRLINNYWGFRPRSLRVKIFNIQVKEVTVQDSTTTIANNLTSTVQVFTDDDYQLPYVV GNGTEGCLPAFPPQVFTLPQYGYATLNRDNTENPTERSSFFCLEYFPSKMLRTGNNFEFT YNFEEVPFHSSFAPSQNLFKLANPLVDQYLYRFVSTNNTGGVQFNKNLAGRYANTYKN WFPGPMGRTQGWNLGSGVNRASVSAFATTNRMELEGASYQVPPQPNGMTNNLQGSN TYALENTMIFNSQPANPGTTATYLEGNMLITSESETQPVNRVAYNVGGQMATNNQSST TAPAAGTYNLQEIVPGSVWMERDVYLQGPIWAKIPETGAHFHPSPAMGGFGLKHPPPM MLIKNTPVPGNIT SFSDVPVSSFITQYSTGQVTVEMEWELKKENSKRWNPEIQYTNNYN DPQFVDFAPDSTGEYRTTRPIGTRYLTRPL), SEQ ID NO: 7 (MSFVDHPPDWLEEVGEGLREFLGLEAGPPKPKPNQQHQDQARGLVLPGYNYLGPGNG LDRGEPVNRADEVAREHDISYNEQLEAGDNPYLKYNHADAEFQEKLADDTSFGGNLG KAVFQAKKRVLEPFGLVEEGAKTAPTGKRIDDHFPKRKKARTEEDSKPSTSSDAEAGPS GSQQLQIPAQPASSLGADTMSAGGGGPLGDNNQGADGVGNASGDWHCDSTWMGDRV VTKSTRTWVLPSYNNHQYREIKSGSVDGSNANAYFGYSTPWGYFDFNRFHSHWSPRD WQRLINNYWGFRPRSLRVKIFNIQVKEVTVQDSTTTIANNLTSTVQVFTDDDYQLPYVV GNGTEGCLPAFPPQVFTLPQYGYATLNRDNTENPTERSSFFCLEYFPSKMLRTGNNFEFT YNFEEVPFHSSFAPSQNLFKLANPLVDQYLYRFVSTNNTGGVQFNKNLAGRYANTYKN WFPGPMGRTQGWNLGSGVNRASVSAFATTNRMELEGASYQVPPQPNGMTNNLQGSN TYALENTMIFNSQPANPGTTATYLEGNMLITSESETQPVNRVAYNVGGQMATNNQSST TAPATGAYNLQEIVPGSVWMERDVYLQGPIWAKIPETGAHFHPSPAMGGFGLKHPPPM MLIKNTPVPGNITSFSDVPVSSFITQYSTGQVTVEMEWELKKENSKRWNPEIQYTNNYN DPQFVDFAPDSTGEYRTTRPIGTRYLTRPL), or SEQ ID NO: 8 (MSFVDHPPDWLEEVGEGLREFLGLEAGPPKPKPNQQHQDQARGLVLPGYNYLGPGNG LDRGEPVNRADEVAREHDISYNEQLEAGDNPYLKYNHADAEFQEKLADDTSFGGNLG KAVFQAKKRVLEPFGLVEEGAKTAPTGKRIDDHFPKRKKARTEEDSKPSTSSDAEAGPS GSQQLQIPAQPASSLGADTMSAGGGGPLGDNNQGADGVGNASGDWHCDSTWMGDRV VTKSTRTWVLPSYNNHQYREIKSGSVDGSNANAYFGYSTPWGYFDFNRFHSHWSPRD WQRLINNYWGFRPRSLRVKIFNIQVKEVTVQDSTTTIANNLTSTVQVFTDDDYQLPYVV GNGTEGCLPAFPPQVFTLPQYGYATLNRDNTENPTERSSFFCLEYFPSKMLRTGNNFEFT YNFEEVPFHSSFAPSQNLFKLANPLVDQYLYRFVSTNNTGGVQFNKNLAGRYANTYKN WFPGPMGRTQGWNLGSGVNRASVSAFATTNRMELEGASYQVPPQPNGMTNNLQGSN TYALENTMIFNSQPANPGTTATYLEGNMLITSESETQPVNRVAYNVGGQMATNNQSST TAPATGTVNTQEIVPGSVWMERDVYLQGPIWAKIPETGAHFHPSPAMGGFGLKHPPPM MLIKNTPVPGNIT SFSDVPVSSFITQYSTGQVTVEMEWELKKENSKRWNPEIQYTNNYN DPQFVDFAPDSTGEYRTTRPIGTRYLTRPL). In some embodiments, the VP1 capsid polypeptide comprises a variation in the 581-589 region. For example, a VP1 capsid polypeptide comprising a variant 581-589 region may comprise a sequence of SEQ ID NO: 1 in which residues 581 to residue 589 are replaced with a sequence of X¹X²X³X⁴X⁵X⁶X⁷X⁸X⁹, wherein X¹, X², X³, X⁴, X⁵, X⁶, X⁷, X⁸, and X⁹are each independently selected from A, R, N, D, C, E, Q, G, H, I, L, K, M, F, P, S, T, W, Y, and V, and wherein the VP1 capsid polypeptide does not have the sequence of any of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, or SEQ ID NO: 8.

In some embodiments, the polynucleotide encodes an AAV VP1 capsid polypeptide that further comprises one or more mutations at an amino acid residue outside of the 581-589 region, with reference to SEQ ID NO: 1, wherein the resulting recombinant capsid is capable of forming an assembled virion that exhibits desired tissue targeting as compared to wild type AAV5 (SEQ ID NO: 1). The one or more mutations may confer increased tissue tropism (e.g., increased CNS tissue tropism or increased muscle tissue tropism) or tissue preference (e.g., increased CNS tissue preference or increased muscle tissue preference) on the assembled virion as compared to a wild type AAV5 capsid polypeptide.

In another aspect, a vector capable of replication in prokaryotic cells is provided, wherein the vector comprises the polynucleotide described immediately above. In typical embodiments, the vector is a plasmid encoding a replication competent AAV genome.

In a further aspect, a library is provided. The library comprises a plurality of vectors comprising the AAV capsid-encoding polynucleotides. In some embodiments, the vectors are plasmids, and the plurality of plasmids comprise a plurality of different AAV VP-encoding polynucleotides. The library may comprise a plurality of vectors encoding AAV VP1 capsid polypeptides having the amino acid sequence of SEQ ID NO: 2 comprising one or more amino acid variations in the 581-589 region.

In various embodiments, the library encodes at least 1×10⁹(1e9) different AAV VP capsid polypeptides, at least 2.5×10⁹(2.5e9) different AAV VP capsid polypeptides, at least 5×10⁹(5e9) different AAV VP capsid polypeptides, at least 7.5×10⁹(7.5e9) different AAV VP capsid polypeptides, at least 1×10¹⁰(1e10) different AAV VP capsid polypeptides, at least 2.5×10¹⁰(2.5e10) different AAV VP capsid polypeptides, at least 5×10¹⁰(5e10) different AAV VP capsid polypeptides, at least 7.5×10¹⁰(7.5e10) different AAV VP capsid polypeptides, at least 1×10¹¹(1e11) different AAV VP capsid polypeptides, at least 2.5×10¹¹(2.5e11) different AAV VP capsid polypeptides, or at least 5×10¹¹(5e11) different AAV VP capsid polypeptides. The library may encode VP capsid polypeptides comprising 581-589 region variants.

In another aspect, prokaryotic cells comprising the vectors are provided. In some embodiments, the prokaryotic cell is an E. coli cell and the vector is a plasmid.

In a related aspect, libraries are provided, the library comprising a plurality of E. coli cells, wherein the plurality of cells comprise a plurality of plasmids, wherein the plurality of plasmids comprise a plurality of different AAV VP-encoding polynucleotides.

In some embodiments, the library encodes at least 1×10⁹(1e9) different AAV VP capsid polypeptides, at least 2.5×10⁹(2.5e9) different AAV VP capsid polypeptides, at least 5×10⁹(5e9) different AAV VP capsid polypeptides, at least 7.5×10⁹(7.5e9) different AAV VP capsid polypeptides, at least 1×10¹⁰(1e10) different AAV VP capsid polypeptides, at least 5×10¹⁰(5e10) different AAV VP capsid polypeptides, at least 7.5×10¹⁰(7.5e10) different AAV VP capsid polypeptides, at least 1×10¹¹(1e11) different AAV VP capsid polypeptides, at least 2.5×10¹¹(2.5e11) different AAV VP capsid polypeptides, or at least 5×10¹¹(5e1l) different AAV VP capsid polypeptides. The library may encode VP capsid polypeptides comprising 581-589 region variants.

VP Polypeptides, Peptide Libraries

In another aspect, AAV VP1 capsid polypeptides are provided. The polypeptide has the amino acid sequence of SEQ ID NO: 2, wherein X¹, X², X³, X⁴, X⁵, X⁶, X⁷, X⁸, and X⁹are each independently selected from A, R, N, D, C, E, Q, G, H, I, L, K, M, F, P, S, T, W, Y, and V. The polypeptide includes at least one mutation as compared to native AAV VP1, and thus does not have the sequence of SEQ ID NO: 1. In addition, the polypeptide does not have the sequence of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, or SEQ ID NO: 8.

In some embodiments, the polypeptide further comprises one or more mutations at an amino acid residue outside of the 581-589 region, with reference to SEQ ID NO: 1, wherein the resulting recombinant capsid is capable of forming an assembled virion that exhibits desired tissue targeting as compared to wild type AAV5 (SEQ ID NO: 1).

In a further aspect, libraries are provided, the libraries comprising a plurality of polypeptides as described immediately above, the plurality having different primary amino acid sequences. A library may comprise a plurality of polypeptides of SEQ ID NO: 2 comprising one or more amino acid variations in the 581-589 region.

In some embodiments, library comprises at least 1×10⁹(1e9) different AAV VP capsid polypeptides, at least 2.5×10⁹(2.5e9) different AAV VP capsid polypeptides, at least 5×10⁹(5e9) different AAV VP capsid polypeptides, at least 7.5×10⁹(7.5e9) different AAV VP capsid polypeptides, at least 1×10¹⁰(1e10) different AAV VP capsid polypeptides, at least 2.5×10¹⁰(2.5e10) different AAV VP capsid polypeptides, at least 5×10¹⁰(5e10) different AAV VP capsid polypeptides, at least 7.5×10¹⁰(7.5e10) different AAV VP capsid polypeptides, at least 1×10¹¹(1e11) different AAV VP capsid polypeptides, at least 2.5×10¹¹(2.5e11) different AAV VP capsid polypeptides, or at least 5×10¹¹(5e11) different AAV VP capsid polypeptides. The library may comprise VP capsid polypeptides comprising 581-589 region variants.

In certain embodiments, the library comprises at least from about 1×10⁵(1e5) to at least about 5×10¹¹(5e11) different AAV VP capsid polypeptides. In certain embodiments, the library comprises at least about 1×10⁵(1e5), at least about 2×10⁵(2e5), at least about 3×10⁵(3e5), at least about 4×10⁵(4e5), at least about 5×10⁵(5e5), at least about 6×10⁵(6e5), at least about 7×10⁵(7e5), at least about 8×10⁵(8e5), at least about 9×10⁵(9e5), at least about 1×10⁶(1e6), at least about 2×10⁶(2e6), at least about 3×10⁶(3e6), at least about 4×10⁶(4e6), at least about 5×10⁶(5e6), at least about 6×10⁶(6e6), at least about 7×10⁶(7e6), at least about 8×10⁶(8e6), at least about 9×10⁶(9e6), at least about 1×10⁷(1e7), at least about 2×10⁷(2e7), at least about 3×10⁷(3e7), at least about 4×10⁷(4e7), at least about 5×10⁷(5e7), at least about 6×10⁷(6e7), at least about 7×10⁷(7e7), at least about 8×10⁷(8e7), at least about 9×10⁷(9e7), at least about 1×10⁸(1e8), at least about 2×10⁸(2e8), at least about 3×10⁸(3e8), at least about 4×10⁸(4e8), at least about 5×10⁸(5e8), at least about 6×10⁸(6e8), at least about 7×10⁸(7e8), at least about 8×10⁸(8e8), at least about 9×10⁸(9e8), at least about 1×109 (1e9), at least about 2×10⁹(2e9), at least about 3×10⁹(3e9), at least about 4×10⁹(4e9), at least about 5×10⁹(5e9), at least about 6×10⁹(6e9), at least about 7×10⁹(7e9), at least about 8×10⁹(8e9), at least about 9×10⁹(9e9), at least about 1×10¹⁰(1e10), at least about 2×10¹⁰(2e10), at least about 3×10¹⁰(3e10), at least about 4×10¹⁰(4e10), at least about 5×10¹⁰(5e10), at least about 6×10¹⁰(6e10), at least about 7×10¹⁰(7e10), at least about 8×10¹⁰(8e10), at least about 9×10¹⁰(9e10), at least about 1×10¹¹(1e11), at least about 2×10¹¹(2e11), at least about 3×10¹¹(3e11), at least about 4×10¹¹(4e11), or at least about 5×10¹¹(5e1l) AAV VP capsid polypeptides.

In certain embodiments, provided herein is a recombinant adeno-associated virus AAV VP1 capsid polypeptide having at least one mutation in a residue of region 581 to residue 589 in SEQ ID NO: 1, inclusive, wherein the mutation confers at least about 1.1-fold, at least about 1.2-fold, at least about 1.3-fold, at least about 1.4-fold, at least about 1.5-fold, at least about 1.6-fold, at least about 1.7-fold, at least about 1.8-fold, at least about 1.9-fold, at least about 2-fold, at least about 2.2-fold, at least about 2.4-fold, at least about 2.6-fold, at least about 2.8-fold, at least about 3-fold, at least about 3.5-fold, at least about 4-fold, at least about 4.5-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 20-fold, at least about 30-fold, at least about 40-fold, at least about 50-fold, at least about 100-fold, at least about 200-fold, at least about 300-fold, at least about 400-fold, or at least about 500-fold increased accumulation of an AAV virion having said AAV VP1 capsid polypeptide in a target tissue (e.g., a CNS tissue or a muscle tissue) relative to a non-target tissue, as compared to wild type AAV virion having a wild type AAV5 VP1 capsid polypeptide, and wherein the AAVVP1 capsid polypeptide does not have the sequence of any of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, or SEQ ID NO: 8. In some embodiments, a VP1 capsid polypeptide comprises a variation in the 581-589 region. For example, a VP1 capsid polypeptide comprising a 581-589 region may comprise a sequence of SEQ ID NO: 1 in which residues 581 to residue 589 are replaced with a sequence of X¹X²X³X⁴X⁵X⁶X⁷X⁸X⁹, wherein X¹, X², X³, X⁴, X⁵, X⁶, X⁷, X⁸, and X⁹are each independently selected from A, R, N, D, C, E, Q, G, H, I, L, K, M, F, P, S, T, W, Y, and V, and wherein the VP1 capsid polypeptide does not have the sequence of any of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, or SEQ ID NO: 8. rAAV virions, virion libraries

In another aspect, recombinant AAV virions (rAAV) are provided. The virion comprises an AAV VP capsid polypeptide as described above. In some embodiments, the rAAV has increased tropism for primate and human CNS tissue as compared to a rAAV having the native AAV5 VP1 capsid polypeptide (SEQ ID NO:1). In some embodiments, the rAAV has increased tropism for primate and human muscle tissue as compared to a rAAV having the native AAV5 VP1 capsid polypeptide (SEQ ID NO:1). In some embodiments, the rAAV has increased ability to assemble, or exhibits greater virion stability, as compared to a rAAV having the native AAV5 VP1 capsid polypeptide (SEQ ID NO: 1).

In some embodiments, the rAAV has increased ability to cross the blood-brain barrier following intravenous administration as compared to a rAAV having the native AAV5 VP1 capsid polypeptide (SEQ ID NO: 1).

In certain of these embodiments, the rAAV has increased ability to infect one or more brain regions selected from hippocampus, dentate gyrus, cerebral cortex, temporal cortex, occipital cortex, thalamus, forebrain, substantia nigra, hypothalamus, cerebellum, and combinations thereof following systemic administration, as compared to a rAAV having the native AAV5 VP1 capsid polypeptide (SEQ ID NO: 1).

In some embodiments, the rAAV has increased ability to infect one or more brain regions selected from hippocampus, dentate gyrus, cerebral cortex, temporal cortex, occipital cortex, thalamus, forebrain, substantia nigra, hypothalamus, cerebellum, and combinations thereof following systemic administration, and also has reduced tropism for non-CNS tissues, as compared to a rAAV having the native AAV5 VP1 capsid polypeptide (SEQ ID NO: 1).

In some embodiments, the rAAV has increased ability to infect muscle tissue, including aorta, esophagus, heart (e.g., atrium, ventricle, valves), skeletal muscle (e.g., biceps, brachii, femoris, diaphragm, gastrocnemius, tibialis anterior, triceps, quadriceps, including vastus lateralis)), or muscle fibers, including type I (slow oxidative) fibers, type IC fibers, type II fibers, type IIC fibers, type IIA (fast oxidative/glycolytic) fibers, type IIAX fibers, type IIXA fibers, type IIX (fast glycolytic) fibers, or combinations thereof, following systemic administration, as compared to a rAAV having the native AAV5 VP1 capsid polypeptide (SEQ ID NO: 1).

In some embodiments, the rAAV has increased ability to infect muscle tissue, including aorta, esophagus, heart (e.g., atrium, ventricle, valves), skeletal muscle (e.g., biceps, brachii, femoris, diaphragm, gastrocnemius, tibialis anterior, triceps, quadriceps, including vastus lateralis)), or muscle fibers, including type I (slow oxidative) fibers, type IC fibers, type II fibers, type IIC fibers, type IIA (fast oxidative/glycolytic) fibers, type IIAX fibers, type IIXA fibers, type IIX (fast glycolytic) fibers, or combinations thereof, following systemic administration, and also has reduced tropism for non-muscle tissues, as compared to a rAAV having the native AAV5 VP1 capsid polypeptide (SEQ ID NO: 1).

Additionally, provided are polynucleotide sequences encoding the rAAV capsid VP proteins described herein.

In a further aspect, libraries are provided that comprise a plurality of rAAV as described above. The plurality of rAAVs comprise a plurality of VP capsid polypeptides having different primary amino acid sequences. An rAAV of the plurality of rAAVs may comprise a polypeptide of SEQ ID NO: 2 comprising one or more amino acid variations in the 581-589 region.

In some embodiments, the library comprises at least 1×10⁹(1e5) different AAV VP capsid polypeptides, at least 2.5×10⁹(2.5e9) different AAV VP capsid polypeptides, at least 5×10⁹(5e9) different AAV VP capsid polypeptides, at least 7.5×10⁹(7.5e9) different AAV VP capsid polypeptides, at least 1×10¹⁰(1e10) different AAV VP capsid polypeptides, at least 2.5×10¹⁰(2.5e10) different AAV VP capsid polypeptides, at least 5×10¹⁰(5e10) different AAV VP capsid polypeptides, at least 7.5×10¹⁰(7.5e10) different AAV VP capsid polypeptides, at least 1×10¹¹(1e11) different AAV VP capsid polypeptides, at least 2.5×10¹¹(2.5e11) different AAV VP capsid polypeptides, or at least 5×10¹¹(5e11) different AAV VP capsid polypeptides. The library may comprise AAV VP capsid polypeptides comprising 581-589 region variants. For example, a VP1 capsid polypeptide comprising a 581-589 region may comprise a sequence of SEQ ID NO: 1 in which residues 581 to residue 589 are replaced with a sequence of X¹X²X³X⁴X⁵X⁶X⁷X⁸X⁹, wherein X¹, X², X³, X⁴, X⁵, X⁶, X⁷, X⁸, and X⁹are each independently selected from A, R, N, D, C, E, Q, G, H, I, L, K, M, F, P, S, T, W, Y, and V, and wherein the VP1 capsid polypeptide does not have the sequence of any of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, or SEQ ID NO: 8.

Pharmaceutical Compositions

In another aspect, pharmaceutical compositions are provided. The pharmaceutical composition comprises a rAAV as described above and a pharmaceutically acceptable carrier.

A pharmaceutical composition can comprise a first active ingredient. The first active ingredient can comprise a viral vector as described herein and/or any payload as described herein. The pharmaceutical composition can be formulated in unit dose form. The pharmaceutical composition can comprise a pharmaceutically acceptable excipient, diluent, or carrier. The pharmaceutical composition can comprise a second, third, or fourth active ingredient—such as to facilitate enhanced gene replacement, RNA editing, DNA editing, or imaging.

A pharmaceutical composition described herein can compromise an excipient. An excipient can comprise a cryo-preservative, such as DMSO, glycerol, polyvinylpyrrolidone (PVP), or any combination thereof. An excipient can comprise a cryo-preservative, such as a sucrose, a trehalose, a starch, a salt of any of these, a derivative of any of these, or any combination thereof Δn excipient can comprise a pH agent (to minimize oxidation or degradation of a component of the composition), a stabilizing agent (to prevent modification or degradation of a component of the composition), a buffering agent (to enhance temperature stability), a solubilizing agent (to increase protein solubility), or any combination thereof. An excipient can comprise a surfactant, a sugar, an amino acid, an antioxidant, a salt, a non-ionic surfactant, a solubilizer, a triglyceride, an alcohol, or any combination thereof. An excipient can comprise sodium carbonate, acetate, citrate, phosphate, poly-ethylene glycol (PEG), human serum albumin (HSA), sorbitol, sucrose, trehalose, polysorbate 80, sodium phosphate, sucrose, disodium phosphate, mannitol, polysorbate 20, histidine, citrate, albumin, sodium hydroxide, glycine, sodium citrate, trehalose, arginine, sodium acetate, acetate, HCl, disodium edetate, lecithin, glycerol, xanthan rubber, soy isoflavones, polysorbate 80, ethyl alcohol, water, teprenone, or any combination thereof.

Compositions and methods provided herein can utilize pharmaceutical compositions. The compositions described throughout can be formulated into a pharmaceutical and be used to treat a human or mammal, in need thereof, diagnosed with a disease. In some cases, pharmaceutical compositions can be used prophylactically.

The compositions provided herein can be utilized in methods provided herein. Any of the provided compositions provided herein can be utilized in methods provided herein. In some cases, a method comprises at least partially preventing, reducing, ameliorating, and/or treating a disease or condition, or a symptom of a disease or condition. A subject can be a human or non-human. A subject can be a mammal (e.g., rat, mouse, cow, dog, pig, sheep, horse). A subject can be a vertebrate or an invertebrate. A subject can be a laboratory animal. A subject can be a patient. A subject can be suffering from a disease. A subject can display symptoms of a disease. A subject may not display symptoms of a disease, but still have a disease. A subject can be under medical care of a caregiver (e.g., the subject is hospitalized and is treated by a physician).

Methods of Treatment or Detection

In some aspects, the present disclosure provides for methods of treatment using an rAAV virion having any one of the engineered AAV VP capsid polypeptide sequences disclosed herein. In some aspects, the present disclosure provides for methods of detection using an rAAV virion having any one of the engineered AAV VP capsid polypeptide sequences disclosed herein. A method of treatment may comprise administering to a subject an effective amount of a pharmaceutical composition comprising rAAV virions assembled from VP polypeptides comprising a 581-589 region that convers tissue tropism (e.g., CNS tissue tropism or muscle tropism) to the rAAV. The rAAV virions may comprise a VP polypeptide 581-589 region described herein (e.g., comprising a variant 581-589 region that confers tissue tropism or tissue preference). In some embodiments, the rAAV may be assembled from VP capsid polypeptides comprising a 581-589 region of any one of SEQ ID NO: 12-SEQ ID NO: 3937, SEQ ID NO: 3938-SEQ ID NO: 4237, SEQ ID NO: 4238-SEQ ID NO: 4933, SEQ ID NO: 4934-SEQ ID NO: 5233, SEQ ID NO: 5234-SEQ ID NO: 5807, or SEQ ID NO: 5808-SEQ ID NO: 5811. The rAAV virions may encapsidate any payload, including those payloads disclosed herein.

A method of treatment may comprise administering an rAAV encapsidating a DNA molecule and enriching the DNA in a tissue of interest (e.g., a CNS tissue, a muscle tissue, a cardiac muscle tissue, or a skeletal muscle tissue) with a DNA enrichment that is at least 1-fold, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 10-fold, at least 15-fold, at least 25-fold, at least 30-fold, at least 40-fold, at least 50-fold, at least 100-fold, at least 200-fold, at least 300-fold, at least 500-fold, or at least 1000-fold a DNA enrichment of a wild type AAV9.

A method of treatment may comprise administering an rAAV encapsidating a DNA molecule and expressing an RNA encoded by the DNA in a tissue of interest (e.g., a CNS tissue, a muscle tissue, a cardiac muscle tissue, or a skeletal muscle tissue) with an RNA enrichment that is at least 1-fold, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 10-fold, at least 15-fold, at least 25-fold, at least 30-fold, at least 40-fold, at least 50-fold, at least 100-fold, at least 200-fold, at least 300-fold, at least 500-fold, or at least 1000-fold an RNA enrichment of a wild type AAV9.

In some embodiments, the effective amount is at least 1×10⁸(1e8) viral genomes per dose. In some embodiments, the effective amount is at least 5×10⁸(5e8) viral genomes/dose, 7.5×10⁸(7.5e8) viral genomes/dose, at least 1×10⁹(1e9) viral genomes/dose, at least 2.5×10⁹(2.5e9) viral genomes/dose, at least 5×10⁹(5e9) viral genomes/dose. In some embodiments, an effective amount of an rAAV assembled from VP polypeptides comprising a 581-589 region of any one of SEQ ID NO: 12-SEQ ID NO: 3937, SEQ ID NO: 3938-SEQ ID NO: 4237, SEQ ID NO: 4238-SEQ ID NO: 4933, SEQ ID NO: 4934-SEQ ID NO: 5233, SEQ ID NO: 5234-SEQ ID NO: 5807, or SEQ ID NO: 5808-SEQ ID NO: 5811 may be lower than an effective amount of an AAV assembled from VP1, VP2, and VP3 polypeptides of SEQ ID NO: 1, SEQ ID NO: 10, and SEQ ID NO: 11, respectively.

In some embodiments, the effective amount is at least 1×10¹¹(1e11) viral genomes/kg patient weight, at least 5×10¹¹(5e1l) viral genomes/kg, at least 1×10¹²(1e12) viral genomes/kg, at least 5×10¹²(5e12) viral genomes/kg, at least 1×10¹³(1e13) viral genomes/kg, at least 1×10¹⁴(1e14) viral genomes/kg, or at least 5×10¹⁴(5e14). In some embodiments, an effective amount of an rAAV assembled from VP polypeptides comprising a 581-589 region of any one of SEQ ID NO: 12-SEQ ID NO: 3937, SEQ ID NO: 3938-SEQ ID NO: 4237, SEQ ID NO: 4238-SEQ ID NO: 4933, SEQ ID NO: 4934-SEQ ID NO: 5233, SEQ ID NO: 5234-SEQ ID NO: 5807, or SEQ ID NO: 5808-SEQ ID NO: 5811 may be lower than an effective amount of an AAV assembled from VP1, VP2, and VP3 polypeptides of SEQ ID NO: 1, SEQ ID NO: 10, and SEQ ID NO: 11, respectively.

In some embodiments, the rAAV virion is administered via a systemic administration route including enteral routes of administration and parenteral routes of administration. The rAAV virion may be administered intravenously. In some embodiments, the rAAV may be administered intramuscularly. In some embodiments, the rAAV may be administered intraperitoneally. In some embodiments, the rAAV may be administered topically. In some embodiments, the rAAV may be administered orally. In particular embodiments, the rAAV virion is administered intravenously. In some embodiments, the rAAV is administered intrathecally. In some embodiments, the rAAV is administered by intracerebroventricular injection. In some embodiments, the rAAV is administered by intracerebral ventricular injection. In some embodiments, the rAAV is administered by intracisternal magna administration. In some embodiments, the rAAV is administered by intravitreal injection. In some embodiments, the rAAV is administered by parenchymal injection. In some embodiments, the rAAV is administered by intraparenchymal injection. In some embodiments, the rAAV is administered by intramyocardial injection. In some embodiments, the rAAV is administered by intracoronary injection. In some embodiments, the rAAV is administered by intrapericardial injection. For example, an rAAV virion with CNS tissue tropism may be administered via intrathecal injection, intracisternal magna injection, intracerebroventricular injection, intraparenchymal injection, or intravenous injection. In another example, an rAAV virion with muscle tissue tropism may be administered via intramuscular injection, intracoronary injection, intrapericardial injection, intramyocardial injection, or intravenous injection. In another example, an rAAV virion with cardiac tissue tropism may be administered via intramyocardial injection, intrapericardial injection, intracoronary injection, or intravenous injection.

In various embodiments, the patient suffers from one of the conditions listed in TABLE 1, below. In particular embodiments, the patient suffers from one of the conditions listed in TABLE 1 and the rAAV includes a payload comprising the transgene product associated therewith in TABLE 1. In some embodiments, an rAAV may be selected to specifically target the primary gene delivery target (e.g., CNS or muscle). For example, an rAAV selected to target the CNS may comprise VP capsid polypeptides comprising a 581-589 region with increased likelihood of conferring CNS tissue tropism (e.g., any one of SEQ ID NO: 12-SEQ ID NO: 3937, SEQ ID NO: 3938-SEQ ID NO: 4237, or SEQ ID NO: 5234-SEQ ID NO: 5807). In another example, an rAAV selected to target muscle may comprise VP capsid polypeptides comprising a 581-589 region with increased likelihood of conferring muscle tropism (e.g., any one of SEQ ID NO: 4238-SEQ ID NO: 4933, SEQ ID NO: 4934-SEQ ID NO: 5233, or SEQ ID NO: 5808-SEQ ID NO: 5811).

In some embodiments, an rAAV virion of the present disclosure, having any of the engineered AAV VP capsid polypeptide sequences disclosed herein, comprises a vector genome, the vector genome comprising a therapeutic polynucleotide or payload. In further embodiments, said payload may be under control of regulatory sequences that direct expression in infected human cells. In some embodiments, the payload comprises a therapeutic polynucleotide encoding any genetically encodable payload, such as an RNA (e.g., a guide RNA), a suppressor tRNA, a transgene, or a genome modifying entity. In some embodiments, the payload may be under control of a promoter. The promoter may be a ubiquitous promoter, or the promoter may be cell or tissue specific. For example, a payload may be under control of a neuronal promoter for expression in neurons. In another example, a payload may be under control of a muscle promoter for expression in muscle cells.

In some embodiments, the therapeutic polynucleotide encodes a guide RNA, a tRNA, a suppressor tRNA, a siRNA, a miRNA, an mRNA, a shRNA, a circular RNA, or an antisense oligonucleotide (ASO), a ribozyme, a DNAzyme, an aptamer, or any combination thereof. In some embodiments, the therapeutic polynucleotide encodes a linear therapeutic polynucleotide or a circular therapeutic polynucleotide.

In some embodiments, the therapeutic polynucleotide is a transgene, encoding a therapeutic protein. In particular embodiments, the transgene encodes a protein selected from the targets suitable for modification or transgene products of TABLE 1. In some embodiments, a transgene encoding a CNS target is encapsidated by an rAAV comprising VP capsid polypeptides comprising a 581-589 region with increased likelihood of conferring CNS tissue tropism or preference (e.g., any one of SEQ ID NO: 12-SEQ ID NO: 3937, SEQ ID NO: 3938-SEQ ID NO: 4237, or SEQ ID NO: 5234-SEQ ID NO: 5807). In some embodiments, a transgene encoding a muscle target is encapsidated by an rAAV comprising VP capsid polypeptides comprising a 581-589 region with increased likelihood of conferring muscle tropism or preference (e.g., any one of SEQ ID NO: 4238-SEQ ID NO: 4933, SEQ ID NO: 4934-SEQ ID NO: 5233, or SEQ ID NO: 5808-SEQ ID NO: 5811).

TABLE 1

Suitable Therapeutic Targets

Primary Gene

Target of a Therapeutic

Delivery Target
Condition
Polynucleotide

Brain/CNS
AADC deficiency
AADC

Alzheimer's Disease
Multiple, including APP, SNCA,

MAPT, ApoE, NGF, TERT

Tauopathies
MAPT

Synucleinopathies
SNCA

Batten disease (CLN2)
CLN2

Batten disease (CLN3)
CLN3

Batten disease (CLN6)
CLN6

MPS-IIIB
NAGLU

Frontotemporal dementia with GRN
GRN

mutations (FTD-GRN)

Parkinson's Disease with GBA1 mutations
GBA1

(PD-GBA) and neuronpathic Gaucher's

disease

Corticobasal Degeneration (CBD)
MAPT

Progressive Supranuclear Palsy (PSP)
MAPT

Chronic Traumatic Encephalopathy (CTE)
MAPT

Synucleinopathies
GBA1 + alpha-synuclein

Gaucher disease type 2
GBA

Canavan Disease
ASPA

Parkinson disease
AADC

Parkinson disease
GDNF

Parkinson disease
Neurturin

Parkinson disease
GAD

Parkinson disease
NTN

Parkinson disease
hFOXG1

Parkinson disease
hKCNQ2

Parkinson disease
hFMR1

Parkinson disease
anti-Tau/miRNA

Parkinson disease
EPM2A or EPM2B

Parkinson disease
LRRK2

Parkinson's Disease
LRRK2

Parkinson's Disease
SNCA

Tay-Sachs Disease
HEXA

Huntington's disease
IT15

Huntington's disease
CYP46A1

Huntington's disease
HTT

Protocki-Lupski Syndrome
IT15

Amyotrophic lateral sclerosis
C9orf72

Amyotrophic lateral sclerosis
SOD1

Down syndrome
DYRK1A

Sanfilippo disease type A
SGSH

Sanfilippo disease type B
hNAGLU

(Nervous system)
HEXB and HEXA

(Nervous system)
human codon-optimized CLN1

complementary DNA

(Nervous system)
SURF1

(Nervous system)
anti-UBE3A-ATS shRNA

(Nervous system)
hSLC6A1

Rett syndrome
MECP2

Muscle
Charcot-Marie-Tooth disease type 1A
NTF3

(CMT1A)

Duchenne muscular dystrophy (DMD)
Micro-dystrophin

Duchenne muscular dystrophy (DMD)
Mini-dystrophin

Facioscapulohumeral muscular dystrophy-
DUX4

1 (FSHD)

Dysferlinopathy
DYSF

Pompe disease
GAA

Limb-girdle muscular dystrophies (LGMD)
FKRP

(2i/R9)

Duchenne muscular dystrophy (DMD)
DMD

Facioscapulohumeral Dystrophy
DUX4

Myotonic Dystrophy
DMPK

Glycogen storage disorders
anti-GYS1 miRNA

X-linked myotubular myopathy (X-linked
MTM1

MTM)

euchromatic histone-lysine N-
anti-EHMT2 shRNA

methyltransferase 2

In some embodiments, the therapeutic polynucleotide encodes a therapeutic RNA. In some embodiments the therapeutic polynucleotide encodes an RNA, such as a guide RNA (including an engineered or synthetic guide RNA) for genome editing or for RNA editing. The guide RNA may target a gene, such as those provided in TABLE 1 or encoding a protein nucleotide provided in TABLE 1, to promote editing of the target gene. In some embodiments, editing of the target gene may treat a condition in a subject, such as a condition provided in TABLE 1.

In some embodiments, the therapeutic polynucleotide encodes a tRNA or a modified tRNA (engineered or synthetic tRNA). For example, the tRNA or modified tRNA can be a suppressor tRNA. The suppressor tRNA can be engineered to have an anticodon region that recognizes a stop codon, such as any premature stop codon (opal, ochre, or amber stop codons). A suppressor tRNA may target a premature stop codon in a gene, such as those provided in TABLE 1 or encoding a protein nucleotide provided in TABLE 1, to promote readthrough of the gene. In some embodiments, suppressing the premature stop codon may treat a condition in a subject, such as a condition provided in TABLE 1.

In some embodiments, the therapeutic polynucleotide (e.g., a therapeutic RNA, a tRNA, or a genome modifying entity) can target a gene listed in TABLE 1 or any gene associated with a neurologic disease, Parkinson's disease, Alzheimer's disease, a Tauopathy, Stargardt disease, alpha-1 antitrypsin deficiency, Duchenne's muscular dystrophy, Rett syndrome, cystic fibrosis, or any genetic disease. In some embodiments, the targeted gene may be ABCA4, AAT, SERPINA1, SERPINA1 E342K, HEXA, LRRK2, SNCA, DMD, APP, Tau, GBA, PINK1, RAB7A, CFTR, ALAS1, ATP7B, ATP7B G1226R, HFE C282Y, LIPA c.894 G>A, PCSK9 start site, or SCNN1A start site, a fragment any of these, or any combination thereof. In some embodiments, the therapeutic polynucleotide is a gene therapy payload (e.g., a transgene) and, thus, may itself be one of the genes listed in TABLE 1 or any gene associated with a neurologic disease, Parkinson's disease, Alzheimer's disease, a Tauopathy, Stargardt disease, alpha-1 antitrypsin deficiency, Duchenne's muscular dystrophy, Rett syndrome, cystic fibrosis, or any genetic disease. In some embodiments, the transgene may be ABCA4, AAT, SERPINA1, SERPINA1 E342K, HEXA, LRRK2, SNCA, DMD, APP, Tau, GBA, PINK1, RAB7A, CFTR, ALAS1, ATP7B, ATP7B G1226R, HFE C282Y, LIPA c.894 G>A, PCSK9 start site, or SCNN1A start site, a fragment any of these, or any combination thereof.

An engineered AAV comprising a VP capsid polypeptide comprising a CNS tissue-tropic 581-589 region may be used to deliver a payload to treat a neurological condition, or a condition of the central nervous system. For example, an engineered AAV comprising a VP capsid polypeptide comprising a 581-589 region of any one of SEQ ID NO: 12-SEQ ID NO: 3937 or SEQ ID NO: 3938-SEQ ID NO: 4237 may be used to treat a neurological condition (e.g., an aromatic l-amino acid decarboxylase (AADC) deficiency, Alzheimer's disease, a tauopathy, a synucleinopathy, Batten disease, mucopolysaccharidosis type III, frontotemporal dementia, Parkinson's disease, Gaucher disease, Canavan disease, Tay-Sachs disease, Huntington's disease, Protocki-Lupski syndrome, amyotrophic lateral sclerosis, Down syndrome, Sanfilippo disease type A, Sanfilippo disease type B, or Rett syndrome). In some embodiments, the engineered AAV comprising a VP capsid polypeptide comprising a CNS tissue-tropic 581-589 region may be used to deliver a transgene encoding a protein associated with a neurological condition (e.g., aromatic l-amino acid decarboxylase (AADC), amyloid precursor protein (APP), α-synuclein, microtubule associated protein tau (MAPT), ApoE, nerve growth factor (NGF), telomerase reverse transcriptase (TERT), CLN2, CLN3, CLN6, N-acetyl-α-glucosaminidase (NAGLU), granulin, glucosylceramidase 13 (GBA), aspartoacylase, GDNF, neurturin (NTN), glutamate decarboxylase (GAD), FOXG1, K_v7.2, laforin, leucine rich repeat kinase 2 (LRRK2), hexosaminidase A (HEXA), hexosaminidase B (HEXB), huntingtin, cholesterol 24-hydroxylase, C9orf72, superoxide dismutase 1 (SOD1), dual specificity tyrosine-phosphorylation-regulated kinase 1A (DYRKA1), N-sulfoglucosamine sulfohydrolase (SGSH), surfeit locus protein 1 (SURF1), GABA transporter 1 (GAT1), and methyl-CpG binding protein 2 (MECP2)). In some embodiments, the engineered AAV comprising a VP capsid polypeptide comprising a CNS tissue-tropic 581-589 region may be used to deliver a therapeutic polynucleotide that alters expression or promotes editing of a gene encoding a protein associated with a neurological condition (e.g., aromatic l-amino acid decarboxylase (AADC), amyloid precursor protein (APP), α-synuclein, microtubule associated protein tau (MAPT), ApoE, nerve growth factor (NGF), telomerase reverse transcriptase (TERT), CLN2, CLN3, CLN6, N-acetyl-α-glucosaminidase (NAGLU), granulin, glucosylceramidase 13 (GBA), aspartoacylase, GDNF, neurturin (NTN), glutamate decarboxylase (GAD), FOXG1, K_v7.2, laforin, leucine rich repeat kinase 2 (LRRK2), hexosaminidase A (HEXA), hexosaminidase B (HEXB), huntingtin, cholesterol 24-hydroxylase, C9orf72, superoxide dismutase 1 (SOD1), dual specificity tyrosine-phosphorylation-regulated kinase 1A (DYRKA1), N-sulfoglucosamine sulfohydrolase (SGSH), surfeit locus protein 1 (SURF1), GABA transporter 1 (GAT1), and methyl-CpG binding protein 2 (MECP2)).

An engineered AAV comprising a VP capsid polypeptide comprising a muscle-tropic 581-589 region may be used to deliver a payload to treat a muscular condition. For example, an engineered AAV comprising a VP capsid polypeptide comprising a 581-589 region of any one of SEQ ID NO: 4238-SEQ ID NO: 4933 or SEQ ID NO: 4934-SEQ ID NO: 5233 may be used to treat a muscular condition (e.g., Charcot-Marie-Tooth disease, Duchenne muscular dystrophy, dysferlinopathy, Pompe disease, Limb-girdle muscular dystrophies, facioscapulohumeral dystrophy, myotonic dystrophy, a glycogen storage disorder, X-linked myotubular myopathy, or euchromatic histone-lysine N-methyltransferase 2). In some embodiments, the engineered AAV comprising a VP capsid polypeptide comprising a muscle-tropic 581-589 region may be used to deliver a transgene encoding a protein associated with a muscular condition (e.g., neurotrophin 3 (NFT3), dystrophin, mini-dystrophin, micro-dystrophin, dysferlin, acid α-glucosidase (GAA), fukutin-related protein (FKRP), double homeobox 4 (DUX4), glycogen synthase 1 (GYS1), myotubularin, or euchromatic histone-lysine N-methyltransferase 2 (EHMT2)). In some embodiments, the engineered AAV comprising a VP capsid polypeptide comprising a muscle-tropic 581-589 region may be used to deliver a therapeutic polynucleotide that alters expression or promotes editing of a gene encoding a protein associated with a muscular condition (e.g., neurotrophin 3 (NFT3), dystrophin, mini-dystrophin, micro-dystrophin, dysferlin, acid α-glucosidase (GAA), fukutin-related protein (FKRP), double homeobox 4 (DUX4), glycogen synthase 1 (GYS1), myotubularin, or euchromatic histone-lysine N-methyltransferase 2 (EHMT2)).

In some embodiments, the therapeutic polynucleotide encodes genome modifying entities. For example, a genome modifying entity may be a DNA editing enzyme, an RNA editing enzyme, a transcriptional activator, or a transcriptional repressor. The DNA editing enzyme may be any DNA editing enzyme, including any CRISPR/Cas systems, meganucleases, zinc-finger nucleases, (ZFNs), TALE Nucleases (TALENs and megaTALENS). The CRISPR/Cas system can be a Cas3, Cas8, Cas10, Cas9, Cas4, Cas12, or Cas13. The RNA editing enzyme may be ADAR. In some embodiments, the ADAR is a human ADAR1 or human ADAR2. The transcriptional activator may be VP64. A transcriptional repressor may be KRAB. Such genome modifying entities may target any gene listed in TABLE 1 for editing.

In some embodiments, the present disclosure provides for rAAV virions having an engineered AAV VP capsid polypeptide (e.g., comprising a 581-589 region variant), where the virion encapsidates any one of or any combination of the therapeutic payloads disclosed herein. In some embodiments, multiple copies of the therapeutic payload are encapsidated.

In some embodiments, the therapeutic polynucleotide is a polynucleotide capable of serving as a homology template for homology-directed repair. In some embodiments, the therapeutic polynucleotide may be a guide polynucleotide for a CRISPR/Cas system or an ADAR enzyme. For example, the therapeutic polynucleotide may be a CRISPR/Cas guide RNA or an ADAR guide RNA.

In some embodiments, an rAAV virion of the present disclosure, having any of the engineered AAV VP capsid polypeptide sequences disclosed herein, comprises a vector genome, the vector genome comprising a detectable polynucleotide or payload. In further embodiments, said payload may be under control of regulatory sequences that direct expression in infected human cells. Examples of detectable polynucleotides include, but are not limited to, any genetically encodable detectable moiety. For example, a genetically encodable detectable moiety may be a fluorescent protein such as EGFP, GFP, YFP, RFP, CFP, or any variants thereof. In some embodiments, the present disclosure provides for rAAV virions having an engineered AAV VP capsid polypeptide, where the virion encapsidates any one of or any combination of the detectable payloads disclosed herein. In some embodiments, multiple copies of the detectable payload are encapsidated.

In some embodiments, the present disclosure provides for rAAV virions having an engineered AAV VP capsid polypeptide, where the virion encapsidates any one of or any combination of the therapeutic payloads and detectable payloads disclosed herein. For example, an rAAV of the present disclosure having an engineered AAV VP capsid polypeptide may encapsidate a transgene and a fluorescent protein. As another example, an rAAV of the present disclosure having an engineered AAV VP capsid polypeptide may encapsidate a therapeutic RNA (e.g., a guide RNA) and a fluorescent protein.

Delivering a payload (e.g., a payload encoding a therapeutic polypeptide or a therapeutic polynucleotide) using the CNS specific or muscle specific AAVs described herein may produce lower toxicity in a subject compared to non-specific AAVs (e.g., AAVs comprising a VP1 polypeptide of SEQ ID NO: 1). Tissue specific AAVs (e.g., CNS specific AAVs or muscle specific AAVs) may be effective at lower doses compared to non-specific AAVs since a larger fraction of the administered AAVs infect the relevant tissue (e.g., CNS tissue or muscle tissue). As a result, a therapeutically effective dose of tissue specific AAVs may be lower than a therapeutically effect dose of non-specific AAVs, leading to lower toxicity due administering a lower dose. For example, administration of a therapeutically effective dose of tissue specific AAVs may result in lower liver toxicity than administration of a therapeutically effective dose of non-specific AAVs. Administration of a lower dose may also lead to lower production of neutralizing antibodies in the subject. Neutralizing antibodies may decrease the efficacy of the AAV therapy by inhibiting infection of the target tissue or may cause severe side effects in the subject due to the immune response. Additionally, tissue specific AAVs (e.g., CNS specific AAVs or muscle specific AAVs) may produce fewer off-target effects compared to non-specific AAVs when administered at the same dose since fewer of the AAVs infect off target tissues (e.g., non-CNS tissues or non-muscle tissues). Off-target effects may include increased gene expression in an off-target tissue, decreased gene expression in an off-target tissue, gene editing in an off-target tissue, immune response, or liver toxicity.

In Vivo Selected VP Polypeptides

In a further aspect, engineered (synonymously, recombinant) adeno-associated virus (AAV) VP capsid polypeptides identified using the methods described herein are provided.

In some embodiments, the engineered adeno-associated virus (AAV) viral protein (VP) capsid polypeptide has an amino acid sequence at least 70% identical to SEQ ID NO: 1, wherein the engineered AAV VP capsid polypeptide has at least one substitution as compared to SEQ ID NO: 1 in the 581-589 region, corresponding to residue 581 to residue 589 of SEQ ID NO: 1, inclusive, wherein the capsid polypeptide is capable of assembling into a recombinant AAV virion (rAAV), and wherein the VP capsid polypeptide does not have the sequence of any of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, or SEQ ID NO: 8. In some embodiments, the VP capsid polypeptide comprises a variant 581-589 region (e.g., comprising one or more amino acid substitutions relative to SEQ ID NO: 1 in the region from residue 581 to residue 589, inclusive). The VP capsid polypeptide may comprise a sequence of SEQ ID NO: 2, wherein the 581-589 region has a sequence of any one of SEQ ID NO: 12-SEQ ID NO: 3937, SEQ ID NO: 3938-SEQ ID NO: 4237, SEQ ID NO: 4238-SEQ ID NO: 4933, SEQ ID NO: 4934-SEQ ID NO: 5233, SEQ ID NO: 5234-SEQ ID NO: 5807, or SEQ ID NO: 5808-SEQ ID NO: 5811.

In some embodiments, the engineered adeno-associated virus (AAV) viral protein (VP) capsid polypeptide is CNS tissue-tropic and has an amino acid sequence at least 70% identical to SEQ ID NO: 1, wherein the engineered AAV VP capsid polypeptide has at least one substitution as compared to SEQ ID NO: 1 in the 581-589 region, corresponding to residue 581 to residue 589 of SEQ ID NO: 1, inclusive, wherein the capsid polypeptide is capable of assembling into a recombinant AAV virion (rAAV), wherein the at least one substitution confers higher tropism for a central nervous system (CNS) tissue on the rAAV as compared to an rAAV virion having an AAV5 VP capsid polypeptide of SEQ ID NO: 1, and wherein the VP capsid polypeptide does not have the sequence of any of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, or SEQ ID NO: 8. In some embodiments, the CNS-tropic VP capsid polypeptide comprises a variant 581-589 region (e.g., comprising one or more amino acid substitutions relative to SEQ ID NO: 1 in the region from residue 581 to residue 589, inclusive). The CNS-tropic VP capsid polypeptide may comprise a sequence of SEQ ID NO: 2, wherein the 581-589 region has a sequence of any one of SEQ ID NO: 12-SEQ ID NO: 3937, SEQ ID NO: 3938-SEQ ID NO: 4237, or SEQ ID NO: 5234-SEQ ID NO: 5807.

In some embodiments, the engineered adeno-associated virus (AAV) viral protein (VP) capsid polypeptide is muscle-tropic and has an amino acid sequence at least 70% identical to SEQ ID NO: 1, wherein the engineered AAV VP capsid polypeptide has at least one substitution as compared to SEQ ID NO: 1 in the 581-589 region, corresponding to residue 581 to residue 589 of SEQ ID NO: 1, inclusive, wherein the capsid polypeptide is capable of assembling into a recombinant AAV virion (rAAV), wherein the at least one substitution confers higher tropism for a muscle tissue on the rAAV as compared to an rAAV virion having an AAV5 VP capsid polypeptide of SEQ ID NO: 1, and wherein the VP capsid polypeptide does not have the sequence of any of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, or SEQ ID NO: 8. In some embodiments, the muscle-tropic VP capsid polypeptide comprises a variant 581-589 region (e.g., comprising one or more amino acid substitutions relative to SEQ ID NO: 1 in the region from residue 581 to residue 589, inclusive). The muscle-tropic VP capsid polypeptide may comprise a sequence of SEQ ID NO: 2, wherein the 581-589 region has a sequence of any one of SEQ ID NO: 4238-SEQ ID NO: 4933, SEQ ID NO: 4934-SEQ ID NO: 5233, or SEQ ID NO: 5808-SEQ ID NO: 5811.

In particular embodiments, the engineered adeno-associated virus (AAV) viral protein (VP) capsid polypeptide (e.g., a CNS tissue-tropic VP capsid polypeptide or a muscle-tropic VP capsid polypeptide) has an amino acid sequence at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% identical to the sequence of SEQ ID NO: 1.

In some embodiments, the AAV VP capsid polypeptide has an amino acid sequence of SEQ ID NO: 2, wherein X¹, X², X³, X⁴, X⁵, X⁶, X⁷, X⁸, and X⁹are each independently selected from any amino acid, wherein the capsid polypeptide is capable of assembling into a recombinant AAV virion (rAAV), and wherein the VP capsid polypeptide does not have the sequence of any of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, or SEQ ID NO: 8, optionally with further mutations elsewhere in the VP capsid polypeptide. In some embodiments, the X¹X²X³X⁴X⁵X⁶X⁷X⁸X⁹portion corresponds to a sequence selected from any one of SEQ ID NO: 12-SEQ ID NO: 3937, SEQ ID NO: 3938-SEQ ID NO: 4237, SEQ ID NO: 4238-SEQ ID NO: 4933, SEQ ID NO: 4934-SEQ ID NO: 5233, SEQ ID NO: 5234-SEQ ID NO: 5807, or SEQ ID NO: 5808-SEQ ID NO: 5811.

In some embodiments, the AAV VP capsid polypeptide is a CNS tissue-tropic capsid polypeptide and has an amino acid sequence of SEQ ID NO: 2, wherein X¹, X², X³, X⁴, X⁵, X⁶, X⁷, X⁸, and X⁹are each independently selected from any amino acid, wherein the capsid polypeptide is capable of assembling into a recombinant AAV virion (rAAV), wherein the at least one substitution confers higher tropism for a central nervous system (CNS) tissue on the rAAV as compared to an rAAV virion having an AAV5 VP capsid polypeptide of SEQ ID NO: 1, and wherein the VP capsid polypeptide does not have the sequence of any of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, or SEQ ID NO: 8, optionally with further mutations elsewhere in the VP capsid polypeptide. In some embodiments, X¹X²X³X⁴X⁵X⁶X⁷X⁸X⁹of the CNS tissue-tropic VP capsid polypeptide correspond to a sequence of any one of SEQ ID NO: 12-SEQ ID NO: 3937, SEQ ID NO: 3938-SEQ ID NO: 4237, or SEQ ID NO: 5234-SEQ ID NO: 5807.

In some embodiments, the AAV VP capsid polypeptide is a muscle-tropic capsid polypeptide and has an amino acid sequence of SEQ ID NO: 2, wherein X¹, X², X³, X⁴, X⁵, X⁶, X⁷, X⁸, and X⁹are each independently selected from any amino acid, wherein the capsid polypeptide is capable of assembling into a recombinant AAV virion (rAAV), wherein the at least one substitution confers higher tropism for a muscle tissue on the rAAV as compared to an rAAV virion having an AAV5 VP capsid polypeptide of SEQ ID NO: 1, and wherein the VP capsid polypeptide does not have the sequence of any of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, or SEQ ID NO: 8, optionally with further mutations elsewhere in the VP capsid polypeptide. In some embodiments, X¹X²X³X⁴X⁵X⁶X⁷X⁸X⁹of the muscle-tropic VP capsid polypeptide correspond to a sequence of any one of SEQ ID NO: 4238-SEQ ID NO: 4933, SEQ ID NO: 4934-SEQ ID NO: 5233, or SEQ ID NO: 5808-SEQ ID NO: 5811.

In some embodiments, the engineered adeno-associated virus (AAV) viral protein (VP) capsid polypeptide (e.g., a CNS tissue-tropic VP capsid polypeptide or a muscle-tropic VP capsid polypeptide) has an amino acid sequence of SEQ ID NO: 2, wherein amino acid residues X¹, X², X³, X⁴, X⁵, X⁶, X⁷, X⁸, and X⁹are each independently selected from A, R, N, D, C, E, Q, G, H, I, L, K, M, F, P, S, T, W, Y, and V; wherein the engineered AAV VP capsid polypeptide is capable of assembling into a recombinant AAV virion (rAAV); and wherein the rAAV VP capsid polypeptide does not have the sequence of any of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, or SEQ ID NO: 8.

In some embodiments, the 581-589 region of the engineered VP capsid polypeptide, corresponding to residues 581 to 589, inclusive, with reference to SEQ ID NO: 1, has a sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of SEQ ID NO: 12-SEQ ID NO: 3937, SEQ ID NO: 3938-SEQ ID NO: 4237, SEQ ID NO: 4238-SEQ ID NO: 4933, SEQ ID NO: 4934-SEQ ID NO: 5233, SEQ ID NO: 5234-SEQ ID NO: 5807, or SEQ ID NO: 5808-SEQ ID NO: 5811. In some embodiments, the 581-589 region of the engineered VP capsid polypeptide comprises 1 amino acid substitution relative to any one of SEQ ID NO: SEQ ID NO: 12-SEQ ID NO: 3937, SEQ ID NO: 3938-SEQ ID NO: 4237, SEQ ID NO: 4238-SEQ ID NO: 4933, SEQ ID NO: 4934-SEQ ID NO: 5233, SEQ ID NO: 5234-SEQ ID NO: 5807, or SEQ ID NO: 5808-SEQ ID NO: 5811. In some embodiments, the 581-589 region of the engineered VP capsid polypeptide comprises 2 amino acid substitutions relative to any one of SEQ ID NO: SEQ ID NO: 12-SEQ ID NO: 3937, SEQ ID NO: 3938-SEQ ID NO: 4237, SEQ ID NO: 4238-SEQ ID NO: 4933, SEQ ID NO: 4934-SEQ ID NO: 5233, SEQ ID NO: 5234-SEQ ID NO: 5807, or SEQ ID NO: 5808-SEQ ID NO: 5811. In particular embodiments, the 581-589 region of the engineered VP capsid polypeptide has a sequence that is identical to any one of SEQ ID NO: SEQ ID NO: 12-SEQ ID NO: 3937, SEQ ID NO: 3938-SEQ ID NO: 4237, SEQ ID NO: 4238-SEQ ID NO: 4933, SEQ ID NO: 4934-SEQ ID NO: 5233, SEQ ID NO: 5234-SEQ ID NO: 5807, or SEQ ID NO: 5808-SEQ ID NO: 5811.

In some embodiments, the engineered adeno-associated virus (AAV) viral protein (VP) capsid polypeptide is an engineered AAV5 viral capsid protein, wherein the engineered AAV VP5 capsid polypeptide has at least one substitution as compared to SEQ ID NO: 1 in the 581-589 region, corresponding to residues 581 to 589, inclusive, of SEQ ID NO: 1, inclusive; wherein the capsid polypeptide is capable of assembling into a recombinant AAV virion (rAAV); wherein the at least one substitution confers higher tropism for a central nervous system (CNS) tissue on the rAAV as compared to an rAAV virion having an AAV5 VP capsid polypeptide of SEQ ID NO: 1, and wherein the VP capsid polypeptide does not have the sequence of any of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, or SEQ ID NO: 8, optionally with further mutations elsewhere in the VP protein.

In some embodiments, the AAV VP capsid polypeptides have an amino acid sequence of SEQ ID NO: 2, wherein X¹, X², X³, X⁴, X⁵, X⁶, X⁷, X⁸, and X⁹are each independently selected from A, R, N, D, C, E, Q, G, H, I, L, K, M, F, P, S, T, W, Y, and V; and wherein the polypeptide does not have the sequence of any of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, or SEQ ID NO: 8.

In some embodiments, the engineered AAV VP capsid polypeptide comprises a polypeptide sequence represented by the formula: (A)-(X)—(B), wherein: (A) is the polypeptide sequence of SEQ ID NO: 5812 (VAYNVGGQMATNNQSSTTAP, corresponding to residues 561 to 580 of SEQ ID NO: 2); (X) is the 581-589 region having a polypeptide sequence comprising amino acid residues X¹, X², X³, X⁴, X⁵, X⁶, X⁷, X⁸, and X⁹of SEQ ID NO: 2; and (B) is the polypeptide sequence of SEQ ID NO: 5813 (IVPGSVWMERDVYLQGPIWA, corresponding to residues 590 to 609 of SEQ ID NO: 2); wherein X¹, X², X³, X⁴, X⁵, X⁶, X⁷, X⁸, and X⁹are each independently selected from A, R, N, D, C, E, Q, G, H, I, L, K, M, F, P, S, T, W, Y, and V; and wherein the capsid polypeptide is capable of assembling into a recombinant AAV virion (rAAV); and wherein the polypeptide does not have the sequence of any of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 8.

In some embodiments, the engineered AAV VP capsid polypeptide comprises a polypeptide sequence represented by the formula: (A)-(X)—(B) wherein: (A) is the polypeptide sequence of SEQ ID NO: 5812 (VAYNVGGQMATNNQSSTTAP, corresponding to residues 561 to 580 of SEQ ID NO: 2); (X) is the 581-589 region having a polypeptide sequence selected from the list of polypeptides in TABLE 9 that is expected to confer CNS tissue tropism or preference on a recombinant AAV virion (rAAV), based on a secondary screen; and (B) is the polypeptide sequence of SEQ ID NO: 5813 (IVPGSVWMERDVYLQGPIWA, corresponding to residues 590 to 609 of SEQ ID NO: 2); and wherein the capsid polypeptide is capable of assembling into the rAAV and, the capsid does not have the sequence of any of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, or SEQ ID NO: 8. Also encompassed herein are rAAVs composed of engineered AAV5 VP2 capsid polypeptides and engineered AAV5 VP3 capsid polypeptides comprising a 581-589 region having a sequence selected from the list of polypeptides in TABLE 9 at the region corresponding to amino acid residues 445 to 453 in AAV5 VP2 or the region corresponding to amino acid residues 389 to 397 in AAV5 VP3.

In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence that is at least 75%, at least 77.7%, at least 85%, at least 88.8%, or 100% identical to a sequence provided in TABLE 9. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence that is at least 75%, at least 77.7%, at least 85%, at least 88.8% or 100% identical to SEQ ID NO: 18. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence that is at least 75%, at least 77.7%, at least 85%, at least 88.8% or 100% identical to SEQ ID NO: 3472. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence that is at least 75%, at least 77.7%, at least 85%, at least 88.8% or 100% identical to SEQ ID NO: 262. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence that is at least 75%, at least 77.7%, at least 85%, at least 88.8% or 100% identical to SEQ ID NO: 3306. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence that is at least 75%, at least 77.7%, at least 85%, at least 88.8% or 100% identical to SEQ ID NO: 2028. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence that is at least 75%, at least 77.7%, at least 85%, at least 88.8% or 100% identical to SEQ ID NO: 2791. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence that is at least 75%, at least 77.7%, at least 85%, at least 88.8% or 100% identical to SEQ ID NO: 424. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence that is at least 75%, at least 77.7%, at least 85%, at least 88.8% or 100% identical to SEQ ID NO: 2536. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence that is at least 75%, at least 77.7%, at least 85%, at least 88.8% or 100% identical to SEQ ID NO: 1971. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence that is at least 75%, at least 77.7%, at least 85%, at least 88.8% or 100% identical to SEQ ID NO: 415. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence that is at least 75%, at least 77.7%, at least 85%, at least 88.8% or 100% identical to SEQ ID NO: 3846. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence that is at least 75%, at least 77.7%, at least 85%, at least 88.8% or 100% identical to SEQ ID NO: 3283. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence that is at least 75%, at least 77.7%, at least 85%, at least 88.8% or 100% identical to SEQ ID NO: 1956. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence that is at least 75%, at least 77.7%, at least 85%, at least 88.8% or 100% identical to SEQ ID NO: 3297. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence that is at least 75%, at least 77.7%, at least 85%, at least 88.8% or 100% identical to SEQ ID NO: 4545. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence that is at least 75%, at least 77.7%, at least 85%, at least 88.8% or 100% identical to SEQ ID NO: 2661. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence that is at least 75%, at least 77.7%, at least 85%, at least 88.8% or 100% identical to SEQ ID NO: 1576. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence that is at least 75%, at least 77.7%, at least 85%, at least 88.8% or 100% identical to SEQ ID NO: 425. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence that is at least 75%, at least 77.7%, at least 85%, at least 88.8% or 100% identical to SEQ ID NO: 709. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence that is at least 75%, at least 77.7%, at least 85%, at least 88.8% or 100% identical to SEQ ID NO: 2168. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence that is at least 75%, at least 77.7%, at least 85%, at least 88.8% or 100% identical to SEQ ID NO: 54. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence that is at least 75%, at least 77.7%, at least 85%, at least 88.8% or 100% identical to SEQ ID NO: 429. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence that is at least 75%, at least 77.7%, at least 85%, at least 88.8% or 100% identical to SEQ ID NO: 708. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence that is at least 75%, at least 77.7%, at least 85%, at least 88.8% or 100% identical to SEQ ID NO: 428. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence that is at least 75%, at least 77.7%, at least 85%, at least 88.8% or 100% identical to SEQ ID NO: 4119. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence that is at least 75%, at least 77.7%, at least 85%, at least 88.8% or 100% identical to SEQ ID NO: 3906. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence that is at least 75%, at least 77.7%, at least 85%, at least 88.8% or 100% identical to SEQ ID NO: 2456. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence that is at least 75%, at least 77.7%, at least 85%, at least 88.8% or 100% identical to SEQ ID NO: 2278. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence that is at least 75%, at least 77.7%, at least 85%, at least 88.8% or 100% identical to SEQ ID NO: 5006. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence that is at least 75%, at least 77.7%, at least 85%, at least 88.8% or 100% identical to SEQ ID NO: 426.

In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence of SEQ ID NO: 18. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence of SEQ ID NO: 3472. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence of SEQ ID NO: 262. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence of SEQ ID NO: 3306. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence of SEQ ID NO: 2028. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence of SEQ ID NO: 2791. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence of SEQ ID NO: 424. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence of SEQ ID NO: 2536. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence of SEQ ID NO: 1971. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence of SEQ ID NO: 415. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence of SEQ ID NO: 3846. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence of SEQ ID NO: 3283. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence of SEQ ID NO: 1956. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence of SEQ ID NO: 3297. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence of SEQ ID NO: 4545. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence of SEQ ID NO: 2661. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence of SEQ ID NO: 1576. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence of SEQ ID NO: 425. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence of SEQ ID NO: 709. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence of SEQ ID NO: 2168. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence of SEQ ID NO: 54. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence of SEQ ID NO: 429. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence of SEQ ID NO: 708. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence of SEQ ID NO: 428. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence of SEQ ID NO: 4119. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence of SEQ ID NO: 3906. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence of SEQ ID NO: 2456. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence of SEQ ID NO: 2278. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence of SEQ ID NO: 5006. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence of SEQ ID NO: 426.

In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence of SEQ ID NO: 18. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence of SEQ ID NO: 2028. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence of SEQ ID NO: 424. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence of SEQ ID NO: 3306. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence of SEQ ID NO: 3472. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence of SEQ ID NO: 415. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence of SEQ ID NO: 709. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence of SEQ ID NO: 1971. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence of SEQ ID NO: 2791. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence of SEQ ID NO: 1956. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence of SEQ ID NO: 262. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence of SEQ ID NO: 425. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence of SEQ ID NO: 2536. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence of SEQ ID NO: 708. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence of SEQ ID NO: 54. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence of SEQ ID NO: 3283. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence of SEQ ID NO: 430. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence of SEQ ID NO: 3297. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence of SEQ ID NO: 2661. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence of SEQ ID NO: 428. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence of SEQ ID NO: 885. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence of SEQ ID NO: 429. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence of SEQ ID NO: 569. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence of SEQ ID NO: 724. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence of SEQ ID NO: 1576. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence of SEQ ID NO: 4545. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence of SEQ ID NO: 426. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence of SEQ ID NO: 1887. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence of SEQ ID NO: 3906. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence of SEQ ID NO: 3935.

In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence of SEQ ID NO: 18 with 1 or 2 conservative substitutions. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence of SEQ ID NO: 3472 with 1 or 2 conservative substitutions. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence of SEQ ID NO: 262 with 1 or 2 conservative substitutions. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence of SEQ ID NO: 3306 with 1 or 2 conservative substitutions. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence of SEQ ID NO: 2028 with 1 or 2 conservative substitutions. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence of SEQ ID NO: 2791 with 1 or 2 conservative substitutions. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence of SEQ ID NO: 424 with 1 or 2 conservative substitutions. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence of SEQ ID NO: 2536 with 1 or 2 conservative substitutions. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence of SEQ ID NO: 1971 with 1 or 2 conservative substitutions. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence of SEQ ID NO: 415 with 1 or 2 conservative substitutions. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence of SEQ ID NO: 3846 with 1 or 2 conservative substitutions. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence of SEQ ID NO: 3283 with 1 or 2 conservative substitutions. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence of SEQ ID NO: 1956 with 1 or 2 conservative substitutions. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence of SEQ ID NO: 3297 with 1 or 2 conservative substitutions. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence of SEQ ID NO: 4545 with 1 or 2 conservative substitutions. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence of SEQ ID NO: 2661 with 1 or 2 conservative substitutions. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence of SEQ ID NO: 1576 with 1 or 2 conservative substitutions. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence of SEQ ID NO: 425 with 1 or 2 conservative substitutions. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence of SEQ ID NO: 709 with 1 or 2 conservative substitutions. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence of SEQ ID NO: 2168 with 1 or 2 conservative substitutions. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence of SEQ ID NO: 54 with 1 or 2 conservative substitutions. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence of SEQ ID NO: 429 with 1 or 2 conservative substitutions. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence of SEQ ID NO: 708 with 1 or 2 conservative substitutions. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence of SEQ ID NO: 428 with 1 or 2 conservative substitutions. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence of SEQ ID NO: 4119 with 1 or 2 conservative substitutions. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence of SEQ ID NO: 3906 with 1 or 2 conservative substitutions. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence of SEQ ID NO: 2456 with 1 or 2 conservative substitutions. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence of SEQ ID NO: 2278 with 1 or 2 conservative substitutions. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence of SEQ ID NO: 5006 with 1 or 2 conservative substitutions. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence of SEQ ID NO: 426 with 1 or 2 conservative substitutions. The conservative substitutions may be at any position in the 581-589 region.

In some embodiments, an AAV VP capsid polypeptide preferentially targets a CNS tissue at a DNA enrichment of at least 100-fold greater than a wild type AAV9. For example, an AAV VP capsid polypeptide comprising a 581-589 region of SEQ ID NO: 18, SEQ ID NO: 3472, SEQ ID NO: 262, SEQ ID NO: 3306, SEQ ID NO: 2028, SEQ ID NO: 2791, SEQ ID NO: 424, SEQ ID NO: 2536, SEQ ID NO: 1971, SEQ ID NO: 415, SEQ ID NO: 3846, SEQ ID NO: 3283, SEQ ID NO: 1956, SEQ ID NO: 3297, SEQ ID NO: 4545, SEQ ID NO: 2661, SEQ ID NO: 1576, SEQ ID NO: 425, SEQ ID NO: 709, SEQ ID NO: 2168, SEQ ID NO: 54, SEQ ID NO: 429, SEQ ID NO: 708, SEQ ID NO: 428, SEQ ID NO: 4119, SEQ ID NO: 3906, SEQ ID NO: 2456, SEQ ID NO: 2278, SEQ ID NO: 5006, SEQ ID NO: 426, SEQ ID NO: 307, SEQ ID NO: 5155, SEQ ID NO: 2640, SEQ ID NO: 4317, SEQ ID NO: 1145, SEQ ID NO: 430, or SEQ ID NO: 885 may preferentially target a CNS tissue at a DNA enrichment of at least 100-fold greater than a wild type AAV9.

In some embodiments, an AAV VP capsid polypeptide preferentially targets a CNS tissue at a DNA enrichment of at least 200-fold greater than a wild type AAV9. For example, an AAV VP capsid polypeptide comprising a 581-589 region of SEQ ID NO: 18, SEQ ID NO: 3472, SEQ ID NO: 262, SEQ ID NO: 3306, SEQ ID NO: 2028, SEQ ID NO: 2791, SEQ ID NO: 424, SEQ ID NO: 2536, SEQ ID NO: 1971, SEQ ID NO: 415, SEQ ID NO: 3846, SEQ ID NO: 3283, SEQ ID NO: 1956, or SEQ ID NO: 3297 may preferentially target a CNS tissue at a DNA enrichment of at least 200-fold greater than a wild type AAV9.

In some embodiments, an AAV VP capsid polypeptide preferentially targets a CNS tissue at a DNA enrichment of at least 1000-fold greater than a wild type AAV9. For example, an AAV VP capsid polypeptide comprising a 581-589 region of SEQ ID NO: 18 may preferentially target a CNS tissue at a DNA enrichment of at least 1000-fold greater than a wild type AAV9.

In some embodiments, an AAV VP capsid polypeptide preferentially targets a CNS tissue at an RNA enrichment of at least 100-fold greater than a wild type AAV9. For example, an AAV VP capsid polypeptide comprising a 581-589 region of SEQ ID NO: 18, SEQ ID NO: 3472, SEQ ID NO: 262, SEQ ID NO: 3306, SEQ ID NO: 2028, SEQ ID NO: 2791, SEQ ID NO: 424, SEQ ID NO: 2536, SEQ ID NO: 1971, SEQ ID NO: 415, SEQ ID NO: 3283, SEQ ID NO: 1956, SEQ ID NO: 3297, SEQ ID NO: 4545, SEQ ID NO: 2661, SEQ ID NO: 1576, SEQ ID NO: 425, SEQ ID NO: 709, SEQ ID NO: 54, SEQ ID NO: 429, SEQ ID NO: 428, SEQ ID NO: 426, or SEQ ID NO: 430 may preferentially target a CNS tissue at an RNA enrichment of at least 100-fold greater than a wild type AAV9.

In some embodiments, an AAV VP capsid polypeptide preferentially targets a CNS tissue at an RNA enrichment of at least 200-fold greater than a wild type AAV9. For example, an AAV VP capsid polypeptide comprising a 581-589 region of SEQ ID NO: 18, SEQ ID NO: 3472, SEQ ID NO: 262, SEQ ID NO: 3306, SEQ ID NO: 2028, SEQ ID NO: 2791, SEQ ID NO: 424, SEQ ID NO: 2536, SEQ ID NO: 1971, SEQ ID NO: 415, SEQ ID NO: 3283, SEQ ID NO: 1956, SEQ ID NO: 2661, SEQ ID NO: 1576, SEQ ID NO: 425, SEQ ID NO: 709, SEQ ID NO: 54, SEQ ID NO: 430, SEQ ID NO: 569, and SEQ ID NO: 719 may preferentially target a CNS tissue at an RNA enrichment of at least 200-fold greater than a wild type AAV9.

In some embodiments, an AAV VP capsid polypeptide preferentially targets a CNS tissue at an RNA enrichment of at least 500-fold greater than a wild type AAV9. For example, an AAV VP capsid polypeptide comprising a 581-589 region of SEQ ID NO: 18, SEQ ID NO: 3472, SEQ ID NO: 262, and SEQ ID NO: 2028 may preferentially target a CNS tissue at an RNA enrichment of at least 500-fold greater than a wild type AAV9.

In some embodiments, an AAV VP capsid polypeptide preferentially targets a CNS tissue at a DNA enrichment of at least 5-fold greater than a wild type AAV9. For example, an AAV VP capsid polypeptide comprising a 581-589 region of SEQ ID NO: 18, SEQ ID NO: 3472, SEQ ID NO: 262, SEQ ID NO: 3306, SEQ ID NO: 2028, SEQ ID NO: 2791, SEQ ID NO: 424, SEQ ID NO: 2536, SEQ ID NO: 1971, SEQ ID NO: 415, SEQ ID NO: 3846, SEQ ID NO: 3283, SEQ ID NO: 1956, SEQ ID NO: 3297, SEQ ID NO: 4545, SEQ ID NO: 2661, or SEQ ID NO: 1576 may preferentially target a CNS tissue at a DNA enrichment of at least 5-fold greater than a wild type AAV5.

In some embodiments, an AAV VP capsid polypeptide preferentially targets a CNS tissue at a DNA enrichment of at least 10-fold greater than a wild type AAV9. For example, an AAV VP capsid polypeptide comprising a 581-589 region of SEQ ID NO: 18, SEQ ID NO: 3472, SEQ ID NO: 262, SEQ ID NO: 3306, SEQ ID NO: 2028, SEQ ID NO: 2791, or SEQ ID NO: 424 may preferentially target a CNS tissue at a DNA enrichment of at least 10-fold greater than a wild type AAV5.

In some embodiments, an AAV VP capsid polypeptide preferentially targets a CNS tissue at an RNA enrichment of at least 5-fold greater than a wild type AAV9. For example, an AAV VP capsid polypeptide comprising a 581-589 region of SEQ ID NO: 18, SEQ ID NO: 3472, SEQ ID NO: 262, SEQ ID NO: 3306, SEQ ID NO: 2028, SEQ ID NO: 2791, SEQ ID NO: 424, SEQ ID NO: 2536, SEQ ID NO: 1971, SEQ ID NO: 415, SEQ ID NO: 3283, SEQ ID NO: 1956, SEQ ID NO: 3297, SEQ ID NO: 4545, SEQ ID NO: 2661, SEQ ID NO: 1576, SEQ ID NO: 425, SEQ ID NO: 709, SEQ ID NO: 54, SEQ ID NO: 429, SEQ ID NO: 708, SEQ ID NO: 428, SEQ ID NO: 426, SEQ ID NO: 307, SEQ ID NO: 4317, SEQ ID NO: 430, or SEQ ID NO: 885 may preferentially target a CNS tissue at an RNA enrichment of at least 5-fold greater than a wild type AAV5.

In some embodiments, an AAV VP capsid polypeptide preferentially targets a CNS tissue at an RNA enrichment of at least 20-fold greater than a wild type AAV9. For example, an AAV VP capsid polypeptide comprising a 581-589 region of SEQ ID NO: 18, SEQ ID NO: 3472, SEQ ID NO: 262, SEQ ID NO: 3306, SEQ ID NO: 2028, SEQ ID NO: 2791, SEQ ID NO: 424, SEQ ID NO: 2536, SEQ ID NO: 1971, SEQ ID NO: 415, SEQ ID NO: 3283, SEQ ID NO: 1956, SEQ ID NO: 2661, SEQ ID NO: 1576, SEQ ID NO: 425, SEQ ID NO: 709, SEQ ID NO: 54, SEQ ID NO: 430, SEQ ID NO: 569, and SEQ ID NO: 719 may preferentially target a CNS tissue at an RNA enrichment of at least 20-fold greater than a wild type AAV5.

