Adeno-associated virus compositions with increased cardiac enrichment

Abstract

Described herein are compositions and kits that include recombinant adeno-associated viruses (rAAVs) that have increased transduction enrichment in the heart. The rAAV compositions described herein encoat transgenes, such as therapeutic nucleic acids. Gene therapies using these rAAVs are described. Methods of treating cardiovascular related diseases and conditions are also described.

Description

Background Art

[0001] Recombinant adeno-associated virus (rAAV) is widely used as a carrier for gene delivery in therapeutic applications due to its ability to transduce both dividing and non-dividing cells, its ability to exist in infected cells for a long time in the form of free DNA, and its low immunogenicity. These characteristics make it attractive for applications in therapeutic applications (such as gene therapy). However, it is necessary to significantly improve the performance of existing AAV serotypes to selectively and effectively express in different cell types when delivered to subjects throughout the body. When AAV must be expressed in heart and vascular tissues, this need is particularly urgent. Summary of the invention

[0002] Disclosed herein are rAAVs engineered for transduction in capsid structures through iterative rounds of selection in non-human primates (NHPs), resulting in variants with increased transduction when measured in the heart.

[0003] The present invention provides rAAVs with broad transduction into the heart and vasculature.

[0004] In one aspect, the present invention provides a peptide insertion sequence comprising Table 1-2, Figure 1 and and / or the amino acid sequence shown in any one of Formulae I-VIII or consisting thereof.

[0005] Another aspect of the present invention is a modified capsid protein, wherein the capsid protein has the following characteristics: Figure 1 The AAV capsid protein having an amino acid sequence as shown in any one of Formulas I-VIII or consisting thereof inserted therein is characterized by increased cardiac enrichment in a subject.

[0006] Another aspect of the present invention is a modified capsid protein, wherein the capsid protein has the following characteristics: Figure 1 The AAV capsid protein having an amino acid sequence as shown in or consisting of any one of Formulas I-VIII and / or inserted therein is characterized by increased enrichment in cardiac tissue of a subject.

[0007] In addition, the present disclosure also includes pharmaceutical compositions, which include Tables 1-2, Figure 1 and / or a peptide having an amino acid sequence shown in any one of Formulas I to VII or consisting thereof, and a pharmaceutically acceptable excipient.

[0008] Aspects disclosed herein provide methods of treating a disease or condition in a subject, comprising administering a therapeutically effective amount of a pharmaceutical formulation comprising an AAV capsid protein or AAV capsid disclosed herein. In some embodiments, the disease or condition is a disease or condition of the heart and cardiovascular tissue of the subject. Relatedly, the invention encompasses the use of rAAV in the preparation of a medicament for treating or preventing the disease or medical condition.

[0009] Other aspects of the invention will be apparent from the ensuing detailed description and claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the invention will be obtained by reference to the following detailed description which sets forth illustrative embodiments in which the principles of the invention are utilized, and in the accompanying drawings:

[0011] Figure 1 Shown are AAV capsid protein insertions and substitutions encoding amino acid sequences found in non-human primate heart tissue after two rounds of evaluation of the engineered AAV library. DETAILED DESCRIPTION

[0012] In one aspect, the present disclosure provides rAAVs with high expression levels in the heart.

[0013] In one aspect, the present disclosure provides a method comprising: Figure 1 and / or rAAV in which the amino acid sequence shown in any one of Formulas I-VIII or a peptide consisting thereof is inserted and / or substituted.

[0014] Aspects disclosed herein provide an AAV capsid comprising an AAV capsid protein comprising the amino acid sequence of Formula I:

[0015] X 1 -GGX 2 -X 3 -X 4 -X 5 (I)

[0016] Where X 1 is an amino acid selected from G, I, K, L and V; X 2 is an amino acid selected from the group consisting of E, G, H, L, N, S and Q; X 3 is an amino acid selected from the group consisting of A, F, I, L, P, T and Y; X 4 is an amino acid selected from the group consisting of F, L, S and Y; and X 5 is an amino acid selected from the group consisting of E, H, I, K, P, S and T.

[0017] In some embodiments, the AAV capsid protein comprises the amino acid sequence of Formula I, wherein X 1 is I, K or 1. In some embodiments, the AAV capsid protein comprises the amino acid sequence of Formula I, wherein X 1 is K. In some embodiments, the AAV capsid protein comprises the amino acid sequence of Formula I, wherein X 1 It's I.

[0018] In some embodiments, the AAV capsid protein comprises the amino acid sequence of Formula I, wherein X 2 is L, H or Q. In some embodiments, the AAV capsid protein comprises the amino acid sequence of Formula I, wherein X 2 is L. In some embodiments, the AAV capsid protein comprises the amino acid sequence of Formula I, wherein X 2 It's H.

[0019] In some embodiments, the AAV capsid protein comprises the amino acid sequence of Formula I, wherein X 3 is I, P or Y. In some embodiments, the AAV capsid protein comprises the amino acid sequence of Formula I, wherein X 3 In some embodiments, the AAV capsid protein comprises the amino acid sequence of Formula I, wherein X 3 It is Y.

[0020] In some embodiments, the AAV capsid protein comprises the amino acid sequence of Formula I, wherein X 4 It is F or L.

[0021] In some embodiments, the AAV capsid protein comprises the amino acid sequence of Formula I, wherein X 5 It is I or H.

[0022] In some embodiments, Figure 1 The sequence described in is selected from KGGLPFI, IGGHYLH, LGGQILI, LGGGFSE, IGGNLLK, KGGETSS, VGGSTYP and GGGLAST.

[0023] In some embodiments, Figure 1 The sequence described in is selected from AQKGGLPFIAQ, AQIGGHYLHAQ, AQLGGQILIAQ, AQLGGGFSEAQ, AQIGGNLLKAQ, AQKGGETSSAQ, AQVGGSTYPAQ and AQGGGLASTAQ.

