Novel adeno-associated virus compositions having increased brain enrichment

Abstract

Described herein are compositions and kits comprising recombinant adeno-associated viruses (rAAVs) with increased transduction enrichment in the brain. The rAAV compositions described herein encapsidate a transgene, such as a therapeutic nucleic acid. Gene therapy using the rAAVs is described. Also described are methods of treating brain-related diseases and conditions.

Description

NOVEL ADENO-ASSOCIATED VIRUS COMPOSITIONS HAVING INCREASED BRAIN ENRICHMENT BACKGROUND

[0001] Recombinant adeno-associated viruses (rAAVs) are widely used as vectors for gene delivery in therapeutic applications because of their ability to transduce both dividing and non- dividing cells, their long-term persistence as episomal DNA in infected cells, and their low immunogenicity. These characteristics make them appealing for therapeutic applications, such as gene therapy. However, there is a need to significantly improve the performance of existing AAV serotypes to selectively and efficiently express in distinct cell-types, upon systemic delivery to a subject. This need is especially acute when the AAV must be expressed in the brain. SEQUENCE LISTING

[0002] The present application is being filed along with a Sequence Listing in electronic format. The Sequence Listing is provided as a file named CAPS-039-01WO-Seq-Listing.xml, created on August 25, 2025, which is 7 MB in size. The information in the electronic format of the Sequence Listing is incorporated herein by reference in its entirety. SUMMARY OF THE INVENTION

[0003] Disclosed herein are recombinant adeno-associated viruses (rAAVs) with engineered mutations introduced into the capsid structure through iterative rounds of selection in non-human primates (NHPs), yielding variants having increased transduction when measured in the brain.

[0004] The present invention provides rAAVs derived from the iterative rounds of selection with widespread transduction to the brain. Furthermore, the present disclosure includes rAAVs having capsid proteins with more than one mutation at different locations on the capsid protein amino acid sequence. At each location the mutations are based on iterative rounds of selection to find target- specific rAAV capsid proteins. These mutations are then combined in some rAAVs of the disclosure.

[0005] The present disclosure provides an AAV capsid protein in which the capsid protein amino acid sequence has been modified to incorporate an amino acid substitution and / or insertion sequence. Specifically, the capsid proteins of the disclosure comprise an amino acid substitution and / or insertion sequence at a position between positions 449 and 460 and having at least 80% identity to a sequence provided in Table 2. Preferred capsids of the disclosure comprise an aminoacid substitution and / or insertion sequence at a position between positions 449 and 460 and having 100% identity to a sequence provided in Table 2.

[0006] The present disclosure includes AAV capsid proteins comprising an amino acid substitution and / or insertion sequence at position 452 having at least 80% identity to a sequence provided in Table 1. The present disclosure includes AAV capsid proteins comprising an amino acid substitution and / or insertion sequence at position 453 having at least 80% identity to a sequence provided in Table 1. The present disclosure includes AAV capsid proteins comprising an amino acid substitution and / or insertion sequence at position 454 having at least 80% identity to a sequence provided in Table 1. The present disclosure includes AAV capsid proteins comprising an amino acid substitution and / or insertion sequence at position 455 having at least 80% identity to a sequence provided in Table 1.

[0007] The present disclosure includes AAV capsid proteins that comprise an amino acid substitution and / or insertion sequence at position 452, 453, 454, or 455 as set forth in Table 1. The present disclosure includes AAV capsid proteins comprising an amino acid substitution and / or insertion sequence at positions 452-457 having at least 80% identity to a sequence provided in Table 1. The present disclosure includes AAV capsid proteins comprising an amino acid substitution and / or insertion sequence at positions 453-458 having at least 80% identity to a sequence provided in Table 1. The present disclosure includes AAV capsid proteins comprising an amino acid substitution and / or insertion sequence at positions 453-456 having at least 80% identity to a sequence provided in Table 1. The present disclosure includes AAV capsid proteins comprising an amino acid substitution and / or insertion sequence at positions 454-456 having at least 80% identity to a sequence provided in Table 1. The present disclosure includes AAV capsid proteins comprising an amino acid substitution and / or insertion sequence at positions 454-455 having at least 80% identity to a sequence provided in Table 1. The present disclosure includes AAV capsid proteins comprising an amino acid substitution and / or insertion sequence at positions 455-456 having at least 80% identity to a sequence provided in Table 1.

[0008] In certain aspects, the present disclosure includes one or more AAV capsid proteins comprising an amino acid substitution and / or insertion sequence at a position between positions 449 and 460, wherein said amino acid sequence starting at position 449 has at least 80% identity to a sequence provided in Table 2. In certain aspects, the present disclosure includes one or more AAV capsid proteins comprising an amino acid substitution and / or insertion sequence at a positionbetween positions 449 and 460, wherein said amino acid sequence starting at position 449 has at least 100% identity to a sequence provided in Table 2.

[0009] The present disclosure includes one or more AAV capsid protein comprising an insertion and / or substitution sequence of Formula A between positions 452-457, and / or wherein the capsid protein comprises an insertion and / or substitution sequence having at least 80% identity to a sequence having Formula A, Xa-Xb-Xc-Xd-Xe-Xf(A) wherein: Xais an amino acid selected from E, D, N, S, and T; Xbis an amino acid selected from R, G, K, T, and S; Xcis an amino acid selected from S, G, T, E, and A; Xdis an amino acid selected from S, G, and T; Xeis an amino acid selected from S, T, G, Q, and A; and Xfis an amino acid selected from S, T, A, and K. In preferred aspects, Xais E, Xbis R, Xcis S, Xdis S, Xeis S, and / or Xfis S.

[0010] The present disclosure includes one or more AAV capsid protein comprising an insertion and / or substitution sequence of Formula B between positions 453-458, and / or wherein the capsid protein comprises an insertion and / or substitution sequence having at least 80% identity to a sequence having Formula B, Xa1-Xb1-Xc1-Xd1-Xe1-Xf1(B) wherein: Xa1is an amino acid selT; Xb1is an amino acid selected from G, T, S, and D; Xc1is an amino acid selected from S, G, T, A, P and V; Xd1is an amino acid selected from S, Q, A and K; Xe1is an amino acid selected from T, S, N, A and G; and Xf1is an amino acid selected from S, G, E, and T. In some preferred aspects, Xa1is K, Xb1is G, Xc1is S, Xd1is S, Xe1is T, and / or Xf1is S.

[0011] The present disclosure includes AAV capsid proteins comprising an insertion and / or substitution sequence of Formula C between positions 453 and 456, and / or wherein the capsid protein comprises an insertion and / or substitution sequence having at least 80% identity to a sequence having Formula C, Xa2-Xb2-Xc2-Xd2-Xe2-Xf2(C) wherein: Xa2is an amino acid selected from S, K, G, and A; Xb2is an amino acid selected from G, S, A, and T; Xc2is an amino acid selected from G, S, A and T; Xd2is an amino acid selected from S, G, T, and R; Xe2is an amino acid selected from S, G, A, R and T; and Xf2is an amino acid selected from G, S, A, and E. In some preferred aspects, Xa2is K, Xb2is G, Xc2is S, Xd2is S, Xe2isT, and / or Xf2is G.

[0012] The present disclosure includes one or more AAV capsid protein comprising an insertion and / or substitution sequence of Formula D between positions 454 and 456, and / or wherein the capsid protein comprises an insertion and / or substitution sequence having at least 80% identity to a sequence having Formula D, Xa3-Xb3-Xc3-Xd3-Xe3-Xf3(D) wherein: Xa3is an amino acid selected from T, S, G, and V; Xb3is an amino acid selected from G, S, A, and T; Xc3is an amino acid selected from S, G, and P; Xd3is an amino acid selected from S, G, A and R; Xe3is an amino acid selected from G, S, R A, and T; and Xf3is an amino acid selected from S, Q, T, E, and A. In some preferred aspects, Xa3is T, Xb3is G, Xc3is S, Xd3is S, Xe3is G, and / or Xf3is S.

[0013] The present disclosure includes one or more AAV capsid protein comprising an insertion and / or substitution sequence of Formula E between positions 454 and 455 or positions 455 and 456, and / or wherein the capsid protein comprises an insertion and / or substitution sequence having at least 80% identity to a sequence having Formula E, Xa4-Xb4-Xc4-Xd4-Xe4-Xf4(E) wherein: Xa4is an amino acid selected from T, A, P, S, G, E, and V; Xb4is an amino acid selected from G, S, A, P, and T; Xc4is an amino acid selected from S, G, A, K, and T; Xd4is an amino acid selected from S, G, A and R; Xe4is an amino acid selected from G, S, R A, and T; and Xf4is an amino acid selected from S, Q, T, E, and A. In some preferred aspects, Xa4is T, Xb4is G, Xc4is S, Xd4is S, Xe4is G and / or Xf4is S.

[0014] The present disclosure includes one or more AAV capsid protein comprising an insertion and / or substitution sequence of Formula F at position 455, and / or wherein the capsid protein insertion and / or substitution sequence comprises a sequence having at least 80% identity to a sequence having Formula F, Xa5-Xb5-Xc5-Xd5-Xe5-Xf5(F) wherein: Xa5is an amino acid selected from A, P, T, and V; Xb5is an amino acid selected from G, S, T, and A; Xc5is an amino acid selected from G, A, T, and V; Xd5is an amino acid selected from G, S, A and K; Xe5is an amino acid selected from A, S, T, G, and R; and Xf5is an amino acid selected from G, A, K, S, and T. In some preferred aspects Xa5is A, Xb5is G, Xc5is G, Xd5is G, Xe5is A, and / or Xf5is G.

[0015] In certain aspects, the AAV capsid proteins of the disclosure having an amino acid substitution and / or insertion sequence between positions 449 and 460 further comprise one or more additional amino acid substitution and / or insertion sequences. In certain preferred aspects, the AAV capsid proteins further comprise an amino acid insertion and / or substitution sequence at a position between 583-599. The present disclosure includes AAV capsid proteins comprising: (i) an amino acid substitution and / or insertion sequence at a position between 583-599 having at least 80% identity to a sequence provided in Table 3; and (ii) an amino acid substitution and / or insertion sequence between positions 449 and 460 having at least 80% identity to a sequence as provided in Table 2 and / or Table 1.

[0016] In some preferred aspects, the amino acid insertion and / or substitution sequence at a position between 583-599 is at position 587-597.

[0017] In some preferred aspects, the amino acid insertion and / or substitution sequence at position 587-597 comprises Formula I: X1-X2-X3-X4-X5-X6-X7-X8-X9-X10-X11(I) wherein: X1is an amino acid selected from A, Q, H, K, T, S, N, and M, and preferably selected from A or Q; X2is an amino acid selected from Q, R, T, Y and M, and preferably from Q, R, and T; X3is an amino acid selected from R, E, L, and T, and is preferably R; X4is an amino acid selected from D, N, and S, and preferably from D and N; X5is an amino acid selected from G and H, and is preferably G; X6is an amino acid selected from L, V, A, and Q, and preferably from L and V; X7is an amino acid selected from I, H, and R, and is preferably I; X8is an amino acid selected from L, F, and S, and is preferably L; X9is an amino acid selected from I, K, V, Q, and A, and is preferably I; X10is an amino acid selected from A, S, E, Q, and T, and is preferably A; and X11is an amino acid selected from Q, P, and K.

[0018] In certain embodiments, in the AAV capsid protein comprising an amino acid insertion and / or substitution sequence of Formula I at position 587-597, X1may be a substitution or the parental amino acid at 587, X2may be a substitution or parental amino acid at position 588, X3, X4, X5, X6, X7, X8, and X9may be insertions, X10may be a substitution or parenteral amino acid at position 589 (i.e., now at position 596 in view of the insertion), and X11and may be a substitution or the parental amino acid at position 590 (i.e., now at position at 597 in view of the insertion).

[0019] In some preferred aspects, the amino acid insertion and / or substitution sequence at a position between 583-599 is at position 583-599.

[0020] In certain aspects, the disclosure provides AAV capsids comprising an AAV capsid protein comprising an amino acid sequence of Formula (II) at positions 583-599: X1-X2-X3-X4-X5-X6-X7-X8-X9-X10-X11-X12-X13-X14-X15-X16-X17(II) wherein: X1 is an amino acid selected from N, S, G, and T, and preferably selected from N; X2 is an amino acid selected from H, N, A, S, V, C, Q, I, T, L, W, M, K, F, R, G, and Y, and preferably H; X3is amino acid Q; X4 is an amino acid selected from S, A, T, G, C, L, H, K, N, Q, R, Y, and M, and preferably from S, M, or G; X5 is an amino acid selected from A, Q, N, S, M, Y, G, H, D, W, T, F, V, I, C, L, T, and E, and is preferably A, Q, or S; X6is an amino acid selected from T, V, Q, S, M, I, N, K, A, H, F, E, R, Y, and L, and preferably from T, V, or S; X7is an amino acid selected from R, K, L, M, Q, V, F, H, A, S, Y, T, W, and I, and is preferably R or K; X8 is an amino acid selected from N, D, G, Q, A, H, M, E, R, L, K, T, V, and S, and is preferably N or D; X9is an amino acid selected from G, S, Q, A, D, K, E, T, H, M, V, I, L, and N, and is preferably G; X10is an amino acid selected from E, H, Q, A, T, G, D, S, K, V, R, and N, and is preferably E or H; X11 is an amino acid selected from V, I, T, H, M, Q, A, S, P, N, R, K, and L, and is preferably V or I; X12 is an amino acid selected from F, L, Y, M, R, K, and H, and is preferably F, L, or Y; X13is an amino acid selected from I, Y, V, L, M, T, H, E, Q, S, A, N, and F, and is preferably I or Y; X14 is an amino acid selected from A, and S, and is preferably A; X15 is an amino acid selected from Q, M, S, G, N, T, A, I, H, L, R, Y, and V, and is preferably Q, M, S, or N; X16is an amino acid selected from A, P, F, T, C, G, H, Q, V, Y, or S, and is preferably A; and X17is an amino acid selected from Q, A, S, L, M, N, D, T, V, H, I, or G, and is preferably Q.

[0021] In certain embodiments, in the capsids of AAV capsids comprising an AAV capsid protein comprising an amino acid sequence of Formula (II), X1is an amino acid selected from N, S, G, and T, and preferably selected from N; X2 is an amino acid selected from H, N, A, and S, and preferably from H; X3is amino acid Q; X4is an amino acid selected from S, A, T, G, and M, and preferably from S, M, or G; X5is an amino acid selected from A, Q, N, S, M, Y, and E, and is preferably A, Q, or S; X6 is an amino acid selected from T, V, Q, S, M and L, and preferably from T, V, or S; X7 is an amino acid selected from R, K, L, M, and I, and is preferably R or K; X8 is an amino acid selected from N, D, G, and S, and is preferably N or D; X9is an amino acid selected from G, S, Q, A, and N, and is preferably G; X10is an amino acid selected from E, H, Q, A, and N, and is preferably E or H; X11 is an amino acid selected from V, I, T, and L, and is preferably Vor I; X12 is an amino acid selected from F, L, Y, M, and H, and is preferably F, L, or Y; X13 is an amino acid selected from I, Y, V, and F, and is preferably I or Y; X14is an amino acid selected from A, and S, and is preferably A; X15is an amino acid selected from Q, M, S, G, N, T, A and V, and is preferably Q, M, S, or N; X16 is an amino acid selected from A or S, and is preferably A; and X17 is an amino acid selected from Q, A, or G, and is preferably Q.

[0022] In certain embodiments, in the AAV capsid protein comprising an amino acid insertion and / or substitution sequence of Formula II at position 583-599, X1 may be a substitution or the parental amino acid at 583, X2 may be a substitution or parental amino acid at position 584, X3 may be a substitution or parental amino acid at position 585, X4 may be a substitution or parental amino acid at position 586, X5 may be a substitution or parental amino acid at position 587, X6 may be a substitution or parental amino acid at position 587, X7, X8, X9, X10, X11, X12, and X13 may be insertions, X14 may be a substitution or parenteral amino acid at position 589 (i.e., now at position 596 in view of the insertion), X15 and may be a substitution or the parental amino acid at position 590 (i.e., now at position at 597 in view of the insertion), X16 and may be a substitution or the parental amino acid at position 591 (i.e., now at position at 598 in view of the insertion), and X17 and may be a substitution or the parental amino acid at position 592 (i.e., now at position at 599 in view of the insertion).

[0023] In certain embodiments, the AAV capsid protein comprises an amino acid sequence of Formula II, wherein X1 is N.

[0024] In certain embodiments, the AAV capsid protein comprises an amino acid sequence of Formula II, wherein X2 is H.

[0025] In certain embodiments, the AAV capsid protein comprises an amino acid sequence of Formula II, wherein X3 is Q

[0026] In certain embodiments, the AAV capsid protein comprises an amino acid sequence of Formula II, wherein X4 is S or G.

[0027] In certain embodiments, the AAV capsid protein comprises an amino acid sequence of Formula II, wherein X5 is A or S.

[0028] In certain embodiments, the AAV capsid protein comprises an amino acid sequence of Formula II, wherein X6 is T or S.

[0029] In certain embodiments, the AAV capsid protein comprises an amino acid sequence of Formula II, wherein X7 is R or K.

[0030] In certain embodiments, the AAV capsid protein comprises an amino acid sequence of Formula II, wherein X8 is N or D.

[0031] In certain embodiments, the AAV capsid protein comprises an amino acid sequence of Formula II, wherein X9 is G.

[0032] In certain embodiments, the AAV capsid protein comprises an amino acid sequence of Formula II, wherein X10 is E or H.

[0033] In certain embodiments, the AAV capsid protein comprises an amino acid sequence of Formula II, wherein X11 is V or I.

[0034] In certain embodiments, the AAV capsid protein comprises an amino acid sequence of Formula II, wherein X12 is F, L, or Y.

[0035] In certain embodiments, the AAV capsid protein comprises an amino acid sequence of Formula II, wherein X13 is I or Y.

[0036] In certain embodiments, the AAV capsid protein comprises an amino acid sequence of Formula II, wherein X14 is A.

[0037] In certain embodiments, the AAV capsid protein comprises an amino acid sequence of Formula II, wherein X15 is Q, M, or N.

[0038] In certain embodiments, the AAV capsid protein comprises an amino acid sequence of Formula II, wherein X16 is A or S.

[0039] In certain embodiments, the AAV capsid protein comprises an amino acid sequence of Formula II, wherein X17is Q.

[0040] In certain embodiments, the AAV capsid protein comprises one or more amino acid sequence as set forth in Tables 1, 2, 3, 4, 4A, 5, 5A, 5B, 6, 6A and / or 6B.

[0041] In some aspects, the AAV is AAV9. In preferred aspects, the AAV is provided in SEQ ID NO: 1. In certain embodiments, 60 copies of the AAV capsid protein are assembled into the AAV capsid. In certain embodiments, the AAV capsid protein is present in VP1, VP2, and VP3 of the AAV capsid. In certain embodiments, the AAV capsid protein is characterized by at least one of an increased transduction enrichment when measured in a brain in a subject when delivered to the subject systemically.

[0042] In certain embodiments, the AAV capsid protein further comprises an amino acid substitution comprising A589N or Q590P.

[0043] Aspects disclosed herein provide methods of treating a disease or condition in a subjectcomprising administering a therapeutically effective amount of a pharmaceutical formulation comprising the AAV capsid protein or the AAV capsid of the present disclosure. In some embodiments, the disease or the condition is a disease or a condition of the brain, and brain of the subject. Relatedly, the invention includes use of the rAAVs in the manufacture of a medicament for treating or preventing the disease or medical condition.

[0044] Other aspects of the invention will be apparent from the detailed description and claims that follow. DETAILED DESCRIPTION OF THE DISCLOSURE

[0045] Disclosed herein are recombinant adeno-associated viruses (rAAVs) with engineered mutations introduced into the capsid structure through iterative rounds of selection in non-human primates (NHPs), yielding variants having increased transduction when measured in the brain.

[0046] In one aspect the disclosure provides rAAVs with high expression levels in the brain.

[0047] In one aspect, the disclosure provides rAAVs with a peptide insertion and / or substitution comprising or consisting of an amino-acid sequence set forth in any one of Tables 1-6, Formulas A-F, and / or Formulas I-II.

[0048] The present disclosure provides an AAV capsid protein in which the capsid protein amino acid sequence has been modified to incorporate an amino acid substitution and / or insertion sequence. Specifically, the capsid proteins of the disclosure comprise an amino acid substitution and / or insertion sequence at a position between positions 449 and 460 and having at least 80% identity to a sequence provided in Tables 1 and / or 2.

[0049] In certain aspects, the present disclosure includes one or more AAV capsid proteins comprising an amino acid substitution and / or insertion sequence at a position between positions 449 and 460, wherein said amino acid sequence starting at position 449 has at least 80% identity to a sequence provided in Table 2. In certain aspects, the present disclosure includes one or more AAV capsid proteins comprising an amino acid substitution and / or insertion sequence at a position between positions 449 and 460, wherein said amino acid sequence starting at position 449 has at least 100% identity to a sequence provided in Table 2.

[0050] The present disclosure includes AAV capsid proteins comprising an amino acid substitution and / or insertion sequence at position 452 having at least 80% identity to a sequence provided in Table 1. The present disclosure includes AAV capsid proteins comprising an amino acid substitution and / or insertion sequence at position 453 having at least 80% identity to asequence provided in Table 1. The present disclosure includes AAV capsid proteins comprising an amino acid substitution and / or insertion sequence at position 454 having at least 80% identity to a sequence provided in Table 1. The present disclosure includes AAV capsid proteins comprising an amino acid substitution and / or insertion sequence at position 455 having at least 80% identity to a sequence provided in Table 1. In some aspects, the present disclosure includes AAV capsid proteins that comprise an amino acid substitution and / or insertion sequence at position 452, 453, 454, or 455 as set forth in Table 1.

[0051] The present disclosure includes AAV capsid proteins comprising an amino acid substitution and / or insertion sequence at positions 452-457 having at least 80% identity to a sequence provided in Table 1. The present disclosure includes AAV capsid proteins comprising an amino acid substitution and / or insertion sequence at positions 453-458 having at least 80% identity to a sequence provided in Table 1. The present disclosure includes AAV capsid proteins comprising an amino acid substitution and / or insertion sequence at positions 453-456 having at least 80% identity to a sequence provided in Table 1. The present disclosure includes AAV capsid proteins comprising an amino acid substitution and / or insertion sequence at positions 454-456 having at least 80% identity to a sequence provided in Table 1. The present disclosure includes AAV capsid proteins comprising an amino acid substitution and / or insertion sequence at positions 454-455 having at least 80% identity to a sequence provided in Table 1. The present disclosure includes AAV capsid proteins comprising an amino acid substitution and / or insertion sequence at positions 455-456 having at least 80% identity to a sequence provided in Table 1. TABLE 1 – sequences showing SEQ ID NO: 11ERGASSsubstituted and / or inserted amino acids SEQ ID NO: 12ERGGSTSEQ ID NO: 13ERGTASSEQ ID NO: 14ERSSATSEQ ID NO: 15ERSSTSSEQ ID NO: 16DPTQSKSEQ ID NO: 17ERGSTASEQ ID NO: 18ERSGSTSEQ ID NO: 19ERTSGSSEQ ID NO: 20TAPGQNSEQ ID NO: 21DRSQSSSEQ ID NO: 22ERTSSTSEQ ID NO: 23ETSSKSSEQ ID NO: 24AGSGQGSEQ ID NO: 25RTESPSSEQ ID NO: 26DRAGTSSEQ ID NO: 67GSTGDRSEQ ID NO: 27DRTGNASEQ ID NO: 68STTSPTSEQ ID NO: 105NTGSTGSEQ ID NO: 142VGGTRESEQ ID NO: 106GGSKNNSEQ ID NO: 143ANGGKSSEQ ID NO: 183SSGSRESEQ ID NO: 224KTVSATSEQ ID NO: 184KGSAGSSEQ ID NO: 225VAGAKDSEQ ID NO: 265APGSASSEQ ID NO: 306NQGSGSSEQ ID NO: 266TGSGSGSEQ ID NO: 307GTGRGESEQ ID NO: 347GSQRGDSEQ ID NO: 388SSVRDSSEQ ID NO: 348SGGSQLSEQ ID NO: 389AAGSRESEQ ID NO: 425VGGSNTSEQ ID NO: 466GAGKTQSEQ ID NO: 426VQAQKPSEQ ID NO: 467GGGRGESEQ ID NO: 507SGVASSSEQ ID NO: 548VSPGQSSEQ ID NO: 508ESRGSSSEQ ID NO: 549PGPVTQSEQ ID NO: 589TSSTRDSEQ ID NO: 630PSSPKSSEQ ID NO: 590TSGSRDSEQ ID NO: 631LSTSHVSEQ ID NO: 671GASRDASEQ ID NO: 708VSGSRDSEQ ID NO: 672STSSGQSEQ ID NO: 709AGGHSASEQ ID NO: 749PQKSTSSEQ ID NO: 790EASRAGSEQ ID NO: 750VQSSAKSEQ ID NO: 791MTGGRESEQ ID NO: 831DTARSTSEQ ID NO: 868SAKGQSSEQ ID NO: 832ERTTAGSEQ ID NO: 869TGAGRESEQ ID NO: 909ATKQSGSEQ ID NO: 950DGTRSSSEQ ID NO: 910PGTGAASEQ ID NO: 951ERASGQSEQ ID NO: 987PGVGSHSEQ ID NO: 995PSVGSVSEQ ID NO: 988EVSGTRSEQ ID NO: 996ETTSRGre compnsertionsequence at a position between positions 449 and 460 and having 80% identity to a sequence provided in Table 2. Preferred capsids of the disclosure comprise an amino acid substitution and / or insertion sequence at a position between positions 449 and 460 and having 90% identity to a sequence provided in Table 2. Preferred capsids of the disclosure comprise an amino acid substitution and / or insertion sequence at a position between positions 449 and 460 and having 95% identity to a sequence provided in Table 2. Preferred capsids of the disclosure comprise an amino acid substitution and / or insertion sequence at a position between positions 449 and 460 and having 100% identity to a sequence provided in Table 2. TABLE 2 – insertion and / or substationSEQ ID NO: 1018RTIERSGSTQQTsequences found within positions 449-4601 1SEQ ID NO: 1039RTIERGSGNQQTSEQ ID NO: 1077RTINKTGSNTQTSEQ ID NO: 1040RTIQKGVASQQTSEQ ID NO: 1078RTINRGSSNEQT456, yielding: RTINXXXXXXNQQTSEQ ID NO: 1156RTINGASSRENQQTSEQ ID NOSequenceSEQ ID NO: 1157RTINNNAASGNQQTSEQ ID NO: 1117RTINKGSSSANQSEQ ID NO: 1197RTINTAAPGSNQQTSEQ ID NO: 1238RTINNTGSSGNQQTSEQ ID NO: 1198RTINKTGSAANQQTSEQ ID NO: 1239RTINPGTGSGNQQTSEQ ID NO: 1279RTINSGSGAGNQQTSEQ ID NO: 1320RTINSASRADNQQTSEQ ID NO: 1280RTINNTGTSTNQQTSEQ ID NO: 1321RTINAGARGENQQTSEQ ID NO: 1361RTINGRSTTQNQQTSubstitution of three amino acids and SEQ ID NO: 1362RTINGNERAQNQQTinsertion of three amino acids between 454- 456 i ldi RTINGXXXXXXN TSEQ ID NO: 1440RTINGTSGRGDNQQTSEQ ID NO: 1481RTINGTASRESNQQTSEQ ID NO: 1441RTINGSVSGRENQQTSEQ ID NO: 1482RTINGIGGSQQNQQTSEQ ID NO: 1522RTINGGASSHSNQQTSEQ ID NO: 1563RTINGVGGSATNQQTSEQ ID NO: 1523RTINGTQGQRENQQTSEQ ID NO: 1564RTINGTVSSGPNQQTSEQ ID NO: 1604RTINGKGPTGVNQQTSEQ ID NO: 1645RTINGRGGSSGNQQTSEQ ID NO: 1605RTINGSSREVSNQQTSEQ ID NO: 1646RTINGDSRSSGNQQTnSEQ ID NO: 1683RTINGTPSVGAQNQQTSEQ ID NO: 1724RTINGHAGSGAQNQQTSEQ ID NO: 1684RTINGRPNGTSQNQQTSEQ ID NO: 1725RTINGERSAGSQNQQTTSEQ ID NO: 1765RTINGAAERSSQNQQTSEQ ID NO: 1806RTINGRSDSHGQNQQTSEQ ID NO: 1766RTINGLGSGAGQNQQTSEQ ID NO: 1807RTINGEGRATSQNQQTnSEQ ID NO: 1844RTINGSGGKSGQNQQTSEQ ID NO: 1885RTINGSNGKGTTNQQTSEQ ID NO: 1845RTINGSAKSVSSNQQTSEQ ID NO: 1886RTINGSTPSPKGNQQTSEQ ID NO: 1926RTINGSKPSPTQNQQTSEQ ID NO: 1963RTINGSESSGRGQNQQTSEQ ID NO: 1927RTINGSSTHGAQNQQTSEQ ID NO: 1964RTINGSTGSQKGQNQQTTTTTTTTTTTTTTTTTTTT,Tyielding: RTINGSXXXXXXQNQQT SEQ ID NOSequence TTTTTTTTTTTTT

[0053] The present disclosure includes one or more AAV capsid protein comprising an insertion and / or substitution sequence of Formula A between positions 452-457, and / or wherein the capsid protein comprises an insertion and / or substitution sequence having at least 80% identity to a sequence having Formula A, Xa-Xb-Xc-Xd-Xe-Xf(A) wherein: Xais an amino acid selected from E D N S and T; Xbis an amino acid selected fromR, G, K, T, and S; Xcis an amino acid selected from S, G, T, E, and A; Xdis an amino acid selected from S, G, and T; Xeis an amino acid selected from S, T, G, Q, and A; and Xfis an amino acid selected from S, T, A, and K. In preferred aspects, Xais E, Xbis R, Xcis S, Xdis S, Xeis S, and / or Xfis S.