In some embodiments, the engineered AAV VP capsid polypeptide comprises a polypeptide sequence represented by the formula: (A)-(X)—(B) wherein: (A) is the polypeptide sequence of SEQ ID NO: 5812 (VAYNVGGQMATNNQSSTTAP, corresponding to residues 561 to 580 of SEQ ID NO: 2); (X) is the 581-589 region having a polypeptide sequence selected from the list of polypeptides in TABLE 2 (SEQ ID NO: 12-SEQ ID NO: 3937) that is expected to confer CNS tissue tropism or preference on a recombinant AAV virion (rAAV), based on a primary screen; and (B) is the polypeptide sequence of SEQ ID NO: 5813 (IVPGSVWMERDVYLQGPIWA, corresponding to residues 590 to 609 of SEQ ID NO: 2); and wherein the capsid polypeptide is capable of assembling into the rAAV and, the capsid does not have the sequence of any of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, or SEQ ID NO: 8. Also encompassed herein are rAAVs composed of engineered AAV5 VP2 capsid polypeptides and engineered AAV5 VP3 capsid polypeptides comprising a 581-589 region having a sequence selected from the list of polypeptides in TABLE 2 (SEQ ID NO: 12-SEQ ID NO: 3937) at the region corresponding to amino acid residues 445 to 453 in AAV5 VP2 or the region corresponding to amino acid residues 389 to 397 in AAV5 VP3.