[0024] Aspects disclosed herein provide AAV capsids having greater expression in the heart, the AAV capsids comprising an AAV capsid protein comprising an amino acid sequence of Formula II:

[0025] RSX 6 -X 7 -X 8 -X 9 -X 10 (II)

[0026] Where X 6 is an amino acid selected from the group consisting of E, G, I, N, Q and T; X 7 is an amino acid selected from L, H, Q, S and T; X 8 is an amino acid selected from D, E, H, P and S; X 9 is an amino acid selected from the group consisting of A, F, L, R, S and T; and X 10 is an amino acid selected from the group consisting of E, I, M, S and V.

[0027] In some embodiments, the AAV capsid protein comprises the amino acid sequence of Formula II, wherein X 10 is I, V or M. In some embodiments, the AAV capsid protein comprises the amino acid sequence of Formula II, wherein X 10 It’s M.

[0028] In some embodiments, the AAV capsid protein comprises the amino acid sequence of Formula II, wherein X 9 is F or L. In some embodiments, the AAV capsid protein comprises the amino acid sequence of Formula II, wherein X 9 It's F.

[0029] In some embodiments, the AAV capsid protein comprises the amino acid sequence of Formula II, wherein X 8 is P or H. In some embodiments, the AAV capsid protein comprises the amino acid sequence of Formula II, wherein X 8 In some embodiments, the AAV capsid protein comprises the amino acid sequence of Formula II, wherein X 8 It's H.

[0030] In some embodiments, the AAV capsid protein comprises the amino acid sequence of Formula II, wherein X 7 is H or L. In some embodiments, the AAV capsid protein comprises the amino acid sequence of Formula II, wherein X 7 is H. In some embodiments, the AAV capsid protein comprises the amino acid sequence of Formula II, wherein X 7 It's L.

[0031] In some embodiments, the AAV capsid protein comprises the amino acid sequence of Formula II, wherein X 6 is E or G. In some embodiments, the AAV capsid protein comprises the amino acid sequence of Formula II, wherein X 6is E. In some embodiments, the AAV capsid protein comprises the amino acid sequence of Formula II, wherein X 7 It's G.

[0032] In some embodiments, Figure 1 The insertion sequence described in is selected from RSGHPFV, RSGLHLM, RSELHFI, RSQTSTE, RSISDRV, RSTTDAS and RSNQESV.

[0033] In some embodiments, Figure 1 The insertion sequence described in is selected from AQRSGHPFVAQ, AQRSGLHLMAQ, AQRSELHFIAQ, AQRSQTSTEAQ, AQRSISDRVAQ, AQRSTTDASAQ and AQRSNQESVAQ.

[0034] Aspects disclosed herein provide AAV capsids having greater expression in the heart, the AAV capsids comprising an AAV capsid protein comprising an amino acid sequence of Formula III:

[0035] RX 11 -GX 12 -X 13 -X 14 -X 15 (III)

[0036] Where X 11 is an amino acid selected from the group consisting of A, D, E, I, N, V ​​and S; X 12 is an amino acid selected from E, H, L, M, S and V; X 13 is an amino acid selected from the group consisting of F, H, L, P, V and Y; X 14 is an amino acid selected from F, L and T; and X 15 is an amino acid selected from the group consisting of D, H, K, L, M, Q, S and V.

[0037] In some embodiments, the AAV capsid protein comprises the amino acid sequence of Formula III, wherein X 11 is an amino acid selected from A, D, N and S. In some embodiments, the AAV capsid protein comprises an amino acid sequence of Formula III, wherein X 11 A. In some embodiments, the AAV capsid protein comprises the amino acid sequence of Formula III, wherein X 11 is D. In some embodiments, the AAV capsid protein comprises the amino acid sequence of Formula III, wherein X 11 is N. In some embodiments, the AAV capsid protein comprises the amino acid sequence of Formula III, wherein X 11 It’s S.

[0038] In some embodiments, the AAV capsid protein comprises the amino acid sequence of Formula III, wherein X 12 is an amino acid selected from E, H, L, and V. In some embodiments, the AAV capsid protein comprises an amino acid sequence of Formula III, wherein X 12 is H. In some embodiments, the AAV capsid protein comprises the amino acid sequence of Formula III, wherein X 12 is L. In some embodiments, the AAV capsid protein comprises the amino acid sequence of Formula III, wherein X 12 is E. In some embodiments, the AAV capsid protein E comprises the amino acid sequence of formula III, wherein X 12 It's V.

[0039] In some embodiments, the AAV capsid protein comprises the amino acid sequence of Formula III, wherein X 13 is an amino acid selected from the group consisting of F, H, P and Y.

[0040] In some embodiments, the AAV capsid protein comprises the amino acid sequence of Formula III, wherein X 14 It is F or L.

[0041] In some embodiments, the AAV capsid protein comprises the amino acid sequence of Formula II, wherein X 15 is an amino acid selected from K, L, M and V. In some embodiments, the AAV capsid protein comprises an amino acid sequence of Formula III, wherein X 15 In some embodiments, the AAV capsid protein comprises the amino acid sequence of Formula III, wherein X 15 I. In some embodiments, the AAV capsid protein comprises the amino acid sequence of Formula III, wherein X 15 is K. In some embodiments, the AAV capsid protein comprises the amino acid sequence of Formula III, wherein X 15 It's L.

[0042] In some embodiments, Figure 1 The insertion sequence described in is selected from RSGHPFV, RNGEHFK, RDGHYLL, RNGEFFH, REGMPFL, RAGVHFV, RSGLHLM, RIGVHTS, RIGSVTQ and RVGSLTD.