[0054] The present disclosure includes one or more AAV capsid protein comprising an insertion and / or substitution sequence of Formula B between positions 453-458, and / or wherein the capsid protein comprises an insertion and / or substitution sequence having at least 80% identity to a sequence having Formula B, Xa1-Xb1-Xc1-Xd1-Xe1-Xf1(B) wherein: Xa1is an amino acid selected from K, R, G, N, and T; Xb1is an amino acid selected from G, T, S, and D; Xc1is an amino acid selected from S, G, T, A, P and V; Xd1is an amino acid selected from S, Q, A and K; Xe1is an amino acid selected from T, S, N, A and G; and Xf1is an amino acid selected from S, G, E, and T. In some preferred aspects, Xa1is K, Xb1is G, Xc1is S, Xd1is S, Xe1is T, and / or Xf1is S.

[0055] The present disclosure includes AAV capsid proteins comprising an insertion and / or substitution sequence of Formula C between positions 453 and 456, and / or wherein the capsid protein comprises an insertion and / or substitution sequence having at least 80% identity to a sequence having Formula C, Xa2-Xb2-Xc2-Xd2-Xe2-Xf2(C) wherein: Xa2is an amino acid selXb2is an amino acid selected from G, S, A, and T; Xc2is an amino acid selected from G, S, A and T; Xd2is an amino acid selected from S, G, T, and R; Xe2is an amino acid selected from S, G, A, R and T; and Xf2is an amino acid selected from G, S, A, and E. In some preferred aspects, Xa2is K, Xb2is G, Xc2is S, Xd2is S, Xe2is T, and / or Xf2is G.

[0056] The present disclosure includes one or more AAV capsid protein comprising an insertion and / or substitution sequence of Formula D between positions 454 and 456, and / or wherein the capsid protein comprises an insertion and / or substitution sequence having at least 80% identity to a sequence having Formula D, Xa3-Xb3-Xc3-Xd3-Xe3-Xf3(D) wherein: Xa3is an amino acid selected from T, S, G, and V; Xb3is an amino acid selected from G, S, A, and T; Xc3is an amino acid selected from S, G, and P; Xd3is an amino acid selected from S, G, A and R; Xe3is an amino acid selected from G, S, R A, and T; and Xf3is an amino acid selected from S, Q, T, E, and A. In some preferred aspects, Xa3is T, Xb3is G, Xc3is S, Xd3is S, Xe3is G, and / or Xf3is S.

[0057] The present disclosure includes one or more AAV capsid protein comprising an insertion and / or substitution sequence of Formula E between positions 454 and 455 or positions 455 and 456, and / or wherein the capsid protein comprises an insertion and / or substitution sequence having at least 80% identity to a sequence having Formula E, Xa4-Xb4-Xc4-Xd4-Xe4-Xf4(E) wherein: Xa4is an amino acid selected from T, A, P, S, G, E, and V; Xb4is an amino acid selected from G, S, A, P, and T; Xc4is an amino acid selected from S, G, A, K, and T; Xd4is an amino acid selected from S, G, A and R; Xe4is an amino acid selected from G, S, R A, and T; and Xf4is an amino acid selected from S, Q, T, E, and A. In some preferred aspects, Xa4is T, Xb4is G, Xc4is S, Xd4is S, Xe4is G and / or Xf4is S.

[0058] The present disclosure includes one or more AAV capsid protein comprising an insertion and / or substitution sequence of Formula F at position 455, and / or wherein the capsid protein insertion and / or substitution sequence comprises a sequence having at least 80% identity to a sequence having Formula F, Xa5-Xb5-Xc5-Xd5-Xe5-Xf5(F) wherein: Xa5is an amino acid selected from A, P, T, and V; Xb5is an amino acid selected from G, S, T, and A; Xc5is an amino acid selected from G, A, T, and V; Xd5is an amino acid selected from G, S, A and K; Xe5is an amino acid selected from A, S, T, G, and R; and Xf5is an amino acid selected from G, A, K, S, and T. In some preferred aspects Xa5is A, Xb5is G, Xc5is G, Xd5is G, Xe5is A, and / or Xf5is G.

[0059] In certain aspects, the AAV capsid proteins of the disclosure having an amino acid substitution and / or insertion sequence between positions 449 and 460 further comprise one or more additional amino acid substitution and / or insertion sequences. In certain preferred aspects, the AAV capsid proteins further comprise an amino acid insertion and / or substitution sequence at one or more position between 583-599. The present disclosure includes AAV capsid proteins comprising: (i) an amino acid substitution and / or insertion sequence at one or more position between 583-599 having at least 80% identity to a sequence provided in Table 3; and (ii) an amino acid substitution and / or insertion sequence between positions 449 and 460 having at least 80% identity to a sequence as provided in Table 2 and / or Table 1. The present disclosure includes AAV capsid proteins comprising: (i) an amino acid substitution and / or insertion sequence at one or more position between 583-599 having at least 90% identity to a sequence provided in Table 3; and (ii) an amino acid substitution and / or insertion sequence between positions 449 and 460 having at least 90% identity to a sequence as provided in Table 2 and / or Table 1. The present disclosure includes AAV capsid proteins comprising: (i) an amino acid substitution and / or insertion sequence at one or more position between 583-599 having 100% identity to a sequence provided in Table 3; and (ii) an amino acid substitution and / or insertion sequence between positions 449 and 460 having 100% identity to a sequence as provided in Table 2 and / or Table 1.

[0060] In some preferred aspects, the amino acid insertion and / or substitution sequence at a position between 583-599 is at position 587-597.

[0061] In some preferred aspects, the amino acid insertion and / or substitution sequence at position 587-597 comprises Formula I: X1-X2-X3-X4-X5-X6-X7-X8-X9-X10-X11(I) wherein: X1is an amino acid selected from A, Q, H, K, T, S, N, and M, and preferably selected from A or Q; X2is an amino acid selected from Q, R, T, Y and M, and preferably from Q, R, and T; X3is an amino acid selected from R, E, L, and T, and is preferably R; X4is an amino acid selected from D, N, and S, and preferably from D and N; X5is an amino acid selected from G and H, and is preferably G; X6is an amino acid selected from L, V, A, and Q, and preferably from L and V; X7is an amino acid selected from I, H, and R, and is preferably I; X8is an amino acid selected from L, F, and S, and is preferably L; X9is an amino acid selected from I, K, V, Q, and A, and is preferably I; X10is an amino acid selected from A, S, E, Q, and T, and is preferably A; and X11is an amino acid selected from Q, P, and K.

[0062] In certain embodiments, in the AAV capsid protein comprising an amino acid insertion and / or substitution sequence of Formula I at position 587-597, X1may be a substitution or the parental amino acid at 587, X2may be a substitution or parental amino acid at position 588, X3, X4, X5, X6, X7, X8, and X9may be insertions, X10may be a substitution or parenteral amino acid at position 589 (i.e., now at position 596 in view of the insertion), and X11and may be a substitution or the parental amino acid at position 590 (i.e., now at position at 597 in view of the insertion).

[0063] In some preferred aspects, the amino acid insertion and / or substitution sequence at position 583-599 comprises Formula (II): X1-X2-X3-X4-X5-X6-X7-X8-X9-X10-X11-X12-X13-X14-X15-X16-X17(II) wherein: X1 is an amino acid selected from N, S, G, and T, and preferably selected from N; X2 is an amino acid selected from H, N, A, S, V, C, Q, I, T, L, W, M, K, F, R, G, and Y, and preferably H; X3is amino acid Q; X4is an amino acid selected from S, A, T, G, C, L, H, K, N, Q, R, Y, and M, and preferably from S, M, or G; X5 is an amino acid selected from A, Q, N, S, M, Y, G, H, D, W, T, F, V, I, C, L, T, and E, and is preferably A, Q, or S; X6 is an amino acid selected from T, V, Q, S, M, I, N, K, A, H, F, E, R, Y, and L, and preferably from T, V, or S; X7is an amino acid selected from R, K, L, M, Q, V, F, H, A, S, Y, T, W, and I, and is preferably R or K; X8 is an amino acid selected from N, D, G, Q, A, H, M, E, R, L, K, T, V, and S, and is preferably N or D; X9 is an amino acid selected from G, S, Q, A, D, K, E, T, H, M, V, I, L, and N, and is preferably G; X10is an amino acid selected from E, H, Q, A, T, G, D, S, K, V, R, and N, and is preferably E or H; X11is an amino acid selected from V, I, T, H, M, Q, A, S, P, N, R, K, and L, and is preferably V or I; X12 is an amino acid selected from F, L, Y, M, R, K, and H, and is preferably F, L, or Y; X13is an amino acid selected from I, Y, V, L, M, T, H, E, Q, S, A, N, and F, and is preferably I or Y; X14is an amino acid selected from A, and S, and is preferably A; X15 is an amino acid selected from Q, M, S, G, N, T, A, I, H, L, R, Y, and V, and is preferably Q, M, S, or N; X16is an amino acid selected from A, P, F, T, C, G, H, Q, V, Y, or S, and is preferably A; and X17is an amino acid selected from Q, A, S, L, M, N, D, T, V, H, I, or G, and is preferably Q.

[0064] In certain embodiments, in the capsids of AAV capsids comprising an AAV capsid protein comprising an amino acid sequence of Formula (II), X1is an amino acid selected from N, S, G, and T, and preferably selected from N; X2is an amino acid selected from H, N, A, and S, and preferably from H; X3 is amino acid Q; X4 is an amino acid selected from S, A, T, G, and M, andpreferably from S, M, or G; X5 is an amino acid selected from A, Q, N, S, M, Y, and E, and is preferably A, Q, or S; X6is an amino acid selected from T, V, Q, S, M and L, and preferably from T, V, or S; X7is an amino acid selected from R, K, L, M, and I, and is preferably R or K; X8is an amino acid selected from N, D, G, and S, and is preferably N or D; X9 is an amino acid selected from G, S, Q, A, and N, and is preferably G; X10 is an amino acid selected from E, H, Q, A, and N, and is preferably E or H; X11is an amino acid selected from V, I, T, and L, and is preferably V or I; X12 is an amino acid selected from F, L, Y, M, and H, and is preferably F, L, or Y; X13 is an amino acid selected from I, Y, V, and F, and is preferably I or Y; X14 is an amino acid selected from A, and S, and is preferably A; X15is an amino acid selected from Q, M, S, G, N, T, A and V, and is preferably Q, M, S, or N; X16is an amino acid selected from A or S, and is preferably A; and X17 is an amino acid selected from Q, A, or G, and is preferably Q.

[0065] In certain embodiments, in the AAV capsid protein comprising an amino acid insertion and / or substitution sequence of Formula II at position 583-599, X1 may be a substitution or the parental amino acid at 583, X2 may be a substitution or parental amino acid at position 584, X3 may be a substitution or parental amino acid at position 585, X4 may be a substitution or parental amino acid at position 586, X5 may be a substitution or parental amino acid at position 587, X6 may be a substitution or parental amino acid at position 587, X7, X8, X9, X10, X11, X12, and X13 may be insertions, X14 may be a substitution or parenteral amino acid at position 589 (i.e., now at position 596 in view of the insertion), X15 and may be a substitution or the parental amino acid at position 590 (i.e., now at position at 597 in view of the insertion), X16and may be a substitution or the parental amino acid at position 591 (i.e., now at position at 598 in view of the insertion), and X17 and may be a substitution or the parental amino acid at position 592 (i.e., now at position at 599 in view of the insertion).

[0066] In certain embodiments, the AAV capsid protein comprises an amino acid sequence of Formula II, wherein X1 is N.

[0067] In certain embodiments, the AAV capsid protein comprises an amino acid sequence of Formula II, wherein X2 is H.

[0068] In certain embodiments, the AAV capsid protein comprises an amino acid sequence of Formula II, wherein X3 is Q

[0069] In certain embodiments, the AAV capsid protein comprises an amino acid sequence of Formula II, wherein X4 is S or G.

[0070] In certain embodiments, the AAV capsid protein comprises an amino acid sequence of Formula II, wherein X5 is A or S.

[0071] In certain embodiments, the AAV capsid protein comprises an amino acid sequence of Formula II, wherein X6 is T or S.

[0072] In certain embodiments, the AAV capsid protein comprises an amino acid sequence of Formula II, wherein X7 is R or K.

[0073] In certain embodiments, the AAV capsid protein comprises an amino acid sequence of Formula II, wherein X8 is N or D.

[0074] In certain embodiments, the AAV capsid protein comprises an amino acid sequence of Formula II, wherein X9 is G.

[0075] In certain embodiments, the AAV capsid protein comprises an amino acid sequence of Formula II, wherein X10 is E or H.

[0076] In certain embodiments, the AAV capsid protein comprises an amino acid sequence of Formula II, wherein X11 is V or I.

[0077] In certain embodiments, the AAV capsid protein comprises an amino acid sequence of Formula II, wherein X12 is F, L, or Y.

[0078] In certain embodiments, the AAV capsid protein comprises an amino acid sequence of Formula II, wherein X13 is I or Y.

[0079] In certain embodiments, the AAV capsid protein comprises an amino acid sequence of Formula II, wherein X14is A.

[0080] In certain embodiments, the AAV capsid protein comprises an amino acid sequence of Formula II, wherein X15 is Q, M, or N.

[0081] In certain embodiments, the AAV capsid protein comprises an amino acid sequence of Formula II, wherein X16 is A or S.

[0082] In certain embodiments, the AAV capsid protein comprises an amino acid sequence of Formula II, wherein X17 is Q.

[0083] In some preferred aspects, the amino acid insertion and / or substitution sequence at one or more position between 583-599, including 587-599, comprises an amino acid sequence having at least 80% identity to a sequence as set forth in Table 3. In some preferred aspects, the amino acid insertion and / or substitution sequence at one or more position between 583-599 comprises an amino acid sequence having at least 90% identity to a sequence as set forth in Table 3. In somepreferred aspects, the amino acid insertion and / or substitution sequence at one or more position between 583-599 comprises an amino acid sequence as set forth in Table 3. TABLE 3 – 587-599 Sequences SEQ ID NO: 2002 QLENHVRSAAQAQ SEQ ID NO: 2041 AQENHVRMSSQAQ SEQ ID NO: 2003 ATRNGEHFHAQAQ SEQ ID NO: 2042 AQENHVRQSNIAQSEQ ID NO: 2080 AQENHVRAPGQAQ SEQ ID NO: 2123 SIRDGHILIASAQ SEQ ID NO: 2081 AEENHVRQSGQAQ SEQ ID NO: 2124 AIRNGEHFKAQAQSEQ ID NO: 2165 ASLGGQILIAMAQ SEQ ID NO: 2208 QHRDGLILIAAAQ SEQ ID NO: 2166 AQRDGQILIAMAQ SEQ ID NO: 2209 AKRDGLILIASAQSEQ ID NO: 2251 AQRNGAIFIAQAQ SEQ ID NO: 2294 QQRNGHDLIAQAQ SEQ ID NO: 2252 ATENQVRSSTLAQ SEQ ID NO: 2295 AQENHVRISSTAQSEQ ID NO: 2337 AQENHVRSTQDAQ SEQ ID NO: 2380 AQENHVRTQADAQ SEQ ID NO: 2338 NQENHVRASATAQ SEQ ID NO: 2381 ANENHVRDSAGAQSEQ ID NO: 2423 MQENHVRSSDGAQ SEQ ID NO: 2466 GQENHVRSDAMAK SEQ ID NO: 2424 AIRDGEHFKAQAQ SEQ ID NO: 2467 AQRNGGIFIAQAQSEQ ID NO: 2509 IQENHVRQSQQAQ SEQ ID NO: 2552 TQENHVRSYDQAQ SEQ ID NO: 2510 AQENHVRHQDQAQ SEQ ID NO: 2553 SQENHVRSNAQAQSEQ ID NO: 2595 AFENHVRSSAGAQ SEQ ID NO: 2638 VNENHVRSSATAQ SEQ ID NO: 2596 AQENHVRSSAMAQ SEQ ID NO: 2639 TQENHVRSSAMAQSEQ ID NO: 2681 AQENHVRKSDSAQ SEQ ID NO: 2724 DSENHVRSSIQAQ SEQ ID NO: 2682 AQENHVRNQANAQ SEQ ID NO: 2725 AYENHVRSQANAQSEQ ID NO: 2767 AVENHVRASNQAQ SEQ ID NO: 2810 EQENHVRSQAGAQ SEQ ID NO: 2768 PQENHVRVSAQAQ SEQ ID NO: 2811 AMRNGQHFQAQAQSEQ ID NO: 2853 AMRNGEHFNAQAQ SEQ ID NO: 2896 ATRDGEHFHAQAQ SEQ ID NO: 2854 AMTNGEHFQAQAQ SEQ ID NO: 2897 ATRDGHILIAVAQSEQ ID NO: 2939 RQRDGGILIATAQ SEQ ID NO: 2982 AQRDGIHFRAQAQ SEQ ID NO: 2940 SQLGGRILIAPAQ SEQ ID NO: 2983 MVRPPGYLLAKAQSEQ ID NO: 3030 MQENHVRSSAVAQ SEQ ID NO: 3079 APENHVRQSAAAQ SEQ ID NO: 3031 SLRNGEHFKAQAQ SEQ ID NO: 3080 AQENHVRILAEAQ 1 A ASEQ ID NO: 3128 AIRDGHILIAQAQ SEQ ID NO: 3177 QQRDGHILIAPAQ SEQ ID NO: 3129 AQRQGEHFAQQAQ SEQ ID NO: 3178 APENHVRQSAFAQ 1 A A A 1 A ASEQ ID NO: 3226 NQENHVRSSPAAQ SEQ ID NO: 3275 ALRDGHILIAMAQ SEQ ID NO: 3227 AMRNGEHFIAVAQ SEQ ID NO: 3276 SQRNGEIFIAQAQ 22 A A 2 A ASEQ ID NO: 3324 AMENHVRSQAEAQ SEQ ID NO: 3373 ATRDGHILIAVAQ SEQ ID NO: 3325 AQRDGHILIANAQ SEQ ID NO: 3374 ATSGGQILISQAQ 2 A A A ASEQ ID NO: 3422 ANENHVRSDATAQ SEQ ID NO: 3471 ASENHVRSSALAQ SEQ ID NO: 3423 AQENHVRSPDRAQ SEQ ID NO: 3472 ASENHVRSSNTAQ 424 A A 4 A A A ASEQ ID NO: 3520 QQRDGEILIKQAQ SEQ ID NO: 3569 NQENHVRSDNAAQ SEQ ID NO: 3521 SQENHVRSSPDAQ SEQ ID NO: 3570 AQENHVRSMPEAQ 22 A A A 1 ASEQ ID NO: 3618 QHLSGQILIAAAQ SEQ ID NO: 3667 AQRDGNILIAPAQ SEQ ID NO: 3619 ATRNGEHFQSQAQ SEQ ID NO: 3668 QQRDGRILIALAQ 2 A AA A ASEQ ID NO: 3716 RQRDGGILIVQAQ SEQ ID NO: 3765 SVRNGEHFIAQAQ SEQ ID NO: 3717 ATRDGSILIAAAQ SEQ ID NO: 3766 HQRDGGILIAAAQ 1 AA A A A ASEQ ID NO: 3814 DRRDGLILIASAQ SEQ ID NO: 3863 YYRDGHILIASAQ SEQ ID NO: 3815 ARLGGQILIMQAQ SEQ ID NO: 3864 YTRDGLILIASAQ 1 A AAASEQ ID NO: 3912 AKENHVRSEATAQ SEQ ID NO: 3950 QQRDGHILIAQAQ SEQ ID NO: 3913 AQENHVRASAGAQ SEQ ID NO: 3951 ANENHVRGSSQAQ 14 A A A A 2 A A A

[0084] Aspects disclosed herein provide AAV capsids comprising an AAV capsid protein comprising an amino acid sequence of any one of Formula A, Formula B, Formula C, Formula D, Formula E, and Formula F and an amino acid sequence of Formula I of Formula II. Aspects disclosed herein provide AAV capsids comprising an AAV capsid protein comprising an amino acid sequence set forth in Table 1 and / or 2 and as set forth in Table 3.

[0085] In some embodiments, the 583-599 insertion / substitution sequence is represented by the peptide sequences listed in Tables 3, 4, 5, and / or 6.

[0086] Generally, the modification at 449-460 comprises a four, five-, six-, or seven-amino acid sequence (4-mer, 5-mer, 6-mer, or 7-mer, respectively) that is inserted and / or substituted in variable region IV (VR-IV), which contains AA449-460 (VP1 numbering RTINGSGQNQQT) in a parental AAV capsid protein. In some preferred aspects, the modification at 449-460 comprises an insertion and / or substitution of a 6-mer at various positions within VR-IV, e.g., at positions 449-460. Aspects provided herein include AAV capsids comprising an AAV capsid protein in which the 449-460 modifications comprise a substitution of six amino acids between amino acids 452-457. Aspects provided herein include AAV capsids comprising an AAV capsid protein in which the 449-460 modifications comprise a substitution of six amino acids between amino acids 453-458. Aspects provided herein include AAV capsids comprising an AAV capsid protein in which the 449-460 modifications comprise a substitution of four amino acids and an insertion of 2 amino acids between amino acids 453-456. Aspects provided herein include AAV capsids comprising an AAV capsid protein in which the 449-460 modifications comprise a substitution of three amino acids and an insertion of three amino acids between amino acids 454-456. Aspects provided herein include AAV capsids comprising an AAV capsid protein in which the 449-460 modifications comprise a substitution of two amino acids and an insertion of four amino acids between amino acids 454-455. Aspects provided herein include AAV capsids comprising an AAV capsid protein in which the 449-460 modifications comprise a substitution of two amino acids and an insertion of four amino acids between amino acids 455-456. Aspects provided herein include AAV capsids comprising an AAV capsid protein in which the 449-460 modifications comprise a substitution of one amino acid and an insertion of five amino acids at position 455.

[0087] Generally, in the capsid proteins of the disclosure further comprising a variation at one or more position between 583-599, the insertion / substitution at 583-599 comprises a five-, six-, or seven-amino acid sequence (5-mer, 6-mer, or 7-mer, respectively) that is inserted or substituted in VR-VIII, which contains AA588 in a parental AAV capsid protein. Aspects provided herein provide amino acid insertions comprising seven amino acid polymer (7-mer) inserted between AA588-589 and may additionally include a substitution of one or two amino acids at amino acid positions flanking the 7-mer sequence (e.g., AA587-588 and / or AA589-590)to produce an eleven amino acid polymer (11-mer) within VR-VIII, preferably at AA588 of a parental AAV capsid protein.