TABLE2

Sequences of 581-589 Regions

SEQ ID

NO
Sequence

12
AAANMYWWS

13
AAAQSPEGV

14
AAAVQWQMM

15
AACHIMSQT

16
AAEPQHCYY

17
AAGQFEWLH

18
AAGRFNYFG

19
AAGTHHVMR

20
AAGVVQRGE

21
AALNTQLGH

22
AALSSRQVL

23
AALSWRSHF

24
AALYSQRAQ

25
AAMPINMCC

26
AAMTQHCPV

27
AANDCQIWF

28
AANYYATSW

29
AAQFDGFHV

30
AAQLEGFHV

31
AAQSMVCDS

32
AARDTMEGK

33
AARIYLMYV

34
AASYWGNMY

35
AATMTNHAW

36
AATQEYWQG

37
AAVYGKFSW

38
AAYLDDQYS

39
AAYPCQLER

40
AAYPYICAD

41
ACGYFTYNV

42
ACHTMVIEN

43
ACIYKIAQE

44
ACKFGQESD

45
ACYQYKCRR

46
ADAIRQPVL

47
ADAMRQPVL

48
ADAPVNASC

49
ADSIKLTEY

50
ADYTMDWHL

51
AEAMMSAVF

52
AEAQFQYWS

53
AEDLSPVPV

54
AEDYWDLGA

55
AEIILPGMQ

56
AEIYVHCQT

57
AEIYVHWQT

58
AEKLYGLST

59
AEREHWFYC

60
AFAQKCRYH

61
AFAYELNIN

62
AFCPISSGI

63
AFEQWAHNE

64
AFHAHEQAG

65
AFHQVHSMG

66
AFNDKPSCC

67
AFQNACKSA

68
AFTYGNHQF

69
AGASTWMQS

70
AGCSIQAWC

71
AGIVQGIWG

72
AGKCTPVTS

73
AGKYYQDYQ

74
AGMPWDGHC

75
AGQREVPSD

76
AHAMLDRNF

77
AHATYNKPT

78
AHDSASRCA

79
AHDWTHICA

80
AHGNSPTAY

81
AHHLGTMGH

82
AHKSPDKDV

83
AHLHNQFMP

84
AHLTPNLHE

85
AHMDEFRSG

86
AHMSTYWGF

87
AHNPCMHHQ

88
AHQTSWAWI

89
AHSDLTGAI

90
AHYQFQALM

91
AICQQHSYA

92
AIDQLSWHG

93
AIEIFGACV

94
AIEMTKYPW

95
AILSIYPSG

96
AILYQSAGE

97
AIMHYHRFK

98
AIQQCHAYA

99
AIQWFQAPG

100
AISYMQLEA

101
AITPRMGMK

102
AITSCICEK

103
AIWGYGMNY

104
AKAYCTSVD

105
AKCEHDHCW

106
AKDCVQSQA

107
AKFVGVAAD

108
AKIHGAWKA

109
AKQHALMIE

110
AKSTMPGYQ

111
AKTFVMSFG

112
AKTSLQIDP

113
ALEASYGYF

114
ALECGHLPY

115
ALFYMNADF

116
ALIQFGQSW

117
ALKSHGCLT

118
ALLNRTACW

119
ALPHIQSEP

120
ALPHIRSEP

121
ALTNWGMSG

122
ALWMFDVFG

123
AMAQSMVQF

124
AMDKIPVFQ

125
AMDPPIREP

126
AMDRIPIFQ

127
AMEQVSATF

128
AMFNGTSMS

129
AMGIGMRID

130
AMGPCNGPM

131
AMGYNGTYP

132
AMKWVEACP

133
AMKWVEAWQ

134
AMKYIGCPM

135
AMLAYFATE

136
AMLKNHCYH

137
AMMLCIWVD

138
AMMNMNVQF

139
AMQCFPAQQ

140
AMQCNQMQD

141
AMQPTRIVG

142
AMQPVRIVV

143
AMRQIIAPC

144
ANAVTEEWM

145
ANGDKVAMD

146
ANITIGPQA

147
ANIYICPQA

148
ANLDSHQFE

149
ANMVAHMMA

150
ANQHFHAES

151
ANTRDNAEL

152
ANTWLAHFE

153
APCHDKDRE

154
APIQGQMMM

155
APKLVGLST

156
AQCQDMWGH

157
AQEPFKTIQ

158
AQFRAIIGN

159
AQGTMDSWY

160
AQIQPAMFP

161
AQMTVCARE

162
AQMYWSSKD

163
AQNNKFDGS

164
AQSFLWKGC

165
AQVLMEMCT

166
AQYCTIMAH

167
ARKFCEGTP

168
ARQVYEMNG

169
ARQWYEMTG

170
ASEMTKGPW

171
ASFMSWEGA

172
ASGWAPCSW

173
ASMQEHHYT

174
ASQHFHAPQ

175
ASQHNHEKQ

176
ASQLIECNA

177
ASQPRGAEQ

178
ASQTTHGPL

179
ASTERAKFG

180
ASTQQGFWC

181
ASVCEHTLE

182
ASVECWQWT

183
ASVPISHSQ

184
ASWLCGRNV

185
ASYNGCMIC

186
ASYPSSQVF

187
ASYSICDAG

188
ATAGIPTAG

189
ATAQFQMEG

190
ATCTFNPGI

191
ATCWVQMGQ

192
ATEGYNMHC

193
ATENYNRRG

194
ATFMPNQSF

195
ATGMRIAVC

196
ATGMRIDVC

197
ATGTYNIQE

198
ATISKLECQ

199
ATKTCAEGG

200
ATLNSMHTQ

201
ATMCHCVVA

202
ATMWFTANG

203
ATMWMQWWD

204
ATMWVQAYG

205
ATNQLNESF

206
ATQGYNMHC

207
ATQLYMHHK

208
ATQMYMHCK

209
ATTFSSASF

210
ATTTVNRQE

211
ATVDVKGAH

212
ATVWSINAD

213
ATWSADVTA

214
AVDLTSKHS

215
AVDPTKDGQ

216
AVEQLNQPI

217
AVGCSQHWK

218
AVKCPESLI

219
AVMPSHQIT

220
AVNACSNSH

221
AVNDCKMWE

222
AVRIMFMQE

223
AVSGCRGSA

224
AVSNAMIAG

225
AVSNWLETS

226
AVSPSNFLA

227
AWCCSSVGE

228
AWFKTMPSD

229
AWFSHHQFC

230
AWHQTQDDM

231
AWREQEWAH

232
AWSEFQWFD

233
AWTPLMSYN

234
AWVPEGHFP

235
AWYHTQDDM

236
AYADMFQCK

237
AYCLTNGPH

238
AYCSKMRGE

239
AYDFVGVQE

240
AYFLLHASG

241
AYGPFAMLY

242
AYNAMHTAW

243
AYNFKVGSD

244
AYNIDYQPV

245
AYNWQQTSA

246
AYRCYACET

247
AYRCYAVEQ

248
CAARCYKAR

249
CADFKNECA

250
CADTAWQYT

251
CAEPKRMEY

252
CAEWRIMWS

253
CAGFMQTCQ

254
CAGGIVCHN

255
CAGGTVHHN

256
CAHSPHSHL

257
CAHYAQWGK

258
CAILAYAHH

259
CAIYSGLHI

260
CAMQCEHAE

261
CANYWQDMG

262
CATRFNIGG

263
CCCTVEREC

264
CCEEYACAE

265
CCTPTECQM

266
CCYPTHCQM

267
CCYQLGLMP

268
CDGSKSVGD

269
CDLCIAACE

270
CECRYFKFA

271
CECSYFKHA

272
CEKVQAHWM

273
CETYTIHIP

274
CEVDICNDR

275
CFLLTNRIS

276
CFTITQGEW

277
CFWVRGMWN

278
CGANYQRFG

279
CGGTYFALP

280
CGVNYQWFG

281
CHAGFPAWP

282
CHCKTDMWA

283
CHHHYHTHL

284
CHNVENCME

285
CHVNLKAWW

286
CIENMFVNS

287
CIGNKSASG

288
CIHECCDSG

289
CIHSVHFAA

290
CIKWYAAST

291
CILPRGIGQ

292
CILYLEEAQ

293
CIMFRSATS

294
CIMTRAATS

295
CIRSKPDYN

296
CISWGGNYG

297
CIVQKGNMV

298
CIYSKTGAG

299
CKAHKQTMS

300
CKELYPQAV

301
CKKTTGVCY

302
CKNPTGVCY

303
CKQEQLKMP

304
CLDHINSLS

305
CLWIYSVMG

306
CMCRVEQVA

307
CMKVKQSQP

308
CMMLMMTGQ

309
CMWVMIHFE

310
CMYNVSHWN

311
CMYTGDGPM

312
CNFSALPKH

313
CNGHYTVIS

314
CNGPNVSEI

315
CNQTIRPII

316
CNQTIRPIK

317
CNSWYAAST

318
CNVPRLVRH

319
CPAKHAQAR

320
CPESSSHQT

321
CPKQANRSG

322
CPTFWGHSE

323
CPWFIKWGI

324
CQAIVNRLN

325
CQASHEHAH

326
CQDAVGRSY

327
CQDAVGRTF

328
CQDCYHCDR

329
CQEDMSSTY

330
CQKWYESFG

331
CQSCISDAG

332
CQYLSFRTV

333
CRGKSHDHT

334
CSAAFQLQP

335
CSALWGGCC

336
CSAQIKAAC

337
CSDVSTIYI

338
CSFQEKAAC

339
CSHETGAMC

340
CSMDEYAMA

341
CSMHMHYVE

342
CSMTGVMFE

343
CSRHDLNGD

344
CSSGFHLFH

345
CSTGFHCEQ

346
CSTNCTANP

347
CSVPGEQAF

348
CTASWMSWD

349
CTCQGTGFS

350
CTCQGTGFT

351
CTERTKCAS

352
CTERTMNAS

353
CTGIQWTGG

354
CTHECNDSG

355
CTHPGHQNQ

356
CTHWFTSLT

357
CTIAYASMA

358
CTILATVHH

359
CTNAYFMSE

360
CTNTYFMSE

361
CTQLKECWL

362
CTTETQCRS

363
CTTPHDCHF

364
CTTPNLQVC

365
CTTSHLIGC

366
CTVKEMANH

367
CTWWGHQSM

368
CVDAKDHCE

369
CVEWRIMWS

370
CVHSKMAFH

371
CVIDVKEAG

372
CVMEYMDWG

373
CVNPDDMAQ

374
CVQQEMGGG

375
CVSFDACFA

376
CVSRAWDEK

377
CVTPKMDLP

378
CVTTKGDLP

379
CVVPLTNYY

380
CWDLVRDCK

381
CWQFMTAHP

382
CWQSEGAMM

383
CWWNFGKDC

384
CYMPFKMQH

385
CYNSGDCDN

386
CYQFWSQYS

387
CYTEPSNKH

388
CYWPMTNCT

389
DACTNKSHM

390
DADDSNIAW

391
DADEYKLDC

392
DAEVTMDAM

393
DAFHMKQHC

394
DAFQYPTIG

395
DAGFTWINC

396
DAGKFGVDI

397
DAGWIMNNS

398
DAGYSMTYH

399
DAHHGSRCG

400
DAHMYMVHQ

401
DAHVAFTDC

402
DAIHFNMKD

403
DAITVCAQE

404
DAIWYGRLG

405
DALHIGRSG

406
DAMGYLKGG

407
DAMQKHSAE

408
DANLYHPME

409
DANMQPVTF

410
DAPLIPQWG

411
DAQCRNTDG

412
DAQGIELFT

413
DAQLLMQAD

414
DAQLSTSYW

415
DARVYRALD

416
DASCATLWS

417
DASCATPWS

418
DASHVKSIE

419
DASIGAGPF

420
DASIMMEMG

421
DASIVWTID

422
DASTQCYMS

423
DATWEHIRL

424
DAWCFISSY

425
DAWLQLKDN

426
DAWMMMWGS

427
DAWQMKEAL

428
DAWQMLSGN

429
DAWSVLQDK

430
DAWSYQCYH

431
DAWYSKSQQ

432
DAYACSSSF

433
DAYPSNIAW

434
DCDAWHANV

435
DCDIGWGCF

436
DCHQSPVFY

437
DCMRNTVFE

438
DCMWMQNGL

439
DCNFDCPGA

440
DCQGQHRVC

441
DCQQILWDA

442
DCRTLEEFI

443
DCSWQTMAF

444
DCVVPMPWP

445
DEMKFNRDD

446
DFAACNSGF

447
DFACHNNFT

448
DFAHHSTGV

449
DFASCDICS

450
DFASFNDCS

451
DFAYHSTGV

452
DFDPWFITT

453
DFELQDTSF

454
DFEYSGQLG

455
DFEYSHDWN

456
DFGGVMGTA

457
DFHTMHQFE

458
DFIHNCHCD

459
DFINMPYWS

460
DFMQNSVHY

461
DFRHVRRSL

462
DFSQHKMHQ

463
DFSVTSHMG

464
DFVTNPNYE

465
DFYTFSFEN

466
DGAVYVKQD

467
DGDAKLVDG

468
DGGWFMQGE

469
DGIVQGVWG

470
DGKLQSTCI

471
DGKLQSTCM

472
DGQPYMTYS

473
DGSFVQMTQ

474
DGYMCMSGG

475
DHCSMCCGN

476
DHKHNVIYP

477
DHMQWGKFD

478
DHRHSFMGF

479
DHVMLRMIN

480
DHWYFLYHG

481
DICEGPKQY

482
DICQVIDNE

483
DICSDSWKV

484
DIDSFMHCL

485
DIGFMDTRA

486
DIICFNWQV

487
DIKCERDAD

488
DIMRLSWPI

489
DIQLYWMNQ

490
DIREMMCKN

491
DIRRAFAHC

492
DISFGEYNP

493
DISSMYWWH

494
DISWGGLME

495
DISYFLYHG

496
DIWIQMVNT

497
DKCCDTGLS

498
DKCYMTSSF

499
DKFPTHFSD

500
DKHEMVNVG

501
DKIFTIACP

502
DKIFYGACP

503
DKLPKVCDW

504
DKLTQSAYQ

505
DKLVYTQDR

506
DKNATAIWE

507
DKQFANHFT

508
DKQWAHHSM

509
DKRICHLNY

510
DKTVQSLAL

511
DKVAVDPYR

512
DKYMFQRDG

513
DKYMFWRDE

514
DLEPITTQA

515
DLFAIKQMW

516
DLGPISSWM

517
DLHRFDADY

518
DLIVKKSTG

519
DLIWNHLRT

520
DLLGIINGG

521
DLQYRLCQQ

522
DLYGQPNYC

523
DLYIMSDAN

524
DMCEQNNMQ

525
DMCVQNNGQ

526
DMEGCCGCA

527
DMHMKDMDS

528
DMHRFDADY

529
DMLPNNEWP

530
DMTEWNFLF

531
DMVFNNEWP

532
DMVMPEMYG

533
DNADLHHEC

534
DNCSDGWKV

535
DNKCYHMLM

536
DNKCYSMAM

537
DNMYPECMQ

538
DNRMWYMCG

539
DNRMWYMYG

540
DNVHPDSQA

541
DNVHRDCQA

542
DNVQISDAE

543
DPEFMAHCE

544
DPEFMTHCE

545
DPHQLPKKS

546
DQGSFNMCR

547
DQLICNMNE

548
DQLNPGVQA

549
DQLPVQYSL

550
DQMFHQFVT

551
DQMPYETNA

552
DQNINIHTS

553
DQQKLECYH

554
DQQSIGDHE

555
DQSLVDSQY

556
DQTMESGKT

557
DQTMFCAWC

558
DRAQESMGP

559
DRCWMPYRN

560
DREIYAHWD

561
DRENAMFFG

562
DRMQKNSAE

563
DRMWIDIAS

564
DRNVCNVCH

565
DRSGNWLWA

566
DRSQSWLWA

567
DRYYMDALM

568
DSAFYDRDN

569
DSASPIMGM

570
DSDCYTLDC

571
DSDIQFGIG

572
DSDYFRQMD

573
DSEETDYYQ

574
DSFFYNFGA

575
DSFILEAMC

576
DSGQNQQAG

577
DSHINWFCD

578
DSHLSFECR

579
DSHTWVADR

580
DSKHQMCID

581
DSKPMYHSD

582
DSKPSVCQE

583
DSKPWYHVD

584
DSMCEHHYY

585
DSNLDMHGC

586
DSPEQMIRH

587
DSQCVNHEE

588
DSQFIGQGE

589
DSQHVCGVN

590
DSQPASEVG

591
DSRFKSEWN

592
DSRPESTVG

593
DSRPFYGSV

594
DSRPKSTNG

595
DSSPFFWHS

596
DSSYFHCCD

597
DSVNSMQCT

598
DSYCCVSTH

599
DSYTTWLDD

600
DTCCFVANP

601
DTCNSMQCT

602
DTCVMYTQD

603
DTDYNHNSA

604
DTFTGIMHQ

605
DTGKPITTA

606
DTHHTHWQY

607
DTHIFIRHA

608
DTHKYTENR

609
DTHLQMQVI

610
DTITFKQQH

611
DTMNHRMTP

612
DTMPTFSTM

613
DTMQQVKFN

614
DTPLCNILC

615
DTQQYWMQD

616
DTRCWNHNA

617
DTRCWNVNC

618
DTRDCNFFN

619
DTTDCKKAS

620
DVAELKSLF

621
DVAHMQWRG

622
DVAKFMQML

623
DVALKNMFS

624
DVAVKKEYE

625
DVCAFRLMN

626
DVCCWTLAP

627
DVCDEHILY

628
DVCERMGFT

629
DVCNFMYSG

630
DVCRKNVSD

631
DVDFNELCE

632
DVDHPCMMF

633
DVDLNELCP

634
DVFSWHKDQ

635
DVHDSQLCY

636
DVIHTTHSG

637
DVKMKLSEQ

638
DVLIATMTC

639
DVLISTMAC

640
DVNKHSFNG

641
DVVKPTLMQ

642
DVVTPKLMQ

643
DVWLMRMCA

644
DVYFIRNAE

645
DWAEKVWGF

646
DWCEMKVGD

647
DWFMAFCGH

648
DWFQAIIHA

649
DWGFWMKHE

650
DWKFNLKDE

651
DWKPSVCQC

652
DWMLHADLL

653
DWRTFQAIA

654
DWRTPQAIA

655
DWVPEGHFP

656
DYAIENSAF

657
DYDHLDTWT

658
DYDSIQRQE

659
DYHPWWVTS

660
DYILPVIAI

661
DYILPVTAM

662
DYLSQFFGI

663
DYLWMNQEK

664
DYNRNNRYL

665
DYQPLSCYP

666
DYQTMTEHE

667
DYRDSHVAG

668
DYSPIDCEQ

669
DYSYRHIGE

670
DYTGMHQNM

671
DYTMMHQNC

672
DYTTNCMVE

673
DYVSLINEI

674
DYVYTNAEA

675
DYWLMHKID

676
EACFKDFWF

677
EACGMMTYC

678
EACGMTTMC

679
EACHPDLTP

680
EACMVNAAF

681
EACYWGHCP

682
EADEWWTIL

683
EADSRANAQ

684
EAEHMNWLM

685
EAEPFKTIQ

686
EAFNAFAGE

687
EAFNAFATR

688
EAFVHYCIN

689
EAGSCAMRV

690
EAHDDEYQC

691
EAHTNSICK

692
EAIPHIMDM

693
EAITDMYQP

694
EAITQFAVI

695
EAKWKECPS

696
EAMEQPYMF

697
EAMGYQAVE

698
EAMPVQKGW

699
EANVREHFC

700
EARTTRDCG

701
EARYSEEVW

702
EASMPYASM

703
EASWKMCPS

704
EATWLQRIM

705
EAVMWTCNS

706
EAWFYHADD

707
EAWMPSASG

708
EAWMYHQFH

709
EAWSKLEQP

710
EAWSMLDAL

711
EAWWYNFYF

712
EAYLYNIPM

713
ECDCGHVHL

714
ECHPYKTVA

715
ECLARQRHV

716
ECTQYVMCN

717
ECTWNMDAW

718
ECVSHIPGK

719
ECWSYQMVD

720
EDEQHLWND

721
EEDPLNGWQ

722
EEEPIWWQV

723
EEEWIWWQC

724
EEEWLNGWQ

725
EELETWIPY

726
EEMADGDAQ

727
EEMADGDAR

728
EEMEGLASE

729
EEPWQCMTA

730
EFDTTPCLF

731
EFEHGNAAS

732
EFFMVANSP

733
EFHMMGHFF

734
EFHYWFRPS

735
EFISLKSYE

736
EFLCINHGE

737
EFLVSGDSG

738
EFNALSFNV

739
EFNKLSFNH

740
EFNNTMMGQ

741
EFRHLTSMP

742
EFRHNQMNT

743
EFSPCVKMR

744
EFSYNVVHF

745
EFTKYSNAV

746
EFTMNQHMW

747
EFTTNYCHG

748
EFWVTECDQ

749
EGCQIPWTA

750
EGDPFKLQK

751
EGKQTNKAV

752
EGLQGNNYY

753
EGVQLNTDE

754
EGYMCMSGH

755
EGYSTHALG

756
EHAWLWMQS

757
EHCWYQFGA

758
EHFKAGCLH

759
EHGYMRCTK

760
EHHVTTGCT

761
EHKHMGCMN

762
EHKPCHASD

763
EHMQAKSAW

764
EHNHMGCMN

765
EHNWVQRAE

766
EHQLVGDWF

767
EHQLVGDWL

768
EHSWFISYN

769
EHSWINNMN

770
EHTFGPVYR

771
EHTQRGAAP

772
EHTQRGCAG

773
EHVQPMQWN

774
EHWHQMSPS

775
EHYSAHFFC

776
EIASKMNQL

777
EIDSKMHQL

778
EIGPQPNQD

779
EIGSVKHWE

780
EIHKRIAMT

781
EIKFSAGGI

782
EILKIDYQV

783
EINSLMYVD

784
EISCMHMSM

785
EISFEYCHE

786
EISPQENQD

787
EITEEALCD

788
EITHYYSTD

789
EIWSALAAN

790
EIWVREVNS

791
EKAHVQYAY

792
EKDTPTCKM

793
EKEMISTLE

794
EKIMVNRAF

795
EKLNAVQFC

796
EKMNCLVRW

797
EKNLYWTWE

798
EKRINTSPG

799
EKYRFSNGY

800
ELCTNTMAS

801
ELGTMTHSE

802
ELMSMIIVE

803
ELQEATHYP

804
ELRDYHIGM

805
ELRHAEQSQ

806
ELRISEQSQ

807
ELWDYHIGM

808
ELYMSNWMM

809
ELYSLAEQI

810
EMAKYMWDP

811
EMAMNLKDT

812
EMAVANSTH

813
EMCKGLCTT

814
EMDWTHVSH

815
EMESLNLLQ

816
EMESRNLLQ

817
EMFFYFGSQ

818
EMHKVPSGM

819
EMIHIGHRE

820
EMKTQCHGE

821
EMKTQTHFE

822
EMKTTQDAE

823
EMLPTQLNC

824
EMLVAHFGN

825
EMMCRCWLM

826
EMMQQAFAQ

827
EMNRYWTWE

828
EMSEEFSHA

829
EMYAFWQRG

830
EMYMMQTDQ

831
EMYPLFDAI

832
EMYVTKADD

833
ENAQMECLM

834
ENCAVTFCA

835
ENDQANLYA

836
ENDRCHTCA

837
ENDYVHGCM

838
ENFIQNCPG

839
ENGPLLCAR

840
ENKPVDQDK

841
ENLLEWKKA

842
ENNTWMQWG

843
ENQPANGWY

844
ENQQGNQCV

845
ENRLVQEVQ

846
ENRMFQDSE

847
ENSFYAGAG

848
ENTTTVAPC

849
ENVHINQAM

850
EPVSLQYMD

851
EPWKTHQQN

852
EQAEYDSCD

853
EQAGVASMW

854
EQCAVWDCM

855
EQFVSMNTG

856
EQGFVMFNG

857
EQGPLGSWF

858
EQGQLWSWF

859
EQNEETPFG

860
EQRNMGERA

861
EQRYCNSYV

862
EQTGVNVCL

863
ERELASATH

864
ERHPINQAL

865
ERQAHNNYA

866
ERTVNLRDC

867
ESCKTIRLE

868
ESCLSQNRA

869
ESEPFECCA

870
ESESGHMEG

871
ESGMIAVPH

872
ESHLWLAME

873
ESHSTQTYV

874
ESHYRCMCN

875
ESISSGVEG

876
ESLSFQTLV

877
ESMPFLDEA

878
ESNPVSMDV

879
ESTHMLYEM

880
ESTPQFTSG

881
ESTQRFDKM

882
ESTSGWSMA

883
ESTSNWAMA

884
ESVLKNQFE

885
ESWMKLEWQ

886
ETAIRDLTQ

887
ETCLSMHYL

888
ETDVSWCES

889
ETEFRQLDG

890
ETEVKSHWD

891
ETFIFPGAG

892
ETFVRHADY

893
ETGKTIYHQ

894
ETGPRQDLF

895
ETHFVQGRM

896
ETHILDQFC

897
ETHIVFGQW

898
ETHNKHTDY

899
ETISSGIEG

900
ETLAKLHCA

901
ETLSRSDIC

902
ETNEVMVQQ

903
ETRVSWCEW

904
ETSLSGNQY

905
ETSTSCAWL

906
ETTHYYSTF

907
ETTLHDLWL

908
ETTLQDLWE

909
ETVICDGVT

910
EVCSIHWMG

911
EVEVLEMGH

912
EVEVMVYSW

913
EVEWYYGQS

914
EVFEEYWSL

915
EVHGRNQDN

916
EVHGWNQDN

917
EVHKGAVMD

918
EVHKYCCNS

919
EVHSTQTAV

920
EVIRSPIEG

921
EVKPIGHSQ

922
EVLLSTDEA

923
EVLVAEWQG

924
EVLVAFATN

925
EVLVAVWQG

926
EVMPYHRTH

927
EVNQQMKAK

928
EVRQWQDED

929
EVSFYAGAI

930
EVSRQMYSQ

931
EVTDPCECA

932
EVTTIAQDH

933
EVVRRCYEC

934
EWCLLHVGH

935
EWEQTWRAP

936
EWFQAGICD

937
EWKHDRNDI

938
EWKHYRNDI

939
EWQGLQQFT

940
EWSGLQSCF

941
EWVFQHLSS

942
EWYCFLDSG

943
EYACHNRMP

944
EYCEFRQMD

945
EYCKYSLGL

946
EYEHMMWSQ

947
EYKSYGNCQ

948
EYLWEAIEH

949
EYMQQAFAW

950
EYNIYIATD

951
EYQISKTMC

952
EYQISNTMC

953
EYSTLPMTT

954
EYTKYQLGL

955
EYVSLINEI

956
EYWLNSSQA

957
FACWILYIG

958
FAEIHMQYH

959
FAEPLENWM

960
FAGFALHDS

961
FAGMYRRNS

962
FAILSGQTL

963
FAIWNYESK

964
FALSRNGNG

965
FALTCEEAP

966
FALTCNEAG

967
FAMKVLDIQ

968
FAMLGYWVH

969
FANCSTHQD

970
FAQLNQASM

971
FASRQGDNH

972
FASTTAECM

973
FATAHQKCW

974
FATQNQSPS

975
FAVMTSTEN

976
FCAGCDMAN

977
FCCHLAAHK

978
FCCSKANDC

979
FCDVVKHHL

980
FCDYHNTIE

981
FCITGNACA

982
FCNSYTMLP

983
FCPNSQMDG

984
FCQNPTQAM

985
FCVNQQTCQ

986
FDALMCWKI

987
FDANDCRPS

988
FDKCHMADW

989
FEACVTECL

990
FEKTMVAMH

991
FFGNVMKEQ

992
FFGWFHMNN

993
FFHVAWLSI

994
FFLKLNDVP

995
FFNHDNIGN

996
FFNNISTYF

997
FFNPTTQQN

998
FFPWEWECW

999
FFSHIQAAD

1000
FFSMVENCG

1001
FFTNRNHEE

1002
FFVKMMCSH

1003
FFVTWICRL

1004
FFYFLDILK

1005
FFYSTNFGM

1006
FGAEMHCPM

1007
FGDFFFNHN

1008
FGKWLTRLH

1009
FGNSMMLQS

1010
FHAMKHVLE

1011
FHLHTHRED

1012
FHNSKSEKH

1013
FHSFIHQHE

1014
FHTNYICQE

1015
FHVKVSCDS

1016
FICHVKEGA

1017
FICRHIHGI

1018
FICSSQCGA

1019
FIEFTIGEF

1020
FIGLKNAHL

1021
FIPNKWLGN

1022
FIQMTMSSD

1023
FITQYPNAE

1024
FKFMVDTSS

1025
FKIECGLFQ

1026
FKNDSVDHE

1027
FLEYAVQNQ

1028
FLQWTESGD

1029
FLVGQHVHP

1030
FLVVNHFGH

1031
FMECLKDTK

1032
FMEDKQVFS

1033
FMEYYLWYC

1034
FMSLVNAGE

1035
FMTMLGCSM

1036
FNARRDTWF

1037
FNGYYETQG

1038
FNQFEFVSA

1039
FNRCMEDRG

1040
FNSNLGAAT

1041
FNSSYGTNQ

1042
FNTQLLMSG

1043
FPEKAIVGQ

1044
FPEPFPMWG

1045
FPMPFPMWK

1046
FQAMIMTNY

1047
FQAVCIWIS

1048
FQCSVHKFV

1049
FQECEAPQH

1050
FQEVFSFFN

1051
FQFWRQIME

1052
FQIPLKAVA

1053
FQSMQHDNN

1054
FSADSMCSF

1055
FSAPITAHF

1056
FSAPLFHCA

1057
FSEPLFHLF

1058
FSEVKGMSG

1059
FSFRTARQQ

1060
FSFRTARYL

1061
FSGTVYCWC

1062
FSLPNNCDD

1063
FSNTVYKWC

1064
FSTHLTDAC

1065
FSTSYCHED

1066
FTDNPNWYA

1067
FTERTKCAA

1068
FTEYEHCAS

1069
FTHQRNALT

1070
FTHTFEACG

1071
FTIIKNNCL

1072
FTMDINLND

1073
FTMQSGDYH

1074
FTNFKMNMY

1075
FTNQLPEAN

1076
FTNYKYSFE

1077
FTQQEFHGQ

1078
FTTDYQMIA

1079
FTVDQLNWQ

1080
FTWHIESKF

1081
FTYGFQTIN

1082
FTYNFMNSW

1083
FVDEAFAWQ

1084
FVGFALHDS

1085
FVHASMINA

1086
FVIGHHLFP

1087
FVRGPWQCE

1088
FVRWSPYPI

1089
FVSAMSGPC

1090
FVSKSEEYH

1091
FVSLMSGPN

1092
FVTVMQFQD

1093
FVTVTACDG

1094
FVWMTGLNM

1095
FWELNGPKN

1096
FWGQYHVQG

1097
FWHEYNFTN

1098
FWKMLGHNH

1099
FWMQTEHGK

1100
FWMQTEHGT

1101
FWNFFHSHL

1102
FWNMVDTGG

1103
FWNQQSQCF

1104
FWQQYNEAN

1105
FWRSLDMQY

1106
FWSNIGAAP

1107
FWSQYDLMY

1108
FWTSYDSSH

1109
FYCSKMRGL

1110
FYYNYMHHS

1111
GAAQFGAFY

1112
GAFIIQKTP

1113
GAFMGLMTQ

1114
GAFVGLMTQ

1115
GAHMHDTHL

1116
GAKTYGEME

1117
GASFYEGME

1118
GATECFNPC

1119
GATQCFNPF

1120
GCDKWLGLC

1121
GCELISVGQ

1122
GCTPRQEMP

1123
GDMGCNVKL

1124
GEEVGCIWG

1125
GEKTYGEMY

1126
GEQQMPFIC

1127
GEYNERMHI

1128
GFANFMMNF

1129
GFANFNMTD

1130
GFDNVSFSY

1131
GFEEMDAQM

1132
GFEEQLTSK

1133
GFEMSTSHS

1134
GFFLTKQDG

1135
GFFTQMSQS

1136
GFGTYLSMH

1137
GFIDYQMQD

1138
GFMHYCSHV

1139
GFMLPIYWC

1140
GFTFMEASH

1141
GGAGVHHHT

1142
GGQTIKEHF

1143
GGWSDSNDA

1144
GGYNERMHI

1145
GHCYTQQQH

1146
GHDFTQFQE

1147
GHEDLLAMD

1148
GHESCGLWT

1149
GHLECCWSI

1150
GHSFRYACD

1151
GHVQAECMY

1152
GIADYQQQG

1153
GIFFCQEEK

1154
GIGWLKHFE

1155
GIICVTRDD

1156
GIKCERDAD

1157
GIKLTGTDH

1158
GILELHFGV

1159
GILHLHQCI

1160
GILNITMDQ

1161
GIMCREQCD

1162
GIMKHQETS

1163
GIMMQQMDP

1164
GIQQHAIMA

1165
GIREQEYCP

1166
GIRWADHWN

1167
GIVMMQAMD

1168
GIVNCTRLG

1169
GIVPLGTNY

1170
GIYMNQENA

1171
GIYPWMTYA

1172
GKQFDEQLE

1173
GKQFDEQQM

1174
GKSPMGEMP

1175
GLFWMWVDN

1176
GLIFHQSLS

1177
GLMSASAHS

1178
GLMTQQNAD

1179
GLMTQQNSD

1180
GLNICKNQQ

1181
GLNKFMQGP

1182
GLSCKSAWL

1183
GLYWQCDGM

1184
GMEHLWNVD

1185
GMKFTAQGN

1186
GMKILRTRK

1187
GMNFCLASH

1188
GMRDVFDCY

1189
GMRYHDDLF

1190
GMSFYNFFE

1191
GMSSYTCED

1192
GMTPRMMGM

1193
GMTSWGCWE

1194
GNEGADAEE

1195
GNEKCGTAV

1196
GNKSFQYAS

1197
GNMEQEGCP

1198
GNYLYMHQG

1199
GPFSFLRGA

1200
GPFSFLRGG

1201
GPNGDAARY

1202
GQAVSHAFS

1203
GQFWTNDAA

1204
GQGVAQQCA

1205
GQHFVMLSS

1206
GQHLQIGAE

1207
GQNTTDRSN

1208
GQPVCCVCM

1209
GQQHYGWQQ

1210
GQSFYTCED

1211
GQVHTSYYG

1212
GQVQHECMY

1213
GRCFMPPGR

1214
GRVLANCAE

1215
GSDAIMWDC

1216
GSDAQMWDH

1217
GSEHKGIGE

1218
GSEWRQDMG

1219
GSFMSCEKN

1220
GSFQQMASL

1221
GSFWHNDAA

1222
GSHNTGAWP

1223
GSMHNSICY

1224
GSNDRLDSC

1225
GSTSMITHA

1226
GTADKPTGI

1227
GTASLDVLS

1228
GTCSMTLDN

1229
GTESLAVLS

1230
GTGFWWGCY

1231
GTIHMYCGW

1232
GTLEHTSST

1233
GTMPFWAYD

1234
GTMTCHYHH

1235
GTPQGKQRQ

1236
GTPWKEQFK

1237
GTQFAQGDN

1238
GTQVRHHDY

1239
GTQWQVSHW

1240
GTQWTQWMK

1241
GTSWELWNA

1242
GTVLKMNDI

1243
GTVQAIVCT

1244
GTWINEQMG

1245
GVADKPTGY

1246
GVEGHGQFD

1247
GVFYVGHWQ

1248
GVINMLASD

1249
GVMSCKVIG

1250
GVMSMSFPV

1251
GVQHYQLGM

1252
GVQHYWLGT

1253
GVQYGSQHF

1254
GVTTSNYCP

1255
GVTVCWSNQ

1256
GVTYHHRQC

1257
GVVATQRSG

1258
GVYDTLSSF

1259
GVYQQFHWF

1260
GWDWIMCNK

1261
GWFTYDKQQ

1262
GWHILPVME

1263
GWIFITGAD

1264
GWKHICVHG

1265
GWNNRDSGP

1266
GWNQHHSSE

1267
GWVQSMCMS

1268
GWYSLAVAN

1269
GYCMRLFGI

1270
GYCPCFHAG

1271
GYERADCWM

1272
GYESFLRDQ

1273
GYIPTQSMY

1274
GYISYGTQE

1275
GYKMMDCSM

1276
GYLSFMHNN

1277
GYMQTLEGL

1278
GYQNFLDYF

1279
GYTSMITHV

1280
GYVQQSKGE

1281
GYVWLGAMP

1282
HAALYTNQN

1283
HAAPGMIHS

1284
HACFNHFPC

1285
HACLMYWYD

1286
HAEELGDCY

1287
HAEIQGHQE

1288
HAELLGAFE

1289
HAHVFIWYA

1290
HAHVFIWYT

1291
HAILLDEMR

1292
HAISIWQTN

1293
HAKLFPTMI

1294
HASAMECIS

1295
HASGFMMQS

1296
HASTAIMCP

1297
HATPVCKEY

1298
HATTCDFDF

1299
HAYPILQMD

1300
HCAMTQRFD

1301
HCAVCQWQA

1302
HCETFMMQF

1303
HCNFDIPGA

1304
HCQPFTGHV

1305
HCSLYAQER

1306
HCSNVEFYQ

1307
HCSPNNCLE

1308
HCSPNNCPE

1309
HCVEFMQSN

1310
HDDAQLDNS

1311
HDEAQLDNS

1312
HDFDNPSMY

1313
HDGPSAKPN

1314
HDHTPLQSP

1315
HDNSAMWMV

1316
HDTMEWGCH

1317
HDTMEWGFH

1318
HECWQMTND

1319
HESMAWTLL

1320
HFCQYQSST

1321
HFDNYQRFC

1322
HFFHNMDYH

1323
HFGPTNCSM

1324
HFHQNFHFF

1325
HFMEMLING

1326
HFWMMECSG

1327
HFWVINGYE

1328
HGCPHFASG

1329
HGLTIGAAT

1330
HGSLLSTCH

1331
HHDSCYVVP

1332
HHESHLCGY

1333
HHQHVDNHG

1334
HHQWLRVME

1335
HHWPQTPLL

1336
HIANNFDWE

1337
HIAQCDVYL

1338
HIDDWAHGR

1339
HIDLMQMEY

1340
HIENKYAHI

1341
HIEVCTLGD

1342
HIFMDEMAY

1343
HIGYKDSLS

1344
HILIMQGVS

1345
HIQEELEEQ

1346
HISEFNRCT

1347
HISFCSEYW

1348
HIVQCDVIL

1349
HIYFQEAQS

1350
HKGTHAFVM

1351
HKYCRLSEE

1352
HLAPYCQQE

1353
HLDDIISFN

1354
HLGMWLAPV

1355
HLQVCNIYH

1356
HLSFNNWCR

1357
HMATLAHEA

1358
HMVIHAIPD

1359
HMVTLAHGA

1360
HNASNHECT

1361
HNEDLGDCY

1362
HNENTHWFN

1363
HNGCLMDVA

1364
HPHDTMKLT

1365
HPVCCLEQA

1366
HPYTAKWDV

1367
HQEVNPTPA

1368
HQEYFDANQ

1369
HQISVNNTL

1370
HQMEYSDFL

1371
HQMEYSDLL

1372
HQVQKYRMC

1373
HRDYFFTYD

1374
HSASGICCN

1375
HSCGTYYWA

1376
HSEMSNILM

1377
HSFEKKIPL

1378
HSVVVYEHA

1379
HSWINNQEH

1380
HTAQTYSTN

1381
HTEAHLSAC

1382
HTECDIQQK

1383
HTECKTIMH

1384
HTGCLLDVA

1385
HTIEGKHMN

1386
HTIHYGEAS

1387
HTINMPRWV

1388
HTMDYNTCR

1389
HTNQMWGMF

1390
HTNSYMEIE

1391
HTNYMQKEY

1392
HTQLKNCTG

1393
HTSFTNCEC

1394
HTSHQGHAE

1395
HTSLDSWFN

1396
HTTLCDPCG

1397
HTTNYGEAS

1398
HVAVNGFYD

1399
HVDGIWRDQ

1400
HVDQSLKYG

1401
HVELNMKGV

1402
HVEVPNTDA

1403
HVFQEGCCI

1404
HVGTDCMAA

1405
HVHQALTIS

1406
HVKVPNTDA

1407
HVLNGTGGS

1408
HVMPYMWVG

1409
HVMQYMWPG

1410
HVMRYYEPY

1411
HVNTYQLRG

1412
HVSAMECIS

1413
HVSPIQDNM

1414
HWLSCEKRW

1415
HWTLCDPEG

1416
HWVSTLADY

1417
HYAEITYRQ

1418
HYCPCHNKY

1419
HYDLSTCAL

1420
HYEWADHQW

1421
HYMQRKEWR

1422
HYTEVQKTP

1423
HYVALDAGS

1424
HYVMFQMSN

1425
HYVSMMLDE

1426
HYVVRHEDP

1427
IAAQPITNE

1428
IACYEPVGS

1429
IAEFLQCDY

1430
IAHNNFEPT

1431
IAHVVEQMG

1432
IALSATKDK

1433
IANVNNGTH

1434
IATKSGLYH

1435
IATLHMLSN

1436
IATWMWKYS

1437
IAYHTKSTI

1438
ICACFNKAY

1439
ICGFFCQQQ

1440
ICIQSNGRE

1441
ICLLEAGEK

1442
ICMQNAMSW

1443
ICMWKQYCS

1444
ICQHNGVAY

1445
ICREQIKWM

1446
ICRYALLHA

1447
ICSHSWFQA

1448
IDAWGHFSE

1449
IDDFRIVMH

1450
IDEQCKFGH

1451
IECSPREYI

1452
IEDVWECPY

1453
IEQQGSPLL

1454
IEYCQGMNF

1455
IEYYPTAEQ

1456
IFAHSNCCE

1457
IFALFDRYN

1458
IFENHDAAM

1459
IFILWQCKE

1460
IFLSKIPCQ

1461
IFNTRFLRE

1462
IFSSGGADA

1463
IFSSGGAYE

1464
IFTAKNEEM

1465
IFVIKQQCI

1466
IFVIYHQSG

1467
IFYAGDCTF

1468
IGAEQKVGI

1469
IGCLAVNHI

1470
IGGIIGNWG

1471
IGLSRNSNG

1472
IGQRIAGYA

1473
IGVERDYYA

1474
IGYQIMDME

1475
IHCDIQCQF

1476
IHEFHIAAM

1477
IHNFYYAST

1478
IHNHYTACQ

1479
IHQSFQQWE

1480
IHSQCQQGQ

1481
IHSYKDAHL

1482
IHTQTLISE

1483
IIAQQMRHH

1484
IIGSCQSFY

1485
IIHLPDHCR

1486
IILQNMKSW

1487
IIMQNMMSW

1488
IIPQKCSNP

1489
IISECQFGG

1490
IITCMDGYF

1491
IITMDLCTD

1492
IIWLGSIRN

1493
IIYHRTNAS

1494
IIYQLDCAT

1495
IKDHYIMQE

1496
IKQVFNQIA

1497
IKTNDQALL

1498
IKVQIDQIE

1499
ILAKWEVCE

1500
ILEQFYKMG

1501
ILERHLCGF

1502
ILHLEHPSD

1503
ILPFFCCGL

1504
ILRCVCDYP

1505
ILSFYWGDY

1506
ILTKWEVCE

1507
IMASVDICA

1508
IMCYKMCEF

1509
IMDEIHYMM

1510
IMDLITYAQ

1511
IMDMIYDCG

1512
IMDSRLTVA

1513
IMETADCHM

1514
IMGLGMRAD

1515
IMGLRMRAD

1516
IMGPAMNYD

1517
IMMANQLSC

1518
IMTPLAGGH

1519
IMTPLASGH

1520
IMTTENAAP

1521
IMVERWQHA

1522
INCQTQKEM

1523
INDHAQIVK

1524
INEDAQEKM

1525
INGSRACSY

1526
INMVSMYAY

1527
IPALKEGQI

1528
IPAPIHVNA

1529
IPDLHDHMG

1530
IPGTMSHTA

1531
IPLAMALWT

1532
IPMGWEFPE

1533
IPTMQWWNE

1534
IPWMERSAS

1535
IQEHLNACE

1536
IQLGCSAFH

1537
IQQQMSMCG

1538
IRDMFEQYT

1539
IREPTEPYC

1540
IRLMTPDEN

1541
IRLPMNSAH

1542
IRSMSNIME

1543
IRTLFNITA

1544
IRTNPWFHE

1545
ISDCVVWTY

1546
ISDLGIMWI

1547
ISEEIVSFE

1548
ISEGQAESR

1549
ISELFGAAF

1550
ISELFGEAE

1551
ISFGMEAMS

1552
ISGEGHQAV

1553
ISGHAQVQP

1554
ISGNAWGSL

1555
ISGWRMPCD

1556
ISNLLCWCP

1557
ISNPLYQVS

1558
ISQEYVSLQ

1559
ISQHNTFCS

1560
ISQVDWKCH

1561
ISRSKDAGM

1562
ISSGCWQFD

1563
ISVGQWEFI

1564
ISVIQPQFA

1565
ISYFTMAAQ

1566
ISYSSFDCH

1567
ITCSNHAMM

1568
ITDLLNVWG

1569
ITDQLCHSE

1570
ITEHVNFVT

1571
ITEVRQECA

1572
ITHKSETMM

1573
ITHSMGDNM

1574
ITHVSGSQL

1575
ITIYELDSG

1576
ITLASKSMR

1577
ITMGPEKWG

1578
ITMQSHFQH

1579
ITNKTFNDN

1580
ITNPFFLDN

1581
ITQDANLPL

1582
ITTQQAGQT

1583
IVAVNGFWD

1584
IVCESIQVH

1585
IVCTIAGTQ

1586
IVDYYMEGG

1587
IVHFGSSWK

1588
IVHWLSWMP

1589
IVIWQGEWI

1590
IVKSEGQKA

1591
IVLPENHLG

1592
IVMKGQEIT

1593
IVMNSKQMS

1594
IVMSCFDAD

1595
IVNHVPGRG

1596
IVQAHMMMS

1597
IVQMDGASF

1598
IVQRDGASC

1599
IVSKEVEAD

1600
IVTGHLQEG

1601
IVYCQGSSF

1602
IWRDSLHAN

1603
IYEWMQCSH

1604
IYKWDNKFH

1605
IYLHKDAFG

1606
IYLKAQQDP

1607
IYMSSVCHC

1608
IYPLQSFCN

1609
IYSHFHQGQ

1610
IYTRMDGYF

1611
IYVDLVDSL

1612
IYWFHPKGG

1613
IYYQIAEHF

1614
KAANDHHVA

1615
KACPYYDEW

1616
KADTEHNTN

1617
KAIHNMYGP

1618
KAREDLWRE

1619
KATVEFEWP

1620
KAYSLESDG

1621
KCALYEYEV

1622
KCDVLYTCQ

1623
KCEAGMFCY

1624
KCEELKTME

1625
KCEGHHYVE

1626
KCESGIFCY

1627
KCLEGVNFT

1628
KCWITEDAN

1629
KDHDPGNWQ

1630
KEMDSITCF

1631
KENAFENAA

1632
KENAFENAS

1633
KFELSSMIQ

1634
KFGVTDTSE

1635
KFMTCDLHN

1636
KFMTSDLPN

1637
KFNEETPFG

1638
KGISNWSFY

1639
KGIWQGMDA

1640
KGKSNWSFY

1641
KGPCIMQGN

1642
KGPRIIQGN

1643
KHKWMVYGA

1644
KHKWMVYHG

1645
KHNEMIHWY

1646
KIDVYDCIA

1647
KIEDMMAQE

1648
KIHDCDTQY

1649
KIHDCQTFY

1650
KIHESQTFY

1651
KIIWRAVAC

1652
KKEAKYRGY

1653
KKIMVKRAF

1654
KLAPWQQNC

1655
KLEHNMLNP

1656
KLEHNMLYP

1657
KLELIMQPR

1658
KLHWMCSFL

1659
KMAFGSAGV

1660
KMECFMDAH

1661
KMEMYAKEW

1662
KMESYDYSD

1663
KMQLSEGVP

1664
KMVDLQHSL

1665
KMWIDKYFH

1666
KMYYFYCNS

1667
KNGNTDTSE

1668
KNLTGVAHH

1669
KNWNPWDSP

1670
KPDEVHNLN

1671
KPMEWQQHH

1672
KPTLEWAHE

1673
KQDAKERIW

1674
KQDTEVLLG

1675
KQDVSKWFK

1676
KQDVSKWQC

1677
KQLICDVHH

1678
KQNDHTHMA

1679
KQNIEQVLH

1680
KRQAHNHYA

1681
KSCCIDHQM

1682
KSEPGFKNH

1683
KSGIMENDE

1684
KSGYGELSE

1685
KSSSSYIWG

1686
KSTGCPRGG

1687
KSYPIFNEG

1688
KTDQCNEAT

1689
KTEQKLTPT

1690
KTFLDVQSV

1691
KTKYSNWRM

1692
KTLPWYRPH

1693
KTSHCEAMG

1694
KTTHHWMTE

1695
KTTPPWMTE

1696
KVCSIVADH

1697
KVDMLDIVD

1698
KVEDVVLNE

1699
KVEIKQMDQ

1700
KVGMDQTAV

1701
KVPKRAYFE

1702
KVSQQEGIV

1703
KVTTETGVP

1704
KWDMICGYD

1705
KWDNIMLMN

1706
KWDYQMQMN

1707
KWEWSKCYL

1708
KWMPFDNTF

1709
KWPVMANLH

1710
KWTELCNLY

1711
KWYSFSFRA

1712
KYDTVAHWE

1713
KYGESNQCQ

1714
KYWISQDCA

1715
LAATSAQCQ

1716
LACKGTNPT

1717
LACYKDGGV

1718
LADRCQCCY

1719
LAEFEDGGF

1720
LAGLDSNTA

1721
LAGPNHDHQ

1722
LAHTYGRDT

1723
LAHVSTQFN

1724
LAIGAEIAL

1725
LAIHEATYP

1726
LAIREATYH

1727
LAISLVDSY

1728
LALPYDDFS

1729
LAMYSQYRG

1730
LAPPYTRSN

1731
LASFWLNPQ

1732
LASPSNESH

1733
LATEHMSWD

1734
LATHCYGGR

1735
LATNTYAYS

1736
LATVQTQAP

1737
LAVSHANFF

1738
LAWIETRDG

1739
LAYLLGHKE

1740
LAYYSLAFG

1741
LCAPCGMSV

1742
LCAQELYMC

1743
LCAVCQLQA

1744
LCAVWDKMD

1745
LCELNSAWN

1746
LCERFHEDT

1747
LCFLMIMCD

1748
LCFLMSNCD

1749
LCGWAPCSW

1750
LCMYSQYRS

1751
LCTLVGMES

1752
LCTMEGPYR

1753
LCTNMMDMS

1754
LCVGLSNCP

1755
LDLAHSIPA

1756
LDTKTLANS

1757
LECQYHNFQ

1758
LEDRISATF

1759
LEGSSQFQD

1760
LEHSSGFMN

1761
LEVTNCNAA

1762
LEWFCWIGS

1763
LEWVCWSGS

1764
LFAKPDALL

1765
LFAVHMGYY

1766
LFCNIGKTF

1767
LFELTSVQN

1768
LFEMTYRNW

1769
LFEYYRRMA

1770
LFGIAIWYV

1771
LFHNAQDDR

1772
LFHYIQRTS

1773
LFIKVKDTS

1774
LFKHCAHHN

1775
LFKHMYENA

1776
LFKPTQNEF

1777
LFLGKDAMM

1778
LFLHDNRFL

1779
LFLQLYRHC

1780
LFMPISSAV

1781
LFMPVQRIC

1782
LFMQNNNWM

1783
LFMQSVQQT

1784
LFNSYSTRE

1785
LFQKMTYEP

1786
LFQQTLRWQ

1787
LFQQYNSPF

1788
LFRVMYCQE

1789
LFRYKTSLW

1790
LFSFYNNTS

1791
LFSHELRYY

1792
LFSIMHAME

1793
LFSQNEACS

1794
LFTDTKRPE

1795
LFTLVQSNS

1796
LFVGMSCEL

1797
LFVQRYQES

1798
LFVYDLLFH

1799
LGELGNSFG

1800
LGERALRDL

1801
LGMDCWQWE

1802
LGNHFNNWM

1803
LGNPCNLGD

1804
LGSLCNCWF

1805
LGSPFYECG

1806
LGTLMSQHC

1807
LHFQIDAED

1808
LHHRVYQDA

1809
LHPTFWQEK

1810
LHQDIMMAG

1811
LHQLHIMQA

1812
LIALFMDYR

1813
LIATWVNAN

1814
LICLAQAWK

1815
LIDERFVQG

1816
LIKPFIDKA

1817
LIMMSNWQD

1818
LINHGQTSC

1819
LINQFIACT

1820
LINSTMVSG

1821
LISLAAYIE

1822
LISWKQTNS

1823
LIYSNIKAQ

1824
LKAPCQMDR

1825
LKDELVMLG

1826
LKGYKPSKF

1827
LKNVIHDGC

1828
LKSWFGQSP

1829
LKVEKVMLG

1830
LKWPVVNCD

1831
LLGYAQCST

1832
LLKDYMNST

1833
LLKTCDIFR

1834
LLMGFWMQD

1835
LLNKYENMG

1836
LLRFYGCCM

1837
LMEAASRSY

1838
LMLTYSDHY

1839
LMNRGEAND

1840
LMNYSGSSM

1841
LMQTFPQKC

1842
LMRQGVLFH

1843
LMSSTSAHY

1844
LMTGFMQMG

1845
LMTPQVKQA

1846
LMTTQVKQA

1847
LMVAKTWDC

1848
LMVFKQLAN

1849
LMVHGLVYS

1850
LMWMTPWDN

1851
LMYMIGTGP

1852
LMYMIITGP

1853
LMYQKDCAQ

1854
LNCHLTNST

1855
LNESHPPTD

1856
LNGHYTVQS

1857
LNKCCSLFY

1858
LNMDYNKCQ

1859
LNNACLSDE

1860
LNNAGYRPC

1861
LNVMSIHCC

1862
LPANGQLLA

1863
LPHAMQKMY

1864
LPHDKIKDM

1865
LPHFWGHSE

1866
LPHKMQNMY

1867
LPVAMQISH

1868
LPVDKIKDC

1869
LQAFYFHDQ

1870
LQARVGKVF

1871
LQDRCVCCY

1872
LQDTMMANE

1873
LQHLKKYMV

1874
LQITHNGWQ

1875
LQMTHNGCQ

1876
LQPWWLGMV

1877
LQSTYKMFT

1878
LQYSLGQQT

1879
LRAVWAKMD

1880
LRDKVDWYA

1881
LRELYTGMF

1882
LRIGVLSQF

1883
LRIPFGAAL

1884
LRMPFSNGV

1885
LRRECSTCL

1886
LRTLQIDSM

1887
LRTPHNHGL

1888
LRVDTNQQC

1889
LRVTHDVCS

1890
LRVTHNVCS

1891
LSACTVQNM

1892
LSAFVSHMK

1893
LSAKEELSG

1894
LSCKMTNPT

1895
LSEESGYCS

1896
LSFQMVDQQ

1897
LSFYYCGPH

1898
LSFYYPLHY

1899
LSGHTYQDN

1900
LSHETHMGN

1901
LSIPYQGLN

1902
LSKDCVAFR

1903
LSKDCVTFR

1904
LSLHYLHHH

1905
LSLNQKWVH

1906
LSNMVLAAD

1907
LSPLLDCGN

1908
LSPTFNHCE

1909
LSQAMSPGC

1910
LSQPINHQM

1911
LSQPYGAGD

1912
LSSIWYAAN

1913
LSSYDAKYH

1914
LSTMTQGSG

1915
LSVHTCQEQ

1916
LSVLAGLGG

1917
LSVQFNNQS

1918
LSWETKGCG

1919
LSWHCGEEN

1920
LTADPQERF

1921
LTAVKQEGY

1922
LTCNCNYWQ

1923
LTCSKHLQP

1924
LTEFIQAAQ

1925
LTEKGMLDC

1926
LTEKGMLDW

1927
LTEWLYQEM

1928
LTGWFNTRD

1929
LTHILDEFC

1930
LTISHNVSG

1931
LTLGMSSHE

1932
LTLGMSSHK

1933
LTLPFKPEY

1934
LTLTWGQTY

1935
LTMCYESNN

1936
LTMVGEYVN

1937
LTRFLNCQR

1938
LTRWNEEKE

1939
LTSFVDCVQ

1940
LTSRYNVTH

1941
LTSSKEFEH

1942
LTSYENWGW

1943
LTTHFVEED

1944
LTTMVKKAA

1945
LTTSLNGFD

1946
LTVGHYVYD

1947
LTVPNESAF

1948
LTVSKEFSH

1949
LTVWQQKME

1950
LTYLLGHKE

1951
LVADMYERS

1952
LVCMQGRSN

1953
LVDQSGDGV

1954
LVECSNLVA

1955
LVGERKNAY

1956
LVGWTLQHV

1957
LVITKMLQP

1958
LVKLATYTH

1959
LVLPVEFYM

1960
LVNLATYTH

1961
LVTTEMGTN

1962
LVTWAANEQ

1963
LVVEDWIRC

1964
LWAAVQIDH

1965
LWCPKEIER

1966
LWDTSMLKE

1967
LWEGLATCL

1968
LWEKCEFDQ

1969
LWFAITKER

1970
LWGSFGASS

1971
LWGWTLQHQ

1972
LWIKQHVKG

1973
LWKGYLKSN

1974
LWMNTGQYH

1975
LWNIYGTME

1976
LWNQQDWCR

1977
LWNTRGIYN

1978
LWQYSHAWQ

1979
LWTPCNHLM

1980
LWYQMLHGL

1981
LYCVWPAGT

1982
LYERYWVKQ

1983
LYFFMGTQA

1984
LYFGFPMSG

1985
LYFNMVQGL

1986
LYFRMGTVA

1987
LYGWVDLEG

1988
LYLQHQIDT

1989
LYRKIMSIV

1990
LYSYMLHDR

1991
LYVMKGIQV

1992
LYVQSHCAV

1993
LYWFYLEIH

1994
MAAEYESCS

1995
MAFFKSNLH

1996
MAGHYFNSR

1997
MAGQWCCPE

1998
MAHPDQQHY

1999
MAIEMPLHW

2000
MAKQEINWI

2001
MALPVTSIR

2002
MAMNQPEWS

2003
MAMPIHDRI

2004
MAQHAITTH

2005
MAQKREVGV

2006
MASPYTHVW

2007
MATCYMYAD

2008
MATHGNSKT

2009
MATVNLRDC

2010
MAVEFCQSD

2011
MAYCNHFSD

2012
MAYHSAQCI

2013
MAYPTQLIF

2014
MAYTPEFGN

2015
MCDDPRAWQ

2016
MCDEKFSYG

2017
MCDEKNSMG

2018
MCEPNQGMQ

2019
MCGPSLVYA

2020
MCHNYAGVD

2021
MCHPYFSDG

2022
MCLDSIMFG

2023
MCNGFCGIW

2024
MCNIHNIWD

2025
MCNYWITDC

2026
MCSVYQACM

2027
MCTADQKSI

2028
MDDICEFYA

2029
MDHPCQQHY

2030
MDNCPPMWN

2031
MEDLWTVGP

2032
MEFLIMADE

2033
MENEANIRG

2034
MESCKLWGV

2035
MEVPDFMSY

2036
MFDLYHDFK

2037
MFEKRNHWG

2038
MFEMQYCSP

2039
MFERMYVVN

2040
MFHAQIHCM

2041
MFNMFTEWR

2042
MFNMLTDSA

2043
MFQFMECLA

2044
MFTISGLHA

2045
MGAEHMCVQ

2046
MGARHMNQE

2047
MGATKSDDF

2048
MGDWLGAPT

2049
MGEGLSFYA

2050
MGGVANGSS

2051
MGHWGAEMP

2052
MGNHLCHYW

2053
MGNICNVMF

2054
MGPLLFLFD

2055
MGSMRIQTD

2056
MGSPAAAVG

2057
MGSQQIQTD

2058
MGTVEKICH

2059
MGVQLPIVE

2060
MGWHYGGWS

2061
MHDSCYVMP

2062
MHGPSLAYA

2063
MHHSVVAQF

2064
MHLEKNEMM

2065
MHMEVLWGH

2066
MHMLYKFNC

2067
MHMSGQSWH

2068
MHMSKQSWL

2069
MHMTATIFG

2070
MHMVKGGWT

2071
MHMVPGGWM

2072
MHNAHGIIE

2073
MHNYYEMCS

2074
MHQTLQCIG

2075
MHSFYGLSG

2076
MHTVYMESI

2077
MHVNFNEQR

2078
MHWFYNTGE

2079
MIAINGGEQ

2080
MIDLGVIDN

2081
MIFHSTGAA

2082
MIHHRDHSI

2083
MIHPLQSHY

2084
MIHQMQTLD

2085
MILLHNQGT

2086
MIMGYNFEF

2087
MIMQGVMQA

2088
MIMQGVVQA

2089
MINCTAELA

2090
MINCTAEVA

2091
MINFNSGTC

2092
MINFPSMIN

2093
MINWKAPRA

2094
MINWKAQCA

2095
MIQQMMFYT

2096
MIRGQDNWL

2097
MIRIMTGQY

2098
MISAPPDTC

2099
MITMFDGMC

2100
MITMYDGMC

2101
MIYSRGNQP

2102
MKDIKQVDC

2103
MKFPNHAEW

2104
MKGEMEQNA

2105
MKHPMVNNE

2106
MKMNYGLVC

2107
MKSEQQILA

2108
MKTTMVMAP

2109
MKWFVFDSN

2110
MLCKCNADT

2111
MLLLIPFAP

2112
MLSTYMPIS

2113
MLYVNMGDR

2114
MMAVIRQQS

2115
MMEQSNWHY

2116
MMGFKDISC

2117
MMHWIDMCT

2118
MMIPWIGIA

2119
MMLKQGPIY

2120
MMNPNTEPH

2121
MMNTWGFMP

2122
MMNYNDHLH

2123
MMPGETVRG

2124
MMQTIQADV

2125
MMSSNVLAE

2126
MMVKRDLND

2127
MMYISCSCT

2128
MNADVCLTG

2129
MNEMAQHDR

2130
MNEQYRHGV

2131
MNEYKGENY

2132
MNEYKHGNY

2133
MNGFTMTYH

2134
MNGRPEQMA

2135
MNGRPNQML

2136
MNINDETQC

2137
MNMMLMSNS

2138
MNMTMQYIT

2139
MNPLGPLLW

2140
MNRFTHNDQ

2141
MNSMKPNSV

2142
MNSWRNTSD

2143
MPHGWCNTI

2144
MPHHTSDPF

2145
MPNQYHSMD

2146
MQDFMFCIT

2147
MQDYWTLGA

2148
MQEKIHMCT

2149
MQELITFDE

2150
MQELITFGE

2151
MQHQNQSVC

2152
MQKTLCGSY

2153
MQNYLQHCE

2154
MQSLYETFG

2155
MQVMYEASE

2156
MRDLGVIWN

2157
MRELMGAYC

2158
MRIPSGVGQ

2159
MRNTFNYLC

2160
MSAARVYDL

2161
MSAECLHSD

2162
MSAIHDWGQ

2163
MSAIPTNGM

2164
MSAQVDCQF

2165
MSATLEAAF

2166
MSDDSTFDF

2167
MSDDSTVDS

2168
MSDLVPKWA

2169
MSDPCVDNH

2170
MSDPCVDNQ

2171
MSDQKEHMS

2172
MSDTCVANQ

2173
MSEPFNYCG

2174
MSERCYAGG

2175
MSEVCPNDI

2176
MSEVCSNDI

2177
MSFQKPKWS

2178
MSGMGDDFP

2179
MSHEYLFYG

2180
MSINMPLHW

2181
MSIPSFMQK

2182
MSLLIPLAP

2183
MSNNSVGSY

2184
MSQFKAYLF

2185
MSQLVHRCE

2186
MSQSTYHAG

2187
MSRHDLNWD

2188
MSVHFIYMY

2189
MSVHTWDHY

2190
MTAILQDNW

2191
MTAQIGHPI

2192
MTDPYAGAK

2193
MTDVHKHLG

2194
MTELKTFGE

2195
MTGEMIQNA

2196
MTGQASDLI

2197
MTGYGQHDA

2198
MTITHHVNY

2199
MTKHNMIVP

2200
MTLPWGQQY

2201
MTLTNMAYL

2202
MTMDENTCR

2203
MTMPVMINQ

2204
MTMTATTFG

2205
MTQRVVHWG

2206
MTQYLQDSV

2207
MTQYNKKVN

2208
MTRCEGLFT

2209
MTSHVQHRF

2210
MTVLICDYE

2211
MVALVDGHR

2212
MVAVYNRQR

2213
MVCPSNCAN

2214
MVCTSLRFK

2215
MVDFGNMQP

2216
MVEYCYTQE

2217
MVILSVETE

2218
MVKQGGDWR

2219
MVLKICTAK

2220
MVMPVNIVD

2221
MVMQDNASM

2222
MVMSKQADL

2223
MVNHHYMTG

2224
MVQMDVHWE

2225
MVSLITSRT

2226
MVTEAHDYY

2227
MVVQSPIVE

2228
MWDVAHQSC

2229
MWISRDQDH

2230
MWNPMYIWN

2231
MWREFTMQQ

2232
MWTEFNEYV

2233
MWWRESAMR

2234
MYADFQSSY

2235
MYCMKPNRQ

2236
MYDSTHAVQ

2237
MYDSVHAVQ

2238
MYGQQSLWN

2239
MYHEANSGF

2240
MYHSKMTEA

2241
MYHSYQNSD

2242
MYKLLNDFS

2243
MYKQMGMAD

2244
MYLRDHNGL

2245
MYRERRAGY

2246
MYVSYMTHC

2247
MYYHCITAQ

2248
NAAMQDINH

2249
NACFFQNDP

2250
NACMIERAK

2251
NACMIEWAK

2252
NACNAYGRG

2253
NACQNDLYW

2254
NADHRFDAV

2255
NADKIGMLD

2256
NADMLMNGS

2257
NADPQQYQE

2258
NAEANWEYQ

2259
NAELQQADI

2260
NAENYQRLY

2261
NAERYQKLY

2262
NAESEDMLE

2263
NAESRFAQM

2264
NAFSEYVYD

2265
NAGSHAIQS

2266
NALPCAFNE

2267
NAMLFHHAM

2268
NAMYSHFLY

2269
NANSHAIHS

2270
NANWLHTQI

2271
NAPPCAFNE

2272
NASLERKWN

2273
NASQYQLSA

2274
NATDMGISF

2275
NATTIHMWF

2276
NAVFVQNLR

2277
NAVLQQYQE

2278
NAYSPESDG

2279
NCAERMKFC

2280
NCAEYFDHC

2281
NCAIQLDQQ

2282
NCEPIPFFP

2283
NCIINHGRD

2284
NCLEVVRAC

2285
NCLPMPSLH

2286
NCLQIGMQF

2287
NCNIKEAMD

2288
NCPREVATT

2289
NCQNTTHYA

2290
NCQPFMDYH

2291
NCQPIMDTH

2292
NCQRVNWTE

2293
NCTLFQDSH

2294
NCVQSFAVD

2295
NCVSRKKFA

2296
NDFMGAPEP

2297
NDVIYIYQG

2298
NEAHLWGHV

2299
NEAILFAYH

2300
NEAPYSTWQ

2301
NESNPQNGW

2302
NFAQFTKFQ

2303
NFCKQGNTC

2304
NFDQIGGCR

2305
NFDQMKCAQ

2306
NFEQINPLG

2307
NFLSSCSSA

2308
NFNQSSSTM

2309
NFRHTDQCG

2310
NFTFQCGIH

2311
NFVHIQSGE

2312
NFVTSGQMG

2313
NFYMIQSSN

2314
NFYMSGQAS

2315
NGEQWHKGG

2316
NGGSYIPCM

2317
NGILMHTTF

2318
NGPSYISWV

2319
NGQHFDMVG

2320
NGTGVVECG

2321
NHAPLLAFW

2322
NHASTGAGT

2323
NHCPVQKAT

2324
NHCSMCCGN

2325
NHDEKLWAP

2326
NHDYVKQTS

2327
NHDYYCQTS

2328
NHELYAQVA

2329
NHGQLKFPV

2330
NHGSREIDI

2331
NHILKMEFW

2332
NHNCVEMHG

2333
NHPYDIGQY

2334
NHRVNYEMY

2335
NHSWVEMFG

2336
NHVAYYMAK

2337
NHYPLMVEW

2338
NIAANWMFD

2339
NIAGYWMFD

2340
NICNLNQVE

2341
NICTWNEAC

2342
NICVKNKLH

2343
NIELKTYFF

2344
NIERKTYAF

2345
NIFDWQFYA

2346
NIHCKLHWY

2347
NIICFNWQV

2348
NIIHLMDYA

2349
NIIQLMDYA

2350
NINCIQIGK

2351
NINHYNEGE

2352
NIRCGNDAS

2353
NISEPSQDY

2354
NISFGTPPN

2355
NISFGTPSN

2356
NITQMYKHI

2357
NIVSQTHQE

2358
NIYHESHCG

2359
NKFNGMHCG

2360
NKFWSIDTS

2361
NKNTHAAVW

2362
NLFLTNGMG

2363
NLFLWLMMD

2364
NLHPRCHWI

2365
NLKCIMIQC

2366
NLVPFQTCE

2367
NMMDLLTHC

2368
NMMDLRTHC

2369
NMMPRVCWQ

2370
NMNTFGKMN

2371
NMTMAHQAS

2372
NMYHVQRFS

2373
NNAIMWASP

2374
NNGPGNIGM

2375
NNGVHIFCE

2376
NNLPLLTSV

2377
NPDTMSHTA

2378
NPEMDLRCA

2379
NPEMDLWCA

2380
NPIPGYFDQ

2381
NPIVMNCLS

2382
NPQPWLGMW

2383
NPTVLMAHL

2384
NQEEGHCLY

2385
NQGPQMHGF

2386
NQHVIYDVT

2387
NQKAMCWMY

2388
NQNFLGNWG

2389
NQVLLDDGM

2390
NRCDFSLFE

2391
NRCFTTIEP

2392
NRCFTTTEP

2393
NREFQMIHC

2394
NREMIYAAF

2395
NRLVVNGPH

2396
NRVAKIWRW

2397
NSAKKEQGV

2398
NSCTDNSSS

2399
NSEHEGMWD

2400
NSEQKQHLQ

2401
NSFAFDMTE

2402
NSGIPNNLA

2403
NSGYECHAL

2404
NSGYEWHAL

2405
NSIPYQFAV

2406
NSNSQARVY

2407
NSQWVQMFG

2408
NSTYATWAC

2409
NSYQIGGTR

2410
NTDHKMWDS

2411
NTEHSPATN

2412
NTGLENNIP

2413
NTGYYNQQC

2414
NTHHVTQWH

2415
NTHQMAVVY

2416
NTIHRESPA

2417
NTMLKEMGP

2418
NTMPLQPME

2419
NTMYWWPRE

2420
NTNFYQQMS

2421
NTQLHDNFG

2422
NTSHGCTYY

2423
NTSMLNNGS

2424
NTTIYQQQP

2425
NVDGQCTVA

2426
NVDGRHPMT

2427
NVDHRFDAV

2428
NVDQKCQAN

2429
NVGYCKWQS

2430
NVISLLNCR

2431
NVNAPKKQM

2432
NVQFHMMMS

2433
NVQQQCNFQ

2434
NVSFTEIGW

2435
NVSGYAAIQ

2436
NVSWYAAIQ

2437
NVTHISVGT

2438
NVVHHPVAP

2439
NVVHVENIG

2440
NVWTCNYQP

2441
NVYMRTCNM

2442
NWAGVQRIM

2443
NWEMAQHDR

2444
NWFLTHPIL

2445
NWLVQHAFQ

2446
NWTQTQAEA

2447
NWYSTNTCF

2448
NYDFLHHVE

2449
NYELYKQVA

2450
NYGPKMLTP

2451
NYMQTTNDD

2452
NYNHFFAAD

2453
NYQSEMINW

2454
NYQSFGLFA

2455
NYYANGNWP

2456
PACYSDVLN

2457
PADLCDTCQ

2458
PCFFRHNYW

2459
PCPTAINQH

2460
PDDDFGSCE

2461
PDDYFGTCE

2462
PDECGFGLT

2463
PDMVNDWWY

2464
PDNQFSRVV

2465
PDQYLVIGD

2466
PDSPGVCSC

2467
PEFYVKKFD

2468
PEFYVQKFD

2469
PFAEKAPDV

2470
PGHSDKSSN

2471
PGLLIPNGK

2472
PHATYNKPT

2473
PHPDSAVQL

2474
PIIKGEPAD

2475
PIIKRVSWT

2476
PILNVMCAE

2477
PKHSDKSHN

2478
PKMIKQPVS

2479
PKQYWQHWF

2480
PKRIRYRTK

2481
PKRYQAWTW

2482
PKSCFQCTA

2483
PKWWPIQYD

2484
PLCCTHWGT

2485
PLQNWFSAQ

2486
PLRAECQWA

2487
PLRPECPWA

2488
PMSVTNSAY

2489
PMVHGLVYS

2490
PPDMQSAKW

2491
PPFKAAKYS

2492
PPMISHCEP

2493
PPPLKAPMY

2494
PPPPPPPPP

2495
PQTLWDKSG

2496
PRHTMLGPS

2497
PRNPLQVQN

2498
PRPLKAPMY

2499
PSYTMHACP

2500
PTAAFMQFM

2501
PTDGINISG

2502
PTEFHINVQ

2503
PTEKTHGPL

2504
PTHNLCMRV

2505
PTQFYGPAF

2506
PTRLTVYRC

2507
PTYPYEWFQ

2508
PVEVGQNQC

2509
PVHFWTQAK

2510
PVTHPQTSK

2511
PWRTRVGAR

2512
PWTEMPNKI

2513
PWTEMSNKI

2514
PYCWLSTAF

2515
PYKTQEGDG

2516
PYKTQEWYG

2517
PYQPLSCPP

2518
QACNITNSG

2519
QADNARSVH

2520
QADTLQHWD

2521
QAEGHFTNC

2522
QAEHRLNAD

2523
QAGFIQNNQ

2524
QAGGSGFNE

2525
QAGSSFHQA

2526
QAHISPSMC

2527
QAHTNMQVH

2528
QAHWWHAHH

2529
QAIANNAMQ

2530
QAIHMPLFT

2531
QAILRQNLP

2532
QAIPRENPM

2533
QAIPYSFCE

2534
QAIQVGMCH

2535
QAITGDRSN

2536
QALMLTRQQ

2537
QAMHKADHS

2538
QAMPNNMCC

2539
QAMTNGIAC

2540
QANFGMTQL

2541
QANPCSFGN

2542
QANPRNRTA

2543
QANSNIHQP

2544
QANWTECID

2545
QAPDQSYMP

2546
QASWHSVDQ

2547
QATLEWAHE

2548
QATLRSSCS

2549
QATMIHHDD

2550
QATNEHIFW

2551
QATVCHFHA

2552
QATWEGIRD

2553
QATWQCRYF

2554
QAVCSSNAM

2555
QAVFEDADN

2556
QAWNFTIFH

2557
QAWSYDNER

2558
QAYAYLNSS

2559
QAYQYRSCK

2560
QCANGYQSQ

2561
QCAPCDICC

2562
QCDFMIIEY

2563
QCDFRRKDY

2564
QCEPIPFEP

2565
QCEQDAQQE

2566
QCGEIHRAY

2567
QCGQQMKMC

2568
QCHHGYIYE

2569
QCHQNYGLI

2570
QCITGNAGA

2571
QCLQTNPYN

2572
QCMFRIDWD

2573
QCMFRIDWY

2574
QCNWIHNTM

2575
QCQHDMHCE

2576
QCQYPSHFM

2577
QCSGYHWMK

2578
QCSWHGVDQ

2579
QCTQMVCCG

2580
QCVEPPQNC

2581
QCVEPPQNW

2582
QCVKFMQYN

2583
QCVMDGCMA

2584
QDDLIYQTG

2585
QDFYIMAMG

2586
QDMSAWPDG

2587
QDVVNIEAA

2588
QEIPAMQQQ

2589
QETMIGQSF

2590
QEVFELDNM

2591
QEVRQFCSS

2592
QFAFRGSHI

2593
QFAIQEGAI

2594
QFCTSCASN

2595
QFEGQSKHE

2596
QFEGWVHNE

2597
QFGLINEHV

2598
QFHQQVIWS

2599
QFILTPRDF

2600
QFKNNWQIE

2601
QFLDNQQAF

2602
QFLEAPMMH

2603
QFLSMMNCF

2604
QFMQHPQMQ

2605
QFNPLQDQC

2606
QFNYVMIGD

2607
QFQWSMEHK

2608
QFRYMGHQI

2609
QFSEFVRWN

2610
QFSHIQAAP

2611
QFSPCVKMF

2612
QFTIHERID

2613
QFTYFNCWS

2614
QFVNWQRLF

2615
QGAMKQTCK

2616
QGDFDQTVL

2617
QGDPDQTTL

2618
QGEFLIGQS

2619
QGESRFMFQ

2620
QGFVVHSPS

2621
QGGFNYAAS

2622
QGGYASFHS

2623
QGHKTMHVF

2624
QGQHVTLAW

2625
QGTHTHWLG

2626
QGTTDMPMY

2627
QGVSTRCHC

2628
QHCQGLFWS

2629
QHCQGLSWS

2630
QHDFTSCYW

2631
QHFNMVQGL

2632
QHIWAPMGM

2633
QHLHSEWGS

2634
QHLHSMWGY

2635
QHMHTTMQY

2636
QHMPSKRLD

2637
QHQTIDEQF

2638
QHRMGFSWM

2639
QHSTYWTGV

2640
QHVATQNLG

2641
QHVSVGDCV

2642
QHWFVKCCV

2643
QHYHTCMQY

2644
QIAHCQYNE

2645
QIANEDTYA

2646
QIDIQMAWP

2647
QIELFMDMD

2648
QIFGIQFAG

2649
QIIPGGFKH

2650
QIKLSPMDP

2651
QIMQHPQMQ

2652
QIQCTMISQ

2653
QIQDFFTLP

2654
QISNWGFGA

2655
QITDNLFEY

2656
QITDWYQFF

2657
QIVYHLLNG

2658
QIYSLASTW

2659
QKCPINQPR

2660
QKGMMLDCA

2661
QKGPHNMSE

2662
QKIPFDHDA

2663
QKIYSWGPI

2664
QKQQGTCGN

2665
QLDTMMYTN

2666
QLELFNSAY

2667
QLEPQVNSD

2668
QLFCWVTSK

2669
QLFVWVTAK

2670
QLGGVEFYT

2671
QLHCVQMQS

2672
QLKEVMLQY

2673
QLMVAFLCL

2674
QLNQWMFAG

2675
QLSQSSTAY

2676
QLTAFCWQE

2677
QLTAFGVQE

2678
QLVWNQKQQ

2679
QMAEFEAEA

2680
QMDHRHTFE

2681
QMDHRHTVC

2682
QMEDTFQNS

2683
QMEQFGIPG

2684
QMFCKNTDM

2685
QMFIFQANN

2686
QMFLWYGHN

2687
QMGYREERM

2688
QMHICMYEG

2689
QMHIVHCTG

2690
QMHTGATWG

2691
QMHTQATNG

2692
QMKPLINDE

2693
QMMPAYHTE

2694
QMMYRDTSE

2695
QMMYWSVIE

2696
QMNQFGVGL

2697
QMNQLFCSS

2698
QMRDIRAMG

2699
QMRIQTQDP

2700
QMVQYQWWN

2701
QMVRFNNSA

2702
QMVSGDMED

2703
QMVSYEAYR

2704
QNAIVTQFA

2705
QNAPYMENA

2706
QNCFTYEDQ

2707
QNENFNTGC

2708
QNGHTGVAI

2709
QNICNQQQD

2710
QNICNQQQY

2711
QNILAGDCI

2712
QNKHLNGLM

2713
QNMLAGDCM

2714
QNNTTRGFT

2715
QNQSADVDA

2716
QNTPQHNFM

2717
QNVCINQAM

2718
QNYYYPPMH

2719
QPAMENTTP

2720
QPKALELVP

2721
QPYQQSQMD

2722
QPYQQSQMG

2723
QQAMWLHSS

2724
QQCIPLTWE

2725
QQECKHKMQ

2726
QQEQLDFSD

2727
QQFTIQDHK

2728
QQLGPKFEK

2729
QQQQTNFQN

2730
QQRFCCPWD

2731
QQSFSVDVN

2732
QQSFYCANV

2733
QQSNLNLGK

2734
QQSVCQVQN

2735
QQTMMEAFD

2736
QQVIANLAG

2737
QRCTWDQAY

2738
QREEANSGC

2739
QRGHGMDSG

2740
QRTTALKDQ

2741
QRVEVSKCR

2742
QRYVQEACP

2743
QRYVQEACS

2744
QSADEAACS

2745
QSADFMFMG

2746
QSAEWYLND

2747
QSAIFNTSH

2748
QSASQVLFP

2749
QSAVWNGCE

2750
QSCWFSHNP

2751
QSDNARSVH

2752
QSFPCMFKD

2753
QSFPHQQAQ

2754
QSFPMQSMQ

2755
QSGAFEKHQ

2756
QSGCREIDW

2757
QSGCTLQFP

2758
QSGNAWGSL

2759
QSHDFANCA

2760
QSHHFLNSD

2761
QSHPSVSNM

2762
QSHSKHDSK

2763
QSHSWVSNM

2764
QSICNQWCQ

2765
QSLNFNQHC

2766
QSLPRYGFD

2767
QSMGKAMNT

2768
QSMQKMMDF

2769
QSMWSAFKH

2770
QSNHFGAGV

2771
QSNLTEAGQ

2772
QSNQVIDQF

2773
QSPYGRKQA

2774
QSQGVVDTS

2775
QSQGVVINS

2776
QSRCLFACI

2777
QSSSSYLWG

2778
QSTDDNICH

2779
QSTHRVGSY

2780
QSTNMDFCG

2781
QSTPILGPK

2782
QSTPTILPK

2783
QSTTYWNAM

2784
QSVHRNTRN

2785
QSVTDNYFY

2786
QSWNVGFTS

2787
QSWSQVAFP

2788
QSWSQVTFP

2789
QSYIMGSFH

2790
QTADIETNQ

2791
QTAEFKRHQ

2792
QTAQIELYH

2793
QTAQINCSM

2794
QTDKPVYSA

2795
QTEEYKAVE

2796
QTELARENT

2797
QTERKFSAL

2798
QTFSTMGTD

2799
QTGMQWTGG

2800
QTGSAQSAF

2801
QTGTFGINS

2802
QTILDVQSV

2803
QTKDWGFVH

2804
QTKVLFERD

2805
QTLSRDDWC

2806
QTLSRLDIC

2807
QTMTQQYPE

2808
QTNMVDRIF

2809
QTNPLGCMC

2810
QTNTREGWG

2811
QTQMGQYAS

2812
QTRLWNNYC

2813
QTSEEQSDW

2814
QTSLTDSIL

2815
QTSPKFCMC

2816
QTSYSYADP

2817
QTTDMFQSF

2818
QTTPYVWVF

2819
QTTQETAQC

2820
QTTVEKICH

2821
QTVEWTTQQ

2822
QTVIQWHDW

2823
QTVKNVYSA

2824
QTWFKGFAC

2825
QTYQYRSCK

2826
QVEGSNMHW

2827
QVESRCMFQ

2828
QVEVGEKYH

2829
QVEYGNHDM

2830
QVFCKHEYT

2831
QVHAINDGH

2832
QVHHNMQVH

2833
QVHLPVEQG

2834
QVIPAMMKQ

2835
QVMHTLRTY

2836
QVNQGFKAS

2837
QVQSGQFIF

2838
QVSCVIMTP

2839
QVSQQEGIV

2840
QVTFVHCEN

2841
QVTHKWFAM

2842
QVTNQNKTR

2843
QVTPNFSCD

2844
QVVDMQDWC

2845
QVVPRYYSD

2846
QWAKMFADN

2847
QWAPIMPLS

2848
QWARWSVQD

2849
QWCESNSHL

2850
QWDCCVQDR

2851
QWDKVDGMM

2852
QWDLVGAVG

2853
QWDNKNQFP

2854
QWDTAQDCQ

2855
QWDYARQNF

2856
QWEAFEFIL

2857
QWEAFGHID

2858
QWEIYGKMN

2859
QWEKQNAWG

2860
QWENQPAWG

2861
QWHPKHSFA

2862
QWKHLHWTI

2863
QWMLKDSLK

2864
QWMQNQRYM

2865
QWMSKISDL

2866
QWNKNQNAP

2867
QWNLVGKVG

2868
QWPEYTTEA

2869
QWPHYTTEC

2870
QWPPTHDMG

2871
QWQDDNSQF

2872
QWQLTGHLN

2873
QWQRFNHGV

2874
QWQSFLTED

2875
QWRIMTQDP

2876
QWSQARSQY

2877
QWSQMDRMQ

2878
QWVMKHSYH

2879
QYAITYQEG

2880
QYAMFEKMS

2881
QYAMSFTQM

2882
QYDPYIQGD

2883
QYDQASDML

2884
QYFLSMDTE

2885
QYFQSNNQC

2886
QYFQTSNSR

2887
QYGLYKDTQ

2888
QYRSLDSDH

2889
QYRTPDEAI

2890
QYRTQDEAL

2891
QYVGMYTQQ

2892
QYVHMLGEC

2893
RAAGNSIDV

2894
RACMEGWPK

2895
RADNRCGQD

2896
RADSNKQFE

2897
RAEHVLNAD

2898
RAEPINHYN

2899
RAGMCENFG

2900
RAGMEPNFG

2901
RAIDCHRLN

2902
RANAESDQP

2903
RATLGASLH

2904
RATQWDSYP

2905
RAVQLEYCQ

2906
RCDSNDGQM

2907
RCDSVEVQY

2908
RCFFKMEAV

2909
RCIGKEDMT

2910
RCVAEPNEP

2911
RCVDLSMAS

2912
RCYEQHMWP

2913
RDDELLEPG

2914
REAYLNYVA

2915
REEMFVYMP

2916
REFELSIGH

2917
REGKVFDCY

2918
REMCSTGHF

2919
REQMVFRDQ

2920
RFADSKLHD

2921
RFDFLQSWF

2922
RFDLMLKDL

2923
RFDLQMGGG

2924
RFDMQHCFY

2925
RFDMSHQGR

2926
RFDNTTQYG

2927
RFDQFLMHM

2928
RFDQYTFLE

2929
RFEEMDFDM

2930
RFELLMEDH

2931
RFELTQNSL

2932
RFEMSHEQW

2933
RFEPSMKHC

2934
RFEPYMKIC

2935
RFEVSMREA

2936
RFIQMEITQ

2937
RFKMQFVTS

2938
RFSVRDYAP

2939
RFVPNCMSI

2940
RFYFTAEMN

2941
RGDKIHACF

2942
RGGLQFVEA

2943
RGHETYGPI

2944
RGKPTWTEK

2945
RGLQRVRTC

2946
RHDEQPFCK

2947
RHGFKDSGE

2948
RHGNFEAMA

2949
RIDDWAHGR

2950
RIDHAHFQW

2951
RIETPCHGS

2952
RIQQQEDLG

2953
RKKVEVVVE

2954
RKMWQSHCE

2955
RLNYRHYSK

2956
RMAEPVENE

2957
RMDTSSRAE

2958
RMDTSVRCE

2959
RMEGCWGCA

2960
RMKWYLPCV

2961
RMMWQSHCD

2962
RNAKEVPLP

2963
RNDVWEHVF

2964
RNLNLQTTI

2965
RNWLPENYT

2966
RPEAHPHYA

2967
RPEANKHYA

2968
RQDIFPHQD

2969
RRDCIIPMM

2970
RSAWMDYTC

2971
RSDFIAHMH

2972
RSEHFEHIG

2973
RSFMCGLIG

2974
RSGQGEGHM

2975
RSMLVERNT

2976
RSTPEAQQY

2977
RSWTACEID

2978
RTESWSSLA

2979
RTEWMKGVD

2980
RTEYKEGYF

2981
RTFMNDMCE

2982
RTFYVMGTE

2983
RTKYCSAFG

2984
RTSVEMCTM

2985
RVDAFNRGT

2986
RVDIGMGWK

2987
RVDPFPIAH

2988
RVDQRHLPT

2989
RVDVFSYCG

2990
RVGFMEGGM

2991
RVLDNPAEY

2992
RVMEYAAEW

2993
RVQMSCYTE

2994
RVQSGHQRG

2995
RVTMTSETC

2996
RVVQRYLPT

2997
RVWECVCSQ

2998
RWAEMHVQC

2999
RWDFVWYIQ

3000
RWDVDHQSC

3001
RWDVEQMVG

3002
RWDVMLEDR

3003
RWEAYGSQN

3004
RWEIRQMDM

3005
RWEPVQIFK

3006
RWHQGPCHP

3007
RWNTVFKID

3008
RYDEQPFCK

3009
RYDSQHQFK

3010
RYESFTKHP

3011
RYPCTIVEQ

3012
RYQPDPMLM

3013
RYTEMSCDD

3014
SAAAWWANG

3015
SAAEKAMDQ

3016
SADFLFKSF

3017
SADRLDLVL

3018
SAHHSHCNP

3019
SAINMHYIE

3020
SAKDPLQAN

3021
SALAHILSL

3022
SAMCSGSLL

3023
SAMDSLGVH

3024
SAMSVMDNM

3025
SAMTAIRMS

3026
SAMWFTANG

3027
SANNDAFLC

3028
SASHGGLAY

3029
SATCIPHSR

3030
SATDMDGAF

3031
SATFCGINV

3032
SATLHPLSL

3033
SATPHLYCH

3034
SATPHWYCH

3035
SAVASILNH

3036
SAVGGCWGA

3037
SAVGSHSSS

3038
SAWQSSVCM

3039
SCCFQIADR

3040
SCEQDGMDR

3041
SCFCMQHPI

3042
SCFLDHASG

3043
SCFRFTQAW

3044
SCGCMHEDF

3045
SCGCSLYQV

3046
SCGHSLGCF

3047
SCHTDGQWP

3048
SCKAHAAAP

3049
SCLAMDHVY

3050
SCLQMFSWH

3051
SCNFEFLWM

3052
SCNIGQYWP

3053
SCQHAIANQ

3054
SCQHPHDWE

3055
SCQLAIGHE

3056
SCQPVNEHE

3057
SCVQNMINC

3058
SCYSPIADR

3059
SDGMVHNCW

3060
SDLFKMFIE

3061
SDQPMHCWR

3062
SDQPMSCWG

3063
SDQPTMCNV

3064
SELYKCPYF

3065
SETICEWAP

3066
SFCKMCSFG

3067
SFCLFENIT

3068
SFDRGMWTV

3069
SFDRMMWTQ

3070
SFDTCNYFV

3071
SFEKYAGSD

3072
SFFYGTGSE

3073
SFGPLQNCG

3074
SFGTGCKCL

3075
SFHPLMHIA

3076
SFHPTPNDA

307
SFIGTNRDY

3078
SFIHMSHND

3079
SFLLCHQET

3080
SFLPIGIQE

3081
SFNTNQASV

3082
SFQSTEKCG

3083
SFSFYKCMQ

3084
SFSGIGPQH

3085
SFSKSFAAR

3086
SFSVFNCSH

3087
SFSVVQEQS

3088
SFSWIQSGD

3089
SFSYCDEPV

3090
SFWQIDDFN

3091
SGAGNNYHQ

3092
SGDSKHCHL

3093
SGEPMASCQ

3094
SGEPMTSCQ

3095
SGERWFVYP

3096
SGHMYQEGL

3097
SGISDHCHL

3098
SGPMSDMIS

3099
SGTGYVHGG

3100
SHASISHML

3101
SHASISHVL

3102
SHAVKHEQA

3103
SHDTWCVCT

3104
SHGFIDLQL

3105
SHLDFPVGR

3106
SHLWPNLHE

3107
SHMMQVAQF

3108
SHNNNPSGE

3109
SHQQYPAFW

3110
SHSCDGCGQ

3111
SHSSCCMTF

3112
SHTFINRHT

3113
SHTGVFKHY

3114
SHTQMELHN

3115
SIDSFMPIY

3116
SIEHWCCCQ

3117
SIFQIGQSP

3118
SIGQAWQSP

3119
SIHNAKEYR

3120
SIHPKQSRY

3121
SIIFLVSSG

3122
SIKYEDHGQ

3123
SILFNPNGR

3124
SIMPMNCGS

3125
SIMWRSDWT

3126
SINNALTIN

3127
SIQHCRDYY

3128
SIQLTNQCE

3129
SIQPCRDYC

3130
SISHMQARA

3131
SISSIEGYP

3132
SIYQLSWHG

3133
SKNTVGIGH

3134
SLADQVHVA

3135
SLDSIFACG

3136
SLGMDGMGV

3137
SLGNKLHIT

3138
SLRSYVDSF

3139
SLSEMRWAA

3140
SLSQYFESF

3141
SLTSSFAVL

3142
SLYNRYIHF

3143
SMAICMHDN

3144
SMDVTHFFE

3145
SMEPYIFGS

3146
SMFTHQLAQ

3147
SMFYVRLVC

3148
SMGYSEVFG

3149
SMHCSGYAL

3150
SMHHWAKGP

3151
SMHYYDTFE

3152
SMLQTTEQC

3153
SMLQTTSQM

3154
SMMCRCWLM

3155
SMNPNLTGD

3156
SMSFYQACH

3157
SMTNVPNLA

3158
SMWVPPNAA

3159
SMYMRVCYT

3160
SMYTMCTNH

3161
SMYTTEDIQ

3162
SNAMMFFCS

3163
SNDRVKKMD

3164
SNEANISVE

3165
SNMHYMRGS

3166
SNMPLMNMA

3167
SNSFYQTLG

3168
SNSHPFQSH

3169
SNSLYKKER

3170
SNWAIVKCD

3171
SNWQIVKTD

3172
SPISVNNTL

3173
SPLPMYNQE

3174
SPMFYGSAH

3175
SPNMFCEMQ

3176
SPYQYQLAE

3177
SQCVMQFPL

3178
SQGAGHCGN

3179
SQGPKHCGD

3180
SQIDLCVDM

3181
SQILEDLMG

3182
SQKQFSMQH

3183
SQKTICGSY

3184
SQMNCYGGC

3185
SQNMSVTQY

3186
SQNYHSMYA

3187
SQQHCQHDQ

3188
SQSFYFNHD

3189
SQTYVDMYA

3190
SRCMSSMCY

3191
SREYFATTF

3192
SREYFETTF

3193
SRGYRESDS

3194
SRKFCEGTP

3195
SRSPMMDMQ

3196
SSAAIYADF

3197
SSACYYAKG

3198
SSASSSEIC

3199
SSDCNRRCH

3200
SSEWLESAE

3201
SSFPEQQCH

3202
SSFYVQQCP

3203
SSGDSYQDY

3204
SSGHYNQLI

3205
SSGIMCADN

3206
SSGITAQCT

3207
SSHEICHDG

3208
SSHQNIGQP

3209
SSILMQMNC

3210
SSIQMQMQC

3211
SSISFQALG

3212
SSLYKFNHH

3213
SSMQQQLTW

3214
SSQHVNGVW

3215
SSQRITCEW

3216
SSQWSSGMY

3217
SSQYNTCEW

3218
SSTGEIQYG

3219
SSTGFDCEQ

3220
SSTGYPRGG

3221
SSTRDVCFC

3222
SSTSSSEIC

3223
SSVEFPWQT

3224
SSVPSMQCG

3225
SSYPESGWQ

3226
STAACTTWC

3227
STAHIIPDN

3228
STCHQWDAG

3229
STDDRMDAA

3230
STEQCGEWY

3231
STEVRWTGA

3232
STGWSAIAN

3233
STHQPCAQN

3234
STLQNIQTH

3235
STLTPVKLE

3236
STMQVIDIS

3237
STNEVWASM

3238
STNHVDDPL

3239
STQNNDMYC

3240
STRLIEQFG

3241
STRVMEQFG

3242
STSPYHRQN

3243
STTHRDNYE

3244
STTPQMHVS

3245
STTRFNFWN

3246
STWMSWEAI

3247
SVADQVQVA

3248
SVAQRPEMI

3249
SVAQRSEMF

3250
SVCQTTQHQ

3251
SVDLGTMEQ

3252
SVEQMRKIP

3253
SVHMLDDFM

3254
SVIPKHKNS

3255
SVIQTEHFD

3256
SVLCLSMGG

3257
SVNTMWYPD

3258
SVRMFDAQY

3259
SVRVQTWIG

3260
SVSPCFCNS

3261
SVSYESTIF

3262
SVTHDLHSF

3263
SVVFSAAEV

3264
SVVVVQTGK

3265
SVYMMTEGW

3266
SWAMISTCS

3267
SWDHLFTDQ

3268
SWISKGHCE

3269
SWLFSQDLK

3270
SWMWITNPN

3271
SWQSFILCE

3272
SWSERCSFE

3273
SWVKQDTMA

3274
SWVMLLPIH

3275
SYCHIMGVQ

3276
SYDAEQRNG

3277
SYESFRSMH

3278
SYGVIMTEE

3279
SYNPWNQHA

3280
SYQFAQYWL

3281
SYQRWDSCD

3282
SYSPCFCRS

3283
TAAFAYKYE

3284
TAALFEGSK

3285
TAAQKFMAN

3286
TAAYGDRYD

3287
TACDFISLT

3288
TADEFSCCQ

3289
TADTRVNHY

3290
TAEAFQKFI

3291
TAENIQTHN

3292
TAEVRWPCP

3293
TAFECMICG

3294
TAFECMIDG

3295
TAFNAQCGQ

3296
TAGGSDAEK

3297
TAGRFNYFD

3298
TAHLETDQN

3299
TAHLHMGNI

3300
TAIPMNELV

3301
TAITGFNIH

3302
TAKAQNAFE

3303
TAMASMWLS

3304
TAMHSAYHS

3305
TANKCDCSD

3306
TANVYRSGQ

3307
TASHCSFNQ

3308
TASMLMMTP

3309
TASNGCINS

3310
TASPMVEHW

3311
TATFSDFGF

3312
TATLSMWLS

3313
TAVEKNTLW

3314
TAVKLEQGS

3315
TAVNYWNTS

3316
TAYAFDCNC

3317
TCAPSSSMD

3318
TCCMWTFIW

3319
TCCSFSFYS

3320
TCCTVERES

3321
TCDHASDIE

3322
TCDYNVDTT

3323
TCEAAQAQH

3324
TCGKAMEQY

3325
TCGLWKNLV

3326
TCGTTVRCG

3327
TCIHVAGVP

3328
TCISNHHTC

3329
TCLPNPMQL

3330
TCQQMGDIG

3331
TCQYEMVCD

3332
TCQYVMLCD

3333
TCTSGGSWL

3334
TCVEFMQWN

3335
TCVFTDHQC

3336
TCYFFGEQE

3337
TDAGMFAHD

3338
TDDPIHNFE

3339
TDEQCKFGH

3340
TEGPMHRAW

3341
TELSYDAFL

3342
TEQFCGIML

3343
TESMVMYER

3344
TFCCMDMIN

3345
TFDLMQAIR

3346
TFDPFTTSQ

3347
TFEPCQSGV

3348
TFHQSIVGE

3349
TFISLQQSS

3350
TFLMLVDDQ

3351
TFNIAYVQS

3352
TFSQEGVSI

3353
TFVIVDDQN

3354
TFWSSSNYE

3355
TGGMYYVQN

3356
TGKWLVYQA

3357
TGLFIRTMK

3358
TGLLIPNGK

3359
TGNDHHEVI

3360
TGNYVHSNQ

3361
TGRIKDAGP

3362
THAQIGTFD

3363
THAVTGILY

3364
THECYQMWH

3365
THEMVNENG

3366
THGYMQSHN

3367
THMNVVRSF

3368
THNTVPVER

3369
THQNTWLFY

3370
THRHSDMGF

3371
THTQYWAMQ

3372
TIAVIGSAA

3373
TICKTEIGH

3374
TICNCMFGA

3375
TICQTEFGH

3376
TIDGMMGDH

3377
TIEAKWECM

3378
TIEANWEYQ

3379
TIECEQRET

3380
TIEPGCQCT

3381
TIEWKLHMG

3382
TIFYYGGNI

3383
TIGEMVNPS

3384
TIHQSQFIC

3385
TIHSSVSAS

3386
TILCDQQQA

3387
TIMNWNDAK

3388
TIMSDGHTH

3389
TINLTTQSV

3390
TIRLTEQSV

3391
TISELQSEF

3392
TISIFTNCR

3393
TISLEYCTF

3394
TISWLGGCH

3395
TIVNMQMGN

3396
TIVWMCVNW

3397
TKEPCTVGV

3398
TKEPTTVGF

3399
TKFHMMIQT

3400
TKNCVASTF

3401
TKQFELKDE

3402
TKQFETKSE

3403
TKQHYDSPA

3404
TLCGLDSEY

3405
TLCGNTQCP

3406
TLEGSDSEY

3407
TLNNLHRVA

3408
TLPLFWRLK

3409
TLQMVMNKG

3410
TLRDSECYS

3411
TLTYYEEGE

3412
TLVPCQTCE

3413
TLVRGMHQI

3414
TLVRSDYDP

3415
TLVRSDYYP

3416
TLYMHEKLG

3417
TMANLCWRT

3418
TMCPHGIRC

3419
TMCPKMWQH

3420
TMDQGHCMI

3421
TMIHHQNAD

3422
TMLHSQNME

3423
TMMCDQQQA

3424
TMPPFPCHM

3425
TMPYSQWAM

3426
TMSDRQQDT

3427
TMSGYCCSF

3428
TMSVHYGQG

3429
TMTSTLQST

3430
TMVAGKMRC

3431
TMWEDNQNM

3432
TMYFNANTQ

3433
TMYTKTSMF

3434
TNEGVLHHT

3435
TNHELAEKP

3436
TNHELAMYP

3437
TNHPKCKKD

3438
TNIQKAMGA

3439
TNKTLPATF

3440
TNLVYQFLE

3441
TNQSQNCMF

3442
TPAMEISMF

3443
TPCISHCEP

3444
TPEEFTMSP

3445
TPGTLQGHH

3446
TPLMMCQAS

3447
TPNSFNCAS

3448
TPTLESNCR

3449
TQAECITVP

3450
TQCHDMWGH

3451
TQDFCSVCD

3452
TQECYYDQF

3453
TQEPIHHQN

3454
TQFPVEYMS

3455
TQGFYNVGV

3456
TQQNCMTGN

3457
TQSGYHFWC

3458
TQTILNCGY

3459
TQVLMEMCT

3460
TQWTEKRQA

3461
TRCFIDQGI

3462
TRIEQEWNH

3463
TSDASVQQS

3464
TSDMTMENQ

3465
TSDWWCQKC

3466
TSEGGIIYN

3467
TSFGISWLH

3468
TSFNMICNS

3469
TSGQPGISW

3470
TSHINIWGQ

3471
TSHWSAVQM

3472
TSHYITFTP

3473
TSLGQWTHF

3474
TSLHSSMGH

3475
TSLHYQFFA

3476
TSMSFDSTH

3477
TSNFEPFSP

3478
TSQCAHQWS

3479
TSQRNIEAG

3480
TSQRNIEVA

3481
TSQWLMNMQ

3482
TSSFMDCVD

3483
TSTIEELAQ

3484
TSTMAGPCI

3485
TSTQRQDVF

3486
TSTRLYPVD

3487
TSVMCNQTR

3488
TSVTIQFWE

3489
TSVWWCKKE

3490
TSVWWCQKE

3491
TSWIQTWGS

3492
TTAIRDLTM

3493
TTEKYWEHR

3494
TTFIIPIPS

3495
TTFRNINPA

3496
TTGPNPNCY

3497
TTGWRYQTS

3498
TTHQLAVVY

3499
TTKTCAEGG

3500
TTLAHILSL

3501
TTNGYMSMD

3502
TTNLPQPEM

3503
TTNQWMNYY

3504
TTQFCGSML

3505
TTSTSIIYH

3506
TTTGINYHH

3507
TTTIYQQQP

3508
TTVDNGFHS

3509
TTYQSGWHG

3510
TTYYCPTSQ

3511
TVAPHWIDH

3512
TVAPSNCAN

3513
TVDMLHMAS

3514
TVDQFDKYN

3515
TVDQYCVRA

3516
TVDSEVNAD

3517
TVENMPTYP

3518
TVENNYGGY

3519
TVKMIHVDW

3520
TVMHDHCDN

3521
TVMLESNCR

3522
TVNAESKGC

3523
TVNHWQQIC

3524
TVNLCDIYK

3525
TVNQSVADM

3526
TVQQDGAQM

3527
TVQQDGHQM

3528
TVTHMSQHC

3529
TVYKNEQVA

3530
TWAPSMYWM

3531
TWCQIQGMG

3532
TWDSCQDFY

3533
TWDSYIQRD

3534
TWEHCQYFE

3535
TWHETDDFI

3536
TWIEQEWNH

3537
TWIQEGHYG

3538
TWIQHMGTF

3539
TWISSQHCH

3540
TWMHMMSYR

3541
TWNCAGLLP

3542
TWNMIGQLP

3543
TWQFYQHGC

3544
TWSFMGCVD

3545
TWSHCSYFE

3546
TWSNYQWYQ

3547
TWTEQEWNH

3548
TWVTTMAMQ

3549
TWYQIEGAA

3550
TWYQKTGAA

3551
TWYTKRTEG

3552
TYDNEMSET

3553
TYDQMQTEF

3554
TYEQSQVLR

3555
TYEWYMGDW

3556
TYFLLHASG

3557
TYGKAMEQY

3558
TYGQWMEKA

3559
TYISNQQSS

3560
TYKTHTIGG

3561
TYLFSNQSR

3562
TYLYRKTVY

3563
TYPLAWLKQ

3564
TYSPMSGIE

3565
TYSPMSGIV

3566
TYSPMSGKA

3567
TYSPMSGKD

3568
TYSQMQTFF

3569
TYSSKYQGD

3570
TYSSWYQGE

3571
TYVKFLVHP

3572
TYVYNHKAF

3573
VAANTYCYG

3574
VADSSWNQP

3575
VADYTACPM

3576
VAGNATPSL

3577
VAGPMWVQT

3578
VAHCHVATE

3579
VAHPDMKYI

3580
VAILGLNPT

3581
VAIPVDYHT

3582
VAIYKTECI

3583
VAIYPGIIE

3584
VALIIDEWT

3585
VALSRNSIG

3586
VANLDYDPS

3587
VANPINRTA

3588
VANQNYAIC

3589
VAQSIQEVA

3590
VARPTMEEM

3591
VATALMMSL

3592
VATHIQVMH

3593
VAVVAEHCA

3594
VAYAFQCNC

3595
VAYLIQDAL

3596
VCEQWNEHL

3597
VCETIPIKE

3598
VCMLHVNCE

3599
VCMLHVNFE

3600
VCQRILELR

3601
VCRQMNDER

3602
VCTLIQDAH

3603
VCTPIIDCS

3604
VCVPIIHET

3605
VCYNADVPG

3606
VDAWDNTAC

3607
VDTHMNQKQ

3608
VDTLNQDAH

3609
VDTVWMLPG

3610
VEMQANKGL

3611
VESPVLTMG

3612
VESPVYNMG

3613
VFEGGFPYI

3614
VFFSVYQDK

3615
VFGIMPCGF

3616
VFGIMPCWQ

3617
VFHSMYNSM

3618
VFNKAIEWK

3619
VFNLAQNWH

3620
VFNPYIDVG

3621
VFNTFDESM

3622
VFNTFDQQM

3623
VFRSASNKH

3624
VFRSVDVVD

3625
VFRVKCAAD

3626
VFWMQLRDH

3627
VFYLSSQSC

3628
VGDHKNCMN

3629
VGEKTHGPL

3630
VGEPHFHSD

3631
VGGYSNQWV

3632
VGHWKVQSC

3633
VGHWKVQSY

3634
VGIMRNDTG

3635
VGIQTQSCF

3636
VGKGIEKVQ

3637
VGLALNCES

3638
VGNPYHHGR

3639
VGQGIEKVQ

3640
VGQHIWAQD

3641
VGQREVPSD

3642
VGRWHYCTE

3643
VGTSFACSN

3644
VGVQCSNGG

3645
VHAPNNLVA

3646
VHDKWNAHT

3647
VHDMCQWSD

3648
VHELFMDMD

3649
VHKCADYMC

3650
VHQPTQMHH

3651
VHQPTQSHH

3652
VHSFSEVTM

3653
VHTTYCQRE

3654
VHYRLCEHW

3655
VIDRLMACS

3656
VIEFFKAQC

3657
VIFSMYTCF

3658
VIGLCMGIV

3659
VIGLCTGIV

3660
VIIEEQDNS

3661
VIKHALAEI

3662
VIMYWHHCS

3663
VIQSYQAVF

3664
VKESDNWQQ

3665
VKFAWQKIN

3666
VKMEYQGGR

3667
VKQLRQDGF

3668
VLAMRVRHK

3669
VLSNWEVAA

3670
VMAPYMWDP

3671
VMDCVNAYW

3672
VMDFYMSGP

3673
VMDQYKLKV

3674
VMEFYKAQC

3675
VMEFYMSPP

3676
VMEHWGSQY

3677
VMEWWGSLY

3678
VMGNSMCLF

3679
VMGPCMNYD

3680
VMHINEQAM

3681
VMMHYGGFY

3682
VMQGFNYGF

3683
VMQMINLAH

3684
VMSDEWQTI

3685
VMTICQSQE

3686
VNADITFGA

3687
VNGREPRWK

3688
VNGREPRWQ

3689
VNITYTLQP

3690
VNMMCGHES

3691
VNRIRDMSV

3692
VNVDCNVQQ

3693
VPEISWSNH

3694
VPICCNLPM

3695
VPLGFNFAG

3696
VQHMSQSDY

3697
VQHMSQSGT

3698
VQHQEEFQT

3699
VQIRTIRQE

3700
VQKQEHAKY

3701
VQMWTIRQE

3702
VQNWKLDKI

3703
VQQFCFQHQ

3704
VRIQFSQAL

3705
VRQMIDHLA

3706
VRSGQQAIF

3707
VSAAWGQME

3708
VSAFGHKAG

3709
VSASFQQHV

3710
VSAVVGQME

3711
VSCEQGKIK

3712
VSDLWQSQE

3713
VSEHCQEGN

3714
VSEHCQEVF

3715
VSEPFEHCD

3716
VSEPHFHKD

3717
VSERDKIYG

3718
VSHNQIYGM

3719
VSLGTHAHH

3720
VSMNTPFCL

3721
VSMWYDRDD

3722
VSSAMTWMT

3723
VSSQYNHLE

3724
VSTRFPMAQ

3725
VSTRMYACS

3726
VSVNEDRAG

3727
VTAATGAST

3728
VTAPSNLVA

3729
VTCRQEGGG

3730
VTDQIWVHN

3731
VTDRADQNY

3732
VTDRIEQYH

3733
VTEHCVRQP

3734
VTEIRPSFG

3735
VTELNMAAW

3736
VTEPLEHCF

3737
VTESWNEHL

3738
VTGFGPATP

3739
VTHLSASSD

3740
VTIPVDYFT

3741
VTNVCQCEA

3742
VTQFCLSGK

3743
VTQSIQEVA

3744
VTSCGQENC

3745
VTTGMQIEE

3746
VTVAVQPCS

3747
VTVNGNAGW

3748
VVCDMLTSL

3749
VVCQPVPCV

3750
VVDNPMMGQ

3751
VVGLEGACK

3752
VVHEITINS

3753
VVHIEWRPT

3754
VVIPMPCCS

3755
VVKCKPQFC

3756
VVPPCMDCG

3757
VVSARVNVQ

3758
VVTAEFEFV

3759
VVVLEGSCK

3760
VWEIFDAMS

3761
VWSGQQAIF

3762
VWVCQHWVQ

3763
VYDEWTKNV

3764
VYEQMAYCP

3765
VYGHMQNWF

3766
VYLPYYWQF

3767
VYPDSAVQL

3768
VYQHWWRVH

3769
VYQHYFVDQ

3770
VYSFFYQNE

3771
VYTMRSALE

3772
VYVLASAQK

3773
VYYCEIREH

3774
WADDKKHYG

3775
WAECRYQTT

3776
WAGVEQRGE

3777
WAGWIPSSI

3778
WAIPMPHCQ

3779
WCCRPINAN

3780
WCDSVEPPG

3781
WCEHVIMNP

3782
WDAYYTNGI

3783
WDGQKMWYC

3784
WDSPKNFWI

3785
WDVRDHAFY

3786
WEVDVCNDR

3787
WEWNAKHKC

3788
WFEHASHVF

3789
WFKGVDHRQ

3790
WFMYLTNAL

3791
WFQTMDHVM

3792
WFYRHNHVE

3793
WGDARMIGP

3794
WGDKQATDR

3795
WHDYQLAAE

3796
WHFKIHVQP

3797
WHFLMIFYI

3798
WHGFGKWSR

3799
WHSHYLLHH

3800
WHVSHWPKL

3801
WHYPWDDWH

3802
WIGSKNSCE

3803
WITSKIKSD

3804
WITSKINSD

3805
WIYPFSFGL

3806
WKAIVLEYW

3807
WKWWADYLA

3808
WLFMHRWAD

3809
WLIEIERHG

3810
WLPLCFPCA

3811
WLPLCFPWA

3812
WLPMVMNKG

3813
WMEYINHEA

3814
WMMQYIIFP

3815
WMSEQMFKG

3816
WNNVELSTP

3817
WNQHKRDQR

3818
WNRELQPPG

3819
WNSCHQTWI

3820
WNYWLLPDD

3821
WPDSESEEV

3822
WPHTMDHVM

3823
WPKQGNAME

3824
WPQQGNAME

3825
WQAVIYANQ

3826
WQKLQGNRG

3827
WRFCIKIGM

3828
WSQSVAKML

3829
WSTGCMQGM

3830
WTANYDYYE

3831
WTCWLLGWF

3832
WVAHTTGHA

3833
WVEIKVMDQ

3834
WVYSLGRAE

3835
WWNHWLWNG

3836
WWRASHRDD

3837
WWWLSTGNQ

3838
YADMFQCEK

3839
YADMFQCVK

3840
YAGPNMHWG

3841
YAHLLNQQA

3842
YAILHQTSK

3843
YAITQSCFT

3844
YAITQSRST

3845
YAIWNMHFT

3846
YALQITRSQ

3847
YALVMGIHN

3848
YAQLMYIDP

3849
YASPQSGAW

3850
YATERYCVC

3851
YATSMHAHK

3852
YCCNQEPFA

3853
YCEPVQDNF

3854
YCISLIYHH

3855
YCQPVDQFY

3856
YCYLSSASC

3857
YCYVCQMTT

3858
YDNSVGHSL

3859
YDTNQIVCP

3860
YEDNNVMQI

3861
YEGRPSDAQ

3862
YESCNLTRG

3863
YFDWFYNHM

3864
YFDYHERKG

3865
YFGTSLRAQ

3866
YFHWHDCIG

3867
YFTTMHVMG

3868
YFWSNHFFR

3869
YGNLMGVGP

3870
YGTLCGVGI

3871
YHAPMLMCN

3872
YHIWCVYWA

3873
YIELRHREA

3874
YIFACGVKG

3875
YKPNYEIME

3876
YKQRISHWL

3877
YLGMWLAPV

3878
YLIPWPRLG

3879
YLMPHWFII

3880
YLSDMCWRG

3881
YLSFMCAIQ

3882
YLTLQGMQE

3883
YMDPVVAMN

3884
YMDRGNIGE

3885
YMERGNIGE

3886
YMSNIIHHN

3887
YMTTQTGYP

3888
YMTYIQSQE

3889
YNKQMMNCP

3890
YPAPYYPAE

3891
YPIQCSVSF

3892
YPYQGHNWT

3893
YQAHYMSMT

3894
YQDIHQCIF

3895
YQFCKPSDD

3896
YQFCKPSDE

3897
YQGEVERAF

3898
YQGYEPHMH

3899
YQHHYHLPW

3900
YQMQMTYVW

3901
YQQQMFERQ

3902
YQSQGIWIE

3903
YQTLCMHQD

3904
YQTVFTLYF

3905
YSAHWCKGD

3906
YSGVRVTGY

3907
YSKHQMCID

3908
YSNSTIMNL

3909
YSQFFSAEE

3910
YSQLMHGGH

3911
YSQSWNRLG

3912
YSQSWNRLW

3913
YSSELECQW

3914
YSTICNFSI

3915
YSTIHAGHP

3916
YSTTMHVMG

3917
YSVSFGCQS

3918
YTDPVLHNP

3919
YTGMSCTMK

3920
YTHEESAHF

3921
YTKTYVDCF

3922
YTQHLVGQY

3923
YTTFTEHWQ

3924
YVAEAHCSY

3925
YVCPSVNWN

3926
YVEDRNGWH

3927
YVENRNGWH

3928
YVHDHLHQQ

3929
YVNIHNRHG

3930
YVNIHNRNG

3931
YVNKDEFRF

3932
YVSVTLQNS

3933
YVTASWHGF

3934
YVYIRKNAL

3935
YWQSMLWQG

3936
YYCLRFVLE

3937
YYTQFSSDY

In some embodiments, the engineered AAV VP capsid polypeptide comprises a polypeptide sequence represented by the formula: (A)-(X)—(B) wherein: (A) is the polypeptide sequence of SEQ ID NO: 5812 (VAYNVGGQMATNNQSSTTAP, corresponding to residues 561 to 580 of SEQ ID NO: 2); (X) is the 581-589 region having a polypeptide sequence selected from the list of polypeptides in TABLE 3 (SEQ ID NO: 3938-SEQ ID NO: 4237) that is expected to confer CNS tissue tropism or preference on a recombinant AAV virion (rAAV), based on a primary screen; and (B) is the polypeptide sequence of SEQ ID NO: 5813 (IVPGSVWMERDVYLQGPIWA, corresponding to residues 590 to 609 of SEQ ID NO: 2); and wherein the capsid polypeptide is capable of assembling into the rAAV and, the capsid does not have the sequence of any of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, or SEQ ID NO: 8. Also encompassed herein are rAAVs composed of engineered AAV5 VP2 capsid polypeptides and engineered AAV5 VP3 capsid polypeptides comprising a 581-589 region having a sequence selected from the list of polypeptides in TABLE 3 (SEQ ID NO: 3938-SEQ ID NO: 4237) at the region corresponding to amino acid residues 445 to 453 in AAV5 VP2 or the region corresponding to amino acid residues 389 to 397 in AAV5 VP3.

TABLE 3

Additional Sequences of 581-589 Regions

SEQ ID

NO
Sequence

3938
EAKWSGLGR

3939
QAYFYLMSS

3940
LFRQNQQFY

3941
QWRGYLSKA

3942
SFGWFDAHI

3943
DANQYHVSY

3944
QAFTNLDQM

3945
LGVYNQSFE

3946
HQSFVQCVH

3947
QYQSQWMSF

3948
HYSQYQNFT

3949
GNQQVNQQM

3950
LFNSMHQFD

3951
QASVYFWQQ

3952
RQSWVFVNH

3953
DAYKVQCYC

3954
EFMTSMTMM

3955
QFALINFDQ

3956
DASMIIMML

3957
QFSLVSLEA

3958
QHLFSNIIP

3959
TWFDMVMCD

3960
HAEYKDAGS

3961
QNSFYSQMG

3962
ELQQIYCAG

3963
SFEHLDCLL

3964
FYAFNMSDN

3965
LSWMQYEFL

3966
QSFNYLFYG

3967
RWQFIHLSK

3968
TFLMNFALD

3969
RWDTYGSWM

3970
GQQQSFSYQ

3971
LFTHCILNE

3972
PTDNINFQM

3973
SFVTFNNSW

3974
FTSSYKCYF

3975
QAHHYNILN

3976
LFDQFYSWS

3977
QWMGLIRWK

3978
HYLPFMYMQ

3979
LFNRSLRYE

3980
FWQLKHGAC

3981
LSLNELALL

3982
INFEMMDTD

3983
LSHTYIEIA

3984
DVAKYMYML

3985
PINSGIYWH

3986
ECSTSMGQF

3987
TSVFYQSNG

3988
TYSSFNFTC

3989
CSSFYFESK

3990
EATYTNAAC

3991
DAWQSNGWQ

3992
MIILILPYS

3993
EADTHWHHN

3994
RHLFWDILE

3995
EFMNASHHF

3996
QFQSVMQQY

3997
MWNQQDCKD

3998
RSEPCKNYG

3999
SIIQANSSF

4000
DATTSYVYC

4001
RFDINLDQF

4002
QWTENVMAR

4003
DSFYSSQQA

4004
PVTQYMSFM

4005
HSQVQQMQI

4006
FVQFWVGYQ

4007
YSKWIGIDQ

4008
YFSQYNVQS

4009
CAAHATAHQ

4010
VHSQITCED

4011
VIFNFYSMG

4012
LFSLRMIDR

4013
SWYCMQAIE

4014
LFLTLSTFN

4015
QAIGYRIWC

4016
QILLFNACR

4017
RTMDTNMFD

4018
LIQNCSSFG

4019
QWMVQHTNF

4020
LFSYVQNVH

4021
SMMFYRHCQ

4022
DATWIMQDN

4023
FAFQSNIEA

4024
SFDLMFRFY

4025
IAITWTSQC

4026
HGFYVFFFI

4027
MWMLKIDCT

4028
GFDYQGLSE

4029
RWFLQEQSN

4030
WFCSFYALQ

4031
HFMSYNSFF

4032
RSFLSYGLK

4033
NPMVTLANR

4034
TAQSIGSQH

4035
HFLSSMHQG

4036
LAMSSMFFH

4037
RVSNMLNCT

4038
DAQMIMSIE

4039
KIFTIHQFE

4040
DNDFFQAQF

4041
NIHYYGSGY

4042
RFDLFHEQY

4043
QFMLIEARF

4044
LFLYYHSAT

4045
SCSSHSQCC

4046
NFSSIGCHF

4047
IASWNQYHF

4048
EASSMQFWA

4049
QYEKSMTAF

4050
DAHHIMEGN

4051
LQIWYTEMF

4052
QMGNIYSSV

4053
NQSFYLHMS

4054
TSESSGVGG

4055
ELSFYQQFF

4056
MMVYFMQQA

4057
QAWFYQFDK

4058
GSDIHHEQF

4059
CAYMLGISQ

4060
EAMWIHFFG

4061
FFAANGQGC

4062
RFDLQNLMK

4063
QLRMWCCIH

4064
VDSTSSAFM

4065
RYQFWLNFT

4066
RFDSVQQYF

4067
IIHRYARWC

4068
DATHMITQS

4069
LHFFYSHSN

4070
TAAPIHVWM

4071
QALHIDCLH

4072
KFSTMLHAT

4073
NYSSSFVQM

4074
TFHQTSHWF

4075
SRLLNFCCI

4076
QADYSYSFL

4077
DYNRSDSYR

4078
LYGHKMQEN

4079
RFENSLTNE

4080
VAMPNMEYM

4081
AMCFYYMLE

4082
KGEQSIIFI

4083
FAMYVWMQA

4084
LFHSWMRYD

4085
FFTLKWHQF

4086
YAYAYQFCI

4087
AFIKHYLFE

4088
LTSNMLARD

4089
FSIMLMSSG

4090
QIQQYLSQF

4091
MALLKDGIE

4092
RYENFHQMD

4093
CAATNYHIH

4094
LFREISMNA

4095
QWHLFDAGV

4096
SANGGSIQM

4097
ATMSIMWAH

4098
DVSSTEAQF

4099
FLTSYTEKM

4100
SFSSLSGVA

4101
QHVQFTCLN

4102
AAQPWEHYY

4103
QMVSGDMGD

4104
LFSQYNGFM

4105
SGHQISDLF

4106
FMQIMQMQF

4107
LKLQLFNCG

4108
RFEQSMSVQ

4109
GNFFNNAIW

4110
FLMQYGDSC

4111
FAGVLLMCC

4112
FAFTSLINA

4113
LYSNLKRRR

4114
TISFCEFFN

4115
TFSLIFQMS

4116
NSGVVQSCF

4117
TGLEKIHFL

4118
FYYMYNSVV

4119
HTSHLGSQF

4120
QSVHVFYSW

4121
RYEPTSFMP

4122
LWAQYCKSE

4123
RFDQQGEYD

4124
QALQMDFLY

4125
DFTSTMTNG

4126
RQACAQNFQ

4127
AQYQWQMQF

4128
QWVVYHWQI

4129
LFQFQHRCR

4130
YTIMMHHLM

4131
LYQLFKRCY

4132
HAYQSITTS

4133
FFADFNKHC

4134
HSGFILALG

4135
TSWMMLQTH

4136
HIMYIGQYP

4137
VYNFLLDLG

4138
SFLPINLYA

4139
QSYQIQWIG

4140
DHFTFNFKC

4141
QDFSTFQSY

4142
RFDSKMENS

4143
AAVTAIMGE

4144
SGTSFGAML

4145
WSNPFIHYQ

4146
HVAQYNFAK

4147
RIIIHQFNE

4148
QAILAQNCM

4149
SFCMQMSSW

4150
GSQSNYHTA

4151
SMDPFSQNQ

4152
HGYFCQSFD

4153
DRHFFFFGI

4154
QYMSSQQCF

4155
NFTSNQQSY

4156
DCSLLNIWG

4157
MATSSSSSL

4158
NIMVYAQAR

4159
NAYTSIHNN

4160
LMQLFGVLR

4161
DSGSGITWH

4162
LFMHFNNMC

4163
HATSVAHHY

4164
ISEGHKNVL

4165
MNIVIEDKI

4166
AAQHSMLDS

4167
FWQYVNQQE

4168
RALDHLFSI

4169
QWEKYLKIM

4170
EAYNYQEMS

4171
LAYHTSWCQ

4172
NFCNCDFIL

4173
HHLFLFDSI

4174
FYQSAHFIF

4175
QGSYVACAE

4176
QAYCSHSCE

4177
QYRTTQDQN

4178
MNAPVFAQE

4179
ISSFQWAIA

4180
IMDLMQGCH

4181
QYQQNGTQH

4182
LYMHRGLYR

4183
CGYLWSSFA

4184
QSGESLSLM

4185
QNGQMQACN

4186
QFTSQSVQY

4187
LCSQYFDRI

4188
LIAPMFTDL

4189
RYCEKNQGK

4190
YSNSLWDSI

4191
FYALTMSCQ

4192
DTCYLQHAE

4193
LSLLAYNHI

4194
IWQLFNDCM

4195
LIGMQPFCN

4196
ESLSAYDKQ

4197
SFNQCQSTC

4198
RWSLIQQFE

4199
LFSSWMCFY

4200
GGISMHFFD

4201
QIGTIQAHS

4202
EWMYMQSGE

4203
QFYQNSQMP

4204
LATCSGQSI

4205
LSFDSNAHL

4206
SILFHQLIR

4207
SWQFSKQYL

4208
YALFELHND

4209
IICHIHTMM

4210
IFDPKIAWF

4211
LGHTQACML

4212
VMDSMHYFM

4213
RWDQLMVGE

4214
LFQFWKNCH

4215
LYAFYMSSE

4216
LFMSWQSTA

4217
KYENYQMLR

4218
DAILQSTHI

4219
LFNFYTVDW

4220
QAVSVTASI

4221
NHVSFQQSM

4222
FWASTGAYS

4223
KSQSTVYQD

4224
QTDHHHHCH

4225
QSFPIQAPA

4226
QMQQMSVMI

4227
QIHIQFDHC

4228
HYRGSKQWE

4229
CFDFQFEHF

4230
RASQMSGSM

4231
QASILQKNH

4232
QVSQQNQHF

4233
QYQQCNFGC

4234
ASFSNPAAT

4235
EAMYNNSFC

4236
DAYTSMSDW

4237
GNANFGNLL

In some embodiments, the engineered AAV VP capsid polypeptide comprises a polypeptide sequence represented by the formula: (A)-(X)—(B) wherein: (A) is the polypeptide sequence of SEQ ID NO: 5812 (VAYNVGGQMATNNQSSTTAP, corresponding to residues 561 to 580 of SEQ ID NO: 2); (X) is the 581-589 region having a polypeptide sequence selected from the list of polypeptides in TABLE 4 (SEQ ID NO: 5234-SEQ ID NO: 5807) that is expected to confer CNS tissue tropism or preference on a recombinant AAV virion (rAAV), based on a primary screen; and (B) is the polypeptide sequence of SEQ ID NO: 5813 (IVPGSVWMERDVYLQGPIWA, corresponding to residues 590 to 609 of SEQ ID NO: 2); and wherein the capsid polypeptide is capable of assembling into the rAAV and, the capsid does not have the sequence of any of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, or SEQ ID NO: 8. Also encompassed herein are rAAVs composed of engineered AAV5 VP2 capsid polypeptides and engineered AAV5 VP3 capsid polypeptides comprising a 581-589 region having a sequence selected from the list of polypeptides in TABLE 4 (SEQ ID NO: 5234-SEQ ID NO: 5807) at the region corresponding to amino acid residues 445 to 453 in AAV5 VP2 or the region corresponding to amino acid residues 389 to 397 in AAV5 VP3.

TABLE 4

Additional Sequences of 581-589 Regions

for CNS Tropism

SEQ ID

NO
Sequence

5234
AADGYVAQD

5235
AAMSSSHSY

5236
AARMVGEFY

5237
ACSQIGQDD

5238
ADYSMMWHM

5239
AEHMVVDHE

5240
AERDHKTIY

5241
AFARQEYAN

5242
AGMYKCQED

5243
AHQTAWAWI

5244
AHQTPWAWI

5245
AIGPALCLT

5246
AIGPDRSNN

5247
AKCTILNFQ

5248
AKRECTHIP

5249
ALQYCQGKD

5250
ANGTRECQS

5251
ANLISQGHC

5252
ANRMIWYPY

5253
AQCHDMWGH

5254
ARKNCDHLN

5255
ARSGINGHE

5256
ASMHEETCL

5257
ASRSCMTQE

5258
ATDMIFVSN

5259
ATGTYNLQE

5260
AVAEIRPEP

5261
AVHWHRKCE

5262
AVNEAQDNR

5263
AVTQLSVER

5264
AVYLDHVFS

5265
AWSYTGACY

5266
AYAPSKNAM

5267
CAGMAWSHS

5268
CAWHMKQGN

5269
CCCVPRSIY

5270
CCLWSYWAF

5271
CHILDGLHD

5272
CIMLNGCFQ

5273
CIQPTWDSY

5274
CIRFMMREG

5275
CKQLFCELG

5276
CNEVCRNYL

5277
CNYPIIWDK

5278
CRMITMMNE

5279
CVDTNRMWQ

5280
CVFSRNNYQ

5281
CVKPFLTGW

5282
CVNLMAVNN

5283
CVRIGMREP

5284
CWGPIMHPF

5285
DARKTYICY

5286
DATRAQTGE

5287
DCRCYDCHD

5288
DEAKKSCWQ

5289
DEGYIMFAP

5290
DGEFLHEVL

5291
DHCHCIAHP

5292
DHIHIERAN

5293
DHLSICCQN

5294
DIATMRNTW

5295
DICQKQSPN

5296
DIEFLDCEN

5297
DKMSLQVQI

5298
DLVDLRSWM

5299
DMTLQSVSS

5300
DNYWIVPFA

5301
DPGWYGLAP

5302
DQFCPFDHY

5303
DQRMLLKEL

5304
DQSMMTIGQ

5305
DRKQADQIY

5306
DSAKQWHHQ

5307
DTAFMHHHV

5308
DTHHVHGWQ

5309
DVCRKPVSH

5310
DVIRDMIAE

5311
DVKSWAVCD

5312
DVMQYQNHM

5313
DVRACMEDL

5314
DVTQTRYCY

5315
DWRYFCVSL

5316
EAYITYNKA

5317
ECIFAPRMS

5318
EDGLIVYGP

5319
EERPWEWET

5320
EFDWLLGTI

5321
EFKCVSDCL

5322
EGATAWQDR

5323
EHSSPREDN

5324
EICMRSGFI

5325
EIIQAHLIN

5326
EITQHFVKH

5327
EKRLEGWVY

5328
ELPDTEVPW

5329
ELRMSFTKV

5330
EPLVCISAG

5331
EPMPMPVWN

5332
EQLRDLNIE

5333
EQPHISIKG

5334
ERFKTFCEE

5335
ERKTTADWK

5336
ERQAHNHYA

5337
ERTACKMKS

5338
ERYYPISSF

5339
ESRQVQDCY

5340
ETEIRQMDG

5341
ETHRGTVGL

5342
EVRSTRKNH

5343
EWEGVSQFR

5344
EWHARHHYW

5345
EYCEFMSEA

5346
EYSRCCHQE

5347
FAESMWCDR

5348
FAPEFDNHC

5349
FAQQIINAS

5350
FCVKSPRVD

5351
FKPFAQQWN

5352
FLTCDQFEW

5353
FNTPYAHIG

5354
FQTSINYYG

5355
FRAVYYYHS

5356
FRDVCMHDV

5357
FSYQIQCQN

5358
FTEWKPVFF

5359
FTHSCCCLS

5360
GAGPVQSEE

5361
GAKYIQNTD

5362
GCLTSECAY

5363
GCMCIRHAY

5364
GDMCEDMCT

5365
GDVSYDAEA

5366
GEMTRCFHN

5367
GFQRTHVDQ

5368
GFTPDRCRN

5369
GGCCGGELN

5370
GGDGRVHNK

5371
GGDRMATDR

5372
GGSYVSAPD

5373
GHMFETFWG

5374
GIDLMRQIN

5375
GIHQLIHHC

5376
GLFHPQFGQ

5377
GMSDCEVSF

5378
GNECFHNVE

5379
GNKSPDECF

5380
GPARSFEAC

5381
GQPVWCVCM

5382
GREITQVMS

5383
GRWMCNEAE

5384
GSCCLNNET

5385
GSKIIMHTE

5386
GSQQTTLSC

5387
GSYIVQNHC

5388
GTEPIMWRD

5389
GTIRMPEHR

5390
GTNAYMAHK

5391
GVHHRCRMT

5392
GVITVDAKG

5393
GWDMLSVAK

5394
GYKFIGRSR

5395
GYNYYWQHA

5396
GYTQQDEHF

5397
HEGLIMRLE

5398
HEVRRYGQN

5399
HFEVRSVQR

5400
HGFKCQYSF

5401
HHRVNYEMY

5402
HIGWMGWLR

5403
HLMPRNQCF

5404
HLWMCPPRY

5405
HMGIAYNDH

5406
HMSQAGVVS

5407
HPLPNTEIC

5408
HQSPIHTNT

5409
HSLVQQLHP

5410
HSTNLFEAK

5411
HTLQWEAVR

5412
HTRKEGENS

5413
HVTMNQLYS

5414
IADDYFMCH

5415
IAKIGNCVW

5416
IAMRCWGNE

5417
IATHNHILN

5418
IATKSNCHK

5419
ICNMDFRCA

5420
IECSPRESI

5421
IEHCSNRYA

5422
IGQYFNFHF

5423
IHFTSQSCL

5424
IHGIDCYEI

5425
IHPMMRENG

5426
IIRQSEQYH

5427
ILINDREWG

5428
IMCHARMMA

5429
IMNPTAQNN

5430
IPAEWDSWR

5431
IPRPFFTMH

5432
IQAVMASGW

5433
IRPQYAKMD

5434
IRRSMQQQN

5435
ISQRGTLWC

5436
ISRMLRQSS

5437
ISTKFGKEI

5438
KAIWQGTEN

5439
KALHYAMIK

5440
KARQDDVNT

5441
KATHCDKYV

5442
KATVEGSWR

5443
KAVLFLKDC

5444
KCADLIIRS

5445
KCLPYMEMF

5446
KCNDIVLYH

5447
KCNVITYHG

5448
KCTMITPCF

5449
KDGYREAWM

5450
KDNQSCEWN

5451
KEAYTDMSC

5452
KENKIMQAS

5453
KFGFMYNEF

5454
KGEIEGGAK

5455
KGLITENNY

5456
KGTPNWWGP

5457
KHITWNHCL

5458
KHIVGQDGG

5459
KHTLAEMYG

5460
KIDAHSNME

5461
KIGCMYSSQ

5462
KIIFELGMD

5463
KISPWDGFY

5464
KIWAGVNLG

5465
KIYMQCDQE

5466
KKTECEMIV

5467
KKTLYEMAC

5468
KMAGIGCWY

5469
KMCWWPESM

5470
KMHGNERCF

5471
KMLHKVWID

5472
KMMAFEKQC

5473
KMNTWDQAR

5474
KNELENVIN

5475
KPAGKNTCW

5476
KPDHAMAQQ

5477
KPLTYTVGS

5478
KQKTQNRNA

5479
KQLFMKFWN

5480
KRTDLHAAD

5481
KRVQQMETM

5482
KRWLHMYQG

5483
KSAPRENFN

5484
KSGWGAPYE

5485
KSMTCVYYP

5486
KSTYYNLKW

5487
KSVHYETCE

5488
KSWVWVDTE

5489
KTCLCIFTK

5490
KTDPIAEYC

5491
KTEKWASHE

5492
KTFTKEVTP

5493
KTLPFAIYH

5494
KTMADMYNR

5495
KTQTHTSEP

5496
KTSNVMDQM

5497
KTVETYAAH

5498
KVAEGQNML

5499
KVAGGHETT

5500
KVDTVRNPK

5501
KVESTTQLC

5502
KWDDMHTQM

5503
KYMQENVLS

5504
LAAIQHCAM

5505
LACTPRCIF

5506
LADCMLNHH

5507
LAESKKRNK

5508
LATTTSYHC

5509
LCTSTERQG

5510
LEQLDRCMY

5511
LFMTYSARN

5512
LFYSPMHTH

5513
LILQRNVKD

5514
LLWRRVKRT

5515
LNELPRAEY

5516
LNHPIADYD

5517
LPPPEEKWL

5518
LPTQVYGHN

5519
LTCMHASTP

5520
LTPRSEGLP

5521
LTQYKALAQ

5522
LVGRTYQHQ

5523
LWDRFCCPV

5524
LWSRFISMD

5525
LYMMALLPT

5526
MAAITQNVA

5527
MAGHIGHHS

5528
MAQPYGRTA

5529
MARQCHSFH

5530
MCRPVMMIE

5531
MCSQTISMR

5532
MDGHFSVGC

5533
MDGRDEIIH

5534
MDPDIPHCR

5535
MDVQQIISG

5536
MEHTMDFGV

5537
MFDMMSVLK

5538
MFVVNQNWA

5539
MGQRSAANY

5540
MGRPHAWWG

5541
MIKPLLVVG

5542
MIRPSINGS

5543
MKNQPTGAS

5544
MKTIMQDYF

5545
MLFDFEEHN

5546
MLGVAPGHL

5547
MLPLEMKNL

5548
MMVRELMSS

5549
MPSWYIAQA

5550
MQHLPPAMT

5551
MQLRIHTNS

5552
MQYRGMSAS

5553
MRQRTYICQ

5554
MRRKTPEDD

5555
MRYHWDEAG

5556
MSLQHHFCT

5557
MSNLEYYEA

5558
MTDNNGPLF

5559
MTGIGKCWN

5560
MTRSCQTEG

5561
MYTGMRCWR

5562
NAGMKGVDV

5563
NAGVVKVWP

5564
NAIPYFVQA

5565
NAKYSMLYG

5566
NAQRLAVGM

5567
NARVMAKRE

5568
NCPMYDMIS

5569
NERDQCQWP

5570
NHSLCWDSK

5571
NLERCNTMM

5572
NMWYSFVMI

5573
NQREWHGLA

5574
NQRGFCKDE

5575
NRCGVSKCC

5576
NRIQMTDFQ

5577
NSTRNTSYY

5578
NSTRWYHTG

5579
NTAKLGWGL

5580
NTCEGWSHF

5581
NTGVKFCAC

5582
NTQYSAKND

5583
NTRIVCDLS

5584
NTSELLMAQ

5585
NVCDNGHEI

5586
NVFPAETHV

5587
NVMGTQAKE

5588
NVTTMFGQK

5589
NWEVIGRWH

5590
NWNHCHQWR

5591
NWTGNMQWA

5592
NYCKYNKMR

5593
PAWRRYCWT

5594
PEKWIHMHE

5595
PIHTYKRHP

5596
PIKGVAVQE

5597
PLNQEQIPQ

5598
PRNLVAMYW

5599
PVKPIIHAS

5600
PWMHTMLNP

5601
QAAVTKEYW

5602
QAKMILDGT

5603
QANPYNHGI

5604
QAVIAVDGR

5605
QCAHFYQTR

5606
QCGCAPMDS

5607
QCGFIRLNE

5608
QCQSQEWFH

5609
QDCERQVFA

5610
QEAGYTRHN

5611
QFCICCDGM

5612
QGENAQHHN

5613
QGFSTDVKP

5614
QGQHVMVHS

5615
QHLMTFSHE

5616
QHQAFFVFT

5617
QIKTHSNVF

5618
QKPTIWDHF

5619
QKSMLNSIQ

5620
QLKRNEQTS

5621
QMKGATEFC

5622
QMNAIQLCV

5623
QMVTQLNWE

5624
QNKHNWWIM

5625
QPKMWMMAS

5626
QPRVYEGFY

5627
QPYWVAVDP

5628
QQTWEQMQR

5629
QRGPLQHQQ

5630
QRNNLFVHD

5631
QSAHPEPMC

5632
QSGTCHYHD

5633
QSRQMMLLH

5634
QSTPTLGPK

5635
QSWWHSDTC

5636
QTDILRDVQ

5637
QTGTGHWFE

5638
QTGVMAAFG

5639
QTLGGHMWT

5640
QVACHMTNP

5641
QVHIGSWPP

5642
QVMQEKVRC

5643
QVNMFMMPC

5644
QVRFEQNPA

5645
QVSFVGCNH

5646
QVSHISEHW

5647
QWAPLQTWA

5648
QWEEVWWEA

5649
QYLLIPHLP

5650
QYQSTGVVR

5651
QYWWPATHD

5652
RAERYFKDE

5653
RAIYNDMTE

5654
RCVQAHFHK

5655
RDIWERPCV

5656
REEWKNASN

5657
REMMIVNCD

5658
RGKPILNQQ

5659
RITQGESGM

5660
RKWMNAKAT

5661
RLAWIEQSP

5662
RMMWQSHCG

5663
RMVLAYGNF

5664
RNDWQQVFS

5665
RQTTYDCLD

5666
RREAMDDSP

5667
RSDEMYVTP

5668
RTRISQMFG

5669
RTVPTQEMS

5670
RVAGEGIQP

5671
RVWSLEWHL

5672
RYNNCPNLW

5673
RYQSHCWLH

5674
SADRTAMFH

5675
SALYRWGHV

5676
SANQRYHDW

5677
SAPRNEITR

5678
SCQLLNDGG

5679
SFKSVSQYN

5680
SFPMILVNC

5681
SIAQDQVWS

5682
SIRYDFYVP

5683
SIYQKWVTN

5684
SKGDLCHQQ

5685
SKGWADPCP

5686
SMQYFFKTE

5687
SMRDLIAEA

5688
SMSHNGQCL

5689
SNRYYSRIF

5690
SPNGRGLCG

5691
SQIQIRQAD

5692
SQMKYAHNQ

5693
SQSRTIIQE

5694
SRDIAKSST

5695
SRLYKKRLF

5696
SRTRMYMDH

5697
SRVMIHVYV

5698
SSVVMAKQP

5699
STLEDEKPY

5700
STQKVPVLA

5701
STRPIACHS

5702
SVETARCSG

5703
SVGYIYDQS

5704
SVKDPMDWG

5705
SVTPDSITT

5706
TAGQQPTFM

5707
TARDREHAM

5708
TARSTSDKC

5709
TCKWYEESI

5710
TCTNSEPHP

5711
TCYCHRAHE

5712
TEEVQFVVH

5713
TFMFNSEWS

5714
TFMRKICGM

5715
TFVRKPSLM

5716
THSSCWYQD

5717
TISQIPWVR

5718
TKLSMMGCE

5719
TKQFFTAHC

5720
TKYHYHVKV

5721
TLEPAYRHE

5722
TLLLVGKDR

5723
TMAQKGLWF

5724
TMPDRDPTY

5725
TMRLFWDMG

5726
TNFPTSMHG

5727
TPKSEGICC

5728
TQAKIECCS

5729
TQEACHHDP

5730
TQNACKVRT

5731
TRHPTIAMH

5732
TRILNEFFC

5733
TSEPHCSYA

5734
TSERHSCMV

5735
TSMEFTQHQ

5736
TSNHNMALE

5737
TTAYHRSDH

5738
TTTITNRLC

5739
TTTNQFMFQ

5740
TVCGSFHGC

5741
TVIRDSRSY

5742
TVRHQMDAY

5743
TVTHMSTWG

5744
TWHKQLVGV

5745
TYASTRNFH

5746
TYDTRNHCL

5747
TYLHQPEDS

5748
TYWCPLMMN

5749
VAADSRMMI

5750
VADTWQVHC

5751
VADWIGHTP

5752
VAYPIQTDV

5753
VCGWWHQYW

5754
VCHNPDPFT

5755
VFGSTRSAA

5756
VFNLMLQDK

5757
VGIWFIPCR

5758
VHVHCMIGS

5759
VICRMKKHS

5760
VIKVNTVNP

5761
VKAWWHDHQ

5762
VKHTAVDVP

5763
VLKSCDRHG

5764
VMFYWRYNS

5765
VPMWAHWQN

5766
VPTGRRVKL

5767
VPVATMIHH

5768
VRADNMPVM

5769
VSAYIERQW

5770
VSKFIAASW

5771
VSRCGIDRS

5772
VSVQSRAQL

5773
VTMTTKSVW

5774
VWWMIIHTS

5775
WAIFGDIHH

5776
WAYYIRLQP

5777
WCEAVGDYA

5778
WCESWGPQG

5779
WCRCKRFIE

5780
WDGRPDLST

5781
WFSIIMSKL

5782
WINEANCLM

5783
WIRSAQRPG

5784
WKDDLRSTY

5785
WKWYGLPHH

5786
WPNIKHQPI

5787
WPTMLSAHM

5788
WSCRSICPP

5789
WVEPEQCFG

5790
WVKQKDVLC

5791
WVYSHEKHY

5792
YDLWGWSTR

5793
YDYWMQAPT

5794
YEHPIMWPV

5795
YEICKHGHF

5796
YFADAWLKT

5797
YFPSIQCYE

5798
YGGSEHWGP

4799
YGNSVTCTS

5800
YHCCFAMQS

5801
YMSWYQIRN

5802
YPAQYTAFS

5803
YPQPCSCHS

5804
YPVHMMPPL

5805
YRDRCWNRI

5806
YTELMKKAC

5807
YTNHYYNAR

In some embodiments, the engineered AAV VP capsid polypeptide confers CNS tissue tropism or preference, wherein the CNS tissue is selected from the group consisting of hippocampus: (dentate gyrus, CA1 and CA3); cerebellum, hypothalamus, cortex: (occipital, temporal and forebrain); substantia nigra, thalamus, and any combination thereof.

In some embodiments, the engineered AAV VP capsid polypeptide comprises a polypeptide sequence represented by the formula: (A)-(X)—(B) wherein: (A) is the polypeptide sequence of SEQ ID NO: 5812 (VAYNVGGQMATNNQSSTTAP, corresponding to residues 561 to 580 of SEQ ID NO: 2); (X) is the 581-589 region having a polypeptide sequence selected from the list of polypeptides in TABLE 10 that is expected to confer cardiac muscle tissue tropism or preference on a recombinant AAV virion (rAAV), based on a secondary screen; and (B) is the polypeptide sequence of SEQ ID NO: 5813 (IVPGSVWMERDVYLQGPIWA, corresponding to residues 590 to 609 of SEQ ID NO: 2); and wherein the capsid polypeptide is capable of assembling into the rAAV and, the capsid does not have the sequence of any of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, or SEQ ID NO: 8. Also encompassed herein are rAAVs composed of engineered AAV5 VP2 capsid polypeptides and engineered AAV5 VP3 capsid polypeptides comprising a 581-589 region having a sequence selected from the list of polypeptides in TABLE 10 at the region corresponding to amino acid residues 445 to 453 in AAV5 VP2 or the region corresponding to amino acid residues 389 to 397 in AAV5 VP3.

In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence that is at least 75%, at least 77.7%, at least 85%, at least 88.8%, or 100% identical to a sequence provided in TABLE 10. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence that is at least 75%, at least 77.7%, at least 85%, at least 88.8% or 100% identical to SEQ ID NO: 348. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence that is at least 75%, at least 77.7%, at least 85%, at least 88.8% or 100% identical to SEQ ID NO: 2536. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence that is at least 75%, at least 77.7%, at least 85%, at least 88.8% or 100% identical to SEQ ID NO: 5607. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence that is at least 75%, at least 77.7%, at least 85%, at least 88.8% or 100% identical to SEQ ID NO: 1576. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence that is at least 75%, at least 77.7%, at least 85%, at least 88.8% or 100% identical to SEQ ID NO: 4955. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence that is at least 75%, at least 77.7%, at least 85%, at least 88.8% or 100% identical to SEQ ID NO: 5017. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence that is at least 75%, at least 77.7%, at least 85%, at least 88.8% or 100% identical to SEQ ID NO: 4349. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence that is at least 75%, at least 77.7%, at least 85%, at least 88.8% or 100% identical to SEQ ID NO: 4964. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence that is at least 75%, at least 77.7%, at least 85%, at least 88.8% or 100% identical to SEQ ID NO: 293. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence that is at least 75%, at least 77.7%, at least 85%, at least 88.8% or 100% identical to SEQ ID NO: 4314. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence that is at least 75%, at least 77.7%, at least 85%, at least 88.8% or 100% identical to SEQ ID NO: 4995. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence that is at least 75%, at least 77.7%, at least 85%, at least 88.8% or 100% identical to SEQ ID NO: 4366. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence that is at least 75%, at least 77.7%, at least 85%, at least 88.8% or 100% identical to SEQ ID NO: 4961. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence that is at least 75%, at least 77.7%, at least 85%, at least 88.8% or 100% identical to SEQ ID NO: 4952. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence that is at least 75%, at least 77.7%, at least 85%, at least 88.8% or 100% identical to SEQ ID NO: 5075. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence that is at least 75%, at least 77.7%, at least 85%, at least 88.8% or 100% identical to SEQ ID NO: 4354. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence that is at least 75%, at least 77.7%, at least 85%, at least 88.8% or 100% identical to SEQ ID NO: 5060. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence that is at least 75%, at least 77.7%, at least 85%, at least 88.8% or 100% identical to SEQ ID NO: 5138. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence that is at least 75%, at least 77.7%, at least 85%, at least 88.8% or 100% identical to SEQ ID NO: 5206. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence that is at least 75%, at least 77.7%, at least 85%, at least 88.8% or 100% identical to SEQ ID NO: 4288. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence that is at least 75%, at least 77.7%, at least 85%, at least 88.8% or 100% identical to SEQ ID NO: 5092. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence that is at least 75%, at least 77.7%, at least 85%, at least 88.8% or 100% identical to SEQ ID NO: 4059. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence that is at least 75%, at least 77.7%, at least 85%, at least 88.8% or 100% identical to SEQ ID NO: 4295. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence that is at least 75%, at least 77.7%, at least 85%, at least 88.8% or 100% identical to SEQ ID NO: 5030. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence that is at least 75%, at least 77.7%, at least 85%, at least 88.8% or 100% identical to SEQ ID NO: 4938. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence that is at least 75%, at least 77.7%, at least 85%, at least 88.8% or 100% identical to SEQ ID NO: 2661. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence that is at least 75%, at least 77.7%, at least 85%, at least 88.8% or 100% identical to SEQ ID NO: 5215. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence that is at least 75%, at least 77.7%, at least 85%, at least 88.8% or 100% identical to SEQ ID NO: 4960. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence that is at least 75%, at least 77.7%, at least 85%, at least 88.8% or 100% identical to SEQ ID NO: 4969. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence that is at least 75%, at least 77.7%, at least 85%, at least 88.8% or 100% identical to SEQ ID NO: 5023.

In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence of SEQ ID NO: 348. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence of SEQ ID NO: 2536. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence of SEQ ID NO: 5607. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence of SEQ ID NO: 1576. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence of SEQ ID NO: 4955. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence of SEQ ID NO: 5017. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence of SEQ ID NO: 4349. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence of SEQ ID NO: 4964. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence of SEQ ID NO: 293. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence of SEQ ID NO: 4314. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence of SEQ ID NO: 4995. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence of SEQ ID NO: 4366. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence of SEQ ID NO: 4961. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence of SEQ ID NO: 4952. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence of SEQ ID NO: 5075. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence of SEQ ID NO: 4354. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence of SEQ ID NO: 5060. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence of SEQ ID NO: 5138. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence of SEQ ID NO: 5206. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence of SEQ ID NO: 4288. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence of SEQ ID NO: 5092. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence of SEQ ID NO: 4059. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence of SEQ ID NO: 4295. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence of SEQ ID NO: 5030. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence of SEQ ID NO: 4938. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence of SEQ ID NO: 2661. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence of SEQ ID NO: 5215. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence of SEQ ID NO: 4960. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence of SEQ ID NO: 4969. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence of SEQ ID NO: 5023. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence that is at least 75%, at least 77.7%, at least 85%, at least 88.8% or 100% identical to SEQ ID NO: 4934.