[0043] Aspects disclosed herein provide AAV capsids having greater expression in the heart, the AAV capsids comprising an AAV capsid protein comprising an amino acid sequence of Formula IV:

[0044] X 16 -X 17 -GHX 18 -X19 -X 20 (IV)

[0045] Where X 16 is an amino acid selected from I, R and L; X 17 is an amino acid selected from D, G, Q and S; X 18 is an amino acid selected from I, P and Y; X 19 is an amino acid selected from the group consisting of F, I, L, S and Y; and X 20 is an amino acid selected from the group consisting of H, L and V.

[0046] In some embodiments, the AAV capsid protein comprises the amino acid sequence of Formula IV, wherein X 16 is an amino acid selected from the group consisting of I, L and R.

[0047] In some embodiments, the AAV capsid protein comprises the amino acid sequence of Formula IV, wherein X 17 Selected from G, Q and S.

[0048] In some embodiments, the AAV capsid protein comprises the amino acid sequence of Formula IV, wherein X 19 is an amino acid selected from the group consisting of F, I, L, S and Y.

[0049] In some embodiments, the AAV capsid protein comprises the amino acid sequence of Formula IV, wherein X 20 is L. In some embodiments, the AAV capsid protein comprises the amino acid sequence of Formula IV, wherein X 20 is H. In some embodiments, the AAV capsid protein comprises the amino acid sequence of Formula IV, wherein X 20 It's V.

[0050] In some embodiments, the AAV capsid protein comprises the amino acid sequence of Formula IV, wherein X 18 I. In some embodiments, the AAV capsid protein comprises the amino acid sequence of Formula IV, wherein X 18 In some embodiments, the AAV capsid protein comprises the amino acid sequence of Formula IV, wherein X 18 It is Y.

[0051] In some embodiments, Figure 1 The insertion sequence described in is selected from RSGHPFV, RDGHYLL, LQGHILL and IGGHYLH.

[0052] In some embodiments, Figure 1 The insertion sequence described in is selected from AQRSGHPFVAQ, AQRDGHYLLAQ, AQLQGHILLAQ and AQIGGHYLHAQ.

[0053] Aspects disclosed herein provide AAV capsids having greater expression in the heart, the AAV capsids comprising an AAV capsid protein comprising an amino acid sequence of Formula V:

[0054] RX 21 -X 22 -X 23 -HFX 24 (V)

[0055] Where X 21 is an amino acid selected from A, N and S; X 22 is an amino acid selected from D, E and G; X 23 is an amino acid selected from E, L and V; and X 24 is an amino acid selected from the group consisting of I, K, Q and V.

[0056] In some embodiments, the AAV capsid protein comprises the amino acid sequence of Formula V, wherein X 21 A. In some embodiments, the AAV capsid protein comprises an amino acid sequence of Formula V, wherein X 21 is N. In some embodiments, the AAV capsid protein comprises an amino acid sequence of Formula V, wherein X 21 It’s S.

[0057] In some embodiments, the AAV capsid protein comprises the amino acid sequence of Formula V, wherein X 22 is D. In some embodiments, the AAV capsid protein comprises the amino acid sequence of Formula V, wherein X 22 is G. In some embodiments, the AAV capsid protein comprises an amino acid sequence of Formula V, wherein X 22 It's E.

[0058] In some embodiments, the AAV capsid protein comprises the amino acid sequence of Formula V, wherein X 23 is E. In some embodiments, the AAV capsid protein comprises the amino acid sequence of Formula V, wherein X 23 is L. In some embodiments, the AAV capsid protein comprises an amino acid sequence of Formula V, wherein X 23 It's V.

[0059] In some embodiments, the AAV capsid protein comprises the amino acid sequence of Formula V, wherein X 24 is Q. In some embodiments, the AAV capsid protein comprises an amino acid sequence of Formula V, wherein X 24 is K. In some embodiments, the AAV capsid protein comprises an amino acid sequence of Formula V, wherein X 24 It's V.

[0060] In some embodiments, Figure 1The insertion sequence described in is selected from RADLHFQ, RAGVHFV, RNGEHFK, RSELHFI.

[0061] In some embodiments, Figure 1 The insertion sequence described in is selected from AQRADLHFQAQ, AQRAGVHFVAQ, AQRNGEHFKAQ and AQRSELHFIAQ.

[0062] Aspects disclosed herein provide AAV capsids having greater expression in the heart, the AAV capsids comprising an AAV capsid protein comprising an amino acid sequence of Formula VI:

[0063] X 25 -X 26 -X 27 -X 28 -X 29 -LL(VI)

[0064] Where X 25 is an amino acid selected from A, L, R and Y; X 26 is an amino acid selected from the group consisting of Q, E, D, T, K and F; X 27 is an amino acid selected from G, Q and A; X 28 is an amino acid selected from the group consisting of H, G, T, V and D; and X 29 is an amino acid selected from the group consisting of I, S, T and Y.

[0065] In some embodiments, the AAV capsid protein comprises the amino acid sequence of Formula VI, wherein X 25 It is L or R.

[0066] In some embodiments, the AAV capsid protein comprises the amino acid sequence of Formula VI, wherein X 26 It is Q, E or D.

[0067] In some embodiments, the AAV capsid protein comprises the amino acid sequence of Formula VI, wherein X 27 It is G or Q.

[0068] In some embodiments, the AAV capsid protein comprises the amino acid sequence of Formula VI, wherein X 28 It is H or G.

[0069] In some embodiments, the AAV capsid protein comprises the amino acid sequence of Formula VI, wherein X 29 It is I or Y.

[0070] In some embodiments, Figure 1The insertion sequence described in is selected from LQGHILL, REQGYLL, RDGHYLL, YTGTSLL, AKAVTLL and AFGDSLL.