[0088] Aspects disclosed herein provide AAV capsids comprising an AAV capsid protein comprising an amino acid substitution and / or insertion sequence at amino acids 587-590 selected from AAAQ, AADQ, AAEQ, ACAF, ADAA, ADAF, ADAH, ADAL, ADAQ, ADRK, ADSQ, ADTQ, AEAC, AEAQ, AFAI, AGAQ, AHAD, AHAR, AHEQ, AKAQ, ALAD, ALAQ, ALNQ, AMDQ, AMGF, AMNQ, AMNY, AMWQ, ANAQ, ANAS, ANRQ, ANSD, APDQ, AQAD, AQAK, AQAM, AQAN, AQAP, AQAR, AQAW, AQCG, AQDD, AQDG, AQDL, AQDQ, AQEQ, AQFF, AQFG, AQFQ, AQGQ, AQIK, AQIQ, AQND, AQNN, AQNT, AQPG, AQWD, ARDQ, ARGK, ASWW, ATAQ, AVAQ, AYAQ, AYSQ, CQAG, DGAQ, DNNQ, DNYQ, DQAE, DQAQ, DQLD, DQNS, DTAQ, DVAQ, DYAQ, ELAG, ELAQ, EMGQ, EQAF, EQAS, EQAY, EQGQ, EQNQ, ERAE, ESAQ, GQAQ, GQAY, GQPM, HMAQ, HQAD, HQAE, HQAR, HQDQ, IDAN, IEVQ, IHPQ, IMEQ, INAP, IQAN, IQAQ, KNHQ, KQRQ, LETQ, LPAN, LQAQ, LTAP, MDAQ, MEAQ, MFAQ, MLAV, MLDQ, MQAD, MQAQ, MQAS, MQMQ, MQPD, MTAQ, MTPQ, NDAH, NDAS, NQAQ, PAAD, PDAQ, PLAQ, PQAD, PQAE, QAAQ, RQPQ, SDAN, TMAQ, TNAN, TNAQ, TQAQ, TQDG, TQGQ, TQQQ, VEAG, VEAQ, VLAQ, VQAQ, VQDA, VQEQ, WQAQ, WQQQ, YQAQ, YQNQ, and YWAQ.

[0089] In some preferred embodiments, such a substitution is selected from ACAF, TNAQ, AMAF, SCAQ, SNAQ, FQAR, AQLC, AQAK, AQGT, AQEG, AQGQ, APDQ, TQAV, EQAS, MFAQ VEAQ, EQAY, DVAQ and MQMQ.

[0090] In some aspects, the insertion amino acid sequence is at least about 85% identical to an amino acid sequence provided in Tables 1-2 and / or Formulas A, B, C, D, E, and F and another insertion amino acid sequence that is at least 85% identical to an amino acid sequence provided in Tables 3-6 and / or Formulas I and II. In some aspects, the insertion amino acid sequence is at least about 85% identical to an amino acid sequence provided in Tables 1-2 and / or Formulas A, B, C, D, E, and F and another insertion amino acid sequence that is at least 90% identical to an amino acid sequence provided in Tables 3-6 and / or Formulas I and II. In some aspects, the insertion amino acid sequence is identical to an amino acid sequence provided in Tables 1-2 and / or Formulas A, B, C, D, E, and F and another insertion amino acid sequence that is identical to an amino acid sequence provided in Tables 3-6 and / or Formulas I and II.

[0091] Also disclosed herein are methods and kits for producing therapeutic recombinant AAV (rAAV) particles, as well as methods and pharmaceutical compositions or formulations comprising the rAAV particles, for the treatment of a disease or condition affecting the brain.

[0092] Disclosed herein are AAV capsids engineered with increased viral transduction in the brain. The AAV capsids can encapsidate a viral vector with a heterologous nucleic acid encoding, for example, a therapeutic gene expression product. Transduction of the heterologous nucleic acid in the brain can be achieved upon systemic delivery to a subject of the AAV capsid of the present disclosure encapsidating a heterologous nucleic acid. The AAV capsids disclosed herein are advantageous for many applications of gene therapy to treat human disease, including, but not limited to, disorders of the central nervous system.

[0093] The recombinant AAV vectors comprising a nucleic acid sequence encoding the AAV capsid proteins of the present disclosure as also provided herein. For example, the viral vectors of the present disclosure comprise a nucleic acid sequence comprising the AAV viral Cap (Capsid) encoding VP1, VP2, and VP3, at least one of which is modified to produce the AAV capsid proteins of the present disclosure. The recombinant AAV vector provided can be derived from an AAV serotype (e.g., AAV9) or a variant AAV serotype including an insertion of the present invention. AAV CAPSIDS

[0094] Provided herein are modified adeno-associated (AAV) virus capsid compositions useful for integrating a transgene into a target cell or environment (in a subject when they are administered systemically).

[0095] An rAAV comprises an AAV capsid that can be engineered to encapsidate a heterologous nucleic acid (e.g., therapeutic nucleic acid, gene editing machinery). The AAV capsid is made up of three AAV capsid protein monomers, VP1, VP2, and VP3. Sixty copies of these three VP proteins interact in a 1:1:10 ratio to form the viral capsid. VP1 covers the whole of VP2 protein in addition to a ~137 amino acid N-terminal region (VP1u), VP2 covers the whole of VP3 in addition to ~65 amino acid N-terminal region (VP1 / 2 common region). The three capsid proteins share a conserved amino acid sequence of VP3, which in some cases is the region beginning at amino acid position 138 (e.g., AA139-736).

[0096] While not wishing to be bound by theory, it is understood that a parent AAV capsid sequence comprises a VP1 region. In certain embodiments, a parent AAV capsid sequencecomprises a VP1, VP2 and / or VP3 region, or any combination thereof. A parent VP1 sequence may be considered synonymous with a parent AAV capsid sequence.

[0097] The AAV VP3 structure contains highly conserved regions that are common to all serotypes, a core eight-stranded β-barrel motif (βB-βI) and a small α-helix (αA). The loop regions inserted between the β-strands consist of the distinctive HI loop between β-strands H and I, the DE loop between β-strands D and E, and nine variable regions (VRs), which form the top of the loops. These VRs, such as the VR-VIII, which contains AA588 in AAV9, are found on the capsid surface and can be associated with specific functional roles in the AAV life cycle including receptor binding, transduction, and antigenic specificity.

[0098] In some aspects, the rAAV variant of the present invention comprises an AAV capsid protein having a peptide insertion at the residues corresponding to amino acids 449-460 and / or 588-589 of the AAV9 native sequence of SEQ ID NO: 1.

[0099] The AAV capsids comprise AAV capsid proteins (e.g., VP1, VP2, and VP3), each with an insertion, such as in the VR-VIII containing AA588 of a parental AAV capsid protein structure (AAV9 VP1 numbering). VR-VIII contains the site of heparan sulfate binding of AAV2 and is amenable to peptide display. The only known receptors for AAV9 are N-linked terminal galactose and AAV receptor (AAVR), but many indications point toward there being others. Modifications to AAV9 within VR-VIII are shown herein to confer increased transduction in target in vivo environments.

[0100] Disclosed herein are AAV capsids comprising AAV capsid proteins with insertions and substitutions in both VR-IV and VR-VIII that confer a higher transduction in brain cell types (e.g., brain endothelial cells, neurons, astrocytes). In particular, the AAV capsid proteins diosed herein enable rAAV-mediated transduction of a heterologous nucleic acid (e.g., transgene) in the brain of a subject. The AAV capsids of the present disclosure may be formulated as a pharmaceutical composition. In addition, the AAV capsids can be isolated and purified to be used for a variety of applications.

[0101] In some embodiments, the rAAV capsid of the present disclosure are generated using the methods disclosed herein. In some instances, the rAAV capsid is chimeric. In some instances, the rAAV, or variant AAV protein comprises therein, confer an increase in a localization of the rAAV within the target tissue, as compared to the parental AAV capsid or capsid protein. AAV Capsid Proteins

[0102] Disclosed herein are recombinant AAV (rAAV) capsids which comprise AAV capsid proteins that are engineered with a modified capsid protein (e.g., VP1, VP2, VP3). In some embodiments, the rAAV capsid proteins of the present disclosure are generated using the methods disclosed herein. In some embodiments, the AAV capsid proteins are used in the methods of delivering a therapeutic nucleic acid (e.g., a transgene) to a subject. In some instances, the rAAV capsid proteins have desired AAV expression rendering them particularly suitable for certain therapeutic applications, e.g., the treatment of a disease or disorder in a subject such as those disclosed herein.

[0103] The rAAV capsid proteins are engineered for optimized expression in the brain, for example the brain, of a subject upon systemic administration of the rAAV to the subject. The rAAV capsid proteins are engineered to include the insertions / substitutions provided in Tables 1, 2, 3, 4, 5, or 6, and / or Formulas A, B, C, D, E, and F and / or Formulas I and II. The rAAV capsid proteins including the sequences provided in Tables 1, 2, 3, 4, 5, or 6, and / or Formulas A, B, C, D, E, F, I, and / or II are engineered to achieve efficient transduction of an encapsidated transgene. In particular, the rAAV capsid proteins have increased expression in the brain of a subject.

[0104] The engineered AAV capsid proteins described herein have, in some cases, a capsid protein comprising a variation at position 449-460 further comprises an insertion and / or substitution of an amino acid that is heterologous to the parental AAV capsid protein at amino acid positions in the 588 loop. In some embodiments, the amino acid is not endogenous to the parental AAV capsid protein at the amino acid position of the insertion. The amino acid may be a naturally occurring amino acid in the same or equivalent amino acid position as the insertion of the substitution in a different AAV capsid protein.

[0105] The 6-mers and 7-mers described herein were advantageously generated using polymerase chain reaction (PCR) with degenerate primers, where each of the seven amino acids is encoded by a deoxyribose nucleic acid (DNA) sequence N-N-K. “N” is any of the four DNA nucleotides and K is guanine (G) or thymine (T). This method of generating random sequences, with reference to 7-mer amino acid sequences, enables upwards of 1.28 billion possible combinations at the protein level.

[0106] The rAAV capsid proteins of the present disclosure comprise an insertion of an amino acid in an amino acid sequence of an AAV capsid protein. The AAV capsid, from which an engineered AAV capsid protein of the present disclosure is produced, is referred to as a “parental” AAVcapsid. The complete genome of AAV1 is provided in GenBank Accession No. NC_002077; the complete genome of AAV2 is provided in GenBank Accession No. NC_001401 and Srivastava et al., J. Virol., 45: 555-564 (1983); the complete genome of AAV3 is provided in GenBank Accession No. NC_1829; the complete genome of AAV4 is provided in GenBank Accession No. NC_001829; the AAV5 genome is provided in GenBank Accession No. AF085716; the complete genome of AAV6 is provided in GenBank Accession No. NC_001862; at least portions of AAV7 and AAV8 genomes are provided in GenBank Accession Nos. AX753246 and AX753249, respectively; the AAV9 genome is provided in Gao et al., J. Virol., 78: 6381-6388 (2004); the AAV10 genome is provided in Mol. Ther., 13(1): 67-76 (2006); the AAV11 genome is provided in Virology, 330(2): 375-383 (2004); portions of the AAV12 genome are provided in Genbank Accession No. DQ813647; portions of the AAV13 genome are provided in Genbank Accession No. EU285562.

[0107] In some cases, the parental AAV is derived from an AAV with a serotype selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11 and AAV12. The AAV capsid protein that is “derived” from another may be a variant AAV capsid protein. A variant may include, for example, a heterologous amino acid in an amino acid sequence of the AAV capsid protein. The heterologous amino acid may be non-naturally occurring in the AAV capsid protein. The heterologous amino acid may be naturally occurring in a different AAV capsid protein. In some instances, the parental AAV capsid is described in US Pat Publication 2020 / 0165576 and U.S. Pat. App. Ser. No.62 / 832,826 and PCT / US20 / 20778; the content of each of which is incorporated herein.

[0108] In some instances, the parental AAV is AAV9. In some instances, the amino acid sequence of the AAV9 capsid protein comprises SEQ ID NO: 1. The amino acid sequence of AAV9 VP1 capsid protein (>tr|Q6JC40|Q6JC40_9VIRU Capsid protein VP1 OS=Adeno-associated virus 9 OX=235455 GN=cap PE=1 SV=1) is provided in SEQ ID NO: 1: (MAADGYLPDWLEDNLSEGIREWWALKPGAPQPKANQQHQDNARGLVLPGYKYLGPG NGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLKYNHADAEFQERLKEDTSFGG NLGRAVFQAKKRLLEPLGLVEEAAKTAPGKKRPVEQSPQEPDSSAGIGKSGAQPAKKRL NFGQTGDTESVPDPQPIGEPPAAPSGVGSLTMASGGGAPVADNNEGADGVGSSSGNWH CDSQWLGDRVITTSTRTWALPTYNNHLYKQISNSTSGGSSNDNAYFGYSTPWGYFDFN RFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTDNNGVKTIANNLTSTVQVFTDSDYQLPYVLGSAHEGCLPPFPADVFMIPQYGYLTLNDGSQAVGRSSFYCLEYFPSQML RTGNNFQFSYEFENVPFHSSYAHSQSLDRLMNPLIDQYLYYLSKTINGSGQNQQTLKFSV AGPSNMAVQGRNYIPGPSYRQQRVSTTVTQNNNSEFAWPGASSWALNGRNSLMNPGP AMASHKEGEDRFFPLSGSLIFGKQGTGRDNVDADKVMITNEEEIKTTNPVATESYGQVA TNHQSAQAQAQTGWVQNQGILPGMVWQDRDVYLQGPIWAKIPHTDGNFHPSPLMGGF GMKHPPPQILIKNTPVPADPPTAFNKDKLNSFITQYSTGQVSVEIEWELQKENSKRWNPE IQYTSNYYKSNNVEFAVNTEGVYSEPRPIGTRYLTRNL).

[0109] In some instances, the parental AAV capsid protein sequence is 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homologous to SEQ ID NO: 1.

[0110] AAV capsid proteins from naturally occurring AAV serotypes, such as AAV9, display peripheral organ tropism (including the liver) and activate the innate immune response. In some cases, transduction of peripheral organs can cause a severe inflammatory response in a subject, which can lead to multi-organ failure. By improving transduction of a native AAV serotype for a target in vivo tissue (e.g., brain), the rAAV particles of the present disclosure reduce the immunogenic properties of AAV-mediated transgene delivery and prevent activation of the innate immune response.

[0111] In some instances, the parental AAV capsid protein comprises the entire VPl region provided in SEQ ID NO: 1 (e.g., amino acids 1-736). In some instances, the parental AAV capsid protein comprises amino acids 217-736 in SEQ ID NO: 1, which is the common region found in VPl, VP2 and VP3 AAV9 capsid proteins. In some instances, the AAV capsid protein comprises amino acids 64-736 in SEQ ID NO: 1, which is the common region found in VPl and VP2. The parental AAV capsid protein sequence may comprise amino acids selected from 1-736, 10-736, 20-736, 30-736, 40-736, 50-736, 60-736, 70-736, 80-736, 90-736, 100-736, 110-736, 120-736, 130-736, 140-736, 150-736, 160-736, 170-736, 180-736, 190-736, 200- 736, 210-736, 220-736, 230-736, 240-736, 250-736, 260-736, 270-736, 280-736, 290-736, 300-736, 310-736, 320-736, 330-736, 340-736, 350-736, 360-736, 370-736, 380-736, 390-736, 400-736, 410-736, 420-736, 430-736, 440-736, and 450-736, from SEQ ID NO: 1. In some aspects, the rAAV variant comprises an AAV capsid protein comprising an amino acid sequence that is at least 98% identical to amino acid 217 to amino acid 736 of SEQ ID NO: 1. Insome instances, the amino acid insertion is at a three (3)-fold axis of symmetry of a corresponding parental AAV capsid protein.

[0112] Disclosed herein are insertions of an amino acid sequence in an AAV capsid protein. Where the sequence numbering designation “453-454”, for example, is noted for AAV9, for example AAV VP1, the invention also includes insertions in similar locations in the other AAV serotypes. As used herein, “AA453-454” indicates that the insertion and / or substitution of the amino acid (or amino acid sequence) is immediately after an amino acid (AA) at position 453 and immediately before an AA at position 455 within an amino acid sequence of a parental AAV VP capsid protein (VP1 numbering). Exemplary AAV capsid protein sequences are provided in Table A. For example, ENHTRSS is inserted at AA588-589 in an AAV9 capsid amino acid sequence and provides variant A. It is envisioned that the sequences disclosed herein (Tables 1, 2, 3, 4, 5, or 6, and Formulas A-F and I-II) may be inserted at one or more position between AA583-590, AA587-597 and / or AA449-460 in an amino acid sequence of a parental AAV9, a variant thereof, or equivalent amino acid position of a parental AAV of a different serotype (e.g., AAV1, AAV2, AAV3, and the like). In some AAV capsid protein sequences disclosed herein, the amino acid at position 449 is preferably R or K and more preferably R. TABLE A. Exemplary AAV Capsid Protein Sequences SEQ ID Identifier Sequence NO V G T G L R S K P PPPQILIKNTPVPADPPTAFNKDKLNSFITQYSTGQVSVEIEWE LQKENSKRWNPEIQYTSNYYKSNNVEFAVNTEGVYSEPRPI V G T G L R S V E P G P V G T G L R S V E P P V G TMASGGGAPVADNNEGADGVGSSSGNWHCDSQWLGDRVIT TSTRTWALPTYNNHLYKQISNSTSGGSSNDNAYFGYSTPWG K L R S V E P G P V G T G L R S V E P P V G T G L R S V EGEDRFFPLSGSLIFGKQGTGRDNVDADKVMITNEEEIKTTNP VATESYGQVATNHQSAQGNGTRLTAQAQTGWVQNQGILP P V G T G L R S V E P P V G T G L R S V E P PYDQQLKAGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAV FQAKKRLLEPLGLVEEAAKTAPGKKRPVEQSPQEPDSSAGIG T G L R S V E P G P V G T G L R S V E P P

[0113] The insertions described herein may, in some cases, comprise a 6-mer insertion and / or substitution sequence at one or more position between 449-460 and optionally, a 7-mer substitution and / or insertion at AA588-589. It is envisioned that a 6-mer and / or 7-mer insertion / substitution disclosed herein in addition to a substitution with any amino acid at an amino acid position between 449-460 and / or 587-597 of SEQ ID NO: 1 may be a portion of an 11-mer.

[0114] Disclosed herein are AAV capsid proteins with an insertion described above in a parental AAV capsid protein that confers an increased transduction in the brain in a subject, even when delivered systemically. One of the many advantages of the AAV capsid proteins described herein is their ability to target tissue and cells within the brain. The tissue can be the brain. Non-limiting examples of brain cells include a neuron and a glial cell. Glial cells can be selected from an oligodendrocyte, an ependymal cell, an astrocyte, and a microglia.

[0115] In some instances, the AAV capsid protein comprises an insertion of at least or about six, seven, eight, nine, ten or eleven amino acids of an amino acid sequence of Tables 1-2 and / or Formulas A-F and / or Tables 3-6 and / or Formulas I-II at an amino acid position 449-460 and / or 583-590 in a parental AAV9 capsid protein (SEQ ID NO: 1). In some cases, the AAV capsid protein has an increased viral transduction enrichment in brain.

[0116] In some instances, the AAV capsid protein comprises an insertion and / or substitution of at least or about five, six, or seven, amino acids of an amino acid sequence of Table 1 and / or 2 at an amino acid position 449-460 in a parental AAV9 capsid protein (SEQ ID NO: 1). In some cases, the AAV capsid protein has an increased viral transduction enrichment in brain.

[0117] The rAAV capsid proteins described herein may be isolated and purified. The AAV may be isolated and purified by methods standard in the art such as by column chromatography, iodixanol gradients, or cesium chloride gradients. Methods for purifying AAV from helper virus are known in the art and may include methods disclosed in, for example, Clark et al., Hum. Gene Ther., 10(6): 1031-1039 (1999); Schenpp and Clark, Methods Mol. Med., 69: 427-443 (2002); U.S. Patent No.6,566,118 and WO 98 / 09657.

[0118] In addition, the AAV capsid proteins disclosed herein, either isolated and purified, or not, may be formulated into a pharmaceutical formulation, which in some cases, further comprises a pharmaceutically acceptable carrier.

[0119] The rAAV capsid protein can be conjugated to a nanoparticle, a second molecule, or a viral capsid protein. In some cases, the nanoparticle or viral capsid protein would encapsidate the therapeutic nucleic acid described herein. In some instances, the second molecule is a therapeutic agent, e.g., a small molecule, antibody, antigen-binding fragment, peptide, or protein, such as those described herein.

[0120] “Percent identity” or “identity” is the percent of the symbols that actually match. Percent Similarity is the percent of the symbols that are similar. Symbols that are across from gaps are ignored. A similarity is scored when the scoring matrix value for a pair of symbols is greater than or equal to 0.50, the similarity threshold. The scoring matrix used in Version 10 of the Wisconsin Genetics Software Package is BLOSUM62 (see: Henikoff and Henikoff, (1989) Proc. Natl. Acad. Sci. USA 89: 10915).

[0121] Sequence identity / similarity values provided herein can refer to the value obtained using the BLAST+ 2.5.0 suite of programs using default settings (blast.ncbi.nlm.nih.gov) (Camacho, C., et al. (2009) BLAST+: architecture and applications. BMC Bioinformatics 10:421).

[0122] As those of ordinary skill in the art will understand, BLAST searches assume that proteins can be modeled as random sequences. However, many real proteins comprise regions of nonrandom sequences, which may be homopolymeric tracts, short-period repeats, or regions enriched in one or more amino acids. Such low-complexity regions may be aligned between unrelated proteins even though other regions of the protein are entirely dissimilar. A number of low-complexity filter programs can be employed to reduce such low-complexity alignments. For example, the SEG (Wooten and Federhen, (1993) Comput. Chem.17: 149-63) and XNU (Ci-ayerie and States (1993) Comput. Chem.17: 191-201) low-complexity filters can be employed alone or in combination.

[0123] The terms “substantial identity” and “substantially identical” indicate that a polypeptide or nucleic acid comprises a sequence with between 55-100% sequence identity to a reference sequence, with at least 55% sequence identity, or at least 60%, or at least 65%, or at least 70%, or at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 95%, or at least 99% sequence identity or any percentage of value within the range of 55-100% sequence identity relative to the reference sequence. The percent sequence identity may occur over a specified comparison window. Optimal alignment may be ascertained or conducted using the homology alignment algorithm of Needleman and Wunsch, supra.

[0124] For example, the insertion sequences may include, but are not limited to, sequences that are not exactly the same as the sequences disclosed herein, but which have, in addition to the substitutions explicitly described for various sequences listed herein, additional substitutions of amino acid residues which substantially do not impair the activity or properties of the sequences described herein, such as those predicted by homology software e.g. BLOSUM62 matrices.AAV PARTICLES

[0125] The rAAV particles with the insertion sequences described herein have an increased transduction enrichment in the brain. In some instances, the increased transduction enrichment comprises a 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold or 10-fold increase, or more. In some instances, the increased transduction enrichment is at least 1-fold. In some instances, the increased transduction enrichment is at least 2-fold. In some instances, the increased transduction enrichment is at least 4-fold.

[0126] The rAAV particles with the insertion sequences described herein have an increased expression enrichment in the brain. Detecting whether a rAAV possesses more or less specificity for a target in vivo environment, includes measuring a level of gene expression product (e.g., RNA or protein) expressed from the heterologous nucleic acid encapsidated by the rAAV in a tissue sample obtained from a subject. Suitable methods for measuring expression of a gene expression product include next-generation sequencing (NGS) and quantitative polymerase chain reaction (qPCR). HETEROLOGOUS NUCLEIC ACIDS

[0127] Disclosed herein are therapeutic nucleic acids useful for the treatment or prevention of a disease or condition, or symptom of the disease or condition. In some embodiments, the therapeutic nucleic acids encode a therapeutic gene expression product. Non-limiting examples of gene expression products include proteins, polypeptides, peptides, enzymes, antibodies, antigen binding fragments, nucleic acid (RNA, DNA, antisense oligonucleotide, siRNA, and the like), and gene editing components, for use in the treatment, prophylaxis, and / or amelioration of the disease or disorder, or symptoms of the disease or disorder. In some instances, the therapeutic nucleic acids are placed in an organism, cell, tissue or organ of a subject by way of a rAAV, such as those disclosed herein.

[0128] Disclosed herein are rAAVs, each comprising a viral vector (e.g., a single stranded DNA molecule (ssDNA)). In some instances, the viral vector comprises two inverted terminal repeat (ITR) sequences that are about 145 bases each, flanking a transgene. In some embodiments, the transgene comprises a therapeutic nucleic acid, and in some cases, a promoter in cis with the therapeutic nucleic acid in an open reading frame (ORF). The promoter is capable of initiating transcription of therapeutic nucleic acid in the nucleus of the target cell. The ITR sequences canbe from any AAV serotype. Non-limiting examples of AAV serotypes include AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, and AAV12. In some cases, an ITR is from AAV2. In some cases, an ITR is from AAV9.

[0129] Disclosed herein are transgenes that can comprise any number of nucleotides. In some cases, a transgene can comprise less than about 100 nucleotides. In some cases, a transgene can comprise at least about 100 nucleotides. In some cases, a transgene can comprise at least about 200 nucleotides. In some cases, a transgene can comprise at least about 300 nucleotides. In some cases, a transgene can comprise at least about 400 nucleotides. In some cases, a transgene can comprise at least about 500 nucleotides. In some cases, a transgene can comprise at least about 1000 nucleotides. In some cases, a transgene can comprise at least about 5000 nucleotides. In some cases, a transgene can comprise over 5,000 nucleotides. In some cases, a transgene can comprise between about 500 and about 5000 nucleotides. In some cases, a transgene comprises about 5000 nucleotides. In any of the cases disclosed herein, the transgene can comprise DNA, RNA, or a hybrid of DNA and RNA. In some cases, the transgene can be single stranded. In some cases, the transgene can be double stranded.

[0130] Disclosed herein are transgenes useful for modulating the expression or activity of a target gene or gene expression product thereof. In some instances, the transgene is encapsidated by an rAAV capsid protein of an rAAV particle described herein. In some instances, the rAAV particle is delivered to a subject to treat a disease or condition disclosed herein in the subject. In some instances, the delivery is systemic.