In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence of SEQ ID NO: 348 with 1 or 2 conservative substitutions. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence of SEQ ID NO: 2536 with 1 or 2 conservative substitutions. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence of SEQ ID NO: 5607 with 1 or 2 conservative substitutions. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence of SEQ ID NO: 1576 with 1 or 2 conservative substitutions. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence of SEQ ID NO: 4955 with 1 or 2 conservative substitutions. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence of SEQ ID NO: 5017 with 1 or 2 conservative substitutions. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence of SEQ ID NO: 4349 with 1 or 2 conservative substitutions. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence of SEQ ID NO: 4964 with 1 or 2 conservative substitutions. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence of SEQ ID NO: 293 with 1 or 2 conservative substitutions. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence of SEQ ID NO: 4314 with 1 or 2 conservative substitutions. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence of SEQ ID NO: 4995 with 1 or 2 conservative substitutions. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence of SEQ ID NO: 4366 with 1 or 2 conservative substitutions. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence of SEQ ID NO: 4961 with 1 or 2 conservative substitutions. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence of SEQ ID NO: 4952 with 1 or 2 conservative substitutions. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence of SEQ ID NO: 5075 with 1 or 2 conservative substitutions. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence of SEQ ID NO: 4354 with 1 or 2 conservative substitutions. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence of SEQ ID NO: 5060 with 1 or 2 conservative substitutions. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence of SEQ ID NO: 5138 with 1 or 2 conservative substitutions. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence of SEQ ID NO: 5206 with 1 or 2 conservative substitutions. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence of SEQ ID NO: 4288 with 1 or 2 conservative substitutions. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence of SEQ ID NO: 5092 with 1 or 2 conservative substitutions. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence of SEQ ID NO: 4059 with 1 or 2 conservative substitutions. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence of SEQ ID NO: 4295 with 1 or 2 conservative substitutions. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence of SEQ ID NO: 5030 with 1 or 2 conservative substitutions. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence of SEQ ID NO: 4938 with 1 or 2 conservative substitutions. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence of SEQ ID NO: 2661 with 1 or 2 conservative substitutions. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence of SEQ ID NO: 5215 with 1 or 2 conservative substitutions. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence of SEQ ID NO: 4960 with 1 or 2 conservative substitutions. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence of SEQ ID NO: 4969 with 1 or 2 conservative substitutions. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence of SEQ ID NO: 5023 with 1 or 2 conservative substitutions. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence of SEQ ID NO: 4934 with 1 or 2 conservative substitutions. The conservative substitutions may be at any position in the 581-589 region.

In some embodiments, an AAV VP capsid polypeptide preferentially targets a cardiac muscle tissue at a DNA enrichment of at least 25-fold greater than a wild type AAV9. For example, an AAV VP capsid polypeptide comprising a 581-589 region of SEQ ID NO: 348, SEQ ID NO: 2536, SEQ ID NO: 1576, SEQ ID NO: 4955, SEQ ID NO: 5017, SEQ ID NO: 4349, SEQ ID NO: 4964, SEQ ID NO: 293, SEQ ID NO: 4314, SEQ ID NO: 4995, SEQ ID NO: 4366, SEQ ID NO: 4961, SEQ ID NO: 4952, SEQ ID NO: 5075, SEQ ID NO: 4354, SEQ ID NO: 5060, SEQ ID NO: 5138, SEQ ID NO: 5206, SEQ ID NO: 4288, SEQ ID NO: 5092, SEQ ID NO: 4059, SEQ ID NO: 4295, SEQ ID NO: 5030, SEQ ID NO: 4938, SEQ ID NO: 2661, SEQ ID NO: 5215, SEQ ID NO: 4960, SEQ ID NO: 4969, SEQ ID NO: 5023, SEQ ID NO: 4934, SEQ ID NO: 4545, SEQ ID NO: 4963, SEQ ID NO: 4363, SEQ ID NO: 5159, or SEQ ID NO: 4973 may preferentially target a cardiac muscle tissue at a DNA enrichment of at least 25-fold greater than a wild type AAV9.

In some embodiments, an AAV VP capsid polypeptide preferentially targets a cardiac muscle tissue at a DNA enrichment of at least 30-fold greater than the wild type AAV9. For example, an AAV VP capsid polypeptide comprising a 581-589 region of SEQ ID NO: 348, SEQ ID NO: 2536, SEQ ID NO: 1576, SEQ ID NO: 4955, SEQ ID NO: 5017, SEQ ID NO: 4349, SEQ ID NO: 4964, SEQ ID NO: 293, SEQ ID NO: 4314, SEQ ID NO: 4995, or SEQ ID NO: 4366 may preferentially target a cardiac muscle tissue at a DNA enrichment of at least 30-fold greater than the wild type AAV9.

In some embodiments, an AAV VP capsid polypeptide preferentially targets a cardiac muscle tissue at a DNA enrichment of at least 50-fold greater than the wild type AAV9. For example, an AAV VP capsid polypeptide comprising a 581-589 region of SEQ ID NO: 348 or SEQ ID NO: 2536 may preferentially target a cardiac muscle tissue at a DNA enrichment of at least 50-fold greater than the wild type AAV9.

In some embodiments, an AAV VP capsid polypeptide preferentially targets a cardiac muscle tissue at an RNA enrichment of at least 10-fold greater than a wild type AAV9. For example, an AAV VP capsid polypeptide comprising a 581-589 region of SEQ ID NO: 348, SEQ ID NO: 5052, SEQ ID NO: 5193, SEQ ID NO: 5138, SEQ ID NO: 5163, SEQ ID NO: 4952, SEQ ID NO: 293, SEQ ID NO: 4317, SEQ ID NO: 5075, SEQ ID NO: 386, SEQ ID NO: 4288, SEQ ID NO: 4995, SEQ ID NO: 5030, SEQ ID NO: 4986, SEQ ID NO: 5206, SEQ ID NO: 4338, SEQ ID NO: 5159, SEQ ID NO: 4346, SEQ ID NO: 5060, SEQ ID NO: 4949, SEQ ID NO: 5017, SEQ ID NO: 5215, SEQ ID NO: 4314, SEQ ID NO: 5080, SEQ ID NO: 4964, SEQ ID NO: 4379, SEQ ID NO: 4304, SEQ ID NO: 18, SEQ ID NO: 4363, SEQ ID NO: 5027, SEQ ID NO: 4349, SEQ ID NO: 5208, or SEQ ID NO: 4938 may preferentially target a cardiac muscle tissue at an RNA enrichment of at least 10-fold greater than a wild type AAV9.

In some embodiments, an AAV VP capsid polypeptide preferentially targets a cardiac muscle tissue at an RNA enrichment of at least 20-fold or greater than the wild type AAV9. For example, an AAV VP capsid polypeptide comprising a 581-589 region of SEQ ID NO: 348, SEQ ID NO: 5052, SEQ ID NO: 5193, SEQ ID NO: 5138, or SEQ ID NO: 5163 may preferentially target a cardiac muscle tissue at an RNA enrichment of at least 20-fold or greater than the wild type AAV9.

In some embodiments, an AAV VP capsid polypeptide preferentially targets a cardiac muscle tissue at an RNA enrichment of at least 40-fold or greater than the wild type AAV9. For example, an AAV VP capsid polypeptide comprising a 581-589 region of SEQ ID NO: 348 may preferentially target a cardiac muscle tissue at an RNA enrichment of at least 40-fold or greater than the wild type AAV9.

In some embodiments, an AAV VP capsid polypeptide preferentially targets a cardiac muscle tissue at at a DNA enrichment of at least 5-fold greater than a wild type AAV5. For example, an AAV VP capsid polypeptide comprising a 581-589 region of SEQ ID NO: 348, SEQ ID NO: 2536, SEQ ID NO: 1576, SEQ ID NO: 4955, SEQ ID NO: 5017, SEQ ID NO: 4349, SEQ ID NO: 4964, SEQ ID NO: 293, SEQ ID NO: 4314, SEQ ID NO: 4995, SEQ ID NO: 4366, SEQ ID NO: 4961, SEQ ID NO: 4952, SEQ ID NO: 5075, SEQ ID NO: 4354, SEQ ID NO: 5060, SEQ ID NO: 5138, SEQ ID NO: 5206, SEQ ID NO: 4288, SEQ ID NO: 5092, SEQ ID NO: 4059, SEQ ID NO: 4295, SEQ ID NO: 5030, SEQ ID NO: 4938, SEQ ID NO: 2661, SEQ ID NO: 5215, SEQ ID NO: 4960, SEQ ID NO: 4969, SEQ ID NO: 5023, SEQ ID NO: 4934, SEQ ID NO: 4545, SEQ ID NO: 4963, SEQ ID NO: 4363, SEQ ID NO: 5159, or SEQ ID NO: 4973 may preferentially target a cardiac muscle tissue at a DNA enrichment of at least 5-fold greater than a wild type AAV5.

In some embodiments, an AAV VP capsid polypeptide preferentially targets a cardiac muscle tissue at a DNA enrichment of at least 10-fold greater than the wild type AAV5. For example, an AAV VP capsid polypeptide comprising a 581-589 region of SEQ ID NO: 348 or SEQ ID NO: 2536 may preferentially target a cardiac muscle tissue at a DNA enrichment of at least 10-fold greater than the wild type AAV5.

In some embodiments, an AAV VP capsid polypeptide preferentially targets a cardiac muscle tissue at an RNA enrichment of at least 5-fold greater than a wild type AAV5. For example, an AAV VP capsid polypeptide comprising a 581-589 region of SEQ ID NO: 348, SEQ ID NO: 5052, SEQ ID NO: 5193, SEQ ID NO: 5138, SEQ ID NO: 5163, SEQ ID NO: 4952, SEQ ID NO: 293, or SEQ ID NO: 4317 may preferentially target a cardiac muscle tissue at an RNA enrichment of at least 5-fold greater than a wild type AAV5.

In some embodiments, an AAV VP capsid polypeptide preferentially targets a cardiac muscle tissue at an RNA enrichment of at least 10-fold greater than the wild type AAV5. For example, an AAV VP capsid polypeptide comprising a 581-589 region of SEQ ID NO: 348 may preferentially target a cardiac muscle tissue at an RNA enrichment of at least 10-fold greater than the wild type AAV5.

In some embodiments, the engineered AAV VP capsid polypeptide comprises a polypeptide sequence represented by the formula: (A)-(X)—(B) wherein: (A) is the polypeptide sequence of SEQ ID NO: 5812 (VAYNVGGQMATNNQSSTTAP, corresponding to residues 561 to 580 of SEQ ID NO: 2); (X) is the 581-589 region having a polypeptide sequence selected from the list of polypeptides in TABLE 11 that is expected to confer skeletal muscle tissue tropism or preference on a recombinant AAV virion (rAAV), based on a secondary screen; and (B) is the polypeptide sequence of SEQ ID NO: 5813 (IVPGSVWMERDVYLQGPIWA, corresponding to residues 590 to 609 of SEQ ID NO: 2); and wherein the capsid polypeptide is capable of assembling into the rAAV and, the capsid does not have the sequence of any of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, or SEQ ID NO: 8. Also encompassed herein are rAAVs composed of engineered AAV5 VP2 capsid polypeptides and engineered AAV5 VP3 capsid polypeptides comprising a 581-589 region having a sequence selected from the list of polypeptides in TABLE 11 at the region corresponding to amino acid residues 445 to 453 in AAV5 VP2 or the region corresponding to amino acid residues 389 to 397 in AAV5 VP3.

In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence that is at least 75%, at least 77.7%, at least 85%, at least 88.8%, or 100% identical to a sequence provided in TABLE 11. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence that is at least 75%, at least 77.7%, at least 85%, at least 88.8% or 100% identical to SEQ ID NO: 3472. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence that is at least 75%, at least 77.7%, at least 85%, at least 88.8% or 100% identical to SEQ ID NO: 3297. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence that is at least 75%, at least 77.7%, at least 85%, at least 88.8% or 100% identical to SEQ ID NO: 2661. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence that is at least 75%, at least 77.7%, at least 85%, at least 88.8% or 100% identical to SEQ ID NO: 4545. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence that is at least 75%, at least 77.7%, at least 85%, at least 88.8% or 100% identical to SEQ ID NO: 348. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence that is at least 75%, at least 77.7%, at least 85%, at least 88.8% or 100% identical to SEQ ID NO: 18. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence that is at least 75%, at least 77.7%, at least 85%, at least 88.8% or 100% identical to SEQ ID NO: 4349. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence that is at least 75%, at least 77.7%, at least 85%, at least 88.8% or 100% identical to SEQ ID NO: 293. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence that is at least 75%, at least 77.7%, at least 85%, at least 88.8% or 100% identical to SEQ ID NO: 5017. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence that is at least 75%, at least 77.7%, at least 85%, at least 88.8% or 100% identical to SEQ ID NO: 4955. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence that is at least 75%, at least 77.7%, at least 85%, at least 88.8% or 100% identical to SEQ ID NO: 4366. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence that is at least 75%, at least 77.7%, at least 85%, at least 88.8% or 100% identical to SEQ ID NO: 5092. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence that is at least 75%, at least 77.7%, at least 85%, at least 88.8% or 100% identical to SEQ ID NO: 210. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence that is at least 75%, at least 77.7%, at least 85%, at least 88.8% or 100% identical to SEQ ID NO: 4059. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence that is at least 75%, at least 77.7%, at least 85%, at least 88.8% or 100% identical to SEQ ID NO: 2536. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence that is at least 75%, at least 77.7%, at least 85%, at least 88.8% or 100% identical to SEQ ID NO: 5206. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence that is at least 75%, at least 77.7%, at least 85%, at least 88.8% or 100% identical to SEQ ID NO: 1971. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence that is at least 75%, at least 77.7%, at least 85%, at least 88.8% or 100% identical to SEQ ID NO: 262. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence that is at least 75%, at least 77.7%, at least 85%, at least 88.8% or 100% identical to SEQ ID NO: 4343. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence that is at least 75%, at least 77.7%, at least 85%, at least 88.8% or 100% identical to SEQ ID NO: 4995. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence that is at least 75%, at least 77.7%, at least 85%, at least 88.8% or 100% identical to SEQ ID NO: 4009. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence that is at least 75%, at least 77.7%, at least 85%, at least 88.8% or 100% identical to SEQ ID NO: 5190. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence that is at least 75%, at least 77.7%, at least 85%, at least 88.8% or 100% identical to SEQ ID NO: 724. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence that is at least 75%, at least 77.7%, at least 85%, at least 88.8% or 100% identical to SEQ ID NO: 4288. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence that is at least 75%, at least 77.7%, at least 85%, at least 88.8% or 100% identical to SEQ ID NO: 4973. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence that is at least 75%, at least 77.7%, at least 85%, at least 88.8% or 100% identical to SEQ ID NO: 4314. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence that is at least 75%, at least 77.7%, at least 85%, at least 88.8% or 100% identical to SEQ ID NO: 1561. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence that is at least 75%, at least 77.7%, at least 85%, at least 88.8% or 100% identical to SEQ ID NO: 5147. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence that is at least 75%, at least 77.7%, at least 85%, at least 88.8% or 100% identical to SEQ ID NO: 4238. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence that is at least 75%, at least 77.7%, at least 85%, at least 88.8% or 100% identical to SEQ ID NO: 4961.

In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence of SEQ ID NO: 3472. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence of SEQ ID NO: 3297. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence of SEQ ID NO: 2661. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence of SEQ ID NO: 4545. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence of SEQ ID NO: 348. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence of SEQ ID NO: 18. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence of SEQ ID NO: 4349. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence of SEQ ID NO: 293. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence of SEQ ID NO: 5017. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence of SEQ ID NO: 4955. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence of SEQ ID NO: 4366. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence of SEQ ID NO: 5092. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence of SEQ ID NO: 210. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence of SEQ ID NO: 4059. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence of SEQ ID NO: 2536. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence of SEQ ID NO: 5206. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence of SEQ ID NO: 1971. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence of SEQ ID NO: 262. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence of SEQ ID NO: 4343. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence of SEQ ID NO: 4995. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence of SEQ ID NO: 4009. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence of SEQ ID NO: 5190. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence of SEQ ID NO: 724. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence of SEQ ID NO: 4288. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence of SEQ ID NO: 4973. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence of SEQ ID NO: 4314. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence of SEQ ID NO: 1561. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence of SEQ ID NO: 5147. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence of SEQ ID NO: 4238. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence of SEQ ID NO: 4961.

In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence of SEQ ID NO: 3472 with 1 or 2 conservative substitutions. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence of SEQ ID NO: 3297 with 1 or 2 conservative substitutions. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence of SEQ ID NO: 2661 with 1 or 2 conservative substitutions. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence of SEQ ID NO: 4545 with 1 or 2 conservative substitutions. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence of SEQ ID NO: 348 with 1 or 2 conservative substitutions. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence of SEQ ID NO: 18 with 1 or 2 conservative substitutions. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence of SEQ ID NO: 4349 with 1 or 2 conservative substitutions. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence of SEQ ID NO: 293 with 1 or 2 conservative substitutions. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence of SEQ ID NO: 5017 with 1 or 2 conservative substitutions. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence of SEQ ID NO: 4955 with 1 or 2 conservative substitutions. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence of SEQ ID NO: 4366 with 1 or 2 conservative substitutions. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence of SEQ ID NO: 5092 with 1 or 2 conservative substitutions. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence of SEQ ID NO: 210 with 1 or 2 conservative substitutions. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence of SEQ ID NO: 4059 with 1 or 2 conservative substitutions. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence of SEQ ID NO: 2536 with 1 or 2 conservative substitutions. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence of SEQ ID NO: 5206 with 1 or 2 conservative substitutions. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence of SEQ ID NO: 1971 with 1 or 2 conservative substitutions. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence of SEQ ID NO: 262 with 1 or 2 conservative substitutions. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence of SEQ ID NO: 4343 with 1 or 2 conservative substitutions. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence of SEQ ID NO: 4995 with 1 or 2 conservative substitutions. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence of SEQ ID NO: 4009 with 1 or 2 conservative substitutions. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence of SEQ ID NO: 5190 with 1 or 2 conservative substitutions. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence of SEQ ID NO: 724 with 1 or 2 conservative substitutions. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence of SEQ ID NO: 4288 with 1 or 2 conservative substitutions. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence of SEQ ID NO: 4973 with 1 or 2 conservative substitutions. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence of SEQ ID NO: 4314 with 1 or 2 conservative substitutions. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence of SEQ ID NO: 1561 with 1 or 2 conservative substitutions. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence of SEQ ID NO: 5147 with 1 or 2 conservative substitutions. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence of SEQ ID NO: 4238 with 1 or 2 conservative substitutions. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence of SEQ ID NO: 4961 with 1 or 2 conservative substitutions. The conservative substitutions may be at any position in the 581-589 region.

In some embodiments, an AAV VP capsid polypeptide preferentially targets a skeletal muscle tissue at a DNA enrichment of at least 15-fold greater than a wild type AAV9. For example, an AAV VP capsid polypeptide comprising a 581-589 region of SEQ ID NO: 3472, SEQ ID NO: 3297, SEQ ID NO: 2661, SEQ ID NO: 4545, SEQ ID NO: 348, SEQ ID NO: 18, SEQ ID NO: 4349, SEQ ID NO: 293, SEQ ID NO: 5017, SEQ ID NO: 4955, SEQ ID NO: 4366, SEQ ID NO: 5092, SEQ ID NO: 210, SEQ ID NO: 4059, SEQ ID NO: 2536, SEQ ID NO: 5206, or SEQ ID NO: 1971 may preferentially target a skeletal muscle tissue at a DNA enrichment of at least 15-fold greater than a wild type AAV9.

In some embodiments, an AAV VP capsid polypeptide preferentially targets a skeletal muscle tissue at a DNA enrichment of at least 20-fold greater than the wild type AAV9. For example, an AAV VP capsid polypeptide comprising a 581-589 region of SEQ ID NO: 3472, SEQ ID NO: 3297, SEQ ID NO: 2661, SEQ ID NO: 4545, or SEQ ID NO: 348 may preferentially target a skeletal muscle tissue at a DNA enrichment of at least 20-fold greater than the wild type AAV9.

In some embodiments, an AAV VP capsid polypeptide preferentially targets a skeletal muscle tissue at a DNA enrichment of at least 30-fold greater than the wild type AAV9. For example, an AAV VP capsid polypeptide comprising a 581-589 region of SEQ ID NO: 3472 or SEQ ID NO: 3297 may preferentially target a skeletal muscle tissue at a DNA enrichment of at least 30-fold greater than the wild type AAV9.

In some embodiments, an AAV VP capsid polypeptide preferentially targets a skeletal muscle tissue at an RNA enrichment of at least 2-fold greater than a wild type AAV9. For example, an AAV VP capsid polypeptide comprising a 581-589 region of SEQ ID NO: 5138, SEQ ID NO: 3472, SEQ ID NO: 1934, SEQ ID NO: 3283, SEQ ID NO: 5817 (KTGTRDSAR), SEQ ID NO: 278, SEQ ID NO: 1634, SEQ ID NO: 1391, or SEQ ID NO: 1537 may preferentially target a skeletal muscle tissue at an RNA enrichment of at least 2-fold greater than a wild type AAV9.

In some embodiments, an AAV VP capsid polypeptide preferentially targets a skeletal muscle tissue at an RNA enrichment of at least 10-fold or greater than the wild type AAV9. For example, an AAV VP capsid polypeptide comprising a 581-589 region of SEQ ID NO: 5138, SEQ ID NO: 3472, SEQ ID NO: 1934, SEQ ID NO: 3283, SEQ ID NO: 5817, or SEQ ID NO: 278 may preferentially target a skeletal muscle tissue at an RNA enrichment of at least 10-fold or greater than the wild type AAV9.

In some embodiments, an AAV VP capsid polypeptide preferentially targets a skeletal muscle tissue at an RNA enrichment of at least 25-fold or greater than the wild type AAV9. For example, an AAV VP capsid polypeptide comprising a 581-589 region of SEQ ID NO: 5138, SEQ ID NO: 3472, SEQ ID NO: 1934, or SEQ ID NO: 3283 may preferentially target a skeletal muscle tissue at an RNA enrichment of at least 25-fold or greater than the wild type AAV9.

In some embodiments, an AAV VP capsid polypeptide preferentially targets a skeletal muscle tissue at a DNA enrichment of at least 4-fold greater than a wild type AAV5. For example, an AAV VP capsid polypeptide comprising a 581-589 region of SEQ ID NO: 3472, SEQ ID NO: 3297, SEQ ID NO: 2661, SEQ ID NO: 4545, SEQ ID NO: 348, SEQ ID NO: 18, SEQ ID NO: 4349, SEQ ID NO: 293, SEQ ID NO: 5017, SEQ ID NO: 4955, or SEQ ID NO: 4366 may preferentially target a skeletal muscle tissue at a DNA enrichment of at least 4-fold greater than a wild type AAV5.

In some embodiments, an AAV VP capsid polypeptide preferentially targets a skeletal muscle tissue at a DNA enrichment of at least 5-fold greater than the wild type AAV5. For example, an AAV VP capsid polypeptide comprising a 581-589 region of SEQ ID NO: 3472, SEQ ID NO: 3297, SEQ ID NO: 2661, SEQ ID NO: 4545, or SEQ ID NO: 348 may preferentially target a skeletal muscle tissue at a DNA enrichment of at least 5-fold greater than the wild type AAV5.

In some embodiments, an AAV VP capsid polypeptide preferentially targets a skeletal muscle tissue at an RNA enrichment of at least that of a wild type AAV5. For example, an AAV VP capsid polypeptide comprising a 581-589 region of SEQ ID NO: 5138, SEQ ID NO: 3472, SEQ ID NO: 1934, SEQ ID NO: 3283, SEQ ID NO: 5817, or SEQ ID NO: 278 may preferentially target a skeletal muscle tissue at an RNA enrichment of at least that of a wild type AAV5.

In some embodiments, an AAV VP capsid polypeptide preferentially targets a skeletal muscle tissue at an RNA enrichment of at least 3-fold greater than the wild type AAV5. For example, an AAV VP capsid polypeptide comprising a 581-589 region of SEQ ID NO: 5138, SEQ ID NO: 3472, SEQ ID NO: 1934, or SEQ ID NO: 3283 may preferentially target a skeletal muscle tissue at an RNA enrichment of at least 3-fold greater than the wild type AAV5.

In some embodiments, the engineered AAV VP capsid polypeptide comprises a polypeptide sequence represented by the formula: (A)-(X)—(B) wherein: (A) is the polypeptide sequence of SEQ ID NO: 5812 (VAYNVGGQMATNNQSSTTAP, corresponding to residues 561 to 580 of SEQ ID NO: 2); (X) is the 581-589 region having a polypeptide sequence selected from the list of polypeptides in TABLE 12 that is expected to confer muscle tissue tropism or preference on a recombinant AAV virion (rAAV), based on a secondary screen; and (B) is the polypeptide sequence of SEQ ID NO: 5813 (IVPGSVWMERDVYLQGPIWA, corresponding to residues 590 to 609 of SEQ ID NO: 2); and wherein the capsid polypeptide is capable of assembling into the rAAV and, the capsid does not have the sequence of any of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, or SEQ ID NO: 8. Also encompassed herein are rAAVs composed of engineered AAV5 VP2 capsid polypeptides and engineered AAV5 VP3 capsid polypeptides comprising a 581-589 region having a sequence selected from the list of polypeptides in TABLE 12 at the region corresponding to amino acid residues 445 to 453 in AAV5 VP2 or the region corresponding to amino acid residues 389 to 397 in AAV5 VP3.

In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence that is at least 75%, at least 77.7%, at least 85%, at least 88.8%, or 100% identical to a sequence provided in TABLE 12. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence that is at least 75%, at least 77.7%, at least 85%, at least 88.8% or 100% identical to SEQ ID NO: 348. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence that is at least 75%, at least 77.7%, at least 85%, at least 88.8% or 100% identical to SEQ ID NO: 2536. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence that is at least 75%, at least 77.7%, at least 85%, at least 88.8% or 100% identical to SEQ ID NO: 4955. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence that is at least 75%, at least 77.7%, at least 85%, at least 88.8% or 100% identical to SEQ ID NO: 5017. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence that is at least 75%, at least 77.7%, at least 85%, at least 88.8% or 100% identical to SEQ ID NO: 4349. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence that is at least 75%, at least 77.7%, at least 85%, at least 88.8% or 100% identical to SEQ ID NO: 293. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence that is at least 75%, at least 77.7%, at least 85%, at least 88.8% or 100% identical to SEQ ID NO: 1576. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence that is at least 75%, at least 77.7%, at least 85%, at least 88.8% or 100% identical to SEQ ID NO: 2661. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence that is at least 75%, at least 77.7%, at least 85%, at least 88.8% or 100% identical to SEQ ID NO: 4964. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence that is at least 75%, at least 77.7%, at least 85%, at least 88.8% or 100% identical to SEQ ID NO: 4314. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence that is at least 75%, at least 77.7%, at least 85%, at least 88.8% or 100% identical to SEQ ID NO: 4995. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence that is at least 75%, at least 77.7%, at least 85%, at least 88.8% or 100% identical to SEQ ID NO: 4366. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence that is at least 75%, at least 77.7%, at least 85%, at least 88.8% or 100% identical to SEQ ID NO: 4545. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence that is at least 75%, at least 77.7%, at least 85%, at least 88.8% or 100% identical to SEQ ID NO: 4961. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence that is at least 75%, at least 77.7%, at least 85%, at least 88.8% or 100% identical to SEQ ID NO: 5206. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence that is at least 75%, at least 77.7%, at least 85%, at least 88.8% or 100% identical to SEQ ID NO: 5092. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence that is at least 75%, at least 77.7%, at least 85%, at least 88.8% or 100% identical to SEQ ID NO: 3472. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence that is at least 75%, at least 77.7%, at least 85%, at least 88.8% or 100% identical to SEQ ID NO: 5075. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence that is at least 75%, at least 77.7%, at least 85%, at least 88.8% or 100% identical to SEQ ID NO: 4059. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence that is at least 75%, at least 77.7%, at least 85%, at least 88.8% or 100% identical to SEQ ID NO: 4354. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence that is at least 75%, at least 77.7%, at least 85%, at least 88.8% or 100% identical to SEQ ID NO: 5060. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence that is at least 75%, at least 77.7%, at least 85%, at least 88.8% or 100% identical to SEQ ID NO: 4288. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence that is at least 75%, at least 77.7%, at least 85%, at least 88.8% or 100% identical to SEQ ID NO: 4952. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence that is at least 75%, at least 77.7%, at least 85%, at least 88.8% or 100% identical to SEQ ID NO: 5138. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence that is at least 75%, at least 77.7%, at least 85%, at least 88.8% or 100% identical to SEQ ID NO: 18. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence that is at least 75%, at least 77.7%, at least 85%, at least 88.8% or 100% identical to SEQ ID NO: 5030. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence that is at least 75%, at least 77.7%, at least 85%, at least 88.8% or 100% identical to SEQ ID NO: 3297. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence that is at least 75%, at least 77.7%, at least 85%, at least 88.8% or 100% identical to SEQ ID NO: 4295. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence that is at least 75%, at least 77.7%, at least 85%, at least 88.8% or 100% identical to SEQ ID NO: 4363. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence that is at least 75%, at least 77.7%, at least 85%, at least 88.8% or 100% identical to SEQ ID NO: 4963.

In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence of SEQ ID NO: 348. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence of SEQ ID NO: 2536. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence of SEQ ID NO: 4955. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence of SEQ ID NO: 5017. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence of SEQ ID NO: 4349. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence of SEQ ID NO: 293. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence of SEQ ID NO: 1576. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence of SEQ ID NO: 2661. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence of SEQ ID NO: 4964. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence of SEQ ID NO: 4314. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence of SEQ ID NO: 4995. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence of SEQ ID NO: 4366. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence of SEQ ID NO: 4545. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence of SEQ ID NO: 4961. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence of SEQ ID NO: 5206. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence of SEQ ID NO: 5092. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence of SEQ ID NO: 3472. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence of SEQ ID NO: 5075. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence of SEQ ID NO: 4059. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence of SEQ ID NO: 4354. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence of SEQ ID NO: 5060. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence of SEQ ID NO: 4288. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence of SEQ ID NO: 4952. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence of SEQ ID NO: 5138. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence of SEQ ID NO: 18. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence of SEQ ID NO: 5030. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence of SEQ ID NO: 3297. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence of SEQ ID NO: 4295. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence of SEQ ID NO: 4363. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence of SEQ ID NO: 4963.

In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence of SEQ ID NO: 348 with 1 or 2 conservative substitutions. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence of SEQ ID NO: 2536 with 1 or 2 conservative substitutions. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence of SEQ ID NO: 4955 with 1 or 2 conservative substitutions. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence of SEQ ID NO: 5017 with 1 or 2 conservative substitutions. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence of SEQ ID NO: 4349 with 1 or 2 conservative substitutions. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence of SEQ ID NO: 293 with 1 or 2 conservative substitutions. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence of SEQ ID NO: 1576 with 1 or 2 conservative substitutions. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence of SEQ ID NO: 2661 with 1 or 2 conservative substitutions. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence of SEQ ID NO: 4964 with 1 or 2 conservative substitutions. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence of SEQ ID NO: 4314 with 1 or 2 conservative substitutions. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence of SEQ ID NO: 4995 with 1 or 2 conservative substitutions. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence of SEQ ID NO: 4366 with 1 or 2 conservative substitutions. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence of SEQ ID NO: 4545 with 1 or 2 conservative substitutions. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence of SEQ ID NO: 4961 with 1 or 2 conservative substitutions. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence of SEQ ID NO: 5206 with 1 or 2 conservative substitutions. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence of SEQ ID NO: 5092 with 1 or 2 conservative substitutions. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence of SEQ ID NO: 3472 with 1 or 2 conservative substitutions. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence of SEQ ID NO: 5075 with 1 or 2 conservative substitutions. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence of SEQ ID NO: 4059 with 1 or 2 conservative substitutions. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence of SEQ ID NO: 4354 with 1 or 2 conservative substitutions. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence of SEQ ID NO: 5060 with 1 or 2 conservative substitutions. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence of SEQ ID NO: 4288 with 1 or 2 conservative substitutions. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence of SEQ ID NO: 4952 with 1 or 2 conservative substitutions. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence of SEQ ID NO: 5138 with 1 or 2 conservative substitutions. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence of SEQ ID NO: 18 with 1 or 2 conservative substitutions. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence of SEQ ID NO: 5030 with 1 or 2 conservative substitutions. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence of SEQ ID NO: 3297 with 1 or 2 conservative substitutions. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence of SEQ ID NO: 4295 with 1 or 2 conservative substitutions. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence of SEQ ID NO: 4363 with 1 or 2 conservative substitutions. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence of SEQ ID NO: 4963 with 1 or 2 conservative substitutions. The conservative substitutions may be at any position in the 581-589 region.

In some embodiments, an AAV VP capsid polypeptide preferentially targets a muscle tissue at a DNA enrichment of at least 20-fold greater than a wild type AAV9. For example, an AAV VP capsid polypeptide comprising a 581-589 region of SEQ ID NO: 348, SEQ ID NO: 2536, SEQ ID NO: 4955, SEQ ID NO: 5017, SEQ ID NO: 4349, SEQ ID NO: 293, SEQ ID NO: 1576, SEQ ID NO: 2661, SEQ ID NO: 4964, SEQ ID NO: 4314, SEQ ID NO: 4995, SEQ ID NO: 4366, SEQ ID NO: 4545, SEQ ID NO: 4961, SEQ ID NO: 5206, SEQ ID NO: 5092, SEQ ID NO: 3472, SEQ ID NO: 5075, SEQ ID NO: 4059, SEQ ID NO: 4354, SEQ ID NO: 5060, SEQ ID NO: 4288, SEQ ID NO: 4952, SEQ ID NO: 5138, SEQ ID NO: 18, SEQ ID NO: 5030, SEQ ID NO: 3297, SEQ ID NO: 4295, SEQ ID NO: 4363, SEQ ID NO: 4963, SEQ ID NO: 4973, SEQ ID NO: 5159, SEQ ID NO: 4960, SEQ ID NO: 4938, SEQ ID NO: 5157, SEQ ID NO: 5023, or SEQ ID NO: 4969 may preferentially target a muscle tissue at a DNA enrichment of at least 20-fold greater than a wild type AAV9.

In some embodiments, an AAV VP capsid polypeptide preferentially targets a muscle tissue at a DNA enrichment of at least 25-fold greater than the wild type AAV9. For example, an AAV VP capsid polypeptide comprising a 581-589 region of SEQ ID NO: 348, SEQ ID NO: 2536, SEQ ID NO: 4955, SEQ ID NO: 5017, SEQ ID NO: 4349, SEQ ID NO: 293, SEQ ID NO: 1576, SEQ ID NO: 2661, SEQ ID NO: 4964, SEQ ID NO: 4314, SEQ ID NO: 4995, or SEQ ID NO: 4366 may preferentially target a muscle tissue at a DNA enrichment of at least 25-fold greater than the wild type AAV9.

In some embodiments, an AAV VP capsid polypeptide preferentially targets a muscle tissue at a DNA enrichment of at least 30-fold greater than the wild type AAV9. For example, an AAV VP capsid polypeptide comprising a 581-589 region of SEQ ID NO: 348 or SEQ ID NO: 2536 may preferentially target a muscle tissue at a DNA enrichment of at least 30-fold greater than the wild type AAV9.

In some embodiments, an AAV VP capsid polypeptide preferentially targets a muscle tissue at an RNA enrichment of at least 5-fold greater than a wild type AAV9. For example, an AAV VP capsid polypeptide comprising a 581-589 region of SEQ ID NO: 5138, SEQ ID NO: 3472, SEQ ID NO: 348, SEQ ID NO: 5052, SEQ ID NO: 5193, SEQ ID NO: 5026, SEQ ID NO: 278, SEQ ID NO: 5163, SEQ ID NO: 293, SEQ ID NO: 4943, SEQ ID NO: 4952, SEQ ID NO: 4317, SEQ ID NO: 5075, SEQ ID NO: 386, SEQ ID NO: 4986, SEQ ID NO: 4995, SEQ ID NO: 4288, SEQ ID NO: 5030, SEQ ID NO: 5206, SEQ ID NO: 5159, SEQ ID NO: 4949, SEQ ID NO: 4338, SEQ ID NO: 5060, SEQ ID NO: 4346, SEQ ID NO: 5215, SEQ ID NO: 5017, SEQ ID NO: 4314, SEQ ID NO: 5080, SEQ ID NO: 4964, SEQ ID NO: 4363, SEQ ID NO: 4379, SEQ ID NO: 5027, SEQ ID NO: 4304, SEQ ID NO: 18, SEQ ID NO: 4349, SEQ ID NO: 4938, or SEQ ID NO: 2536 may preferentially target a muscle tissue at an RNA enrichment of at least 5-fold greater than a wild type AAV9.

In some embodiments, an AAV VP capsid polypeptide preferentially targets a muscle tissue at an RNA enrichment of at least 10-fold or greater than the wild type AAV9. For example, an AAV VP capsid polypeptide comprising a 581-589 region of SEQ ID NO: 5138, SEQ ID NO: 3472, SEQ ID NO: 348, SEQ ID NO: 5052, SEQ ID NO: 5193, SEQ ID NO: 5026, SEQ ID NO: 278, or SEQ ID NO: 5163 may preferentially target a muscle tissue at an RNA enrichment of at least 10-fold or greater than the wild type AAV9.

In some embodiments, an AAV VP capsid polypeptide preferentially targets a muscle tissue at an RNA enrichment of at least 100-fold or greater than the wild type AAV9. For example, an AAV VP capsid polypeptide comprising a 581-589 region of SEQ ID NO: 5138 may preferentially target a muscle tissue at an RNA enrichment of at least 100-fold or greater than the wild type AAV9.

In some embodiments, an AAV VP capsid polypeptide preferentially targets a muscle tissue at a DNA enrichment of at least 5-fold greater than a wild type AAV5. For example, an AAV VP capsid polypeptide comprising a 581-589 region of SEQ ID NO: 348, SEQ ID NO: 2536, SEQ ID NO: 4955, SEQ ID NO: 5017, SEQ ID NO: 4349, SEQ ID NO: 293, SEQ ID NO: 1576, SEQ ID NO: 2661, SEQ ID NO: 4964, SEQ ID NO: 4314, SEQ ID NO: 4995, SEQ ID NO: 4366, SEQ ID NO: 4545, SEQ ID NO: 4961, SEQ ID NO: 5206, SEQ ID NO: 5092, SEQ ID NO: 3472, SEQ ID NO: 5075, SEQ ID NO: 4059, SEQ ID NO: 4354, or SEQ ID NO: 5060 may preferentially target a muscle tissue at a DNA enrichment of at least 5-fold greater than a wild type AAV5.

In some embodiments, an AAV VP capsid polypeptide preferentially targets a muscle tissue at a DNA enrichment of at least 10-fold greater than the wild type AAV5. For example, an AAV VP capsid polypeptide comprising a 581-589 region of SEQ ID NO: 348 may preferentially target a muscle tissue at a DNA enrichment of at least 10-fold greater than the wild type AAV5.

In some embodiments, an AAV VP capsid polypeptide preferentially targets a muscle tissue at an RNA enrichment of at least that of a wild type AAV5. For example, an AAV VP capsid polypeptide comprising a 581-589 region of SEQ ID NO: 5138, SEQ ID NO: 3472, SEQ ID NO: 348, SEQ ID NO: 5052, SEQ ID NO: 5193, SEQ ID NO: 5026, SEQ ID NO: 278, SEQ ID NO: 5163, SEQ ID NO: 293, SEQ ID NO: 4943, SEQ ID NO: 4952, SEQ ID NO: 4317, SEQ ID NO: 5075, SEQ ID NO: 386, SEQ ID NO: 4986, or SEQ ID NO: 4995 may preferentially target a muscle tissue at an RNA enrichment of at least that of a wild type AAV5.

In some embodiments, an AAV VP capsid polypeptide preferentially targets a muscle tissue at an RNA enrichment of at least 2-fold greater than the wild type AAV5. For example, an AAV VP capsid polypeptide comprising a 581-589 region of SEQ ID NO: 5138, SEQ ID NO: 3472, SEQ ID NO: 348, and SEQ ID NO: 5052 may preferentially target a muscle tissue at an RNA enrichment of at least 2-fold greater than the wild type AAV5.

In some embodiments, the engineered AAV VP capsid polypeptide comprises a polypeptide sequence represented by the formula: (A)-(X)—(B) wherein: (A) is the polypeptide sequence of SEQ ID NO: 5812 (VAYNVGGQMATNNQSSTTAP, corresponding to residues 561 to 580 of SEQ ID NO: 2); (X) is the 581-589 region having a polypeptide sequence selected from the list of polypeptides in TABLE 5 (SEQ ID NO: 4238-SEQ ID NO: 4933) that is expected to confer muscle tissue tropism or preference on a recombinant AAV virion (rAAV), based on a primary screen; and (B) is the polypeptide sequence of SEQ ID NO: 5813 (IVPGSVWMERDVYLQGPIWA, corresponding to residues 590 to 609 of SEQ ID NO: 2); and wherein the capsid polypeptide is capable of assembling into the rAAV and, the capsid does not have the sequence of any of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, or SEQ ID NO: 8. Also encompassed herein are rAAVs composed of engineered AAV5 VP2 capsid polypeptides and engineered AAV5 VP3 capsid polypeptides comprising a 581-589 region having a sequence selected from the list of polypeptides in TABLE 5 (SEQ ID NO: 4238-SEQ ID NO: 4933) at the region corresponding to amino acid residues 445 to 453 in AAV5 VP2 or the region corresponding to amino acid residues 389 to 397 in AAV5 VP3.

TABLE 5

Sequences of 581-589 Regions for Muscle

Tropism

SEQ ID

NO
Sequence

4238
AACFKQLMQ

4239
AACQWQSQR

4240
AACSFAIQR

4241
AADKPAHNP

4242
AAIQAMACG

4243
AALSINDQP

4244
AAMPHQKRE

4245
AAQYVMSLA

4246
AAREGVKLE

4247
ACATYQMTW

4248
ADCSIIYPE

4249
ADPRPAKGS

4250
AFQRTYQFH

4251
AGPFMVGFW

4252
AGTWENMSG

4253
AHILMGHNP

4254
AHLWYHQCA

4255
AIEQGLTMA

4256
AIFCAHYSY

4257
AKMYWDGHT

4258
AMFPQLWAQ

4259
AMNPKQMCF

4260
ANIPQTAWN

4261
APYCPTYIS

4262
AQGCMHRWQ

4263
AQIMLPSSK

4264
AQSTARHIE

4265
AQTEFGFME

4266
ARCLSVIEQ

4267
ARQLESFWT

4268
ASFYQLLIP

4269
ASIVLRDSF

4270
ASNPLNCPN

4271
ASRFEDLWE

4272
ASTEWDMHN

4273
ASVELMKVC

4274
ASYLWCTMP

4275
ATDRIRTMM

4276
ATELGKDSG

4277
ATGRAAYHE

4278
ATIWPVESW

4279
ATLCTQRGS

4280
ATLFYNAFW

4281
ATTAVGDAL

4282
ATWISHNAS

4283
AVALSFVSS

4284
AVCEYLRMD

4285
AVTQWMLYA

4286
AYPERLYSD

4287
AYRQMCIER

4288
CACKVSMSG

4289
CACSIGHHR

4290
CAFQMYDSP

4291
CAKQYDKGI

4292
CAMLLQAVQ

4293
CAQPQHNWC

4294
CATFVNKLS

4295
CAVKFHAYD

4296
CDGELGRKC

4297
CDGRGIPGC

4298
CDGSCKYYQ

4299
CDWMMANLG

4300
CEPEGWIRA

4301
CEPFNINWW

4302
CESHEYRGH

4303
CFMPMMYYE

4304
CFRLHEMHQ

4305
CFRNILDDS

4306
CGRCNWRGY

4307
CHGYTWERP

4308
CHWFRCSCA

4309
CIAFVKQCC

4310
CIARTDYQP

4311
CIGKWPISR

4312
CIHRLIEDN

4313
CIMSMMWSQ

4314
CIMYRSEAS

4315
CIQATKAYC

4316
CIRHAQDCY

4317
CITHQQMAV

4318
CKEWYVRDR

4319
CKPANWVRY

4320
CKTHIWGFP

4321
CKVIVNIGA

4322
CKWIPWQCD

4323
CKWPCTWNS

4324
CKYCCDSHE

4325
CLMEKISDF

4326
CLPFFINRW

4327
CMESYSISE

4328
CMINLFDAC

4329
CMNESGQFH

4330
CMQRPHTAP

4331
CMSQFHLER

4332
CNETPWHKG

4333
CPAILNVAC

4334
CPKWSNHDQ

4335
CPMKHIQDR

4336
CPQQDDWPG

4337
CPWFGFLGW

4338
CQATFSRAY

4339
CQCNWWGRP

4340
CQQRYGHGK

4341
CRCPLSIMS

4342
CSDLWRIPF

4343
CSHKKEQYD

4344
CSICHYDHA

4345
CSIYQQMFQ

4346
CSMSTQRGA

4347
CSSLFMFNT

4348
CSSNWPMGI

4349
CSSYLVTAN

4350
CSTARGYDL

4351
CSTESSNSM

4352
CSTTYGRKE

4353
CSWYTHAGA

4354
CTFMVYSQG

4355
CTFQSTSAW

4356
CTKTFDRTE

4357
CTNIQSVFH

4358
CTPTQWGSA

4359
CVATKSRML

4360
CVAVPLSMS

4361
CVGFWTQMN

4362
CVGTVFHTA

4363
CVHIKVDGT

4364
CVHVKEQSQ

4365
CVIYNDMQI

4366
CVLFRHVDQ

4367
CVMPLGMAA

4368
CVNMKYVDS

4369
CVQSEWGSK

4370
CVQWQMNDA

4371
CVSLHSISM

4372
CVYQFDFTQ

4373
CWELLTWQW

4374
CWFDFMITE

4375
CWGYTPDWN

4376
CYAPKFIGD

4377
CYIDYRMSY

4378
CYLEIPNNF

4379
CYNLFNSMY

4380
CYSLRPDGP

4381
DAKAHIFSF

4382
DASECKWYN

4383
DDLIPINPW

4384
DDVAEKQCC

4385
DEHTIDVMQ

4386
DENGTWPHG

4387
DEPENMYSS

4388
DFFWPRPKA

4389
DFPRPENHW

4390
DHECVHGSG

4391
DHELKGACS

4392
DHLWFSSPG

4393
DHNPIMMWC

4394
DHVECWPQG

4395
DIYPWVCYY

4396
DKCHWASEG

4397
DLGNDRWRF

4398
DMHSYNNAK

4399
DMKGRQDGA

4400
DQSPLRTQS

4401
DRMMYARNG

4402
DRNQLYSSF

4403
DSACAGWGL

4404
DSDYVLVDY

4405
DSIAKGVAH

4406
DSPFGWFNC

4407
DSPWSKWQP

4408
DTKDYPKQS

4409
DTKPVEEVK

4410
DTLHKLSDC

4411
DTRCYGEMF

4412
DTVQMLCQY

4413
DVHCICRYD

4414
DVHKWSCAK

4415
DVICRHHGM

4416
DVILSQEYN

4417
DVLKVRIQE

4418
DVRIPCCQH

4419
DVTMYAAWS

4420
DVVLCNQWR

4421
DVWNDLNSM

4422
DWKLTYPQG

4423
DWMVVRKEG

4424
DWPHIMFNY

4425
DYTFKRYVC

4426
EAEQCMIGQ

4427
EAGWFNHST

4428
EAHDLAGNM

4429
EAHKMISTR

4430
EAKASAHTC

4431
EAQLYPDML

4432
EASCMSIWT

4433
ECGHIFYPA

4434
ECIDVMQNR

4435
ECQLVKTVP

4436
ECTVICSSS

4437
ECVGGNIWG

4438
EERKCNLGC

4439
EFRNYWSPY

4440
EFWSVKNHD

4441
EHHICSKSA

4442
EHMKLFLSE

4443
EIATHITFQ

4444
EIGMVMDMN

4445
EIMALDSPC

4446
EKEWGTPMW

4447
EKFPPHGDV

4448
ELKWQVAQF

4449
EMGPYPYPS

4450
EMKIDEHWP

4451
EMLLQPMYG

4452
ENCVISVHH

4453
ENGFIHKQS

4454
ENQPYWQVY

4455
EPEGIYQPM

4456
EQDDYMIPP

4457
EQESWEMGP

4458
ESESANHEG

4459
ESFAKHDQN

4460
ESGSQSGQA

4461
ESSQVNWHR

4462
ESTGESTLG

4463
ETQPHADYE

4464
ETTKWEAQH

4465
EVGCSWMWG

4466
EVGNYPRIP

4467
EVGRQQWMD

4468
EVKADIAQM

4469
EVKSANATW

4470
EVPAQTMQS

4471
EVQKRLVAE

4472
EVTCESKIE

4473
FCDLAHHYE

4474
FCRDFVKDM

4475
FDKWPKPQC

4476
FDPADVQKW

4477
FFESWQYPY

4478
FHWREPYVE

4479
FKRHEIPEY

4480
FMWITISNY

4481
FRQPYVWDK

4482
FSCSLYRHG

4483
FTTSQTLAE

4484
FVGSRNCID

4485
FYFQNIHPW

4486
GCYQSNVIP

4487
GDCPYSMQP

4488
GDGHGMWKM

4489
GDPKSKHDC

4490
GEHHCKFPM

4491
GEKWELYPM

4492
GEPRPNYVD

4493
GFEFMLNCP

4494
GGDRWLGYW

4495
GGEDEKPGC

4496
GGHFGHITW

4497
GHDFTNQDR

4498
GHFLDRWRD

4499
GKFCMPRNY

4500
GKKCGWPND

4501
GKSDMDCVE

4502
GKWQQQMVL

4503
GNPNCCLTG

4504
GPCCKWCTE

4505
GQNHVIVGA

4506
GRADCSAHP

4507
GSAQNMFGW

4508
GSCLKIAQE

4509
GSGLHMGYK

4510
GSRDSPISF

4511
GTAPPGTYS

4512
GTATMMHTH

4513
GVDLDNDRE

4514
GVFPMLMHS

4515
GVGWDSCHN

4516
GVIYRHHDP

4517
GVNFEDGAY

4518
GVTERFCPD

4519
GVWSKNTPF

4520
GWMSHEYPF

4521
GWWPGKPMD

4522
GYPRDPHET

4523
HAAMSGEGM

4524
HAEWACSGR

4525
HASYMCGLW

4526
HCEAVLVHG

4527
HDIRNVRML

4528
HDRWPKNCM

4529
HEPFRISGE

4530
HHWLGDCNS

4531
HIGTQYRQY

4532
HMRKTLIWK

4533
HPQFKSHEY

4534
HQYWMIPDG

4535
HSEPKLDIP

4536
HTAQLMAHE

4537
HTIAANMVA

4538
HTPTDAHVG

4539
HTTPAAFHN

4540
HVDPGFASL

4541
HWPFPWMRI

4542
IAFMKSVEG

4543
IAMAYKGQI

4544
IAYYEKMWS

4545
ICMSRTQMK

4546
IDPQGWLTH

4547
IDSFGELRE

4548
IGVRYAVQE

4549
IIRVQVDGS

4550
IMCKCPVWC

4551
IMEIVGIGN

4552
INSRTNSMC

4553
IPWPPVKGD

4554
IQKMWDHFQ

4555
IQRTMEANG

4556
IQVVKMAHG

4557
ISADQSMQD

4558
ISAWQDFDA

4559
ISEIHYTQC

4560
ITGGFNKDK

4561
ITNWFQQDK

4562
ITQVWCSDG

4563
ITTCYPNQT

4564
IVCQSHVMN

4565
IVTQRHYQI

4566
IYRPVDSFR

4567
KACDSKESW

4568
KANEDLKQF

4569
KCDYEHYFA

4570
KCKCPKMPQ

4571
KEHVYGMCP

4572
KFMSQEFGP

4573
KGCLLDSDH

4574
KGMDTTIVP

4575
KHERDPPQY

4576
KKFQRALTC

4577
KMEPVELFT

4578
KMQFIDACA

4579
KMRDGTEQK

4580
KNALYVSYQ

4581
KPPWINYPR

4582
KQAQALEYF

4583
KQDCYFWHT

4584
KSWDTIQYT

4585
KSYICEDFF

4586
KTEWLMWYN

4587
KTVIMEDFK

4588
KVTDCNQSG

4589
KWMHYDSIC

4590
LACQIDRFH

4591
LAELYLNMR

4592
LAGNQMDAD

4593
LAHGRCFHE

4594
LALSANLGG

4595
LAPPITWTD

4596
LASLAMAKA

4597
LATHALMAH

4598
LAWSQLLTP

4599
LCEPIAIRD

4600
LCIRSLANG

4601
LCMRYGSKA

4602
LDRPRFTDW

4603
LGQPVDDYS

4604
LHNLYEMSP

4605
LIEPDSFRG

4606
LKAEKNTMS

4607
LMVDVQRCK

4608
LMYCRYCPS

4609
LNAEWDEWT

4610
LNFLWAMLW

4611
LPCSLLRGD

4612
LPIPPVPEY

4613
LPRLNFESK

4614
LPSAVVMHE

4615
LQGLSWLFP

4616
LQNLFSYQE

4617
LQQQTHWYG

4618
LRSFIDTMD

4619
LSFCLGHND

4620
LSMIPMAQW

4621
LSYWSGNAA

4622
LTAPMEEDN

4623
LTCLQDGDA

4624
LTCQYQGGQ

4625
LTMGHTMQA

4626
LVHEPGAVT

4627
LVHQKWECK

4628
LVHREECQI

4629
LVHTIMMGS

4630
LYHAYSSGF

4631
MAANVSEEK

4632
MACAFGTNF

4633
MACWKNATE

4634
MAQIGPLHH

4635
MASTCTQQD

4636
MATRLFDSG

4637
MDFNIDWHT

4638
MEASNGFGL

4639
MEDSCRPGF

4640
MHMPCRVGK

4641
MIEENEWFG

4642
MILPIHHQQ

4643
MKQWPVFPS

4644
MLGVSTWEW

4645
MMRIDGLGP

4646
MNATQMYGQ

4647
MNGYYHVMN

4648
MNLGKNMWN

4649
MNMEYCQFG

4650
MPRLTTYTW

4651
MQGFFTRGF

4652
MQNKQASTP

4653
MSDLWPFSY

4654
MTACHTMGR

4655
MTCDFSAAE

4656
MTCHFQHMH

4657
MTCNWGLNG

4658
MTKPGIPNF

4659
MTMDWQFQG

4660
MTSIVMENA

4661
MTSRVDRTD

4662
MVENWAKDM

4663
MVEQFACLP

4664
MVLCYNQMV

4665
MVNMHVFHS

4666
MVSVVRQGS

4667
MVTEYYHDV

4668
MWTMFNGQF

4669
MYDTACVIG

4670
MYWPDISRN

4671
NATPWNFGT

4672
NAVEKMAFN

4673
NAYMQQAQG

4674
NCMQMHWDG

4675
NCNCDEYHY

4676
NDGFDFWTC

4677
NELNNGREF

4678
NEPWPDHLP

4679
NFCYKNIYA

4680
NFMCSAGHT

4681
NGPWDWWIN

4682
NHFCGEWLQ

4683
NHWILSCAE

4684
NIERTVKHM

4685
NIKHEEMWN

4686
NIMTVDMWP

4687
NIPFKERSW

4688
NKPCWPCPH

4689
NKSDMSCYG

4690
NMSVYMKLG

4691
NPHARTVNG

4692
NPMPDWNFT

4693
NPSMGYFGF

4694
NQRIMDAGL

4695
NRHLGNNPW

4696
NRMNYDAWG

4697
NRWSVYRAE

4698
NTGCLKQAT

4699
NTLRHCVHT

4700
NTMQAMDVH

4701
NTQWNQKVP

4702
NVATQFWDM

4703
NVGMYGQKA

4704
NVHCQAASQ

4705
NVIEYQNRD

4706
NVPPPKFMA

4707
NYALLGDWE

4708
PCDVNHTSG

4709
PCEFSTCVR

4710
PCKGPPDIS

4711
PCNDHPCAG

4712
PCPCKGDAA

4713
PDAERKPTW

4714
PDCEKYENE

4715
PDCFPWDHN

4716
PDCGEWYPG

4717
PDGILNTWW

4718
PDICMQSGW

4719
PDLWTWQSD

4720
PDPWFMMRP

4721
PEAELPDTS

4722
PEKMRWMHH

4723
PELTLVNPG

4724
PFFPIDELG

4725
PFNCKWGMF

4726
PFNDHESEK

4727
PGDQNSYPG

4728
PGDRQEKMY

4729
PGKEFQPKW

4730
PGLLDNIPS

4731
PGPRPGEMC

4732
PHMPPFHCD

4733
PIAPHNDYW

4734
PIDQNPKFG

4735
PIPSPYCGF

4736
PKGSKYHEW

4737
PKIEPPFHS

4738
PKRGDSLPN

4739
PLKSGWTRT

4740
PLRTEISPW

4741
PNECARGDC

4742
PNFFNYHTS

4743
PNGLAELKP

4744
PPHCL WNTI

4745
PPIMMTIAN

4746
PPISHYWFD

4747
PPKCHQWQW

4748
PPLDTEHYF

4749
PPLISGFET

4750
PPNDRRFYD

4751
PPPCLEAPY

4752
PPPMQGSRM

4753
PPPWHQFDE

4754
PPRCEMLCM

4755
PPRMPYHPY

4756
PPVRMLFKQ

4757
PPWYHKGNS

4758
PQYEQYWQE

4759
PREYHPEEP

4760
PRFCMWCCG

4761
PRLICHERS

4762
PSIHGWELG

4763
PVKSEMWPT

4764
PWERWEHKG

4765
PYHPPTSGN

4766
PYLPDGNPM

4767
PYSCIPDGC

4768
QAACKIADL

4769
QCEAVRRCR

4770
QCLLIHEVG

4771
QCTKLHDWH

4772
QEKAMDNFQ

4773
QEYSQWWDN

4774
QFGTHHRHN

4775
QFTHMNWLQ

4776
QGCHLMTGQ

4777
QGDFCQQMN

4778
QGGWNSTCE

4779
QGKENNVEE

4780
QHDSKVAWM

4781
QHVHKDGIE

4782
QHYQPGPHN

4783
QIEYAQMVG

4784
QIQPVSCPN

4785
QKQYQDCVT

4786
QMAHVQYQQ

4787
QNIQMHFLG

4788
QNRETIVGG

4789
QPMTREMPP

4790
QQHQYTSKQ

4791
QSCMKIHHA

4792
QSISYTHND

4793
QSNLKKEDH

4794
QSSIENRFN

4795
QSSYYYAQS

4796
QSVKYNWGH

4797
QTDVTQSMY

4798
QTECLKWMR

4799
QTGLYNVMV

4800
QTHTQTYSW

4801
QVDDGKGPE

4802
QVIAVQDFR

4803
QVVECQHIG

4804
QVWDYERGI

4805
QYCPNSLEF

4806
QYQQSTVSN

4807
QYRNIDEDY

4808
RDMCQWDQP

4809
RGVHVEVEE

4810
RMQPINARI

4811
RMVGLEKAY

4812
RNQLKIDDM

4813
RSTGILEHC

4814
RSTSDPAML

4815
RTAWDQPFS

4816
RTNEAMMWN

4817
RVDYCYYFQ

4818
SACHNNTLQ

4819
SAGYLQKVH

4820
SASEAIAHQ

4821
SCCQQFNSG

4822
SDNGEYYRI

4823
SDNIQVWPW

4824
SDRQRFIPW

4825
SFCGLLTGR

4826
SGCPYWWPS

4827
SGKEQDSWN

4828
SHDVSGSCE

4829
SHFCYSMQE

4830
SISWKHSEA

4831
SITVQHEIS

4832
SKQCTGKEA

4833
SLAWNRVIQ

4834
SLPNEGYYP

4835
SMAWRNVIP

4836
SMCYVHGKE

4837
SMEKPSHYQ

4838
SPWYDQLCW

4839
SSADNVCWS

4840
SSCQFVADL

4841
SSDQYEEMN

4842
SSLCCNVGW

4843
STDQYNQDF

4844
STISEGSKN

4845
STLRHALIE

4846
SVAHSPMQC

4847
SVAMKQIEE

4848
SVGDNSFEH

4849
SVLTPHVTF

4850
SVTFKHQHQ

4851
SYKEYLHDE

4852
SYMWLQKGQ

4853
SYSIKQFHP

4854
TAAFMTTRD

4855
TACKWVHAC

4856
TANPTQGMQ

4857
TAVDHGLFH

4858
TCEPAVFGN

4859
TCEVYRQEC

4860
TCMRYNQHH

4861
TCQIAHQEA

4862
TECISKYPT

4863
TGHQYEWGY

4864
THHSSNQFF

4865
TKTATDFWQ

4866
TLQWMNAYR

4867
TMGMKHACC

4868
TPEHSVPGH

4869
TPKCGLWKD

4870
TQDEQRTMP

4871
TQHPYMKWH

4872
TSCEWDMMV

4873
TSMCYRTEE

4874
TTAFIGMHF

4875
TTAVLRENG

4876
TTLAVAHTG

4877
TVAGWMSHN

4878
TVAQSEAEF

4879
TVDMVNREQ

4880
TVFRVVQCA

4881
TVGHPGHAC

4882
TVHMWDQHA

4883
TWRMKGDDK

4884
TYNLCTLQP

4885
VAGIHDVVP

4886
VAKQLDCLG

4887
VATELPADY

4888
VCDQMQSFN

4889
VGIEMMGSG

4890
VHTHIIQDL

4891
VIKNCYEAQ

4892
VKAELHKTY

4893
VKEHSHFCP

4894
VKNTVHEMQ

4895
VKQLFTKDN

4896
VKSQSNGWN

4897
VNFQYNLGI

4898
VRAVKDTEG

4899
VSNHRPLMP

4900
VSQGIPKCE

4901
VTQMGHMMV

4902
VTQPKTDQT

4903
VTYGNMEDR

4904
VVCRMSGQD

4905
VVGNANEQK

4906
VVSKCEEMG

4907
WDEKPYFGF

4908
WDWWEVHRA

4909
WENQGDLTR

4910
WFQQSIMFI

4911
WGMPPKDNM

4912
WGPQNLFQG

4913
WLWWEEPLW

4914
WNIAPWDCS

4915
WTYMEASDF

4916
YCPKDDMVY

4917
YCPSKLPRN

4918
YEIKPEIVG

4919
YEQNSMWMC

4920
YGAPATWCD

4921
YGKIKIYEW

4922
YGPDLMRNE

4923
YIDRLTYSF

4924
YKESDWKIC

4925
YMPVLKDQA

4926
YPDLKEPMS

4927
YPGLRYTRS

4928
YPPVDQTPY

4929
YQPRNECIQ

4930
YQYPHELHY

4931
YSNVGSRYA

4932
YTHKLEAGN

4933
YWDNQWLKI

In some embodiments, the engineered AAV VP capsid polypeptide comprises a polypeptide sequence represented by the formula: (A)-(X)—(B) wherein: (A) is the polypeptide sequence of SEQ ID NO: 5812 (VAYNVGGQMATNNQSSTTAP, corresponding to residues 561 to 580 of SEQ ID NO: 2); (X) is the 581-589 region having a polypeptide sequence selected from the list of polypeptides in TABLE 6 (SEQ ID NO: 4934-SEQ ID NO: 5233) with increased likelihood of conferring muscle tissue tropism or preference on a recombinant AAV virion (rAAV); and (B) is the polypeptide sequence of SEQ ID NO: 5813 (IVPGSVWMIERDVYLQGPIWA, corresponding to residues 590 to 609 of SEQ ID NO: 2); and wherein the capsid polypeptide is capable of assembling into the rAAV and, the capsid does not have the sequence ofany of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, or SEQ ID NO: 8. Also encompassed herein are rAAVs composed of engineered AAV5 VP2 capsid polypeptides and engineered AAV5 VP3 capsid polypeptides comprising a 581-589 region having a sequence selected from the list of polypeptides in TABLE 6 (SEQ ID NO: 4934-SEQ ID NO: 5233) at the region corresponding to amino acid residues 445 to 453 in AAV5 VP2 or the region corresponding to amino acid residues 389 to 397 in AAV5 CP3.

TABLE 6

Additional Sequences of 581-589 Regions

for Muscle Tropism

SEQ ID

NO
Sequence

4934
CVMSLLKVP

4935
CWVSYPFWM

4936
CVMPYSNHP

4937
PPIKDMLFK

4938
CVLNWHVGS

4939
PFPPPHKTL

4940
GDPPPLSFK

4941
CVRSLFWVF

4942
CIVMYSNKY

4943
CIVFYMKNQ

4944
CWKWQMMFG

4945
CVQQQFSAM

4946
CIVPNKFMW

4947
CVNYPNQAF

4948
CVKYSNQQG

4949
CYHQVFSQG

4950
CVMNPMVFK

4951
CVKQRGQRS

4952
CVILSTRDK

4953
CWKPWYLHG

4954
CVMGSYLSM

4955
CVWSQILGG

4956
CIKHWFVKF

4957
CIQLKRKMH

4958
CIQQLMWQN

4959
CVGPYRFGG

4960
CWREFSFQN

4961
CVYSVWKTS

4962
CVKLHQFEE

4963
CIMRWNSYG

4964
CVGKYGGHS

4965
CVQLVKEIG

4966
CVAMLHHYY

4967
CVMVPKGAG

4968
EDWMDMDLK

4969
CIGMKMQNG

4970
CINFIMKLG

4971
RIQEPPFPW

4972
CVLVFSNSG

4973
CAYMQQMHG

4974
CYFLIQQNF

4975
CAVPQVYQS

4976
RIKEPPDPK

4977
CVYLKGSLC

4978
CVYHKEMTS

4979
CVNFKTRQN

4980
CDDFKKKMT

4981
CISNLRMHG

4982
CWSVMPMAG

4983
CVNKVHSYN

4984
CSVLKHHGW

4985
CIKFFPIQG

4986
CVFSTSFGE

4987
CVSLYMDKL

4988
CVSHSHHGS

4989
CQMMPMKMD

4990
CMVPYNMYQ

4991
CWSQNQQGH

4992
CVFTIFEAK

4993
CVKQYMEMN

4994
CVMTTPLWF

4995
CVGYRVHSP

4996
CVLSIRIIS

4997
CVEYFLLHP

4998
CPRPLMKNF

4999
CVGLLEHSV

5000
CISPVMMYH

5001
CVVMSQMWM

5002
CYNMYKMWS

5003
QRPHRFPRH

5004
CVMGKTVFN

5005
CVHFKQFTW

5006
CVRWKHFGY

5007
CGNYIMYYK

5008
CFICIMPKT

5009
CVIYTRKHE

5010
CITRQQNEQ

5011
CILQMIILG

5012
MVEEPVGPL

5013
CVSMLGNHP

5014
CIMWKKPAD

5015
CVASYTLKP

5016
CVFQDMLFK

5017
CFYKIQHKG

5018
CTVFPIQSG

5019
CWVYMQGPS

5020
CISQNPMVY

5021
CTSPPRMWD

5022
CVQMMWPFP

5023
CVYTWGMQG

5024
CVHVHQLYG

5025
CIKLGNIIM

5026
CTRSLLGMG

5027
CVIRSSDQE

5028
WHPGRNEKC

5029
CSFNIHKHT

5030
CVSQMGKWP

5031
PIENPDQRI

5032
CITFYGMWW

5033
CVYTNVTSN

5034
CVKQLSYWQ

5035
CWGYPQVLM

5036
CIKFKSPSD

5037
CVTVSGRDY

5038
CVVWPVLGQ

5039
CVSWKNSGQ

5040
CVLRMGTFC

5041
CVGEFDSHR

5042
CIMWYTNNP

5043
CVIWQQITL

5044
CVVVYLKMC

5045
CVFPQVGKQ

5046
CVTQLFHQL

5047
HDESKSEPE

5048
CTRHMFRLG

5049
CIHKHPVSP

5050
CHFHQMIFY

5051
CTIPMQIIQ

5052
CVMMMNTKF

5053
CIMLHPVSF

5054
CYKYWPMHL

5055
CVVQNLFSG

5056
CVLNYFQFG

5057
CWYYRFKKN

5058
PPLSFRPIP

5059
CWKSMQFQY

5060
CVVQWGKWN

5061
DVEDDMEDN

5062
CFIPYYKMG

5063
KPEWWGILK

5064
CWNLFQISR

5065
CVKEFMMGC

5066
ADDDEDMEI

5067
CFWIKKNEI

5068
CVGSQNHIT

5069
CAMPYHFYW

5070
CVQMKVLFG

5071
CWMNMFSKH

5072
CVVNQHKFI

5073
CIKFEWRHN

5074
PRTPPREQR

5075
CINKYSGNT

5076
FHPKPHPQN

5077
CVGSQLHAM

5078
CIFMNGFMG

5079
REPNPLKSL

5080
CVGRQLMYS

5081
CVSIHQKNS

5082
QNGINNFPD

5083
CVSHSTFQS

5084
CWPSMQLNY

5085
CWILMLQMY

5086
CVGAGGKVT

5087
CVQHFMEHI

5088
CVIHIMMYD

5089
CWGYIHNFL

5090
HFRQPWPFK

5091
PHGEPRKIS

5092
CISNYLKQG

5093
CVWLKLFWY

5094
CTHMNLLHM

5095
CVMFREGSR

5096
CTIFMKSSN

5097
CVMTLVMMC

5098
CITKTKGQS

5099
CVIEILLWH

5100
CIMMRIPCG

5101
CVRSQLCNE

5102
CVGLKGIFE

5103
CVFWLGIRS

5104
CVGLLLFHG

5105
CVHPFVSWK

5106
CVQQTMFNL

5107
CVKTQGFIH

5108
CWQFFMSLV

5109
CIVPNQTRM

5110
RNDWKWPQE

5111
CRLYYIQHP

5112
MPFFDWIFK

5113
CVTHIQTGY

5114
PHINKWKMY

5115
IIMDQDSPK

5116
CWQKRDILF

5117
MVIFKNGKP

5118
CFWIHKMAA

5119
CAFPLNRQK

5120
LMMCPIHQT

5121
CMGRQFLQS

5122
WKNRDEKNI

5123
CIILLGSRD

5124
RTNYRPPAC

5125
CIMQYIKMY

5126
NIRQYMLQN

5127
CVFQKMNSD

5128
CVQLHKNHG

5129
CVFKMEPTE

5130
CSNIWVPKM

5131
CNVSYMFMG

5132
CVRRSLGLP

5133
CWYSTFKRG

5134
CVAMHRHNS

5135
CIIVKISKG

5136
WFVWIYPLK

5137
CGPYIVSLR

5138
CVSLFLHSG

5139
CMHQLRNRF

5140
HNYLPRNRP

5141
CVGIIQMGF

5142
CIKLNHHHK

5143
CWRHNIISP

5144
WIFFKDFMM

5145
LPRWMINPY

5146
PPTHDCKPK

5147
PPYCLDMKR

5148
CVHVYNFQM

5149
CVDSYIRWM

5150
CFLRLKKHH

5151
CKVLYIQIG

5152
CIVNTKKYH

5153
CVRGIFSDH

5154
CVGRILRSC

5155
CVIWYHKYE

5156
YICFVSYRN

5157
CWTKLVQHQ

5158
CVMNWDFFI

5159
CVMFAHNTQ

5160
CVLYFTFGK

5161
CTHMMGKSP

5162
WDTQLLPNI

5163
CFIHMQKFP

5164
CSMPLYSYY

5165
CIQSFGSGP

5166
YWGEFIKKS

5167
CISKKGRWY

5168
CIKQIKTYE

5169
CTQPFNRQD

5170
CVSWLLGQG

5171
DPEDDPGNE

5172
CVGHGSHQK

5173
CWKMFSLHG

5174
CIMKPDNYP

5175
CVQQLVQIE

5176
EEPEPYLKN

5177
CIRFQNPPF

5178
CIKWVDIFP

5179
NGPFGEWKR

5180
YNPDDKRVI

5181
DEGHKKFLK

5182
CVRSYQIVG

5183
PPRHQWNFY

5184
CISSKLRGF

5185
CVTFQLGWQ

5186
ATYLKMSSP

5187
CVSGSNFPH

5188
CVSFYNHME

5189
CFHVMMKYE

5190
CVWRITQQE

5191
DVDELDIHI

5192
CWAKIMPLF

5193
CWSHLYFQT

5194
EVLDWIETE

5195
CHYQNLKTH

5196
CVTQQHRDG

5197
CVVMMQIWP

5198
PRKYPWKKK

5199
CGCRFIYYQ

5200
QRWKHDPLP

5201
CVSLLEMSI

5202
HNIKPWKPY

5203
QENETFKKL

5204
DVDEEQDIF

5205
CVACKMKTG

5206
CFFYPMSMS

5207
PDERLFNKK

5208
CSTPMRMYM

5209
CFWQYGLVT

5210
CWLPFQMFH

5211
CTRQSIIVY

5212
CISVYFMWP

5213
DPRKLEPRI

5214
CTIGTMGPF

5215
CVRTLQSPD

5216
QDRFNHKPW

5217
CVSTVLNMG

5218
GDFPDEDDW

5219
CVWIWQNSY

5220
ETEYYQPFL

5221
YCPHRDRTH

5222
FEDEPEPWK

5223
PFDEGEPEI

5224
CVLKGKHEG

5225
CVQVLQLGG

5226
CTISQKMIW

5227
PKMSLMKFN

5228
CWSMYNSRY

5229
CWVMLYRGW

5230
CWTRQSNWP

5231
CVSPYTGLL

5232
PPPFFKYRG

5233
CTLYFGFIY

In some embodiments, the engineered AAV VP capsid polypeptide comprises a polypeptide sequence represented by the formula: (A)-(X)—(B) wherein: (A) is the polypeptide sequence of SEQ ID NO: 5812 (VAYNVGGQMATNNQSSTTAP, corresponding to residues 561 to 580 of SEQ ID NO: 2); (X) is the 581-589 region having a polypeptide sequence selected from the list of polypeptides in TABLE 7 (SEQ ID NO: 5808-SEQ ID NO: 5811) with increased likelihood of conferring CNS tissue tropism or preference on a recombinant AAV virion (rAAV); and (B) is the polypeptide sequence of SEQ ID NO: 5813 (IVPGSVWMERDVYLQGPIWA, corresponding to residues 590 to 609 of SEQ ID NO: 2); and wherein the capsid polypeptide is capable of assembling into the rAAV and, the capsid does not have the sequence of any of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, or SEQ ID NO: 8. Also encompassed herein are rAAVs composed of engineered AAV5 VP2 capsid polypeptides and engineered AAV5 VP3 capsid polypeptides comprising a 581-589 region having a sequence selected from the list of polypeptides in TABLE 7 (SEQ ID NO: 5808-SEQ ID NO: 5811) at the region corresponding to amino acid residues 445 to 453 in AAV5 VP2 or the region corresponding to amino acid residues 389 to 397 in AAV5 VP3.

TABLE 7

Additional Sequences of 581-589 Regions

for Muscle Tropism

SEQ ID NO
Sequence

5808
DDFGTLPRM

5809
GIIQVRMEW

5810
GQFEDLLVL

5811
RKEGCTSHG

In some embodiments, the engineered AAV VP capsid polypeptide confers muscle tissue tropism or preference, wherein the muscle tissue is selected from the group consisting of aorta, esophagus, heart (e.g., atrium, ventricle, valves), skeletal muscle (e.g., biceps, brachii, femoris, diaphragm, gastrocnemius, tibialis anterior, triceps, quadriceps, including vastus lateralis)), muscle fibers including type I (slow oxidative) fibers, type IC fibers, type II fibers, type IIC fibers, type IIA (fast oxidative/glycolytic) fibers, type IIAX fibers, type IIXA fibers, type IIX (fast glycolytic) fibers, and any combination thereof.

Described below are engineered mutated AAV5 VP1 polypeptide sequences that confer stable or improved virion assembly, tissue tropism, or both. In some embodiments, the present disclosure provides an AAV5 VP1 capsid polypeptide having a sequence homology of no more than 98.7% to SEQ ID NO: 1, wherein the AAV5 capsid polypeptide sequence has at least one mutation in a region from a position corresponding to 581 to a position corresponding to 589 of SEQ ID NO: 1. In some embodiments, an engineered AAV5 polypeptide comprises a variant 581-589 region (e.g., comprising at least one amino acid substitution relative to residues 561 to 580 of SEQ ID NO: 1).