[0071] In some embodiments, Figure 1 The insertion sequence described in is selected from AQLQGHILLAQ, AQREQGYLLAQ, AQRDGHYLLAQ, AQYTGTSLLAQ, AQAKAVTLLAQ and AQAFGDSLLAQ.

[0072] Aspects disclosed herein provide AAV capsids having greater expression in the heart, the AAV capsids comprising an AAV capsid protein comprising the amino acid sequence of Formula VII:

[0073] X 30 -X 31 -X 32 -X 33 -X 34 -SS(VII)

[0074] Where X 30 is an amino acid selected from the group consisting of A, E, F, H, I, K, L, M, N, P, Q, S, T, V and Y; X 31 is an amino acid selected from the group consisting of F, G, H, I, K, L, M, P, Q, R, S, T and V; X 32 is an amino acid selected from the group consisting of A, G, H, L, P, Q, S and V; X 33 is an amino acid selected from the group consisting of E, G, H, I, L, P, Q, R, S, T and V; and X 34 is an amino acid selected from the group consisting of D, G, I, L, M, N, R, S, T and V.

[0075] In some embodiments, the AAV capsid protein comprises the amino acid sequence of Formula VII, wherein X 30 It is V, Q or H.

[0076] In some embodiments, the AAV capsid protein comprises the amino acid sequence of Formula VII, wherein X 31 It is F, L or M.

[0077] In some embodiments, the AAV capsid protein comprises the amino acid sequence of Formula VII, wherein X 32 It is S, A or Q.

[0078] In some embodiments, the AAV capsid protein comprises the amino acid sequence of Formula VII, wherein X 33 It is G, H or S.

[0079] In some embodiments, the AAV capsid protein comprises the amino acid sequence of Formula VII, wherein X 34 It is G, V or M.

[0080] In some embodiments, Figure 1 The insertion sequence described in is selected from VFSGVSS, QLQHGSS, HMASMSS, SSLTLSS, SVPSSS, ITPSVSS, MTVQVSS, SHLSVSS, YPHQTSS, PQGGSSS, FRSSLSS, ISVSISS, ETQRGSS, IHPPSSS, TSAITSS, FKSQSSS, ASHSTSS, AVMRDSS, KGGETSS, VVSVTSS, LISIRSS, NSLGLSS and HLAQNSS.

[0081] In some embodiments, Figure 1 The insertion sequence described in is selected from AQVFSGVSSAQ, AQQLQHGSSAQ, AQHMASMSSAQ, AQSSLTLSSAQ, AQSVPSSSSAQ, AQITPSVSSAQ, AQMTVQVSSAQ, AQSHLSVSSAQ, AQYPHQTSSAQ, AQPQGGSSSAQ, AQFRSSLSSAQ, AQISVSISSAQ, AQETQRGSSAQ, AQIHPPSSSAQ, AQTSAITSSAQ, AQFKSQSSSAQ, AQASHSTSSAQ, AQAVMRDSSAQ, AQKGGETSSAQ, AQVVSVTSSAQ, AQLISIRSSAQ, AQNSLGLSSAQ and AQHLAQNSSAQ.

[0082] Aspects disclosed herein provide AAV capsids having greater expression in the heart, the AAV capsids comprising an AAV capsid protein comprising the amino acid sequence of Formula VIII:

[0083] DLX 35 -X 36 -X 37 -X 38 -X 39 (VIII)

[0084] Where X 35 is an amino acid selected from G, H, Q, S and T; X 36 is an amino acid selected from K, N, P, R and S; X 37 is an amino acid selected from H, L, R and S; X 38 is an amino acid selected from the group consisting of D, K, R, S, T and V; and X 39is an amino acid selected from the group consisting of A, D, E, L and V.

[0085] In some embodiments, the AAV capsid protein comprises the amino acid sequence of Formula VIII, wherein X 35 It is H or S.

[0086] In some embodiments, the AAV capsid protein comprises the amino acid sequence of Formula VIII, wherein X 36 It is K or R.

[0087] In some embodiments, the AAV capsid protein comprises the amino acid sequence of Formula VIII, wherein X 37 It is L or S.

[0088] In some embodiments, the AAV capsid protein comprises the amino acid sequence of Formula VIII, wherein X 38 It is S or V.

[0089] In some embodiments, the AAV capsid protein comprises the amino acid sequence of Formula VIII, wherein X 39 It is A or L.

[0090] In some embodiments, Figure 1 The insertion sequence described in is selected from DLSRLS, DLHKSVA, DLGNLKV, DLTPHKD, DLQRSDL and DLGSRRE.

[0091] In some embodiments, Figure 1 The insertion sequence described in is selected from AQDLSRLSLAQ, AQDLHKSVAAQ, AQDLGNLKVAQ, AQDLTPHKDAQ, AQDLQRSDLAQ and AQDLGSRREAQ.

[0092] In some embodiments, the insert sequence is represented by the peptide sequences listed in Tables 1-2.

[0093] Table 1.

[0094] sequence sequence DLSRLSL AQDLSRLSLAQ KLDPLLM AQKLDPLLMAQ TPKHLLI AQTPKHLLIAQ QDQ AQPDQRRTDAQ RHDPLVM AQRHDPLVMAQ SHFSGRN AQSHFSGRNAQ VSAVRDR AQVSAVRDRAQ VGVPVVL AQVGVPVVLAQ LLPSNYL AQLLPSNYLAQ QLQITKE AQQLQITKEAQ LQTSV AQLQVTSVLAQ VFSGVSS AQVFSGVSSAQ

[0095] Table 2.