[0131] The transgenes disclosed herein are useful for expressing an endogenous gene at a level similar to that of a healthy or normal individual. This is particularly useful in the treatment of a disease or condition related to the under-expression, or lack of expression, of a gene expression product. In some embodiments, the transgenes disclosed herein are useful for overexpressing an endogenous gene, such that an expression level of the endogenous gene is above the expression level of a healthy or normal individual. Additionally, transgenes can be used to express exogenous genes (e.g., active agent such as an antibody, peptide, nucleic acid, or gene editing components). In some embodiments, the therapeutic gene expression product is capable of altering, enhancing, increasing, or inducing the activity of one or more endogenous biological processes in the cell. In some embodiments, the transgenes disclosed herein are useful for reducing expression of an endogenous gene, for example, a dominant negative gene. In some embodiments, the therapeuticgene expression product is capable of altering, inhibiting, reducing, preventing, eliminating, or impairing the activity of one or more endogenous biological processes in the cell. In some aspects, the increase of gene expression refers to an increase by at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95% and 100%. In one aspect, the protein product of the targeted gene may be increased by at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95% and 100%. In some aspects, the decrease of gene expression refers to an increase by at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95% and 100%. In one aspect, the protein product of the targeted gene may be decreased by at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95% and 100%.

[0132] When endogenous sequences (endogenous or part of a transgene) are expressed with a transgene, the endogenous sequences can be full-length sequences (wild-type or mutant) or partial sequences. The endogenous sequences can be functional. Non-limiting examples of the function of these full length or partial sequences include increasing the serum half-life of the polypeptide expressed by a transgene (e.g., therapeutic gene) and / or acting as a carrier.

[0133] A transgene can be inserted into an endogenous gene such that all, some or none of the endogenous gene is expressed. For example, a transgene as described herein can be inserted into an endogenous locus such that some (N-terminal and / or C-terminal to a transgene) or none of the endogenous sequences are expressed, for example as a fusion with a transgene. In other cases, a transgene (e.g., with or without additional coding sequences of the endogenous gene) is integrated into any endogenous locus, for example a safe-harbor locus. For example, a Frataxin (FXN) transgene can be inserted into an endogenous FXN gene. A transgene can be inserted into any gene, e.g., the genes as described herein.

[0134] At least one advantage of the present disclosure is that virtually any therapeutic nucleic acid may be used to express any therapeutic gene expression product. In some instances, the therapeutic gene expression product is a therapeutic protein or a peptide (e.g., antibody, antigen- binding fragment, peptide, or protein). In one embodiment the protein encoded by the therapeutic nucleic acid is between 50-5000 amino acids in length. In some embodiments the protein encoded is between 50-2000 amino acids in length. In some embodiments the protein encoded is between 50-1000 amino acids in length. In some embodiments the protein encoded is between 50-1500 amino acids in length. In some embodiments the protein encoded is between 50-800 amino acids in length. In some embodiments the protein encoded is between 50-600 amino acids in length. Insome embodiments the protein encoded is between 50-400 amino acids in length. In some embodiments the protein encoded is between 50-200 amino acids in length. In some embodiments the protein encoded is between 50-100 amino acids in length. In some embodiments the peptide encoded is between 4-50 amino acids in length. In some embodiments, the protein encoded is a tetrapeptide, a pentapeptide, a hexapeptide, a heptapeptide, an octapeptide, a nonapeptide, or a decapeptide. In some embodiments, the protein encoded comprises a peptide of 2-30 amino acids, such as for example 5-30, 10-30, 2-25, 5-25, 10-25, or 10-20 amino acids. In some embodiments, the protein encoded comprises a peptide of at least 11, 12, 13, 14, 15, 17, 20, 25 or 30 amino acids, or a peptide that is no longer than 50 amino acids, e.g., no longer than 35, 30, 25, 20, 17, 15, 14, 13, 12, 11 or 10 amino acids.

[0135] Non-limiting examples of therapeutic protein or peptides include an adrenergic agonist, an anti-apoptosis factor, an apoptosis inhibitor, a cytokine receptor, a cytokine, a cytotoxin, an erythropoietic agent, a glutamic acid decarboxylase, a glycoprotein, a growth factor, a growth factor receptor, a hormone, a hormone receptor, an interferon, an interleukin, an interleukin receptor, a kinase, a kinase inhibitor, a nerve growth factor, a netrin, a neuroactive peptide, a neuroactive peptide receptor, a neurogenic factor, a neurogenic factor receptor, a neuropilin, a neurotrophic factor, a neurotrophin, a neurotrophin receptor, an N-methyl-D-aspartate antagonist, a plexin, a protease, a protease inhibitor, a protein decarboxylase, a protein kinase, a protein kinsase inhibitor, a proteolytic protein, a proteolytic protein inhibitor, a semaphoring, a semaphorin receptor, a serotonin transport protein, a serotonin uptake inhibitor, a serotonin receptor, a serpin, a serpin receptor, and a tumor suppressor. In certain embodiments, the therapeutic protein or peptide is selected from brain-derived neurotrophic factor (BDNF), ciliary neurotrophic factor (CNTF), macrophage colony-stimulating factor (CSF), epidermal growth factor (EGF), fibroblast growth factor (FGF), gonadotropin, interferon-gamma (IFN), insulin-like growth factor 1 (IFG-1), nerve growth factor (NGF), platelet-derived growth factor (PDGF), pigment epithelium-derived factor (PEDF), transforming growth factor (TGF), transforming growth factor-beta (TGF-B), tumor necrosis factor (TNF), vascular endothelial growth factor (VEGF), prolactin, somatotropin, X-linked inhibitor of apoptosis protein 1 (XIAP1), interleukin 1 (IL-1), IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-10, viral IL-10, IL-11, IL-12, IL- 13, IL-14, IL-15, IL-16, IL-17, and IL-18.

[0136] A therapeutic gene expression product can comprise gene editing components. Non- limiting examples of gene editing components include those required for CRISPR / Cas, artificial site-specific RNA endonuclease (ASRE), zinc finger endonuclease (ZFN), and transcription factor like effector nuclease (TALEN). In a non-limiting example, a subject having Huntington's disease is identified. The subject is then systemically administered a first amount of a rAAV encapsidating a viral vector encoding ZFN engineered to represses the transcription of the Huntingtin (HTT) gene. The rAAV will include a modified AAV capsid protein that includes an amino acid sequence provided in any one of Tables 1, 2, 3, 4, 4A, 5, 5A, 5B, 6, 6A and / or 6B, Formulas A-F, and Formulas I-II, so as to allow proper targeting of the ZFN to the nervous system, while reducing expression in off-target organs, such as the liver. If needed, the subject is administered a second or third dose of the rAAV, until a therapeutically effective amount of the ZFN is expressed in the subject’s nervous system.

[0137] A therapeutic nucleic acid can comprise a non-protein coding gene e.g., sequences encoding antisense RNAs, RNAi, shRNAs and micro RNAs (miRNAs), miRNA sponges or decoys, recombinase delivery for conditional gene deletion, conditional (recombinase-dependent) expression, includes those required for the gene editing components described herein. The non- protein coding gene may also encode a tRNA, rRNA, tmRNA, piRNA, double stranded RNA, snRNA, snoRNA, and / or long non-coding RNA (IncRNA). In some cases, the non-protein coding gene can modulate the expression or the activity of a target gene or gene expression product. For example, the RNAs described herein may be used to inhibit gene expression in the brain. In some cases, inhibition of gene expression refers to an inhibition by at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95% and 100%. In some cases, the protein product of the targeted gene may be inhibited by at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95% and 100%. The gene can be either a wild type gene or a gene with at least one mutation. The targeted protein may be either a wild-type protein or a protein with at least one mutation.

[0138] A therapeutic nucleic acid can modulate the expression or activity of a gene or gene expression product expressed from the gene that is implicated in a disease or disorder of the brain. For example, the therapeutic nucleic acid, in some cases is a gene or a modified version of the gene described herein. In some instances, the gene or gene expression product is inhibited. In some instances, the gene or gene expression product is enhanced.

[0139] In another example, the therapeutic nucleic acid comprises an effector gene expression product such as a gene editing component specific to target a gene therein. Non-limited examples of genes include target gene or gene expression product selected from ATP1A2, CACNAIA, SETD5, SHANK3, NF2, DNMTl, TCF4, RAI1, PEXl, ARSA, EIF2B5, EIF2B1, EIF2B2, NPCl, ADAR, MFSD8, STXBPl, PRICKLE2, PRRT2, IDUA, STX1B, Sarcoglycan Alpha (SGCA), glutamic acid decarboxylase 65 (GAD65), glutamic acid decarboxylase 67 (GAD67), CLN2, Nerve Growth Factor (NGF), glial cell derived neurotrophic factor (GDNF), Survival Of Motor Neuron 1 , Telomeric (SMNl), Factor X (FIX), Retinoid Isomerohydrolase (RPE65), sarco / endoplasmic reticulum Ca2+-ATPase (SERCA2a), Glucocerebrosidase (GCase), galactocerebrosidase (GALC), CDKL5, Frataxin (FXN), Huntingtin (HTT), methyl-CpG binding protein 2 (MECP2), a peroxisomal biogenesis factor (PEX), progranulin (GRN), an antitubulin agent, copper-zinc superoxide dismutase (SODl), iduronate 2 sulfatase (hIDS), Glucosylceramidase Beta (GBA), fragile X mental retardation 1 (FMR1), NPC Intracellular Cholesterol Transporter 1 (NPCl), SCN1A, C9orf72, NPS3 and a NLRP3 inflammasome. In some embodiments, the peroxisomal biogenesis factor (PEX) is selected from PEX1, PEX2, PEX3, PEX4, PEX5, PEX6, PEX7, PEX10, PEX11β, PEX12, PEX13, PEX14, PEX16, PEX19, and PEX26. In some instances, the gene or gene expression product is inhibited. In some instances, the gene or gene expression product is enhanced. AAV Vectors

[0140] Aspects disclosed herein comprise plasmid vectors comprising a nucleic acid sequence encoding the AAV capsids and AAV capsid proteins described herein. AAV vectors described herein are useful for the assembly of a rAAV and viral packaging of a heterologous nucleic acid. In addition, an AAV vector may encode a transgene comprising the heterologous nucleic acid.

[0141] An AAV vector can comprise a transgene, which in some cases encodes a heterologous gene expression product (e.g., therapeutic gene expression product, recombinant capsid protein, and the like). The transgene is in cis with two inverted terminal repeats (ITRs) flanking the transgene. The transgene may comprise a therapeutic nucleic acid encoding a therapeutic gene expression product. Due to the limited packaging capacity of the rAAV (~5kB), in some cases, a longer transgene may be split between two AAV vectors, the first with 3’ splice donor and the second with a 5’ splice acceptor. Upon co-infection of a cell, concatemers form, which are spliced together to express a full-length transgene.

[0142] A transgene is generally inserted so that its expression is driven by the endogenous promoter at the integration site, namely the promoter that drives expression of the endogenous gene into which a transgene is inserted. In some instances, a transgene comprises a promoter and / or enhancer, for example a constitutive promoter or an inducible or tissue / cell specific promoter. As a non-limiting example, the promoter may be CMV promoter, a CMV-β-Actin- intron-β-Globin hybrid promoter (CAG), CBA promoter, FRDA or FXN promoter, UBC promoter, GUSB promoter, NSE promoter, Synapsin promoter, MeCP2 promoter, GFAP promoter, H1 promoter, U6 promoter, NFL promoter, NFH promoter, SCN8A promoter, or PGK promoter. As a non-limiting example, promoters can be tissue-specific expression elements include, but are not limited to, human elongation factor 1α-subunit (EF1α), immediate-early cytomegalovirus (CMV), chicken β-actin (CBA) and its derivative CAG, the β glucuronidase (GUSB), and ubiquitin C (UBC). The transgene may include a tissue-specific expression elements for neurons such as, but not limited to, neuron-specific enolase (NSE), platelet-derived growth factor (PDGF), platelet-derived growth factor B-chain (PDGF-β), the synapsin (Syn), the methyl- CpG binding protein 2 (MeCP2), Ca2+ / calmodulin-dependent protein kinase II (CaMKII), metabotropic glutamate receptor 2 (mGluR2), NFL, NFH, np32, PPE, Enk and EAAT2 promoters. The transgene may comprise a tissue-specific expression element for astrocytes such as, but not limited to, the glial fibrillary acidic protein (GFAP) and EAAT2 promoters. The transgene may comprise tissue-specific expression elements for oligodendrocytes such as, but not limited to, the myelin basic protein (MBP) promoter.

[0143] In some embodiments, the promoter is less than 1 kb. The promoter may have a length of 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800 or more than 800. The promoter may have a length between 200-300, 200-400, 200-500, 200-600, 200-700, 200-800, 300-400, 300-500, 300-600, 300-700, 300-800, 400-500, 400-600, 400-700, 400-800, 500-600, 500-700, 500-800, 600-700, 600-800 or 700-800. The promoter may provide expression of the therapeutic gene expression product for a period of time in targeted tissues such as, but not limited to, the brain. Expression of the therapeutic gene expression product may be for a period of 1 hour, 2, hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 2 weeks, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 3 weeks, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days, 31 days, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year, 13 months, 14 months, 15 months, 16 months, 17 months, 18 months, 19 months, 20 months, 21 months, 22 months, 23 months, 2 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, 10 years, 11 years, 12 years, 13 years, 14 years, 15 years, 16 years, 17 years, 18 years, 19 years, 20 years, 21 years, 22 years, 23 years, 24 years, 25 years, 26 years, 27 years, 28 years, 29 years, 30 years, 31 years, 32 years, 33 years, 34 years, 35 years, 36 years, 37 years, 38 years, 39 years, 40 years, 41 years, 42 years, 43 years, 44 years, 45 years, 46 years, 47 years, 48 years, 49 years, 50 years, 55 years, 60 years, 65 years, or more than 65 years. Expression of the payload may be for 1-5 hours, 1-12 hours, 1-2 days, 1-5 days, 1-2 weeks, 1-3 weeks, 1-4 weeks, 1-2 months, 1-4 months, 1-6 months, 2-6 months, 3-6 months, 3-9 months, 4-8 months, 6-12 months, 1-2 years, 1-5 years, 2-5 years, 3-6 years, 3-8 years, 4-8 years or 5-10 years or 10-15 years, or 15-20 years, or 20-25 years, or 25-30 years, or 30-35 years, or 35-40 years, or 40-45 years, or 45-50 years, or 50-55 years, or 55-60 years, or 60-65 years.

[0144] An AAV vector can comprise a genome of a helper virus. Helper virus proteins are required for the assembly of a recombinant AAV (rAAV), and packaging of a transgene containing a heterologous nucleic acid into the rAAV. The helper virus genes are adenovirus genes E4, E2a and VA, that when expressed in the cell, assist with AAV replication. In some embodiments, an AAV vector comprises E2. In some embodiments, an AAV vector comprises E4. In some embodiments, an AAV vector comprises VA. In some instances, the AAV vector comprises one of helper virus proteins, or any combination.

[0145] The target gene or gene expression product for use in a transgene can be selected from ATP1A2, CACNAIA, SETD5, SHANK3, NF2, DNMTl, TCF4, RAI1, PEXl, ARSA, EIF2B5, EIF2B1, EIF2B2, NPCl, ADAR, MFSD8, STXBPl, PRICKLE2, PRRT2, IDUA, STX1B, Sarcoglycan Alpha (SGCA), glutamic acid decarboxylase 65 (GAD65), glutamic acid decarboxylase 67 (GAD67), CLN2, Nerve Growth Factor (NGF), glial cell derived neurotrophic factor (GDNF), Survival Of Motor Neuron 1, Telomeric (SMNl), Factor X (FIX), Retinoid Isomerohydrolase (RPE65), sarco / endoplasmic reticulum Ca2+-ATPase(SERCA2a), Glucocerebrosidase (GCase), galactocerebrosidase (GALC), CDKL5, Frataxin (FXN), Huntingtin (HTT), methyl-CpG binding protein 2 (MECP2), a peroxisomal biogenesis factor (PEX), progranulin (GRN), an antitubulin agent, copper-zinc superoxide dismutase (SODl), iduronate 2 sulfatase (hIDS), Glucosylceramidase Beta (GBA), fragile X mental retardation 1 (FMR1), NPC Intracellular Cholesterol Transporter 1 (NPCl), SCN1A, C9orf72, NPS3 and a NLRP3 inflammasome. In some embodiments, the peroxisomal biogenesis factor (PEX) is selected from PEX1, PEX2, PEX3, PEX4, PEX5, PEX6, PEX7, PEX10, PEX11β, PEX12, PEX13, PEX14, PEX16, PEX19, and PEX26.

[0146] An AAV vector can comprise a viral genome comprising a nucleic acid encoding the recombinant AAV (rAAV) capsid protein described herein. The viral genome can comprise a Replication (Rep) gene encoding a Rep protein, and Capsid (Cap) gene encoding an AAP protein in the first open reading frame (ORF1) or a Cap protein in the second open reading frame (ORF2). The Rep protein is selected from Rep78, Rep68, Rep52, and Rep40. In some instances, the Cap gene is modified encoding a modified AAV capsid protein described herein. A wild-type Cap gene encodes three proteins, VP1, VP2, and VP3. In some cases, VP1 is modified. In some cases, VP2 is modified. In some cases, VP3 is modified. In some cases, all three VP1-VP3 are modified. The AAV vector can comprise nucleic acids encoding wild-type Rep78, Rep68, Rep52, Rep40 and AAP proteins. Methods of Producing rAAVs

[0147] Disclosed herein are methods of producing the AAV capsids comprising the AAV capsid proteins and viral vector encoding a therapeutic nucleic acid. The AAV capsid proteins are produced by introduction into a cell (e.g., immortalized stem cell) a first vector containing a transgene cassette flanked by inverted terminal repeat (ITR) sequences from a parental AAV virus (the transgene cassette has a promoter sequence that drives transcription of a heterologous nucleic acid in the nucleus of the target cell), a second vector encoding the AAV genome with a AAV capsid protein (encoding the AAV Rep gene as well as the modified Cap gene for the variant being produced), and a third vector encoding helper virus proteins, required for assembly of the AAV capsid structure and packaging of the transgene in the modified AAV capsid structure. The assembled AAV capsid can be isolated and purified from the cell using suitable methods known in the art.

[0148] The transgenes contained in a recombinant AAV (rAAV) vector and encapsidated by the AAV capsid proteins of the present disclosure are also provided herein. The transgenes disclosed herein are delivered to a subject for a variety of purposes, such as to treat a disease or condition in the subject. The transgene can be gene editing components that modulate the activity or expression of a target gene or gene expression product. Alternatively, the transgene is a gene encoding a therapeutic gene expression product that is effective to modulate the activity or expression of itself, or another target gene or gene expression product.

[0149] Aspects disclosed herein provide methods of manufacturing rAAV virus or virus particles comprising: (a) introducing into a cell a nucleic acid comprising: (i) first vector containing a transgene cassette flanked by inverted terminal repeat (ITR) sequences from a parental AAV virus (the transgene cassette has a promoter sequence that drives transcription of a heterologous nucleic acid in the nucleus of the target cell); (ii) a second vector encoding the AAV genome with a AAV capsid protein of the present invention; and (iii) a vector encoding helper virus proteins, required for assembly of the AAV capsid structure and packaging of the transgene in the modified AAV capsid structure; (b) expressing in the cell the AAV capsid protein described herein; (c) assembling an AAV particle comprising the AAV capsid proteins disclosed herein; and (d) packaging the AAV particle. In some instances, the cell is mammalian. In some instances, the cell is immortalized. In some instances, the immortalized cell is an embryonic stem cell. In some instances, the embryonic stem cell is a human embryonic stem cell. In some instances, the human embryonic stem cell is a human embryonic kidney 293 (HEK-293) cell. In some instances, the Cap gene is derived from the deoxyribose nucleic acid (DNA). In some instances, the 5’ ITR and the 3’ ITR are derived from an AAV2 serotype. In some instances, the 5’ ITR and the 3’ ITR are derived from an AAV5 serotype. In some instances, the 5’ ITR and the 3’ ITR are derived from an AAV9 serotype. In some instances, the 5’ ITR and / or the 3’ ITR originate from another natural serotype or have been engineered for improved transduction or transgene expression efficiency. In some instances, the first nucleic acid sequence and the second nucleic acid sequence are in trans. In some instances, the first nucleic acid sequence and the second nucleic acid sequence are in cis. In some instances, the first nucleic acid sequence, the second nucleic acid sequence and the third nucleic acid sequence, are in trans.

[0150] In some instances, the methods comprise packing the first nucleic acid sequence encoding the therapeutic gene expression product such that it becomes encapsidated by the modified AAVcapsid protein. In some embodiments, the rAAV particles are isolated, concentrated, and purified using suitable viral purification methods, such as those described herein.

[0151] In some cases, rAAVs of the present disclosure are generated using the methods described in Challis, R. C. et al. Nat. Protoc. 14, 379 (2019). Briefly, triple transfection of HEK293T cells (ATCC) using polyethylenimine (PEI) is performed, viruses are collected after 120 hours from both cell lysates and media and purified over iodixanol. In a non-limiting example, the rAAVs are generated by triple transfection of precursor cells (e.g., HEK293T) cells using a standard transfection protocol (e.g., PEI). Viral particles are harvested from the media after a period of time (e.g., 72 h post transfection) and from the cells and media at a later point in time (e.g., 120 h post transfection). Virus present in the media is concentrated by precipitation with 8% polyethylene glycol (PEG) and 500 mM sodium chloride and the precipitated virus is added to the lysates prepared from the collected cells. The viruses are purified over iodixanol (Optiprep, Sigma) step gradients (15%, 25%, 40% and 60%). Viruses are concentrated and formulated in PBS. Virus titers are determined by measuring the number of DNaseI-resistant vector genome copies (VGs) using qPCR and the linearized genome plasmid as a control.

[0152] The cell can be selected from a human, a primate, a murine, a feline, a canine, a porcine, an ovine, a bovine, an equine, an epine, a caprine and a lupine host cell. In some instances, the cell is a progenitor or precursor cell, such as a stem cell. In some instances, the stem cell is a mesenchymal cell, embryonic stem cell, induced pluripotent stem cell (iPSC), fibroblast or other tissue specific stem cell. The cell can be immortalized. In some cases, the immortalized cell is a HEK293 cell. In some instances, the cell is a differentiated cell. Based on the disclosure provided, it is expected that this system can be used in conjunction with any transgenic line expressing a recombinase in the target cell type of interest to develop AAV capsids that more efficiently transduce that target cell population. Methods of Treatment

[0153] Disclosed herein are methods of treating a disease or condition, or a symptom of the disease or condition, in a subject, comprising administrating of therapeutically effective amount of one or more compositions (e.g., rAAV particle, AAV vector, pharmaceutical composition) disclosed herein to the subject. In some embodiments, the composition is a rAAV capsid protein described herein. In some embodiments, the composition is an isolated and purified rAAV capsid protein described herein. In some embodiments, the rAAV particle encapsidates an AAV vectorcomprising a transgene (e.g., therapeutic nucleic acid). In some embodiments, the composition is a rAAV capsid protein described herein conjugated with a therapeutic agent disclosed herein. In some embodiments, the composition is a pharmaceutical composition comprising the rAAV particle and a pharmaceutically acceptable carrier. In some embodiments, the one or more compositions are administered to the subject alone (e.g., stand-alone therapy). In some embodiments, the composition is a first-line therapy for the disease or condition. In some embodiments, the composition is a second-line, third-line, or fourth-line therapy, for the disease or condition.

[0154] Recombinant adeno-associated virus (rAAV) mediated gene delivery leverages the AAV mechanism of viral transduction for nuclear expression of an episomal heterologous nucleic acid (e.g., a transgene, therapeutic nucleic acid). For example, upon delivery to a host in vivo environment, a rAAV may (1) bind or attach to cellular surface receptors on the target cell, (2) endocytose, (3) traffic to the nucleus, (4) uncoat the virus to release the encapsidated heterologous nucleic acid, (5) convert of the heterologous nucleic acid from single-stranded to double-stranded DNA as a template for transcription in the nucleus, and (6) transcribe of the episomal heterologous nucleic acid in the nucleus of the host cell. rAAVs engineered to have an increased specificity (binding to cellular surface receptors on the target cell), transduction efficiency (the effectiveness of a virus, engineered or naturally occurring, at delivering its DNA component to a host cell), and transgene expression (transcription of the episomal heterologous nucleic acid in the host cell) are desirable for gene therapy applications.

[0155] Aspects disclosed herein provide methods of treating a disease or condition in a subject, the method comprising administering to the subject a therapeutically effective amount of the rAAV of the present disclosure, or the pharmaceutical formulation of the present disclosure, wherein the gene product is a therapeutic gene product. In some embodiments, the administering is by intracranial, intraventricular, intracerebroventricular, intravenous, intraarterial, intranasal, intrathecal, intracisternae magna, or subcutaneous.

[0156] Provided here, are methods of treating a disease or a condition associated with an aberrant expression or activity of a target gene or gene expression product thereof, the method comprising modulating the expression or the activity of a target gene or gene expression product in a subject by administering a rAAV encapsidating a heterologous nucleic acid of the present disclosure. In some instances, the expression or the activity of the target gene or gene expression product isdecreased, relative to that in a normal (non-diseased) individual; and administering the rAAV to the subject is sufficient to increase the expression of the activity of the target gene or gene expression product. In some instances, the expression or the activity of the gene or gene expression product is increased, relative to that in a normal individual; and administering the rAAV to the subject is sufficient to decrease the expression or the activity of the target gene or gene expression product. In a non-limiting example, a subject diagnosed with Alzheimer’s disease, which is caused, in some cases, by a gain-of-function of a Presenilin 1 and / or Presenilin 2 (encoded by the gene PSEN1 and PSEN2, respectively) is administered a rAAV disclosed herein encapsidating a therapeutic nucleic acid that is a silencing RNA (siRNA), or other RNAi with a loss-of-function effect on PSEN1 mRNA.

[0157] Also provided are methods of preventing a disease or condition disclosed herein in a subject comprising administering to the subject a therapeutically effective amount of an rAAV vector comprising a nucleic acid sequence encoding a therapeutic gene expression product described herein. The rAAV vector may be encapsidated in the modified capsid protein or rAAV viral particle described herein. In some instances, the therapeutic gene expression product is effective to modulate the activity or expression of a target gene or gene expression product.

[0158] Disclosed herein are methods of treating a disease or condition in a subject by administering a composition comprising a rAAV disclosed herein. An advantage of the rAAVs disclosed herein, is that the rAAV may be used to treat virtually any disease or condition that would benefit from a transgene therapy, including but not limited to spinal muscular atrophy (SMA), amyotrophic lateral sclerosis (ALS), Parkinson's disease, Pompe disease, mucopolysaccharidosis type II, fragile X syndrome, STXBP1 encephalopathy, Krabbe disease, Huntington's disease, Alzheimer's disease, Battens disease, lysosomal storage disorders, glioblastoma multiforme, Rett syndrome, Leber's congenital amaurosis, Late infantile neuronal ceroid lipofuscinosis (LINCL), chronic pain, stroke, spinal cord injury, traumatic brain injury and lysosomal storage disorders.