Also encompassed herein are rAAVs composed of engineered AAV5 VP2 capsid polypeptides and engineered AAV5 VP3 capsid polypeptides comprising a 581-589 region having a sequence selected from the list of polypeptides in TABLE 2, TABLE 3, TABLE 4, TABLE 5, TABLE 6, or TABLE 7 (SEQ ID NO: 12-SEQ ID NO: 3937, SEQ ID NO: 3938-SEQ ID NO: 4237, SEQ ID NO: 4238-SEQ ID NO: 4933, SEQ ID NO: 4934-SEQ ID NO: 5233, SEQ ID NO: 5234-SEQ ID NO: 5807, or SEQ ID NO: 5808-SEQ ID NO: 5811) at the region corresponding to amino acid residues 445 to 453 in AAV5 VP2 or the region corresponding to amino acid residues 389 to 397 in AAV5 VP3. Also encompassed herein are rAAVs composed of engineered AAV5 VP2 capsid polypeptides and engineered AAV5 VP3 capsid polypeptides comprising a 581-589 region having a sequence selected from the list of polypeptides in TABLE 9, TABLE 10, TABLE 11, or TABLE 10 at the region corresponding to amino acid residues 445 to 453 in AAV5 VP2 or the region corresponding to amino acid residues 389 to 397 in AAV5 VP3.

Engineered VP Polypeptides Competent for rAAV Assembly

In various preferred embodiments, the mutated (engineered, recombinant) VP capsid polypeptides of the present disclosure (e.g., comprising a variant 581-589 region) are capable of forming an assembled virion, and in some instances that exhibit similar or improved stability when compared to a virion that comprises the AAV5 VP1 capsid polypeptide of SEQ ID NO: 1.

The frequency of a given amino acid residue occurring in assembled, purified viruses at a specified position within the 581-589 region, corresponding to position 581 to position 589 of SEQ ID NO: 1, or corresponding to X¹to X⁹as generalized in SEQ ID NO: 2, over the frequency of that given amino acid residue occurring at the specified position in the entire plasmid library was analyzed to identify sequence rules for capsid polypeptide sequences that favor viral capsid assembly. Based on the determined amino acid residue frequency, the contribution of each amino acid at each position of the 581-589 region to capsid assembly was determined, and sequences of the 581-589 region (X¹X²X³X⁴X⁵X⁶X⁷X⁸X⁹of SEQ ID NO: 2) that favor capsid assembly, and rules for selecting sequences that favor capsid assembly, were identified.

Disclosed herein are engineered AAV5 VP capsid polypeptides capable of forming an assembled viral capsid that may exhibit similar or improved stability as compared to wild type AAV5 VP capsid polypeptide, wherein the engineered variant AAV5 VP capsid polypeptide sequence has one or more mutations, wherein the VP1 polypeptide sequence has said one or more mutations in a 581-589 region (corresponding to position 581 to position 589 in SEQ ID NO: 2), and wherein X¹is selected from A, D, E, G, L, M, N, Q, S, T, or V, or X¹is selected from A, D, E, M, or T. In some embodiments, X¹is E; or X²is selected from A, C, D, E, G, H, I, N, P, Q, S, T, or V, or X²is selected from A, S, T, or V, or X²is A; or wherein X³is selected from A, D, E, G, H, M, N, Q, S, T, or V, or X³is selected from D, E, N, Q or T, or X³is D or T; or wherein X⁴is selected from A, D, E, G, H, N, P, Q, S, or T, or X⁴is selected from D, E, P, or Q, or X⁴is E; or wherein X⁵is selected from A, C, D, E, G, H, N, Q, S, T, or Y, or X⁵is selected from D, E, N, Q or T, or X⁵is N; or wherein X⁶is selected from A, D, E, G, H, N, P, Q, S, or T, or X⁶is selected from D, N, or Q, or X⁶is D; or wherein X⁷is selected from A, C, D, E, G, H, N, Q, S, or T, or X⁷is selected from A, D, E or G, or X⁷is A; or wherein X⁸is selected from A, C, D, E, G, H, N, Q, S, or T, or X⁸comprises A, D, G, or S, or X⁸is G; or wherein X⁹is selected from A, D, E, G, H, N, P, Q, S, or T, or X⁹is selected from A, D, G, or P, or X⁹is G.

In various embodiments, the VP polypeptide is capable of forming an assembled viral capsid, and in some instances exhibits similar or improved stability when compared to a virion that comprises the AAV5 VP1 capsid polypeptide of SEQ ID NO:1.

Examples of amino acid substitutions within a 581-589 region that may favor viral capsid assembly are provided in TABLE 8. In some embodiments, the following amino acids can be independently mutated, in any combination, at any one or more positions X¹to X⁹, with reference to SEQ ID NO: 2, to provide an AAV VP1 capsid that is capable of assembling. Additionally, one or more mutations outside of the X¹to X⁹region can be allowed, as long as the capsid is still capable of assembling.

TABLE 8

581-589 Region Amino Acid Variations for Viral Assembly

Viral Assembly Options

X¹is A, D, E, G, L, M, N, Q, S, T, or V

X¹is A, D, E, M, or T

X¹is E

X²is A, C, D, E, G, H, I, N, P, Q, S, T, or V

X²is A, S, T, or V

X²is A

X³is A, D, E, G, H, M, N, Q, S, T, or V

X³is D, E, N, Q or T

X³is D or T

X⁴is A, D, E, G, H, N, P, Q, S, or T

X⁴is D, E, P, or Q

X⁴is E

X⁵is A, C, D, E, G, H, N, Q, S, T, or Y

X⁵is D, E, N, Q or T

X⁵is N.

X⁶is A, D, E, G, H, N, P, Q, S, or T;

X⁶is D, N, or Q; or

X⁶is D.

X⁷is A, C, D, E, G, H, N, Q, S, or T;

X⁷is A, D, E or G; or

X⁷is A.

X⁸is A, C, D, E, G, H, N, Q, S, or T;

X⁸is A, D, G, or S; or

X⁸is G.

X⁹is A, D, E, G, H, N, P, Q, S, or T;

X⁹is A, D, G, or P; or

X⁹is G.

Variant 581-589 Regions for CNS Tissue Tropism

The present disclosure provides AAV5 virions with a VP capsid polypeptide having at least one mutation in a region with residues that interact with target cells (e.g., a target CNS cell in a target CNS tissue of interest), where the mutation confers increased CNS tissue tropism or preference as compared to a wild type VP capsid polypeptide (e.g., a VP capsid polypeptide of SEQ ID NO: 1, SEQ ID NO: 10, or SEQ ID NO: 11). In some embodiments, provided herein are AAV5 VP1 capsid polypeptide having a sequence homology of at least 80% to SEQ ID NO: 1, wherein the AAV5 VP1 capsid polypeptide has at least one mutation in a 581-589 region, corresponding to residues 581 to 589 of SEQ ID NO: 1, and wherein said at least one mutation drives increased central nervous system (CNS) tropism or preference as compared to the wild type VP capsid polypeptide (SEQ ID NO: 1). The following sequences rules and sequences also apply to the 581-589 region in AAV5 VP2 (amino acid residues 445 to 453; VP2 sequence shown in SEQ ID NO: 10) and in AAV5 VP3 (amino acid residues 389 to 397; VP3 sequences shown in SEQ ID NO: 11). Thus, the present disclosure encompasses AAV5 VP2 capsid polypeptides and AAV5 VP3 capsid polypeptides having one or more amino acid substitutions in the 581-589 regions of VP2 and VP3, corresponding to the AAV5 VP1 amino acid residues of the 581 to 589, where the one or more mutations comport to the rules or sequences in the following section.

VP capsid polypeptides (e.g., V1, VP2, or VP3 capsid polypeptides, or combinations thereof) comprising a CNS tissue-tropic 581-589 region (e.g., any one of SEQ ID NO: 12-SEQ ID NO: 3937 or SEQ ID NO: 3938-SEQ ID NO: 4237, or any 581-589 region adhering to the rules identified herein) may assemble to form an rAAV with altered surface properties compared to a wild type AAV (e.g., comprising a VP1 polypeptide of SEQ ID NO: 1). The surface may form an interface that forms interactions with a target tissue, and the altered surface properties of the rAAV may promote tissue specific interactions (e.g., CNS tissue-specific interactions) between the rAAV and the target tissue. In some embodiments, the altered surface properties may be an altered charge distribution, increased or decreased hydrophobicity, altered availability or distribution of hydrogen bond donors or acceptors, or formation or reshaping of binding pockets. Such altered surface properties may favor interactions between the rAAV and CNS tissue while disfavoring interactions between the rAAV and non-CNS tissue.

A CNS-tropic rAAV assembled from VP capsid polypeptides (e.g., V1, VP2, or VP3 capsid polypeptides, or combinations thereof) comprising a 581-589 region (e.g., any one of SEQ ID NO: 12-SEQ ID NO: 3937 or SEQ ID NO: 3938-SEQ ID NO: 4237, or any 581-589 region adhering to the rules identified herein) may preferentially infect a CNS tissue (e.g., cortex forebrain, cortex occipital, cortex temporal, thalamus, hypothalamus, substantia nigra, hippocampus DG, hippocampus CA1, hippocampus CA3, cerebellum, or combinations thereof) as compared to non-CNS tissue (e.g., liver, skeletal muscle, heart, lung, spleen, lymph node, bone marrow, mammary gland, skin, adrenal gland, thyroid, colon, sciatic nerve, spinal cord, or combinations thereof) at a higher level as compared to wild type AAV5. The CNS-tropic AAV may infect the CNS tissue at a rate that is at least about 1.1-fold, at least about 1.2-fold, at least about 1.3-fold, at least about 1.4-fold, at least about 1.5-fold, at least about 1.6-fold, at least about 1.7-fold, at least about 1.8-fold, at least about 1.9-fold, at least about 2-fold, at least about 2.2-fold, at least about 2.4-fold, at least about 2.6-fold, at least about 2.8-fold, at least about 3-fold, at least about 3.5-fold, at least about 4-fold, at least about 4.5-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 20-fold, at least about 30-fold, at least about 40-fold, at least about 50-fold, at least about 100-fold, at least about 200-fold, at least about 300-fold, at least about 400-fold, or at least about 500-fold an infection rate for the non-CNS tissue, as compared to wild type AAV5. In some embodiments, the non-CNS tissue is a liver tissue.

In some embodiments, the CNS-tropic rAAV assembled from VP capsid polypeptides (e.g., V1, VP2, or VP3 capsid polypeptides, or combinations thereof) comprising a 581-589 region (e.g., any one of SEQ ID NO: 12-SEQ ID NO: 3937 or SEQ ID NO: 3938-SEQ ID NO: 4237, or any 581-589 region adhering to the rules identified herein) may deliver a payload to the tissue infected by the rAAV. The payload (e.g., a payload encoding a therapeutic peptide or a therapeutic polynucleotide) may be expressed in the tissue. In some embodiments, an expression level of the payload in a CNS tissue may be at least about 2.5-fold, at least about 2.8-fold, at least about 3-fold, at least about 3.2-fold, at least about 3.5-fold, at least about 4-fold, at least about 4.5-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 20-fold, at least about 50-fold, at least about 75-fold, at least about 100-fold, at least about 200-fold, at least about 500-fold higher, or at least about 1000-fold an expression level in a non-CNS tissue (e.g., a liver tissue). In some embodiments, payload delivery to a CNS tissue using a CNS-tropic AAV may have at least about 1.1-fold, at least about 1.2-fold, at least about 1.3-fold, at least about 1.4-fold, at least about 1.5-fold, at least about 1.6-fold, at least about 1.7-fold, at least about 1.8-fold, at least about 1.9-fold, at least about 2-fold, at least about 2.2-fold, at least about 2.4-fold, at least about 2.6-fold, at least about 2.8-fold, at least about 3-fold, at least about 3.5-fold, at least about 4-fold, at least about 4.5-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 20-fold, at least about 30-fold, at least about 40-fold, at least about 50-fold, at least about 100-fold, at least about 200-fold, at least about 300-fold, at least about 400-fold, or at least about 500-fold higher specificity for CNS tissue as compared to a wild type AAV5.

CNS-Tropic Sequences

Recombinant AAV viral capsids with specificity for central nervous system (CNS) tissues (CNS-tropic rAAVs) may be identified by screening rAAV libraries comprising VP capsid polypeptides with 581-589 regions, as described herein. The libraries may be screened in non-human primates (NHPs) by systemically administering the rAAV library and identifying sequences of 581-589 regions in VP capsid polypeptides that conferred tissue-tropic accumulation in or infection of CNS tissues (e.g., cortex forebrain, cortex occipital, cortex temporal, thalamus, hypothalamus, substantia nigra, hippocampus DG, hippocampus CA1, hippocampus CA3, cerebellum, or combinations thereof). The CNS-tropic rAAVs may preferentially accumulate in or infect CNS tissues as compared to non-CNS tissues (e.g., liver, skeletal muscle, heart, lung, spleen, lymph node, bone marrow, mammary gland, skin, adrenal gland, thyroid, colon, sciatic nerve, spinal cord, or combinations thereof). Exemplary 581-589 region sequences identified in a primary screen as conferring CNS tissue tropism to an rAAV are provided in TABLE 2 and TABLE 4. Exemplary 581-589 region sequences identified in a secondary screen as conferring CNS tissue tropism to an rAAV are provided in TABLE 9.

A CNS-tropic rAAV assembled from VP capsid polypeptides (e.g., V1, VP2, or VP3 capsid polypeptides, or combinations thereof) comprising a 581-589 region (e.g., any one of SEQ ID NO: 12-SEQ ID NO: 3937) may preferentially infect a CNS tissue (e.g., cortex forebrain, cortex occipital, cortex temporal, thalamus, hypothalamus, substantia nigra, hippocampus DG, hippocampus CA1, hippocampus CA3, cerebellum, or combinations thereof) as compared to non-CNS tissue (e.g., liver, skeletal muscle, heart, lung, spleen, lymph node, bone marrow, mammary gland, skin, adrenal gland, thyroid, colon, sciatic nerve, spinal cord, or combinations thereof).

Disclosed herein are engineered AAV5 VP capsid polypeptides capable of forming an assembled virion that exhibits increased central nervous system tissue tropism as compared to a wild type AAV5 VP capsid polypeptide (e.g., a VP1 of SEQ ID NO: 1, a VP2 of SEQ ID NO: 10, or a VP3 of SEQ ID NO: 11), wherein the engineered variant AAV5 VP capsid polypeptide sequence has one or more amino acid substitutions in a 581-589 region of the VP capsid polypeptide, corresponding to positions 581 to 589 in SEQ ID NO: 2 (X¹X²X³X⁴X⁵X⁶X⁷X⁸X⁹). In some embodiments, X¹is selected from A, R, N, D, C, E, Q, G, H, I, L, K, M, F, P, S, T, W, Y, or V. In some embodiments, X²is selected from A, R, N, D, C, E, Q, G, H, I, L, K, M, F, P, S, T, W, Y, or V. In some embodiments, X³is selected from A, R, N, D, C, E, Q, G, H, I, L, K, M, F, P, S, T, W, Y, or V. In some embodiments, X⁴is selected from A, R, N, D, C, E, Q, G, H, I, L, K, M, F, P, S, T, W, Y, or V. In some embodiments, X⁵is selected from A, R, N, D, C, E, Q, G, H, I, L, K, M, F, P, S, T, W, Y, or V. In some embodiments, X⁶is selected from A, R, N, D, C, E, Q, G, H, I, L, K, M, F, P, S, T, W, Y, or V. In some embodiments, X⁷is selected from A, R, N, D, C, E, Q, G, H, I, L, K, M, F, P, S, T, W, Y, or V. In some embodiments, X⁸is selected A, R, N, D, C, E, Q, G, H, I, L, K, M, F, P, S, T, W, Y, or V. In some embodiments, X⁹is selected from A, R, N, D, C, E, Q, G, H, I, L, K, M, F, P, S, T, W, Y, or V.

In some embodiments, a 581-589 region of a CNS tissue-tropic VP capsid polypeptide may comprise a sequence of any one of SEQ ID NO: 12-SEQ ID NO: 3937.

In some embodiments, provided herein are AAV5 VP capsid polypeptide having at least one mutation in a 581-589 region, corresponding to residues 581 to 589 of AAV5 VP1 (e.g., SEQ ID NO: 1) and having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 99%, or 100% sequence identity to any sequence selected from SEQ ID NO: 12-SEQ ID NO: 3937, wherein said at least one mutation drives increased central nervous system tissue tropism as compared to a wild type AAV5 capsid polypeptide.

CNS-Tropic Sequences Generated by Machine Learning

Additional 581-589 regions that confer CNS tissue tropism may be generated using machine learning algorithms trained with sequence identified in CNS tissue in in vivo screens. These patterns may be used to generate new sequences of 581-589 regions that increase the likelihood of conferring CNS tissue tropism to VP capsid polypeptides and rAAVs assembled from the VP capsid polypeptides. In some embodiments, new sequences of 581-589 regions may be generated by randomly sampling amino acid residues at each position of 581-589 regions that favored CNS tissue tropism in an in vivo screen. The generated sequences may be tested using random forests or gradient boosting classifiers to predicted CNS tissue-specificity for each 581-589 region. The 581-589 regions with the highest predicted CNS tissue-specificity may be selected as CNS tissue-tropic sequences. For example, a 581-589 region of any one of SEQ ID NO: 3938-SEQ ID NO: 4237 may be selected. 581-589 region sequences generated using machine learning for increased likelihood of conferring CNS tissue tropism to an rAAV are provided in TABLE 3.

Favored amino acid residues and disfavored amino acid residues at each position in the 581-589 region of an engineered AAV5 capsid polypeptide that increase the likelihood of conferring increased CNS tissue tropism as compared to a wild type AAV5 capsid polypeptide may be determined using in vivo data, machine learning (ML) models, or combinations thereof. Recombinant AAV viral capsid libraries may be screened in non-human primates (NHPs) by systemically administering the rAAV library and identifying sequences of 581-589 regions in VP capsid polypeptides that increased the likelihood of conferring tissue-tropic accumulation in or infection of CNS tissues (e.g., cortex forebrain, cortex occipital, cortex temporal, thalamus, hypothalamus, substantia nigra, hippocampus DG, hippocampus CA1, hippocampus CA3, cerebellum, or combinations thereof).

A CNS-tropic rAAV assembled from VP capsid polypeptides (e.g., V1, VP2, or VP3 capsid polypeptides, or combinations thereof) comprising a 581-589 region may preferentially infect a CNS tissue (e.g., cortex forebrain, cortex occipital, cortex temporal, thalamus, hypothalamus, substantia nigra, hippocampus DG, hippocampus CA1, hippocampus CA3, cerebellum, or combinations thereof) as compared to non-CNS tissue (e.g., liver, skeletal muscle, heart, lung, spleen, lymph node, bone marrow, mammary gland, skin, adrenal gland, thyroid, colon, sciatic nerve, spinal cord, or combinations thereof), as compared to a wild type AAV5 capsid polypeptide.

Disclosed herein are engineered AAV5 VP capsid polypeptides capable of forming an assembled virion that exhibits increased central nervous system tissue tropism or preference as compared to a wild type AAV5 VP capsid polypeptide (e.g., a VP1 of SEQ ID NO: 1, a VP2 of SEQ ID NO: 10, or a VP3 of SEQ ID NO: 11), wherein the engineered variant AAV5 VP capsid polypeptide sequence has one or more amino acid substitutions in a 581-589 region of the VP capsid polypeptide, corresponding to positions 581 to 589 in SEQ ID NO: 2 (X¹X²X³X⁴X⁵X⁶X⁷X⁸X⁹). In some embodiments, X¹is selected from A, R, N, D, C, E, Q, G, H, I, L, K, M, F, P, S, T, W, Y, or V. In some embodiments, X²is selected from A, R, N, D, C, E, Q, G, H, I, L, K, M, F, P, S, T, W, Y, or V. In some embodiments, X³is selected from A, R, N, D, C, E, Q, G, H, I, L, K, M, F, P, S, T, W, Y, or V. In some embodiments, X⁴is selected from A, R, N, D, C, E, Q, G, H, I, L, K, M, F, P, S, T, W, Y, or V. In some embodiments, X⁵is selected from A, R, N, D, C, E, Q, G, H, I, L, K, M, F, P, S, T, W, Y, or V. In some embodiments, X⁶is selected from A, R, N, D, C, E, Q, G, H, I, L, K, M, F, P, S, T, W, Y, or V. In some embodiments, X⁷is selected from A, R, N, D, C, E, Q, G, H, I, L, K, M, F, P, S, T, W, Y, or V. In some embodiments, X⁸is selected A, R, N, D, C, E, Q, G, H, I, L, K, M, F, P, S, T, W, Y, or V. In some embodiments, X⁹is selected from A, R, N, D, C, E, Q, G, H, I, L, K, M, F, P, S, T, W, Y, or V.

In some embodiments, a 581-589 region of a CNS tissue-tropic VP capsid polypeptide may comprise a sequence of any one of SEQ ID NO: 3938-SEQ ID NO: 4237.

In some embodiments, provided herein are AAV5 VP capsid polypeptide having at least one mutation in a 581-589 region, corresponding to residues 581 to 589 of AAV5 VP1 (e.g., SEQ ID NO: 1) and having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 99%, or 100% sequence identity to any sequence selected from SEQ ID NO: 3938-SEQ ID NO: 4237, wherein said at least one mutation drives increased central nervous system tissue tropism as compared to a wild type AAV5 capsid polypeptide.

The present disclosure provides a tissue-tropic rAAV comprising a VP capsid polypeptide with a 581-589 variant region, relative to WT AAV5, of SEQ ID NO: 18. The present disclosure provides a tissue-tropic rAAV comprising a VP capsid polypeptide with a 581-589 variant region, relative to WT AAV5, of SEQ ID NO: 54. The present disclosure provides a tissue-tropic rAAV comprising a VP capsid polypeptide with a 581-589 variant region, relative to WT AAV5, of SEQ ID NO: 2028. The present disclosure provides a tissue-tropic rAAV comprising a VP capsid polypeptide with a 581-589 variant region, relative to WT AAV5, of SEQ ID NO: 424. The present disclosure provides a tissue-tropic rAAV comprising a VP capsid polypeptide with a 581-589 variant region, relative to WT AAV5, of SEQ ID NO: 3306. The present disclosure provides a tissue-tropic rAAV comprising a VP capsid polypeptide with a 581-589 variant region, relative to WT AAV5, of SEQ ID NO: 3472. The present disclosure provides a tissue-tropic rAAV comprising a VP capsid polypeptide with a 581-589 variant region, relative to WT AAV5, of SEQ ID NO: 415. The present disclosure provides a tissue-tropic rAAV comprising a VP capsid polypeptide with a 581-589 variant region, relative to WT AAV5, of SEQ ID NO: 709. The present disclosure provides a tissue-tropic rAAV comprising a VP capsid polypeptide with a 581-589 variant region, relative to WT AAV5, of SEQ ID NO: 1971. The present disclosure provides a tissue-tropic rAAV comprising a VP capsid polypeptide with a 581-589 variant region, relative to WT AAV5, of SEQ ID NO: 2791. The present disclosure provides a tissue-tropic rAAV comprising a VP capsid polypeptide with a 581-589 variant region, relative to WT AAV5, of SEQ ID NO: 1956. The present disclosure provides a tissue-tropic rAAV comprising a VP capsid polypeptide with a 581-589 variant region, relative to WT AAV5, of SEQ ID NO: 262. The present disclosure provides a tissue-tropic rAAV comprising a VP capsid polypeptide with a 581-589 variant region, relative to WT AAV5, of SEQ ID NO: 425. The present disclosure provides a tissue-tropic rAAV comprising a VP capsid polypeptide with a 581-589 variant region, relative to WT AAV5, of SEQ ID NO: 2536. The present disclosure provides a tissue-tropic rAAV comprising a VP capsid polypeptide with a 581-589 variant region, relative to WT AAV5, of SEQ ID NO: 708. The present disclosure provides a tissue-tropic rAAV comprising a VP capsid polypeptide with a 581-589 variant region, relative to WT AAV5, of SEQ ID NO: 3283. The present disclosure provides a tissue-tropic rAAV comprising a VP capsid polypeptide with a 581-589 variant region, relative to WT AAV5, of SEQ ID NO: 430. The present disclosure provides a tissue-tropic rAAV comprising a VP capsid polypeptide with a 581-589 variant region, relative to WT AAV5, of SEQ ID NO: 3297. The present disclosure provides a tissue-tropic rAAV comprising a VP capsid polypeptide with a 581-589 variant region, relative to WT AAV5, of SEQ ID NO: 2661. The present disclosure provides a tissue-tropic rAAV comprising a VP capsid polypeptide with a 581-589 variant region, relative to WT AAV5, of SEQ ID NO: 428. The present disclosure provides a tissue-tropic rAAV comprising a VP capsid polypeptide with a 581-589 variant region, relative to WT AAV5, of SEQ ID NO: 885. The present disclosure provides a tissue-tropic rAAV comprising a VP capsid polypeptide with a 581-589 variant region, relative to WT AAV5, of SEQ ID NO: 429. The present disclosure provides a tissue-tropic rAAV comprising a VP capsid polypeptide with a 581-589 variant region, relative to WT AAV5, of SEQ ID NO: 569. The present disclosure provides a tissue-tropic rAAV comprising a VP capsid polypeptide with a 581-589 variant region, relative to WT AAV5, of SEQ ID NO: 724. The present disclosure provides a tissue-tropic rAAV comprising a VP capsid polypeptide with a 581-589 variant region, relative to WT AAV5, of SEQ ID NO: 1576. The present disclosure provides a tissue-tropic rAAV comprising a VP capsid polypeptide with a 581-589 variant region, relative to WT AAV5, of SEQ ID NO: 4545. The present disclosure provides a tissue-tropic rAAV comprising a VP capsid polypeptide with a 581-589 variant region, relative to WT AAV5, of SEQ ID NO: 426. The present disclosure provides a tissue-tropic rAAV comprising a VP capsid polypeptide with a 581-589 variant region, relative to WT AAV5, of SEQ ID NO: 1887. The present disclosure provides a tissue-tropic rAAV comprising a VP capsid polypeptide with a 581-589 variant region, relative to WT AAV5, of SEQ ID NO: 3906. The present disclosure provides a tissue-tropic rAAV comprising a VP capsid polypeptide with a 581-589 variant region, relative to WT AAV5, of SEQ ID NO: 3935. The present disclosure provides a tissue-tropic rAAV comprising a VP capsid polypeptide with a 581-589 variant region, relative to WT AAV5, of SEQ ID NO: 3846. The present disclosure provides a tissue-tropic rAAV comprising a VP capsid polypeptide with a 581-589 variant region, relative to WT AAV5, of SEQ ID NO: 2168. The present disclosure provides a tissue-tropic rAAV comprising a VP capsid polypeptide with a 581-589 variant region, relative to WT AAV5, of SEQ ID NO: 4119. The present disclosure provides a tissue-tropic rAAV comprising a VP capsid polypeptide with a 581-589 variant region, relative to WT AAV5, of SEQ ID NO: 2456. The present disclosure provides a tissue-tropic rAAV comprising a VP capsid polypeptide with a 581-589 variant region, relative to WT AAV5, of SEQ ID NO: 2278. The present disclosure provides a tissue-tropic rAAV comprising a VP capsid polypeptide with a 581-589 variant region, relative to WT AAV5, of SEQ ID NO: 5006. The present disclosure provides a tissue-tropic rAAV comprising a VP capsid polypeptide with a 581-589 variant region, relative to WT AAV5, of SEQ ID NO: 307. The present disclosure provides a tissue-tropic rAAV comprising a VP capsid polypeptide with a 581-589 variant region, relative to WT AAV5, of SEQ ID NO: 5155. The present disclosure provides a tissue-tropic rAAV comprising a VP capsid polypeptide with a 581-589 variant region, relative to WT AAV5, of SEQ ID NO: 2640. The present disclosure provides a tissue-tropic rAAV comprising a VP capsid polypeptide with a 581-589 variant region, relative to WT AAV5, of SEQ ID NO: 4317. The present disclosure provides a tissue-tropic rAAV comprising a VP capsid polypeptide with a 581-589 variant region, relative to WT AAV5, of SEQ ID NO: 1145. The present disclosure provides a tissue-tropic rAAV comprising a VP capsid polypeptide with a 581-589 variant region, relative to WT AAV5, of SEQ ID NO: 23. The present disclosure provides a tissue-tropic rAAV comprising a VP capsid polypeptide with a 581-589 variant region, relative to WT AAV5, of SEQ ID NO: 103. The present disclosure provides a tissue-tropic rAAV comprising a VP capsid polypeptide with a 581-589 variant region, relative to WT AAV5, of SEQ ID NO: 22. The present disclosure provides a tissue-tropic rAAV comprising a VP capsid polypeptide with a 581-589 variant region, relative to WT AAV5, of SEQ ID NO: 4031. The present disclosure provides a tissue-tropic rAAV comprising a VP capsid polypeptide with a 581-589 variant region, relative to WT AAV5, of SEQ ID NO: 1008. The present disclosure provides a tissue-tropic rAAV comprising a VP capsid polypeptide with a 581-589 variant region, relative to WT AAV5, of SEQ ID NO: 4790. The present disclosure provides a tissue-tropic rAAV comprising a VP capsid polypeptide with a 581-589 variant region, relative to WT AAV5, of SEQ ID NO: 2522. The present disclosure provides a tissue-tropic rAAV comprising a VP capsid polypeptide with a 581-589 variant region, relative to WT AAV5, of SEQ ID NO: 1432. The present disclosure provides a tissue-tropic rAAV comprising a VP capsid polypeptide with a 581-589 variant region, relative to WT AAV5, of SEQ ID NO: 2914. The present disclosure provides a tissue-tropic rAAV comprising a VP capsid polypeptide with a 581-589 variant region, relative to WT AAV5, of SEQ ID NO: 5042. The present disclosure provides a tissue-tropic rAAV comprising a VP capsid polypeptide with a 581-589 variant region, relative to WT AAV5, of SEQ ID NO: 2865. The present disclosure provides a tissue-tropic rAAV comprising a VP capsid polypeptide with a 581-589 variant region, relative to WT AAV5, of SEQ ID NO: 4264. The present disclosure provides a tissue-tropic rAAV comprising a VP capsid polypeptide with a 581-589 variant region, relative to WT AAV5, of SEQ ID NO: 964. The present disclosure provides a tissue-tropic rAAV comprising a VP capsid polypeptide with a 581-589 variant region, relative to WT AAV5, of SEQ ID NO: 1268. The present disclosure provides a tissue-tropic rAAV comprising a VP capsid polypeptide with a 581-589 variant region, relative to WT AAV5, of SEQ ID NO: 5065. The present disclosure provides a tissue-tropic rAAV comprising a VP capsid polypeptide with a 581-589 variant region, relative to WT AAV5, of SEQ ID NO: 706. The present disclosure provides a tissue-tropic rAAV comprising a VP capsid polypeptide with a 581-589 variant region, relative to WT AAV5, of SEQ ID NO: 4704. The present disclosure provides a tissue-tropic rAAV comprising a VP capsid polypeptide with a 581-589 variant region, relative to WT AAV5, of SEQ ID NO: 2994. The present disclosure provides a tissue-tropic rAAV comprising a VP capsid polypeptide with a 581-589 variant region, relative to WT AAV5, of SEQ ID NO: 829. The present disclosure provides a tissue-tropic rAAV comprising a VP capsid polypeptide with a 581-589 variant region, relative to WT AAV5, of SEQ ID NO: 1171. The present disclosure provides a tissue-tropic rAAV comprising a VP capsid polypeptide with a 581-589 variant region, relative to WT AAV5, of SEQ ID NO: 1041. The present disclosure provides a tissue-tropic rAAV comprising a VP capsid polypeptide with a 581-589 variant region, relative to WT AAV5, of SEQ ID NO: 5070. The present disclosure provides a tissue-tropic rAAV comprising a VP capsid polypeptide with a 581-589 variant region, relative to WT AAV5, of SEQ ID NO: 139. The present disclosure provides a tissue-tropic rAAV comprising a VP capsid polypeptide with a 581-589 variant region, relative to WT AAV5, of SEQ ID NO: 3304. The present disclosure provides a tissue-tropic rAAV comprising a VP capsid polypeptide with a 581-589 variant region, relative to WT AAV5, of SEQ ID NO: 2431. The present disclosure provides a tissue-tropic rAAV comprising a VP capsid polypeptide with a 581-589 variant region, relative to WT AAV5, of SEQ ID NO: 4288. The present disclosure provides a tissue-tropic rAAV comprising a VP capsid polypeptide with a 581-589 variant region, relative to WT AAV5, of SEQ ID NO: 5795. The present disclosure provides a tissue-tropic rAAV comprising a VP capsid polypeptide with a 581-589 variant region, relative to WT AAV5, of SEQ ID NO: 1300. The present disclosure provides a tissue-tropic rAAV comprising a VP capsid polypeptide with a 581-589 variant region, relative to WT AAV5, of SEQ ID NO: 1770. The present disclosure provides a tissue-tropic rAAV comprising a VP capsid polypeptide with a 581-589 variant region, relative to WT AAV5, of SEQ ID NO: 2830. The present disclosure provides a tissue-tropic rAAV comprising a VP capsid polypeptide with a 581-589 variant region, relative to WT AAV5, of SEQ ID NO: 1972. The present disclosure provides a tissue-tropic rAAV comprising a VP capsid polypeptide with a 581-589 variant region, relative to WT AAV5, of SEQ ID NO: 1649. The present disclosure provides a tissue-tropic rAAV comprising a VP capsid polypeptide with a 581-589 variant region, relative to WT AAV5, of SEQ ID NO: 2515. The present disclosure provides a tissue-tropic rAAV comprising a VP capsid polypeptide with a 581-589 variant region, relative to WT AAV5, of SEQ ID NO: 2849. The present disclosure provides a tissue-tropic rAAV comprising a VP capsid polypeptide with a 581-589 variant region, relative to WT AAV5, of SEQ ID NO: 3796. The present disclosure provides a tissue-tropic rAAV comprising a VP capsid polypeptide with a 581-589 variant region, relative to WT AAV5, of SEQ ID NO: 5815 (SYKMIHNTA). The present disclosure provides a tissue-tropic rAAV comprising a VP capsid polypeptide with a 581-589 variant region, relative to WT AAV5, of SEQ ID NO: 118. The present disclosure provides a tissue-tropic rAAV comprising a VP capsid polypeptide with a 581-589 variant region, relative to WT AAV5, of SEQ ID NO: 4766. The present disclosure provides a tissue-tropic rAAV comprising a VP capsid polypeptide with a 581-589 variant region, relative to WT AAV5, of SEQ ID NO: 770. The present disclosure provides a tissue-tropic rAAV comprising a VP capsid polypeptide with a 581-589 variant region, relative to WT AAV5, of SEQ ID NO: 1069. The present disclosure provides a tissue-tropic rAAV comprising a VP capsid polypeptide with a 581-589 variant region, relative to WT AAV5, of SEQ ID NO: 3061. The present disclosure provides a tissue-tropic rAAV comprising a VP capsid polypeptide with a 581-589 variant region, relative to WT AAV5, of SEQ ID NO: 4290. The present disclosure provides a tissue-tropic rAAV comprising a VP capsid polypeptide with a 581-589 variant region, relative to WT AAV5, of SEQ ID NO: 4261. The present disclosure provides a tissue-tropic rAAV comprising a VP capsid polypeptide with a 581-589 variant region, relative to WT AAV5, of SEQ ID NO: 4239. The present disclosure provides a tissue-tropic rAAV comprising a VP capsid polypeptide with a 581-589 variant region, relative to WT AAV5, of SEQ ID NO: 3478. The present disclosure provides a tissue-tropic rAAV comprising a VP capsid polypeptide with a 581-589 variant region, relative to WT AAV5, of SEQ ID NO: 2671. The present disclosure provides a tissue-tropic rAAV comprising a VP capsid polypeptide with a 581-589 variant region, relative to WT AAV5, of SEQ ID NO: 256. The present disclosure provides a tissue-tropic rAAV comprising a VP capsid polypeptide with a 581-589 variant region, relative to WT AAV5, of SEQ ID NO: 1256. The present disclosure provides a tissue-tropic rAAV comprising a VP capsid polypeptide with a 581-589 variant region, relative to WT AAV5, of SEQ ID NO: 719. The present disclosure provides a tissue-tropic rAAV comprising a VP capsid polypeptide with a 581-589 variant region, relative to WT AAV5, of SEQ ID NO: 1448. The present disclosure provides a tissue-tropic rAAV comprising a VP capsid polypeptide with a 581-589 variant region, relative to WT AAV5, of SEQ ID NO: 280. The present disclosure provides a tissue-tropic rAAV comprising a VP capsid polypeptide with a 581-589 variant region, relative to WT AAV5, of SEQ ID NO: 4338. The present disclosure provides a tissue-tropic rAAV comprising a VP capsid polypeptide with a 581-589 variant region, relative to WT AAV5, of SEQ ID NO: 5037. The present disclosure provides a tissue-tropic rAAV comprising a VP capsid polypeptide with a 581-589 variant region, relative to WT AAV5, of SEQ ID NO: 1901. The present disclosure provides a tissue-tropic rAAV comprising a VP capsid polypeptide with a 581-589 variant region, relative to WT AAV5, of SEQ ID NO: 438. The present disclosure provides a tissue-tropic rAAV comprising a VP capsid polypeptide with a 581-589 variant region, relative to WT AAV5, of SEQ ID NO: 2834. The present disclosure provides a tissue-tropic rAAV comprising a VP capsid polypeptide with a 581-589 variant region, relative to WT AAV5, of SEQ ID NO: 5491. The present disclosure provides a tissue-tropic rAAV comprising a VP capsid polypeptide with a 581-589 variant region, relative to WT AAV5, of SEQ ID NO: 4591. The present disclosure provides a tissue-tropic rAAV comprising a VP capsid polypeptide with a 581-589 variant region, relative to WT AAV5, of SEQ ID NO: 4936.

Variant 581-589 Regions for CNS Targeting

In some embodiments, provided herein are AAV5 VP capsid polypeptide having at least one mutation in a 581-589 region, corresponding to residues 581 to 589 of AAV5 VP1, and having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 99%, or 100% sequence identity to any sequence selected from SEQ ID NO: 12-SEQ ID NO: 3937, SEQ ID NO: 3938-SEQ ID NO: 4237, or SEQ ID NO: 5234-SEQ ID NO: 5807, wherein said at least one mutation drives increased CNS tissue tropism as compared to a wild type AAV5 capsid polypeptide.

Variant 581-589 Regions for Muscle Tissue Tropism

The present disclosure provides AAV5 virions with a VP capsid polypeptide having at least one mutation in a region with residues that interact with target cells (e.g., a target muscle cell in a target muscle tissue of interest), where the at least one mutation confers increased muscle tissue tropism or preference as compared to a wild type VP capsid polypeptide (e.g., a VP capsid polypeptide of SEQ ID NO: 1, SEQ ID NO: 10, or SEQ ID NO: 11). In some embodiments, provided herein are AAV5 VP1 capsid polypeptide having a sequence homology of at least 80% to SEQ ID NO: 1, wherein the AAV5 VP1 capsid polypeptide has at least one mutation in a 581-589 region, corresponding to residues 581 to 589 of SEQ ID NO: 1, and wherein said at least one mutation drives increased muscle tropism or preference as compared to the wild type VP capsid polypeptide of SEQ ID NO: 1. The following sequences rules and sequences also apply to the 581-589 region in AAV5 VP2 (amino acid residues 445 to 453; VP2 sequence shown in SEQ ID NO: 10) and in AAV5 VP3 (amino acid residues 389 to 397; VP3 sequences shown in SEQ ID NO: 11). Thus, the present disclosure encompasses AAV5 VP2 capsid polypeptides and AAV5 VP3 capsid polypeptides having one or more amino acid substitutions in the 581-589 regions of VP2 and VP3, corresponding to the AAV5 VP1 amino acid residues of the 581 to 589, where the one or more mutations comport to the rules or sequences in the following section.

VP capsid polypeptides (e.g., V1, VP2, or VP3 capsid polypeptides, or combinations thereof) comprising a muscle-tropic 581-589 region (e.g., any one of SEQ ID NO: 4238-SEQ ID NO: 4933 or SEQ ID NO: 4934-SEQ ID NO: 5233, or any 581-589 region adhering to the rules identified herein) may assemble to form an rAAV with altered surface properties compared to a wild type AAV (e.g., comprising a VP1 polypeptide of SEQ ID NO: 1). The surface may form an interface that forms interactions with a target tissue, and the altered surface properties of the rAAV may promote tissue specific interactions (e.g., muscle-specific interactions) between the rAAV and the target tissue.

A muscle-tropic rAAV assembled from VP capsid polypeptides (e.g., V1, VP2, or VP3 capsid polypeptides, or combinations thereof) comprising a 581-589 region (e.g., any one of SEQ ID NO: 4238-SEQ ID NO: 4933 or SEQ ID NO: 4934-SEQ ID NO: 5233, or any 581-589 region adhering to the rules identified herein) may preferentially infect a muscle tissue (e.g., aorta, esophagus, heart (including atrium, ventricle, valves), skeletal muscle (including biceps, brachii, femoris, diaphragm, gastrocnemius, tibialis anterior, triceps, quadriceps, including vastus lateralis)), or muscle fibers (e.g., type I (slow oxidative) fibers, type IC fibers, type II fibers, type IIC fibers, type IIA (fast oxidative/glycolytic) fibers, type IIAX fibers, type IIXA fibers, type IIX (fast glycolytic) fibers, or combinations thereof) as compared to non-muscle tissue (e.g., liver, CNS, heart, lung, spleen, lymph node, bone marrow, mammary gland, skin, adrenal gland, thyroid, colon, sciatic nerve, spinal cord, or combinations thereof) at a higher level as compared to wild type AAV5. The muscle-tropic AAV may infect the muscle tissue at a rate that is at least about 1.1-fold, at least about 1.2-fold, at least about 1.3-fold, at least about 1.4-fold, at least about 1.5-fold, at least about 1.6-fold, at least about 1.7-fold, at least about 1.8-fold, at least about 1.9-fold, at least about 2-fold, at least about 2.2-fold, at least about 2.4-fold, at least about 2.6-fold, at least about 2.8-fold, at least about 3-fold, at least about 3.5-fold, at least about 4-fold, at least about 4.5-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 20-fold, at least about 30-fold, at least about 40-fold, at least about 50-fold, at least about 100-fold, at least about 200-fold, at least about 300-fold, at least about 400-fold, or at least about 500-fold an infection rate for the non-muscle tissue, as compared to wild type AAV5. In some embodiments, the non-muscle tissue is a liver tissue.

In some embodiments, the muscle-tropic rAAV assembled from VP capsid polypeptides (e.g., V1, VP2, or VP3 capsid polypeptides, or combinations thereof) comprising a 581-589 region (e.g., any one of SEQ ID NO: 4238-SEQ ID NO: 4933 or SEQ ID NO: 4934-SEQ ID NO: 5233, or any 581-589 region adhering to the rules identified herein) may deliver a payload to the tissue infected by the rAAV. The payload (e.g., a payload encoding a therapeutic peptide or a therapeutic polynucleotide) may be expressed in the tissue. In some embodiments, an expression level of the payload in a muscle tissue may be at least about 2.5-fold, at least about 2.8-fold, at least about 3-fold, at least about 3.2-fold, at least about 3.5-fold, at least about 4-fold, at least about 4.5-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 20-fold, at least about 50-fold, at least about 75-fold, at least about 100-fold, at least about 200-fold, at least about 500-fold higher, or at least about 1000-fold an expression level in a non-muscle tissue (e.g., a liver tissue). In some embodiments, payload delivery to a muscle tissue using a CNS-tropic AAV may have at least about 1.1-fold, at least about 1.2-fold, at least about 1.3-fold, at least about 1.4-fold, at least about 1.5-fold, at least about 1.6-fold, at least about 1.7-fold, at least about 1.8-fold, at least about 1.9-fold, at least about 2-fold, at least about 2.2-fold, at least about 2.4-fold, at least about 2.6-fold, at least about 2.8-fold, at least about 3-fold, at least about 3.5-fold, at least about 4-fold, at least about 4.5-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 20-fold, at least about 30-fold, at least about 40-fold, at least about 50-fold, at least about 100-fold, at least about 200-fold, at least about 300-fold, at least about 400-fold, or at least about 500-fold higher specificity for muscle tissue as compared to a wild type AAV5.

Muscle-Tropic Sequences

Recombinant AAV viral capsids with specificity for muscle tissue (muscle-tropic rAAVs) may be identified by screening rAAV libraries comprising VP capsid polypeptides with variant 581-589 regions, as described herein. The libraries may be screened in non-human primates (NHPs) by systemically administering the rAAV library and identifying sequences of 581-589 regions in VP capsid polypeptides that conferred tissue-tropic accumulation in or infection of muscle tissues (e.g., aorta, esophagus, heart (including atrium, ventricle, valves), skeletal muscle (including biceps, brachii, femoris, diaphragm, gastrocnemius, tibialis anterior, triceps, quadriceps, including vastus lateralis)), or muscle fibers (e.g., type I (slow oxidative) fibers, type IC fibers, type II fibers, type IIC fibers, type IIA (fast oxidative/glycolytic) fibers, type IIAX fibers, type IIXA fibers, type IIX (fast glycolytic) fibers, or combinations thereof) to the rAAVs. The muscle-tropic rAAVs may preferentially accumulate in or infect muscle tissues as compared to non-muscle tissues (e.g., liver, CNS, heart, lung, spleen, lymph node, bone marrow, mammary gland, skin, adrenal gland, thyroid, colon, sciatic nerve, spinal cord, or combinations thereof). Exemplary region sequences identified in a primary screen as conferring muscle tropism to an rAAV are provided in TABLE 5 and TABLE 7. Exemplary region sequences identified in a secondary screen as conferring cardiac muscle tropism to an rAAV are provided in TABLE 10. Exemplary region sequences identified in a secondary screen as conferring skeletal muscle tropism to an rAAV are provided in TABLE 11. Exemplary region sequences identified in a secondary screen as conferring muscle tropism to an rAAV are provided in TABLE 12.

A muscle-tropic rAAV assembled from VP capsid polypeptides (e.g., V1, VP2, or VP3 capsid polypeptides, or combinations thereof) comprising a 581-589 region (e.g., any one of SEQ ID NO: 4238-SEQ ID NO: 4933) may preferentially infect a muscle tissue (e.g., aorta, esophagus, heart (including atrium, ventricle, valves), skeletal muscle (including biceps, brachii, femoris, diaphragm, gastrocnemius, tibialis anterior, triceps, quadriceps, including vastus lateralis)), or muscle fibers (e.g., type I (slow oxidative) fibers, type IC fibers, type II fibers, type IIC fibers, type IIA (fast oxidative/glycolytic) fibers, type IIAX fibers, type IIXA fibers, type IIX (fast glycolytic) fibers, or combinations thereof) as compared to non-muscle tissue (e.g., liver, CNS, heart, lung, spleen, lymph node, bone marrow, mammary gland, skin, adrenal gland, thyroid, colon, sciatic nerve, spinal cord, or combinations thereof).

Disclosed herein are engineered AAV5 VP capsid polypeptides capable of forming an assembled virion that exhibits increased muscle tropism as compared to a wild type AAV5 VP capsid polypeptide (e.g., a VP1 of SEQ ID NO: 1, a VP2 of SEQ ID NO: 10, or a VP3 of SEQ ID NO: 11), wherein the engineered variant AAV5 VP capsid polypeptide sequence has one or more amino acid substitutions in a 581-589 region of the VP capsid polypeptide, corresponding to positions 581 to 589 in SEQ ID NO: 2 (X¹X²X³X⁴X⁵X⁶X⁷X⁸X⁹). In some embodiments, X¹is selected from A, R, N, D, C, E, Q, G, H, I, L, K, M, F, P, S, T, W, Y, or V. In some embodiments, X²is selected from A, R, N, D, C, E, Q, G, H, I, L, K, M, F, P, S, T, W, Y, or V. In some embodiments, X³is selected from A, R, N, D, C, E, Q, G, H, I, L, K, M, F, P, S, T, W, Y, or V. In some embodiments, X⁴is selected from A, R, N, D, C, E, Q, G, H, I, L, K, M, F, P, S, T, W, Y, or V. In some embodiments, X⁵is selected from A, R, N, D, C, E, Q, G, H, I, L, K, M, F, P, S, T, W, Y, or V. In some embodiments, X⁶is selected from A, R, N, D, C, E, Q, G, H, I, L, K, M, F, P, S, T, W, Y, or V. In some embodiments, X⁷is selected from A, R, N, D, C, E, Q, G, H, I, L, K, M, F, P, S, T, W, Y, or V. In some embodiments, X⁸is selected A, R, N, D, C, E, Q, G, H, I, L, K, M, F, P, S, T, W, Y, or V. In some embodiments, X⁹is selected from A, R, N, D, C, E, Q, G, H, I, L, K, M, F, P, S, T, W, Y, or V.

In some embodiments, a 581-589 region of a muscle-tropic VP capsid polypeptide may comprise a sequence of any one of SEQ ID NO: 4238-SEQ ID NO: 4933.

In some embodiments, provided herein are AAV5 VP capsid polypeptide having at least one mutation in a 581-589 region, corresponding to residues 581 to 589 of AAV5 VP1 (e.g., SEQ ID NO: 1) and having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 99%, or 100% sequence identity to any sequence selected from SEQ ID NO: 4238-SEQ ID NO: 4933, wherein said at least one mutation drives increased muscle tissue tropism as compared to a wild type AAV5 capsid polypeptide.

Muscle-Tropic Sequences Generated by Machine Learning

Additional 581-589 regions that confer muscle tissue tropism or preference may be generated using machine learning algorithms trained with sequence identified in muscle tissue in in vivo screens. These patterns may be used to generate new sequences of 581-589 regions that increase the likelihood of conferring muscle tissue tropism to VP capsid polypeptides and rAAVs assembled from the VP capsid polypeptides. In some embodiments, new sequences of 581-589 regions may be generated by randomly sampling amino acid residues at each position of 581-589 regions that favored muscle tissue tropism in an in vivo screen. The generated sequences may be tested using random forests or gradient boosting classifiers to predicted muscle-specificity for each 581-589 region. The 581-589 regions with the highest predicted muscle-specificity may be selected as muscle-tropic sequences. For example, a 581-589 region of any one of SEQ ID NO: 4934-SEQ ID NO: 5233 may be selected. 581-589 region sequences generated using machine learning for increased likelihood of conferring muscle tropism to an rAAV are provided in TABLE 6.

Recombinant AAV viral capsids libraries may be screened in non-human primates (NHPs) by systemically administering the rAAV library and identifying sequences of 581-589 regions in VP capsid polypeptides that increased the likelihood of conferring tissue-tropic accumulation in or infection of muscle tissues (e.g., aorta, esophagus, heart (including atrium, ventricle, valves), skeletal muscle (including biceps, brachii, femoris, diaphragm, gastrocnemius, tibialis anterior, triceps, quadriceps, including vastus lateralis)), or muscle fibers (e.g., type I (slow oxidative) fibers, type IC fibers, type II fibers, type IIC fibers, type IIA (fast oxidative/glycolytic) fibers, type IIAX fibers, type IIXA fibers, type IIX (fast glycolytic) fibers, or combinations thereof.

A muscle-tropic rAAV assembled from VP capsid polypeptides (e.g., V1, VP2, or VP3 capsid polypeptides, or combinations thereof) comprising a 581-589 region may preferentially infect a muscle tissue (e.g., aorta, esophagus, heart (including atrium, ventricle, valves), skeletal muscle (including biceps, brachii, femoris, diaphragm, gastrocnemius, tibialis anterior, triceps, quadriceps, including vastus lateralis)), or muscle fibers (e.g., type I (slow oxidative) fibers, type IC fibers, type II fibers, type IIC fibers, type IIA (fast oxidative/glycolytic) fibers, type IIAX fibers, type IIXA fibers, type IIX (fast glycolytic) fibers, or combinations thereof) as compared to non-muscle tissue (e.g., liver, CNS, heart, lung, spleen, lymph node, bone marrow, mammary gland, skin, adrenal gland, thyroid, colon, sciatic nerve, spinal cord, or combinations thereof), as compared to a wild type AAV5 capsid polypeptide.

Disclosed herein are engineered AAV5 VP capsid polypeptides capable of forming an assembled virion that exhibits increased muscle tissue tropism as compared to a wild type AAV5 VP capsid polypeptide (e.g., a VP1 of SEQ ID NO: 1, a VP2 of SEQ ID NO: 10, or a VP3 of SEQ ID NO: 11), wherein the engineered variant AAV5 VP capsid polypeptide sequence has one or more amino acid substitutions in a 581-589 region of the VP capsid polypeptide, corresponding to positions 581 to 589 in SEQ ID NO: 2 (X¹X²X³X⁴X⁵X⁶X⁷X⁸X⁹). In some embodiments, X¹is selected from A, R, N, D, C, E, Q, G, H, I, L, K, M, F, P, S, T, W, Y, or V. In some embodiments, X²is selected from A, R, N, D, C, E, Q, G, H, I, L, K, M, F, P, S, T, W, Y, or V. In some embodiments, X³is selected from A, R, N, D, C, E, Q, G, H, I, L, K, M, F, P, S, T, W, Y, or V. In some embodiments, X⁴is selected from A, R, N, D, C, E, Q, G, H, I, L, K, M, F, P, S, T, W, Y, or V. In some embodiments, X⁵is selected from A, R, N, D, C, E, Q, G, H, I, L, K, M, F, P, S, T, W, Y, or V. In some embodiments, X⁶is selected from A, R, N, D, C, E, Q, G, H, I, L, K, M, F, P, S, T, W, Y, or V. In some embodiments, X⁷is selected from A, R, N, D, C, E, Q, G, H, I, L, K, M, F, P, S, T, W, Y, or V. In some embodiments, X⁸is selected A, R, N, D, C, E, Q, G, H, I, L, K, M, F, P, S, T, W, Y, or V. In some embodiments, X⁹is selected from A, R, N, D, C, E, Q, G, H, I, L, K, M, F, P, S, T, W, Y, or V.

In some embodiments, a 581-589 region of a muscle-tropic VP capsid polypeptide may comprise a sequence of any one of SEQ ID NO: 4934-SEQ ID NO: 5233.

In some embodiments, provided herein are AAV5 VP capsid polypeptide having at least one mutation in a 581-589 region, corresponding to residues 581 to 589 of AAV5 VP1 (e.g., SEQ ID NO: 1) and having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 99%, or 100% sequence identity to any sequence selected from SEQ ID NO: 4934-SEQ ID NO: 5233, wherein said at least one mutation drives increased muscle tissue tropism as compared to a wild type AAV5 capsid polypeptide.

Variant 581-589 Regions for Muscle Targeting

In some embodiments, provided herein are AAV5 VP capsid polypeptide having at least one mutation in a 581-589 region, corresponding to residues 581 to 589 of AAV5 VP1, and having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 99%, or 100% sequence identity to any sequence selected from SEQ ID NO: 4238-SEQ ID NO: 4933, SEQ ID NO: 4934-SEQ ID NO: 5233, or SEQ ID NO: 5808-SEQ ID NO: 5811, wherein said at least one mutation drives increased muscle tissue tropism as compared to a wild type AAV5 capsid polypeptide.

The present disclosure provides a tissue-tropic rAAV comprising a VP capsid polypeptide with a 581-589 variant region, relative to WT AAV5, of SEQ ID NO: 348. The present disclosure provides a tissue-tropic rAAV comprising a VP capsid polypeptide with a 581-589 variant region, relative to WT AAV5, of SEQ ID NO: 2536. The present disclosure provides a tissue-tropic rAAV comprising a VP capsid polypeptide with a 581-589 variant region, relative to WT AAV5, of SEQ ID NO: 5607. The present disclosure provides a tissue-tropic rAAV comprising a VP capsid polypeptide with a 581-589 variant region, relative to WT AAV5, of SEQ ID NO: 1576. The present disclosure provides a tissue-tropic rAAV comprising a VP capsid polypeptide with a 581-589 variant region, relative to WT AAV5, of SEQ ID NO: 4955. The present disclosure provides a tissue-tropic rAAV comprising a VP capsid polypeptide with a 581-589 variant region, relative to WT AAV5, of SEQ ID NO: 5017. The present disclosure provides a tissue-tropic rAAV comprising a VP capsid polypeptide with a 581-589 variant region, relative to WT AAV5, of SEQ ID NO: 4349. The present disclosure provides a tissue-tropic rAAV comprising a VP capsid polypeptide with a 581-589 variant region, relative to WT AAV5, of SEQ ID NO: 4964. The present disclosure provides a tissue-tropic rAAV comprising a VP capsid polypeptide with a 581-589 variant region, relative to WT AAV5, of SEQ ID NO: 293. The present disclosure provides a tissue-tropic rAAV comprising a VP capsid polypeptide with a 581-589 variant region, relative to WT AAV5, of SEQ ID NO: 4314. The present disclosure provides a tissue-tropic rAAV comprising a VP capsid polypeptide with a 581-589 variant region, relative to WT AAV5, of SEQ ID NO: 4995. The present disclosure provides a tissue-tropic rAAV comprising a VP capsid polypeptide with a 581-589 variant region, relative to WT AAV5, of SEQ ID NO: 4366. The present disclosure provides a tissue-tropic rAAV comprising a VP capsid polypeptide with a 581-589 variant region, relative to WT AAV5, of SEQ ID NO: 4961. The present disclosure provides a tissue-tropic rAAV comprising a VP capsid polypeptide with a 581-589 variant region, relative to WT AAV5, of SEQ ID NO: 4952. The present disclosure provides a tissue-tropic rAAV comprising a VP capsid polypeptide with a 581-589 variant region, relative to WT AAV5, of SEQ ID NO: 5075. The present disclosure provides a tissue-tropic rAAV comprising a VP capsid polypeptide with a 581-589 variant region, relative to WT AAV5, of SEQ ID NO: 4354. The present disclosure provides a tissue-tropic rAAV comprising a VP capsid polypeptide with a 581-589 variant region, relative to WT AAV5, of SEQ ID NO: 5060. The present disclosure provides a tissue-tropic rAAV comprising a VP capsid polypeptide with a 581-589 variant region, relative to WT AAV5, of SEQ ID NO: 5138. The present disclosure provides a tissue-tropic rAAV comprising a VP capsid polypeptide with a 581-589 variant region, relative to WT AAV5, of SEQ ID NO: 5206. The present disclosure provides a tissue-tropic rAAV comprising a VP capsid polypeptide with a 581-589 variant region, relative to WT AAV5, of SEQ ID NO: 4288. The present disclosure provides a tissue-tropic rAAV comprising a VP capsid polypeptide with a 581-589 variant region, relative to WT AAV5, of SEQ ID NO: 5283. The present disclosure provides a tissue-tropic rAAV comprising a VP capsid polypeptide with a 581-589 variant region, relative to WT AAV5, of SEQ ID NO: 5092. The present disclosure provides a tissue-tropic rAAV comprising a VP capsid polypeptide with a 581-589 variant region, relative to WT AAV5, of SEQ ID NO: 4059. The present disclosure provides a tissue-tropic rAAV comprising a VP capsid polypeptide with a 581-589 variant region, relative to WT AAV5, of SEQ ID NO: 4295. The present disclosure provides a tissue-tropic rAAV comprising a VP capsid polypeptide with a 581-589 variant region, relative to WT AAV5, of SEQ ID NO: 5030. The present disclosure provides a tissue-tropic rAAV comprising a VP capsid polypeptide with a 581-589 variant region, relative to WT AAV5, of SEQ ID NO: 4938. The present disclosure provides a tissue-tropic rAAV comprising a VP capsid polypeptide with a 581-589 variant region, relative to WT AAV5, of SEQ ID NO: 2661. The present disclosure provides a tissue-tropic rAAV comprising a VP capsid polypeptide with a 581-589 variant region, relative to WT AAV5, of SEQ ID NO: 5215. The present disclosure provides a tissue-tropic rAAV comprising a VP capsid polypeptide with a 581-589 variant region, relative to WT AAV5, of SEQ ID NO: 4960. The present disclosure provides a tissue-tropic rAAV comprising a VP capsid polypeptide with a 581-589 variant region, relative to WT AAV5, of SEQ ID NO: 4969. The present disclosure provides a tissue-tropic rAAV comprising a VP capsid polypeptide with a 581-589 variant region, relative to WT AAV5, of SEQ ID NO: 5023. The present disclosure provides a tissue-tropic rAAV comprising a VP capsid polypeptide with a 581-589 variant region, relative to WT AAV5, of SEQ ID NO: 4934. The present disclosure provides a tissue-tropic rAAV comprising a VP capsid polypeptide with a 581-589 variant region, relative to WT AAV5, of SEQ ID NO: 4545. The present disclosure provides a tissue-tropic rAAV comprising a VP capsid polypeptide with a 581-589 variant region, relative to WT AAV5, of SEQ ID NO: 4963. The present disclosure provides a tissue-tropic rAAV comprising a VP capsid polypeptide with a 581-589 variant region, relative to WT AAV5, of SEQ ID NO: 4363. The present disclosure provides a tissue-tropic rAAV comprising a VP capsid polypeptide with a 581-589 variant region, relative to WT AAV5, of SEQ ID NO: 5159. The present disclosure provides a tissue-tropic rAAV comprising a VP capsid polypeptide with a 581-589 variant region, relative to WT AAV5, of SEQ ID NO: 4973. The present disclosure provides a tissue-tropic rAAV comprising a VP capsid polypeptide with a 581-589 variant region, relative to WT AAV5, of SEQ ID NO: 5157. The present disclosure provides a tissue-tropic rAAV comprising a VP capsid polypeptide with a 581-589 variant region, relative to WT AAV5, of SEQ ID NO: 4972. The present disclosure provides a tissue-tropic rAAV comprising a VP capsid polypeptide with a 581-589 variant region, relative to WT AAV5, of SEQ ID NO: 4345. The present disclosure provides a tissue-tropic rAAV comprising a VP capsid polypeptide with a 581-589 variant region, relative to WT AAV5, of SEQ ID NO: 5039. The present disclosure provides a tissue-tropic rAAV comprising a VP capsid polypeptide with a 581-589 variant region, relative to WT AAV5, of SEQ ID NO: 5163. The present disclosure provides a tissue-tropic rAAV comprising a VP capsid polypeptide with a 581-589 variant region, relative to WT AAV5, of SEQ ID NO: 5040. The present disclosure provides a tissue-tropic rAAV comprising a VP capsid polypeptide with a 581-589 variant region, relative to WT AAV5, of SEQ ID NO: 4304. The present disclosure provides a tissue-tropic rAAV comprising a VP capsid polypeptide with a 581-589 variant region, relative to WT AAV5, of SEQ ID NO: 18. The present disclosure provides a tissue-tropic rAAV comprising a VP capsid polypeptide with a 581-589 variant region, relative to WT AAV5, of SEQ ID NO: 5193. The present disclosure provides a tissue-tropic rAAV comprising a VP capsid polypeptide with a 581-589 variant region, relative to WT AAV5, of SEQ ID NO: 5077. The present disclosure provides a tissue-tropic rAAV comprising a VP capsid polypeptide with a 581-589 variant region, relative to WT AAV5, of SEQ ID NO: 5814. The present disclosure provides a tissue-tropic rAAV comprising a VP capsid polypeptide with a 581-589 variant region, relative to WT AAV5, of SEQ ID NO: 386. The present disclosure provides a tissue-tropic rAAV comprising a VP capsid polypeptide with a 581-589 variant region, relative to WT AAV5, of SEQ ID NO: 5081. The present disclosure provides a tissue-tropic rAAV comprising a VP capsid polypeptide with a 581-589 variant region, relative to WT AAV5, of SEQ ID NO: 308. The present disclosure provides a tissue-tropic rAAV comprising a VP capsid polypeptide with a 581-589 variant region, relative to WT AAV5, of SEQ ID NO: 22. The present disclosure provides a tissue-tropic rAAV comprising a VP capsid polypeptide with a 581-589 variant region, relative to WT AAV5, of SEQ ID NO: 4338. The present disclosure provides a tissue-tropic rAAV comprising a VP capsid polypeptide with a 581-589 variant region, relative to WT AAV5, of SEQ ID NO: 3846. The present disclosure provides a tissue-tropic rAAV comprising a VP capsid polypeptide with a 581-589 variant region, relative to WT AAV5, of SEQ ID NO: 278. The present disclosure provides a tissue-tropic rAAV comprising a VP capsid polypeptide with a 581-589 variant region, relative to WT AAV5, of SEQ ID NO: 5127. The present disclosure provides a tissue-tropic rAAV comprising a VP capsid polypeptide with a 581-589 variant region, relative to WT AAV5, of SEQ ID NO: 5029. The present disclosure provides a tissue-tropic rAAV comprising a VP capsid polypeptide with a 581-589 variant region, relative to WT AAV5, of SEQ ID NO: 5037. The present disclosure provides a tissue-tropic rAAV comprising a VP capsid polypeptide with a 581-589 variant region, relative to WT AAV5, of SEQ ID NO: 5143. The present disclosure provides a tissue-tropic rAAV comprising a VP capsid polypeptide with a 581-589 variant region, relative to WT AAV5, of SEQ ID NO: 4983. The present disclosure provides a tissue-tropic rAAV comprising a VP capsid polypeptide with a 581-589 variant region, relative to WT AAV5, of SEQ ID NO: 4361. The present disclosure provides a tissue-tropic rAAV comprising a VP capsid polypeptide with a 581-589 variant region, relative to WT AAV5, of SEQ ID NO: 4317. The present disclosure provides a tissue-tropic rAAV comprising a VP capsid polypeptide with a 581-589 variant region, relative to WT AAV5, of SEQ ID NO: 5080. The present disclosure provides a tissue-tropic rAAV comprising a VP capsid polypeptide with a 581-589 variant region, relative to WT AAV5, of SEQ ID NO: 23. The present disclosure provides a tissue-tropic rAAV comprising a VP capsid polypeptide with a 581-589 variant region, relative to WT AAV5, of SEQ ID NO: 4343. The present disclosure provides a tissue-tropic rAAV comprising a VP capsid polypeptide with a 581-589 variant region, relative to WT AAV5, of SEQ ID NO: 4986. The present disclosure provides a tissue-tropic rAAV comprising a VP capsid polypeptide with a 581-589 variant region, relative to WT AAV5, of SEQ ID NO: 4311. The present disclosure provides a tissue-tropic rAAV comprising a VP capsid polypeptide with a 581-589 variant region, relative to WT AAV5, of SEQ ID NO: 5102. The present disclosure provides a tissue-tropic rAAV comprising a VP capsid polypeptide with a 581-589 variant region, relative to WT AAV5, of SEQ ID NO: 5280. The present disclosure provides a tissue-tropic rAAV comprising a VP capsid polypeptide with a 581-589 variant region, relative to WT AAV5, of SEQ ID NO: 5185. The present disclosure provides a tissue-tropic rAAV comprising a VP capsid polypeptide with a 581-589 variant region, relative to WT AAV5, of SEQ ID NO: 964. The present disclosure provides a tissue-tropic rAAV comprising a VP capsid polypeptide with a 581-589 variant region, relative to WT AAV5, of SEQ ID NO: 4935. The present disclosure provides a tissue-tropic rAAV comprising a VP capsid polypeptide with a 581-589 variant region, relative to WT AAV5, of SEQ ID NO: 4379. The present disclosure provides a tissue-tropic rAAV comprising a VP capsid polypeptide with a 581-589 variant region, relative to WT AAV5, of SEQ ID NO: 5052. The present disclosure provides a tissue-tropic rAAV comprising a VP capsid polypeptide with a 581-589 variant region, relative to WT AAV5, of SEQ ID NO: 5027. The present disclosure provides a tissue-tropic rAAV comprising a VP capsid polypeptide with a 581-589 variant region, relative to WT AAV5, of SEQ ID NO: 5173. The present disclosure provides a tissue-tropic rAAV comprising a VP capsid polypeptide with a 581-589 variant region, relative to WT AAV5, of SEQ ID NO: 5055. The present disclosure provides a tissue-tropic rAAV comprising a VP capsid polypeptide with a 581-589 variant region, relative to WT AAV5, of SEQ ID NO: 5123. The present disclosure provides a tissue-tropic rAAV comprising a VP capsid polypeptide with a 581-589 variant region, relative to WT AAV5, of SEQ ID NO: 4335. The present disclosure provides a tissue-tropic rAAV comprising a VP capsid polypeptide with a 581-589 variant region, relative to WT AAV5, of SEQ ID NO: 4936. The present disclosure provides a tissue-tropic rAAV comprising a VP capsid polypeptide with a 581-589 variant region, relative to WT AAV5, of SEQ ID NO: 5208. The present disclosure provides a tissue-tropic rAAV comprising a VP capsid polypeptide with a 581-589 variant region, relative to WT AAV5, of SEQ ID NO: 4633. The present disclosure provides a tissue-tropic rAAV comprising a VP capsid polypeptide with a 581-589 variant region, relative to WT AAV5, of SEQ ID NO: 4359. The present disclosure provides a tissue-tropic rAAV comprising a VP capsid polypeptide with a 581-589 variant region, relative to WT AAV5, of SEQ ID NO: 289. The present disclosure provides a tissue-tropic rAAV comprising a VP capsid polypeptide with a 581-589 variant region, relative to WT AAV5, of SEQ ID NO: 4353. The present disclosure provides a tissue-tropic rAAV comprising a VP capsid polypeptide with a 581-589 variant region, relative to WT AAV5, of SEQ ID NO: 4316. The present disclosure provides a tissue-tropic rAAV comprising a VP capsid polypeptide with a 581-589 variant region, relative to WT AAV5, of SEQ ID NO: 5210. The present disclosure provides a tissue-tropic rAAV comprising a VP capsid polypeptide with a 581-589 variant region, relative to WT AAV5, of SEQ ID NO: 4949. The present disclosure provides a tissue-tropic rAAV comprising a VP capsid polypeptide with a 581-589 variant region, relative to WT AAV5, of SEQ ID NO: 4947. The present disclosure provides a tissue-tropic rAAV comprising a VP capsid polypeptide with a 581-589 variant region, relative to WT AAV5, of SEQ ID NO: 2838. The present disclosure provides a tissue-tropic rAAV comprising a VP capsid polypeptide with a 581-589 variant region, relative to WT AAV5, of SEQ ID NO: 378. The present disclosure provides a tissue-tropic rAAV comprising a VP capsid polypeptide with a 581-589 variant region, relative to WT AAV5, of SEQ ID NO: 5013. The present disclosure provides a tissue-tropic rAAV comprising a VP capsid polypeptide with a 581-589 variant region, relative to WT AAV5, of SEQ ID NO: 5278. The present disclosure provides a tissue-tropic rAAV comprising a VP capsid polypeptide with a 581-589 variant region, relative to WT AAV5, of SEQ ID NO: 1471. The present disclosure provides a tissue-tropic rAAV comprising a VP capsid polypeptide with a 581-589 variant region, relative to WT AAV5, of SEQ ID NO: 4346. The present disclosure provides a tissue-tropic rAAV comprising a VP capsid polypeptide with a 581-589 variant region, relative to WT AAV5, of SEQ ID NO: 297. The present disclosure provides a tissue-tropic rAAV comprising a VP capsid polypeptide with a 581-589 variant region, relative to WT AAV5, of SEQ ID NO: 4993. The present disclosure provides a tissue-tropic rAAV comprising a VP capsid polypeptide with a 581-589 variant region, relative to WT AAV5, of SEQ ID NO: 384. The present disclosure provides a tissue-tropic rAAV comprising a VP capsid polypeptide with a 581-589 variant region, relative to WT AAV5, of SEQ ID NO: 5062. The present disclosure provides a tissue-tropic rAAV comprising a VP capsid polypeptide with a 581-589 variant region, relative to WT AAV5, of SEQ ID NO: 4945. The present disclosure provides a tissue-tropic rAAV comprising a VP capsid polypeptide with a 581-589 variant region, relative to WT AAV5, of SEQ ID NO: 3472. The present disclosure provides a tissue-tropic rAAV comprising a VP capsid polypeptide with a 581-589 variant region, relative to WT AAV5, of SEQ ID NO: 3297. The present disclosure provides a tissue-tropic rAAV comprising a VP capsid polypeptide with a 581-589 variant region, relative to WT AAV5, of SEQ ID NO: 210. The present disclosure provides a tissue-tropic rAAV comprising a VP capsid polypeptide with a 581-589 variant region, relative to WT AAV5, of SEQ ID NO: 1971. The present disclosure provides a tissue-tropic rAAV comprising a VP capsid polypeptide with a 581-589 variant region, relative to WT AAV5, of SEQ ID NO: 262. The present disclosure provides a tissue-tropic rAAV comprising a VP capsid polypeptide with a 581-589 variant region, relative to WT AAV5, of SEQ ID NO: 4009. The present disclosure provides a tissue-tropic rAAV comprising a VP capsid polypeptide with a 581-589 variant region, relative to WT AAV5, of SEQ ID NO: 5190. The present disclosure provides a tissue-tropic rAAV comprising a VP capsid polypeptide with a 581-589 variant region, relative to WT AAV5, of SEQ ID NO: 724. The present disclosure provides a tissue-tropic rAAV comprising a VP capsid polypeptide with a 581-589 variant region, relative to WT AAV5, of SEQ ID NO: 1561. The present disclosure provides a tissue-tropic rAAV comprising a VP capsid polypeptide with a 581-589 variant region, relative to WT AAV5, of SEQ ID NO: 5147. The present disclosure provides a tissue-tropic rAAV comprising a VP capsid polypeptide with a 581-589 variant region, relative to WT AAV5, of SEQ ID NO: 4238. The present disclosure provides a tissue-tropic rAAV comprising a VP capsid polypeptide with a 581-589 variant region, relative to WT AAV5, of SEQ ID NO: 5527. The present disclosure provides a tissue-tropic rAAV comprising a VP capsid polypeptide with a 581-589 variant region, relative to WT AAV5, of SEQ ID NO: 3283. The present disclosure provides a tissue-tropic rAAV comprising a VP capsid polypeptide with a 581-589 variant region, relative to WT AAV5, of SEQ ID NO: 4355. The present disclosure provides a tissue-tropic rAAV comprising a VP capsid polypeptide with a 581-589 variant region, relative to WT AAV5, of SEQ ID NO: 5125. The present disclosure provides a tissue-tropic rAAV comprising a VP capsid polypeptide with a 581-589 variant region, relative to WT AAV5, of SEQ ID NO: 4108. The present disclosure provides a tissue-tropic rAAV comprising a VP capsid polypeptide with a 581-589 variant region, relative to WT AAV5, of SEQ ID NO: 2948. The present disclosure provides a tissue-tropic rAAV comprising a VP capsid polypeptide with a 581-589 variant region, relative to WT AAV5, of SEQ ID NO: 3821. The present disclosure provides a tissue-tropic rAAV comprising a VP capsid polypeptide with a 581-589 variant region, relative to WT AAV5, of SEQ ID NO: 4632. The present disclosure provides a tissue-tropic rAAV comprising a VP capsid polypeptide with a 581-589 variant region, relative to WT AAV5, of SEQ ID NO: 294. The present disclosure provides a tissue-tropic rAAV comprising a VP capsid polypeptide with a 581-589 variant region, relative to WT AAV5, of SEQ ID NO: 4943. The present disclosure provides a tissue-tropic rAAV comprising a VP capsid polypeptide with a 581-589 variant region, relative to WT AAV5, of SEQ ID NO: 1391. The present disclosure provides a tissue-tropic rAAV comprising a VP capsid polypeptide with a 581-589 variant region, relative to WT AAV5, of SEQ ID NO: 5078. The present disclosure provides a tissue-tropic rAAV comprising a VP capsid polypeptide with a 581-589 variant region, relative to WT AAV5, of SEQ ID NO: 3299. The present disclosure provides a tissue-tropic rAAV comprising a VP capsid polypeptide with a 581-589 variant region, relative to WT AAV5, of SEQ ID NO: 2897. The present disclosure provides a tissue-tropic rAAV comprising a VP capsid polypeptide with a 581-589 variant region, relative to WT AAV5, of SEQ ID NO: 1537. The present disclosure provides a tissue-tropic rAAV comprising a VP capsid polypeptide with a 581-589 variant region, relative to WT AAV5, of SEQ ID NO: 5255. The present disclosure provides a tissue-tropic rAAV comprising a VP capsid polypeptide with a 581-589 variant region, relative to WT AAV5, of SEQ ID NO: 141. The present disclosure provides a tissue-tropic rAAV comprising a VP capsid polypeptide with a 581-589 variant region, relative to WT AAV5, of SEQ ID NO: 5038. The present disclosure provides a tissue-tropic rAAV comprising a VP capsid polypeptide with a 581-589 variant region, relative to WT AAV5, of SEQ ID NO: 5153. The present disclosure provides a tissue-tropic rAAV comprising a VP capsid polypeptide with a 581-589 variant region, relative to WT AAV5, of SEQ ID NO: 1181. The present disclosure provides a tissue-tropic rAAV comprising a VP capsid polypeptide with a 581-589 variant region, relative to WT AAV5, of SEQ ID NO: 307. The present disclosure provides a tissue-tropic rAAV comprising a VP capsid polypeptide with a 581-589 variant region, relative to WT AAV5, of SEQ ID NO: 1204. The present disclosure provides a tissue-tropic rAAV comprising a VP capsid polypeptide with a 581-589 variant region, relative to WT AAV5, of SEQ ID NO: 4404. The present disclosure provides a tissue-tropic rAAV comprising a VP capsid polypeptide with a 581-589 variant region, relative to WT AAV5, of SEQ ID NO: 5106. The present disclosure provides a tissue-tropic rAAV comprising a VP capsid polypeptide with a 581-589 variant region, relative to WT AAV5, of SEQ ID NO: 2779. The present disclosure provides a tissue-tropic rAAV comprising a VP capsid polypeptide with a 581-589 variant region, relative to WT AAV5, of SEQ ID NO: 1824. The present disclosure provides a tissue-tropic rAAV comprising a VP capsid polypeptide with a 581-589 variant region, relative to WT AAV5, of SEQ ID NO: 5116. The present disclosure provides a tissue-tropic rAAV comprising a VP capsid polypeptide with a 581-589 variant region, relative to WT AAV5, of SEQ ID NO: 3179. The present disclosure provides a tissue-tropic rAAV comprising a VP capsid polypeptide with a 581-589 variant region, relative to WT AAV5, of SEQ ID NO: 3306. The present disclosure provides a tissue-tropic rAAV comprising a VP capsid polypeptide with a 581-589 variant region, relative to WT AAV5, of SEQ ID NO: 98. The present disclosure provides a tissue-tropic rAAV comprising a VP capsid polypeptide with a 581-589 variant region, relative to WT AAV5, of SEQ ID NO: 1872. The present disclosure provides a tissue-tropic rAAV comprising a VP capsid polypeptide with a 581-589 variant region, relative to WT AAV5, of SEQ ID NO: 1268. The present disclosure provides a tissue-tropic rAAV comprising a VP capsid polypeptide with a 581-589 variant region, relative to WT AAV5, of SEQ ID NO: 1634. The present disclosure provides a tissue-tropic rAAV comprising a VP capsid polypeptide with a 581-589 variant region, relative to WT AAV5, of SEQ ID NO: 1060. The present disclosure provides a tissue-tropic rAAV comprising a VP capsid polypeptide with a 581-589 variant region, relative to WT AAV5, of SEQ ID NO: 4941. The present disclosure provides a tissue-tropic rAAV comprising a VP capsid polypeptide with a 581-589 variant region, relative to WT AAV5, of SEQ ID NO: 4981. The present disclosure provides a tissue-tropic rAAV comprising a VP capsid polypeptide with a 581-589 variant region, relative to WT AAV5, of SEQ ID NO: 5274. The present disclosure provides a tissue-tropic rAAV comprising a VP capsid polypeptide with a 581-589 variant region, relative to WT AAV5, of SEQ ID NO: 5816. The present disclosure provides a tissue-tropic rAAV comprising a VP capsid polypeptide with a 581-589 variant region, relative to WT AAV5, of SEQ ID NO: 2842. The present disclosure provides a tissue-tropic rAAV comprising a VP capsid polypeptide with a 581-589 variant region, relative to WT AAV5, of SEQ ID NO: 1934. The present disclosure provides a tissue-tropic rAAV comprising a VP capsid polypeptide with a 581-589 variant region, relative to WT AAV5, of SEQ ID NO: 5817. The present disclosure provides a tissue-tropic rAAV comprising a VP capsid polypeptide with a 581-589 variant region, relative to WT AAV5, of SEQ ID NO: 3998. The present disclosure provides a tissue-tropic rAAV comprising a VP capsid polypeptide with a 581-589 variant region, relative to WT AAV5, of SEQ ID NO: 5026. The present disclosure provides a tissue-tropic rAAV comprising a VP capsid polypeptide with a 581-589 variant region, relative to WT AAV5, of SEQ ID NO: 4290.