[0096] sequence sequence KGGLPFI AQKGGLPFIAQ DLSRLSL AQDLSRLSLAQ RSGHPFV AQRSGHPFVAQ RADLHFQ AQRADLHFQAQ LQG AQLQGHILLAQ KLDPLLM AQKLDPLLMAQ IGGHYLH AQIGGHYLHAQ RAGVHFV AQRAGVHFVAQ LGGQILI AQLGGQILIAQ TPKHLLI AQTPKHLLIAQ RNGEHFK AQRNGEHFKA KDSIPFR AQKDSIPFRAQ QDQ AQPDQRRTDAQ RHDPLVM AQRHDPLVMAQ DNYTRHT AQDNYTRHTAQ RSGLHLM AQRSGLHLMAQ TNSVRVV AQTNSVRVVAQ

[0097] In some aspects, the inserted amino acid sequence is consistent with Table 1-2, Figure 1 and / or the amino acid sequences provided in Formulas I-VII are at least 71.4% identical. In some aspects, the inserted amino acid sequence is identical to Tables 1-2, Figure 1 and / or the amino acid sequences provided in Formulas I-VII are at least 86.7% identical.

[0098] Also disclosed herein are methods and kits for producing therapeutic recombinant AAV (rAAV) particles, as well as methods and pharmaceutical compositions or formulations comprising these rAAV particles for treating diseases or conditions affecting the heart.

[0099] Disclosed herein are AAV capsids engineered to increase viral transduction in the heart. AAV capsids can encapsidate viral vectors having heterologous nucleic acids encoding, for example, therapeutic gene expression products. Transduction of heterologous nucleic acids in the heart can be achieved by systemically delivering the AAV capsids of the present disclosure that encapsidate heterologous nucleic acids to a subject. The AAV capsids disclosed herein are advantageous for many applications of gene therapy for treating human diseases (including, but not limited to, disorders of the cardiovascular system).

[0100] Also provided herein is a recombinant AAV vector comprising a nucleic acid sequence encoding an AAV capsid protein of the present disclosure. For example, a viral vector of the present disclosure comprises a nucleic acid sequence comprising an AAV viral Cap (capsid) encoding VP1, VP2, and VP3, at least one of which is modified to produce an AAV capsid protein of the present disclosure. The recombinant AAV vector provided can be derived from an AAV serotype (e.g., AAV9) or a variant AAV serotype comprising an insertion of the present invention.

[0101] AAV capsid

[0102] Provided herein are modified adeno-associated (AAV) viral capsid compositions that can be used to integrate a transgene into target cells or the environment of a subject when administered systemically to the subject.

[0103] rAAV includes AAV capsids that can be engineered to encapsidate heterologous nucleic acids (e.g., therapeutic nucleic acids, gene editing machines). The AAV capsid is composed of three AAV capsid protein monomers VP1, VP2 and VP3. Sixty copies of these three VP proteins interact with each other at a ratio of 1:1:10 to form a viral capsid. In addition to the approximately 137 amino acid N-terminal region (VP1u), VP1 also covers the entire VP2 protein, and in addition to the approximately 65 amino acid N-terminal region (VP1 / 2 common region), VP2 also covers the entire VP3. These three capsid proteins share the conservative amino acid sequence of VP3, and in some cases, the conservative amino acid sequence is a region starting from amino acid position 138 (e.g., AA139-736).

[0104] Although not wishing to be bound by theory, it is understood that the parent AAV capsid sequence includes the VP1 region. In certain embodiments, the parent AAV capsid sequence includes the VP1, VP2 and / or VP3 regions or any combination thereof. The parent VP1 sequence can be considered synonymous with the parent AAV capsid sequence.

[0105] The AAV VP3 structure contains highly conserved regions common to all serotypes, a core eight-stranded β-barrel motif (βB-βI) and a small α-helix (αA). The loop region inserted between the β strands consists of a unique HI loop between β strands H and I, a DE loop between β strands D and E, and nine variable regions (VRs) forming the top of the loop. These VRs, such as the AA588 loop, are present on the capsid surface and can be associated with specific functional roles in the AAV life cycle, including receptor binding, transduction, and antigen specificity.

[0106] In some aspects, the rAAV variants of the invention include an AAV capsid protein having a peptide insertion at residues corresponding to amino acids 588-589 of the AAV9 native sequence of SEQ ID NO:1.

[0107] The AAV capsid includes AAV capsid proteins (e.g., VP1, VP2, and VP3), each of which has an insertion in the 588 loop of the parent AAV capsid protein structure (AAV9 VP1 numbering). The 588 loop contains a site for heparan sulfate that binds to AAV2 and is suitable for peptide display. The only known receptor for AAV9 is the N-linked terminal galactose and AAV receptor (AAVR), but there are many indications that other receptors exist. Modifications to the AAV9 588 loop are shown herein to confer increased transgene transduction in the target in vivo environment.

[0108] In one aspect, the present invention provides a peptide insertion at the AAV 588 loop, the peptide insertion comprising Table 1-2, Figure 1 and and / or the amino acid sequence shown in any one of Formulae I-VII or consisting thereof.

[0109] AAV capsids are disclosed herein, comprising an AAV capsid protein having an insertion at loop 588 that is more transducing cardiomyocyte types (e.g.,). Specifically, the AAV capsid proteins disclosed herein enable rAAV-mediated transduction of heterologous nucleic acids (e.g., transgenes) in the heart of a subject. The AAV capsids disclosed herein can be formulated into pharmaceutical compositions. In addition, the AAV capsids can be isolated and purified for various applications.

[0110] In some embodiments, the rAAV capsids of the present disclosure are produced using the methods disclosed herein. In some cases, the rAAV capsids are chimeric. In some cases, the rAAV or variant AAV proteins included therein increase the localization of the rAAV within the target tissue compared to the parent AAV capsid or capsid protein.