[0159] In some cases, the disease or condition is localized to a particular in vivo environment in the subject, e.g., the brain. The compositions of the present disclosure are particularly useful for the treatment of the diseases or conditions described herein because they specifically or more efficiently target the in vivo environment and deliver a therapeutic nucleic acid engineered tomodulate the activity or the expression of a target gene expression product involved with the pathogenesis or pathology of the disease or condition.

[0160] Provided herein are methods of treating a disease or a condition, or a symptom of the disease or condition, in a subject, comprising: (a) diagnosing a subject with a disease or a condition affecting a target in vivo environment; and (b) treating the disease or the condition by administering to the subject a therapeutically effective amount of a composition disclosed herein (e.g., rAAV particle, AAV vector, pharmaceutical composition), wherein the composition is engineered with an increased specificity for the target in vivo environment.

[0161] Disclosed herein are methods of treating a disease or a condition, or a symptom of the disease or the condition, afflicting a target in a subject comprising: (a) administering to the subject a composition (e.g., rAAV particle, AAV vector, pharmaceutical composition); and (b) expressing the therapeutic nucleic acid into a target in vivo environment in the subject with an increased transduction enrichment.

[0162] In some embodiments, methods further comprise reducing or ablating delivery of the heterologous nucleic acid in an off-target in vivo environment, such as the liver. In some embodiments, delivery is characterized by an increase in enrichment of transduction (e.g., of the heterologous nucleic acid) in the brain.

[0163] In some embodiments, methods of treating a disease or condition affecting the brain comprise administering a rAAV particle to a brain in a subject, the rAAV particle comprising an rAAV capsid protein comprising an insertion and / or substitution (combined) of about, five, six, or seven amino acids of an amino acid sequence provided in Tables 1-2 and 3, 4, 4A, 5, 5A, 5B, 6, 6A and / or 6B, and / or Formulas A-F and I-II in a parental AAV capsid protein. In some embodiments, methods of treating a disease or condition affecting the brain comprise administering a rAAV particle to a brain in a subject, the rAAV particle comprising an rAAV capsid protein comprising an insertion of about, five, six, or seven amino acids of an amino acid sequence as well as one or more substitution at amino acid found at amino acid positions 449-460 such as provided in Tables 1-2 and Formulas A-F. In some embodiments, methods of treating a disease or condition affecting the brain comprise administering a rAAV particle to a brain in a subject, the rAAV particle comprising an rAAV with a variation at 449-460 in the capsid protein further comprises an insertion of about, five, six, or seven amino acids of an amino acid sequence as well as one or more substitution at amino acid found at amino acid positions 587-590 [AQAQ]such as provided in Tables 3, 4, 4A, 5, 5A, 5B, 6, 6A and / or 6B, and Formulas I-II. In preferred embodiments, the parental AAV capsid protein is AAV9 capsid protein (e.g., provided in SEQ ID NO: 1).

[0164] Also provided are methods of modulating a target gene expression product, the methods comprising administering to a subject in need thereof a composition (e.g., rAAV particle, AAV vector, pharmaceutical composition) disclosed herein. For example, methods provided herein comprise administering to a subject a rAAV with a rAAV capsid protein encapsidating a viral vector comprising a heterologous nucleic acid that modulates the expression or the activity of the target gene expression product.

[0165] The term “normal individual” refers to an individual that is not afflicted with the disease or the condition characterized by the variation in expression or activity of the gene or gene expression product thereof.

[0166] In some embodiments, the disease or condition of the brain selected from Absence of the Septum Pellucidum, Acid Lipase Disease, Acid Maltase Deficiency, Acquired Epileptiform Aphasia, Acute Disseminated Encephalomyelitis, Attention Deficit-Hyperactivity Disorder (ADHD), Adie's Pupil, Adie's Syndrome, Adrenoleukodystrophy, Agenesis of the Corpus Callosum, Agnosia, Aicardi Syndrome, Aicardi-Goutieres Syndrome Disorder, AIDS - Neurological Complications, Alexander Disease, Alpers' Disease, Alternating Hemiplegia, Alzheimer's Disease, Amyotrophic Lateral Sclerosis (ALS), Anencephaly, Aneurysm, Angelman Syndrome, Angiomatosis, Anoxia, Antiphospholipid Syndrome, Aphasia, Apraxia, Arachnoid Cysts, Arachnoiditis, Arnold-Chiari Malformation, Arteriovenous Malformation, Asperger Syndrome, Ataxia, Ataxia Telangiectasia, Ataxias and Cerebellar or Spinocerebellar Degeneration, Atrial Fibrillation and Stroke, Attention Deficit-Hyperactivity Disorder, Autism Spectrum Disorder, Autonomic Dysfunction, Back Pain, Barth Syndrome, Batten Disease, Becker's Myotonia, Behcet's Disease, Bell's Palsy, Benign Essential Blepharospasm, Benign Focal Amyotrophy, Benign Intracranial Hypertension, Bernhardt-Roth Syndrome, Binswanger's Disease, Blepharospasm, Bloch-Sulzberger Syndrome, Brachial Plexus Birth Injuries, Brachial Plexus Injuries, Bradbury-Eggleston Syndrome, Brain and Spinal Tumors, Brain Aneurysm, Brain Injury, Brown-Sequard Syndrome, Bulbospinal Muscular Atrophy, Cerebral Autosomal Dominant Arteriopathy with Subcortical Infarcts and Leukoencephalopathy (CADASIL), Canavan Disease, Carpal Tunnel Syndrome, Causalgia, Cavernomas, Cavernous Angioma, CavernousMalformation, Central Cervical Cord Syndrome, Central Cord Syndrome, Central Pain Syndrome, Central Pontine Myelinolysis, Cephalic Disorders, Ceramidase Deficiency, Cerebellar Degeneration, Cerebellar Hypoplasia, Cerebral Aneurysms, Cerebral Arteriosclerosis, Cerebral Atrophy, Cerebral Beriberi, Cerebral Cavemous Malformation, Cerebral Gigantism, Cerebral Hypoxia, Cerebral Palsy, Cerebro-Oculo-Facio-Skeletal Syndrome (COFS), Charcot-Marie-Tooth Disease, Charcot-Marie-Tooth syndrome, classical rhizomelic chondrodysplasia punctata (RCDP), Chiari Malformation, Cholesterol Ester Storage Disease, Chorea, Choreoacanthocytosis, Chronic Inflammatory Demyelinating Polyneuropathy (CIDP), Chronic Orthostatic Intolerance, Chronic Pain, Cockayne Syndrome, Cockayne Syndrome Type II, Coffin Lowry Syndrome, Colpocephaly, Coma, Complex Regional Pain Syndrome, Congenital Facial Diplegia, Congenital Myasthenia, Congenital Myopathy, Congenital Vascular Cavernous Malformations, Corticobasal Degeneration, Cranial Arteritis, Craniosynostosis, Cree encephalitis, Creutzfeldt- Jakob Disease, Cumulative Trauma Disorders, Cushing's Syndrome, Cytomegalic Inclusion Body Disease, Cytomegalovirus Infection, Dancing Eyes-Dancing Feet Syndrome, Dandy-Walker Syndrome, Dawson Disease, Deafness, De Morsier's Syndrome, Dejerine-Klumpke Palsy, Dementia, Dementia -Multi -Infarct, Dementia-Semantic, Dementia-Subcortical, Dementia With Lewy Bodies, Dentate Cerebellar Ataxia, Dentatorubral Atrophy, Dermatomyositis, Developmental Dyspraxia, Devic's Syndrome, Diabetic Neuropathy, Diffuse Sclerosis, Dravet Syndrome, Duchenne muscular dystrophy, Dysautonomia, Dysgraphia, Dyslexia, Dysphagia, Dyspraxia, Dyssynergia Cerebellaris Myoclonica, Dyssynergia Cerebellaris Progressiva, Dystonias, Early Infantile Epileptic Encephalopathy, Empty Sella Syndrome, Encephalitis, Encephalitis Lethargica, Encephaloceles, Encephalopathy, Encephalopathy (familial infantile), Encephalotrigeminal Angiomatosis, Epilepsy, Epileptic Hemiplegia, Erb's Palsy, Erb-Duchenne and Dejerine-Klumpke Palsies, Essential Tremor, Extrapontine Myelinolysis, Fabry Disease, Fahr's Syndrome, Fainting, Familial Dysautonomia, Familial Hemangioma, Familial Idiopathic Basal Ganglia Calcification, Familial Periodic Paralyses, Familial Spastic Paralysis, Farber's Disease, Febrile Seizures, Fibromuscular Dysplasia, Fisher Syndrome, Floppy Infant Syndrome, Foot Drop, Fragile X syndrome, Friedreich's Ataxia, Frontotemporal Dementia (FTD), Gaucher Disease, Generalized Gangliosidoses, Gerstmann's Syndrome, Gerstmann-Straussler-Scheinker Disease, Giant Axonal Neuropathy, Giant Cell Arteritis, Giant Cell Inclusion Disease, glioblastoma, Globoid Cell Leukodystrophy, Glossopharyngeal Neuralgia, Glycogen Storage Disease, Guillain-BarreSyndrome, Hallervorden-Spatz Disease, Head Injury, Headache, Hemicrania Continua, Hemifacial Spasm, Hemiplegia Alterans, Hereditary Neuropathies, Hereditary Spastic Paraplegia, Heredopathia Atactica Polyneuritiformis, Herpes Zoster, Herpes Zoster Oticus, Hirayama Syndrome, Holmes-Adie syndrome, Holoprosencephaly, HTLV-1 Associated Myelopathy, Hughes Syndrome, Huntington's Disease, Hydranencephaly, Hydrocephalus, Hydrocephalus - Normal Pressure, Hydromyelia, Hypercortisolism, Hypersomnia, Hypertonia, Hypotonia, Hypoxia, Immune-Mediated Encephalomyelitis, Inclusion Body Myositis, Incontinentia Pigmenti, Infantile Hypotonia, Infantile Neuroaxonal Dystrophy, Infantile Phytanic Acid Storage Disease, Infantile Refsum Disease (IRD) , Infantile Spasms, Inflammatory Myopathies, Iniencephaly, Intestinal Lipodystrophy, Intracranial Cysts, Intracranial Hypertension, Isaacs' Syndrome, Joubert Syndrome, Kearns-Sayre Syndrome, Kennedy's Disease, Kinsbourne syndrome, Kleine-Levin Syndrome, Klippel-Feil Syndrome, Klippel-Trenaunay Syndrome (KTS), Kliiver-Bucy Syndrome, Korsakoff s Amnesic Syndrome, Krabbe Disease, Kugelberg-Welander Disease, Kuru, Lambert-Eaton Myasthenic Syndrome, Landau-Kleffner Syndrome, Lateral Femoral Cutaneous Nerve Entrapment, Lateral Medullary Syndrome, Learning Disabilities, Leigh's Disease, Lennox- Gastaut Syndrome, Lesch-Nyhan Syndrome, Leukodystrophy, Levine-Critchley Syndrome, Lewy Body Dementia, Lipid Storage Diseases, Lipoid Proteinosis, Lissencephaly, Locked-In Syndrome, Lou Gehrig's Disease, Lupus -Neurological Sequelae, Lyme Disease - Neurological Complications, Machado-Joseph Disease, Macrencephaly, Maple syrup urine disease, Megalencephaly, Melkersson-Rosenthal Syndrome, Meningitis, Meningitis and Encephalitis, Menkes Disease, Menkes syndrome, Meralgia Paresthetica, Metachromatic Leukodystrophy, Microcephaly, Migraine, Miller Fisher Syndrome, Mini Stroke, Mitochondrial Myopathy, Moebius Syndrome, Monomelic Amyotrophy, Motor Neuron Diseases, Moyamoya Disease, Mucolipidoses, Mucopolysaccharidosis, Mucopolysaccharidosis II, Multi-Infarct Dementia, Multifocal Motor Neuropathy, Multiple Sclerosis, Multiple System Atrophy, Multiple System Atrophy with Orthostatic Hypotension, Muscular Dystrophy, Myasthenia -Congenital, Myasthenia Gravis, Myelinoclastic Diffuse Sclerosis, Myoclonic Encephalopathy of Infants, Myoclonus, Myopathy, Myopathy-Congenital, Myopathy-Thyrotoxic, Myotonia, Myotonia Congenita, Myotonic dystrophy, Narcolepsy, Neuroacanthocytosis, Neurodegeneration with Brain Iron Accumulation, Neurofibromatosis, Neuroleptic Malignant Syndrome, Neurological Complications of AIDS, Neurological Complications of Lyme Disease, NeurologicalConsequences of Cytomegalovirus Infection, Neurological Manifestations of Pompe Disease, Neurological Sequelae Of Lupus, Neuromyelitis Optica, Neuromyotonia, Neuronal Ceroid Lipofuscinosis, Neuronal Migration Disorders, Neuropathy- Hereditary, Neurosarcoidosis, Neurosyphilis, Neurotoxicity, Nevus Cavernosus, Niemann-Pick Disease, O'Sullivan-McLeod Syndrome, Occipital Neuralgia, Ohtahara Syndrome, Olivopontocerebellar Atrophy, Opsoclonus Myoclonus, Orthostatic Hypotension, Overuse Syndrome, Pain -Chronic, Pantothenate Kinase- Associated Neurodegeneration, Paraneoplastic Syndromes, Paresthesia, Parkinson's Disease, Paroxysmal Choreoathetosis, Paroxysmal Hemicrania, Parry -Romberg, Pelizaeus-Merzbacher Disease, Pena Shokeir II Syndrome, Perineural Cysts, Periodic Paralyses, Peripheral Neuropathy, Periventricular Leukomalacia, Persistent Vegetative State, Pervasive Developmental Disorders, Phenylketonuria, Phytanic Acid Storage Disease, Pick's Disease, Pinched Nerve, Piriformis Syndrome, Pituitary Tumors, Polymyositis, Pompe Disease, Porencephaly, Post-Polio Syndrome, Postherpetic Neuralgia, Postinfectious Encephalomyelitis, Postural Hypotension, Postural Orthostatic Tachycardia Syndrome, Postural Tachycardia Syndrome, Prader-Willi syndrome, Primary Dentatum Atrophy, Primary Lateral Sclerosis, Primary Progressive Aphasia, Prion Diseases, Progressive Hemifacial Atrophy, Progressive Locomotor Ataxia, Progressive Multifocal Leukoencephalopathy, Progressive Sclerosing Poliodystrophy, Progressive Supranuclear Palsy, Prosopagnosia, Pseudo-Torch syndrome, Pseudotoxoplasmosis syndrome, Pseudotumor Cerebri, Psychogenic Movement, Ramsay Hunt Syndrome I, Ramsay Hunt Syndrome II, Rasmussen's Encephalitis, Reflex Sympathetic Dystrophy Syndrome, Refsum Disease, Refsum Disease - Infantile, Repetitive Motion Disorders, Repetitive Stress Injuries, Restless Legs Syndrome, Retrovirus-Associated Myelopathy, Rett Syndrome, Reye's Syndrome, Rheumatic Encephalitis, Riley-Day Syndrome, Sacral Nerve Root Cysts, Saint Vitus Dance, Salivary Gland Disease, Sandhoff Disease, Schilder's Disease, Schizencephaly, Seitelberger Disease, Seizure Disorder, Semantic Dementia, Septo-Optic Dysplasia, Severe Myoclonic Epilepsy of Infancy (SMEI), Shaken Baby Syndrome, Shingles, Shy-Drager Syndrome, Sjogren's Syndrome, Sleep Apnea, Sleeping Sickness, Sotos Syndrome, Spasticity, Spina Bifida, Spinal Cord Infarction, Spinal Cord Injury, Spinal Cord Tumors, Spinal Muscular Atrophy, Spinocerebellar ataxia, Spinocerebellar Atrophy, Spinocerebellar Degeneration, Steele-Richardson-Olszewski Syndrome, Stiff-Person Syndrome, Striatonigral Degeneration, Stroke, Sturge-Weber Syndrome, STXBP1 encephalopathy, Subacute Sclerosing Panencephalitis, Subcortical ArterioscleroticEncephalopathy, Short-lasting, Unilateral, Neuralgiform (SUNCT) Headache, Swallowing Disorders, Sydenham Chorea, Syncope, Syphilitic Spinal Sclerosis, Syringohydromyelia, Syringomyelia, Systemic Lupus Erythematosus, Tabes Dorsalis, Tangier disease, Tardive Dyskinesia, Tarlov Cysts, Tay-Sachs Disease, Temporal Arteritis, Tethered Spinal Cord Syndrome, Thomsen's Myotonia, Thoracic Outlet Syndrome, Thyrotoxic Myopathy, Tic Douloureux, Todd's Paralysis, Tourette Syndrome, Transient Ischemic Attack, Transmissible Spongiform Encephalopathies, Transverse Myelitis, Traumatic Brain Injury, Tremor, Trigeminal Neuralgia, Tropical Spastic Paraparesis, Troyer Syndrome, Tuberous Sclerosis, Vascular Erectile Tumor, Vasculitis Syndromes of the Central Nervous Systems, Von Economo's Disease, Von Hippel-Lindau Disease (VHL), Von Hippel-Lindau syndrome, Von Recklinghausen's Disease, Wallenberg's Syndrome, Werdnig-Hoffman Disease, Wernicke-Korsakoff Syndrome, West Syndrome, Whiplash, Whipple's Disease, Williams Syndrome, Wilson Disease, Wolman's Disease, X-Linked Spinal and Bulbar Muscular Atrophy and Zellweger syndrome.

[0167] In some embodiments, the pharmaceutical formulation comprises a therapeutic nucleic acid encoding a therapeutic gene expression product. In some instances, the therapeutic gene expression product is effective to modulate an activity or an expression of a target gene or gene expression product selected from ATP1A2, CACNAIA, SETD5, SHANK3, NF2, DNMTl, TCF4, RAI1, PEXl, ARSA, EIF2B5, EIF2B1, EIF2B2, NPCl, ADAR, MFSD8, STXBPl, PRICKLE2, PRRT2, IDUA, STX1B, Sarcoglycan Alpha (SGCA), glutamic acid decarboxylase 65 (GAD65), glutamic acid decarboxylase 67 (GAD67), CLN2, Nerve Growth Factor (NGF), glial cell derived neurotrophic factor (GDNF), Survival Of Motor Neuron 1, Telomeric (SMNl), Factor X (FIX), Retinoid Isomerohydrolase (RPE65), sarco / endoplasmic reticulum Ca2+- ATPase (SERCA2a), Glucocerebrosidase (GCase), galactocerebrosidase (GALC), CDKL5, Frataxin (FXN), Huntingtin (HTT), methyl-CpG binding protein 2 (MECP2), a peroxisomal biogenesis factor (PEX), progranulin (GRN), an antitubulin agent, copper-zinc superoxide dismutase (SODl), iduronate 2 sulfatase (hIDS), Glucosylceramidase Beta (GBA), fragile X mental retardation 1 (FMR1), NPC Intracellular Cholesterol Transporter 1 (NPCl), SCN1A, C9orf72, NPS3 and a NLRP3 inflammasome. In some embodiments, the peroxisomal biogenesis factor (PEX) is selected from PEX1, PEX2, PEX3, PEX4, PEX5, PEX6, PEX7, PEX10, PEX11β, PEX12, PEX13, PEX14, PEX16, PEX19, and PEX26.

[0168] In some aspects, other examples of genes involved in neurologic or brain diseases or disorders include MAPT, IDUA, SNCA, ATXN2, Ube3a, GNS, HGSNAT, NAGLU, SGSH, CLN1, CLN3, CLN4, CLN5, CLN6, CLN7, CLN8, CTSD, ABCD1, HEXA, HEXB, ASM, ASPA, GLB1, AADC, MFN2, GNAO1, SYNGAP1, GRIN2A, GRIN2B, KCNQ2, EPM2A, NHLRC1, SLC6A1, SLC13A5, SURF1, GBE1, ATXN1, ATXN3, and ATXN7.

[0169] In some instances, the therapeutic gene expression product comprises gene editing components. In some instances, the gene editing components are selected from an artificial site-specific RNA endonuclease (ASRE), a zinc finger endonuclease (ZFN), a transcription factor like effector nuclease (TALEN), a clustered regularly interspaced short palindromic repeats (CRISPR) / Cas enzyme, and a CRISPR / Cas guide RNA.

[0170] In some instances, the expression of a gene or expression or activity of a gene expression product is inhibited by the administration of the composition to the subject. In some instances, the expression of a gene or the expression or the activity of a gene expression product is enhanced by the administration of the composition to the subject. FORMULATIONS, DOSAGES, AND ROUTES OF ADMINISTRATION

[0171] Disclosed herein are methods comprising delivering a rAAV particle encapsidating a heterologous nucleic acid to the brain in a subject, the rAAV particle comprising (i) an increased transduction of the heterologous nucleic acid in the brain, wherein the rAAV particle has an rAAV capsid protein comprising an insertion and / or substitution of five, six, or seven amino acids of an amino acid sequence provided in Tables 1-2 and Formulas A-F, at an amino acid position 449-640 in a parental AAV capsid protein as well as one or more insertion and / or substitution at amino acid found at amino acid positions 583-590 such as provided in Tables 3, 4, 5, or 6, and Formulas I-II. In various embodiments, the rAAV capsid protein may comprise one or more insertion and / or substitutions at amino acid positions 449-460 alone or in combination with the modifications above.

[0172] In general, methods disclosed herein comprise administering a therapeutic rAAV composition by systemic administration. In some instances, methods comprise administering a therapeutic rAAV composition by intravenous (“i.v.”) administration. One may administer therapeutic rAAV compositions by additional routes, such as subcutaneous injection, intramuscular injection, intradermal injection, transdermal injection, percutaneous administration, intranasal administration, intralymphatic injection, rectal administration intragastricadministration, intraocular administration, intracerebroventricular administration, intrathecally, intracisternal, or any other suitable parenteral administration. Routes, dosage, time points, and duration of administrating therapeutics may be adjusted. In some embodiments, administration of therapeutics is prior to, or after, onset of either, or both, acute and chronic symptoms of the disease or condition. Other routes of delivery to the brain include, but are not limited to intracranial administration, lateral cerebroventricular administration, and endovascular administration.

[0173] An effective dose and dosage of pharmaceutical compositions to prevent or treat the disease or condition disclosed herein is defined by an observed beneficial response related to the disease or condition, or symptom of the disease or condition. Beneficial response comprises preventing, alleviating, arresting, or curing the disease or condition, or symptom of the disease or condition. In some embodiments, the beneficial response may be measured by detecting a measurable improvement in the presence, level, or activity, of biomarkers, transcriptomic risk profile, or intestinal microbiome in the subject. An “improvement,” as used herein refers to shift in the presence, level, or activity towards a presence, level, or activity, observed in normal individuals (e.g. individuals who do not suffer from the disease or condition). In instances wherein the therapeutic rAAV composition is not therapeutically effective or is not providing a sufficient alleviation of the disease or condition, or symptom of the disease or condition, then the dosage amount and / or route of administration may be changed, or an additional agent may be administered to the subject, along with the therapeutic rAAV composition. In some embodiments, as a patient is started on a regimen of a therapeutic rAAV composition, the patient is also weaned off (e.g., step-wise decrease in dose) a second treatment regimen.

[0174] In some cases, a dose of the pharmaceutical composition may comprise a concentration of infectious particles of at least or about 107, 108, 109, 1010, 1011, 1012, 1013, 1014, 1015, 1016, or 1017. In some cases, the concentration of infectious particles is 2x107, 2x108, 2x109, 2x1010, 2x1011, 2x1012, 2x1013, 2x1014, 2x1015, 2x1016, or 2x1017. In some cases, the concentration of the infectious particles is 3x107, 3x108, 3x109, 3x1010, 3x1011, 3x1012, 3x1013, 3x1014, 3x1015, 3x1016, or 3x1017. In some cases, the concentration of the infectious particles is 4x107, 4x108, 4x109, 4x1010, 4x1011, 4x1012, 4x1013, 4x1014, 4x1015, 4x1016, or 4x1017. In some cases, the concentration of the infectious particles is 5x107, 5x108, 5x109, 5x1010, 5x1011, 5x1012, 5x1013, 5x1014, 5x1015, 5x1016, or 5x1017. In some cases, the concentration of the infectious particles is 6x107, 6x108, 6x109, 6x1010, 6x1011, 6x1012, 6x1013, 6x1014, 6x1015, 6x1016, or 6x1017. In some cases, the concentration of the infectiousparticles is 7x107, 7x108, 7x109, 7x1010, 7x1011, 7x1012, 7x1013, 7x1014, 7x1015, 7x1016, or 7x1017. In some cases, the concentration of the infectious particles is 8x107, 8x108, 8x109, 8x1010, 8x1011, 8x1012, 8x1013, 8x1014, 8x1015, 8x1016, or 8x1017. In some cases, the concentration of the infectious particles is 9x107, 9x108, 9x109, 9x1010, 9x1011, 9x1012, 9x1013, 9x1014, 9x1015, 9x1016, or 9x1017.

[0175] Disclosed herein, in some embodiments are formulations of pharmaceutically-acceptable excipients and carrier solutions suitable for delivery of the rAAV compositions described herein, as well as suitable dosing and treatment regimens for using the particular compositions described herein in a variety of treatment regimens. In some embodiments, the amount of therapeutic gene expression product in each therapeutically-useful composition may be prepared in such a way that a suitable dosage will be obtained in any given unit dose of the compound. Factors such as solubility, bioavailability, biological half-life, route of administration, product shelf life, as well as other pharmacological considerations will be contemplated by one skilled in the art of preparing such pharmaceutical formulations, and as such, a variety of dosages and treatment regimens may be desirable.

[0176] In some embodiments, the pharmaceutical forms of the rAAV-based viral compositions suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and / or vegetable oils. Proper fluidity may be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride.

[0177] In some cases, for administration of an injectable aqueous solution, the solution may be suitably buffered, if necessary, and the liquid diluent first rendered isotonic with sufficient saline or glucose. These particular aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration. Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject.Moreover, for human administration, preparations should meet sterility, pyrogenicity, and the general safety and purity standards as required by FDA Office of Biologics standards.

[0178] Disclosed herein are sterile injectable solutions comprising the rAAV compositions disclosed herein, which are prepared by incorporating the rAAV compositions disclosed herein in the required amount in the appropriate solvent with several of the other ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof. Injectable solutions may be advantageous for systemic administration, for example by intravenous or intrathecal administration.