Variant 581-589 Regions for CNS and Cardiac Muscle Dual Tissue Tropism

Variant 581-589 Regions for CNS and Skeletal Muscle Dual Tissue Tropism

Variant 581-589 Regions for CNS and Muscle Dual Tissue Tropism

Variant 581-589 Regions for Cardiac Muscle and Skeletal Muscle Dual Tissue Tropism

The present disclosure provides AAV5 virions with a VP capsid polypeptide having at least one mutation in a region with residues that interact with target cells, where the at least one mutation confers tissue tropism for cardiac muscle and skeletal muscle as compared to a wild-type VP capsid polypeptide (e.g., a VP capsid polypeptide of SEQ ID NO: 1, SEQ ID NO: 10, or SEQ ID NO: 11). In some embodiments, provided herein are AAV5 VP capsid polypeptide having at least one mutation in a 581-589 region, corresponding to residues 581 to 589 of AAV5 VP1, and having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 99%, or 100% sequence identity to any sequence selected from SEQ ID NO: 348, SEQ ID NO: 2536, SEQ ID NO: 5607, SEQ ID NO: 4955, SEQ ID NO: 5017, SEQ ID NO: 4349, SEQ ID NO: 4964, SEQ ID NO: 293, SEQ ID NO: 4314, SEQ ID NO: 4995, SEQ ID NO: 4366, SEQ ID NO: 4961, SEQ ID NO: 4952, SEQ ID NO: 5075, SEQ ID NO: 4354, SEQ ID NO: 5060, SEQ ID NO: 5138, SEQ ID NO: 5206, SEQ ID NO: 4288, SEQ ID NO: 5283, SEQ ID NO: 5092, SEQ ID NO: 4059, SEQ ID NO: 4295, SEQ ID NO: 5030, SEQ ID NO: 2661, SEQ ID NO: 4545, SEQ ID NO: 4963, SEQ ID NO: 4363, SEQ ID NO: 5159, SEQ ID NO: 4973, SEQ ID NO: 5157, SEQ ID NO: 4345, SEQ ID NO: 5039, SEQ ID NO: 4304, SEQ ID NO: 18, SEQ ID NO: 386, SEQ ID NO: 308, SEQ ID NO: 278, SEQ ID NO: 5037, SEQ ID NO: 4361, SEQ ID NO: 4317, SEQ ID NO: 4343, SEQ ID NO: 4311, SEQ ID NO: 5055, SEQ ID NO: 4359, SEQ ID NO: 4353, or SEQ ID NO: 4346, wherein said at least one mutation drives cardiac muscle and skeletal muscle tissue tropism. The present disclosure provides an rAAV having a VP capsid polypeptide with a 581-589 variant region of SEQ ID NO: 348, wherein the rAAV has tissue tropism for cardiac muscle tissue and skeletal muscle tissue. The present disclosure provides an rAAV having a VP capsid polypeptide with a 581-589 variant region of SEQ ID NO: 2536, wherein the rAAV has tissue tropism for cardiac muscle tissue and skeletal muscle tissue. The present disclosure provides an rAAV having a VP capsid polypeptide with a 581-589 variant region of SEQ ID NO: 5607, wherein the rAAV has tissue tropism for cardiac muscle tissue and skeletal muscle tissue. The present disclosure provides an rAAV having a VP capsid polypeptide with a 581-589 variant region of SEQ ID NO: 4955, wherein the rAAV has tissue tropism for cardiac muscle tissue and skeletal muscle tissue. The present disclosure provides an rAAV having a VP capsid polypeptide with a 581-589 variant region of SEQ ID NO: 5017, wherein the rAAV has tissue tropism for cardiac muscle tissue and skeletal muscle tissue. The present disclosure provides an rAAV having a VP capsid polypeptide with a 581-589 variant region of SEQ ID NO: 4349, wherein the rAAV has tissue tropism for cardiac muscle tissue and skeletal muscle tissue. The present disclosure provides an rAAV having a VP capsid polypeptide with a 581-589 variant region of SEQ ID NO: 4964, wherein the rAAV has tissue tropism for cardiac muscle tissue and skeletal muscle tissue. The present disclosure provides an rAAV having a VP capsid polypeptide with a 581-589 variant region of SEQ ID NO: 293, wherein the rAAV has tissue tropism for cardiac muscle tissue and skeletal muscle tissue. The present disclosure provides an rAAV having a VP capsid polypeptide with a 581-589 variant region of SEQ ID NO: 4314, wherein the rAAV has tissue tropism for cardiac muscle tissue and skeletal muscle tissue. The present disclosure provides an rAAV having a VP capsid polypeptide with a 581-589 variant region of SEQ ID NO: 4995, wherein the rAAV has tissue tropism for cardiac muscle tissue and skeletal muscle tissue. The present disclosure provides an rAAV having a VP capsid polypeptide with a 581-589 variant region of SEQ ID NO: 4366, wherein the rAAV has tissue tropism for cardiac muscle tissue and skeletal muscle tissue. The present disclosure provides an rAAV having a VP capsid polypeptide with a 581-589 variant region of SEQ ID NO: 4961, wherein the rAAV has tissue tropism for cardiac muscle tissue and skeletal muscle tissue. The present disclosure provides an rAAV having a VP capsid polypeptide with a 581-589 variant region of SEQ ID NO: 4952, wherein the rAAV has tissue tropism for cardiac muscle tissue and skeletal muscle tissue. The present disclosure provides an rAAV having a VP capsid polypeptide with a 581-589 variant region of SEQ ID NO: 5075, wherein the rAAV has tissue tropism for cardiac muscle tissue and skeletal muscle tissue. The present disclosure provides an rAAV having a VP capsid polypeptide with a 581-589 variant region of SEQ ID NO: 4354, wherein the rAAV has tissue tropism for cardiac muscle tissue and skeletal muscle tissue. The present disclosure provides an rAAV having a VP capsid polypeptide with a 581-589 variant region of SEQ ID NO: 5060, wherein the rAAV has tissue tropism for cardiac muscle tissue and skeletal muscle tissue. The present disclosure provides an rAAV having a VP capsid polypeptide with a 581-589 variant region of SEQ ID NO: 5138, wherein the rAAV has tissue tropism for cardiac muscle tissue and skeletal muscle tissue. The present disclosure provides an rAAV having a VP capsid polypeptide with a 581-589 variant region of SEQ ID NO: 5206, wherein the rAAV has tissue tropism for cardiac muscle tissue and skeletal muscle tissue. The present disclosure provides an rAAV having a VP capsid polypeptide with a 581-589 variant region of SEQ ID NO: 4288, wherein the rAAV has tissue tropism for cardiac muscle tissue and skeletal muscle tissue. The present disclosure provides an rAAV having a VP capsid polypeptide with a 581-589 variant region of SEQ ID NO: 5283, wherein the rAAV has tissue tropism for cardiac muscle tissue and skeletal muscle tissue. The present disclosure provides an rAAV having a VP capsid polypeptide with a 581-589 variant region of SEQ ID NO: 5092, wherein the rAAV has tissue tropism for cardiac muscle tissue and skeletal muscle tissue. The present disclosure provides an rAAV having a VP capsid polypeptide with a 581-589 variant region of SEQ ID NO: 4059, wherein the rAAV has tissue tropism for cardiac muscle tissue and skeletal muscle tissue. The present disclosure provides an rAAV having a VP capsid polypeptide with a 581-589 variant region of SEQ ID NO: 4295, wherein the rAAV has tissue tropism for cardiac muscle tissue and skeletal muscle tissue. The present disclosure provides an rAAV having a VP capsid polypeptide with a 581-589 variant region of SEQ ID NO: 5030, wherein the rAAV has tissue tropism for cardiac muscle tissue and skeletal muscle tissue. The present disclosure provides an rAAV having a VP capsid polypeptide with a 581-589 variant region of SEQ ID NO: 2661, wherein the rAAV has tissue tropism for cardiac muscle tissue and skeletal muscle tissue. The present disclosure provides an rAAV having a VP capsid polypeptide with a 581-589 variant region of SEQ ID NO: 4545, wherein the rAAV has tissue tropism for cardiac muscle tissue and skeletal muscle tissue. The present disclosure provides an rAAV having a VP capsid polypeptide with a 581-589 variant region of SEQ ID NO: 4963, wherein the rAAV has tissue tropism for cardiac muscle tissue and skeletal muscle tissue. The present disclosure provides an rAAV having a VP capsid polypeptide with a 581-589 variant region of SEQ ID NO: 4363, wherein the rAAV has tissue tropism for cardiac muscle tissue and skeletal muscle tissue. The present disclosure provides an rAAV having a VP capsid polypeptide with a 581-589 variant region of SEQ ID NO: 5159, wherein the rAAV has tissue tropism for cardiac muscle tissue and skeletal muscle tissue. The present disclosure provides an rAAV having a VP capsid polypeptide with a 581-589 variant region of SEQ ID NO: 4973, wherein the rAAV has tissue tropism for cardiac muscle tissue and skeletal muscle tissue. The present disclosure provides an rAAV having a VP capsid polypeptide with a 581-589 variant region of SEQ ID NO: 5157, wherein the rAAV has tissue tropism for cardiac muscle tissue and skeletal muscle tissue. The present disclosure provides an rAAV having a VP capsid polypeptide with a 581-589 variant region of SEQ ID NO: 4345, wherein the rAAV has tissue tropism for cardiac muscle tissue and skeletal muscle tissue. The present disclosure provides an rAAV having a VP capsid polypeptide with a 581-589 variant region of SEQ ID NO: 5039, wherein the rAAV has tissue tropism for cardiac muscle tissue and skeletal muscle tissue. The present disclosure provides an rAAV having a VP capsid polypeptide with a 581-589 variant region of SEQ ID NO: 4304, wherein the rAAV has tissue tropism for cardiac muscle tissue and skeletal muscle tissue. The present disclosure provides an rAAV having a VP capsid polypeptide with a 581-589 variant region of SEQ ID NO: 18, wherein the rAAV has tissue tropism for cardiac muscle tissue and skeletal muscle tissue. The present disclosure provides an rAAV having a VP capsid polypeptide with a 581-589 variant region of SEQ ID NO: 386, wherein the rAAV has tissue tropism for cardiac muscle tissue and skeletal muscle tissue. The present disclosure provides an rAAV having a VP capsid polypeptide with a 581-589 variant region of SEQ ID NO: 308, wherein the rAAV has tissue tropism for cardiac muscle tissue and skeletal muscle tissue. The present disclosure provides an rAAV having a VP capsid polypeptide with a 581-589 variant region of SEQ ID NO: 278, wherein the rAAV has tissue tropism for cardiac muscle tissue and skeletal muscle tissue. The present disclosure provides an rAAV having a VP capsid polypeptide with a 581-589 variant region of SEQ ID NO: 5037, wherein the rAAV has tissue tropism for cardiac muscle tissue and skeletal muscle tissue. The present disclosure provides an rAAV having a VP capsid polypeptide with a 581-589 variant region of SEQ ID NO: 4361, wherein the rAAV has tissue tropism for cardiac muscle tissue and skeletal muscle tissue. The present disclosure provides an rAAV having a VP capsid polypeptide with a 581-589 variant region of SEQ ID NO: 4317, wherein the rAAV has tissue tropism for cardiac muscle tissue and skeletal muscle tissue. The present disclosure provides an rAAV having a VP capsid polypeptide with a 581-589 variant region of SEQ ID NO: 4343, wherein the rAAV has tissue tropism for cardiac muscle tissue and skeletal muscle tissue. The present disclosure provides an rAAV having a VP capsid polypeptide with a 581-589 variant region of SEQ ID NO: 4311, wherein the rAAV has tissue tropism for cardiac muscle tissue and skeletal muscle tissue. The present disclosure provides an rAAV having a VP capsid polypeptide with a 581-589 variant region of SEQ ID NO: 5055, wherein the rAAV has tissue tropism for cardiac muscle tissue and skeletal muscle tissue. The present disclosure provides an rAAV having a VP capsid polypeptide with a 581-589 variant region of SEQ ID NO: 4359, wherein the rAAV has tissue tropism for cardiac muscle tissue and skeletal muscle tissue. The present disclosure provides an rAAV having a VP capsid polypeptide with a 581-589 variant region of SEQ ID NO: 4353, wherein the rAAV has tissue tropism for cardiac muscle tissue and skeletal muscle tissue. The present disclosure provides an rAAV having a VP capsid polypeptide with a 581-589 variant region of SEQ ID NO: 4346, wherein the rAAV has tissue tropism for cardiac muscle tissue and skeletal muscle tissue.

Research Tools

An engineered VP capsid polypeptide of the present disclosure, or a recombinant AAV capsid comprising an engineered VP capsid polypeptide, may be used as a research tool in mice. In some embodiments, a recombinant capsid may exhibit tissue tropism in both humans and in a model organism (e.g., non-human primates or mice). In some embodiments, a recombinant capsid may exhibit tissue tropism in both non-human primates and in a second model organism (e.g., mice). A recombinant AAV capsid exhibiting tissue tropism a first organism (e.g., humans or non-human primates) and a second organism (e.g., non-human primates or mice) may be used as a research tool in the second organism to represent behavior of the recombinant AAV capsid, or a recombinant virus comprising the recombinant AAV capsid, in the second organism. For example, a recombinant AAV capsid having 581-589 region sequences of SEQ ID NO: 5027, SEQ ID NO: 4633, SEQ ID NO: 4943, SEQ ID NO: 386, SEQ ID NO: 334, SEQ ID NO: 4309, SEQ ID NO: 4288, SEQ ID NO: 4964, SEQ ID NO: 5017, SEQ ID NO: 5814 (MACKVHLQP), SEQ ID NO: 4335, SEQ ID NO: 348, SEQ ID NO: 4936, SEQ ID NO: 5206, SEQ ID NO: 4238, SEQ ID NO: 5077, or SEQ ID NO: 5603 may be used as a research tool in mice to represent behavior of the recombinant AAV capsid, or a recombinant virus comprising the recombinant AAV capsid in humans or non-human primates. In some embodiments, a recombinant AAV capsid having 581-589 region sequences of SEQ ID NO: 5027, SEQ ID NO: 4633, SEQ ID NO: 4943, SEQ ID NO: 386, SEQ ID NO: 334, SEQ ID NO: 4309, SEQ ID NO: 4288, SEQ ID NO: 4964, SEQ ID NO: 5017, SEQ ID NO: 5814, SEQ ID NO: 4335, SEQ ID NO: 348, SEQ ID NO: 4936, SEQ ID NO: 5206, SEQ ID NO: 4238, SEQ ID NO: 5077, or SEQ ID NO: 5603 exhibits cardiac muscle tissue tropism in both non-human primates and mice.

In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence that is at least 75%, at least 77.7%, at least 85%, at least 88.8% or 100% identical to a sequence provided in TABLE 13.

In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence that is at least 75%, at least 77.7%, at least 85%, at least 88.8% or 100% identical to SEQ ID NO: 5027. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence that is at least 75%, at least 77.7%, at least 85%, at least 88.8% or 100% identical to SEQ ID NO: 4633. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence that is at least 75%, at least 77.7%, at least 85%, at least 88.8% or 100% identical to SEQ ID NO: 4943. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence that is at least 75%, at least 77.7%, at least 85%, at least 88.8% or 100% identical to SEQ ID NO: 386. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence that is at least 75%, at least 77.7%, at least 85%, at least 88.8% or 100% identical to SEQ ID NO: 334. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence that is at least 75%, at least 77.7%, at least 85%, at least 88.8% or 100% identical to SEQ ID NO: 4309. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence that is at least 75%, at least 77.7%, at least 85%, at least 88.8% or 100% identical to SEQ ID NO: 4288. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence that is at least 75%, at least 77.7%, at least 85%, at least 88.8% or 100% identical to SEQ ID NO: 4964. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence that is at least 75%, at least 77.7%, at least 85%, at least 88.8% or 100% identical to SEQ ID NO: 5017. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence that is at least 75%, at least 77.7%, at least 85%, at least 88.8% or 100% identical to SEQ ID NO: 5814. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence that is at least 75%, at least 77.7%, at least 85%, at least 88.8% or 100% identical to SEQ ID NO: 4335. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence that is at least 75%, at least 77.7%, at least 85%, at least 88.8% or 100% identical to SEQ ID NO: 348. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence that is at least 75%, at least 77.7%, at least 85%, at least 88.8% or 100% identical to SEQ ID NO: 4936. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence that is at least 75%, at least 77.7%, at least 85%, at least 88.8% or 100% identical to SEQ ID NO: 5206. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence that is at least 75%, at least 77.7%, at least 85%, at least 88.8% or 100% identical to SEQ ID NO: 4238. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence that is at least 75%, at least 77.7%, at least 85%, at least 88.8% or 100% identical to SEQ ID NO: 5077. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence that is at least 75%, at least 77.7%, at least 85%, at least 88.8% or 100% identical to SEQ ID NO: 5603.

In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence of SEQ ID NO: 5027. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence of SEQ ID NO: 4633. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence of SEQ ID NO: 4943. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence of SEQ ID NO: 386. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence of SEQ ID NO: 334. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence of SEQ ID NO: 4309. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence of SEQ ID NO: 4288. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence of SEQ ID NO: 4964. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence of SEQ ID NO: 5017. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence of SEQ ID NO: 5814. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence of SEQ ID NO: 4335. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence of SEQ ID NO: 348. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence of SEQ ID NO: 4936. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence of SEQ ID NO: 5206. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence of SEQ ID NO: 4238. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence of SEQ ID NO: 5077. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence of SEQ ID NO: 5603.

In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence of SEQ ID NO: 5027 with 1 or 2 conservative substitutions. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence of SEQ ID NO: 4633 with 1 or 2 conservative substitutions. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence of SEQ ID NO: 4943 with 1 or 2 conservative substitutions. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence of SEQ ID NO: 386 with 1 or 2 conservative substitutions. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence of SEQ ID NO: 334 with 1 or 2 conservative substitutions. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence of SEQ ID NO: 4309 with 1 or 2 conservative substitutions. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence of SEQ ID NO: 4288 with 1 or 2 conservative substitutions. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence of SEQ ID NO: 4964 with 1 or 2 conservative substitutions. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence of SEQ ID NO: 5017 with 1 or 2 conservative substitutions. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence of SEQ ID NO: 5814 with 1 or 2 conservative substitutions. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence of SEQ ID NO: 4335 with 1 or 2 conservative substitutions. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence of SEQ ID NO: 348 with 1 or 2 conservative substitutions. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence of SEQ ID NO: 4936 with 1 or 2 conservative substitutions. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence of SEQ ID NO: 5206 with 1 or 2 conservative substitutions. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence of SEQ ID NO: 4238 with 1 or 2 conservative substitutions. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence of SEQ ID NO: 5077 with 1 or 2 conservative substitutions. In some embodiments, a 581-589 region of an AAV5 VP capsid polypeptide has a sequence of SEQ ID NO: 5603 with 1 or 2 conservative substitutions. The conservative substitutions may be at any position in the 581-589 region.

NUMBERED EMBODIMENTS

The following embodiments recite non-limiting permutations of combinations of features disclosed herein. Other permutations of combinations of features are also contemplated. In particular, each of these numbered embodiments is contemplated as depending from or relating to every previous or subsequent numbered embodiment, independent of their order as listed. 1. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 a VP1 polypeptide, wherein the 581-589 region comprises at least one amino acid substitution as compared to SEQ ID NO: 9, and wherein a recombinant viral capsid comprising the VP capsid polypeptide is capable of preferentially targeting a cardiac muscle tissue at a DNA enrichment of at least 25-fold greater than a wild type AAV9. 2. The VP capsid polypeptide of embodiment 1, wherein the 581-589 region has a sequence selected from the group consisting of SEQ ID NO: 348, SEQ ID NO: 2536, SEQ ID NO: 1576, SEQ ID NO: 4955, SEQ ID NO: 5017, SEQ ID NO: 4349, SEQ ID NO: 4964, SEQ ID NO: 293, SEQ ID NO: 4314, SEQ ID NO: 4995, SEQ ID NO: 4366, SEQ ID NO: 4961, SEQ ID NO: 4952, SEQ ID NO: 5075, SEQ ID NO: 4354, SEQ ID NO: 5060, SEQ ID NO: 5138, SEQ ID NO: 5206, SEQ ID NO: 4288, SEQ ID NO: 5092, SEQ ID NO: 4059, SEQ ID NO: 4295, SEQ ID NO: 5030, SEQ ID NO: 4938, SEQ ID NO: 2661, SEQ ID NO: 5215, SEQ ID NO: 4960, SEQ ID NO: 4969, SEQ ID NO: 5023, SEQ ID NO: 4934, SEQ ID NO: 4545, SEQ ID NO: 4963, SEQ ID NO: 4363, SEQ ID NO: 5159, and SEQ ID NO: 4973. 3. The VP capsid polypeptide of any one of embodiments 1-2, wherein the recombinant viral capsid is capable of preferentially targeting the cardiac muscle tissue at a DNA enrichment of at least 30-fold greater than the wild type AAV9. 4. The VP capsid polypeptide of embodiment 3, wherein the 581-589 region has a sequence selected from the group consisting of SEQ ID NO: 348, SEQ ID NO: 2536, SEQ ID NO: 1576, SEQ ID NO: 4955, SEQ ID NO: 5017, SEQ ID NO: 4349, SEQ ID NO: 4964, SEQ ID NO: 293, SEQ ID NO: 4314, SEQ ID NO: 4995, and SEQ ID NO: 4366. 5. The VP capsid polypeptide of any one of embodiments 1-4, wherein the recombinant viral capsid is capable of preferentially targeting the cardiac muscle tissue at a DNA enrichment of at least 50-fold greater than the wild type AAV9. 6. The VP capsid polypeptide of embodiment 5, wherein the 581-589 region has a sequence of SEQ ID NO: 348 or SEQ ID NO: 2536. 7. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 a VP1 polypeptide, wherein the 581-589 region comprises at least one amino acid substitution as compared to SEQ ID NO: 9, and wherein a recombinant viral capsid comprising the VP capsid polypeptide is capable of preferentially targeting a cardiac muscle tissue at an RNA enrichment of at least 10-fold greater than a wild type AAV9. 8. The VP capsid polypeptide of embodiment 7, wherein the 581-589 region has a sequence selected from the group consisting of SEQ ID NO: 348, SEQ ID NO: 5052, SEQ ID NO: 5193, SEQ ID NO: 5138, SEQ ID NO: 5163, SEQ ID NO: 4952, SEQ ID NO: 293, SEQ ID NO: 4317, SEQ ID NO: 5075, SEQ ID NO: 386, SEQ ID NO: 4288, SEQ ID NO: 4995, SEQ ID NO: 5030, SEQ ID NO: 4986, SEQ ID NO: 5206, SEQ ID NO: 4338, SEQ ID NO: 5159, SEQ ID NO: 4346, SEQ ID NO: 5060, SEQ ID NO: 4949, SEQ ID NO: 5017, SEQ ID NO: 5215, SEQ ID NO: 4314, SEQ ID NO: 5080, SEQ ID NO: 4964, SEQ ID NO: 4379, SEQ ID NO: 4304, SEQ ID NO: 18, SEQ ID NO: 4363, SEQ ID NO: 5027, SEQ ID NO: 4349, SEQ ID NO: 5208, and SEQ ID NO: 4938. 9. The VP capsid polypeptide of any one of embodiments 7-8, wherein the recombinant viral capsid is capable of preferentially targeting the cardiac muscle tissue at an RNA enrichment of at least 20-fold or greater than the wild type AAV9. 10. The VP capsid polypeptide of embodiment 9, wherein the 581-589 region has a sequence selected from the group consisting of SEQ ID NO: 348, SEQ ID NO: 5052, SEQ ID NO: 5193, SEQ ID NO: 5138, and SEQ ID NO: 5163. 11. The VP capsid polypeptide of any one of embodiments 7-8, wherein the recombinant viral capsid is capable of preferentially targeting the cardiac muscle tissue at an RNA enrichment of at least 40-fold or greater than the wild type AAV9. 12. The VP capsid polypeptide of embodiment 11, wherein the 581-589 region has a sequence of SEQ ID NO: 348. 13. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 a VP1 polypeptide, wherein the 581-589 region comprises at least one amino acid substitution as compared to SEQ ID NO: 9, and wherein a recombinant viral capsid comprising the VP capsid polypeptide is capable of preferentially targeting a cardiac muscle tissue at a DNA enrichment of at least 5-fold greater than a wild type AAV5. 14. The VP capsid polypeptide of embodiment 13, wherein the 581-589 region has a sequence selected from the group consisting of SEQ ID NO: 348, SEQ ID NO: 2536, SEQ ID NO: 1576, SEQ ID NO: 4955, SEQ ID NO: 5017, SEQ ID NO: 4349, SEQ ID NO: 4964, SEQ ID NO: 293, SEQ ID NO: 4314, SEQ ID NO: 4995, SEQ ID NO: 4366, SEQ ID NO: 4961, SEQ ID NO: 4952, SEQ ID NO: 5075, SEQ ID NO: 4354, SEQ ID NO: 5060, SEQ ID NO: 5138, SEQ ID NO: 5206, SEQ ID NO: 4288, SEQ ID NO: 5092, SEQ ID NO: 4059, SEQ ID NO: 4295, SEQ ID NO: 5030, SEQ ID NO: 4938, SEQ ID NO: 2661, SEQ ID NO: 5215, SEQ ID NO: 4960, SEQ ID NO: 4969, SEQ ID NO: 5023, SEQ ID NO: 4934, SEQ ID NO: 4545, SEQ ID NO: 4963, SEQ ID NO: 4363, SEQ ID NO: 5159, and SEQ ID NO: 4973. 15. The VP capsid polypeptide of any one of embodiments 13-14, wherein the recombinant viral capsid is capable of preferentially targeting the cardiac muscle tissue at a DNA enrichment of at least 10-fold greater than the wild type AAV5. 16. The VP capsid polypeptide of embodiment 15, wherein the 581-589 region has a sequence of SEQ ID NO: 348 or SEQ ID NO: 2536. 17. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 a VP1 polypeptide, wherein the 581-589 region comprises at least one amino acid substitution as compared to SEQ ID NO: 9, and wherein a recombinant viral capsid comprising the VP capsid polypeptide is capable of preferentially targeting a cardiac muscle tissue at an RNA enrichment of at least 5-fold greater than a wild type AAV5. 18. The VP capsid polypeptide of embodiment 17, wherein the 581-589 region has a sequence selected from the group consisting of SEQ ID NO: 348, SEQ ID NO: 5052, SEQ ID NO: 5193, SEQ ID NO: 5138, SEQ ID NO: 5163, SEQ ID NO: 4952, SEQ ID NO: 293, and SEQ ID NO: 4317. 19. The VP capsid polypeptide of any one of embodiments 16-17, wherein the recombinant viral capsid is capable of preferentially targeting the cardiac muscle tissue at an RNA enrichment of at least 10-fold greater than the wild type AAV5. 20. The VP capsid polypeptide of embodiment 19, wherein the 581-589 region has a sequence of SEQ ID NO: 348. 21. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence that is at least 85% identical to: (a) SEQ ID NO: 348, (b) SEQ ID NO: 2536, (c) SEQ ID NO: 1576, (d) SEQ ID NO: 4955, (e) SEQ ID NO: 5017, (f) SEQ ID NO: 4349, (g) SEQ ID NO: 4964, (h) SEQ ID NO: 293, (i) SEQ ID NO: 4314, (j) SEQ ID NO: 4995, (k) SEQ ID NO: 4366, (l) SEQ ID NO: 4961, (m) SEQ ID NO: 4952, (n) SEQ ID NO: 5075, (o) SEQ ID NO: 4354, (p) SEQ ID NO: 5060, (q) SEQ ID NO: 5138, (r) SEQ ID NO: 5206, (s) SEQ ID NO: 4288, (t) SEQ ID NO: 5092, (u) SEQ ID NO: 4059, (v) SEQ ID NO: 4295, (w) SEQ ID NO: 5030, (x) SEQ ID NO: 4938, (y) SEQ ID NO: 2661, (z) SEQ ID NO: 5215, (aa) SEQ ID NO: 4960, (bb) SEQ ID NO: 4969, (cc) SEQ ID NO: 5023, or (dd) SEQ ID NO: 4934. 22. The VP capsid polypeptide of embodiment 21, wherein the 581-589 region has a sequence of: (a) SEQ ID NO: 348, (b) SEQ ID NO: 2536, (c) SEQ ID NO: 1576, (d) SEQ ID NO: 4955, (e) SEQ ID NO: 5017, (f) SEQ ID NO: 4349, (g) SEQ ID NO: 4964, (h) SEQ ID NO: 293, (i) SEQ ID NO: 4314, (j) SEQ ID NO: 4995, (k) SEQ ID NO: 4366, (l) SEQ ID NO: 4961, (m) SEQ ID NO: 4952, (n) SEQ ID NO: 5075, (o) SEQ ID NO: 4354, (p) SEQ ID NO: 5060, (q) SEQ ID NO: 5138, (r) SEQ ID NO: 5206, (s) SEQ ID NO: 4288, (t) SEQ ID NO: 5092, (u) SEQ ID NO: 4059, (v) SEQ ID NO: 4295, (w) SEQ ID NO: 5030, (x) SEQ ID NO: 4938, (y) SEQ ID NO: 2661, (z) SEQ ID NO: 5215, (aa) SEQ ID NO: 4960, (bb) SEQ ID NO: 4969, (cc) SEQ ID NO: 5023, or (dd) SEQ ID NO: 4934. 23. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 a VP1 polypeptide, wherein the 581-589 region comprises at least one amino acid substitution as compared to SEQ ID NO: 9, and wherein a recombinant viral capsid comprising the VP capsid polypeptide is capable of preferentially targeting a skeletal muscle tissue at a DNA enrichment of at least 15-fold greater than a wild type AAV9. 24. The VP capsid polypeptide of embodiment 23, wherein the 581-589 region has a sequence selected from the group consisting of SEQ ID NO: 3472, SEQ ID NO: 3297, SEQ ID NO: 2661, SEQ ID NO: 4545, SEQ ID NO: 348, SEQ ID NO: 18, SEQ ID NO: 4349, SEQ ID NO: 293, SEQ ID NO: 5017, SEQ ID NO: 4955, SEQ ID NO: 4366, SEQ ID NO: 5092, SEQ ID NO: 210, SEQ ID NO: 4059, SEQ ID NO: 2536, SEQ ID NO: 5206, and SEQ ID NO: 1971. 25. The VP capsid polypeptide of any one of embodiments 23-24, wherein the recombinant viral capsid is capable of preferentially targeting the skeletal muscle tissue at a DNA enrichment of at least 20-fold greater than the wild type AAV9. 26. The VP capsid polypeptide of embodiment 25, wherein the 581-589 region has a sequence selected from the group consisting of SEQ ID NO: 3472, SEQ ID NO: 3297, SEQ ID NO: 2661, SEQ ID NO: 4545, and SEQ ID NO: 348. 27. The VP capsid polypeptide of any one of embodiments 23-26, wherein the recombinant viral capsid is capable of preferentially targeting the skeletal muscle tissue at a DNA enrichment of at least 30-fold greater than the wild type AAV9. 28. The VP capsid polypeptide of embodiment 27, wherein the 581-589 region has a sequence of SEQ ID NO: 3472 or SEQ ID NO: 3297. 29. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 a VP1 polypeptide, wherein the 581-589 region comprises at least one amino acid substitution as compared to SEQ ID NO: 9, and wherein a recombinant viral capsid comprising the VP capsid polypeptide is capable of preferentially targeting a skeletal muscle tissue at an RNA enrichment of at least 2-fold greater than a wild type AAV9. 30. The VP capsid polypeptide of embodiment 29, wherein the 581-589 region has a sequence selected from the group consisting of SEQ ID NO: 5138, SEQ ID NO: 3472, SEQ ID NO: 1934, SEQ ID NO: 3283, SEQ ID NO: 5817, SEQ ID NO: 278, SEQ ID NO: 1634, SEQ ID NO: 1391, and SEQ ID NO: 1537. 31. The VP capsid polypeptide of any one of embodiments 29-30, wherein the recombinant viral capsid is capable of preferentially targeting the skeletal muscle tissue at an RNA enrichment of at least 10-fold or greater than the wild type AAV9. 32. The VP capsid polypeptide of embodiment 31, wherein the 581-589 region has a sequence selected from the group consisting of SEQ ID NO: 5138, SEQ ID NO: 3472, SEQ ID NO: 1934, SEQ ID NO: 3283, SEQ ID NO: 5817, and SEQ ID NO: 278. 33. The VP capsid polypeptide of any one of embodiments 29-30, wherein the recombinant viral capsid is capable of preferentially targeting the skeletal muscle tissue at an RNA enrichment of at least 25-fold or greater than the wild type AAV9. 34. The VP capsid polypeptide of embodiment 33, wherein the 581-589 region has a sequence selected from the group consisting of SEQ ID NO: 5138, SEQ ID NO: 3472, SEQ ID NO: 1934, or SEQ ID NO: 3283. 35. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 a VP1 polypeptide, wherein the 581-589 region comprises at least one amino acid substitution as compared to SEQ ID NO: 9, and wherein a recombinant viral capsid comprising the VP capsid polypeptide is capable of preferentially targeting a skeletal muscle tissue at a DNA enrichment of at least 4-fold greater than a wild type AAV5. 36. The VP capsid polypeptide of embodiment 35, wherein the 581-589 region has a sequence selected from the group consisting of SEQ ID NO: 3472, SEQ ID NO: 3297, SEQ ID NO: 2661, SEQ ID NO: 4545, SEQ ID NO: 348, SEQ ID NO: 18, SEQ ID NO: 4349, SEQ ID NO: 293, SEQ ID NO: 5017, SEQ ID NO: 4955, and SEQ ID NO: 4366. 37. The VP capsid polypeptide of any one of embodiments 35-36, wherein the recombinant viral capsid is capable of preferentially targeting the skeletal muscle tissue at a DNA enrichment of at least 5-fold greater than the wild type AAV5. 38. The VP capsid polypeptide of embodiment 37, wherein the 581-589 region has a sequence selected from the group consisting of SEQ ID NO: 3472, SEQ ID NO: 3297, SEQ ID NO: 2661, SEQ ID NO: 4545, and SEQ ID NO: 348. 39. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 a VP1 polypeptide, wherein the 581-589 region comprises at least one amino acid substitution as compared to SEQ ID NO: 9, and wherein a recombinant viral capsid comprising the VP capsid polypeptide is capable of preferentially targeting a skeletal muscle tissue at an RNA enrichment of at least that of a wild type AAV5. 40. The VP capsid polypeptide of embodiment 39, wherein the 581-589 region has a sequence selected from the group consisting of SEQ ID NO: 5138, SEQ ID NO: 3472, SEQ ID NO: 1934, SEQ ID NO: 3283, SEQ ID NO: 5817, and SEQ ID NO: 278. 41. The VP capsid polypeptide of any one of embodiments 39-40, wherein the recombinant viral capsid is capable of preferentially targeting the skeletal muscle tissue at an RNA enrichment of at least 3-fold greater than the wild type AAV5. 42. The VP capsid polypeptide of embodiment 41, wherein the 581-589 region has a sequence selected from the group consisting of SEQ ID NO: 5138, SEQ ID NO: 3472, SEQ ID NO: 1934, and SEQ ID NO: 3283. 43. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence that is at least 85% identical to: (a) SEQ ID NO: 3472, (b) SEQ ID NO: 3297, (c) SEQ ID NO: 2661, (d) SEQ ID NO: 4545, (e) SEQ ID NO: 348, (f) SEQ ID NO: 18, (g) SEQ ID NO: 4349, (h) SEQ ID NO: 293, (i) SEQ ID NO: 5017, (j) SEQ ID NO: 4955, (k) SEQ ID NO: 4366, (l) SEQ ID NO: 5092, (m) SEQ ID NO: 210, (n) SEQ ID NO: 4059, (o) SEQ ID NO: 2536, (p) SEQ ID NO: 5206, (q) SEQ ID NO: 1971, (r) SEQ ID NO: 262, (s) SEQ ID NO: 4343, (t) SEQ ID NO: 4995, (u) SEQ ID NO: 4009, (v) SEQ ID NO: 5190, (w) SEQ ID NO: 724, (x) SEQ ID NO: 4288, (y) SEQ ID NO: 4973, (z) SEQ ID NO: 4314, (aa) SEQ ID NO: 1561, (bb) SEQ ID NO: 5147, (cc) SEQ ID NO: 4238, or (dd) SEQ ID NO: 4961. 44. The VP capsid polypeptide of embodiment 43, wherein the 581-589 region has a sequence of: (a) SEQ ID NO: 3472, (b) SEQ ID NO: 3297, (c) SEQ ID NO: 2661, (d) SEQ ID NO: 4545, (e) SEQ ID NO: 348, (f) SEQ ID NO: 18, (g) SEQ ID NO: 4349, (h) SEQ ID NO: 293, (i) SEQ ID NO: 5017, (j) SEQ ID NO: 4955, (k) SEQ ID NO: 4366, (l) SEQ ID NO: 5092, (m) SEQ ID NO: 210, (n) SEQ ID NO: 4059, (o) SEQ ID NO: 2536, (p) SEQ ID NO: 5206, (q) SEQ ID NO: 1971, (r) SEQ ID NO: 262, (s) SEQ ID NO: 4343, (t) SEQ ID NO: 4995, (u) SEQ ID NO: 4009, (v) SEQ ID NO: 5190, (w) SEQ ID NO: 724, (x) SEQ ID NO: 4288, (y) SEQ ID NO: 4973, (z) SEQ ID NO: 4314, (aa) SEQ ID NO: 1561, (bb) SEQ ID NO: 5147, (cc) SEQ ID NO: 4238, or (dd) SEQ ID NO: 4961. 45. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 a VP1 polypeptide, wherein the 581-589 region comprises at least one amino acid substitution as compared to SEQ ID NO: 9, and wherein a recombinant viral capsid comprising the VP capsid polypeptide is capable of preferentially targeting a muscle tissue at a DNA enrichment of at least 20-fold greater than a wild type AAV9. 46. The VP capsid polypeptide of embodiment 45, wherein the 581-589 region has a sequence selected from the group consisting of SEQ ID NO: 348, SEQ ID NO: 2536, SEQ ID NO: 4955, SEQ ID NO: 5017, SEQ ID NO: 4349, SEQ ID NO: 293, SEQ ID NO: 1576, SEQ ID NO: 2661, SEQ ID NO: 4964, SEQ ID NO: 4314, SEQ ID NO: 4995, SEQ ID NO: 4366, SEQ ID NO: 4545, SEQ ID NO: 4961, SEQ ID NO: 5206, SEQ ID NO: 5092, SEQ ID NO: 3472, SEQ ID NO: 5075, SEQ ID NO: 4059, SEQ ID NO: 4354, SEQ ID NO: 5060, SEQ ID NO: 4288, SEQ ID NO: 4952, SEQ ID NO: 5138, SEQ ID NO: 18, SEQ ID NO: 5030, SEQ ID NO: 3297, SEQ ID NO: 4295, SEQ ID NO: 4363, SEQ ID NO: 4963, SEQ ID NO: 4973, SEQ ID NO: 5159, SEQ ID NO: 4960, SEQ ID NO: 4938, SEQ ID NO: 5157, SEQ ID NO: 5023, and SEQ ID NO: 4969. 47. The VP capsid polypeptide of any one of embodiments 45-46, wherein the recombinant viral capsid is capable of preferentially targeting the muscle tissue at a DNA enrichment of at least 25-fold greater than the wild type AAV9. 48. The VP capsid polypeptide of embodiment 47, wherein the 581-589 region has a sequence selected from the group consisting of SEQ ID NO: 348, SEQ ID NO: 2536, SEQ ID NO: 4955, SEQ ID NO: 5017, SEQ ID NO: 4349, SEQ ID NO: 293, SEQ ID NO: 1576, SEQ ID NO: 2661, SEQ ID NO: 4964, SEQ ID NO: 4314, SEQ ID NO: 4995, and SEQ ID NO: 4366. 49. The VP capsid polypeptide of any one of embodiments 46-48, wherein the recombinant viral capsid is capable of preferentially targeting the muscle tissue at a DNA enrichment of at least 30-fold greater than the wild type AAV9. 50. The VP capsid polypeptide of embodiment 49, wherein the 581-589 region has a sequence of SEQ ID NO: 348 or SEQ ID NO: 2536. 51. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 a VP1 polypeptide, wherein the 581-589 region comprises at least one amino acid substitution as compared to SEQ ID NO: 9, and wherein a recombinant viral capsid comprising the VP capsid polypeptide is capable of preferentially targeting a muscle tissue at an RNA enrichment of at least 5-fold greater than a wild type AAV9. 52. The VP capsid polypeptide of embodiment 51, wherein the 581-589 region has a sequence selected from the group consisting of SEQ ID NO: 5138, SEQ ID NO: 3472, SEQ ID NO: 348, SEQ ID NO: 5052, SEQ ID NO: 5193, SEQ ID NO: 5026, SEQ ID NO: 278, SEQ ID NO: 5163, SEQ ID NO: 293, SEQ ID NO: 4943, SEQ ID NO: 4952, SEQ ID NO: 4317, SEQ ID NO: 5075, SEQ ID NO: 386, SEQ ID NO: 4986, SEQ ID NO: 4995, SEQ ID NO: 4288, SEQ ID NO: 5030, SEQ ID NO: 5206, SEQ ID NO: 5159, SEQ ID NO: 4949, SEQ ID NO: 4338, SEQ ID NO: 5060, SEQ ID NO: 4346, SEQ ID NO: 5215, SEQ ID NO: 5017, SEQ ID NO: 4314, SEQ ID NO: 5080, SEQ ID NO: 4964, SEQ ID NO: 4363, SEQ ID NO: 4379, SEQ ID NO: 5027, SEQ ID NO: 4304, SEQ ID NO: 18, SEQ ID NO: 4349, SEQ ID NO: 4938, and SEQ ID NO: 2536. 53. The VP capsid polypeptide of any one of embodiments 51-52, wherein the recombinant viral capsid is capable of preferentially targeting the muscle tissue at an RNA enrichment of at least 10-fold or greater than the wild type AAV9. 54. The VP capsid polypeptide of embodiment 53, wherein the 581-589 region has a sequence selected from the group consisting of SEQ ID NO: 5138, SEQ ID NO: 3472, SEQ ID NO: 348, SEQ ID NO: 5052, SEQ ID NO: 5193, SEQ ID NO: 5026, SEQ ID NO: 278, and SEQ ID NO: 5163. 55. The VP capsid polypeptide of any one of embodiments 51-52, wherein the recombinant viral capsid is capable of preferentially targeting the muscle tissue at an RNA enrichment of at least 100-fold or greater than the wild type AAV9. 56. The VP capsid polypeptide of embodiment 55, wherein the 581-589 region has a sequence of SEQ ID NO: 5138. 57. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 a VP1 polypeptide, wherein the 581-589 region comprises at least one amino acid substitution as compared to SEQ ID NO: 9, and wherein a recombinant viral capsid comprising the VP capsid polypeptide is capable of preferentially targeting a muscle tissue at a DNA enrichment of at least 5-fold greater than a wild type AAV5. 58. The VP capsid polypeptide of embodiment 57, wherein the 581-589 region has a sequence selected from the group consisting of SEQ ID NO: 348, SEQ ID NO: 2536, SEQ ID NO: 4955, SEQ ID NO: 5017, SEQ ID NO: 4349, SEQ ID NO: 293, SEQ ID NO: 1576, SEQ ID NO: 2661, SEQ ID NO: 4964, SEQ ID NO: 4314, SEQ ID NO: 4995, SEQ ID NO: 4366, SEQ ID NO: 4545, SEQ ID NO: 4961, SEQ ID NO: 5206, SEQ ID NO: 5092, SEQ ID NO: 3472, SEQ ID NO: 5075, SEQ ID NO: 4059, SEQ ID NO: 4354, and SEQ ID NO: 5060. 59. The VP capsid polypeptide of any one of embodiments 57-58, wherein the recombinant viral capsid is capable of preferentially targeting the muscle tissue at a DNA enrichment of at least 10-fold greater than the wild type AAV5. 60. The VP capsid polypeptide of embodiment 59, wherein the 581-589 region has a sequence of SEQ ID NO: 348. 61. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 a VP1 polypeptide, wherein the 581-589 region comprises at least one amino acid substitution as compared to SEQ ID NO: 9, and wherein a recombinant viral capsid comprising the VP capsid polypeptide is capable of preferentially targeting a muscle tissue at an RNA enrichment of at least that of a wild type AAV5. 62. The VP capsid polypeptide of embodiment 61, wherein the 581-589 region has a sequence selected from the group consisting of SEQ ID NO: 5138, SEQ ID NO: 3472, SEQ ID NO: 348, SEQ ID NO: 5052, SEQ ID NO: 5193, SEQ ID NO: 5026, SEQ ID NO: 278, SEQ ID NO: 5163, SEQ ID NO: 293, SEQ ID NO: 4943, SEQ ID NO: 4952, SEQ ID NO: 4317, SEQ ID NO: 5075, SEQ ID NO: 386, SEQ ID NO: 4986, and SEQ ID NO: 4995. 63. The VP capsid polypeptide of any one of embodiments 61-62, wherein the recombinant viral capsid is capable of preferentially targeting the muscle tissue at an RNA enrichment of at least 2-fold greater than the wild type AAV5. 64. The VP capsid polypeptide of embodiment 63, wherein the 581-589 region has a sequence selected from the group consisting of SEQ ID NO: 5138, SEQ ID NO: 3472, SEQ ID NO: 348, and SEQ ID NO: 5052. 65. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence that is at least 85% identical to: (a) SEQ ID NO: 348, (b) SEQ ID NO: 2536, (c) SEQ ID NO: 4955, (d) SEQ ID NO: 5017, (e) SEQ ID NO: 4349, (f) SEQ ID NO: 293, (g) SEQ ID NO: 1576, (h) SEQ ID NO: 2661, (i) SEQ ID NO: 4964, (j) SEQ ID NO: 4314, (k) SEQ ID NO: 4995, (l) SEQ ID NO: 4366, (m) SEQ ID NO: 4545, (n) SEQ ID NO: 4961, (o) SEQ ID NO: 5206, (p) SEQ ID NO: 5092, (q) SEQ ID NO: 3472, (r) SEQ ID NO: 5075, (s) SEQ ID NO: 4059, (t) SEQ ID NO: 4354, (u) SEQ ID NO: 5060, (v) SEQ ID NO: 4288, (w) SEQ ID NO: 4952, (x) SEQ ID NO: 5138, (y) SEQ ID NO: 18, (z) SEQ ID NO: 5030, (aa) SEQ ID NO: 3297, (bb) SEQ ID NO: 4295, (cc) SEQ ID NO: 4363, or (dd) SEQ ID NO: 4963. 66. The VP capsid polypeptide of embodiment 65, wherein the 581-589 region has a sequence of: (a) SEQ ID NO: 348, (b) SEQ ID NO: 2536, (c) SEQ ID NO: 4955, (d) SEQ ID NO: 5017, (e) SEQ ID NO: 4349, (f) SEQ ID NO: 293, (g) SEQ ID NO: 1576, (h) SEQ ID NO: 2661, (i) SEQ ID NO: 4964, (j) SEQ ID NO: 4314, (k) SEQ ID NO: 4995, (l) SEQ ID NO: 4366, (m) SEQ ID NO: 4545, (n) SEQ ID NO: 4961, (o) SEQ ID NO: 5206, (p) SEQ ID NO: 5092, (q) SEQ ID NO: 3472, (r) SEQ ID NO: 5075, (s) SEQ ID NO: 4059, (t) SEQ ID NO: 4354, (u) SEQ ID NO: 5060, (v) SEQ ID NO: 4288, (w) SEQ ID NO: 4952, (x) SEQ ID NO: 5138, (y) SEQ ID NO: 18, (z) SEQ ID NO: 5030, (aa) SEQ ID NO: 3297, (bb) SEQ ID NO: 4295, (cc) SEQ ID NO: 4363, or (dd) SEQ ID NO: 4963. 67. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide, wherein the 581-589 region comprises a sequence having at least 85% identity to any one of any one of SEQ ID NO: 4238-SEQ ID NO: 4933 or SEQ ID NO: 4934-SEQ ID NO: 5233. 68. The VP capsid polypeptide of embodiment 67, wherein the 581-589 region has a sequence of any one of any one of SEQ ID NO: 4238-SEQ ID NO: 4933 or SEQ ID NO: 4934-SEQ ID NO: 5233. 69. The VP capsid polypeptide of any one of embodiments 1-68, wherein the VP capsid polypeptide is capable of assembling into a recombinant viral capsid. 70. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide is capable of assembling into a recombinant viral capsid, and wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region: comprises at least one amino acid substitution as compared to residues 581 to 589 of SEQ ID NO: 1, and confers on the recombinant viral capsid an infection rate for muscle tissue with at least 3-fold higher muscle tissue tropism than a wild type VP capsid polypeptide of SEQ ID NO: 1; wherein the VP capsid polypeptide comprises at least one amino acid substitution as compared to each of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 8. 71. The VP capsid polypeptide of embodiment 70, wherein the 581-589 region comprises a sequence of X¹X²X³X⁴X⁵X⁶X⁷X⁸X⁹, wherein X¹, X², X³, X⁴, X⁵, X⁶, X⁷, X⁸, and X⁹are each individually selected from A, R, N, D, C, E, Q, G, H, I, L, K, M, F, P, S, T, W, Y, and V. 72. The VP capsid polypeptide of embodiment 70 or embodiment 83, wherein the VP capsid polypeptide has a sequence of SEQ ID NO: 2, wherein residues 581 to 589 of SEQ ID NO: 2 (X¹X²X³X⁴X⁵X⁶X⁷X⁸X⁹) correspond to the 581-589 region. 73. The VP capsid polypeptide of any one of embodiments 70-72, wherein the 581-589 region has a sequence that is at least 70%, at least 77%, at least 80%, at least 88%, at least 90%, or at least 95% identical to any one of SEQ ID NO: 4238-SEQ ID NO: 4933. 74. The VP capsid polypeptide of any one of embodiments 70-72, wherein the 581-589 region has a sequence of any one of SEQ ID NO: 4238-SEQ ID NO: 4933. 75. The VP capsid polypeptide of any one of embodiments 82-84, wherein the 581-589 region has a sequence that is at least 70%, at least 77%, at least 80%, at least 88%, at least 90%, or at least 95% identical to any one of SEQ ID NO: 4934-SEQ ID NO: 5233. 76. The VP capsid polypeptide of any one of embodiments 70-72, wherein the 581-589 region has a sequence of any one of SEQ ID NO: 4934-SEQ ID NO: 5233. 77. The VP capsid polypeptide of any one of embodiments 1-76, wherein the muscle tissue comprises cardiac muscle tissue. 78. The VP capsid polypeptide of any one of embodiments 1-77, wherein the muscle tissue comprises skeletal muscle tissue. 79. The VP capsid polypeptide of any one of embodiments 1-78, wherein the muscle tissue comprises skeletal muscle tissue and cardiac muscle tissue. 80. The VP capsid polypeptide of any one of embodiments 1-79, wherein the VP capsid polypeptide comprises an amino acid sequence at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 98.5%, at least 99%, or at least 99.5% identical to SEQ ID NO: 1. 81. The VP capsid polypeptide of any one of embodiments 1-80, wherein the infection rate for muscle tissue is at least 2.5-fold, at least 3-fold, at least 3.5-fold, at least 4-fold, at least 5-fold, at least 10-fold, at least 20-fold, at least 50-fold, at least 75-fold, at least 100-fold, at least 200-fold, at least 500-fold, or at least 1000-fold higher tissue tropism than an infection rate of the wild type VP capsid polypeptide for muscle tissue. 82. The VP capsid polypeptide of any one of embodiments 1-81, wherein the 581-589 region confers on a recombinant viral capsid assembled from the VP capsid polypeptide improved stability compared to a wild type AAV5 capsid comprising a peptide of SEQ ID NO: 1. 83. The VP capsid polypeptide of any one of embodiments 1-82, wherein the 581-589 region confers lower toxicity upon administration to a subject compared to a wild type VP capsid polypeptide of SEQ ID NO: 1. 84. The VP capsid polypeptide of any one of embodiments 1-83, wherein the muscle tissue is selected from aorta, esophagus, heart, skeletal muscle, and combinations thereof. 85. A pharmaceutical composition comprising the VP capsid polypeptide of any one of embodiments 1-84. 86. The pharmaceutical composition of embodiment 85, wherein the VP capsid polypeptide is assembled into a recombinant viral capsid. 87. The pharmaceutical composition of embodiment 85 or embodiment 86, further comprising a payload encapsidated by the recombinant viral capsid. 88. The pharmaceutical composition of embodiment 87, wherein the payload encodes a therapeutic polynucleotide or a therapeutic peptide. 89. The pharmaceutical composition of embodiment 87, wherein the payload encodes a guide RNA, a tRNA, a suppressor tRNA, a siRNA, a miRNA, an mRNA, a shRNA, a circular RNA, or an antisense oligonucleotide (ASO), a ribozyme, a DNAzyme, an aptamer, or any combination thereof 90. The pharmaceutical composition of embodiment 87, wherein the payload encodes a component of a CRISPR/Cas system, an adenosine deaminase acting on RNA (ADAR) enzyme, a transcriptional activator, or a transcriptional repressor. 91. A recombinant adeno-associated virus (rAAV), comprising the VP capsid polypeptide of any one of embodiments 1-84 assembled into a recombinant viral capsid and a payload encapsidated by the recombinant viral capsid. 92. The rAAV of embodiment 91, further comprising a VP2 polypeptide comprising the 581-589 region and a VP3 polypeptide comprising the 581-589 region. 93. The rAAV of embodiment 91 or embodiment 92, wherein the payload encodes a therapeutic polynucleotide or a therapeutic peptide. 94. The rAAV of embodiment 93, wherein the payload encodes a guide RNA, a tRNA, a suppressor tRNA, a siRNA, a miRNA, an mRNA, a shRNA, a circular RNA, or an antisense oligonucleotide (ASO), a ribozyme, a DNAzyme, an aptamer, or any combination thereof 95. The rAAV of embodiment 93, wherein the payload encodes a component of a CRISPR/Cas system, an adenosine deaminase acting on RNA (ADAR) enzyme, a transcriptional activator, or a transcriptional repressor. 96. A method of delivering a payload to a muscle tissue of a subject, the method comprising: administering a recombinant adeno-associated virus (rAAV) to the subject, wherein the rAAV encapsidates the payload, and wherein the rAAV comprises a VP capsid polypeptide comprising a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide, wherein the 581-589 region comprises at least one amino acid substitution as compared to SEQ ID NO: 9; infecting the muscle tissue with the rAAV, wherein the muscle tissue comprises a cardiac muscle tissue; and preferentially delivering the payload to the muscle tissue infected by the rAAV at a DNA enrichment of at least 25-fold greater than a wild type AAV9. 97. The method of embodiment 96, wherein the DNA enrichment is at least 30-fold greater than the wild type AAV9. 98. The method of embodiment 96 or embodiment 97, wherein the DNA enrichment is at least 50-fold greater than the wild type AAV9. 99. A method of transcribing a payload in a muscle tissue of a subject, the method comprising: administering a recombinant adeno-associated virus (rAAV) to the subject, wherein the rAAV encapsidates the payload, and wherein the rAAV comprises a VP capsid polypeptide comprising a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide, wherein the 581-589 region comprises at least one amino acid substitution as compared to SEQ ID NO: 9; infecting the muscle tissue with the rAAV, wherein the muscle tissue comprises a cardiac muscle tissue; and preferentially transcribing the payload to the muscle tissue infected by the rAAV at an RNA enrichment of at least 10-fold greater than a wild type AAV9. 100. The method of embodiment 99, wherein the RNA enrichment is at least 20-fold greater than the wild type AAV9. 101. The method of embodiment 99 or embodiment 100, wherein the RNA enrichment is at least 40-fold greater than the wild type AAV9. 102. A method of delivering a payload to a muscle tissue of a subject, the method comprising: administering a recombinant adeno-associated virus (rAAV) to the subject, wherein the rAAV encapsidates the payload, and wherein the rAAV comprises a VP capsid polypeptide comprising a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide, wherein the 581-589 region comprises at least one amino acid substitution as compared to SEQ ID NO: 9; infecting the muscle tissue with the rAAV, wherein the muscle tissue comprises a cardiac muscle tissue; and preferentially delivering the payload to the muscle tissue infected by the rAAV at a DNA enrichment of at least 5-fold greater than a wild type AAV5. 103. The method of embodiment 102, wherein the DNA enrichment is at least 10-fold greater than the wild type AAV5. 104. A method of transcribing a payload in a muscle tissue of a subject, the method comprising: administering a recombinant adeno-associated virus (rAAV) to the subject, wherein the rAAV encapsidates the payload, and wherein the rAAV comprises a VP capsid polypeptide comprising a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide, wherein the 581-589 region comprises at least one amino acid substitution as compared to SEQ ID NO: 9; infecting the muscle tissue with the rAAV, wherein the muscle tissue comprises a cardiac muscle tissue; and preferentially transcribing the payload to the muscle tissue infected by the rAAV at an RNA enrichment of at least 5-fold greater than a wild type AAV5. 105. The method of embodiment 104, wherein the RNA enrichment is at least 10-fold greater than the wild type AAV5. 106. A method of delivering a payload to a muscle tissue of a subject, the method comprising: administering a recombinant adeno-associated virus (rAAV) to the subject, wherein the rAAV encapsidates the payload, and wherein the rAAV comprises a VP capsid polypeptide comprising a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide, wherein the 581-589 region has a sequence that is at least 85% identical to: (a) SEQ ID NO: 348, (b) SEQ ID NO: 2536, (c) SEQ ID NO: 1576, (d) SEQ ID NO: 4955, (e) SEQ ID NO: 5017, (f) SEQ ID NO: 4349, (g) SEQ ID NO: 4964, (h) SEQ ID NO: 293, (i) SEQ ID NO: 4314, (j) SEQ ID NO: 4995, (k) SEQ ID NO: 4366, (l) SEQ ID NO: 4961, (m) SEQ ID NO: 4952, (n) SEQ ID NO: 5075, (o) SEQ ID NO: 4354, (p) SEQ ID NO: 5060, (q) SEQ ID NO: 5138, (r) SEQ ID NO: 5206, (s) SEQ ID NO: 4288, (t) SEQ ID NO: 5092, (u) SEQ ID NO: 4059, (v) SEQ ID NO: 4295, (w) SEQ ID NO: 5030, (x) SEQ ID NO: 4938, (y) SEQ ID NO: 2661, (z) SEQ ID NO: 5215, (aa) SEQ ID NO: 4960, (bb) SEQ ID NO: 4969, (cc) SEQ ID NO: 5023, or (dd) SEQ ID NO: 4934; infecting the muscle tissue with the rAAV, wherein the muscle tissue comprises cardiac muscle tissue; and delivering the payload to the muscle tissue infected by the rAAV. 107. A method of delivering a payload to a muscle tissue of a subject, the method comprising: administering a recombinant adeno-associated virus (rAAV) to the subject, wherein the rAAV encapsidates the payload, and wherein the rAAV comprises a VP capsid polypeptide comprising a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide, wherein the 581-589 region comprises at least one amino acid substitution as compared to SEQ ID NO: 9; and infecting the muscle tissue with the rAAV, wherein the muscle tissue comprises a skeletal muscle tissue; preferentially delivering the payload to the muscle tissue infected by the rAAV at a DNA enrichment of at least 15-fold greater than a wild type AAV9. 108. The method of embodiment 107, wherein the DNA enrichment is at least 20-fold greater than the wild type AAV9. 109. The method of embodiment 107 or embodiment 108, wherein the DNA enrichment is at least 30-fold greater than the wild type AAV9. 110. A method of transcribing a payload in a muscle tissue of a subject, the method comprising: administering a recombinant adeno-associated virus (rAAV) to the subject, wherein the rAAV encapsidates the payload, and wherein the rAAV comprises a VP capsid polypeptide comprising a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide, wherein the 581-589 region comprises at least one amino acid substitution as compared to SEQ ID NO: 9; infecting the muscle tissue with the rAAV, wherein the muscle tissue comprises a skeletal muscle tissue; and preferentially transcribing the payload to the muscle tissue infected by the rAAV at an RNA enrichment of at least 2-fold greater than a wild type AAV9. 111. The method of embodiment 110, wherein the RNA enrichment is at least 10-fold greater than the wild type AAV9. 112. The method of embodiment 110 or embodiment 111, wherein the RNA enrichment is at least 25-fold greater than the wild type AAV9. 113. A method of delivering a payload to a muscle tissue of a subject, the method comprising: administering a recombinant adeno-associated virus (rAAV) to the subject, wherein the rAAV encapsidates the payload, and wherein the rAAV comprises a VP capsid polypeptide comprising a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide, wherein the 581-589 region comprises at least one amino acid substitution as compared to SEQ ID NO: 9; infecting the muscle tissue with the rAAV, wherein the muscle tissue comprises a skeletal muscle tissue; and preferentially delivering the payload to the muscle tissue infected by the rAAV at a DNA enrichment of at least 4-fold greater than a wild type AAV5. 114. The method of embodiment 113, wherein the DNA enrichment is at least 5-fold greater than the wild type AAV5. 115. A method of transcribing a payload in a muscle tissue of a subject, the method comprising: administering a recombinant adeno-associated virus (rAAV) to the subject, wherein the rAAV encapsidates the payload, and wherein the rAAV comprises a VP capsid polypeptide comprising a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide, wherein the 581-589 region comprises at least one amino acid substitution as compared to SEQ ID NO: 9; infecting the muscle tissue with the rAAV, wherein the muscle tissue comprises a skeletal muscle tissue; and preferentially transcribing the payload to the muscle tissue infected by the rAAV at an RNA enrichment of at that of a wild type AAV5. 116. The method of embodiment 115, wherein the RNA enrichment is at least 3-fold greater than the wild type AAV5. 117. A method of delivering a payload to a muscle tissue of a subject, the method comprising: administering a recombinant adeno-associated virus (rAAV) to the subject, wherein the rAAV encapsidates the payload, and wherein the rAAV comprises a VP capsid polypeptide comprising a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide, wherein the 581-589 region has a sequence that is at least 85% identical to: (a) SEQ ID NO: 3472, (b) SEQ ID NO: 3297, (c) SEQ ID NO: 2661, (d) SEQ ID NO: 4545, (e) SEQ ID NO: 348, (f) SEQ ID NO: 18, (g) SEQ ID NO: 4349, (h) SEQ ID NO: 293, (i) SEQ ID NO: 5017, (j) SEQ ID NO: 4955, (k) SEQ ID NO: 4366, (l) SEQ ID NO: 5092, (m) SEQ ID NO: 210, (n) SEQ ID NO: 4059, (o) SEQ ID NO: 2536, (p) SEQ ID NO: 5206, (q) SEQ ID NO: 1971, (r) SEQ ID NO: 262, (s) SEQ ID NO: 4343, or (t) SEQ ID NO: 4995, (u) SEQ ID NO: 4009, (v) SEQ ID NO: 5190, (w) SEQ ID NO: 724, (x) SEQ ID NO: 4288, (y) SEQ ID NO: 4973, (z) SEQ ID NO: 4314, (aa) SEQ ID NO: 1561, (bb) SEQ ID NO: 5147, (cc) SEQ ID NO: 4238, or (dd) SEQ ID NO: 4961; infecting the muscle tissue with the rAAV, wherein the muscle tissue comprises skeletal muscle tissue; and delivering the payload to the muscle tissue infected by the rAAV. 118. A method of delivering a payload to a muscle tissue of a subject, the method comprising: administering a recombinant adeno-associated virus (rAAV) to the subject, wherein the rAAV encapsidates the payload, and wherein the rAAV comprises a VP capsid polypeptide comprising a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide, wherein the 581-589 region comprises at least one amino acid substitution as compared to SEQ ID NO: 9; infecting the muscle tissue with the rAAV; and preferentially delivering the payload to the muscle tissue infected by the rAAV at a DNA enrichment of at least 20-fold greater than a wild type AAV9. 119. The method of embodiment 118, wherein the DNA enrichment is at least 25-fold greater than the wild type AAV9. 120. The method of embodiment 118 or embodiment 119, wherein the DNA enrichment is at least 30-fold greater than the wild type AAV9. 121. A method of transcribing a payload in a muscle tissue of a subject, the method comprising: administering a recombinant adeno-associated virus (rAAV) to the subject, wherein the rAAV encapsidates the payload, and wherein the rAAV comprises a VP capsid polypeptide comprising a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide, wherein the 581-589 region comprises at least one amino acid substitution as compared to SEQ ID NO: 9; infecting the muscle tissue with the rAAV; and preferentially transcribing the payload to the muscle tissue infected by the rAAV at an RNA enrichment of at least 5-fold greater than a wild type AAV9. 122. The method of embodiment 121, wherein the RNA enrichment is at least 10-fold greater than the wild type AAV9. 123. The method of embodiment 121 or embodiment 122, wherein the RNA enrichment is at least 100-fold greater than the wild type AAV9. 124. A method of delivering a payload to a muscle tissue of a subject, the method comprising: administering a recombinant adeno-associated virus (rAAV) to the subject, wherein the rAAV encapsidates the payload, and wherein the rAAV comprises a VP capsid polypeptide comprising a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide, wherein the 581-589 region comprises at least one amino acid substitution as compared to SEQ ID NO: 9; infecting the muscle tissue with the rAAV; and preferentially delivering the payload to the muscle tissue infected by the rAAV at a DNA enrichment of at least 5-fold greater than a wild type AAV5. 125. The method of embodiment 124, wherein the DNA enrichment is at least 10-fold greater than the wild type AAV5. 126. A method of transcribing a payload in a muscle tissue of a subject, the method comprising: administering a recombinant adeno-associated virus (rAAV) to the subject, wherein the rAAV encapsidates the payload, and wherein the rAAV comprises a VP capsid polypeptide comprising a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide, wherein the 581-589 region comprises at least one amino acid substitution as compared to SEQ ID NO: 9; infecting the muscle tissue with the rAAV; and preferentially transcribing the payload to the muscle tissue infected by the rAAV at an RNA enrichment of at least that of a wild type AAV5. 127. The method of embodiment 126, wherein the RNA enrichment is at least 2-fold greater than the wild type AAV5. 128. A method of delivering a payload to a muscle tissue of a subject, the method comprising: administering a recombinant adeno-associated virus (rAAV) to the subject, wherein the rAAV encapsidates the payload, and wherein the rAAV comprises a VP capsid polypeptide comprising a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide, wherein the 581-589 region has a sequence that is at least 85% identical to: (a) SEQ ID NO: 348, (b) SEQ ID NO: 2536, (c) SEQ ID NO: 4955, (d) SEQ ID NO: 5017, (e) SEQ ID NO: 4349, (f) SEQ ID NO: 293, (g) SEQ ID NO: 1576, (h) SEQ ID NO: 2661, (i) SEQ ID NO: 4964, (j) SEQ ID NO: 4314, (k) SEQ ID NO: 4995, (l) SEQ ID NO: 4366, (m) SEQ ID NO: 4545, (n) SEQ ID NO: 4961, (o) SEQ ID NO: 5206, (p) SEQ ID NO: 5092, (q) SEQ ID NO: 3472, (r) SEQ ID NO: 5075, (s) SEQ ID NO: 4059, (t) SEQ ID NO: 4354, (u) SEQ ID NO: 5060, (v) SEQ ID NO: 4288, (w) SEQ ID NO: 4952, (x) SEQ ID NO: 5138, (y) SEQ ID NO: 18, (z) SEQ ID NO: 5030, (aa) SEQ ID NO: 3297, (bb) SEQ ID NO: 4295, (cc) SEQ ID NO: 4363, or (dd) SEQ ID NO: 4963; infecting the muscle tissue with the rAAV; and delivering the payload to the muscle tissue infected by the rAAV. 129. A method of delivering a payload to a muscle tissue of a subject, the method comprising: administering a recombinant adeno-associated virus (rAAV) to the subject, wherein the rAAV encapsidates the payload, and wherein the rAAV comprises a VP capsid polypeptide comprising a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide, wherein the 581-589 region comprises at least one amino acid substitution as compared to residues 581 to 589 of SEQ ID NO: 1, and wherein the VP capsid polypeptide comprises at least one amino acid substitution as compared to each of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 8; infecting the muscle tissue with the rAAV with at least 3-fold higher muscle tissue tropism as compared to a wild type AAV5 capsid comprising a peptide of SEQ ID NO: 1; and delivering the payload to the muscle tissue infected by the rAAV. 130. A method of delivering a payload to a muscle tissue of a subject, the method comprising: administering a recombinant adeno-associated virus (rAAV) to the subject, wherein the rAAV encapsidates the payload, and wherein the rAAV comprises a VP capsid polypeptide of any one of embodiments 1-84; infecting the muscle tissue with the rAAV; and delivering the payload to the muscle tissue infected by the rAAV. 131. The method of any one of embodiments 128-130, wherein the muscle tissue comprises aorta, esophagus, heart, skeletal muscle, or a combination thereof 132. The method of any one of embodiments 96-131, further comprising expressing a therapeutic polypeptide or a therapeutic polynucleotide encoded by the payload in the muscle tissue infected by the rAAV. 133. The method of any one of embodiments 96-132, further comprising producing a therapeutic effect in the muscle tissue of the subject. 134. The method of embodiment 133, wherein the therapeutic effect is produced upon administration of from 1×10⁵to 5×10¹⁴rAAVs per kg subject weight. 135. The method of embodiment 133 or embodiment 134, comprising administering an amount of the rAAV sufficient to produce the therapeutic effect without producing a toxicity in the subject. 136. A method of treating a condition in a subject, the method comprising: administering a recombinant adeno-associated virus (rAAV) to the subject, wherein the rAAV encapsidates the payload, and wherein the rAAV comprises a VP capsid polypeptide comprising a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide, wherein the 581-589 region comprises at least one amino acid substitution as compared to SEQ ID NO: 9; infecting a muscle tissue of the subject with the rAAV, wherein the muscle tissue comprises a cardiac muscle tissue; preferentially delivering the payload to the muscle tissue infected by the rAAV at a DNA enrichment of at least 25-fold greater than a wild type AAV9; and producing a therapeutic effect in the muscle tissue, thereby treating the condition. 137. The method of embodiment 136, wherein the DNA enrichment is at least 30-fold greater than the wild type AAV9. 138. The method of embodiment 136 or embodiment 137, wherein the DNA enrichment is at least 50-fold greater than the wild type AAV9. 139. A method of treating a condition in a subject, the method comprising: administering a recombinant adeno-associated virus (rAAV) to the subject, wherein the rAAV encapsidates the payload, and wherein the rAAV comprises a VP capsid polypeptide comprising a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide, wherein the 581-589 region comprises at least one amino acid substitution as compared to SEQ ID NO: 9; infecting a muscle tissue of the subject with the rAAV, wherein the muscle tissue comprises a cardiac muscle tissue; preferentially transcribing the payload to the muscle tissue infected by the rAAV at an RNA enrichment of at least 10-fold greater than a wild type AAV9; and producing a therapeutic effect in the muscle tissue, thereby treating the condition. 140. The method of embodiment 139, wherein the RNA enrichment is at least 20-fold greater than the wild type AAV9. 141. The method of embodiment 139 or embodiment 140, wherein the RNA enrichment is at least 40-fold greater than the wild type AAV9. 142. A method of treating a condition in a subject, the method comprising: administering a recombinant adeno-associated virus (rAAV) to the subject, wherein the rAAV encapsidates the payload, and wherein the rAAV comprises a VP capsid polypeptide comprising a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide, wherein the 581-589 region comprises at least one amino acid substitution as compared to SEQ ID NO: 9; infecting a muscle tissue of the subject with the rAAV, wherein the muscle tissue comprises a cardiac muscle tissue; preferentially delivering the payload to the muscle tissue infected by the rAAV at a DNA enrichment of at least 5-fold greater than a wild type AAV5; and producing a therapeutic effect in the muscle tissue, thereby treating the condition. 143. The method of embodiment 142, wherein the DNA enrichment is at least 10-fold greater than the wild type AAV5. 144. A method of treating a condition in a subject, the method comprising: administering a recombinant adeno-associated virus (rAAV) to the subject, wherein the rAAV encapsidates the payload, and wherein the rAAV comprises a VP capsid polypeptide comprising a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide, wherein the 581-589 region comprises at least one amino acid substitution as compared to SEQ ID NO: 9; infecting a muscle tissue of the subject with the rAAV, wherein the muscle tissue comprises a cardiac muscle tissue; preferentially transcribing the payload to the muscle tissue infected by the rAAV at an RNA enrichment of at least 5-fold greater than a wild type AAV5; and producing a therapeutic effect in the muscle tissue, thereby treating the condition. 145. The method of embodiment 144, wherein the RNA enrichment is at least 10-fold greater than the wild type AAV5. 146. A method of treating a condition in a subject, the method comprising: administering a recombinant adeno-associated virus (rAAV) to the subject, wherein the rAAV encapsidates a payload, and wherein the rAAV comprises a VP capsid polypeptide comprising a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide, wherein the 581-589 region has a sequence that is at least 85% identical to: (a) SEQ ID NO: 348, (b) SEQ ID NO: 2536, (c) SEQ ID NO: 1576, (d) SEQ ID NO: 4955, (e) SEQ ID NO: 5017, (f) SEQ ID NO: 4349, (g) SEQ ID NO: 4964, (h) SEQ ID NO: 293, (i) SEQ ID NO: 4314, (j) SEQ ID NO: 4995, (k) SEQ ID NO: 4366, (l) SEQ ID NO: 4961, (m) SEQ ID NO: 4952, (n) SEQ ID NO: 5075, (o) SEQ ID NO: 4354, (p) SEQ ID NO: 5060, (q) SEQ ID NO: 5138, (r) SEQ ID NO: 5206, (s) SEQ ID NO: 4288, (t) SEQ ID NO: 5092, (u) SEQ ID NO: 4059, (v) SEQ ID NO: 4295, (w) SEQ ID NO: 5030, (x) SEQ ID NO: 4938, (y) SEQ ID NO: 2661, (z) SEQ ID NO: 5215, (aa) SEQ ID NO: 4960, (bb) SEQ ID NO: 4969, (cc) SEQ ID NO: 5023, or (dd) SEQ ID NO: 4934; infecting a muscle tissue of the subject with the rAAV, wherein the muscle tissue comprises cardiac muscle tissue; delivering the payload to the muscle tissue infected by the rAAV; and producing a therapeutic effect in the muscle tissue, thereby treating the condition. 147. A method of treating a condition in a subject, the method comprising: administering a recombinant adeno-associated virus (rAAV) to the subject, wherein the rAAV encapsidates the payload, and wherein the rAAV comprises a VP capsid polypeptide comprising a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide, wherein the 581-589 region comprises at least one amino acid substitution as compared to SEQ ID NO: 9; infecting a muscle tissue of the subject with the rAAV, wherein the muscle tissue comprises a skeletal muscle tissue; preferentially delivering the payload to the muscle tissue infected by the rAAV at a DNA enrichment of at least 15-fold greater than a wild type AAV9; and producing a therapeutic effect in the muscle tissue, thereby treating the condition. 148. The method of embodiment 147, wherein the DNA enrichment is at least 20-fold greater than the wild type AAV9. 149. The method of embodiment 147 or embodiment 148, wherein the DNA enrichment is at least 30-fold greater than the wild type AAV9. 150. A method of treating a condition in a subject, the method comprising: administering a recombinant adeno-associated virus (rAAV) to the subject, wherein the rAAV encapsidates the payload, and wherein the rAAV comprises a VP capsid polypeptide comprising a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide, wherein the 581-589 region comprises at least one amino acid substitution as compared to SEQ ID NO: 9; infecting a muscle tissue of the subject with the rAAV, wherein the muscle tissue comprises a skeletal muscle tissue; preferentially transcribing the payload to the muscle tissue infected by the rAAV at an RNA enrichment of at least 2-fold greater than a wild type AAV9. 151. The method of embodiment 150, wherein the RNA enrichment is at least 10-fold greater than the wild type AAV9. 152. The method of embodiment 150 or embodiment 151, wherein the RNA enrichment is at least 25-fold greater than the wild type AAV9. 153. A method of treating a condition in a subject, the method comprising: administering a recombinant adeno-associated virus (rAAV) to the subject, wherein the rAAV encapsidates the payload, and wherein the rAAV comprises a VP capsid polypeptide comprising a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide, wherein the 581-589 region comprises at least one amino acid substitution as compared to SEQ ID NO: 9; infecting a muscle tissue of the subject with the rAAV, wherein the muscle tissue comprises a skeletal muscle tissue; preferentially delivering the payload to the muscle tissue infected by the rAAV at a DNA enrichment of at least 4-fold greater than a wild type AAV5; and producing a therapeutic effect in the muscle tissue, thereby treating the condition. 154. The method of embodiment 153, wherein the DNA enrichment is at least 5-fold greater than the wild type AAV5. 155. A method of treating a condition in a subject, the method comprising: administering a recombinant adeno-associated virus (rAAV) to the subject, wherein the rAAV encapsidates the payload, and wherein the rAAV comprises a VP capsid polypeptide comprising a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide, wherein the 581-589 region comprises at least one amino acid substitution as compared to SEQ ID NO: 9; infecting a muscle tissue of the subject with the rAAV, wherein the muscle tissue comprises a skeletal muscle tissue; preferentially transcribing the payload to the muscle tissue infected by the rAAV at an RNA enrichment of at that of a wild type AAV5; and producing a therapeutic effect in the muscle tissue, thereby treating the condition. 156. The method of embodiment 155, wherein the RNA enrichment is at least 3-fold greater than the wild type AAV5. 157. A method of treating a condition in a subject, the method comprising: administering a recombinant adeno-associated virus (rAAV) to the subject, wherein the rAAV encapsidates a payload, and wherein the rAAV comprises a VP capsid polypeptide comprising a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide, wherein the 581-589 region has a sequence that is at least 85% identical to: (a) SEQ ID NO: 3472, (b) SEQ ID NO: 3297, (c) SEQ ID NO: 2661, (d) SEQ ID NO: 4545, (e) SEQ ID NO: 348, (f) SEQ ID NO: 18, (g) SEQ ID NO: 4349, (h) SEQ ID NO: 293, (i) SEQ ID NO: 5017, (j) SEQ ID NO: 4955, (k) SEQ ID NO: 4366, (l) SEQ ID NO: 5092, (m) SEQ ID NO: 210, (n) SEQ ID NO: 4059, (o) SEQ ID NO: 2536, (p) SEQ ID NO: 5206, (q) SEQ ID NO: 1971, (r) SEQ ID NO: 262, (s) SEQ ID NO: 4343, (t) SEQ ID NO: 4995, (u) SEQ ID NO: 4009, (v) SEQ ID NO: 5190, (w) SEQ ID NO: 724, (x) SEQ ID NO: 4288, (y) SEQ ID NO: 4973, (z) SEQ ID NO: 4314, (aa) SEQ ID NO: 1561, (bb) SEQ ID NO: 5147, (cc) SEQ ID NO: 4238, or (dd) SEQ ID NO: 4961; infecting a muscle tissue of the subject with the rAAV, wherein the muscle tissue comprises skeletal muscle tissue; delivering the payload to the muscle tissue infected by the rAAV; and producing a therapeutic effect in the muscle tissue, thereby treating the condition. 158. A method of treating a condition in a subject, the method comprising: administering a recombinant adeno-associated virus (rAAV) to the subject, wherein the rAAV encapsidates the payload, and wherein the rAAV comprises a VP capsid polypeptide comprising a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide, wherein the 581-589 region comprises at least one amino acid substitution as compared to SEQ ID NO: 9; infecting a muscle tissue of the subject with the rAAV; preferentially delivering the payload to the muscle tissue infected by the rAAV at a DNA enrichment of at least 20-fold greater than a wild type AAV9; and producing a therapeutic effect in the muscle tissue, thereby treating the condition. 159. The method of embodiment 158, wherein the DNA enrichment is at least 25-fold greater than the wild type AAV9. 160. The method of embodiment 158 or embodiment 159, wherein the DNA enrichment is at least 30-fold greater than the wild type AAV9. 161. A method of treating a condition in a subject, the method comprising: administering a recombinant adeno-associated virus (rAAV) to the subject, wherein the rAAV encapsidates the payload, and wherein the rAAV comprises a VP capsid polypeptide comprising a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide, wherein the 581-589 region comprises at least one amino acid substitution as compared to SEQ ID NO: 9; infecting a muscle tissue of the subject with the rAAV; preferentially transcribing the payload to the muscle tissue infected by the rAAV at an RNA enrichment of at least 5-fold greater than a wild type AAV9; and producing a therapeutic effect in the muscle tissue, thereby treating the condition. 162. The method of embodiment 161, wherein the RNA enrichment is at least 10-fold greater than the wild type AAV9. 163. The method of embodiment 161 or embodiment 162, wherein the RNA enrichment is at least 100-fold greater than the wild type AAV9. 164. A method of treating a condition in a subject, the method comprising: administering a recombinant adeno-associated virus (rAAV) to the subject, wherein the rAAV encapsidates the payload, and wherein the rAAV comprises a VP capsid polypeptide comprising a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide, wherein the 581-589 region comprises at least one amino acid substitution as compared to SEQ ID NO: 9; infecting a muscle tissue of the subject with the rAAV; preferentially delivering the payload to the muscle tissue infected by the rAAV at a DNA enrichment of at least 5-fold greater than a wild type AAV5; and producing a therapeutic effect in the muscle tissue, thereby treating the condition. 165. The method of embodiment 164, wherein the DNA enrichment is at least 10-fold greater than the wild type AAV5. 166. A method of treating a condition in a subject, the method comprising: administering a recombinant adeno-associated virus (rAAV) to the subject, wherein the rAAV encapsidates the payload, and wherein the rAAV comprises a VP capsid polypeptide comprising a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide, wherein the 581-589 region comprises at least one amino acid substitution as compared to SEQ ID NO: 9; infecting the muscle tissue with the rAAV; preferentially transcribing the payload to the muscle tissue infected by the rAAV at an RNA enrichment of at least that of a wild type AAV5; and producing a therapeutic effect in the muscle tissue, thereby treating the condition. 167. The method of embodiment 166, wherein the RNA enrichment is at least 2-fold greater than the wild type AAV5. 168. A method of treating a condition in a subject, the method comprising: administering a recombinant adeno-associated virus (rAAV) to the subject, wherein the rAAV encapsidates a payload, and wherein the rAAV comprises a VP capsid polypeptide comprising a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide, wherein the 581-589 region has a sequence that is at least 85% identical to: (a) SEQ ID NO: 348, (b) SEQ ID NO: 2536, (c) SEQ ID NO: 4955, (d) SEQ ID NO: 5017, (e) SEQ ID NO: 4349, (f) SEQ ID NO: 293, (g) SEQ ID NO: 1576, (h) SEQ ID NO: 2661, (i) SEQ ID NO: 4964, (j) SEQ ID NO: 4314, (k) SEQ ID NO: 4995, (l) SEQ ID NO: 4366, (m) SEQ ID NO: 4545, (n) SEQ ID NO: 4961, (o) SEQ ID NO: 5206, (p) SEQ ID NO: 5092, (q) SEQ ID NO: 3472, (r) SEQ ID NO: 5075, (s) SEQ ID NO: 4059, (t) SEQ ID NO: 4354, (u) SEQ ID NO: 5060, (v) SEQ ID NO: 4288, (w) SEQ ID NO: 4952, (x) SEQ ID NO: 5138, (y) SEQ ID NO: 18, (z) SEQ ID NO: 5030, (aa) SEQ ID NO: 3297, (bb) SEQ ID NO: 4295, (cc) SEQ ID NO: 4363, or (dd) SEQ ID NO: 4963; infecting a muscle tissue of the subject with the rAAV; delivering the payload to the muscle tissue infected by the rAAV; and producing a therapeutic effect in the muscle tissue, thereby treating the condition. 169. A method of treating a condition in a subject, the method comprising: administering a recombinant adeno-associated virus (rAAV) to the subject, wherein the rAAV encapsidates a payload, and wherein the rAAV comprises a VP capsid polypeptide a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide, wherein the 581-589 region comprises at least one amino acid substitution as compared to residues 581 to 589 of SEQ ID NO: 1, and wherein the VP capsid polypeptide does not have the sequence of any of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, or SEQ ID NO: 8; infecting a muscle tissue of the subject with the rAAV with at least 3-fold higher muscle tissue tropism as compared to a wild type AAV5 capsid comprising a peptide of SEQ ID NO: 1; delivering the payload to the muscle tissue infected by the rAAV; and producing a therapeutic effect in the muscle tissue, thereby treating the condition. 170. A method of treating a condition in a subject, the method comprising: administering a recombinant adeno-associated virus (rAAV) to the subject, wherein the rAAV encapsidates a payload, and wherein the rAAV comprises a VP capsid polypeptide of any one of embodiments 1-84; infecting a muscle tissue of the subject with the rAAV; delivering the payload to the muscle tissue infected by the rAAV; and producing a therapeutic effect in the muscle tissue, thereby treating the condition. 171. The method of any one of embodiments 136-170, wherein the condition is a muscle condition. 172. The method of embodiment 171, wherein the muscle condition is a vascular condition, a cardiac condition, a skeletal muscle condition, Charcot-Marie-Tooth disease, Duchenne muscular dystrophy, facioscapulohumeral muscular dystrophy, dysferlinopathy, Pompe disease, limb-girdle muscular dystrophy, myotonic dystrophy, a glycogen storage disorder, X-linked myotubular myopathy, or euchromatic histone-lysine N-methyltransferase 2. 173. The method of any one of embodiments 136-172, wherein the condition is facioscapulohumeral muscular dystrophy syndrome. 174. The method of embodiment 173, wherein the payload encodes DUX4 or a guide RNA or miRNA that targets an mRNA encoding DUX4. 175. The method of any one of embodiments 136-172, wherein the condition is Duchenne muscular dystrophy. 176. The method of embodiment 175, wherein the payload encodes dystrophin or a guide RNA or miRNA that targets an mRNA encoding dystrophin. 177. The method of any one of embodiments 136-176, comprising administering from 1×10⁵to 5×10¹⁴rAAVs per kg subject weight. 178. The method of any one of embodiments 96-177, comprising infecting the muscle tissue with at least 2.5-fold, at least 3-fold, at least 3.5-fold, at least 4-fold, at least 5-fold, at least 10-fold, at least 20-fold, at least 50-fold, at least 75-fold, at least 100-fold, at least 200-fold, at least 500-fold, or at least 1000-fold higher tissue tropism than a wild type AAV5 capsid comprising a peptide of SEQ ID NO: 1. 179. The method of any one of embodiments 96-178, wherein the payload encodes a therapeutic protein, therapeutic polynucleotide, a guide RNA, a tRNA, a suppressor tRNA, a siRNA, a miRNA, an mRNA, a shRNA, a circular RNA, an antisense oligonucleotide (ASO), a ribozyme, a DNAzyme, an aptamer, or any combination thereof 180. The method of embodiment 179, wherein the guide RNA is a CRISPR/Cas guide RNA or an ADAR guide RNA. 181. The method of embodiment 179, wherein the therapeutic protein is selected from the group consisting of neurotrophin-3, micro-dystrophin, mini-dystrophin, double homeobox 4 (DUX4), dysferlin, α-glucosidase, FKRP, dystrophin, myotonin-protein kinase, glycogen synthase, myotubularin, and EHMT2. 182. The method embodiment 179, wherein the therapeutic polynucleotide targets an mRNA encoding a protein selected from the group consisting of neurotrophin-3, micro-dystrophin, mini-dystrophin, double homeobox 4 (DUX4), dysferlin, α-glucosidase, FKRP, dystrophin, myotonin-protein kinase, glycogen synthase, myotubularin, and EHMT2. 183. The method of any one of embodiments 96-182, wherein the payload encodes a component of a CRISPR/Cas system, an adenosine deaminase acting on RNA (ADAR) enzyme, a transcriptional activator, or a transcriptional repressor. 184. The method of embodiment 183, wherein the component of the CRISPR/Cas system comprises a Cas3, a Cas8, a Cas10, a Cas9, a Cas4, a Cas12, a Cas13, a guide RNA, or a combination thereof. 185. The method of embodiment 183, wherein the ADAR enzyme is ADAR1 or ADAR2. 186. The method of embodiment 183, wherein the transcriptional activator is VP64. 187. The method of embodiment 183, wherein the transcriptional repressor is KRAB. 188. The method of any one of embodiments 96-187, comprising systemically administering the rAAV to the subject. 189. The method of any one of embodiments 96-188, comprising administering the rAAV via intravenous administration, intramuscular administration, intraperitoneal administration, or oral administration. 190. The method of any one of embodiments 96-189, further comprising expressing a therapeutic polypeptide or a therapeutic polynucleotide encoded by the payload in the muscle tissue infected by the rAAV. 191. The method of embodiment 190, comprising expressing the therapeutic polypeptide or the therapeutic polynucleotide with higher muscle tissue tropism compared to a wild type AAV5 capsid comprising a peptide of SEQ ID NO: 1. 192. The method of embodiment 191, comprising expressing the therapeutic polypeptide or the therapeutic polynucleotide with at least 3-fold, at least 3.5-fold, at least 4-fold, at least 5-fold, at least 10-fold, at least 20-fold, at least 50-fold, at least 75-fold, at least 100-fold, at least 200-fold, at least 500-fold higher, or at least 1000-fold higher tropism compared to the wild type AAV5 capsid. 193. The method of any one of embodiments 96-192, comprising producing a toxicity in the subject that is lower than a toxicity produced upon systemic administration of a comparable number of wild type AAV5 capsids comprising a VP capsid polypeptide of SEQ ID NO: 1. 194. The method of any one of embodiments 96-193, comprising producing a toxicity in the subject that is lower than a toxicity produced upon systemic administration of a number of wild type AAV5 capsids comprising a VP capsid polypeptide of SEQ ID NO: 1 sufficient to deliver a comparable number of payloads to the muscle tissue. 195. The method of any one of embodiments 96-194, comprising producing a concentration of neutralizing antibodies in the subject that is lower than a concentration of neutralizing antibodies produced upon systemic administration of a comparable number of wild type AAV5 capsids comprising a VP capsid polypeptide of SEQ ID NO: 1. 196. The method of any one of embodiments 96-195, comprising producing a concentration of neutralizing antibodies in the subject that is lower than a concentration of neutralizing antibodies produced upon intravitreal administration of a comparable number of wild type AAV2 capsids. 197. The method of any one of embodiments 96-196, comprising producing a concentration of neutralizing antibodies in the subject that is lower than a concentration of neutralizing antibodies produced upon intravitreal administration of a comparable number of wild type AAV8 capsids. 198. The method of any one of embodiments 96-197, comprising producing a concentration of neutralizing antibodies in the subject that is lower than a concentration of neutralizing antibodies produced upon intravitreal administration of a comparable number of wild type AAV9 capsids. 199. The method of any one of embodiments 96-198, wherein the VP capsid polypeptide further comprises one or more mutations outside of the 581-589 region that contributes to reduced production of neutralizing antibodies relative to a wild type AAV capsid. 200. The method of any one of embodiments 96-199, comprising producing a concentration of neutralizing antibodies in the subject that is lower than a concentration of neutralizing antibodies produced upon systemic administration of a number of wild type AAV5 capsids comprising a VP capsid polypeptide of SEQ ID NO: 1 sufficient to deliver a comparable number of payloads to the muscle tissue. 201. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 a VP1 polypeptide, wherein the 581-589 region comprises at least one amino acid substitution as compared to SEQ ID NO: 9, and wherein a recombinant viral capsid comprising the VP capsid polypeptide is capable of preferentially targeting cardiac muscle tissue and a skeletal muscle tissue. 202. The VP capsid polypeptide of embodiment 201, wherein the 581-589 region has a sequence having at least 85% sequence identity to a sequence selected from the group consisting of SEQ ID NO: 348, SEQ ID NO: 2536, SEQ ID NO: 5607, SEQ ID NO: 4955, SEQ ID NO: 5017, SEQ ID NO: 4349, SEQ ID NO: 4964, SEQ ID NO: 293, SEQ ID NO: 4314, SEQ ID NO: 4995, SEQ ID NO: 4366, SEQ ID NO: 4961, SEQ ID NO: 4952, SEQ ID NO: 5075, SEQ ID NO: 4354, SEQ ID NO: 5060, SEQ ID NO: 5138, SEQ ID NO: 5206, SEQ ID NO: 4288, SEQ ID NO: 5283, SEQ ID NO: 5092, SEQ ID NO: 4059, SEQ ID NO: 4295, SEQ ID NO: 5030, SEQ ID NO: 2661, SEQ ID NO: 4545, SEQ ID NO: 4963, SEQ ID NO: 4363, SEQ ID NO: 5159, SEQ ID NO: 4973, SEQ ID NO: 5157, SEQ ID NO: 4345, SEQ ID NO: 5039, SEQ ID NO: 4304, SEQ ID NO: 18, SEQ ID NO: 386, SEQ ID NO: 308, SEQ ID NO: 278, SEQ ID NO: 5037, SEQ ID NO: 4361, SEQ ID NO: 4317, SEQ ID NO: 4343, SEQ ID NO: 4311, SEQ ID NO: 5055, SEQ ID NO: 4359, SEQ ID NO: 4353, and SEQ ID NO: 4346. 203. The VP capsid polypeptide of embodiment 201 or embodiment 202, wherein the 581-589 region has a sequence selected from the group consisting of SEQ ID NO: 348, SEQ ID NO: 2536, SEQ ID NO: 5607, SEQ ID NO: 4955, SEQ ID NO: 5017, SEQ ID NO: 4349, SEQ ID NO: 4964, SEQ ID NO: 293, SEQ ID NO: 4314, SEQ ID NO: 4995, SEQ ID NO: 4366, SEQ ID NO: 4961, SEQ ID NO: 4952, SEQ ID NO: 5075, SEQ ID NO: 4354, SEQ ID NO: 5060, SEQ ID NO: 5138, SEQ ID NO: 5206, SEQ ID NO: 4288, SEQ ID NO: 5283, SEQ ID NO: 5092, SEQ ID NO: 4059, SEQ ID NO: 4295, SEQ ID NO: 5030, SEQ ID NO: 2661, SEQ ID NO: 4545, SEQ ID NO: 4963, SEQ ID NO: 4363, SEQ ID NO: 5159, SEQ ID NO: 4973, SEQ ID NO: 5157, SEQ ID NO: 4345, SEQ ID NO: 5039, SEQ ID NO: 4304, SEQ ID NO: 18, SEQ ID NO: 386, SEQ ID NO: 308, SEQ ID NO: 278, SEQ ID NO: 5037, SEQ ID NO: 4361, SEQ ID NO: 4317, SEQ ID NO: 4343, SEQ ID NO: 4311, SEQ ID NO: 5055, SEQ ID NO: 4359, SEQ ID NO: 4353, and SEQ ID NO: 4346. 204. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence that is at least 85% identical to: (a) SEQ ID NO: 5027, (b) SEQ ID NO: 4633, (c) SEQ ID NO: 4943, (d) SEQ ID NO: 386, (e) SEQ ID NO: 334, (f) SEQ ID NO: 4309, (g) SEQ ID NO: 4288, (h) SEQ ID NO: 4964, (i) SEQ ID NO: 5017, (j) SEQ ID NO: 5814, (k) SEQ ID NO: 4335, (l) SEQ ID NO: 348, (m) SEQ ID NO: 4936, (n) SEQ ID NO: 5206, (o) SEQ ID NO: 4238, (p) SEQ ID NO: 5077, or (q) SEQ ID NO: 5603. 205. The VP capsid polypeptide of embodiment 204, wherein the 581-589 region has a sequence of: (a) SEQ ID NO: 5027, (b) SEQ ID NO: 4633, (c) SEQ ID NO: 4943, (d) SEQ ID NO: 386, (e) SEQ ID NO: 334, (f) SEQ ID NO: 4309, (g) SEQ ID NO: 4288, (h) SEQ ID NO: 4964, (i) SEQ ID NO: 5017, (j) SEQ ID NO: 5814, (k) SEQ ID NO: 4335, (l) SEQ ID NO: 348, (m) SEQ ID NO: 4936, (n) SEQ ID NO: 5206, (o) SEQ ID NO: 4238, (p) SEQ ID NO: 5077, or (q) SEQ ID NO: 5603. 206. The VP capsid polypeptide of embodiment 204 or embodiment 205, wherein the 581-589 region confers cardiac tissue tropism in mice and non-human primates. 207. A research method comprising administering a recombinant adeno-associated virus (rAAV) to a model organism, wherein the rAAV comprises a VP capsid polypeptide of any one of embodiments 204-206. 208. The research method of embodiment 207, further comprising evaluating an effect of the rAAV in the model organism. 209. The research method of embodiment 208, wherein the model organism is a mouse. 210. The research method of embodiment 208 or embodiment 209, further comprising inferring an effect of the rAAV in an organism of interest based on the effect of the rAAV in the model organism. 211. The research method of embodiment 210, wherein the organism of interest is a non-human primate or a human. 212. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence that is at least 85% identical to SEQ ID NO: 18. 213. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence that is at least 85% identical to SEQ ID NO: 3472. 214. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence that is at least 85% identical to SEQ ID NO: 262. 215. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence that is at least 85% identical to SEQ ID NO: 3306. 216. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence that is at least 85% identical to SEQ ID NO: 2028. 217. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence that is at least 85% identical to SEQ ID NO: 2791. 218. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence that is at least 85% identical to SEQ ID NO: 424. 219. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence that is at least 85% identical to SEQ ID NO: 2536. 220. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence that is at least 85% identical to SEQ ID NO: 1971. 221. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence that is at least 85% identical to SEQ ID NO: 415. 222. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence that is at least 85% identical to SEQ ID NO: 3846. 223. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence that is at least 85% identical to SEQ ID NO: 3283. 224. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence that is at least 85% identical to SEQ ID NO: 1956. 225. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence that is at least 85% identical to SEQ ID NO: 3297. 226. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence that is at least 85% identical to SEQ ID NO: 4545. 227. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence that is at least 85% identical to SEQ ID NO: 2661. 228. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence that is at least 85% identical to SEQ ID NO: 1576. 229. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence that is at least 85% identical to SEQ ID NO: 425. 230. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence that is at least 85% identical to SEQ ID NO: 709. 231. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence that is at least 85% identical to SEQ ID NO: 2168. 232. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence that is at least 85% identical to SEQ ID NO: 54. 233. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence that is at least 85% identical to SEQ ID NO: 429. 234. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence that is at least 85% identical to SEQ ID NO: 708. 235. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence that is at least 85% identical to SEQ ID NO: 428. 236. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence that is at least 85% identical to SEQ ID NO: 4119. 237. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence that is at least 85% identical to SEQ ID NO: 3906. 238. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence that is at least 85% identical to SEQ ID NO: 2456. 239. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence that is at least 85% identical to SEQ ID NO: 2278. 240. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence that is at least 85% identical to SEQ ID NO: 5006. 241. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence that is at least 85% identical to SEQ ID NO: 426. 242. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence that is at least 85% identical to SEQ ID NO: 307. 243. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence that is at least 85% identical to SEQ ID NO: 5155. 244. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence that is at least 85% identical to SEQ ID NO: 2640. 245. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence that is at least 85% identical to SEQ ID NO: 4317. 246. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence that is at least 85% identical to SEQ ID NO: 1145. 247. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence that is at least 85% identical to SEQ ID NO: 430. 248. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence that is at least 85% identical to SEQ ID NO: 885. 249. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence that is at least 85% identical to SEQ ID NO: 23. 250. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence that is at least 85% identical to SEQ ID NO: 103. 251. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence that is at least 85% identical to SEQ ID NO: 22. 252. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence that is at least 85% identical to SEQ ID NO: 4031. 253. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence that is at least 85% identical to SEQ ID NO: 1008. 254. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence that is at least 85% identical to SEQ ID NO: 4790. 255. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence that is at least 85% identical to SEQ ID NO: 2522. 256. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence that is at least 85% identical to SEQ ID NO: 1432. 257. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence that is at least 85% identical to SEQ ID NO: 2914. 258. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence that is at least 85% identical to SEQ ID NO: 3935. 259. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence that is at least 85% identical to SEQ ID NO: 5042. 260. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence that is at least 85% identical to SEQ ID NO: 2865. 261. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence that is at least 85% identical to SEQ ID NO: 4264. 262. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence that is at least 85% identical to SEQ ID NO: 964. 263. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence that is at least 85% identical to SEQ ID NO: 1268. 264. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence that is at least 85% identical to SEQ ID NO: 5065. 265. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence that is at least 85% identical to SEQ ID NO: 706. 266. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence that is at least 85% identical to SEQ ID NO: 4704. 267. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence that is at least 85% identical to SEQ ID NO: 569. 268. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence that is at least 85% identical to SEQ ID NO: 2994. 269. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence that is at least 85% identical to SEQ ID NO: 829. 270. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence that is at least 85% identical to SEQ ID NO: 1171. 271. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence that is at least 85% identical to SEQ ID NO: 1041. 272. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence that is at least 85% identical to SEQ ID NO: 5070. 273. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence that is at least 85% identical to SEQ ID NO: 139. 274. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence that is at least 85% identical to SEQ ID NO: 3304. 275. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence that is at least 85% identical to SEQ ID NO: 2431. 276. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence that is at least 85% identical to SEQ ID NO: 4288. 277. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence that is at least 85% identical to SEQ ID NO: 5795. 278. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence that is at least 85% identical to SEQ ID NO: 1300. 279. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence that is at least 85% identical to SEQ ID NO: 724. 280. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence that is at least 85% identical to SEQ ID NO: 1770. 281. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence that is at least 85% identical to SEQ ID NO: 2830. 282. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence that is at least 85% identical to SEQ ID NO: 1972. 283. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence that is at least 85% identical to SEQ ID NO: 1649. 284. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence that is at least 85% identical to SEQ ID NO: 2515. 285. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence that is at least 85% identical to SEQ ID NO: 2849. 286. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence that is at least 85% identical to SEQ ID NO: 3796. 287. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence that is at least 85% identical to SEQ ID NO: 5815. 288. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence that is at least 85% identical to SEQ ID NO: 118. 289. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence that is at least 85% identical to SEQ ID NO: 4766. 290. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence that is at least 85% identical to SEQ ID NO: 770. 291. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence that is at least 85% identical to SEQ ID NO: 1069. 292. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence that is at least 85% identical to SEQ ID NO: 3061. 293. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence that is at least 85% identical to SEQ ID NO: 4290. 294. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence that is at least 85% identical to SEQ ID NO: 4261. 295. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence that is at least 85% identical to SEQ ID NO: 4239. 296. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence that is at least 85% identical to SEQ ID NO: 3478. 297. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence that is at least 85% identical to SEQ ID NO: 1887. 298. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence that is at least 85% identical to SEQ ID NO: 2671. 299. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence that is at least 85% identical to SEQ ID NO: 256. 300. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence that is at least 85% identical to SEQ ID NO: 1256. 301. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence that is at least 85% identical to SEQ ID NO: 719. 302. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence that is at least 85% identical to SEQ ID NO: 1448. 303. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence that is at least 85% identical to SEQ ID NO: 280. 304. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence that is at least 85% identical to SEQ ID NO: 4338. 305. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence that is at least 85% identical to SEQ ID NO: 5037. 306. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence that is at least 85% identical to SEQ ID NO: 1901. 307. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence that is at least 85% identical to SEQ ID NO: 438. 308. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence that is at least 85% identical to SEQ ID NO: 2834. 309. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence that is at least 85% identical to SEQ ID NO: 5491. 310. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence that is at least 85% identical to SEQ ID NO: 4591. 311. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence that is at least 85% identical to SEQ ID NO: 4936. 312. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence that is at least 85% identical to SEQ ID NO: 348. 313. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence that is at least 85% identical to SEQ ID NO: 5607. 314. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence that is at least 85% identical to SEQ ID NO: 4955. 315. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence that is at least 85% identical to SEQ ID NO: 5017. 316. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence that is at least 85% identical to SEQ ID NO: 4349. 317. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence that is at least 85% identical to SEQ ID NO: 4964. 318. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence that is at least 85% identical to SEQ ID NO: 293. 319. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence that is at least 85% identical to SEQ ID NO: 4314. 320. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence that is at least 85% identical to SEQ ID NO: 4995. 321. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence that is at least 85% identical to SEQ ID NO: 4366. 322. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence that is at least 85% identical to SEQ ID NO: 4961. 323. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence that is at least 85% identical to SEQ ID NO: 4952. 324. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence that is at least 85% identical to SEQ ID NO: 5075. 325. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence that is at least 85% identical to SEQ ID NO: 4354. 326. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence that is at least 85% identical to SEQ ID NO: 5060. 327. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence that is at least 85% identical to SEQ ID NO: 5138. 328. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence that is at least 85% identical to SEQ ID NO: 5206. 329. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence that is at least 85% identical to SEQ ID NO: 5283. 330. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence that is at least 85% identical to SEQ ID NO: 5092. 331. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence that is at least 85% identical to SEQ ID NO: 4059. 332. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence that is at least 85% identical to SEQ ID NO: 4295. 333. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence that is at least 85% identical to SEQ ID NO: 5030. 334. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence that is at least 85% identical to SEQ ID NO: 4938. 335. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence that is at least 85% identical to SEQ ID NO: 5215. 336. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence that is at least 85% identical to SEQ ID NO: 4960. 337. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence that is at least 85% identical to SEQ ID NO: 4969. 338. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence that is at least 85% identical to SEQ ID NO: 5023. 339. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence that is at least 85% identical to SEQ ID NO: 4934. 340. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence that is at least 85% identical to SEQ ID NO: 4963. 341. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence that is at least 85% identical to SEQ ID NO: 4363. 342. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence that is at least 85% identical to SEQ ID NO: 5159. 343. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence that is at least 85% identical to SEQ ID NO: 4973. 344. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence that is at least 85% identical to SEQ ID NO: 5157. 345. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence that is at least 85% identical to SEQ ID NO: 4972. 346. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence that is at least 85% identical to SEQ ID NO: 4345. 347. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence that is at least 85% identical to SEQ ID NO: 5039. 348. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence that is at least 85% identical to SEQ ID NO: 5163. 349. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence that is at least 85% identical to SEQ ID NO: 5040. 350. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence that is at least 85% identical to SEQ ID NO: 4304. 351. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence that is at least 85% identical to SEQ ID NO: 5193. 352. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence that is at least 85% identical to SEQ ID NO: 5077. 353. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence that is at least 85% identical to SEQ ID NO: 5814. 354. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence that is at least 85% identical to SEQ ID NO: 386. 355. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence that is at least 85% identical to SEQ ID NO: 5081. 356. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence that is at least 85% identical to SEQ ID NO: 308. 357. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence that is at least 85% identical to SEQ ID NO: 278. 358. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence that is at least 85% identical to SEQ ID NO: 5127. 359. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence that is at least 85% identical to SEQ ID NO: 5029. 360. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence that is at least 85% identical to SEQ ID NO: 5143. 361. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence that is at least 85% identical to SEQ ID NO: 4983. 362. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence that is at least 85% identical to SEQ ID NO: 4361. 363. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence that is at least 85% identical to SEQ ID NO: 5080. 364. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence that is at least 85% identical to SEQ ID NO: 4343. 365. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence that is at least 85% identical to SEQ ID NO: 4986. 366. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence that is at least 85% identical to SEQ ID NO: 4311. 367. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence that is at least 85% identical to SEQ ID NO: 5102. 368. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence that is at least 85% identical to SEQ ID NO: 5280. 369. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence that is at least 85% identical to SEQ ID NO: 5185. 370. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence that is at least 85% identical to SEQ ID NO: 4935. 371. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence that is at least 85% identical to SEQ ID NO: 4379. 372. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence that is at least 85% identical to SEQ ID NO: 5052. 373. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence that is at least 85% identical to SEQ ID NO: 5027. 374. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence that is at least 85% identical to SEQ ID NO: 5173. 375. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence that is at least 85% identical to SEQ ID NO: 5055. 376. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence that is at least 85% identical to SEQ ID NO: 5123. 377. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence that is at least 85% identical to SEQ ID NO: 4335. 378. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence that is at least 85% identical to SEQ ID NO: 5208. 379. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence that is at least 85% identical to SEQ ID NO: 4633. 380. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence that is at least 85% identical to SEQ ID NO: 4359. 381. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence that is at least 85% identical to SEQ ID NO: 289. 382. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence that is at least 85% identical to SEQ ID NO: 4353. 383. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence that is at least 85% identical to SEQ ID NO: 4316. 384. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence that is at least 85% identical to SEQ ID NO: 5210. 385. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence that is at least 85% identical to SEQ ID NO: 4949. 386. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence that is at least 85% identical to SEQ ID NO: 4947. 387. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence that is at least 85% identical to SEQ ID NO: 2838. 388. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence that is at least 85% identical to SEQ ID NO: 378. 389. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence that is at least 85% identical to SEQ ID NO: 5013. 390. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence that is at least 85% identical to SEQ ID NO: 5278. 391. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence that is at least 85% identical to SEQ ID NO: 1471. 392. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence that is at least 85% identical to SEQ ID NO: 4346. 393. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence that is at least 85% identical to SEQ ID NO: 297. 394. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence that is at least 85% identical to SEQ ID NO: 4993. 395. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence that is at least 85% identical to SEQ ID NO: 384. 396. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence that is at least 85% identical to SEQ ID NO: 5062. 397. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence that is at least 85% identical to SEQ ID NO: 4945. 398. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence that is at least 85% identical to SEQ ID NO: 210. 399. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence that is at least 85% identical to SEQ ID NO: 4009. 400. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence that is at least 85% identical to SEQ ID NO: 5190. 401. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence that is at least 85% identical to SEQ ID NO: 1561. 402. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence that is at least 85% identical to SEQ ID NO: 5147. 403. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence that is at least 85% identical to SEQ ID NO: 4238. 404. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence that is at least 85% identical to SEQ ID NO: 5527. 405. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence that is at least 85% identical to SEQ ID NO: 4355. 406. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence that is at least 85% identical to SEQ ID NO: 5125. 407. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence that is at least 85% identical to SEQ ID NO: 4108. 408. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence that is at least 85% identical to SEQ ID NO: 2948. 409. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence that is at least 85% identical to SEQ ID NO: 3821. 410. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence that is at least 85% identical to SEQ ID NO: 4632. 411. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence that is at least 85% identical to SEQ ID NO: 294. 412. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence that is at least 85% identical to SEQ ID NO: 4943. 413. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence that is at least 85% identical to SEQ ID NO: 1391. 414. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence that is at least 85% identical to SEQ ID NO: 5078. 415. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence that is at least 85% identical to SEQ ID NO: 3299. 416. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence that is at least 85% identical to SEQ ID NO: 2897. 417. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence that is at least 85% identical to SEQ ID NO: 1537. 418. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence that is at least 85% identical to SEQ ID NO: 5255. 419. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence that is at least 85% identical to SEQ ID NO: 141. 420. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence that is at least 85% identical to SEQ ID NO: 5038. 421. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence that is at least 85% identical to SEQ ID NO: 5153. 422. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence that is at least 85% identical to SEQ ID NO: 1181. 423. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence that is at least 85% identical to SEQ ID NO: 1204. 424. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence that is at least 85% identical to SEQ ID NO: 4404. 425. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence that is at least 85% identical to SEQ ID NO: 5106. 426. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence that is at least 85% identical to SEQ ID NO: 2779. 427. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence that is at least 85% identical to SEQ ID NO: 1824. 428. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence that is at least 85% identical to SEQ ID NO: 5116. 429. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence that is at least 85% identical to SEQ ID NO: 3179. 430. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence that is at least 85% identical to SEQ ID NO: 98. 431. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence that is at least 85% identical to SEQ ID NO: 1872. 432. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence that is at least 85% identical to SEQ ID NO: 1634. 433. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence that is at least 85% identical to SEQ ID NO: 1060. 434. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence that is at least 85% identical to SEQ ID NO: 4941. 435. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence that is at least 85% identical to SEQ ID NO: 4981. 436. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence that is at least 85% identical to SEQ ID NO: 5274. 437. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence that is at least 85% identical to SEQ ID NO: 5816 (QKMPNNMYG). 438. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence that is at least 85% identical to SEQ ID NO: 2842. 439. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence that is at least 85% identical to SEQ ID NO: 1934. 440. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence that is at least 85% identical to SEQ ID NO: 5817. 441. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence that is at least 85% identical to SEQ ID NO: 3998. 442. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence that is at least 85% identical to SEQ ID NO: 5026. 443. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence of SEQ ID NO: 18. 444. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence of SEQ ID NO: 3472. 445. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence of SEQ ID NO: 262. 446. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence of SEQ ID NO: 3306. 447. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence of SEQ ID NO: 2028. 448. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence of SEQ ID NO: 2791. 449. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence of SEQ ID NO: 424. 450. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence of SEQ ID NO: 2536. 451. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence of SEQ ID NO: 1971. 452. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence of SEQ ID NO: 415. 453. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence of SEQ ID NO: 3846. 454. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence of SEQ ID NO: 3283. 455. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence of SEQ ID NO: 1956. 456. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence of SEQ ID NO: 3297. 457. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence of SEQ ID NO: 4545. 458. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence of SEQ ID NO: 2661. 459. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence of SEQ ID NO: 1576. 460. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence of SEQ ID NO: 425. 461. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence of SEQ ID NO: 709. 462. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence of SEQ ID NO: 2168. 463. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence of SEQ ID NO: 54. 464. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence of SEQ ID NO: 429. 465. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence of SEQ ID NO: 708. 466. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence of SEQ ID NO: 428. 467. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence of SEQ ID NO: 4119. 468. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence of SEQ ID NO: 3906. 469. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence of SEQ ID NO: 2456. 470. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence of SEQ ID NO: 2278. 471. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence of SEQ ID NO: 5006. 472. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence of SEQ ID NO: 426. 473. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence of SEQ ID NO: 307. 474. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence of SEQ ID NO: 5155. 475. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence of SEQ ID NO: 2640. 476. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence of SEQ ID NO: 4317. 477. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence of SEQ ID NO: 1145. 478. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence of SEQ ID NO: 430. 479. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence of SEQ ID NO: 885. 480. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence of SEQ ID NO: 23. 481. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence of SEQ ID NO: 103. 482. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence of SEQ ID NO: 22. 483. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence of SEQ ID NO: 4031. 484. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence of SEQ ID NO: 1008. 485. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence of SEQ ID NO: 4790. 486. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence of SEQ ID NO: 2522. 487. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence of SEQ ID NO: 1432. 488. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence of SEQ ID NO: 2914. 489. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence of SEQ ID NO: 3935. 490. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence of SEQ ID NO: 5042. 491. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence of SEQ ID NO: 2865. 492. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence of SEQ ID NO: 4264. 493. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence of SEQ ID NO: 964. 494. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence of SEQ ID NO: 1268. 495. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence of SEQ ID NO: 5065. 496. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence of SEQ ID NO: 706. 497. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence of SEQ ID NO: 4704. 498. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence of SEQ ID NO: 569. 499. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence of SEQ ID NO: 2994. 500. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence of SEQ ID NO: 829. 501. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence of SEQ ID NO: 1171. 502. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence of SEQ ID NO: 1041. 503. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence of SEQ ID NO: 5070. 504. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence of SEQ ID NO: 139. 505. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence of SEQ ID NO: 3304. 506. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence of SEQ ID NO: 2431. 507. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence of SEQ ID NO: 4288. 508. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence of SEQ ID NO: 5795. 509. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence of SEQ ID NO: 1300. 510. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence of SEQ ID NO: 724. 511. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence of SEQ ID NO: 1770. 512. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence of SEQ ID NO: 2830. 513. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence of SEQ ID NO: 1972. 514. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence of SEQ ID NO: 1649. 515. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence of SEQ ID NO: 2515. 516. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence of SEQ ID NO: 2849. 517. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence of SEQ ID NO: 3796. 518. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence of SEQ ID NO: 5815. 519. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence of SEQ ID NO: 118. 520. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence of SEQ ID NO: 4766. 521. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence of SEQ ID NO: 770. 522. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence of SEQ ID NO: 1069. 523. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence of SEQ ID NO: 3061. 524. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence of SEQ ID NO: 4290. 525. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence of SEQ ID NO: 4261. 526. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence of SEQ ID NO: 4239. 527. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence of SEQ ID NO: 3478. 528. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence of SEQ ID NO: 1887. 529. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence of SEQ ID NO: 2671. 530. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence of SEQ ID NO: 256. 531. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence of SEQ ID NO: 1256. 532. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence of SEQ ID NO: 719. 533. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence of SEQ ID NO: 1448. 534. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence of SEQ ID NO: 280. 535. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence of SEQ ID NO: 4338. 536. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence of SEQ ID NO: 5037. 537. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence of SEQ ID NO: 1901. 538. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence of SEQ ID NO: 438. 539. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence of SEQ ID NO: 2834. 540. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence of SEQ ID NO: 5491. 541. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence of SEQ ID NO: 4591. 542. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence of SEQ ID NO: 4936. 543. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence of SEQ ID NO: 348. 544. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence of SEQ ID NO: 5607. 545. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence of SEQ ID NO: 4955. 546. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence of SEQ ID NO: 5017. 547. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence of SEQ ID NO: 4349. 548. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence of SEQ ID NO: 4964. 549. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence of SEQ ID NO: 293. 550. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence of SEQ ID NO: 4314. 551. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence of SEQ ID NO: 4995. 552. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence of SEQ ID NO: 4366. 553. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence of SEQ ID NO: 4961. 554. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence of SEQ ID NO: 4952. 555. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence of SEQ ID NO: 5075. 556. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence of SEQ ID NO: 4354. 557. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence of SEQ ID NO: 5060. 558. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence of SEQ ID NO: 5138. 559. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence of SEQ ID NO: 5206. 560. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence of SEQ ID NO: 5283. 561. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence of SEQ ID NO: 5092. 562. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence of SEQ ID NO: 4059. 563. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence of SEQ ID NO: 4295. 564. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence of SEQ ID NO: 5030. 565. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence of SEQ ID NO: 4938. 566. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence of SEQ ID NO: 5215. 567. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence of SEQ ID NO: 4960. 568. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence of SEQ ID NO: 4969. 569. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence of SEQ ID NO: 5023. 570. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence of SEQ ID NO: 4934. 571. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence of SEQ ID NO: 4963. 572. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence of SEQ ID NO: 4363. 573. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence of SEQ ID NO: 5159. 574. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence of SEQ ID NO: 4973. 575. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence of SEQ ID NO: 5157. 576. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence of SEQ ID NO: 4972. 577. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence of SEQ ID NO: 4345. 578. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence of SEQ ID NO: 5039. 579. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence of SEQ ID NO: 5163. 580. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence of SEQ ID NO: 5040. 581. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence of SEQ ID NO: 4304. 582. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence of SEQ ID NO: 5193. 583. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence of SEQ ID NO: 5077. 584. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence of SEQ ID NO: 5814. 585. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence of SEQ ID NO: 386. 586. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence of SEQ ID NO: 5081. 587. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence of SEQ ID NO: 308. 588. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence of SEQ ID NO: 278. 589. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence of SEQ ID NO: 5127. 590. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence of SEQ ID NO: 5029. 591. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence of SEQ ID NO: 5143. 592. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence of SEQ ID NO: 4983. 593. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence of SEQ ID NO: 4361. 594. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence of SEQ ID NO: 5080. 595. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence of SEQ ID NO: 4343. 596. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence of SEQ ID NO: 4986. 597. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence of SEQ ID NO: 4311. 598. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence of SEQ ID NO: 5102. 599. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence of SEQ ID NO: 5280. 600. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence of SEQ ID NO: 5185. 601. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence of SEQ ID NO: 4935. 602. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence of SEQ ID NO: 4379. 603. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence of SEQ ID NO: 5052. 604. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence of SEQ ID NO: 5027. 605. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence of SEQ ID NO: 5173. 606. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence of SEQ ID NO: 5055. 607. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence of SEQ ID NO: 5123. 608. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence of SEQ ID NO: 4335. 609. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence of SEQ ID NO: 5208. 610. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence of SEQ ID NO: 4633. 611. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence of SEQ ID NO: 4359. 612. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence of SEQ ID NO: 289. 613. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence of SEQ ID NO: 4353. 614. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence of SEQ ID NO: 4316. 615. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence of SEQ ID NO: 5210. 616. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence of SEQ ID NO: 4949. 617. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence of SEQ ID NO: 4947. 618. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence of SEQ ID NO: 2838. 619. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence of SEQ ID NO: 378. 620. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence of SEQ ID NO: 5013. 621. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence of SEQ ID NO: 5278. 622. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence of SEQ ID NO: 1471. 623. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence of SEQ ID NO: 4346. 624. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence of SEQ ID NO: 297. 625. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence of SEQ ID NO: 4993. 626. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence of SEQ ID NO: 384. 627. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence of SEQ ID NO: 5062. 628. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence of SEQ ID NO: 4945. 629. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence of SEQ ID NO: 210. 630. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence of SEQ ID NO: 4009. 631. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence of SEQ ID NO: 5190. 632. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence of SEQ ID NO: 1561. 633. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence of SEQ ID NO: 5147. 634. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence of SEQ ID NO: 4238. 635. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence of SEQ ID NO: 5527. 636. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence of SEQ ID NO: 4355. 637. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence of SEQ ID NO: 5125. 638. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence of SEQ ID NO: 4108. 639. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence of SEQ ID NO: 2948. 640. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence of SEQ ID NO: 3821. 641. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence of SEQ ID NO: 4632. 642. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence of SEQ ID NO: 294. 643. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence of SEQ ID NO: 4943. 644. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence of SEQ ID NO: 1391. 645. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence of SEQ ID NO: 5078. 646. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence of SEQ ID NO: 3299. 647. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence of SEQ ID NO: 2897. 648. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence of SEQ ID NO: 1537. 649. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence of SEQ ID NO: 5255. 650. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence of SEQ ID NO: 141. 651. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence of SEQ ID NO: 5038. 652. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence of SEQ ID NO: 5153. 653. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence of SEQ ID NO: 1181. 654. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence of SEQ ID NO: 1204. 655. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence of SEQ ID NO: 4404. 656. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence of SEQ ID NO: 5106. 657. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence of SEQ ID NO: 2779. 658. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence of SEQ ID NO: 1824. 659. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence of SEQ ID NO: 5116. 660. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence of SEQ ID NO: 3179. 661. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence of SEQ ID NO: 98. 662. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence of SEQ ID NO: 1872. 663. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence of SEQ ID NO: 1634. 664. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence of SEQ ID NO: 1060. 665. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence of SEQ ID NO: 4941. 666. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence of SEQ ID NO: 4981. 667. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence of SEQ ID NO: 5274. 668. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence of SEQ ID NO: 5816. 669. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence of SEQ ID NO: 2842. 670. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence of SEQ ID NO: 1934. 671. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence of SEQ ID NO: 5817. 672. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence of SEQ ID NO: 3998. 673. A viral protein (VP) capsid polypeptide, wherein the VP capsid polypeptide comprises a 581-589 region corresponding to residues 581 to 589 of a VP1 polypeptide; wherein the 581-589 region has a sequence of SEQ ID NO: 5026.