[0111] AAV capsid protein

[0112] Disclosed herein are recombinant AAV (rAAV) capsids, including AAV capsid proteins engineered to have modified capsid proteins (e.g., VP1, VP2, VP3). In some embodiments, the rAAV capsid proteins of the present disclosure are produced using the methods disclosed herein. In some embodiments, AAV capsid proteins are used in methods for delivering therapeutic nucleic acids (e.g., transgenes) to subjects. In some cases, rAAV capsid proteins have desired AAV expression, making these rAAV capsid proteins particularly suitable for certain therapeutic applications, e.g., treatment of a disease or condition of a subject as disclosed herein.

[0113] The rAAV capsid protein is engineered to achieve optimized expression in the heart of a subject when the rAAV is administered systemically to the subject. The rAAV capsid protein is engineered to contain Table 1-2, Figure 1 and / or the insertion provided in Formula I-VII. Figure 1 The inserted rAAV capsid protein provided in and / or Formula I-VII is engineered to achieve efficient transduction of the encapsidated transgene. Specifically, the expression of the rAAV capsid protein in the heart of the subject is increased.

[0114] In some cases, the engineered AAV capsid proteins described herein have an insertion of an amino acid that is heterologous to the parent AAV capsid protein at an amino acid position in the 588 loop. In some embodiments, at the inserted amino acid position, the amino acid is not endogenous to the parent AAV capsid protein. The amino acid can be a naturally occurring amino acid in the same or equivalent amino acid position as the substituted insertion in a different AAV capsid protein.

[0115] Typically, insertion includes five, six or seven amino acid sequences (respectively 5-mers, 6-mers or 7-mers) inserted or replaced at the 588 loop in the parent AAV capsid protein. The various aspects provided herein provide amino acid insertions, which include seven amino acid polymers (7-mers) inserted at AA588-589, and can additionally be included in the amino acid positions (e.g., AA587-588 and / or AA589-590) of the 7-mer sequences on the sides to produce eleven amino acid polymers (11-mers) at the 588 loop of the parent AAV capsid protein. The 7-mers described herein are advantageously produced using polymerase chain reaction (PCR) using degenerate primers, wherein each of the seven amino acids is encoded by a deoxyribonucleic acid (DNA) sequence NNK. "N" is any one of the four DNA nucleotides, and K is guanine (G) or thymine (T). This method for producing random 7-mer amino acid sequences enables 1.28 billion possible combinations to be achieved at the protein level.

[0116] The rAAV capsid protein of the present disclosure includes the insertion of amino acids in the amino acid sequence of the AAV capsid protein. The AAV capsid that produces the engineered AAV capsid protein of the present disclosure is referred to as the "parent" AAV capsid. The complete genome of AAV-1 is provided in GenBank Accession No. NC_002077; the complete genome of AAV-2 is provided in GenBank Accession No. NC_001401 and Srivastava et al., Journal of Virology (J. Virol.), 45:555-564 (1983); the complete genome of AAV-3 is provided in GenBank Accession No. NC_1829; the complete genome of AAV-4 is provided in GenBank Accession No. NC_001829; the AAV-5 genome is provided in GenBank Accession No. AF085716; the complete genome of AAV-6 is provided in GenBank Accession No. NC_00 1862; at least portions of the AAV-7 and AAV-8 genomes are provided in GenBank Accession Nos. AX753246 and AX753249, respectively; the AAV-9 genome is provided in Gao et al., Journal of Virology, 78:6381-6388 (2004); the AAV-10 genome is provided in Mol. Ther., 13(1):67-76 (2006); the AAV-11 genome is provided in Virology, 330(2):375-383 (2004); portions of the AAV-12 genome are provided in Genbank Accession No. DQ813647; portions of the AAV-13 genome are provided in Genbank Accession No. EU285562.

[0117] In some cases, the parent AAV is derived from an AAV having a serotype selected from the following: AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11 and AAV12. The AAV capsid protein "derived" from another serotype can be a variant AAV capsid protein. The variant can include, for example, heterologous amino acids in the amino acid sequence of the AAV capsid protein. Heterologous amino acids may be non-naturally present in the AAV capsid protein. Heterologous amino acids may be naturally present in different AAV capsid proteins. In some cases, the parent AAV capsid is described in U.S. Patent Publication 2020 / 0165576 and U.S. Patent Application Serial Nos. 62 / 832,826 and PCT / US20 / 20778; the contents of each of these U.S. patents are incorporated herein.

[0118] In some cases, the parent AAV is AAV9. In some cases, the amino acid sequence of the AAV9 capsid protein includes SEQ ID NO: 1. The amino acid sequence of the AAV9 VP1 capsid protein (>tr|Q6JC40|Q6JC40_9VIRU capsid protein VP1 OS=adeno-associated virus 9OX=235455GN=cap PE=1SV=1) is provided in SEQ ID NO: 1 (MAADGYLPDWLEDNLSEGIREWWALKPGAPQPKANQQHQDNARGLVLPGYKYLGP ). In some cases, the parent 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.

[0119] AAV capsid proteins from natural AAV serotypes such as AAV9 with tropism (including liver) activate innate immune responses, which in some cases cause severe inflammatory responses in subjects, which may lead to multi-organ failure. By improving the transduction of target tissues (e.g., heart) in vivo by natural AAV serotypes, the rAAV particles of the present disclosure can reduce undesirable side effects.

[0120] In some cases, the parent AAV capsid protein includes the entire VP1 region (e.g., amino acids 1-736) provided in SEQ ID NO: 1. In some cases, the parent AAV capsid protein includes amino acids 217-736 in SEQ ID NO: 1, which is a common region found in VP1, VP2, and VP3 AAV9 capsid proteins. In some cases, the AAV capsid protein includes amino acids 64-736 in SEQ ID NO: 1, which is a common region found in VP1 and VP2. The parent AAV capsid protein sequence may include amino acids 1-736 from SEQ ID NO: 1. NO: 1 is selected from the following amino acids: 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 In some aspects, the rAAV variant comprises an AAV capsid protein comprising an amino acid sequence at least 98% identical to amino acid 217 to amino acid 736 of SEQ ID NO: 1. In some cases, the amino acid is inserted at the three (3)-fold symmetry axis of the corresponding parent AAV capsid protein.