[0179] Suitable dose and dosage administrated to a subject is determined by factors including, but not limited to, the particular therapeutic rAAV composition, disease condition and its severity, the identity (e.g., weight, sex, age) of the subject in need of treatment, and can be determined according to the particular circumstances surrounding the case, including, e.g., the specific agent being administered, the route of administration, the condition being treated, and the subject or host being treated.

[0180] The amount of rAAV compositions and time of administration of such compositions will be within the purview of the skilled artisan having benefit of the present teachings. It is likely, however, that the administration of therapeutically-effective amounts of the disclosed compositions may be achieved by a single administration, example, a single injection of sufficient numbers of infectious particles to provide therapeutic benefit to the patient undergoing such treatment. This is made possible, at least in part, by the fact that certain target cells (e.g., neurons) do not divide, obviating the need for multiple or chronic dosing.

[0181] In certain embodiments, the data obtained from cell culture assays and animal studies are used in formulating the therapeutically effective daily dosage range and / or the therapeutically effective unit dosage amount for use in mammals, including humans. In certain embodiments, the dosage range and / or the unit dosage amount varies within this range depending upon the dosage form employed and the route of administration utilized.Combination Therapies

[0182] A therapeutic rAAV may be used alone or in combination with an additional therapeutic agent (together, “therapeutic agents”). In some cases, a therapeutic rAAV as used herein is administered alone. The therapeutic agent may be administered together or sequentially in a combination therapy. The combination therapy may be administered within the same day, or may be administered one or more days, weeks, months, or years apart.

[0183] The additional therapeutic agent can comprise a small molecule. The additional therapeutic agent can comprise an antibody, or antigen-binding fragment. The additional therapeutic agent can include lipid nanoparticle-based therapies, anti-sense oligonucleotide therapies, as well as other viral therapies.

[0184] The additional therapeutic agent can comprise a cell-based therapy. Exemplary cell-based therapies include without limitation immune effector cell therapy, chimeric antigen receptor T-cell (CAR-T) therapy, natural killer cell therapy and chimeric antigen receptor natural killer (NK) cell therapy. Either NK cells, or CAR-NK cells, or a combination of both NK cells and CAR-NK cells can be used in combination with the methods disclosed herein. In some embodiments, the NK cells and CAR-NK cells are derived from human induced pluripotent stem cells (iPSC), umbilical cord blood, or a cell line. The NK cells and CAR-NK cells can comprise a cytokine receptor and a suicide gene. The cell-based therapy can comprise a stem cell therapy. The stem cell therapy may be embryonic or somatic stem cells. The stem cells may be isolated from a donor (allogeneic) or isolated from the subject (autologous). The stem cells may be expanded adipose-derived stem cells (eASCs), hematopoietic stem cells (HSCs), mesenchymal stem (stromal) cells (MSCs), or induced pluripotent stem cells (iPSCs) derived from the cells of the subject. KITS

[0185] Disclosed herein are kits comprising compositions disclosed herein. Also disclosed herein are kits for the treatment or prevention of a disease or conditions of the brain. In some instances, the disease or condition is cancer, a pathogen infection, pulmonary disease or condition, neurological disease, muscular disease, or an immune disorder, such as those described herein.

[0186] In one embodiment, a kit can include a therapeutic or prophylactic composition containing an effective amount of a composition of a rAAV particle encapsidating a recombinant AAV vector encoding a therapeutic nucleic acid (e.g., therapeutic nucleic acid) and a recombinant AAV (rAAV) capsid protein of the present disclosure. In another embodiment, a kit can include atherapeutic or prophylactic composition containing an effective amount of cells modified by the rAAV described herein (“modified cell”), in unit dosage form that express therapeutic nucleic acid. In some embodiments, a kit comprises a sterile container which can contain a therapeutic composition; such containers can be boxes, ampules, bottles, vials, tubes, bags, pouches, blister- packs, or other suitable container forms known in the art. Such containers can be made of plastic, glass, laminated paper, metal foil, or other materials suitable for holding medicaments.

[0187] In some instances, the kit further comprises a cell. In some instances, the cell is mammalian. In some instances, the cell is immortalized. In some instances, the immortalized cell is an embryonic stem cell. In some instances, the embryonic stem cell is a human embryonic stem cell. In some instances, the human embryonic stem cell is a human embryonic kidney 293 (HEK- 293) cell. In some instances, the kit further comprises an AAV vector comprising a heterologous nucleic acid encoding a therapeutic gene expression product. In some instances, the AAV vector is an episome.

[0188] In some cases, rAAV are provided together with instructions for administering the rAAV to a subject having or at risk of developing the disease or condition (e.g., disease of the brain). Instructions can generally include information about the use of the composition for the treatment or prevention of the disease or condition.

[0189] In some cases, the instructions include at least one of the following: description of the therapeutic rAAV composition; dosage schedule and administration for treatment or prevention of the disease or condition disclosed herein; precautions; warnings; indications; counter-indications; overdosage information; adverse reactions; animal pharmacology; clinical studies; and / or references. The instructions can be printed directly on the container (when present), or as a label applied to the container, or as a separate sheet, pamphlet, card, or folder supplied in or with the container. In some cases, instructions provide procedures for administering the rAAV to the subject alone. In some instances, the instructions provide that the rAAV is formulated for systemic delivery. DEFINITIONS

[0190] The terminology used herein is for the purpose of describing particular cases only and is not intended to be limiting. As used herein, the singular forms “a”, “an” and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise. Furthermore, to the extent that the terms “including”, “includes”, “having”, “has”, “with”, or variants thereof are usedin either the detailed description and / or the claims, such terms are intended to be inclusive in a manner similar to the term “comprising.”

[0191] The term “about” or “approximately” means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, e.g., the limitations of the measurement system. For example, “about” can mean within 1 or more than 1 standard deviation, per the practice in the given value. Where particular values are described in the application and claims, unless otherwise stated the term “about” should be assumed to mean an acceptable error range for the particular value.

[0192] As used herein “consisting essentially of” when used to define compositions and methods, shall mean excluding other elements of any essential significance to the combination for the stated purpose. Thus, a composition consisting essentially of the elements as defined herein would not exclude other materials or steps that do not materially affect the basic and novel characteristic(s) of the claimed disclosure, such as compositions for treating skin disorders like acne, eczema, psoriasis, and rosacea.

[0193] The terms “homologous,” “homology,” or “percent homology” are used herein to generally mean an amino acid sequence or a nucleic acid sequence having the same, or similar sequence to a reference sequence. Percent homology of sequences can be determined using the most recent version of BLAST, as of the filing date of this application.

[0194] The terms “increased,” or “increase” are used herein to generally mean an increase by a statically significant amount. In some embodiments, the terms “increased,” or “increase,” mean an increase of at least 10% as compared to a reference level, for example an increase of at least about 10%, at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% or up to and including a 100% increase or any increase between 10-100% as compared to a reference level, standard, or control. Other examples of “increase” include an increase of at least 2-fold, at least 5- fold, at least 10-fold, at least 20-fold, at least 50-fold, at least 100-fold, at least 1000-fold or more as compared to a reference level.

[0195] The terms “decreased” or “decrease” are used herein generally to mean a decrease by a statistically significant amount. In some embodiments, “decreased” or “decrease” means a reduction by at least 10% as compared to a reference level, for example a decrease by at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, orat least about 70%, or at least about 80%, or at least about 90% or up to and including a 100% decrease (e.g., absent level or non-detectable level as compared to a reference level), or any decrease between 10-100% as compared to a reference level. In the context of a marker or symptom, by these terms is meant a statistically significant decrease in such level. The decrease can be, for example, at least 10%, at least 20%, at least 30%, at least 40% or more, and is preferably down to a level accepted as within the range of normal for an individual without a given disease.

[0196] The terms “subject” is any organism. In some instances, the organism is a mammal. Non- limiting examples of mammal include, any member of the mammalian class: humans, non–human primates such as chimpanzees, and other apes and monkey species; farm animals such as cattle, horses, sheep, goats, swine; domestic animals such as rabbits, dogs, and cats; laboratory animals including rodents, such as rats, mice and guinea pigs, and the like. In one aspect, the mammal is a human. The term “animal” as used herein comprises human beings and non–human animals. In one embodiment, a “non–human animal” is a mammal, for example a rodent such as rat or a mouse. In one embodiment, a “non–human primate” is a mammal, for example a monkey. In some instances, the subject is a patient, which as used herein, may refer to a subject diagnosed with a particular disease or disorder.

[0197] The term “gene,” as used herein, refers to a segment of nucleic acid that encodes an individual protein or RNA (also referred to as a “coding sequence” or “coding region”), optionally together with associated regulatory region such as promoter, operator, terminator and the like, which may be located upstream or downstream of the coding sequence.

[0198] The term “adeno-associated virus,” or “AAV” as used herein refers to the adeno-associated virus or derivatives thereof. Non-limited examples of AAV’s include AAV type 1 (AAV1), AAV type 2 (AAV2), AAV type 3 (AAV3), AAV type 4 (AAV4), AAV type 5 (AAV5), AAV type 6 (AAV6), AAV type 7 (AAV7), AAV type 8 (AAV8), AAV type 9 (AAV9), AAV type 10 (AAV10), AAV type 11 (AAV11), AAV type 12 (AAV12), avian AAV, bovine AAV, canine AAV, equine AAV, primate AAV, non-primate AAV, and ovine AAV. In some instances, the AAV is described as a “Primate AAV,” which refers to AAV that infect primates. Likewise, an AAV may infect bovine animals (e.g., “bovine AAV”, and the like). In some instances, the AAV is wildtype, or naturally occurring. In some instances, the AAV is recombinant.

[0199] The term “AAV capsid” as used herein refers to a capsid protein or peptide of an adeno- associated virus. In some instances, the AAV capsid protein is configured to encapsidate geneticinformation (e.g., a transgene, therapeutic nucleic acid, viral genome). In some instances, the AAV capsid of the instant disclosure is a modified AAV capsid, relative to a corresponding parental AAV capsid protein.

[0200] The term “tropism” as used herein refers to a quality or characteristic of the AAV capsid that may include specificity for, and / or an increase or a decrease in enrichment of, expressing the encapsidated genetic information into an in vivo environment, relative to a second in vivo environment. An in vivo environment, in some instances, is a cell-type. An in vivo environment, in some instances, is an organ or organ system.

[0201] The term “AAV vector” as used herein refers to nucleic acid polymer encoding genetic information related to the virus. The AAV vector may be a recombinant AAV vector (rAAV), which refers to an AAV vector generated using recombinatorial genetics methods. In some instances, the rAAV vector comprises at least one heterologous polynucleotide (e.g., a polynucleotide other than a wild-type or naturally occurring AAV genome such as a transgene).

[0202] The term “AAV particle” as used herein refers to an AAV virus, virion, AAV capsid protein or component thereof. In some cases, the AAV particle is modified relative to a parental AAV particle.

[0203] The term “gene product” of “gene expression product” refers to an expression product of a polynucleotide sequence such as, for e.g., a polypeptide, peptide, protein or RNA, including interfering RNA (e.g., siRNA, miRNA, shRNA) and messenger RNA (mRNA).

[0204] The term “heterologous” as used herein refers to a genetic element (e.g., coding region) or gene expression product (e.g., RNA, protein) that is derived from a genotypically distinct entity from that of the rest of the entity to which it is being compared.

[0205] The term “endogenous” as used herein refers to a genetic element (e.g., coding region) or gene expression product (e.g., RNA, protein) that is naturally occurring in or associated with an organism or a particular cell within the organism.

[0206] The terms “treat,” “treating,” and “treatment” as used herein refers to alleviating or abrogating a disorder, disease, or condition; or one or more of the symptoms associated with the disorder, disease, or condition; or alleviating or eradicating a cause of the disorder, disease, or condition itself. Desirable effects of treatment can include, but are not limited to, preventing occurrence or recurrence of disease, alleviation of symptoms, diminishing any direct or indirectpathological consequences of the disease, preventing metastasis, decreasing the rate of disease progression, amelioration or palliation of the disease state and remission or improved prognosis.

[0207] The term “therapeutically effective amount” refers to the amount of a compound or therapy that, when administered, is sufficient to prevent development of, or alleviate to some extent, one or more of the symptoms of a disorder, disease, or condition of the disease; or the amount of a compound that is sufficient to elicit biological or medical response of a cell, tissue, system, animal, or human that is being sought by a researcher, veterinarian, medical doctor, or clinician.

[0208] The term “pharmaceutically acceptable carrier,” “pharmaceutically acceptable excipient,” “physiologically acceptable carrier,” or “physiologically acceptable excipient” refers to a pharmaceutically acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, excipient, solvent, or encapsulating material. A component can be “pharmaceutically acceptable” in the sense of being compatible with the other ingredients of a pharmaceutical formulation. It can also be suitable for use in contact with the tissue or organ of humans and animals without excessive toxicity, irritation, allergic response, immunogenicity, or other problems or complications, commensurate with a reasonable benefit / risk ratio. See, Remington: The Science and Practice of Pharmacy, 21st Edition; Lippincott Williams & Wilkins: Philadelphia, PA, 2005; Handbook of Pharmaceutical Excipients, 5th Edition; Rowe et al., Eds., The Pharmaceutical Press and the American Pharmaceutical Association: 2005; and Handbook of Pharmaceutical Additives, 3rd Edition; Ash and Ash Eds., Gower Publishing Company: 2007; Pharmaceutical Preformulation and Formulation, Gibson Ed., CRC Press LLC: Boca Raton, FL, 2004).

[0209] The term “pharmaceutical composition” refers to a mixture of a compound disclosed herein with other chemical components, such as diluents or carriers. The pharmaceutical composition can facilitate administration of the compound to an organism. Multiple techniques of administering a compound exist in the art including, but not limited to, systemic administration.

[0210] Non-limiting examples of “sample” include any material from which nucleic acids and / or proteins can be obtained. As non-limiting examples, this includes whole blood, peripheral blood, plasma, serum, saliva, mucus, urine, semen, lymph, fecal extract, cheek swab, cells or other bodily fluid or tissue, including but not limited to tissue obtained through surgical biopsy or surgical resection. Alternatively, a sample can be obtained through primary patient derived cell lines, or archived patient samples in the form of preserved samples, or fresh frozen samples.

[0211] The term “in vivo” is used to describe an event that takes place in a subject’s body.

[0212] The term “in vitro” is used to describe an event that takes places contained in a container for holding laboratory reagent such that it is separated from the biological source from which the material is obtained. In vitro assays can encompass cell-based assays in which living or dead cells are employed. In vitro assays can also encompass a cell-free assay in which no intact cells are employed.

[0213] The term “brain” means a tissue selected from brain, thalamus, cortex, putamen, lateral ventricles, medulla, the pons, the amygdala, the motor cortex, caudate, hypothalamus, striatum, ventral midbrain, neocortex, basal ganglia, hippocampus, cerebrum, cerebellum, brain stem, and spinal cord. The brain includes a variety of cortical and subcortical areas, including the frontal, temporal, occipital and parietal lobes.

[0214] The term “systemic delivery” is defined as a route of administration of medication or other substance into a circulatory system so that the entire body is affected. Administration can take place via enteral administration (absorption of the drug through the gastrointestinal tract) or parenteral administration (generally injection, infusion, or implantation). “Circulatory system” includes both blood or cerebrospinal fluid circulatory systems. Examples of systemic administration for the brain include intraarterial, intravenous or intrathecal injection. Other examples include administration to the cerebrospinal fluid at any location, in the spine (i.e. but not limited to lumbar) or brain (i.e. but not limited to cisterna magna). The terms “systemic administration” and “systemic delivery” are used interchangeably.

[0215] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0216] EXAMPLES Example 1 Method of Identifying the Modified Capsid Proteins in Cynomolgus Macaques

[0217] Of primary concern for the therapeutic applicability of engineered adeno-associated viruses (AAVs) is how well their transduction profiles translate to human application. While previous engineering efforts have focused on in vitro or in vivo rodent screening platforms due to the ease and flexibility of their use, screening efforts directly in non- human primates (NHPs) are much more likely to identify viruses that translate. Thus,cynomolgus macaques, an old world NHP, were chosen for the engineering efforts underlying the capsid proteins of the disclosure. These experiments initially focused engineering efforts on a region of the AAV9 capsid surface located at amino acid position 588, one of the most exposed loops on the capsid surface that is a variable region between natural AAV serotypes and has a role in receptor binding. Insertion of peptides between positions 588 and 589 and 452 and 458 has been studied in the past by the present inventors and has resulted in novel receptor binding (AAV-PHP.B / AAV-PHP.eB binding of Ly6a on rodent brain endothelium to facilitate blood-brain barrier crossing and high transduction of the brain) and drastically altered capsid tropism.

[0218] A library of viral capsids was created by performing random amino acid insertions and substitutions at these sites within AAV9, hoping for novel tropism toward the NHP brain. Multiple libraries of viral capsids were created according to the following scheme at these sites within AAV9 in hopes of achieving novel tropism toward the NHP brain and / or the central nervous system (CNS), e.g., in the spinal cord.

[0219] First, Adeno-Associated Virus serotype 9 (AAV9) was modified by inserting a stretch of 7 amino acids (7mer) following residue #588 in its capsid coding sequence and substituting randomly chosen residues from region #587-597, leading to seven amino acid sequences for region #587-597 of: AQENHVRSSAQ (SEQ ID NO: 6382) [Parent Capsid 1] AQRDGLILIAQ (SEQ ID NO: 6383) [Parent Capsid 2] AQRDGHILIAK (SEQ ID NO: 6384) [Parent Capsid 3] HQKPPGYLLAK (SEQ ID NO: 6385) [Parent Capsid 4] QQRNGHILIAK (SEQ ID NO: 6386) [Parent Capsid 5] AQLSGQILIAQ (SEQ ID NO: 6387) [Parent Capsid 6] AQRNGEHFKAQ (SEQ ID NO: 6388) [Parent Capsid 7]

[0220] Four libraries were produced from these parent capsids: the substitutions were made to 3 randomly chosen residues from region #587-597 of all seven parent capsids. Further, the libraries were produced with the selected modifications: § Substitutions were made to 4 residues from region #583-599 of parent capsid 1, and region #452-458 was substituted to NVSKSNQ (SEQ ID NO: 6392). § Substitutions were made to 4 residues from region #583-599 of parent capsids 2,4-6. § Substitutions were made to 3 randomly chosen residues from region #587-597 of all seven parent capsids, and all residues in region #452-458 were substituted with random7-mers. § Substitutions were made to 4 residues from region #583-599 of parent capsids 2,4-6, and all residues in region #452-458 were substituted with random 7-mers.

[0221] These four libraries that included hundreds of billions of variants were IV injected into three Cynomologus Macaque Non-Human Primates, then two weeks later the animals were sacrificed and tissue samples were screened with PCR to uncover the best sequences that caused increased brain enrichment relative to the parent capsid, and / or decreased liver enrichment. Following this PCR screening of tissues, the top 60,891 variants identified in the NHP brains were brought forward into a ‘round 2’ screening. This smaller library was IV injected into four Cynomologus Macaque Non-Human Primates, then four weeks later the animals were sacrificed and tissue samples from brain, spinal cord, DRG, heart, and liver, were screened with PCR to uncover the best sequences that caused further improved brain enrichment relative to the parent capsid, and / or decreased liver enrichment. The specific sequences of the enriched variants found as a result are unique. The nature of directed evolution makes these results fundamentally stochastic, thus the same experiment could be repeated yet result in an entirely different list of enriched variants. Enrichment data for Table 3 variant sequences measured in this study are included in Tables 4 and 4A. The data provided in Tables 4 and 4A provide the enrichment in the specific locations. Further Variant Selection:

[0222] Further maturation of engineered AAV variant sequences was performed through substitution at one or two randomly selected positions between #583-599 within engineered capsids: NHQSATRNGEVFIAQAQ (SEQ ID NO: 4739), NHQSAVRDGHILIAMAQ (SEQ ID NO: 4754), NHQSQQRDGQILIAMAQ (SEQ ID NO: 5158), and NQHSQQRNGEHFIAVAQ (SEQ ID NO: 4857).

[0223] The viral library, comprised of 53418 amino acid variants, was IV injected at a dose of 1.5E13 vg / kg into three adult cynomologus macaques for variant selection. After four weeks in- life, animals were sacrificed and brain, spinal cord, DRG, heart, kidney, liver, lung, and pancreas tissue samples were collected. DNA transduction and RNA expression were screened for each tissue via next-generation sequencing to determine which sequences were improved in brainenrichment and liver detargeting. Enrichment data for Table 4 variant sequences measured in this study are included in Tables 5, 5A, and 5B.

[0224] Enrichment data for exemplary sequences discovered in this study are included in Tables 6, 6A, and 6B.

[0225] Plasmids. The first-round viral DNA library was generated by amplification of a section of the AAV9 capsid genome between amino acids 450-599 using NNK degenerate primers (Integrated DNA Technologies, Inc., IDT) to insert seven random amino acids between amino acids 588 and 589 with all possible variations. The resulting library inserts were then introduced into the rAAV-ΔCap-in-rev-RNA plasmid via Gibson assembly as previously described. The resulting capsid DNA library, rAAV-Cap-Cag-GFP11, contained a diversity of ~1.28 billion variants at the amino acid level.

[0226] An AAV2 / 9 REP-AAP-ΔCAP plasmid transfected into HEK293T cells to provide the Rep gene for library viral production prevents production of a wild-type AAV9 capsid during viral library production after a plausible recombination event between this plasmid co-transfected with the library plasmids at each stage containing the library inserts.

[0227] Viral production. Recombinant AAVs were generated according to established protocols. Briefly, immortalized HEK293T cells (ATCC) were quadruple transfected with four vectors using polyethylenimine (PEI). The first vector was the rAAV-Cap-in-cis-Lox library flanked by inverted terminal repeat (ITR) sequences from a parental AAV virus. The second vector was the AAV2 / 9 REP-AAP-ΔCAP plasmid. The third vector contains nucleic acids encoding helper virus proteins needed for viral assembly and packaging of the heterologous nucleic acid into the modified capsid structure. The fourth is a pUC-18 plasmid included to achieve the right PEI / DNA ratio for optimal transfection enrichment. Only 10 ng of rAAV-Cap-in-cis-Lox library DNA was transfected (per 150 mm plate) to decrease the likelihood of multiple library DNAs entering the same cell. Viral particles are harvested from the cells and media after 60 h post transfection. Virus present in the media is concentrated by precipitation with 8% polyethylene glycol and 500mM sodium chloride and the precipitated virus is added to the lysates prepared from the collected cells. The viruses are purified over iodixanol (Optiprep, Sigma) step gradients (15%, 25%, 40%, and 60%). Viruses are concentrated and formulated in PBS. Virus titers are determined by measuring the number of DNaseI-resistant vector genome copies (VGs) using qPCR and the linearized genome plasmid as a control.

[0228] Animals. Cynomolgus Macaque procedures were approved by ACUC of the National Institutes of Mental Health. Cynomolgus Macaques were born and raised in NIMH colonies and housed in family groups under standard conditions of 27oC temperature and 50% humidity. They were fed ad libitum and received enrichment as part of the primate enrichment program for NHPs at the NIH. For AAV infusions, animals were screened for endogenous neutralizing antibodies (Nab). None of the animals that were screened showed any detectible blocking reaction at 1:5 dilution of serum (Penn Vector Core, University of Pennsylvania). They were then housed individually for several days and acclimated to a new room before injections. The day before infusion the animals’ food was removed. Animals were anesthetized with isoflurane in oxygen, the skin over the femoral vein was shaved and sanitized with an isopropanol scrub, and the virus was infused over several minutes. Anesthesia was withdrawn and the animals were monitored until they became active, upon which they were returned to their cages. Activity and behavior were closely monitored over the next three days, with daily observations thereafter.

[0229] DNA / RNA recovery and sequencing. Viral libraries were injected into Macaques at a dose of 1x1013vg / kg animal and rAAV genomes were recovered four weeks post injection. Animals were euthanized and brain, spinal cord and liver were recovered, snap frozen, and placed into long- term storage at -80oC as well as other peripheral tissues such as heart, spleen, adrenal, kidney, and quad. The brain was separated into eleven brain regions, and 20-300mg of each brain section was homogenized in buffer using the MagMAX DNA ULTRA (A25597) and a Bead Ruptor 96 (OMNI, INC) and viral DNA was isolated according to the manufacturers recommended protocol. Recovered viral DNA was treated with RNase, and purified with a Zymo DNA Clean and Concentrator kit (D4033). Viral genomes were enriched by 25 cycles of PCR amplification with primers flanking the 588-589 insertion site in the capsid genome using 50% of the total extracted viral DNA as a template. After Zymo DNA purification, samples were diluted 1:10 to 1:1000 depending on tissue type and each dilution further amplified around the library variable region with 10 cycles of PCR. Subsequently, samples were further amplified using custom primers with Illumina Indices for 10 more cycles. The amplification products were run on a 2% low-melting point agarose gel (ThermoFisher Scientific, 16520050) for better separation and recovery of the 480 bp band.

[0230] Packaged viral library DNA was isolated from the injected viral library by digestion of the viral capsid and purification of the contained ssDNA. These viral genomes were amplified by twoPCR amplification steps, like the viral DNA extracted from tissue, to add adapters and indices for Illumina next-generation sequencing, and purified after gel electrophoresis. This viral library DNA, along with the viral DNA extracted from tissue, was sent for deep sequencing using an Illumina NextSeq 2000 system.

[0231] NGS data alignment and processing. Raw Fastq files from NGS runs were processed with custom-built scripts (Capsida CapSeq Tools). For the first-round library, the pipeline to process these datasets involved filtering to remove low-quality reads, utilizing a quality score for each sequence, and eliminating bias from PCR-induced mutations or high GC-content. The filtered dataset was then aligned by a perfect string match algorithm and trimmed to improve the alignment quality. Read counts for each sequence were pulled out and displayed by tissue, at which point all sequences found in the brain were compiled for formation of the libraries.