EXAMPLES

Below are examples of specific embodiments for carrying out the present invention. The examples are offered for illustrative purposes only and are not intended to limit the scope of the present invention in any way. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperatures, etc.), but some experimental error and deviation should, of course, be allowed for.

The practice of the present invention will employ, unless otherwise indicated, conventional methods of protein chemistry, biochemistry, recombinant DNA techniques and pharmacology, within the skill of the art. Such techniques are explained fully in the literature.

Example 1
High Throughput Capsid Engineering Systems

This example describes the high throughput capsid engineering systems and methods disclosed herein to discover engineered tissue tropic AAV variants, including CNS tissue-tropic variants and muscle-tropic variants. The high throughput capsid engineering system is schematized in FIG. 1. “Detargeting” may be referred to herein as “sparing” with respect to a particular tissue. For example, liver detargeting or liver sparing may both be used herein to refer to variants or properties of variants that preferentially exhibit reduced homing to liver tissue.

Tissues were harvested and transducing capsid genes are labeled with unique molecular identifiers and barcodes. These variant sequences/UMIs/barcodes were parameterized, and machine learning algorithms were used to identify deterministic features of specific tissue targeting/detargeting capsids.

FIG. 2A provides a side view (top panel) and top view (bottom panel) of key residues of known AAV capsids that have been shown to interact with target cells. FIG. 2B illustrates salient features of the AAV capsid library, showing the region of introduced diversity, with residue numbering corresponding to the numbering of amino acids in AAV5 VP1. The library plasmid includes the invariant rep and diversified cap coding sequences between AAV ITRs (a cis-packaging approach).

The variants in the AAV capsid library were able to assemble at levels similar to that of wild type AAVs. FIG. 7A illustrates the normalized counts of plasmid library DNA compared to assembled virus DNA for wild type AAV controls (textured points) and for variant AAV candidates (gray circles). Levels of assembled virus for variant AAV candidates were comparable to wild type AAV controls at 1× spike-in frequency.

Example 2

AAV5 Variants with Tissue Tropism in CNS

This example describes engineered AAV5 variants with tissue tropism in CNS that were discovered using the methods and systems described in EXAMPLE 1.

AAV5 variants found to exhibit CNS tropism included SEQ ID NO: 12-SEQ ID NO: 3937 and SEQ ID NO: 5234-SEQ ID NO: 5807). Variant residues are in the 581-589 region corresponding to positions 581 to 589 of VP1.

TABLE 2 and TABLE 4 provide sequences of 581-589 regions, corresponding to residues 581 to 589 of VP1, for variant AAV VP capsid polypeptides that were found in CNS tissue. The present disclosure, thus, provides for rAAVs composed of engineered AAV5 VP1 capsid polypeptides having a SEQ ID NO: 2, wherein the X¹to X⁹region is any one of SEQ ID NO: SEQ ID NO: 12-SEQ ID NO: 3937 or SEQ ID NO: 5234-SEQ ID NO: 5807, as disclosed in TABLE 2 and TABLE 4. Also encompassed herein are rAAVs composed of engineered AAV5 VP2 capsid polypeptides and engineered AAV5 VP3 capsid polypeptides having the sequences disclosed in the below table at the regions in AAV5 VP2 (amino acid residues 445 to 453) and AAV5 VP3 (amino acid residues 389 to 397) corresponding to the amino acids in the AAV5 VP1 581 to 589 region.

Library members selected as CNS-tropic candidates showed greater enrichment in CNS tissue relative to liver (FIG. 7B) or muscle (FIG. 7C) that wild type AAVs or other AAV library members. FIG. 7B shows DNA levels in liver versus CNS for variant AAV candidates (gray circles), wild type controls (textured points), and select AAV candidates with CNS tropism (open circles). CNS-tropic AAV candidates were enriched in CNS tissue relative to liver tissue. FIG. 7C shows DNA levels in muscle versus CNS for variant AAV candidates (gray circles), wild type controls (textured points), and select AAV candidates with CNS tropism (open circles). CNS-tropic AAV candidates were enriched in CNS tissue relative to muscle tissue.

Example 3
AAV5 Variants with Tissue Tropism in Muscle

This example describes engineered AAV5 variants with tissue tropism in muscle that were discovered using the methods and systems described in EXAMPLE 1.

AAV5 variants found to exhibit muscle tropism included SEQ ID NO: 4238-SEQ ID NO: 4933 and SEQ ID NO: 5808-SEQ ID NO: 5811. Variant residues are in the 581-589 region corresponding to positions 581 to 589 of VP1.

TABLE 5 and TABLE 7 provide sequences of 581-589 regions, corresponding to residues 581 to 589 of VP1, for variant AAV VP capsid polypeptides that were found in muscle tissue. The present disclosure, thus, provides for rAAVs composed of engineered AAV5 VP1 capsid polypeptides having a SEQ ID NO: 2, wherein the X¹to X⁹region is any one of SEQ ID NO: 4238-SEQ ID NO: 4933 or SEQ ID NO: 5808-SEQ ID NO: 5811, as disclosed in TABLE 5 and TABLE 7. Also encompassed herein are rAAVs composed of engineered AAV5 VP2 capsid polypeptides and engineered AAV5 VP3 capsid polypeptides having the sequences disclosed in TABLE 5 and TABLE 7 at the regions in AAV5 VP2 (amino acid residues 445 to 453) and AAV5 VP3 (amino acid residues 389 to 397) corresponding to the amino acids in the AAV5 VP1 581 to 589 region.

As shown in FIG. 5, muscle-tropic AAV candidates showed 4-fold to 11-fold enrichment in muscle tissue relative to other non-muscle tissues compared to wild type AAV9. FIG. 5 is a bar graph showing relative accumulation, expressed as log 2 fold change in muscle accumulation relative to other tissues, of variant AAV capsids comprising 581-589 regions of SEQ ID NO: 4318 (CKEWYVRDR), SEQ ID NO: 4960 (CWREFSFQN), SEQ ID NO: 4371 (CVSLHSISM), SEQ ID NO: 3975 (QAHHYNILN), SEQ ID NO: 5215 (CVRTLQSPD), SEQ ID NO: 4598 (LAWSQLLTP), SEQ ID NO: 4359 (CVATKSRML), SEQ ID NO: 5665 (RQTTYDCLD), SEQ ID NO: 257 (CAHYAQWGK), SEQ ID NO: 344 (CSSGFHLFH), SEQ ID NO: 5607 (QCGFIRLNE), SEQ ID NO: 3528 (TVTHMSQHC), SEQ ID NO: 5155 (CVIWYHKYE), SEQ ID NO: 5363 (GCMCIRHAY), SEQ ID NO: 4633 (MACWKNATE), SEQ ID NO: 2525 (QAGSSFHQA), SEQ ID NO: 386 (CYQFWSQYS), SEQ ID NO: 4970 (CINFIMKLG), SEQ ID NO: 5143 (CWRHNIISP), SEQ ID NO: 5178 (CIKWVDIFP), SEQ ID NO: 2505 (PTQFYGPAF), SEQ ID NO: 3709 (VSASFQQHV), SEQ ID NO: 5056 (CVLNYFQFG), SEQ ID NO: 5017 (CFYKIQHKG), SEQ ID NO: 5040 (CVLRMGTFC), SEQ ID NO: 5161 (CTHMMGKSP), SEQ ID NO: 1128 (GFANFMMNF), SEQ ID NO: 2204 (MTMTATTFG), SEQ ID NO: 4316 (CIRHAQDCY), SEQ ID NO: 5463 (KISPWDGFY), SEQ ID NO: 4944 (CWKWQMMFG), SEQ ID NO: 4955 (CVWSQILGG), SEQ ID NO: 4952 (CVILSTRDK), SEQ ID NO: 3260 (SVSPCFCNS), SEQ ID NO: 384 (CYMPFKMQH), SEQ ID NO: 5038 (CVVWPVLGQ), SEQ ID NO: 2371 (NMTMAHQAS), SEQ ID NO: 289 (CIHSVHFAA), SEQ ID NO: 2985 (RVDAFNRGT), SEQ ID NO: 4508 (GSCLKIAQE), SEQ ID NO: 4292 (CAMLLQAVQ), SEQ ID NO: 4995 (CVGYRVHSP), SEQ ID NO: 4949 (CYHQVFSQG), SEQ ID NO: 5077 (CVGSQLHAM), SEQ ID NO: 2370 (NMNTFGKMN), SEQ ID NO: 5193 (CWSHLYFQT), SEQ ID NO: 4568 (KANEDLKQF), SEQ ID NO: 5206 (CFFYPMSMS), SEQ ID NO: 4215 (LYAFYMSSE), SEQ ID NO: 4841 (SSDQYEEMN), SEQ ID NO: 5092 (CISNYLKQG), SEQ ID NO: 4335 (CPMKHIQDR), SEQ ID NO: 5029 (CSFNIHKHT), SEQ ID NO: 3258 (SVRMFDAQY), SEQ ID NO: 4349 (CSSYLVTAN), SEQ ID NO: 348 (CTASWMSWD), or SEQ ID NO: 2729 (QQQQTNFQN). AAV variants that were generated using machine learning (ML) are denoted with arrows. Accumulation was compared to wild type AAV capsids of AAV9, AAV PHP.B, AAV1, AAV2, and AAV5.

Example 4
Identification of De Novo Variant 581-589 Regions

This example describes identification of de novo variant 581-589 regions using machine learning. Machine learning algorithms were trained using AAV5 variants with CNS tissue tropism identified in EXAMPLE 2 or AAV5 variants with muscle tissue tropism identified in EXAMPLE 3.

Sequences of 581-589 regions that were not identified in the in vivo NHP screen but were predicted by the machine learning analysis to have increased likelihood of either CNS tissue tropism or muscle tropism were generated. After isolating the CNS-specific variants and muscle-specific variants from the in vivo data, novel CNS-specific and muscle-specific sequences were first generated by randomly sampling the amino acid residues at each position of the corresponding population of variants. This created a list of one million variants for each tissue type based on the positional amino acid frequencies observed in the other tissue-specific variants. These one million variants were then tested using random forests classifiers. This calculated a score of predicted tissue-specificity for each variant. The top 300 highest-ranking variants for each tissue type were selected for secondary screening.

TABLE 3 provides sequences of the top 300 581-589 regions, corresponding to residues 581 to 589 of VP1, for variant AAV VP capsid polypeptides that were predicted to be CNS tissue-tropic. The present disclosure, thus, provides for rAAVs composed of engineered AAV5 VP1 capsid polypeptides having a SEQ ID NO: 2, wherein the X¹to X⁹region is any one of SEQ ID NO: 3938-SEQ ID NO: 4237, as disclosed in TABLE 3. Also encompassed herein are rAAVs composed of engineered AAV5 VP2 capsid polypeptides and engineered AAV5 VP3 capsid polypeptides having the sequences disclosed in TABLE 3 at the regions in AAV5 VP2 (amino acid residues 445 to 453) and AAV5 VP3 (amino acid residues 389 to 397) corresponding to the amino acids in the AAV5 VP1 581 to 589 region. FIG. 9 shows the CNS prediction score for CNS targeted candidates identified from multiple non-human primates (NHPs), multiple samples, observational enrichment, frequency enrichment, sequence similarity, or machine learning. CNS targeted candidates generated using machine learning had, on average, higher CNS prediction scores than candidates identified from other sources.

TABLE 6 provides sequences of the top 300 581-589 regions, corresponding to residues 581 to 589 of VP1, for variant AAV VP capsid polypeptides that were predicted to be muscle-tropic. The present disclosure, thus, provides for rAAVs composed of engineered AAV5 VP1 capsid polypeptides having a SEQ ID NO: 2, wherein the X¹to X⁹region is any one of SEQ ID NO: 4934-SEQ ID NO: 5233, as disclosed in TABLE 6. Also encompassed herein are rAAVs composed of engineered AAV5 VP2 capsid polypeptides and engineered AAV5 VP3 capsid polypeptides having the sequences disclosed in TABLE 6 at the regions in AAV5 VP2 (amino acid residues 445 to 453) and AAV5 VP3 (amino acid residues 389 to 397) corresponding to the amino acids in the AAV5 VP1 581 to 589 region. FIG. 8A shows a bar graph of the proportion of candidates from different library sub-sets that were identified as muscle-specific in a secondary screen. Muscle candidates generated using machine learning (“Muscle ML Synthetic”) contained a higher proportion of muscle-specific sequences than candidates identified in muscle in a primary screen (“Muscle Observed”), CNS targeted sequences, or negative control sequences. Muscle candidate sequences generated using machine learning were five times more likely to be heart- and/or skeletal muscle-specific than muscle candidates identified in the primary screen and were 20 times more likely to be heart- and/or skeletal muscle-specific than CNS targeted variants. Candidates were classified as muscle-specific if they were >2-fold enriched in heart and/or skeletal muscle relative to all other tissues and viral input (FDR <0.1, permutations with α-RRA to account for consistency across multiple barcodes for each capsid). FIG. 8B shows a box and whisker plot comparing the predicted probability of muscle specificity from primary screen for candidates that were (“yes”) or were not (“no”) identified as muscle-specific in the secondary screen. Candidates were classified as muscle-specific if they were >2-fold enriched in heart and/or skeletal muscle relative to all other tissues and viral input (FDR <0.1, permutations with α-RRA to account for consistency across multiple barcodes for each capsid).

Example 5
Secondary Screen of Recombinant AAV Capsid Candidates with Variant 581-589 Regions

This example describes a secondary screen of recombinant AAV capsid candidates with variant 581-589 regions. Six thousand AAV capsid variants identified or generated using the methods described in EXAMPLE 1-EXAMPLE 4 were systemically administered to non-human primate. Fifteen wild type spike-in controls representing wild type AAV1, AAV2, AAV5, AAV9, and AAV PHP.B, each at 1×, 10×, and 100× concentrations, along with 100 AAV capsids with stop codons in the 581-589 region, were also administered. Tissue-specific accumulation and tissue-specific transduction of each capsid were evaluated by quantifying capsid DNA and RNA, respectively, in tissues of interest. Barcode sequences for each capsid were extracted from raw sequencing data after filtering based on sequencing quality (Q30) and exact match to the reference library of 48,090 variants representing the 6,000 variant capsid sequences and 15 wild type spike-in controls.

Enrichment of variant capsids relative to wild type AAVs was quantified for the barcodes associated with each capsid. Enrichment was determined for each capsid for both the RNA and DNA fractions from each of the analyzed tissues, as well as for the input viral library injected into each primate. DNA counts were representative of capsid accumulation, and RNA counts were representative of viral transduction in the tissue. This analysis collectively formed 12 distinct analysis strategies to assess the differential expression of capsids. The different analytical outputs were consolidated into a single z-score to evaluate enrichment of each capsid in a tissue of interest relative to the overall distribution of the capsid differential expression data. The z-score was calculated as follows:

$Z_{i} = \frac{X_{i} - mean (X)}{sd (X)}$

where X_iis the enrichment of capsid, i, estimated as the median of the log 2(fold change) across capsid barcodes and tissue comparisons. Superior capsid variants were selected based on parameters such as DNA or RNA abundance in a tissue of interest.

Example 6
Secondary Screen Results to Identify CNS Tissue-Tropic Recombinant AAV Capsids

This example describes secondary screen results to identify CNS tissue-tropic recombinant AAV capsids. The secondary screen and enrichment analysis were performed as described in EXAMPLE 5. Recombinant AAV capsids containing variant 581-589 regions that were enriched in CNS tissue and exhibited functional transduction in CNS tissue were identified. TABLE 9 provides enrichment data for the 581-589 region sequences of the top 100 CNS tissue-tropic AAV capsids, as identified in the secondary screen. Superior capsids were selected based on parameters including relative DNA and RNA abundance in CNS tissue, as well as additional metrics shown in TABLE 9, including average of z-score, liver DNA fold change over AAV5, liver RNA fold change over AAV5, liver DNA fold change over AAV9, and liver RNA fold change over AAV9.

TABLE 9

Secondary Screen Results for CNS Tissue-Tropic Capsids

SEQ ID
DNA FC
RNA FC
DNA FC
RNA FC
DNA FC
RNA FC
Z
Liver DNA
Liver RNA
Liver DNA
Liver RNA

NO
Input
Input
AAV9
AAV9
AAV5
AAV5
Ave.
FC AAV5
FC AAV5
FC AAV9
FC AAV9

18
191.25
274.64
1164.65
984.41
33.20
112.33
8.61
1.34
1.22
2.78
1.25

3472
101.65
174.44
619.05
625.26
17.65
71.35
6.94
1.30
0.62
2.70
0.64

262
74.65
175.05
454.62
627.45
12.96
71.60
6.01
2.15
1.01
4.45
1.04

3306
71.12
123.11
433.11
441.25
12.35
50.35
7.54
1.08
0.49
2.24
0.51

2028
65.57
139.59
399.28
500.34
11.38
57.09
8.53
0.87
0.35
1.79
0.36

2791
64.72
112.35
394.14
402.71
11.24
45.95
6.10
0.72
1.23
1.49
1.27

424
61.14
110.28
372.33
395.30
10.62
45.11
8.12
0.94
0.45
1.95
0.46

2536
57.12
117.60
347.86
421.52
9.92
48.10
5.71
1.04
0.24
2.16
0.25

1971
55.09
71.39
335.48
255.89
9.56
29.20
6.10
0.89
0.62
1.84
0.64

415
51.53
65.45
313.81
234.59
8.95
26.77
6.68
1.64
1.52
3.40
1.56

3846
48.77
1.79
297.02
6.40
8.47
0.73
2.20
1.21
0.63
2.50
0.65

3283
45.30
119.98
275.88
430.06
7.87
49.07
5.43
0.89
0.29
1.84
0.30

1956
44.62
131.11
271.74
469.93
7.75
53.62
6.06
0.98
0.29
2.04
0.30

3297
37.74
40.08
229.83
143.66
6.55
16.39
4.90
1.16
0.77
2.41
0.79

4545
32.45
39.08
197.63
140.07
5.63
15.98
3.50
0.98
0.43
2.03
0.45

2661
31.72
63.55
193.14
227.79
5.51
25.99
4.80
2.18
1.24
4.51
1.27

1576
29.64
62.61
180.47
224.43
5.15
25.61
3.54
1.40
0.99
2.91
1.02

425
27.64
57.62
168.33
206.52
4.80
23.57
5.94
0.82
0.54
1.70
0.56

709
26.54
118.04
161.61
423.11
4.61
48.28
6.36
0.73
1.02
1.52
1.05

2168
26.46
1.72
161.14
6.17
4.59
0.70
−0.67
1.53
0.90
3.18
0.93

54
24.61
81.85
149.88
293.39
4.27
33.48
5.50
0.78
0.89
1.61
0.92

429
24.47
29.17
149.03
104.55
4.25
11.93
4.44
0.78
0.53
1.62
0.55

708
23.66
25.56
144.07
91.62
4.11
10.45
5.65
0.61
0.64
1.26
0.66

428
23.48
38.00
143.01
136.19
4.08
15.54
4.76
0.86
0.75
1.79
0.77

4119
22.63
3.10
137.79
11.11
3.93
1.27
−0.09
1.43
0.60
2.96
0.62

3906
22.06
10.52
134.37
37.69
3.83
4.30
3.41
1.05
0.68
2.17
0.71

2456
21.87
4.38
133.18
15.72
3.80
1.79
0.68
0.93
0.14
1.92
0.15

2278
20.11
0.18
122.45
0.65
3.49
0.07
0.93
0.84
0.28
1.74
0.29

5006
19.89
3.13
121.13
11.21
3.45
1.28
0.20
1.33
0.55
2.76
0.57

426
19.78
27.90
120.48
100.02
3.43
11.41
3.48
0.94
0.35
1.95
0.36

307
18.65
19.51
113.58
69.94
3.24
7.98
2.91
1.68
0.95
3.49
0.98

5155
17.70
0.18
107.81
0.63
3.07
0.07
−0.76
1.17
0.99
2.42
1.02

2640
17.65
0.18
107.51
0.63
3.07
0.07
−0.40
1.54
0.98
3.18
1.02

4317
17.21
25.30
104.82
90.68
2.99
10.35
−0.67
1.16
1.15
2.41
1.19

1145
17.18
4.79
104.62
17.18
2.98
1.96
0.24
1.11
0.43
2.29
0.44

430
17.08
112.52
104.03
403.32
2.97
46.02
5.26
0.85
0.20
1.76
0.20

885
16.85
26.78
102.60
95.98
2.93
10.95
4.49
0.80
0.41
1.65
0.43

23
15.09
5.08
91.89
18.22
2.62
2.08
1.65
1.57
0.79
3.25
0.82

103
14.93
0.37
90.91
1.33
2.59
0.15
2.04
1.02
0.24
2.11
0.25

22
14.24
7.55
86.72
27.06
2.47
3.09
1.16
1.76
0.90
3.65
0.93

4031
14.08
0.08
85.76
0.29
2.44
0.03
−0.83
1.19
0.53
2.46
0.55

1008
13.74
0.20
83.69
0.70
2.39
0.08
0.66
1.40
1.62
2.89
1.67

4790
13.33
0.07
81.15
0.24
2.31
0.03
−0.16
1.34
1.38
2.78
1.43

2522
13.08
0.14
79.63
0.50
2.27
0.06
−0.05
1.28
0.97
2.65
1.00

1432
13.01
2.52
79.26
9.02
2.26
1.03
2.03
0.97
0.86
2.00
0.89

2914
12.99
3.03
79.13
10.84
2.26
1.24
−0.48
1.44
0.54
2.99
0.56

3935
12.91
15.87
78.65
56.88
2.24
6.49
3.37
0.80
0.25
1.65
0.26

5042
12.78
0.09
77.84
0.33
2.22
0.04
0.60
0.82
0.27
1.70
0.28

2865
12.37
22.18
75.33
79.48
2.15
9.07
2.54
0.94
0.51
1.95
0.53

4264
12.27
4.51
74.69
16.17
2.13
1.84
−0.88
2.07
2.54
4.29
2.62

964
11.88
8.67
72.35
31.08
2.06
3.55
1.73
1.03
1.54
2.13
1.59

1268
11.85
22.35
72.18
80.12
2.06
9.14
3.33
0.95
0.91
1.97
0.94

5065
11.85
5.41
72.16
19.38
2.06
2.21
−0.20
1.06
0.81
2.21
0.84

706
11.55
13.76
70.31
49.31
2.00
5.63
2.86
0.78
0.31
1.61
0.32

4704
11.23
2.07
68.39
7.40
1.95
0.84
−0.08
1.13
0.72
2.35
0.74

569
11.17
81.65
68.00
292.66
1.94
33.40
4.36
0.74
0.78
1.53
0.80

2994
11.01
0.10
67.06
0.35
1.91
0.04
0.62
2.01
1.32
4.16
1.36

829
10.81
0.53
65.82
1.90
1.88
0.22
0.96
0.99
0.64
2.05
0.66

1171
10.78
9.90
65.68
35.48
1.87
4.05
2.66
1.09
1.09
2.26
1.13

1041
10.60
0.16
64.53
0.56
1.84
0.06
0.31
0.85
0.73
1.76
0.76

5070
10.34
3.89
62.97
13.95
1.80
1.59
−0.20
1.26
0.56
2.61
0.58

139
10.29
3.51
62.66
12.57
1.79
1.43
−0.44
0.97
0.69
2.01
0.72

3304
10.15
0.12
61.81
0.44
1.76
0.05
−0.20
1.30
0.92
2.70
0.95

2431
9.87
2.16
60.13
7.73
1.71
0.88
0.73
1.11
0.84
2.31
0.87

4288
9.87
14.80
60.10
53.06
1.71
6.05
−1.21
1.57
1.21
3.25
1.25

5795
9.85
0.24
59.96
0.86
1.71
0.10
1.01
0.91
0.56
1.89
0.58

1300
9.79
8.49
59.64
30.45
1.70
3.47
0.49
0.86
0.23
1.78
0.23

724
9.78
15.72
59.58
56.33
1.70
6.43
3.70
0.74
0.17
1.53
0.17

1770
9.77
0.07
59.47
0.26
1.70
0.03
0.37
1.43
0.49
2.96
0.50

2830
9.63
3.28
58.63
11.77
1.67
1.34
0.44
1.26
0.91
2.60
0.94

1972
9.56
3.95
58.19
14.16
1.66
1.62
0.18
0.95
0.36
1.96
0.37

1649
9.53
0.26
58.02
0.93
1.65
0.11
−0.19
1.86
2.25
3.86
2.33

2515
9.50
0.05
57.88
0.20
1.65
0.02
0.42
0.76
0.87
1.58
0.89

2849
9.40
0.05
57.27
0.17
1.63
0.02
−0.06
0.90
0.63
1.86
0.65

3796
9.34
0.05
56.90
0.17
1.62
0.02
0.20
1.13
0.43
2.35
0.44

5815
9.33
0.42
56.83
1.50
1.62
0.17
−0.48
2.37
1.72
4.90
1.78

118
9.27
0.33
56.48
1.19
1.61
0.14
1.79
0.99
0.23
2.05
0.24

4766
9.22
0.12
56.16
0.44
1.60
0.05
0.82
1.12
1.62
2.32
1.67

770
9.17
0.19
55.84
0.67
1.59
0.08
0.56
1.50
0.15
3.11
0.16

1069
9.14
7.40
55.67
26.53
1.59
3.03
2.12
1.07
2.73
2.22
2.82

3061
8.90
0.36
54.18
1.29
1.54
0.15
0.67
1.17
0.42
2.41
0.43

4290
8.86
3.51
53.97
12.58
1.54
1.44
−1.31
0.95
0.59
1.98
0.61

4261
8.83
0.15
53.75
0.52
1.53
0.06
0.65
0.72
0.24
1.48
0.25

4239
8.78
0.47
53.44
1.68
1.52
0.19
0.00
1.11
0.72
2.31
0.75

3478
8.68
5.43
52.83
19.45
1.51
2.22
−0.31
1.32
1.07
2.73
1.11

1887
8.65
39.87
52.71
142.90
1.50
16.31
3.43
1.39
0.89
2.88
0.92

2671
8.57
4.68
52.17
16.79
1.49
1.92
0.67
1.06
0.60
2.19
0.62

256
8.38
3.78
51.03
13.55
1.45
1.55
−0.26
1.33
0.59
2.76
0.61

1256
8.35
6.91
50.87
24.78
1.45
2.83
−0.63
1.82
1.94
3.77
2.00

719
8.31
71.34
50.61
255.71
1.44
29.18
3.02
1.02
0.93
2.11
0.96

1448
8.27
0.14
50.38
0.49
1.44
0.06
0.43
0.85
0.85
1.76
0.87

280
8.25
4.62
50.23
16.57
1.43
1.89
0.22
1.06
0.41
2.20
0.42

4338
8.21
13.98
49.98
50.11
1.42
5.72
−0.64
1.17
0.68
2.43
0.71

5037
8.09
3.86
49.29
13.83
1.41
1.58
−0.89
1.29
0.43
2.66
0.44

1901
8.01
0.69
48.80
2.46
1.39
0.28
−0.82
1.42
1.05
2.93
1.09

438
7.99
6.30
48.67
22.59
1.39
2.58
−0.31
1.10
0.77
2.27
0.79

2834
7.96
9.98
48.50
35.77
1.38
4.08
1.10
1.98
0.92
4.09
0.95

5491
7.96
1.42
48.45
5.10
1.38
0.58
−0.32
3.00
3.06
6.22
3.16

4591
7.94
11.88
48.35
42.57
1.38
4.86
−0.26
1.17
0.92
2.43
0.95

4936
7.94
1.98
48.33
7.08
1.38
0.81
−0.42
1.23
0.91
2.54
0.94

As shown in TABLE 9, “DNA FC Input” represents the fold change of counts per million (CPM) values, normalized by Trimmed Mean of M-values (TMM) of capsid DNA relative to viral input in CNS tissue. “RNA FC Input” represents the fold change of CPM values (normalized by TMM) of capsid RNA relative to viral input in CNS tissue. “DNA FC AAV9” represents the fold change of CPM values (normalized by TMM and to viral input) of capsid DNA relative to wild-type AAV9 in CNS tissue. “RNA FC AAV9” represents the fold change of CPM values (normalized by TMM and to viral input) of capsid RNA relative to wild-type AAV9 in CNS tissue. “DNA FC AAV5” represents the fold change of CPM values (normalized by TMM and to viral input) of capsid DNA relative to wild-type AAV5 in CNS tissue. “RNA FC AAV5” represents the fold change of CPM values (normalized by TMM and to viral input) of capsid RNA relative to wild-type AAV5 in CNS tissue. “Z. Ave.” represents the average of z-scores calculated from the enrichment values of 12 distinct differential expression analyses comparing the tissue of interest to all other tissues. “Liver DNA FC AAV5” represents the fold change of CPM values (normalized by TMM and to viral input) of capsid DNA relative to wild-type AAV5 in the liver. “Liver RNA FC AAV5” represents the fold change of CPM values (normalized by TMM and to viral input) of capsid RNA relative to wild-type AAV5 in the liver. “Liver DNA FC AAV9” represents the fold change of CPM values (normalized by TMM and to viral input) of capsid DNA relative to wild-type AAV9 in the liver. “Liver RNA FC AAV9” represents the fold change of CPM values (normalized by TMM and to viral input) of capsid RNA relative to wild-type AAV9 in the liver.