[0121] Disclosed herein is the insertion of an amino acid sequence in an AAV capsid protein. Noting the sequence number designation "588-589" of AAV9 (e.g., AAV VP1), the present invention also encompasses insertions in similar positions in other AAV serotypes. As used herein, "AA588-589" indicates that the insertion of an amino acid (or amino acid sequence) is immediately after the amino acid (AA) at position 588 within the amino acid sequence of the parent AAV VP capsid protein (VP1 numbering), and immediately before the AA at position 589. Amino acids 587-591 contain a motif including "AQAQA" as shown in SEQ ID NO:1. Exemplary AAV capsid protein sequences are provided in Table 3. For example, KGGLPFI is inserted at AA588-589 in the AAV9 capsid amino acid sequence, and variant A is provided. It is envisioned that the sequences disclosed herein (Tables 1-2, Figure 1 and Formulas I-VII) can be inserted at AA588-589 in the amino acid sequence of the parent AAV9 capsid protein, or at AA587-590 (instead of amino acids AA587-590), a variant thereof, or an equivalent amino acid position of a parent AAV of a different serotype (e.g., AAV1, AAV2, AAV3, etc.). In any AAV capsid protein sequence disclosed herein, the amino acid at position 449

[0122] Table 3. Exemplary AAV capsid protein sequences

[0123]

[0124]

[0125]

[0126] In some cases, the insertions described herein may include a 7-mer insertion at AA588-589. It is contemplated that any 7-mer insertion disclosed herein may also include an 11-mer in addition to substitutions with any amino acid at amino acid positions 587-590 [AQAQ].

[0127] Disclosed herein are AAV capsid proteins having the above-described insertions in a parent AAV capsid protein that confer increased transduction of a subject's heart even when delivered systemically. One of the many advantages of the AAV capsid proteins described herein is their ability to target tissues and cells within the heart.

[0128] In some cases, the AAV capsid protein comprises the amino acid positions 588-589 or 587-590 of the parent AAV9 capsid protein (SEQ ID NO: 1). Figure 1and an insertion of at least or about seven, eight, nine, ten, or eleven amino acids in the amino acid sequence of Formulae I-VII. In some cases, the AAV capsid protein has increased viral transduction enrichment in the heart.

[0129] The rAAV capsid protein of the present disclosure may also have a substitution of the amino acid sequence at amino acid positions 452-458 in the parent AAV9 capsid protein or a variant thereof, as described in WO 2020068990. In some embodiments, the substitution of the amino acid sequence includes KDNTPGR at amino acid positions 452-458 of the parent AAV9 capsid protein. In some embodiments, the substitution of the amino acid sequence includes DGAATKN at amino acid positions 452-458 of the parent AAV9 capsid protein.

[0130] The rAAV capsid protein described herein can be separated and purified. AAV can be separated and purified by standard methods in the art, such as by column chromatography, iodixanol gradient or cesium chloride gradient. Methods for purifying AAV from helper viruses are known in the art and can be included in methods disclosed in, for example, Clark et al., Human Gene Therapy (Hum.Gene Ther.), 10 (6): 1031-1039 (1999); Schenpp and Clark, Methods Mol.Med., 69: 427-443 (2002); U.S. Pat. No. 6,566,118 and WO 98 / 09657.

[0131] In addition, the AAV capsid proteins disclosed herein, whether isolated and purified or not, can be formulated into a pharmaceutical formulation, which in some cases further comprises a pharmaceutically acceptable carrier.

[0132] 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 will encapsidate the therapeutic nucleic acid described herein. In some cases, the second molecule is a therapeutic agent, e.g., a small molecule, an antibody, an antigen binding fragment, a peptide, or a protein, such as those described herein.

[0133] "Percent identity" is the percentage of symbols that actually match. Percent similarity is the percentage of similar symbols. Symbols across gaps are ignored. When the scoring matrix value of a pair of symbols is greater than or equal to 0.50 (similarity threshold), similarity is scored. The scoring matrix used in the 10th version of the Wisconsin Genetics Software Package is BLOSUM62 (see: Henikoff and Henikoff, (1989) Proc. Natl. Acad. Sci. USA 89: 10915).

[0134] The sequence identity / similarity values ​​provided herein may refer to the values ​​obtained using the BLAST+ 2.5.0 program suite (blast.ncbi.nlm.nih.gov) using default settings (Camacho, C. et al., (2009) BLAST+: architecture and applications. BMC Bioinformatics 10:421).

[0135] Those of ordinary skill in the art will appreciate that BLAST searches assume that proteins can be modeled as random sequences. However, many real proteins include regions of non-random sequences, which may be homopolymer segments, short-period repeats, or regions rich in one or more amino acids. Such low-complexity regions can be compared between unrelated proteins, even if other regions of the protein are completely different. Many low-complexity filter programs can be used to reduce such low-complexity comparisons. For example, 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 used alone or in combination.

[0136] The terms "substantial identity" and "substantially identical" indicate that a polypeptide or nucleic acid includes a sequence having between 55%-100% sequence identity with a reference sequence, 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 relative to the reference sequence, or any percentage of values ​​within the range of 55%-100% sequence identity. Percent sequence identity may occur over a specified comparison window. The above-mentioned Needleman and Wunsch homology alignment algorithm may be used to determine or perform optimal alignment.

[0137] For example, the inserted sequences may include, but are not limited to, sequences that are not completely identical to the sequences disclosed herein, but in addition to the substitutions explicitly described for the various sequences listed herein, these inserted sequences also have additional substitutions of amino acid residues that do not substantially impair the activity or properties of the sequences described herein, as predicted by homology software (e.g., the BLOSUM62 matrix).