[0232] Library read counts by tissue were similarly tabulated. Then, a read count of 1 was added to each sequence to remove 0 values, all brain regions for each sequence were summed together, and the read sequences for each codon replicate of a given 7-mer or 11-mer amino acid sequence were summed together to give a single value for each peptide insertion. Finally, the data was log10 counts per million (Cpm) normalized. Enrichment values were calculated with normalized Cpm of brain vs Cpm of virus and converted to log10. Table 4: Enrichment Data: #583-599 amino acid Brain Brain Spinal Spinal DRG DRG R A D A R A D A4011 NHQSAAENHVRSTAAAQ 2.464 1.263 0.007 4.797 0 1.768 4012 NHQSAAENHVRSVAVAQ 2.857 1.09 0.008 1.453 0 0.791 41 AA A 2 1 14060 NHQSAIRAGEHFKAAAQ 0.195 1.128 0 0.163 0 1.609 4061 NHQSAIRDGEHFKAQAQ 1.596 1.848 0.005 0.638 0 0.65 4 2 A A A 111 1 41 2 18 94109 NHQSAKRDGHILIAPAQ 0.881 3.005 0.001 3.437 0 1.404 4110 NHQSAKRDGHILIASAQ 1.546 1.234 0.007 0.405 0 0.297 4111 A A 1 2 2 12 14158 NHQSALRNGEHFQSQAQ 0.995 1.269 0.004 0.488 0 0.003 4159 NHQSALRNGEHFVAMAK 3.7 5.945 0 5.187 0 0 41 A A 14207 NHQSAMRNGIHFKAPAQ 2.682 2.823 0.005 0.362 0 2.778 4208 NHQSAMRNGMHFQAQAQ 0.012 2.893 0 0.906 0 0 42 A A A 42 1 1 1 42 2 1 64256 NHQSAQENHVRAQAQAQ 0.172 1.455 0.015 2.414 0 2.006 4257 NHQSAQENHVRASAGAQ 4.042 1.061 0.01 1.827 0.004 1.947 42 A A A A 2 1 2 24305 NHQSAQENHVRNSMQAQ 1.076 1.647 46.344 2.957 0.019 6.216 4306 NHQSAQENHVRNSQQAQ 0.475 1.258 0.005 1.36 0 2.73 4 A AAA 12 14 1 4 1 184354 NHQSAQENHVRSNADAQ 3.368 0.944 0.01 0.904 0.001 0.517 4355 NHQSAQENHVRSNANAQ 0.909 1.498 0.005 4.48 0 1.273 4 A A A 1 1 4 44 1 454403 NHQSAQENHVRSSQIAQ 2.135 0.078 0.027 0.873 0.785 0.323 4404 NHQSAQENHVRSSSLAQ 10.014 0.903 0.006 0.052 4.827 1.096 44 A A 42 114452 NHQSAQENHVRVSASAQ 2.623 0.293 0.009 0.02 0 0.003 4453 NHQSAQENHVRVYAQAQ 2.388 0.62 0.004 0.447 0 0.598 444 A A A A 4 14501 NHQSAQRDGLILISGAQ 1.503 1.932 0.004 2.242 0 0.596 4502 NHQSAQRDGMHFVAQAQ 2.899 1.934 0.006 1.916 0 4.47 4 A A A 1 144550 NHQSAQRNGEYFHASAQ 0 0.259 0.003 0 0 0 4551 NHQSAQRNGGHFIAAAQ 6.653 0.684 0.017 0.008 0 0.295 4 2 A AAA 24 12 4 134599 NHQSARLGGNILIAPAQ 0 0.878 0 1.023 0 1.006 4600 NHQSARLGGQILIAGAQ 0.002 2.873 0 2.018 0.007 1.42 4 1 A A A 2 1 1 23 2 7 34648 NHQSASENHVRSLAQAQ 0.418 1.304 0.009 2.228 0.001 1.001 4649 NHQSASENHVRSNAQAQ 0.274 1.057 0.004 2.247 0 3.844 4 A A A 2 12 124 12 4 24164697 NHQSATENHVRSSNTAQ 4.526 0.899 0.012 2.207 0.003 0.024 4698 NHQSATENHVRSSTGAQ 3.622 1.023 0.028 1.857 0.003 2.706 4 A A A 1 1 1 1 54746 NHQSATTNGTHFIAQAQ 0.697 0 0.003 0 0 0.401 4747 NHQSAVENHVRASNQAQ 0.465 1.119 0.011 1.1 0.002 0.599 44 A A A 4 144 1 2 2 184795 NHQSAYRNGEHFHAQAQ 0.095 1.154 0 1.011 0 0 4796 NHQSAYRNGEHFKAQAQ 0 1.052 0 0.957 0 0.294 4 A A A 41 1 11 24844 NHQSEQRDGHILIVRAQ 10.947 0.135 0 0 0 2.128 4845 NHQSERENHVRSVAAAQ 0.306 0.487 0.023 0.561 0.012 0 44 A A 14 24 2 144893 NHQSHNKPPGYLLTKAQ 20.65 0 0 0 0 0 4894 NHQSHNLSGQILIASAQ 6.771 0.101 0 0 0 0.738 4 A A 1 12 4 494942 NHQSKARDGHILIAPAQ 6.063 1.374 0.004 1.255 0 1.132 4943 NHQSKEENHVRSSATAQ 2.678 0.722 0.011 0.256 0 1.329 444 A A 2 2 1 24991 NHQSMQENHVRSSAQAQ 0.566 1.239 0.004 2.797 0 3.032 4992 NHQSMQENHVRSSASAQ 5.339 0.612 0.002 1.605 0 1.338 4 A 4 22 4 45040 NHQSNQRPPGYLLTGAQ 30.792 0.063 0 0.016 0 0 5041 NHQSNRLSGQILIANAQ 6.945 0.256 0 0.006 0 0 42 A A 14 2 12 65089 NHQSQHRDGHILIANAQ 11.738 1.087 0.002 0.798 0 1.485 5090 NHQSQHRDGLILIAAAQ 6.203 2.621 0 0.451 0 0 1 A 4 42 14 1 45138 NHQSQQRDGEILIKQAQ 11.004 0.018 0 0 0 0 5139 NHQSQQRDGGILIAMAQ 27.47 1.129 0.009 0.074 0 0 14 A A 1 1 2 35187 NHQSQSRDGHILIAQAQ 5.825 1.569 0.012 3.985 0 5.2 5188 NHQSQSRDGHILIASAQ 5.217 1.602 0.115 0.204 0 0.168 1 A A 1 1 224 2 2 2 4 25236 NHQSSMENHVRASATAQ 8.923 0.835 0.023 2.203 74.066 0.037 5237 NHQSSMENHVRSSGQAQ 0.786 1.143 0.01 1.099 0 0.976 2 A A 4 1 4 213 85285 NHQSSVRDGLILIAAAQ 33.439 0.989 0 0.074 0 10.038 5286 NHQSSVRNGEHFIAQAQ 5.097 0.019 0.004 0.01 0 0 2 A A 4 1 195334 NHQSTQRPPGYLLAQAQ 2.373 1.281 0 0.006 0 0 5335 NHQSTRLSGQILIAGAQ 8.078 0.257 0.035 0.011 0 0 A A A 4 4 1 4 1 13Table 4A: Enrichment Data: SEQ ID NO #583-599 amino acid Heart Heart Liver Liver sequences RNA DNA RNA DNA4042 NHQSAFLGGQILISNAQ 0 0 5.88 0.202 4043 NHQSAFRDGLILIHLAQ 0 0 0 0.252 444 A A 1 2 44091 NHQSAIRNGLHFKAHAQ 0 5.902 0 0 4092 NHQSAIRNGQHFFAQAQ 0 1.534 0 0.161 4 A A A 14 214140 NHQSALRDGEHFKMQAQ 0 0.076 0 0.642 4141 NHQSALRDGEHFKVQAQ 0 2.242 0 0.092 4142 A A A 1 14189 NHQSAMRDGHILIAVAQ 0 0 0 0.146 4190 NHQSAMRDGHILITQAQ 0 0.297 2.859 1.116 411 A A A 2 24238 NHQSANENHVRTSAQAQ 0.005 1.59 2.311 2.932 4239 NHQSANLSGQILIAPAQ 0 0 0 0.173 424 A A A 44287 NHQSAQENHVRMSANAQ 5.954 1.17 1.073 1.992 4288 NHQSAQENHVRMSSGAQ 0.004 1.877 2.211 2.613 42 A AA 141 4 14336 NHQSAQENHVRSFSNAQ 7.964 1.669 0.838 0.564 4337 NHQSAQENHVRSGGAAQ 0 3.446 0 1.17 4 A A 1 2 4 22 1224385 NHQSAQENHVRSSASAQ 5.406 3.2 3.522 1.968 4386 NHQSAQENHVRSSATAQ 2.855 1.958 1.095 2.067 4 A A 4 12 414434 NHQSAQENHVRSVSGAQ 74.452 1.374 0.024 0.853 4435 NHQSAQENHVRSYDQAQ 0.003 1.493 1.029 1.064 44 A AA 1 214 4 144483 NHQSAQRDGHILIAPAQ 0.002 2.497 0 0.16 4484 NHQSAQRDGHILIASAQ 0 1.706 0 0.236 44 A A A 14532 NHQSAQRNGEIFFAAAQ 0 0 0 0.069 4533 NHQSAQRNGEIFFARAQ 0 3.152 0 0.151 4 4 A AA 1 14581 NHQSAQRNGQIFIAQAQ 0 1.793 0.279 1.187 4582 NHQSAQRNGQPFLAQAQ 0.005 0.264 4.004 2.673 4 A A A 1 1 2444630 NHQSARRDGQILIATAQ 0 2.618 0 0.401 4631 NHQSARRDGQILIAVAQ 0 0.529 0 0.307 4 2 A A A 44679 NHQSASRNGEHFVARAQ 0 0.833 0 0.08 4680 NHQSASRNGEHFVSQAQ 0 0.014 0 1.101 4 1 A A A 1 1 14728 NHQSATRNGEHFIALAQ 0 0 0 0.09 4729 NHQSATRNGEHFIAVAQ 0 0.414 0 0.062 4 A AAA 1 44 14777 NHQSAYENHVRSQANAQ 0.002 0.632 0.352 0.957 4778 NHQSAYENHVRSSNGAQ 3.531 1.13 0.118 2.636 4 A AA 14 144826 NHQSEIKPPGYLLTKAQ 0 0.552 0 0.175 4827 NHQSEKENHVRSSADAQ 4.528 2.165 1.786 0.826 42 A A 144875 NHQSGRLSGQILIANAQ 0 0 0 1.836 4876 NHQSGRRDGLILIATAQ 0 0 0 0.035 4 A 21 1 14924 NHQSHTLGGQILISQAQ 0 0.338 0.01 0.398 4925 NHQSHTRDGLILIMQAQ 0 0 0 0.768 42 A 4244973 NHQSLAENHVRSSAAAQ 0 0.407 3.008 0.895 4974 NHQSLIENHVRASAQAQ 0 2.013 3.738 2.145 4 A A 25022 NHQSNQENHVRSADQAQ 0 2.759 2.123 2.713 5023 NHQSNQENHVRSDANAQ 0.006 2.02 1.64 1.127 24 A A 4 2 2 15071 NHQSPQENHVRSSTNAQ 0.002 1.925 1.009 1.201 5072 NHQSPQENHVRSSVQAQ 0 2.5 1.629 2.917 A 24 2 2 215120 NHQSQQENHVRSSATAQ 0 1.777 0.28 2.375 5121 NHQSQQENHVRSSGQAQ 0 1.107 0 1.087 122 A 1 1 11 4 225169 NHQSQQRNGEHFMAVAQ 0 0 0 0.239 5170 NHQSQQRNGEIFIAQAQ 0 0.699 0.614 0.327 11 A A 1 45218 NHQSRQLGGHILIAAAQ 0 0 0 0.335 5219 NHQSRQLGGQILINPAQ 0 0.33 0 0.359 22 A 4 1 15267 NHQSSQENHVRYSAQAQ 0.005 1.242 2.157 1.821 5268 NHQSSQLGGRILIAPAQ 0 0.155 1.622 1.745 2 AAA 1 12 242 225316 NHQSTQENHVRSMEQAQ 0 3.32 2.264 2.2 5317 NHQSTQENHVRSSADAQ 0 3.448 0.53 0.821 1 A A 14 1 15365 NHQSVQENHVRTSANAQ 5.743 0.531 4.718 2.272 5366 NHQSVQLGGQILIAQAK 0 0 0 0.411 A A 4 1 124a e SEQ #583-599 amino acid Brain Brain Spinal Spinal DRG DRG ID sequences RNA DNA Cord Cord RNA DNATable 5A SEQ #583-599 amino acid Heart Heart Kidney Kidney Liver Liver4754 NHQSAVRDGHILIAMAQ 0.699 16.734 2.524 27.577 0.785 0.846 4771 NHQSAVRNGEIFIAQAQ 0.094 12.266 0.004 22.303 0.614 0.881 1 A A 1 24 2 24 2 2SEQ #583-599 amino acid Lung Lung Pancreas Pancreas ID sequences RNA DNA RNA DNATable 6: Enrichment data SEQ #583-599 amino acid Brain Brain Spinal Spinal DRG DRG ID n RNA DNA Cord Cord RNA DNA5397 NHQAALRNGEVFIAQAQ 23.235 10.168 0.183 17.906 7.248 4.896 5398 NHQAAMRNGEVFIAQAQ 23.745 8.767 0.229 15.611 0.008 1.6765442 NHQRQTRNGEVFIAQAQ 10.797 11.73 0.072 74.645 0.002 8.693 5443 NHQSAARNGEVFIAAAQ 31.969 7.623 0.173 28.477 0.002 2.6295487 NHQSALRNGEVFIAHAQ 21.697 13.045 0.114 10.224 27.913 10.707 5488 NHQSALRNGEVFIASAQ 38.942 11.673 0.168 29.243 0 12.6345532 NHQSAQRNGEVFIASAQ 30.043 8.648 0.168 8.776 0.317 10.605 5533 NHQSAQRNGEVFIATAQ 49.626 11.19 1.288 5.162 0.001 3.0065577 NHQSATKNGEIFIAQAQ 72.415 16.966 219.929 19.167 0.41 11.593 5578 NHQSATKNGEVFFAQAQ 20.144 11.126 0.229 16.021 0.004 2.0465622 NHQSATQNGAVFIAQAQ 9.286 4.026 5.527 7.076 0.012 0.425 5623 NHQSATQNGEVFIAAAQ 12.854 4.396 0.057 6.062 0 1.3265667 NHQSATRGIEVFIAQAQ 18.283 8.681 0.099 5.244 0.021 3.13 5668 NHQSATRGKEVFIAQAQ 13.873 10.535 0.103 29.887 0.067 5.542 35712 NHQSATRNGEAFIAIAQ 44.318 9.184 17.257 7.352 0.001 7.18 5713 NHQSATRNGEAFIALAQ 42.979 8.117 3.219 2.319 0.005 0.5785757 NHQSATRNGETYIAQAQ 74.311 16.001 135.554 48.119 0.009 4.583 5758 NHQSATRNGEVFAAIAQ 15.722 2.51 158.332 0.025 11.54 0.0195802 NHQSATRNGEVFIAQPQ 73.312 16.133 264.803 37.059 14.275 8.437 5803 NHQSATRNGEVFIAQQQ 22.858 4.402 0.092 6.763 0 0.0125847 NHQSATRNGEVFVAAAQ 74.716 9.747 48.522 6.795 8.399 3.525 5848 NHQSATRNGEVFVAGAQ 70.731 10.364 38.729 22.414 0.155 7.7665892 NHQSATRNGNVFVAQAQ 18.043 16.835 11.185 6.494 0.235 8.012 5893 NHQSATRNGQLFIAQAQ 17.29 21.936 0.119 23.96 0.148 13.4765937 NHQSATRNSEVYIAQAQ 61.354 18.964 91.69 40.607 1.516 10.487 5938 NHQSATRNTEVFIAGAQ 13.624 3.031 0.083 5.114 0 4.1225982 NHQSAVLNGEVFIAQAQ 30.939 4.895 6.614 9.428 0.236 3.431 5983 NHQSAVMDGHIRIAMAQ 24.316 12.973 0.213 8.845 34.609 4.2716027 NHQSAVRNAEVFIAQAQ 25.495 10.856 52.994 14.437 0.004 5.341 6028 NHQSAVRNGAVFIAQAQ 19.325 92.664 0.14 144.982 0.29 101.2616072 NHQSETRHGEVFIAQAQ 7.335 3.763 0.073 6.492 4.545 1.711 6073 NHQSETRNAEVFIAQAQ 12.393 1.745 0.1 5.389 0.146 2.8496117 NHQSMTRNGEVFIAQAQ 44.337 16.718 29.207 15.309 0.172 5.171 6118 NHQSMTRNGEVFIASAQ 12.913 8.148 0.049 7.554 0.001 1.9716162 NHQSNTRNMEVFIAQAQ 16.873 10.334 0.073 12.305 0 2.477 6163 NHQSNTRNQEVFIAQAQ 27.193 10.824 0.194 13.279 0.577 0.0636207 NHQSQQRNGETFIAHAQ 12.483 5.625 0.094 6.694 2.14 0.747 6208 NHQSQQRNGETFIAVAQ 27.216 9.117 13.567 8.914 0.003 2.416252 NHQSQVRDGQIHIAMAQ 28.35 7.36 4.395 13.625 0.173 4.211 6253 NHQSQVRDGSILIAMAQ 54.503 10.018 0.244 8.862 0 1.4516297 NHQSTTRNGEVFIATAQ 12.602 8.67 0.09 5.261 0 3.471 6298 NHQSTTRNGEVYIAQAQ 76.363 14.693 68.802 25.429 0.012 13.0536342 NKQSATRNGEVFIAQAQ 12.843 31.668 0.144 15.446 0.365 12.83 6343 NLQSATRNGEVFIANAQ 39.878 12.147 44.594 29.945 0 19.06Table 6A: Enrichment dataSEQ #583-599 amino acid Heart Heart Kidney Kidney Liver Liver ID sequences RNA DNA RNA DNA RNA DNA NO5423 NHQKQTRNGEVFIAQAQ 0.073 9.965 0.045 0.884 3.058 6.631 5424 NHQLATRNGEVYIAQAQ 0.33 3.074 0.132 1.748 0.964 3.2955468 NHQSAIRNGEVFIAGAQ 0.058 7.906 0.003 12.836 0.159 0.446 5469 NHQSAIRNGEVFIAQAQ 0.42 5.35 24.709 15.568 0.566 3.8495513 NHQSAMRNKEVFIAQAQ 0.158 18.329 0.031 2.292 5.941 10.625 5514 NHQSAMRNNEVFIAQAQ 0.193 5.423 0.083 3.904 15.042 3.5425558 NHQSATFNGEVFIAQAQ 0.034 1.508 0.017 1.214 0.179 0.73 5559 NHQSATFNGEVFIASAQ 0.053 1.183 0 0.876 0 0.1515603 NHQSATLNGEIFIAQAQ 0.001 2.712 0.025 1.618 0.01 0.418 5604 NHQSATLNGEVFIAQAQ 0.08 3.937 2.963 1.329 0.641 0.5265648 NHQSATRDGNILIAMAQ 0.183 4.278 0.004 1.613 3.303 0.713 5649 NHQSATRDGNVFIAQAQ 0.266 14.573 0.062 22.53 8.173 3.865693 NHQSATRNDEIFIAQAQ 0.082 4.231 0.04 1.855 2.742 0.864 5694 NHQSATRNDEVFIANAQ 0.112 1.573 0.034 0.86 13.572 0.1795738 NHQSATRNGEPFIAVAQ 0.037 2.293 0 1.175 0 0.281 5739 NHQSATRNGEQFIAIAQ 0 3.87 0 1.168 0 0.5585783 NHQSATRNGEVFIANAA 0.171 4.73 0.002 2.376 1.327 0.524 5784 NHQSATRNGEVFIANAQ 0.383 5.991 0.016 1.748 0.136 0.7965828 NHQSATRNGEVFISTAQ 0.07 6.02 0 1.511 0.002 0.247 5829 NHQSATRNGEVFLAAAQ 0.038 4.619 0.003 2.237 0.002 0.3325873 NHQSATRNGEVMIAVAQ 0.056 4.091 0 0.82 0.01 0.292 5874 NHQSATRNGEVRIAIAQ 0.211 13.719 0.093 2.07 4.116 9.5765918 NHQSATRNKEVYIAQAQ 0.134 24.479 0.04 1.175 7.03 15.721 5919 NHQSATRNLEVYIAQAQ 0 7.293 0.121 1.331 1.782 2.4255963 NHQSATVNGEVFIAGAQ 0.023 1.794 0.001 1.169 0.001 0.165 5964 NHQSATVNGEVFIASAQ 0.014 1.637 0.001 1.047 0 0.1486008 NHQSAVRDGQIMIAMAQ 0 6.261 0 1.854 0.606 1.08 6009 NHQSAVRDGQVLIAMAQ 0.001 6.265 0.002 1.788 0.181 1.8396053 NHQSAVRNQEVFIAQAQ 0 4.844 0.004 1.011 1.182 2.682 6054 NHQSAVRNSEVFIAQAQ 0 6.578 0.05 1.933 5.02 6.776098 NHQSHTRNGEVFFAQAQ 0.068 6.387 0.011 1.014 0.405 1.489 6099 NHQSHTRNGEVFIASAQ 0.078 13.024 0.014 36.267 0.001 0.3526143 NHQSNTRGGEVFIAQAQ 0.114 14.553 0.011 2.076 4.95 3.162 6144 NHQSNTRHGEVFIAQAQ 0.644 23.111 345.914 2.909 9.808 5.3916188 NHQSQQRDGGILIAMAQ 0 11.584 0.004 22.626 1.926 0.549 6189 NHQSQQRDGHLLIAMAQ 0.14 17.664 0.002 9.361 0.003 0.816233 NHQSQTRNGEVFLAQAQ 0.494 18.486 0.019 13.141 1.813 7.466 6234 NHQSQTRNGEVFYAQAQ 0.235 22.875 0.012 2.982 5.942 2.3476278 NHQSSTRNGGVFIAQAQ 0.257 27.286 0.01 16.703 1.726 5.695 6279 NHQSSTRNGQVFIAQAQ 0.574 41.91 0.034 23.807 0.599 4.3356323 NHQSYTRNGQVFIAQAQ 0.07 21.246 0.004 3.947 1.119 4.191 6324 NHQSYTRNSEVFIAQAQ 0 4.53 0.174 1.365 2.882 2.9466368 NTQSATRNGEVFIASAQ 0 8.158 0.005 5.134 0.618 0.324 6369 NVQSATRNGEVFIAGAQ 0 7.96 0.001 4.545 0.002 0.378a e : nrc men aa SEQ #583-599 amino acid Lung Lung Pancreas Pancreas ID sequences RNA DNA RNA DNA5405 NHQAMTRNGEVFIAQAQ 0.016 14.499 6.725 4.381 5406 NHQANTRNGEVFIAQAQ 0.016 22.923 3.581 4.85450 NHQSAERNGEVFIARAQ 0 2.109 0.002 2.921 5451 NHQSAERNGEVFIAVAQ 0.004 3.887 0 2.8825495 NHQSAMRDGAILIAMAQ 0 4.17 0.036 8.044 5496 NHQSAMRDGGILIAMAQ 0 5.32 0 5.6785540 NHQSASRDGHVLIAMAQ 0.01 6.433 1.336 6.44 5541 NHQSASRDGKILIAMAQ 0.006 4.622 0.006 2.6825585 NHQSATKNGEVFIAYAQ 0 2.427 0.001 1.424 5586 NHQSATKNGEVFYAQAQ 0.015 17.261 2.305 3.5885630 NHQSATRAGEVFIAGAQ 0.003 3.19 0 2.317 5631 NHQSATRAGEVFIAHAQ 0.009 1.434 1.69 1.2355675 NHQSATRHGEVFIAQAQ 0.199 11.391 6.962 6.186 5676 NHQSATRHGEVFIASAQ 0.005 13.604 0.198 3.7595720 NHQSATRNGEIFYAQAQ 0.009 20.861 0.115 3.537 5721 NHQSATRNGEIYIAQAQ 186.734 38.858 3.565 4.0345765 NHQSATRNGEVFIAAAA 0.004 12.472 0 3.09 5766 NHQSATRNGEVFIAAAG 0.009 4.829 0.586 2.5775810 NHQSATRNGEVFIASAG 0 2.438 0.002 3.629 5811 NHQSATRNGEVFIASAL 0.001 4.341 0.313 2.2385855 NHQSATRNGEVHIALAQ 0.001 2.423 2.5 4.401 5856 NHQSATRNGEVHIAMAQ 0.014 8.571 4.198 4.3835900 NHQSATRNGSVFIAQAQ 0.071 50.891 2.212 15.117 5901 NHQSATRNGTIFIAQAQ 0.033 32.814 0.014 11.4285945 NHQSATRQGEVFIASAQ 0.001 5.789 0 2.739 5946 NHQSATRQGEVFIATAQ 0.002 5.069 0.263 2.6625990 NHQSAVRDGHILIAVAQ 0.026 3.338 0.043 9.132 5991 NHQSAVRDGHILMAVAQ 0.027 3.459 5.715 4.8686035 NHQSAVRNGEVFIAQAQ 0.042 14.414 1.644 4.279 6036 NHQSAVRNGEVFIASAQ 0.002 7.817 0.003 2.1516080 NHQSETRNGHVFIAQAQ 0.029 21.014 5.959 5.906 6081 NHQSETRNGNVFIAQAQ 0.024 22.044 5.734 2.5926125 NHQSMTRNTEVFIAQAQ 0.076 8.558 25.805 5.593 6126 NHQSMTRQGEVFIAQAQ 0.016 3.786 3.564 4.0336170 NHQSNVRDGAILIAMAQ 0.014 3.048 4.704 3.932 6171 NHQSNVRDGHIHIAMAQ 0.002 4.325 0.722 5.5826215 NHQSQTFNGEVFIAQAQ 0.001 3.264 0.183 2.073 6216 NHQSQTHNGEVFIAQAQ 0.007 3.606 0.272 3.8056260 NHQSSTINGEVFIAQAQ 0.039 3.175 0.35 4.261 6261 NHQSSTKNGEVFIAQAQ 0.019 21.35 4.695 5.2826305 NHQSWTKNGEVFIAQAQ 0.005 10.071 1.748 2.146 6306 NHQSWTRNGETFIAQAQ 0.005 13.069 1.202 3.4686350 NNQSATRNGEVFIAHAQ 0.002 3.345 0.008 2.481 6351 NNQSATRNGEVFIANAQ 0.01 3.113 2.901 4.969Example 2 Method of Identifying the Modified Capsid Proteins in Cynomolgus Macaques and Mice

[0233] Following the successful identification of promising variants mutated between amino acid residues 583-599, amino acid substitution and insertion libraries between amino acid residues 449 through 460 were produced and investigated. These variants were combined with capsid proteins that contained insertion and / or substitutions between residues 583-599, described in tables 3, 4, 4A, 5, 5A, 5B, 6, 6A and / or 6B. Iterative rounds of screening and library generation selected capsid proteins with amino acid substitution or insertion in both 449-460 and 583-590 which showedbrain enrichment (e.g., specific transport to and targeting of brain tissue) relative to the parental capsid proteins.