The average Z-score of CNS tissue enrichment for the screened capsid variants, plotted in order of capsid rank, is shown in FIG. 18. High scoring variants corresponding to SEQ ID NO: 18, SEQ ID NO: 2028, SEQ ID NO: 424, SEQ ID NO: 3306, SEQ ID NO: 3472, SEQ ID NO: 415, SEQ ID NO: 709, SEQ ID NO: 1971, SEQ ID NO: 2791, SEQ ID NO: 1956, SEQ ID NO: 262, SEQ ID NO: 425, SEQ ID NO: 2536, SEQ ID NO: 708, SEQ ID NO: 54, SEQ ID NO: 3283, SEQ ID NO: 430, SEQ ID NO: 3297, SEQ ID NO: 2661, SEQ ID NO: 428, SEQ ID NO: 885, SEQ ID NO: 429, SEQ ID NO: 569, SEQ ID NO: 724, SEQ ID NO: 1576, SEQ ID NO: 4545, SEQ ID NO: 426, SEQ ID NO: 1887, SEQ ID NO: 3906, and SEQ ID NO: 3935 are emphasized. The high scoring variant capsid, corresponding to the outlined points in the upper left quadrant, were ranked consistently highly across multiple analysis strategies and ranked consistently higher than wild type AAV5 (wtAAV5) and wild type AAV9 (wtAAV9).

Example 7
Secondary Screen Results to Identify Cardiac Tissue-Tropic Recombinant AAV Capsids

This example describes secondary screen results to identify cardiac tissue-tropic recombinant AAV capsids. The secondary screen and enrichment analysis were performed as described in EXAMPLE 5. Recombinant AAV capsids containing variant 581-589 regions that were enriched in cardiac tissue (including heart tissue and aorta tissue) and exhibited functional transduction in cardiac tissue were identified. TABLE 10 provides enrichment data for the 581-589 region sequences of the top 100 cardiac tissue-tropic AAV capsids, as identified in the secondary screen, and the 15 wild type spike-in controls. Superior capsids were selected based on parameters including relative DNA and RNA abundance in cardiac tissue, as well as additional metrics shown in TABLE 10, including liver DNA fold change over AAV5, liver RNA fold change over AAV5, liver DNA fold change over AAV9, and liver RNA fold change over AAV9.

TABLE 10

Secondary Screen Results for Cardiac Tissue-Tropic Capsids

DNA
RNA
DNA
RNA
DNA
RNA
Liver
Liver
Liver

SEQ
FC
FC
FC
FC
FC
FC
DNA FC
RNA FC
DNA FC

ID NO
Input
Input
AAV9
AAV9
AAV5
AAV5
AAV5
AAV5
AAV9

348
9.32
10.61
62.21
47.20
12.56
14.17
2.85
7.14
2.94

2536
7.74
2.24
51.67
9.97
10.43
2.99
0.24
2.16
0.25

5607
6.03
2.46
40.23
10.95
8.12
3.29
18.66
32.87
19.25

1576
5.53
1.38
36.89
6.15
7.45
1.84
0.99
2.91
1.02

4955
5.37
2.20
35.86
9.78
7.24
2.93
0.38
2.70
0.39

5017
5.22
2.68
34.87
11.94
7.04
3.58
1.94
2.76
2.00

4349
5.12
2.31
34.20
10.28
6.90
3.08
0.46
2.40
0.47

4964
4.95
2.55
33.04
11.35
6.67
3.41
1.28
3.69
1.32

293
4.92
4.10
32.83
18.24
6.63
5.48
0.79
3.06
0.81

4314
4.78
2.66
31.91
11.83
6.44
3.55
0.49
2.21
0.50

4995
4.74
3.17
31.66
14.08
6.39
4.23
0.73
1.95
0.76

4366
4.50
1.99
30.03
8.83
6.06
2.65
1.20
2.86
1.24

4961
4.47
1.64
29.86
7.30
6.03
2.19
1.02
2.25
1.05

4952
4.42
4.11
29.52
18.29
5.96
5.49
0.96
2.80
0.99

5075
4.41
3.45
29.42
15.34
5.94
4.60
1.52
2.70
1.57

4354
4.40
2.03
29.39
9.02
5.93
2.71
1.91
1.81
1.97

5060
4.39
2.88
29.29
12.82
5.91
3.85
0.41
2.62
0.42

5138
4.32
5.91
28.86
26.27
5.82
7.89
0.57
2.04
0.59

5206
4.27
3.04
28.50
13.53
5.75
4.06
0.63
2.58
0.65

4288
4.23
3.18
28.25
14.14
5.70
4.24
1.21
3.25
1.25

5283
4.20
2.02
28.00
8.98
5.65
2.70
5.79
11.85
5.97

5092
4.18
1.66
27.89
7.39
5.63
2.22
0.36
1.88
0.37

4059
4.12
1.97
27.50
8.76
5.55
2.63
0.72
2.34
0.75

4295
4.07
2.17
27.15
9.66
5.48
2.90
1.19
2.89
1.23

5030
4.04
3.17
26.99
14.08
5.45
4.23
3.24
8.80
3.34

4938
4.03
2.25
26.92
10.02
5.43
3.01
0.68
2.83
0.70

2661
4.02
1.99
26.86
8.85
5.42
2.66
1.24
4.51
1.27

5215
4.02
2.68
26.82
11.94
5.41
3.58
1.53
5.08
1.58

4960
4.00
1.79
26.69
7.97
5.39
2.39
0.59
2.26
0.61

4969
3.95
1.63
26.38
7.26
5.33
2.18
0.29
2.49
0.30

5023
3.93
1.43
26.22
6.36
5.29
1.91
0.46
2.39
0.48

4934
3.92
1.92
26.15
8.54
5.28
2.56
0.85
2.00
0.88

4545
3.89
1.82
26.00
8.12
5.25
2.44
0.43
2.03
0.45

4963
3.89
1.47
25.95
6.53
5.24
1.96
3.75
10.01
3.87

4363
3.88
2.40
25.93
10.67
5.23
3.20
0.45
2.20
0.46

5159
3.79
2.96
25.30
13.15
5.11
3.95
1.03
2.57
1.06

4973
3.77
1.86
25.17
8.28
5.08
2.48
0.59
2.63
0.61

5157
3.68
1.60
24.58
7.13
4.96
2.14
5.00
13.10
5.15

4972
3.63
1.62
24.24
7.21
4.89
2.16
0.56
2.57
0.58

4345
3.63
1.68
24.21
7.46
4.89
2.24
1.11
2.15
1.14

5039
3.60
1.83
24.04
8.13
4.85
2.44
0.97
2.22
1.00

5163
3.59
4.67
23.94
20.78
4.83
6.24
0.78
2.10
0.80

5040
3.57
1.27
23.86
5.64
4.81
1.69
0.94
3.36
0.96

4304
3.55
2.44
23.71
10.87
4.79
3.26
0.49
2.44
0.50

18
3.52
2.41
23.50
10.73
4.74
3.22
1.22
2.78
1.25

5193
3.52
6.22
23.48
27.69
4.74
8.31
1.00
2.21
1.03

5077
3.51
1.71
23.42
7.59
4.73
2.28
2.91
1.91
3.01

5814
3.51
1.79
23.42
7.97
4.73
2.39
0.88
2.44
0.91

386
3.51
3.26
23.40
14.49
4.72
4.35
0.77
2.30
0.80

5081
3.49
1.81
23.30
8.04
4.70
2.41
0.86
2.79
0.89

308
3.48
1.69
23.25
7.50
4.69
2.25
0.38
2.37
0.39

22
3.46
1.30
23.12
5.76
4.67
1.73
0.90
3.65
0.93

4338
3.42
3.00
22.83
13.34
4.61
4.00
0.68
2.43
0.71

3846
3.41
1.25
22.79
5.58
4.60
1.68
0.63
2.50
0.65

278
3.40
2.04
22.68
9.07
4.58
2.72
0.92
2.90
0.95

5127
3.39
1.55
22.66
6.91
4.57
2.07
0.70
2.45
0.72

5029
3.39
1.80
22.62
8.00
4.57
2.40
0.42
2.51
0.43

5037
3.38
1.50
22.54
6.67
4.55
2.00
0.43
2.66
0.44

5143
3.33
1.67
22.21
7.42
4.48
2.23
1.65
3.58
1.70

4983
3.31
1.90
22.09
8.46
4.46
2.54
0.60
3.28
0.62

4361
3.31
1.50
22.08
6.67
4.46
2.00
0.86
1.63
0.88

4317
3.30
3.82
22.00
17.01
4.44
5.10
1.15
2.41
1.19

5080
3.27
2.62
21.80
11.67
4.40
3.50
1.05
3.25
1.09

23
3.26
0.85
21.76
3.78
4.39
1.14
0.79
3.25
0.82

4343
3.25
1.60
21.67
7.11
4.37
2.13
0.91
2.77
0.94

4986
3.25
3.11
21.66
13.85
4.37
4.16
0.92
1.96
0.94

4311
3.24
1.76
21.65
7.85
4.37
2.36
2.50
6.05
2.58

5102
3.22
1.90
21.49
8.44
4.34
2.53
0.65
2.60
0.67

5280
3.21
3.51
21.45
15.61
4.33
4.68
2.00
6.31
2.06

5185
3.19
1.80
21.32
8.00
4.30
2.40
0.47
2.63
0.49

964
3.18
1.42
21.26
6.33
4.29
1.90
1.54
2.13
1.59

4935
3.17
1.99
21.18
8.85
4.27
2.65
1.00
2.81
1.03

4379
3.15
2.48
21.00
11.02
4.24
3.31
0.61
1.85
0.63

5052
3.13
6.63
20.92
29.49
4.22
8.85
1.14
2.00
1.17

5027
3.13
2.34
20.91
10.40
4.22
3.12
0.93
2.41
0.96

5173
3.12
1.46
20.86
6.51
4.21
1.95
2.28
5.66
2.35

5055
3.12
2.16
20.81
9.61
4.20
2.88
1.05
2.09
1.08

5123
3.11
1.56
20.77
6.96
4.19
2.09
1.05
2.46
1.08

4335
3.09
1.86
20.65
8.29
4.17
2.49
1.06
2.53
1.10

4936
3.09
1.58
20.64
7.03
4.17
2.11
0.91
2.54
0.94

5208
3.09
2.28
20.60
10.12
4.16
3.04
0.43
2.13
0.44

4633
3.07
1.31
20.52
5.83
4.14
1.75
0.70
1.81
0.72

4359
3.06
1.64
20.42
7.30
4.12
2.19
1.00
2.44
1.03

289
3.05
1.43
20.39
6.35
4.11
1.90
0.60
1.93
0.62

4353
3.02
1.67
20.17
7.44
4.07
2.23
0.24
2.17
0.25

4316
3.02
1.57
20.13
7.00
4.06
2.10
0.12
1.93
0.12

5210
3.01
1.56
20.12
6.95
4.06
2.09
0.99
2.26
1.02

4949
3.00
2.82
20.03
12.56
4.04
3.77
0.84
1.89
0.87

4947
2.99
1.57
19.94
7.00
4.02
2.10
0.83
2.52
0.86

2838
2.98
1.35
19.87
6.01
4.01
1.80
0.69
2.52
0.71

378
2.97
1.75
19.81
7.77
4.00
2.33
0.57
2.70
0.58

5013
2.96
2.12
19.77
9.45
3.99
2.84
0.92
1.87
0.94

5278
2.96
1.90
19.73
8.47
3.98
2.54
0.98
3.14
1.01

1471
2.95
0.98
19.71
4.37
3.98
1.31
0.86
2.15
0.89

4346
2.94
2.93
19.63
13.01
3.96
3.91
0.59
2.13
0.61

297
2.94
1.15
19.61
5.12
3.96
1.54
0.36
2.22
0.37

4993
2.93
1.85
19.58
8.24
3.95
2.47
0.54
2.63
0.56

384
2.91
1.23
19.46
5.49
3.93
1.65
1.35
3.15
1.40

5062
2.91
1.51
19.41
6.73
3.92
2.02
0.94
2.22
0.96

4945
2.90
1.50
19.39
6.67
3.91
2.00
0.96
2.42
0.99

As shown in TABLE 10, “DNA FC Input” represents the fold change of counts per million (CPM) values, normalized by Trimmed Mean of M-values (TMM) of capsid DNA relative to viral input in cardiac muscle tissue. “RNA FC Input” represents the fold change of CPM values (normalized by TMM) of capsid RNA relative to viral input in cardiac muscle tissue. “DNA FC AAV9” represents the fold change of CPM values (normalized by TMM and to viral input) of capsid DNA relative to wild-type AAV9 in cardiac muscle tissue. “RNA FC AAV9” represents the fold change of CPM values (normalized by TMM and to viral input) of capsid RNA relative to wild-type AAV9 in cardiac muscle tissue. “DNA FC AAV5” represents the fold change of CPM values (normalized by TMM and to viral input) of capsid DNA relative to wild-type AAV5 in cardiac muscle tissue. “RNA FC AAV5” represents the fold change of CPM values (normalized by TMM and to viral input) of capsid RNA relative to wild-type AAV5 in cardiac muscle tissue. “Liver DNA FC AAV5” represents the fold change of CPM values (normalized by TMM and to viral input) of capsid DNA relative to wild-type AAV5 in the liver. “Liver RNA FC AAV5” represents the fold change of CPM values (normalized by TMM and to viral input) of capsid RNA relative to wild-type AAV5 in the liver. “Liver DNA FC AAV9” represents the fold change of CPM values (normalized by TMM and to viral input) of capsid DNA relative to wild-type AAV9 in the liver. “Liver RNA FC AAV9” represents the fold change of CPM values (normalized by TMM and to viral input) of capsid RNA relative to wild-type AAV9 in the liver.

Example 8
Secondary Screen Results to Identify Skeletal Muscle Tissue-Tropic Recombinant AAV Capsids

This example describes secondary screen results to identify skeletal muscle tissue-tropic recombinant AAV capsids. The secondary screen and enrichment analysis were performed as described in EXAMPLE 5. Recombinant AAV capsids containing variant 581-589 regions that were enriched in skeletal muscle tissue and exhibited functional transduction in skeletal muscle tissue were identified. TABLE 11 provides enrichment data for the 581-589 region sequences of the top 100 skeletal muscle tissue-tropic AAV capsids, as identified in the secondary screen, and the 15 wild type spike-in controls. Superior capsids were selected based on parameters including relative DNA and RNA abundance in skeletal muscle tissue, as well as additional metrics shown in TABLE 11, including liver DNA fold change over AAV5, liver RNA fold change over AAV5, liver DNA fold change over AAV9, and liver RNA fold change over AAV9.

TABLE 11

Secondary Screen Results for Skeletal Muscle Tissue-Tropic Capsids

DNA
RNA
DNA
RNA
DNA
RNA
Liver
Liver
Liver

SEQ
FC
FC
FC
FC
FC
FC
DNA FC
RNA FC
DNA FC

ID NO
Input
Input
AAV9
AAV9
AAV5
AAV5
AAV5
AAV5
AAV9

3472
7.59
29.12
34.42
48.42
8.24
4.67
0.62
2.70
0.64

3297
6.62
0.29
30.01
0.49
7.18
0.05
0.77
2.41
0.79

2661
5.62
0.14
25.49
0.24
6.10
0.02
1.24
4.51
1.27

4545
5.05
0.27
22.90
0.45
5.48
0.04
0.43
2.03
0.45

348
4.77
0.15
21.66
0.25
5.18
0.02
2.85
7.14
2.94

18
4.37
0.23
19.82
0.38
4.74
0.04
1.22
2.78
1.25

4349
4.18
0.14
18.97
0.23
4.54
0.02
0.46
2.40
0.47

293
4.15
0.55
18.81
0.91
4.50
0.09
0.79
3.06
0.81

5017
4.14
0.19
18.76
0.32
4.49
0.03
1.94
2.76
2.00

4955
4.09
0.14
18.54
0.24
4.44
0.02
0.38
2.70
0.39

4366
3.80
0.23
17.24
0.39
4.13
0.04
1.20
2.86
1.24

5092
3.67
0.28
16.63
0.47
3.98
0.05
0.36
1.88
0.37

210
3.61
0.57
16.38
0.95
3.92
0.09
1.40
3.81
1.44

4059
3.52
0.13
15.95
0.22
3.82
0.02
0.72
2.34
0.75

2536
3.49
0.23
15.85
0.37
3.80
0.04
0.24
2.16
0.25

5206
3.43
0.32
15.55
0.53
3.72
0.05
0.63
2.58
0.65

1971
3.31
0.68
15.00
1.13
3.59
0.11
0.62
1.84
0.64

5607
3.26
0.32
14.79
0.54
3.54
0.05
18.66
32.87
19.25

262
3.24
0.24
14.70
0.40
3.52
0.04
1.01
4.45
1.04

4343
3.20
1.16
14.50
1.93
3.47
0.19
0.91
2.77
0.94

4995
3.16
0.27
14.33
0.45
3.43
0.04
0.73
1.95
0.76

4009
3.12
0.56
14.15
0.93
3.39
0.09
0.71
2.51
0.74

5190
3.09
0.30
14.02
0.50
3.36
0.05
1.15
2.90
1.18

724
3.08
0.19
13.99
0.32
3.35
0.03
0.17
1.53
0.17

4288
3.08
0.09
13.96
0.16
3.34
0.02
1.21
3.25
1.25

4973
3.07
0.79
13.94
1.31
3.34
0.13
0.59
2.63
0.61

4314
3.06
0.18
13.88
0.30
3.32
0.03
0.49
2.21
0.50

1561
3.05
0.60
13.82
1.00
3.31
0.10
1.56
3.81
1.61

5147
3.01
0.15
13.68
0.25
3.27
0.02
0.54
2.71
0.56

4238
3.00
0.10
13.61
0.17
3.26
0.02
0.15
1.86
0.16

4961
2.99
0.64
13.57
1.06
3.25
0.10
1.02
2.25
1.05

4964
2.97
0.23
13.49
0.39
3.23
0.04
1.28
3.69
1.32

5527
2.96
0.87
13.45
1.45
3.22
0.14
1.35
3.25
1.39

3283
2.92
23.44
13.24
38.97
3.17
3.76
0.29
1.84
0.30

5075
2.86
0.12
12.97
0.20
3.11
0.02
1.52
2.70
1.57

4355
2.86
0.24
12.97
0.39
3.10
0.04
1.05
2.51
1.08

5157
2.85
0.19
12.93
0.32
3.09
0.03
5.00
13.10
5.15

5159
2.85
0.33
12.92
0.54
3.09
0.05
1.03
2.57
1.06

4363
2.82
0.53
12.81
0.88
3.07
0.08
0.45
2.20
0.46

4317
2.78
0.54
12.62
0.89
3.02
0.09
1.15
2.41
1.19

4963
2.77
0.18
12.58
0.30
3.01
0.03
3.75
10.01
3.87

5055
2.76
0.21
12.53
0.34
3.00
0.03
1.05
2.09
1.08

5125
2.73
0.25
12.38
0.42
2.96
0.04
0.42
2.88
0.43

4108
2.72
0.43
12.33
0.71
2.95
0.07
0.89
3.87
0.92

308
2.69
0.60
12.22
1.00
2.92
0.10
0.38
2.37
0.39

4311
2.68
0.14
12.17
0.23
2.91
0.02
2.50
6.05
2.58

5060
2.66
0.39
12.06
0.64
2.89
0.06
0.41
2.62
0.42

2948
2.65
0.55
12.01
0.91
2.88
0.09
1.46
4.76
1.51

3821
2.65
0.36
12.00
0.60
2.87
0.06
1.08
2.52
1.12

5138
2.64
422.81
11.96
702.96
2.86
67.77
0.57
2.04
0.59

4354
2.64
0.33
11.96
0.54
2.86
0.05
1.91
1.81
1.97

4353
2.63
0.37
11.93
0.62
2.86
0.06
0.24
2.17
0.25

5030
2.62
0.09
11.88
0.15
2.84
0.01
3.24
8.80
3.34

4346
2.59
0.27
11.77
0.46
2.82
0.04
0.59
2.13
0.61

5283
2.57
0.09
11.65
0.15
2.79
0.01
5.79
11.85
5.97

4632
2.55
0.19
11.55
0.32
2.76
0.03
0.57
2.33
0.59

294
2.53
0.38
11.48
0.63
2.75
0.06
0.92
1.86
0.95

386
2.50
0.20
11.35
0.33
2.72
0.03
0.77
2.30
0.80

4943
2.49
0.35
11.31
0.59
2.71
0.06
0.51
2.85
0.52

1391
2.49
2.22
11.31
3.70
2.71
0.36
0.58
3.88
0.60

5078
2.48
0.43
11.26
0.71
2.69
0.07
0.79
2.10
0.81

3299
2.48
0.87
11.26
1.44
2.69
0.14
0.34
2.91
0.35

2897
2.47
0.55
11.21
0.92
2.68
0.09
1.23
3.90
1.27

1537
2.46
1.24
11.16
2.06
2.67
0.20
0.40
3.00
0.41

5255
2.46
0.36
11.15
0.60
2.67
0.06
1.68
4.90
1.73

141
2.45
0.63
11.14
1.04
2.67
0.10
1.27
3.85
1.31

4345
2.45
0.12
11.12
0.19
2.66
0.02
1.11
2.15
1.14

5038
2.44
0.43
11.06
0.72
2.65
0.07
0.45
2.31
0.47

5153
2.44
0.17
11.06
0.29
2.65
0.03
0.63
3.87
0.65

1181
2.43
0.54
11.03
0.90
2.64
0.09
0.57
3.51
0.58

307
2.42
0.20
10.98
0.33
2.63
0.03
0.95
3.49
0.98

5039
2.42
0.19
10.98
0.31
2.63
0.03
0.97
2.22
1.00

1204
2.41
0.37
10.92
0.61
2.61
0.06
1.13
2.72
1.16

4404
2.41
0.22
10.92
0.36
2.61
0.04
0.25
1.64
0.26

5106
2.40
0.28
10.89
0.47
2.61
0.04
0.59
2.31
0.60

2779
2.40
0.51
10.89
0.85
2.61
0.08
1.56
4.50
1.61

4952
2.40
0.09
10.89
0.15
2.61
0.01
0.96
2.80
0.99

278
2.40
6.97
10.87
11.60
2.60
1.12
0.92
2.90
0.95

1824
2.40
1.12
10.87
1.86
2.60
0.18
1.76
5.93
1.82

5116
2.39
0.23
10.83
0.39
2.59
0.04
0.48
2.64
0.49

4304
2.37
0.16
10.75
0.26
2.57
0.02
0.49
2.44
0.50

4295
2.36
0.06
10.73
0.09
2.57
0.01
1.19
2.89
1.23

3179
2.36
0.41
10.69
0.68
2.56
0.07
0.82
2.94
0.85

3306
2.34
0.48
10.64
0.80
2.55
0.08
0.49
2.24
0.51

98
2.34
0.46
10.64
0.76
2.55
0.07
1.50
3.46
1.54

1872
2.34
0.50
10.63
0.84
2.55
0.08
0.54
2.47
0.56

5037
2.34
0.26
10.62
0.43
2.54
0.04
0.43
2.66
0.44

1268
2.34
0.81
10.60
1.35
2.54
0.13
0.91
1.97
0.94

1634
2.34
2.46
10.60
4.09
2.54
0.39
1.55
3.50
1.60

1060
2.33
0.23
10.57
0.38
2.53
0.04
1.55
2.63
1.59

4941
2.33
0.27
10.55
0.44
2.53
0.04
1.00
3.40
1.04

4361
2.32
0.27
10.53
0.44
2.52
0.04
0.86
1.63
0.88

4981
2.31
0.37
10.49
0.61
2.51
0.06
1.97
4.47
2.03

4359
2.31
0.22
10.47
0.37
2.51
0.04
1.00
2.44
1.03

5274
2.31
0.09
10.47
0.15
2.51
0.01
2.68
5.94
2.76

5816
2.30
0.80
10.44
1.33
2.50
0.13
2.09
5.41
2.16

2842
2.30
0.28
10.44
0.47
2.50
0.05
0.63
2.36
0.65

1934
2.30
28.74
10.43
47.78
2.50
4.61
1.26
2.95
1.30

5817
2.30
10.17
10.43
16.91
2.50
1.63
2.49
5.43
2.57

3998
2.29
0.66
10.41
1.10
2.49
0.11
2.54
5.64
2.62

As shown in TABLE 11, “DNA FC Input” represents the fold change of counts per million (CPM) values, normalized by Trimmed Mean of M-values (TMM) of capsid DNA relative to viral input in skeletal muscle tissue. “RNA FC Input” represents the fold change of CPM values (normalized by TMMN/) of capsid RNA relative to viral input in skeletal muscle tissue. “DNA FC AAV9” represents the fold change of CPM values (normalized by TMM and to viral input) of capsid DNA relative to wild-type AAV9 in skeletal muscle tissue. “RNA FC AAV9” represents the fold change of CPM values (normalized by TMM and to viral input) of capsid RNA relative to wild-type AAV9 in skeletal muscle tissue. “DNA FC AAV5” represents the fold change of CPM values (normalized by TMVM and to viral input) of capsid DNA relative to wild-type AAV5 in skeletal muscle tissue. “RNA FC AAV5” represents the fold change of CPM values (normalized by TMMN and to viral input) of capsid RNA relative to wild-type AAV5 in skeletal muscle tissue. “Liver DNA FC AAV5” represents the fold change of CPM values (normalized by TMMN and to viral input) of capsid DNA relative to wild-type AAV5 in the liver. “Liver RNA FC AAV5” represents the fold change of CPM values (normalized by TMMN and to viral input) of capsid RNA relative to wild-type AAV5 in the liver. “Liver DNA FC AAV9” represents the fold change of CPM values (normalized by TMM and to viral input) of capsid DNA relative to wild-type AAV9 in the liver. “Liver RNA FC AAV9” represents the fold change of CPM values (normalized by TMM and to viral input) of capsid RNA relative to wild-type AAV9 in the liver.

Example 9
Secondary Screen Results to Identify Muscle Tissue-Tropic Recombinant AAV Capsids

This example describes secondary screen results to identify muscle tissue-tropic recombinant AAV capsids. The secondary screen and enrichment analysis were performed as described in EXAMPLE 5. Recombinant AAV capsids containing variant 581-589 regions that were enriched in muscle tissue (including cardiac tissue and skeletal muscle tissue) and exhibited functional transduction in muscle tissue were identified. TABLE 12 provides enrichment data for the 581-589 region sequences of the top 100 muscle tissue-tropic AAV capsids, as identified in the secondary screen, and the 15 wild type spike-in controls. Superior capsids were selected based on parameters including relative DNA and RNA abundance in muscle tissue, as well as additional metrics shown in TABLE 12, including liver DNA fold change over AAV5, liver RNA fold change over AAV5, liver DNA fold change over AAV9, and liver RNA fold change over AAV9.

TABLE 12

Secondary Screen Results for Muscle Tissue-Tropic Capsids

DNA
RNA
DNA
RNA
DNA
RNA
Liver
Liver
Liver

SEQ
FC
FC
FC
FC
FC
FC
DNA FC
RNA FC
DNA FC

ID NO
Input
Input
AAV9
AAV9
AAV5
AAV5
AAV5
AAV5
AAV9

348
8.14
7.62
48.43
22.93
10.32
3.29
2.85
7.14
2.94

2536
6.64
1.67
39.50
5.01
8.42
0.72
0.24
2.16
0.25

5607
5.31
1.85
31.58
5.57
6.73
0.80
18.66
32.87
19.25

4955
5.04
1.61
29.97
4.85
6.39
0.70
0.38
2.70
0.39

5017
4.94
1.97
29.39
5.93
6.27
0.85
1.94
2.76
2.00

4349
4.88
1.69
29.02
5.08
6.19
0.73
0.46
2.40
0.47

293
4.72
3.09
28.06
9.28
5.98
1.33
0.79
3.06
0.81

1576
4.62
1.14
27.51
3.43
5.86
0.49
0.99
2.91
1.02

2661
4.44
1.46
26.39
4.40
5.63
0.63
1.24
4.51
1.27

4964
4.44
1.89
26.39
5.68
5.63
0.82
1.28
3.69
1.32

4314
4.33
1.95
25.78
5.87
5.50
0.84
0.49
2.21
0.50

4995
4.33
2.34
25.77
7.04
5.49
1.01
0.73
1.95
0.76

4366
4.32
1.48
25.68
4.47
5.48
0.64
1.20
2.86
1.24

4545
4.19
1.38
24.94
4.16
5.32
0.60
0.43
2.03
0.45

4961
4.09
1.35
24.32
4.07
5.19
0.58
1.02
2.25
1.05

5206
4.05
2.26
24.10
6.81
5.14
0.98
0.63
2.58
0.65

5092
4.04
1.27
24.06
3.81
5.13
0.55
0.36
1.88
0.37

3472
4.03
9.18
23.96
27.61
5.11
3.96
0.62
2.70
0.64

5075
4.01
2.50
23.83
7.51
5.08
1.08
1.52
2.70
1.57

4059
3.96
1.44
23.57
4.35
5.03
0.62
0.72
2.34
0.75

4354
3.94
1.54
23.46
4.64
5.00
0.67
1.91
1.81
1.97

5060
3.94
2.17
23.43
6.53
5.00
0.94
0.41
2.62
0.42

4288
3.93
2.30
23.39
6.91
4.99
0.99
1.21
3.25
1.25

4952
3.90
2.96
23.19
8.91
4.94
1.28
0.96
2.80
0.99

5138
3.89
125.02
23.11
376.10
4.93
53.95
0.57
2.04
0.59

5283
3.77
1.47
22.45
4.41
4.79
0.63
5.79
11.85
5.97

18
3.74
1.79
22.25
5.38
4.74
0.77
1.22
2.78
1.25

5030
3.67
2.29
21.85
6.88
4.66
0.99
3.24
8.80
3.34

3297
3.67
1.54
21.84
4.63
4.66
0.66
0.77
2.41
0.79

4295
3.63
1.57
21.57
4.72
4.60
0.68
1.19
2.89
1.23

4363
3.61
1.86
21.47
5.61
4.58
0.80
0.45
2.20
0.46

4963
3.60
1.10
21.41
3.31
4.56
0.47
3.75
10.01
3.87

4973
3.59
1.55
21.35
4.67
4.55
0.67
0.59
2.63
0.61

5159
3.55
2.21
21.09
6.63
4.50
0.95
1.03
2.57
1.06

4960
3.50
1.50
20.83
4.52
4.44
0.65
0.59
2.26
0.61

4938
3.47
1.67
20.63
5.03
4.40
0.72
0.68
2.83
0.70

5157
3.47
1.20
20.62
3.61
4.40
0.52
5.00
13.10
5.15

5023
3.45
1.06
20.55
3.18
4.38
0.46
0.46
2.39
0.48

4969
3.40
1.20
20.21
3.61
4.31
0.52
0.29
2.49
0.30

5215
3.34
2.05
19.84
6.17
4.23
0.88
1.53
5.08
1.58

4345
3.32
1.23
19.76
3.70
4.21
0.53
1.11
2.15
1.14

5039
3.29
1.36
19.60
4.09
4.18
0.59
0.97
2.22
1.00

308
3.28
1.38
19.50
4.14
4.16
0.59
0.38
2.37
0.39

4304
3.25
1.79
19.31
5.38
4.12
0.77
0.49
2.44
0.50

386
3.25
2.38
19.30
7.17
4.12
1.03
0.77
2.30
0.80

4934
3.24
1.46
19.29
4.41
4.11
0.63
0.85
2.00
0.88

4343
3.23
1.47
19.23
4.43
4.10
0.64
0.91
2.77
0.94

5163
3.22
3.36
19.14
10.11
4.08
1.45
0.78
2.10
0.80

5081
3.17
1.40
18.86
4.22
4.02
0.61
0.86
2.79
0.89

4317
3.16
2.88
18.81
8.68
4.01
1.24
1.15
2.41
1.19

5814
3.15
1.40
18.72
4.23
3.99
0.61
0.88
2.44
0.91

278
3.14
3.45
18.66
10.37
3.98
1.49
0.92
2.90
0.95

5040
3.12
0.95
18.55
2.85
3.95
0.41
0.94
3.36
0.96

4972
3.11
1.47
18.50
4.41
3.94
0.63
0.56
2.57
0.58

5037
3.11
1.15
18.48
3.45
3.94
0.49
0.43
2.66
0.44

4311
3.10
1.30
18.43
3.91
3.93
0.56
2.50
6.05
2.58

5127
3.08
1.18
18.33
3.56
3.91
0.51
0.70
2.45
0.72

5193
3.07
4.58
18.28
13.79
3.90
1.98
1.00
2.21
1.03

4361
3.05
1.15
18.15
3.45
3.87
0.50
0.86
1.63
0.88

5077
3.04
1.24
18.11
3.73
3.86
0.54
2.91
1.91
3.01

5029
3.03
1.35
18.04
4.05
3.85
0.58
0.42
2.51
0.43

5055
3.03
1.60
18.00
4.82
3.84
0.69
1.05
2.09
1.08

5143
3.01
1.23
17.88
3.69
3.81
0.53
1.65
3.58
1.70

5080
2.98
1.91
17.72
5.74
3.78
0.82
1.05
3.25
1.09

4983
2.94
1.48
17.51
4.46
3.73
0.64
0.60
3.28
0.62

5190
2.93
1.19
17.45
3.59
3.72
0.51
1.15
2.90
1.18

4353
2.92
1.30
17.37
3.91
3.70
0.56
0.24
2.17
0.25

4338
2.92
2.18
17.35
6.56
3.70
0.94
0.68
2.43
0.71

4379
2.91
1.81
17.32
5.44
3.69
0.78
0.61
1.85
0.63

5173
2.89
1.10
17.19
3.32
3.66
0.48
2.28
5.66
2.35

5052
2.88
4.80
17.12
14.43
3.65
2.07
1.14
2.00
1.17

3846
2.87
0.98
17.09
2.94
3.64
0.42
0.63
2.50
0.65

4986
2.87
2.35
17.07
7.07
3.64
1.01
0.92
1.96
0.94

4935
2.87
1.47
17.06
4.42
3.64
0.63
1.00
2.81
1.03

4359
2.86
1.24
17.04
3.72
3.63
0.53
1.00
2.44
1.03

4346
2.85
2.17
16.96
6.52
3.62
0.94
0.59
2.13
0.61

262
2.85
0.84
16.95
2.53
3.61
0.36
1.01
4.45
1.04

289
2.83
1.16
16.84
3.50
3.59
0.50
0.60
1.93
0.62

22
2.82
1.05
16.79
3.16
3.58
0.45
0.90
3.65
0.93

5280
2.82
2.53
16.75
7.61
3.57
1.09
2.00
6.31
2.06

5210
2.82
1.27
16.75
3.82
3.57
0.55
0.99
2.26
1.02

5027
2.81
1.80
16.73
5.41
3.57
0.78
0.93
2.41
0.96

5185
2.80
1.33
16.66
3.99
3.55
0.57
0.47
2.63
0.49

4009
2.79
1.18
16.59
3.56
3.54
0.51
0.71
2.51
0.74

5102
2.78
1.42
16.54
4.26
3.53
0.61
0.65
2.60
0.67

5123
2.78
1.25
16.53
3.75
3.52
0.54
1.05
2.46
1.08

294
2.77
1.07
16.47
3.21
3.51
0.46
0.92
1.86
0.95

4949
2.76
2.20
16.42
6.62
3.50
0.95
0.84
1.89
0.87

5208
2.75
1.66
16.34
4.99
3.48
0.72
0.43
2.13
0.44

4633
2.75
0.97
16.33
2.93
3.48
0.42
0.70
1.81
0.72

4238
2.74
1.01
16.31
3.05
3.48
0.44
0.15
1.86
0.16

5026
2.70
4.30
16.07
12.94
3.43
1.86
1.22
3.16
1.26

23
2.70
0.84
16.04
2.52
3.42
0.36
0.79
3.25
0.82

378
2.69
1.40
16.03
4.22
3.42
0.61
0.57
2.70
0.58

4316
2.68
1.17
15.93
3.51
3.40
0.50
0.12
1.93
0.12

964
2.66
1.29
15.83
3.89
3.38
0.56
1.54
2.13
1.59

5062
2.66
1.14
15.83
3.43
3.37
0.49
0.94
2.22
0.96

4290
2.65
1.22
15.74
3.68
3.35
0.53
0.59
1.98
0.61

4943
2.64
3.00
15.68
9.04
3.34
1.30
0.51
2.85
0.52

4947
2.63
1.25
15.62
3.75
3.33
0.54
0.83
2.52
0.86

As shown in TABLE 12, “DNA FC Input” represents the fold change of counts per million (CPM) values, normalized by Trimmed Mean of M-values (TMM) of capsid DNA relative to viral input in muscle tissue. “RNA FC Input” represents the fold change of CPM values (normalized by TMM) of capsid RNA relative to viral input in muscle tissue. “DNA FC AAV9” represents the fold change of CPM values (normalized by TMM and to viral input) of capsid DNA relative to wild-type AAV9 in muscle tissue. “RNA FC AAV9” represents the fold change of CPM values (normalized by TMM and to viral input) of capsid RNA relative to wild-type AAV9 in muscle tissue. “DNA FC AAV5” represents the fold change of CPM values (normalized by TMM and to viral input) of capsid DNA relative to wild-type AAV5 in muscle tissue. “RNA FC AAV5” represents the fold change of CPM values (normalized by TMM and to viral input) of capsid RNA relative to wild-type AAV5 in muscle tissue. “Liver DNA FC AAV5” represents the fold change of CPM values (normalized by TMM and to viral input) of capsid DNA relative to wild-type AAV5 in the liver. “Liver RNA FC AAV5” represents the fold change of CPM values (normalized by TMM and to viral input) of capsid RNA relative to wild-type AAV5 in the liver. “Liver DNA FC AAV9” represents the fold change of CPM values (normalized by TMM and to viral input) of capsid DNA relative to wild-type AAV9 in the liver. “Liver RNA FC AAV9” represents the fold change of CPM values (normalized by TMM and to viral input) of capsid RNA relative to wild-type AAV9 in the liver.

Example 10
Screening of AAV5 Capsid Variants in Mice

This example describes screening of recombinant AAV capsid candidates with variant 581-589 regions in mice. The goal of this screen was to identify capsid candidates with corresponding tissue specificity in both mice and non-human primates (NHPs) for use as a research tool in mice. The 6,000 AAV capsid variants library using the methods described and administered to NHPs in EXAMPLE 5 was administered to mice. Fifteen wild type spike-in controls representing wild type AAV1, AAV2, AAV5, AAV9, and AAV PHP.B, each at 1×, 10×, and 100× concentrations, along with 100 AAV capsids with stop codons in the 581-589 region, were also administered. Tissue-specific accumulation of each capsid was evaluated by quantifying capsid DNA in heart and CNS tissues. Barcode sequences for each capsid were extracted from raw sequencing data after filtering based on sequencing quality (Q30) and exact match to the reference library of 48,090 variants representing the 6,000 variant capsid sequences and 15 wild type spike-in controls.

Enrichment of variant capsids relative to wild type AAVs was quantified for the barcodes associated with each capsid. Enrichment was determined for each capsid for the DNA fraction from each of the analyzed tissues, as well as for the input viral library injected into each primate and mouse. DNA counts were representative of capsid accumulation

From the secondary screen, 17 capsid variants were identified that exhibited cardiac muscle tissue tropism in both NHPs and in mice. Quantification of cardiac muscle tissue tropism for these 17 variants is provided in TABLE 13. As shown in TABLE 13, capsid variants having 581-589 region sequences of SEQ ID NO: 5027, SEQ ID NO: 4633, SEQ ID NO: 4943, SEQ TD NO: 386, SEQ ID NO: 334, SEQ ID NO: 4309, SEQ ID NO: 4288, SEQ ID NO: 4964, SEQ TD NO: 5017, SEQ ID NO: 5814 SEQ ID NO: 4335, SEQ ID NO:348, SEQ ID NO: 4936, SEQ TD NO: 5206, SEQ ID NO: 4238, SEQ ID NO: 5077, and SEQ ID NO: 5603 were enriched in heart tissue in both NUPs and mice. Additionally, the enrichment of these 17 variants was well correlated between NUPs and mice, as indicated by the median log 2 fold change calculated from the differential expression analysis comparing cardiac tissue to all other tissues. Rho values represent the likelihood that enrichment occurred by chance.

TABLE 13

AAV Capsid Variant Enrichment in Heart Tissue

NHP Median

Mouse Median

SEQ ID

log2 Fold
Mouse
log2 Fold

NO
Capsid
NHP Rho
Change
Rho
Change

5027
CVIRSSDQE
4.69E−08
4.47
0.03
3.69

4633
MACWKNATE
3.08E−07
4.49
0.02
5.66

4943
CIVFYMKNQ
3.93E−05
4.07
0.02
4.95

386
CYQFWSQYS
1.09E−07
4.73
0.00
5.75

334
CSAAFQLQP
1.78E−05
4.45
0.00
4.07

4309
CIAFVKQCC
3.94E−06
4.64
0.03
3.05

4288
CACKVSMSG
2.30E−07
4.49
0.00
4.34

4964
CVGKYGGHS
2.71E−11
4.95
0.01
3.41

5017
CFYKIQHKG
2.18E−08
4.82
0.00
4.46

5814
MACKVHLQP
3.33E−12
4.94
0.00
4.66

4335
CPMKHIQDR
8.53E−10
4.65
0.04
4.95

348
CTASWMSWD
1.28E−07
4.28
0.02
3.10

4936
CVMPYSNHP
4.73E−08
4.65
0.02
4.84

5206
CFFYPMSMS
4.34E−07
4.50
0.04
2.97

4238
AACFKQLMQ
2.12E−07
4.44
0.03
2.79

5077
CVGSQLHAM
2.43E−07
4.63
0.02
5.60

5603
QANPYNHGI
7.85E−05
3.82
0.03
5.26

The number of capsids (“intersection size”) enriched in cardiac muscle tissue in NHPs, mice, or both NHPs and mice is shown in FIG. 16A. Seventeen capsid variants, corresponding to the capsid variants provided in TABLE 13, were enriched in cardiac tissue in both NHPs and mice. A differential expression analysis comparing cardiac muscle enrichment of the AAV capsid variants in mice and NHPs is shown in FIG. 16B. These data further demonstrated that these 17 variants (dark red circles) had significant enrichment in both NHPs (FDR <0.0001, log 2fc>1) and mice (FDR <0.05, log 2fc>1). Wild type spike-in controls are shown added at three different concentrations to the input library.

The number of capsids (“intersection size”) enriched in CNS tissue in NHPs or mice is shown in FIG. 17A. A differential expression analysis comparing CNS tissue enrichment of AAV capsid variants in mice and NHPs is shown in FIG. 17B. The 28 capsid variants enriched in NHP CNS tissue (dark gray circles) did not show enrichment in mouse CNS tissue.

Example 11
Treatment of a Neurological Disease or Condition with an AAV5-Derived Virion Encapsidating a Therapeutic Payload

This example describes treatment of a neurological disease or condition with a variant AAV5-derived virion having any one of the engineered CNS tropic variant AAV5 VP capsid polypeptides disclosed herein, wherein the variant AAV5 virion encapsidates a therapeutic payload. Polynucleotide sequence encoding for AAV Rep, an AAV5-derived variant Cap and helper proteins and a therapeutic payload are transfected in cells to produce variant AAV5 virions, where the polynucleotide sequence encoding for the variant AAV5 Cap comprises at least one mutation in a 581-589 region, corresponding to residues 581 to 589 in the VP1 capsid polypeptide, and where the polynucleotide sequence encodes for a variant AAV5 Cap comprising a 581-589 region amino acid sequence of any one of SEQ ID NO: 12-SEQ ID NO: 3937 or SEQ ID NO: 3938-SEQ ID NO: 4237. The therapeutic payload is a guide RNA targeting an mRNA encoded for by a gene implicated in the neurological disease or condition, or a transgene. The variant AAV5 virion encapsidating the payload is administered to a subject. The subject is a human or non-human animal. The route of administration is a systemic route of administration. The systemic route of administration is intravenous administration. Upon administration to the subject, the variant AAV5 virions encapsidating the therapeutic payload exhibit enhanced CNS tissue tropism as compared to wild type AAV5 virions encapsidating the therapeutic payload. Upon administration to the subject, a lower dose of the variant AAV5 virions encapsidating the therapeutic payload is administered as compared to wild type AAV5 virions encapsidating the therapeutic payload. Upon administration to the subject, at least one symptom of neurological disease or condition is alleviated, or the subject is cured.

Example 12
Treatment of Alzheimer's Disease with an AAV5-Derived Virion Encapsidating a Therapeutic Payload

This example describes treatment of Alzheimer's disease with a variant AAV5-derived virion having any one of the engineered CNS tropic variant AAV5 VP capsid polypeptides disclosed herein, wherein the variant AAV5 virion encapsidates a therapeutic payload. Polynucleotide sequence encoding for AAV Rep, an AAV5-derived variant Cap and helper proteins and a therapeutic payload are transfected in cells to produce variant AAV5 virions, where the polynucleotide sequence encoding for the variant AAV5 Cap comprises at least one mutation in a 581-589 region, corresponding to residues 581 to 589 in the VP1 capsid polypeptide, and where the polynucleotide sequence encodes for a variant AAV5 Cap comprising a 581-589 region amino acid sequence of any one of SEQ ID NO: 12-SEQ ID NO: 3937 or SEQ ID NO: 3938-SEQ ID NO: 4237. Each such variant is detargeted for all non-CNS tissues, including being detargeted for cardiac tissue. The therapeutic payload is a guide RNA targeting an mRNA encoded for by a gene implicated in Alzheimer's disease or a transgene. The mRNA targeted by the guide RNA is an mRNA encoded for by a gene encoding for amyloid precursor protein (APP), alpha-synuclein (SNCA), Tau protein (MAPT), or Apolipoprotein E (ApoE). The transgene is a gene encoding for amyloid precursor protein (APP), alpha-synuclein (SNCA), Tau protein (MAPT), or Apolipoprotein E (ApoE). The variant AAV5 virion encapsidating the payload is administered to a subject. The subject is a human or non-human animal. The route of administration is a systemic route of administration. The systemic route of administration is intravenous administration. Upon administration to the subject, the variant AAV5 virions encapsidating the therapeutic payload exhibit enhanced CNS tissue tropism as compared to wild type AAV5 virions encapsidating the therapeutic payload. Upon administration to the subject, a lower dose of the variant AAV5 virions encapsidating the therapeutic payload is administered as compared to wild type AAV5 virions encapsidating the therapeutic payload. Upon administration to the subject, at least one symptom of Alzheimer's disease is alleviated, or the subject is cured.

Example 13
Treatment of Parkinson's Disease with an AAV5-Derived Virion Encapsidating a Therapeutic Payload

This example describes treatment of Parkinson's disease with a variant AAV5-derived virion having any one of the engineered CNS tropic variant AAV5 VP capsid polypeptides disclosed herein, wherein the variant AAV5 virion encapsidates a therapeutic payload. Polynucleotide sequence encoding for AAV Rep, an AAV5-derived variant Cap and helper proteins and a therapeutic payload are transfected in cells to produce variant AAV5 virions, where the polynucleotide sequence encoding for the variant AAV5 Cap comprises at least one mutation in a 581-589 region, corresponding to residues 581 to 589 in the VP1 capsid polypeptide, and where the polynucleotide sequence encodes for a variant AAV5 Cap comprising a 581-589 region amino acid sequence of any one of SEQ ID NO: 12-SEQ ID NO: 3937 or SEQ ID NO: 3938-SEQ ID NO: 4237. The therapeutic payload is a guide RNA targeting an mRNA encoded for by a gene implicated in Parkinson's disease or a transgene implicated in Parkinson's disease. The mRNA targeted by the guide RNA is an mRNA encoded for by a gene encoding for leucine-rich repeat kinase 2 (LRRK2). The transgene is LRRK2; GBA1; SNCA (alpha-synuclein); AADC; GDNF; Neurturin; GAD; NTN; hFOXG1; hKCNQ2; hFMR1; anti-Tau/miRNA; EPM2A or EPM2B. The variant AAV5 virion encapsidating the payload is administered to a subject. The subject is a human or non-human animal. The route of administration is a systemic route of administration. The systemic route of administration is intravenous administration. Upon administration to the subject, the variant AAV5 virions encapsidating the therapeutic payload exhibit enhanced CNS tissue tropism as compared to wild type AAV5 virions encapsidating the therapeutic payload. Upon administration to the subject, a lower dose of the variant AAV5 virions encapsidating the therapeutic payload is administered as compared to wild type AAV5 virions encapsidating the therapeutic payload. Upon administration to the subject, at least one symptom of Parkinson's disease is alleviated, or the subject is cured.

Example 14
Treatment of a Tauopathy with an AAV5-Derived Virion Encapsidating a Therapeutic Payload

This example describes treatment of a Tauopathy with a variant AAV5-derived virion having any one of the engineered CNS tropic variant AAV5 VP capsid polypeptides disclosed herein, wherein the variant AAV5 virion encapsidates a therapeutic payload. Polynucleotide sequence encoding for AAV Rep, an AAV5-derived variant Cap and helper proteins and a therapeutic payload are transfected in cells to produce variant AAV5 virions, where the polynucleotide sequence encoding for the variant AAV5 Cap comprises at least one mutation in a 581-589 region, corresponding to residues 581 to 589 in the VP1 capsid polypeptide, and where the polynucleotide sequence encodes for a variant AAV5 Cap comprising a 581-589 region amino acid sequence of any one of SEQ ID NO: 12-SEQ ID NO: 3937 or SEQ ID NO: 3938-SEQ ID NO: 4237. The therapeutic payload is a guide RNA targeting an mRNA encoded for by a gene implicated in the Tauopathy or a transgene. The mRNA targeted by the guide RNA is an mRNA encoded for by a gene (MAPT) encoding for Tau protein. The transgene is MAPT. The variant AAV5 virion encapsidating the payload is administered to a subject. The subject is a human or non-human animal. The route of administration is a systemic route of administration. The systemic route of administration is intravenous administration. Upon administration to the subject, the variant AAV5 virions encapsidating the therapeutic payload exhibit enhanced CNS tissue tropism as compared to wild type AAV5 virions encapsidating the therapeutic payload. Upon administration to the subject, a lower dose of the variant AAV5 virions encapsidating the therapeutic payload is administered as compared to wild type AAV5 virions encapsidating the therapeutic payload. Upon administration to the subject, at least one symptom of Tauopathy disease is alleviated or the subject is cured.

Example 15
Treatment of Frontotemporal Dementia with an AAV5-Derived Virion Encapsidating a Therapeutic Payload

This example describes treatment of frontotemporal dementia (FTD) with a variant AAV5-derived virion having any one of the engineered CNS tropic variant AAV5 VP capsid polypeptides disclosed herein, wherein the variant AAV5 virion encapsidates a therapeutic payload. Polynucleotide sequence encoding for AAV Rep, an AAV5-derived variant Cap and helper proteins and a therapeutic payload are transfected in cells to produce variant AAV5 virions, where the polynucleotide sequence encoding for the variant AAV5 Cap comprises at least one mutation in a 581-589 region, corresponding to residues 581 to 589 in the VP1 capsid polypeptide, and where the polynucleotide sequence encodes for a variant AAV5 Cap comprising a 581-589 region amino acid sequence of any one of SEQ ID NO: 12-SEQ ID NO: 3937 or SEQ ID NO: 3938-SEQ ID NO: 4237. The therapeutic payload is a guide RNA targeting an mRNA encoded for by a gene implicated in FTD or a transgene encoding a protein implicated in FTD. The gene or the protein implicated in FTD is MAPT (Tau), GRN (Granulin), or progranulin. The variant AAV5 virion encapsidating the payload is administered to a subject. The subject is a human or non-human animal. The route of administration is a systemic route of administration. The systemic route of administration is intravenous administration. Upon administration to the subject, the variant AAV5 virions encapsidating the therapeutic payload exhibit enhanced CNS tissue tropism as compared to wild type AAV5 virions encapsidating the therapeutic payload. Upon administration to the subject, a lower dose of the variant AAV5 virions encapsidating the therapeutic payload is administered as compared to wild type AAV5 virions encapsidating the therapeutic payload. Upon administration to the subject, at least one symptom of FTD is alleviated, or the subject is cured.

Example 16
Treatment of Progressive Supranuclear Palsy with an AAV5-Derived Virion Encapsidating a Therapeutic Payload

This example describes treatment of progressive supranuclear palsy (PSP) with a variant AAV5-derived virion having any one of the engineered CNS tropic variant AAV5 VP capsid polypeptides disclosed herein, wherein the variant AAV5 virion encapsidates a therapeutic payload. Polynucleotide sequence encoding for AAV Rep, an AAV5-derived variant Cap and helper proteins and a therapeutic payload are transfected in cells to produce variant AAV5 virions, where the polynucleotide sequence encoding for the variant AAV5 Cap comprises at least one mutation in a 581-589 region, corresponding to residues 581 to 589 in the VP1 capsid polypeptide, and where the polynucleotide sequence encodes for a variant AAV5 Cap comprising a 581-589 region amino acid sequence of any one of SEQ ID NO: 12-SEQ ID NO: 3937 or SEQ ID NO: 3938-SEQ ID NO: 4237. The therapeutic payload is a guide RNA targeting an mRNA encoded for by a gene implicated in PSP or a transgene encoding a protein implicated in PSP. The gene or the protein implicated in PSP is MAPT (Tau). The variant AAV5 virion encapsidating the payload is administered to a subject. The subject is a human or non-human animal. The route of administration is a systemic route of administration. The systemic route of administration is intravenous administration. Upon administration to the subject, the variant AAV5 virions encapsidating the therapeutic payload exhibit enhanced CNS tissue tropism as compared to wild type AAV5 virions encapsidating the therapeutic payload. Upon administration to the subject, a lower dose of the variant AAV5 virions encapsidating the therapeutic payload is administered as compared to wild type AAV5 virions encapsidating the therapeutic payload. Upon administration to the subject, at least one symptom of PSP is alleviated, or the subject is cured.

Example 17
Treatment of Corticobasal Degeneration with an AAV5-Derived Virion Encapsidating a Therapeutic Payload

This example describes treatment of corticobasal degeneration (CBD) with a variant AAV5-derived virion having any one of the engineered CNS tropic variant AAV5 VP capsid polypeptides disclosed herein, wherein the variant AAV5 virion encapsidates a therapeutic payload. Polynucleotide sequence encoding for AAV Rep, an AAV5-derived variant Cap and helper proteins and a therapeutic payload are transfected in cells to produce variant AAV5 virions, where the polynucleotide sequence encoding for the variant AAV5 Cap comprises at least one mutation in a 581-589 region, corresponding to residues 581 to 589 in the VP1 capsid polypeptide, and where the polynucleotide sequence encodes for a variant AAV5 Cap comprising a 581-589 region amino acid sequence of any one of SEQ ID NO: 12-SEQ ID NO: 3937 or SEQ ID NO: 3938-SEQ ID NO: 4237. The therapeutic payload is a guide RNA targeting an mRNA encoded for by a gene implicated in CBD or a transgene encoding a protein implicated in CBD. The gene or the protein implicated in FTD is MAPT (Tau). The variant AAV5 virion encapsidating the payload is administered to a subject. The subject is a human or non-human animal. The route of administration is a systemic route of administration. The systemic route of administration is intravenous administration. Upon administration to the subject, the variant AAV5 virions encapsidating the therapeutic payload exhibit enhanced CNS tissue tropism as compared to wild type AAV5 virions encapsidating the therapeutic payload. Upon administration to the subject, a lower dose of the variant AAV5 virions encapsidating the therapeutic payload is administered as compared to wild type AAV5 virions encapsidating the therapeutic payload. Upon administration to the subject, at least one symptom of CBD is alleviated, or the subject is cured.

Example 18
Treatment of Chronic Traumatic Encephalopathy with an AAV5-derived Virion Encapsidating a Therapeutic Payload

This example describes treatment of chronic traumatic encephalopathy (CTE) with a variant AAV5-derived virion having any one of the engineered CNS tropic variant AAV5 VP capsid polypeptides disclosed herein, wherein the variant AAV5 virion encapsidates a therapeutic payload. Polynucleotide sequence encoding for AAV Rep, an AAV5-derived variant Cap and helper proteins and a therapeutic payload are transfected in cells to produce variant AAV5 virions, where the polynucleotide sequence encoding for the variant AAV5 Cap comprises at least one mutation in a 581-589 region, corresponding to residues 581 to 589 in the VP1 capsid polypeptide, and where the polynucleotide sequence encodes for a variant AAV5 Cap comprising a 581-589 region amino acid sequence of any one of SEQ ID NO: 12-SEQ ID NO: 3937 or SEQ ID NO: 3938-SEQ ID NO: 4237. The therapeutic payload is a guide RNA targeting an mRNA encoded for by a gene implicated in CTE or a transgene encoding a protein implicated in CTE. The gene or the protein implicated in CTE is MAPT (Tau). The variant AAV5 virion encapsidating the payload is administered to a subject. The subject is a human or non-human animal. The route of administration is a systemic route of administration. The systemic route of administration is intravenous administration. Upon administration to the subject, the variant AAV5 virions encapsidating the therapeutic payload exhibit enhanced CNS tissue tropism as compared to wild type AAV5 virions encapsidating the therapeutic payload. Upon administration to the subject, a lower dose of the variant AAV5 virions encapsidating the therapeutic payload is administered as compared to wild type AAV5 virions encapsidating the therapeutic payload. Upon administration to the subject, at least one symptom of CTE is alleviated, or the subject is cured.

Example 19
Treatment of a Synucleinopathy with an AAV5-derived Virion Encapsidating a Therapeutic Payload

This example describes treatment of a synucleinopathy with a variant AAV5-derived virion having any one of the engineered CNS tropic variant AAV5 VP capsid polypeptides disclosed herein, wherein the variant AAV5 virion encapsidates a therapeutic payload. Polynucleotide sequence encoding for AAV Rep, an AAV5-derived variant Cap and helper proteins and a therapeutic payload are transfected in cells to produce variant AAV5 virions, where the polynucleotide sequence encoding for the variant AAV5 Cap comprises at least one mutation in a 581-589 region, corresponding to residues 581 to 589 in the VP1 capsid polypeptide, and where the polynucleotide sequence encodes for a variant AAV5 Cap comprising a 581-589 region amino acid sequence of any one of SEQ ID NO: 12-SEQ ID NO: 3937 or SEQ ID NO: 3938-SEQ ID NO: 4237. The therapeutic payload is a guide RNA targeting an mRNA encoded for by a gene implicated in a synucleinopathy or a transgene encoding a protein implicated in a synucleinopathy. The gene or the protein implicated in the synucleinopathy is SNCA (α-synuclein). The variant AAV5 virion encapsidating the payload is administered to a subject. The subject is a human or non-human animal. The route of administration is a systemic route of administration. The systemic route of administration is intravenous administration. Upon administration to the subject, the variant AAV5 virions encapsidating the therapeutic payload exhibit enhanced CNS tissue tropism as compared to wild type AAV5 virions encapsidating the therapeutic payload. Upon administration to the subject, a lower dose of the variant AAV5 virions encapsidating the therapeutic payload is administered as compared to wild type AAV5 virions encapsidating the therapeutic payload. Upon administration to the subject, at least one symptom of the synucleinopathy is alleviated, or the subject is cured.

Example 20
Treatment of Rett Syndrome with an AAV5-derived Virion Encapsidating a Therapeutic Payload

This example describes treatment of Rett syndrome with a variant AAV5-derived virion having any one of the engineered CNS tropic variant AAV5 VP capsid polypeptides disclosed herein, wherein the variant AAV5 virion encapsidates a therapeutic payload. Polynucleotide sequence encoding for AAV Rep, an AAV5-derived variant Cap and helper proteins and a therapeutic payload are transfected in cells to produce variant AAV5 virions, where the polynucleotide sequence encoding for the variant AAV5 Cap comprises at least one mutation in a 581-589 region, corresponding to residues 581 to 589 in the VP1 capsid polypeptide, and where the polynucleotide sequence encodes for a variant AAV5 Cap comprising a 581-589 region amino acid sequence of any one of SEQ ID NO: 12-SEQ ID NO: 3937 or SEQ ID NO: 3938-SEQ ID NO: 4237. The therapeutic payload is a guide RNA targeting an mRNA encoded for by a gene implicated in Rett syndrome, a suppressor tRNA targeting a premature termination codon (PTC) in a gene implicated in Rett syndrome, or a transgene. The mRNA targeted by the guide RNA is an mRNA encoded for by a gene encoding for MECP2. The suppressor tRNA targets a PTC in MECP2. The transgene is MECP2. The variant AAV5 virion encapsidating the payload is administered to a subject. The subject is a human or non-human animal. The route of administration is a systemic route of administration. The systemic route of administration is intravenous administration. Upon administration to the subject, the variant AAV5 virions encapsidating the therapeutic payload exhibit enhanced CNS tissue tropism as compared to wild type AAV5 virions encapsidating the therapeutic payload. Upon administration to the subject, a lower dose of the variant AAV5 virions encapsidating the therapeutic payload is administered as compared to wild type AAV5 virions encapsidating the therapeutic payload. Upon administration to the subject, at least one symptom of Rett syndrome is alleviated or the subject is cured.

Example 21
Treatment of a Cardiac Disorder with an AAV5-derived Virion Encapsidating a Therapeutic Payload

This example describes treatment of a cardiac disorder with a variant AAV5-derived virion having any one of the engineered muscle-tropic variant AAV5 VP capsid polypeptides disclosed herein, wherein the variant AAV5 virion encapsidates a therapeutic payload. Polynucleotide sequence encoding for AAV Rep, an AAV5-derived variant Cap and helper proteins and a therapeutic payload are transfected in cells to produce variant AAV5 virions, where the polynucleotide sequence encoding for the variant AAV5 Cap comprises at least one mutation in a 581-589 region, corresponding to residues 581 to 589 in the VP1 capsid polypeptide, and where the polynucleotide sequence encodes for a variant AAV5 Cap comprising a 581-589 region amino acid sequence of any one of SEQ ID NO: 4238-SEQ ID NO: 4933 or SEQ ID NO: 4934-SEQ ID NO: 5233. The therapeutic payload is a guide RNA targeting an mRNA encoded for by a gene implicated in the cardiac disorder or a transgene implicated in the cardiac disorder. The variant AAV5 virion encapsidating the payload is administered to a subject. The subject is a human or non-human animal. The route of administration is a systemic route of administration. The systemic route of administration is intravenous administration. Upon administration to the subject, the variant AAV5 virions encapsidating the therapeutic payload exhibit enhanced muscle tissue tropism as compared to wild type AAV5 virions encapsidating the therapeutic payload. Upon administration to the subject, a lower dose of the variant AAV5 virions encapsidating the therapeutic payload is administered as compared to wild type AAV5 virions encapsidating the therapeutic payload. Upon administration to the subject, at least one symptom of the cardiac disorder is alleviated, or the subject is cured.

Example 22
Treatment of a Skeletal Muscle Disorder with an AAV5-derived Virion Encapsidating a Therapeutic Payload

This example describes treatment of a skeletal muscle disorder with a variant AAV5-derived virion having any one of the engineered muscle-tropic variant AAV5 VP capsid polypeptides disclosed herein, wherein the variant AAV5 virion encapsidates a therapeutic payload. Polynucleotide sequence encoding for AAV Rep, an AAV5-derived variant Cap and helper proteins and a therapeutic payload are transfected in cells to produce variant AAV5 virions, where the polynucleotide sequence encoding for the variant AAV5 Cap comprises at least one mutation in a 581-589 region, corresponding to residues 581 to 589 in the VP1 capsid polypeptide, and where the polynucleotide sequence encodes for a variant AAV5 Cap comprising a 581-589 region amino acid sequence of any one of SEQ ID NO: 4238-SEQ ID NO: 4933 or SEQ ID NO: 4934-SEQ ID NO: 5233. The therapeutic payload is a guide RNA targeting an mRNA encoded for by a gene implicated in the skeletal muscle disorder or a transgene implicated in the skeletal muscle disorder. The variant AAV5 virion encapsidating the payload is administered to a subject. The subject is a human or non-human animal. The route of administration is a systemic route of administration. The systemic route of administration is intravenous administration. Upon administration to the subject, the variant AAV5 virions encapsidating the therapeutic payload exhibit enhanced muscle tissue tropism as compared to wild type AAV5 virions encapsidating the therapeutic payload. Upon administration to the subject, a lower dose of the variant AAV5 virions encapsidating the therapeutic payload is administered as compared to wild type AAV5 virions encapsidating the therapeutic payload. Upon administration to the subject, at least one symptom of the skeletal muscle disorder is alleviated, or the subject is cured.

Example 23
Treatment of Duchenne Muscular Dystrophy with an AAV5-derived Virion Encapsidating a Therapeutic Payload

This example describes treatment of Duchenne muscular dystrophy with a variant AAV5-derived virion having any one of the engineered muscle-tropic variant AAV5 VP capsid polypeptides disclosed herein, wherein the variant AAV5 virion encapsidates a therapeutic payload. Polynucleotide sequence encoding for AAV Rep, an AAV5-derived variant Cap and helper proteins and a therapeutic payload are transfected in cells to produce variant AAV5 virions, where the polynucleotide sequence encoding for the variant AAV5 Cap comprises at least one mutation in a 581-589 region, corresponding to residues 581 to 589 in the VP1 capsid polypeptide, and where the polynucleotide sequence encodes for a variant AAV5 Cap comprising a 581-589 region amino acid sequence of any one of SEQ ID NO: 4238-SEQ ID NO: 4933 or SEQ ID NO: 4934-SEQ ID NO: 5233. The therapeutic payload is a guide RNA targeting an mRNA encoded for by a gene implicated in Duchenne muscular dystrophy or a transgene Duchenne muscular dystrophy. The mRNA targeted by the guide RNA is an mRNA encoded for by a gene encoding for DMID. The transgene is DMID. The variant AAV5 virion encapsidating the payload is administered to a subject. The subject is a human or non-human animal. The route of administration is a systemic route of administration. The systemic route of administration is intravenous administration. Upon administration to the subject, the variant AAV5 virions encapsidating the therapeutic payload exhibit enhanced muscle tissue tropism as compared to wild type AAV5 virions encapsidating the therapeutic payload. Upon administration to the subject, a lower dose of the variant AAV5 virions encapsidating the therapeutic payload is administered as compared to wild type AAV5 virions encapsidating the therapeutic payload. Upon administration to the subject, at least one symptom of Duchenne muscular dystrophy is alleviated, or the subject is cured.

Example 24
Treatment of Facioscapulohumeral Muscular Dystrophy with an AAV5-derived Virion Encapsidating a Therapeutic Payload

This example describes treatment of facioscapulohumeral muscular dystrophy-1 (FSHD) with a variant AAV5-derived virion having any one of the engineered muscle-tropic variant AAV5 VP capsid polypeptides disclosed herein, wherein the variant AAV5 virion encapsidates a therapeutic payload. Polynucleotide sequence encoding for AAV Rep, an AAV5-derived variant Cap and helper proteins and a therapeutic payload are transfected in cells to produce variant AAV5 virions, where the polynucleotide sequence encoding for the variant AAV5 Cap comprises at least one mutation in a 581-589 region, corresponding to residues 581 to 589 in the VP1 capsid polypeptide, and where the polynucleotide sequence encodes for a variant AAV5 Cap comprising a 581-589 region amino acid sequence of any one of SEQ ID NO: 4238-SEQ ID NO: 4933 or SEQ ID NO: 4934-SEQ ID NO: 5233. The therapeutic payload is a guide RNA targeting an mRNA encoded for by a gene implicated in FSHD or a transgene implicated in FSHD. The mRNA targeted by the guide RNA is an mRNA encoded for by a gene encoding for DUX4. The transgene is DUX4. The variant AAV5 virion encapsidating the payload is administered to a subject. The subject is a human or non-human animal. The route of administration is a systemic route of administration. The systemic route of administration is intravenous administration. Upon administration to the subject, the variant AAV5 virions encapsidating the therapeutic payload exhibit enhanced muscle tissue tropism as compared to wild type AAV5 virions encapsidating the therapeutic payload. Upon administration to the subject, a lower dose of the variant AAV5 virions encapsidating the therapeutic payload is administered as compared to wild type AAV5 virions encapsidating the therapeutic payload. Upon administration to the subject, at least one symptom of FSHD is alleviated, or the subject is cured.

Example 25
Tissue Abundance of Engineered AAV5 Variants by DNA Analysis

This example demonstrates the tissue abundance of engineered AAV5 variants of the present disclosure as compared to control serotypes by DNA analysis. The CNS-targeted secondary library of the present disclosure comprising engineered AAV5 variants was administered intravenously to four cynomolgus macaques. Four weeks post-injection, animals were euthanized, and a diverse set of tissues was collected for DNA (measuring total variant infection) and RNA (measuring functional transduction) analysis.

FIG. 3A shows a next generation sequencing (NGS) analysis of purified secondary screen library virus containing engineered AAV5 variants of the present disclosure and barcoded control serotypes (AAV1, AAV2, AAV5, AAV9, and AAV PHP.B) spiked in at 1×, 10×, and 100× stoichiometric ratios. FIG. 3B is a schematic highlighting identification of AAV spike-in control DNA in CNS, liver, heart, and skeletal muscle by NGS analysis of NHP tissues post-selection. FIG. 3C demonstrates that DNA from 94% of capsid variants predicted to target the CNS were found in the CNS. FIG. 3D demonstrates that in a comparison of DNA abundance in the CNS for three engineered AAV5 variants of the present disclosure (AAGRFNYFG (SEQ ID NO: 18), AEDYWDLGA (SEQ ID NO: 54), and MDDICEFYA (SEQ ID NO: 2028)) versus wild type AAV5 and AAV9 spike-in controls, the three engineered AAV5 variants show much greater infection than the highest (100×) controls. FIG. 3E shows that engineered AAV5 variants of the present disclosure exhibit decreased liver infection compared to wild type (parental) AAV5 control.

As demonstrated in FIG. 3A-FIG. 3E, select engineered AAV5 variants of the present disclosure exhibited greater than 100-fold infectivity for CNS and exhibited liver detuning as compared to control serotypes, including commonly used wild type AAVs.

Example 26
Functional Transduction of RNA Expression in CNS Tissue

This example demonstrates functional transduction of RNA expression in CNS tissue using CNS tissue-tropic variants. Variants of SEQ ID NO: 18 (AAGRFNYFG), SEQ ID NO: 424 (DAWCFISSY), and SEQ ID NO: 2028 (MDDICEFYA) were compared. A library of barcoded AAV variants was systemically administered to non-human primates. Wild type AAV9 was also administered for reference. Tissues were harvested from the non-human primates, and barcoded DNA was quantified to measure accumulation of the AAV variants in each tissue. RNA expressed from the DNA payload was also quantified to measure functional transduction.

Relative accumulation of AAVs and functional transduction of RNA expression for wild type AAV9 and three CNS tissue-tropic AAV variants (SEQ ID NO: 18, SEQ ID NO: 424, and SEQ ID NO: 2028) is shown in FIG. 10. The three AAV variants exhibited increased CNS tissue tropism, as measured by DNA accumulation, compared to wild type AAV9. The three AAV variants also exhibited CNS tissue-specific transduction of RNA expression. The AAV variants also exhibited broad accumulation and functional transduction in various CNS tissues, including occipital cortex, motor cortex, forebrain, thalamus, striatum, hypothalamus, cerebellum, caudate, putamen, pons, midbrain, and substantia nigra, as shown in FIG. 11. The AAV variant comprising a sequence of SEQ ID NO: 2028 showed broad functional transduction in various CNS tissues compared to wild type AAV9, as shown in FIG. 12.

FIG. 13 shows that three AAV variants of the present disclosure (also shown in FIG. 11) functionally transduce neurons. CAG promoter-driven RNA expression highlights functional transduction of any cell type while SYN promoter-driven RNA expression highlights functional transduction of neurons. FIG. 14 shows that some AAV variants of the present disclosure that target CNS also demonstrate dorsal root ganglion (DRG) depletion.

Example 27
Functional Transduction of RNA Expression in CNS Tissue Versus Heart Muscle Tissues

This example demonstrates functional transduction of RNA expression in CNS tissue and heart muscle tissue. A library of barcoded AAV variants was systemically administered to non-human primates. Wild type AAV9 was also administered for reference. Tissues were harvested from the non-human primates, and DNA and RNA were quantified.

FIG. 15 shows that promoter usage differentiates CNS AAV variants and heart muscle AAV variants. For example, certain AAV CNS variants of the present disclosure shown CAG and SYN promoter-driven RNA expression, while certain AAV heart muscle variants show only CAG promoter-driven RNA expression.

Example 28
Singleplex Tertiary Screen to Validate CNS Tissue-Tropic Engineered AAV Capsids

This example describes a tertiary screen, in singleplex, to identify CNS tissue-tropic engineered AAV capsids. Prospective tissue-tropic capsids identified from an in vivo secondary screen, through machine learning, or both are introduced into an in vivo tertiary screen, in singleplex, to confirm tissue-tropic behavior of the identified capsids when individually dosed in NHP. Capsid variant sequences including SEQ ID NO: 2028, SEQ ID NO: 18, SEQ ID NO: 54, and SEQ ID NO: 424 are selected for singleplex screening. Recombinant AAVs of the selected variants encapsidate a model transgene. The transgene is GFP or a tagged frataxin protein. The tertiary screen capsid library is systemically administered to a non-human primate (NHP) by intravenous injection. Following injection, tissues, including CNS and liver, are isolated from the NHP, and the sequences are quantified. Prospective tissue-tropic capsids are compared to control, wild-type AAV to identify superior variant capsids of the present disclosure that target various CNS tissues using DNA and RNA analysis.

Preliminary DNA Enrichment Results. In a pilot study, four male NHPs were intravenously administered, in singleplex, wild-type AAV9 or a capsid variant of the present disclosure of having a 581-589 region of MDDICEFYA (SEQ ID NO: 2028), AAGRFNYFG (SEQ ID NO: 18), or AEDYWDLGA (SEQ ID NO: 54), each carrying a GFP transgene under control of a CMV promoter. Virus was administered at a dose of 3E13 viral genomes (VGs)/kg. Two weeks post-administration, NHPs were euthanized, and various tissues were harvested, including CNS and liver tissues. A kit was used to extract DNA from tissues, and primers against the CMV region of the transgene and GAPDH were used to track vector and genomic counts. DNA was quantitated via droplet digital PCR (ddPCR). In a preliminary analysis of an n=1 regional biopsy (4 mm tissue punches) from several CNS tissue regions (caudate; putamen; cerebellum; cortex, forebrain; cortex, occipital; cortex, temporal; midbrain; pons; substantia nigra; thalamus; motor cortex; Broca's area; hippocampus; hypothalamus; globus pallidus; Wernicke's area; and medulla) and liver tissue regions, the three capsid variants of the present disclosure each exhibited at least about a 7-fold average enrichment across brain regions and at least about a 4-fold average decrease in liver enrichment.

Further analyses are conducted to confirm the preliminary analysis and explore selectivity for CNS tissues as compared to liver and other tissues harvested from NHPs.

Example 29
Singleplex Tertiary Screen to Validate Muscle Tissue-Tropic Engineered AAV Capsids

This example describes a tertiary screen, in singleplex, to identify muscle tissue-tropic engineered AAV capsids. Prospective tissue-tropic capsids identified from an in vivo secondary screen, through machine learning, or both are introduced into an in vivo tertiary screen, in singleplex, to confirm tissue-tropic behavior of the identified capsids when individually dosed in NHP. Capsid variant sequences of the present disclosure are selected for singleplex screening. Recombinant AAVs of the selected variants encapsidate a model transgene. The tertiary screen capsid library is systemically administered to a non-human primate (NHP) by intravenous injection. Following injection, tissues, including muscle and liver, are isolated from the NHP, and the sequences are quantified. Prospective tissue-tropic capsids are compared to control, wild-type AAV to identify superior variant capsids of the present disclosure that target various muscle tissues using DNA and RNA analysis.

While the invention has been particularly shown and described with reference to a preferred embodiment and various alternate embodiments, it is understood by persons skilled in the relevant art that various changes in form and details can be made therein without departing from the spirit and scope of the invention.

Number	Date	Country
63284977	Dec 2021	US
63342032	May 2022	US
63354635	Jun 2022	US
63399164	Aug 2022	US

FUNCTIONAL AAV CAPSIDS FOR SYSTEMIC ADMINISTRATION

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