[0234] A library of viral capsids was created by performing random amino acid insertions and substitutions between amino acid residues 449-460 within AAV9, targeting NHP brain and / or central nervous system (e.g., in the spinal cord) tropism

[0235] Adeno-Associated Virus serotype 9 (AAV9) was modified by inserting and / or substituting six contiguous amino acids (6-mer) at various positions in variable region IV, typically following amino acid residue 452 in its capsid coding sequence. A combination of substitution and insertions were made between amino acid residues 449-460 (VP1 numbering, RTINGSGQNQQT) (SEQ ID NO: 3996), alongside the VR-VIII insertion NHQSATRNGEIFIAQAQ (SEQ ID NO: 4738) at positions 583-599, which was a substitution / insertion produced in Example 1 that showed promising brain enrichment activity.

[0236] Seven libraries were produced off of these parent capsids, where an X indicates diversified residues: Substitution of six amino acids between amino acids 452-457, yielding RTIXXXXXXQQT; Substitution of six amino acids between amino acids 453-458, yielding RTINXXXXXXQT; Substitution of four amino acids and insertion of two amino acids between 453-456; yielding RTINXXXXXXNQQT; Substitution of three amino acids and insertion of three amino acids between 454-456, yielding RTINGXXXXXXNQQT; Substitution of two amino acids and insertion of four amino acids between 454-455, yielding RTINGXXXXXXQNQQT; Substitution of two amino acids and insertion of four amino acids between 455-456, yielding RTINGSXXXXXXNQQT; and Substitution of a single amino acid and insertion five amino acids at position 455, yielding RTINGSXXXXXXQNQQT.

[0237] These seven libraries comprised of billions of variants were IV injected into three Cynomologus Macaque Non-Human Primates. Four weeks later the animals were sacrificed and tissue samples were screened with PCR to uncover the best sequences that caused increased brain enrichment relative to the parent capsid, and / or decreased liver enrichment. Following this PCR screening of tissues, the top 35,000 variants identified in the NHP brains were brought forwardinto a second round of selection paired with VR-VIII insertions NHQSATRNGEIFIAQAQ (SEQ ID NO: 4738) and NHQSATRNGEVFIAQAQ (SEQ ID 4739), which are promising variants from Example 1. This smaller library was IV injected into three Cynomologus Macaque Non-Human Primates, then four weeks later the animals were sacrificed and tissue samples from brain, liver, and heart were screened with PCR to uncover the best sequences that caused further improved brain enrichment relative to the parent capsid, and / or decreased liver enrichment. Enrichment data for variant sequences in Tables 2 measured in this study is provided in Table 8. The same smaller library was also IV injected into five C57BL6 / J mice, then four weeks later the animals were sacrificed and tissue samples from brain, liver, and heart were screened with PCR to uncover sequences that caused further improved brain enrichment relative to the parent capsid, and / or decreased liver enrichment. Enrichment data for variant sequences in Tables 2 measured in this study is provided in Table 7.

[0238] Three variants were selected to be produced individually and pooled for IV injection into three cynomologus macaque non-human primates, then four weeks later the animals were sacrificed and tissue samples from brain, liver, and heart were screened with PCR to determine improved enrichment relative to viral pool input, and / or decreased liver enrichment. Enrichment data for the variant sequences measured in this study are included in Table 9.

[0239] As these modified AAV9 capsid proteins were produced using directed evolution via peptide insertions, the resulting sequences of the enriched variants found are novel as a result. The nature of directed evolution makes results fundamentally stochastic, thus the same experiment could be repeated yet result in an entirely different list of enriched variants. Nonetheless, the variant capsid proteins described herein show statistically significant brain enrichment relative to the underlying parental capsid protein. This is of critical importance, as following IV injection, unmodified AAV9 does not have sufficient tissue enrichment to treat many human diseases that would otherwise be amenable to treatment by delivery from AAV cargo of 4.7kbp DNA. Directed evolution of AAV9 as performed herein provided a means of producing capsids that lead to increasing viral tissue enrichment to levels that enable disease treatment.

[0240] While preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. It should be understood that various alternatives tothe embodiments of the invention described herein may be employed in practicing the invention. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby.

[0241] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. Table 7: SEQ ID VR-IV amino acid sequence RNA Brain RNA RNA DNA NO: Heart Liver Brain1771 RTINGTPGASTQNQQT 0009 0295 001 0689Table 8: SEQ ID VR-IV amino acid RNA RNA RNA DNA DNA DNA1991 RTINGSPAGKGSQNQQT 2.285 0 7.376 1.505 1.604 2.086SEQ VR-IV amino acid RNA RNA RNA DNA DNA DNA Liver ID sequence Brain Heart Liver Brain HeartExample 3 Further Selection of Modified Capsid Proteins in Cynomolgus Macaques

[0242] Following identification of promising variants between amino acid residues 449-460 in Example 2, further maturation was achieved via one or two amino acid substitutions at positions 452-462 in combination with six different peptide insertions at positions 583-599 including insertion NHQSATRNGEIFIAQAQ (SEQ ID NO.4738).

[0243] Six capsids identified in Example 4 with combinations of substitution and insertions at 449- 460 were selected as templates for mutation alongside the VR-VIII insertion NHQSATRNGEIFIAQAQ (SEQ ID NO: 4738) at positions 583-599 from Example 1: RTINGAGQNQQT (SEQ ID NO: 1072), RTINGTGQNQQT (SEQ ID NO: 1074), RTINKGGSAGNQQT (SEQ ID NO: 1222), RTINKGTSSQQT (SEQ ID NO: 1073), RTIGGDSSKQQT (SEQ ID NO.1002), and RTINGPVSSGKQNQQT (SEQ ID NO.1678)).

[0244] Six libraries were produced off of these parent capsids, where X indicate diversified residues: Substitution of a single amino acid between amino acids 452-458, yielding RTIXXXXXXXQT; Substitution of a single amino acid between amino acids 452-460, yielding RTIXXXXXXXXXQT;Substitution of a single amino acid between amino acids 452-462, yielding RTIXXXXXXXXXXXQT;

[0245] These six libraries of viral capsids comprised of tens of thousands of amino acid variants were IV injected into 3 Cynomologus Macaque Non Human Primates. Four weeks later the animals were sacrificed, and brain, heart, liver, spinal cord, and DRG tissue samples were collected. Samples were screened with PCR to uncover the best sequences that caused increased brain enrichment relative to the parent capsid, and / or decreased liver enrichment. Exemplary variant sequences with NHQSATRNGEIFIAQAQ (SEQ ID NO. 4738) at positions 583-599 from this study are included in Tables 10A and 10B. Table 10A: SEQ ID VR-IV amino acid RNA RNA RNA RNA RNA NO: sequence Brain Heart Liver Spine DRGTable 10B: SEQ ID VR-IV amino acid DNA DNA DNA DNA DNA O B i H Li S i DRG1678 RTINGPVSSGKQNQQT 3.584 3.069 0.896 5.974 1.147 1705 RTINGPVSQGKQNQQT 6.793 2.242 1.943 0.027 0Incorporation by Reference References and citations to other documents, such as patents, patent applications, patent publications, journals, books, papers, web contents, publicly accessible databases, have been made throughout this disclosure. All such documents are hereby incorporated herein by reference in their entirety for all purposes. Equivalents Various modifications of the invention and many further embodiments thereof, in addition to those shown and described herein, will become apparent to those skilled in the art from the full contents of this document, including references to the scientific and patent literature cited herein. The subject matter herein contains important information, exemplification and guidance that can be adapted to the practice of this invention in its various embodiments and equivalents thereof.

Claims

CLAIMS WHAT IS CLAIMED IS:

1. An AAV capsid protein comprising an amino acid substitution and / or insertion sequence at position 452 having at least 80% identity to a sequence provided in Table 1.

2. An AAV capsid protein comprising an amino acid substitution and / or insertion sequence at position 453 having at least 80% identity to a sequence provided in Table 1.

3. An AAV capsid protein comprising an amino acid substitution and / or insertion sequence at position 454 having at least 80% identity to a sequence provided in Table 1.

4. An AAV capsid protein comprising an amino acid substitution and / or insertion sequence at position 455 having at least 80% identity to a sequence provided in Table 1.

5. An AAV capsid protein comprising an amino acid substitution and / or insertion sequence between positions 449 and 460, wherein said amino acid sequence starting at position 449 has at least 80% identity to a sequence provided in Table 2.

6. An AAV capsid protein of any one of claims 1-5, wherein the capsid protein further comprises an amino acid substitution and / or insertion sequence at position 587-597 having at least 80% identity to a sequence provided in Table 3.

7. An AAV capsid protein comprising an insertion and / or substitution sequence of Formula A between positions 452-457, and / or wherein the capsid protein comprises an insertion and / or substitution sequence having at least 80% identity to a sequence having Formula A, Xa-Xb-Xc-Xd-Xe-Xf(A) wherein: Xais an amino acid selected from E, D, N, S, and T; Xbis an amino acid selected from R, G, K, T, and S; Xcis an amino acid selected from S, G, T, E, and A; Xdis an amino acid selected from S, G, and T;Xeis an amino acid selected from S, T, G, Q, and A; and Xfis an amino acid selected from S, T, A, and K.

8. The AAV capsid protein of Claim 7, wherein Xais E.

9. The AAV capsid protein of Claim 7, wherein Xbis R.

10. The AAV capsid protein of Claim 7, wherein Xcis S.

11. The AAV capsid protein of Claim 7, wherein Xdis S.

12. The AAV capsid protein of Claim 7, wherein Xeis S.

13. The AAV capsid protein of Claim 7, wherein Xfis S.

14. An AAV capsid protein comprising an insertion and / or substitution sequence of Formula B between positions 453-458, and / or wherein the capsid protein comprises an insertion and / or substitution sequence having at least 80% identity to a sequence having Formula B, Xa1-Xb1-Xc1-Xd1-Xe1-Xf1(B) wherein: Xa1is an amino acid selected from K, R, G, N, and T; Xb1is an amino acid selected from G, T, S, and D; Xc1is an amino acid selected from S, G, T, A, P and V; Xd1is an amino acid selected from S, Q, A and K; Xe1is an amino acid selected from T, S, N, A and G; and Xf1is an amino acid selected from S, G, E, and T.

15. The AAV capsid protein of Claim 14, wherein Xa1is K.

16. The AAV capsid protein of Claim 14, wherein Xb1is G.

17. The AAV capsid protein of Claim 14, wherein Xc1is S.

18. The AAV capsid protein of Claim 14, wherein Xd1is S.

19. The AAV capsid protein of Claim 14, wherein Xe1is T.

20. The AAV capsid protein of Claim 14, wherein Xf1is S.

21. An AAV capsid protein comprising an insertion and / or substitution sequence of Formula C between positions 453 and 456, and / or wherein the capsid protein comprises an insertion and / or substitution sequence having at least 80% identity to a sequence having Formula C, Xa2-Xb2-Xc2-Xd2-Xe2-Xf2(C) wherein: Xa2is an amino acid selected from S, K, G, and A; Xb2is an amino acid selected from G, S, A, and T; Xc2is an amino acid selected from G, S, A and T; Xd2is an amino acid selected from S, G, T, and R; Xe2is an amino acid selected from S, G, A, R and T; and Xf2is an amino acid selected from G, S, A, and E.

22. The AAV capsid protein of Claim 21, wherein Xa2is K.

23. The AAV capsid protein of Claim 21, wherein Xb2is G.

24. The AAV capsid protein of Claim 21, wherein Xc2is S.

25. The AAV capsid protein of Claim 21, wherein Xd2is S.

26. The AAV capsid protein of Claim 21, wherein Xe2is T.

27. The AAV capsid protein of Claim 21, wherein Xf2is G.

28. An AAV capsid protein comprising an insertion and / or substitution sequence of Formula D between positions 454 and 456, and / or wherein the capsid protein comprises an insertion and / or substitution sequence having at least 80% identity to a sequence having Formula D, Xa3-Xb3-Xc3-Xd3-Xe3-Xf3(D) wherein:Xa3is an amino acid selected from T, S, G, and V; Xb3is an amino acid selected from G, S, A, and T; Xc3is an amino acid selected from S, G, and P; Xd3is an amino acid selected from S, G, A and R; Xe3is an amino acid selected from G, S, R A, and T; and Xf3is an amino acid selected from S, Q, T, E, and A.

29. The AAV capsid protein of Claim 28, wherein Xa3is T.

30. The AAV capsid protein of Claim 28, wherein Xb3is G.

31. The AAV capsid protein of Claim 28, wherein Xc3is S.

32. The AAV capsid protein of Claim 28, wherein Xd3is S.

33. The AAV capsid protein of Claim 28, wherein Xe3is G.

34. The AAV capsid protein of Claim 28, wherein Xf3is S.

35. An AAV capsid protein comprising an insertion and / or substitution sequence of Formula E between positions 454 and 456 or positions 454 and 455, and / or wherein the capsid protein comprises an insertion and / or substitution sequence having at least 80% identity to a sequence having Formula E, Xa4-Xb4-Xc4-Xd4-Xe4-Xf4(E) wherein: Xa4is an amino acid selected from T, A, P, S, G, E, and V; Xb4is an amino acid selected from G, S, A, P, and T; Xc4is an amino acid selected from S, G, A, K, and T; Xd4is an amino acid selected from S, G, A and R; Xe4is an amino acid selected from G, S, R A, and T; and Xf4is an amino acid selected from S, Q, T, E, and A.

36. The AAV capsid protein of Claim 35, wherein Xa4is T.

37. The AAV capsid protein of Claim 35, wherein Xb4is G.

38. The AAV capsid protein of Claim 35, wherein Xc4is S.

39. The AAV capsid protein of Claim 35, wherein Xd4is S.

40. The AAV capsid protein of Claim 35, wherein Xe4is G.

41. The AAV capsid protein of Claim 35, wherein Xf4is S.

42. An AAV capsid protein comprising an insertion and / or substitution sequence of Formula F at position 455, and / or wherein the capsid protein insertion and / or substitution sequence comprises a sequence having at least 80% identity to a sequence having Formula F, Xa5-Xb5-Xc5-Xd5-Xe5-Xf5(F) wherein: Xa5is an amino acid selected from A, P, T, and V; Xb5is an amino acid selected from G, S, T, and A; Xc5is an amino acid selected from G, A, T, and V; Xd5is an amino acid selected from G, S, A and K; Xe5is an amino acid selected from A, S, T, G, and R; and Xf5is an amino acid selected from G, A, K, S, and T.

43. The AAV capsid protein of Claim 42, wherein Xa5is A.

44. The AAV capsid protein of Claim 42, wherein Xb5is G.

45. The AAV capsid protein of Claim 42, wherein Xc5is G.

46. The AAV capsid protein of Claim 42, wherein Xd5is G.

47. The AAV capsid protein of Claim 42, wherein Xe5is A.

48. The AAV capsid protein of Claim 42, wherein Xf5is G.

49. The AAV capsid protein of any one of claims 7-48, further comprising an amino acid substitution and / or insertion sequence of Formula I at position 587-597 X1-X2-X3-X4-X5-X6-X7-X8-X9-X10-X11(I) wherein: X1is an amino acid selected from A, Q, H, K, T, S, N, and M, and preferably selected from A or Q; X2is an amino acid selected from Q, R, T, Y and M, and preferably from Q, R, and T; X3is an amino acid selected from R, E, L, and T, and is preferably R; X4is an amino acid selected from D, N, and S, and preferably from D and N; X5is an amino acid selected from G and H, and is preferably G; X6is an amino acid selected from L, V, A, and Q, and preferably from L and V; X7is an amino acid selected from I, H, and R, and is preferably I; X8is an amino acid selected from L, F, and S, and is preferably L; X9is an amino acid selected from I, K, V, Q, and A, and is preferably I; X10is an amino acid selected from A, S, E, Q, and T, and is preferably A; and X11is an amino acid selected from Q, P, and K.

50. The AAV capsid protein of Claim 49, wherein X1is A or Q.

51. The AAV capsid protein of Claim 49 wherein X2is Q, R, or T.

52. The AAV capsid protein of Claim 49 wherein X3is R.

53. The AAV capsid protein of Claim 49 wherein X4is D or N.

54. The AAV capsid protein of Claim 49 wherein X5is G.

55. The AAV capsid protein of Claim 49 wherein X6is L or V.

56. The AAV capsid protein of Claim 49 wherein X7is I.

57. The AAV capsid protein of Claim 49 wherein X8is L.

58. The AAV capsid protein of Claim 49 wherein X9is I.

59. The AAV capsid protein of Claim 49 wherein X10is A.

60. The AAV capsid protein of Claim 49 wherein X11is Q or P.

61. An AAV capsid protein comprising: (i) an amino acid substitution and / or insertion sequence at position 587-597 having at least 80% identity to a sequence provided in Table 3; and (ii) an amino acid substitution and / or insertion sequence between positions 449 and 460 having at least 80% identity to a sequence as provided in Table 1 and / or Table 2.

62. The AAV capsid protein of claim 61, wherein the amino substitution and / or insertion sequence at position 587-597 comprises a sequence having at least 80% identity with a sequence as provided in Table 5.

63. The AAV capsid protein of any one of claims 7-48, further comprising an amino acid substitution and / or insertion sequence of amino acid sequence of Formula (II) at positions 583-599:X1-X2-X3-X4-X5-X6-X7-X8-X9-X10-X11-X12-Xl3-X14-X15-X16-X17 (II) wherein:X1 is an amino acid selected from N, S, G, and T, and preferably selected from N;X2 is an amino acid selected from H, N, A, S, V, C, Q, I, T, L, W, M, K, F, R, G, and Y, and preferably H;X3 is amino acid Q;X4 is an amino acid selected from S, A, T, G, C, L, H, K, N, Q, R, Y, and M, and preferably from S, M, or G;X5 is an amino acid selected from A, Q, N, S, M, Y, G, H, D, W, T, F, V, I, C, L, T, and E, and is preferably A, Q, or S,X6 is an amino acid selected from T, V, Q, S, M, I, N, K, A, H, F, E, R, Y, and L, and preferably from T, V:or S;X7 is an amino acid selected from R, K, L, M, Q, V, F, H, A, S, Y, T, W, and I, and is preferably R or K;X8 is an amino acid selected from N, D, G, Q, A, H, M, E, R, L, K, T, V, and S, and is preferably N or D;X9 is an amino acid selected from G, S, Q, A, D, K, E, T, H, M, V, I, L, and N, and is preferably G:Xio is an amino acid selected from E, H, Q, A, T, G, D, S, K, V, R, and N, and is preferably E or H;Xu is an amino acid selected from V, I, T, II, M, Q, A, S, P, N, R, K, and L, and is preferably V or I;Xi?. is an amino acid selected from F, L, Y, M, R, K, and H, and is preferably F, I.., or Y;Xnis an amino acid selected from I, Y, V, L, M, T, H, E, Q, S, A, N, and F, and is preferably I or Y;Xuis an amino acid selected from A, and S, and is preferably A;Xis is an amino acid selected from Q, M, S, G, N, T, A, I, H, L, R, Y, and V, and is preferably Q, M, S, or N;Xie is an amino acid selected from A, P, F, T, C, G, H, Q, V, Y, or S, and is preferably A; andXnis an amino acid selected from Q, A, S, L, M, N, D, T, V, H, I, or G, and is preferably Q64. The AAV capsid protein of claim 63, wherein X2is an amino acid selected from H, N, A, and S.

65. The AAV capsid protein of claim 63, wherein Xds an amino acid selected from S, A, T, G, and M.

66. The AAV capsid protein of claim 63, wherein Xs is an amino acid selected from A, Q, N, S, M, Y, and E.

67. The AAV capsid protein of claim 63, wherein Xe is an amino acid selected from T, V, Q, S, M and L.

68. The AAV capsid protein of claim 63, wherein X? is an amino acid selected from R, K, L,M, and I69. The AAV capsid protein of claim 63, wherein Xs is an amino acid selected from N, D, G,and S70. The AAV capsid protein of claim 63, wherein Xs is an amino acid selected from G, S, Q, A, and N.

71. The AAV capsid protein of claim 63, wherein Xio is an amino acid selected from E, H, Q, A, and N.

72. The AAV capsid protein of claim 63, wherein Xfi is an amino acid selected from V, I, T, and L.

73. The AAV capsid protein of claim 63, wherein X12 is an amino acid selected from F, L, Y, M, and H.

74. The AAV capsid protein of claim 63, wherein Xi.Gs an amino acid selected from I, Y, V, and F.

75. The AAV capsid protein of claim 63, wherein Xi4is an amino acid selected from A, and S.

76. The AAV capsid protein of claim 63, wherein Xu is an amino acid selected from Q, M, S,G, N, I', A and V.

77. The AAV capsid protein of claim 63, wherein X16 is an amino acid selected from A or S.

78. The AAV capsid protein of claim 63, wherein Xnis an amino acid selected from Q, A, orG.79 The AAV capsid protein of claim 63, wherein Xi is N.80 The AAV capsid protein of claim 63, wherein X?.is II81. The AAV capsid protein of claim 63, wherein X3 is Q.

82. The AAV capsid protein of claim 63, wherein X4 is S or G.

83. The AAV capsid protein of claim 63, wherein Xs is A or S.84, The AAV capsid protein of claim 63, wherein Xt;is T or S.85, The AAV capsid protein of claim 63, wherein X? is R or K.86, The AAV capsid protein of claim 63, wherein Xs is N or D.87, The AAV capsid protein of claim 63, wherein X9 is G.88, The AAV capsid protein of claim 63, wherein X10 is E or H.89, The AAV capsid protein of claim 63, wherein X11 is V or I.90, The AAV capsid protein of claim 63, wherein X12 is F, L, or Y.

91. The AAV capsid protein of claim 63, wherein X13 is [ or Y.

92. The AAV capsid protein of claim 63, wherein X14 is A.

93. The AAV capsid protein of claim 63, wherein X15 is Q, M, or N.

94. The AAV capsid protein of claim 63, wherein Xis is A or S.

95. An AAV capsid protein comprising (i) an amino acid substitution and / or insertion sequence at position 583-599 having at least 80% identity to a sequence provided in Tables 5, 5A, 5B, 6, 6 A, and / or 6B; and (ii) an amino acid substitution and / or insertion sequence between positions 449 and 460 having at least 80% identity to a sequence as provided in Table 1, Table 2 and / or Table 596 The AAV capsid protein of claim 1, wherein the AAV capsid protein comprises (i) an amino acid substitution and / or insertion sequence at position 583-599 provided in Tables 5, 5A, 5B, 6, 6A, and / or 6B; and (ii) an amino acid substitution and / or insertion sequence between positions 449 and 460 provided in Table 1, Table 2 and / or Table 5.

97. The AAV capsid protein of claim 1, wherein the AAV capsid protein comprises (i) an amino acid substitution and / or insertion sequence at position 583-599 provided in Tables 5, 5 A, 5B, 6, 6A, and / or 6B; and (ii) an amino acid substitution and / or insertion sequence betweenpositions 449 and 460 provided in Table 5.

98. The AAV capsid protein of any of claims 1-97 wherein the AAV is AAV9.

99. The AAV capsid protein of any of claims 1-97 wherein the AAV is provided in SEQ ID NO: 1.

100. The AAV capsid protein of any of claims 1-99, wherein the AAV capsid protein is present in VP1, VP2, and VP3 of the AAV capsid.

101. The AAV capsid protein of any of claims 1-100, wherein the AAV capsid protein is characterized by at least one of an increased transduction enrichment when measured in a brain in a subject when delivered to the subject systemically.

102. The AAV capsid protein any of claims 1-101, wherein the AAV capsid protein further comprises an amino acid substitution comprising A589N or Q590P.

103. An AAV capsid comprising a sequence encoding an AAV capsid protein of any of claims 1-102.

104. The .AAV capsid of claim 103, wherein the .AAV capsid is chimeric.

105. The .AAV capsid of any of claims 103-104 that is isolated and purified.

106. The AAV capsid of any one of claims 103-105 formulated as a pharmaceutical formulation for systemic administration to treat a disease or a condition of the brain, the pharmaceutical formulation further comprising a pharmaceutically acceptable carrier.

107. The AAV capsid protein of any of claims 1-99, wherein the capsid protein is expressed in the brain.

108. The AAV capsid protein of claim 107, w'herein the brain comprises a cell-type selected from a neuron, an oligodendrocyte, an astrocyte, and a brain vascular cell.

109. The AAV capsid protein of any one of claims 107-108, wherein the brain comprises a tissue that is selected from brain, thalamus, cortex, putamen, lateral ventricles, medulla, the pons, the amygdala, the motor cortex, caudate, hypothalamus, striatum, ventral midbrain, neocortex, basal ganglia, hippocampus, thalamus, cerebrum, cerebellum, brain stem, and spinal cord.

110. A nucleic acid sequence encoding the AAV capsid protein of any of claims 1-99.

111. A recombinant vector comprising a nucleic acid sequence encoding the AAV capsid protein of any one of claims 1 -99.

112. A ki t com pri si ng : a) a first vector comprising the recombinant vector of claim 111; b) a second vector encoding a helper virus protein; and c) a third vector comprising a therapeutic nucleic acid encoding a therapeutic gene expression product.

113. A method of treating a disease or condition in a subject comprising administering a therapeutically effective amount of a pharmaceutical formulation comprising the AAV capsid protein of any one of claims 1-99.

114. The method of claim 113, wherein the disease or the condition is a disease or a condition of a brain of the subject.

115. A method of manufacturing a recombinant AAV particle from the AAV capsid protein of any one of claims 1-99, the method comprising: a. introducing into a cell a nucleic acid comprising: i. a first nucleic acid sequence encoding a therapeutic gene expression product;ii. a second nucleic acid sequence encoding a recombinant viral genome comprising a capsid (Cap) gene modified to express an AAV capsid comprising the AAV capsid protein; and iii. a third nucleic acid sequence encoding an AAV helper virus genome; and b. assembling the recombinant AAV particle, the recombinant AAV particle comprising the AAV capsid encapsidating the first nucleic acid.

116. The AAV capsid protein of any of claims 1-99 wherein the AAV capsid protein is characterized by an increased transduction enrichment when measured in a tissue in a subject when delivered to the subject systemically.

117. The .AAV capsid protein of any of claims 1-99 wherein the tissue is brain tissue.

118. .An AAV particle comprising an AAV capsid protein of any of claims 1-99 and a viral genome.

Images