[0138] AAV particles

[0139] The rAAV particles having the insert sequences described herein have increased transduction enrichment in the heart. In some cases, the increased transduction enrichment comprises an increase of 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold or more. In some cases, the increased transduction enrichment is at least 1-fold. In some cases, the increased transduction enrichment is at least 2-fold. In some cases, the increased transduction enrichment is at least 4-fold.

[0140] rAAV particles with insertion sequences described herein have increased expression enrichment in the heart. Detecting whether rAAV has more or less specificity to the target in vivo environment includes measuring the level of gene expression products (e.g., RNA or protein) expressed by the heterologous nucleic acid encapsidated by rAAV in tissue samples obtained from subjects. Suitable methods for measuring the expression of gene expression products include next generation sequencing (NGS) and quantitative polymerase chain reaction (qPCR).

[0141] Increased expression in the heart is caused by Figure 1 The enrichment values ​​provided are indicated in .

[0142] Heterologous Nucleic Acid

[0143] Disclosed herein are therapeutic nucleic acids that can be used to treat or prevent a disease or condition or a symptom of the disease or condition. In some embodiments, these therapeutic nucleic acids encode therapeutic gene expression products. Non-limiting examples of gene expression products include proteins, polypeptides, peptides, enzymes, antibodies, antigen binding fragments, nucleic acids (RNA, DNA, antisense oligonucleotides, siRNA, etc.) and gene editing components for treating, preventing and treating and / or improving a disease or condition or a symptom of a disease or condition. In some cases, these therapeutic nucleic acids are placed in an organism, a cell, a tissue or an organ of a subject by rAAVs as disclosed herein.

[0144] Disclosed herein are rAAVs, each of which includes a viral vector (e.g., a single-stranded DNA molecule (ssDNA)). In some cases, the viral vector includes two reverse terminal repeat (ITR) sequences of about 145 bases each, and each of the two ITR sequences is flanked by a transgene. In some embodiments, the transgene includes a therapeutic nucleic acid, and in some cases, includes a promoter arranged in cis with the therapeutic nucleic acid in an open reading frame (ORF). The promoter is capable of initiating transcription of the therapeutic nucleic acid in the nucleus of the target cell. The ITR sequence can be 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, ITR is from AAV2. In some cases, ITR is from AAV9.

[0145] Transgenics that can include any number of nucleotides are disclosed herein. In some cases, the transgenic can include less than about 100 nucleotides. In some cases, the transgenic can include at least about 100 nucleotides. In some cases, the transgenic can include at least about 200 nucleotides. In some cases, the transgenic can include at least about 300 nucleotides. In some cases, the transgenic can include at least about 400 nucleotides. In some cases, the transgenic can include at least about 500 nucleotides. In some cases, the transgenic can include at least about 1000 nucleotides. In some cases, the transgenic can include at least about 5000 nucleotides. In some cases, the transgenic can include about 500 to about 5000 nucleotides. In some cases, the transgenic includes about 5000 nucleotides. In any case disclosed herein, the transgenic can include DNA, RNA, or a hybrid of DNA and RNA. In some cases, the transgenic can be single-stranded. In some cases, the transgenic can be double-stranded.

[0146] Disclosed herein are transgenics that can be used to regulate the expression or activity of a target gene or its gene expression product. In some cases, the transgenic is encapsidated by the rAAV capsid protein of the rAAV particles described herein. In some cases, the rAAV particles are delivered to a subject to treat a disease or condition disclosed herein in the subject. In some cases, the delivery is systemic.

[0147] Transgenics disclosed herein can be used to express endogenous genes at a level similar to that of healthy or normal individuals. This is particularly useful in treating diseases or conditions related to low expression or insufficient expression of gene expression products. In some embodiments, transgenics disclosed herein can be used to overexpress endogenous genes so that the expression level of endogenous genes is higher than that of healthy or normal individuals. Additionally, transgenics can be used to express exogenous genes (e.g., such as antibodies, peptides, nucleic acids, or gene editing components). In some embodiments, therapeutic gene expression products can change, enhance, increase, or induce the activity of one or more endogenous biological processes in cells. In some embodiments, transgenics disclosed herein can be used to reduce endogenous genes, for example, the expression of dominant negative genes. In some embodiments, therapeutic gene expression products can change, inhibit, reduce, prevent, eliminate, or weaken the activity of one or more endogenous biological processes in cells. In some aspects, the increase in gene expression refers to an increase of at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, and 100%. On the one hand, the protein product of the gene targeted can increase by at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95% and 100%. In some respects, the minimizing of gene expression refers to an increase of at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95% and 100%. On the one hand, the protein product of the gene targeted can reduce by at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95% and 100%.

[0148] When an endogenous sequence (endogenous or partial transgene) is expressed with a transgene, the endogenous sequence can be a full-length sequence (wild type or mutant) or a partial sequence. The endogenous sequence can be functional. Non-limiting examples of the functions of these full-length or partial sequences include increasing the serum half-life of a polypeptide expressed by a transgene (e.g., a therapeutic gene) and / or acting as a carrier.

[0149] Transgenics can be inserted into endogenous genes so that all, some, or no endogenous genes are expressed. For example, transgenics described herein can be inserted into endogenous loci so that some endogenous sequences (located at the N-terminus and / or C-terminus of the transgenic) in the endogenous sequences are expressed or no endogenous sequences are expressed, for example as a fusion with a transgenic. In other cases, transgenics (e.g., with or without additional coding sequences of endogenous genes) are integrated into any endogenous loci, such as safe harbor loci. For example, a commissure protein (FXN) transgenic can be inserted into an endogenous FXN gene. Transgenics can be inserted into any gene (e.g., a gene described herein).

[0150] At least one advantage of the present disclosure is that in fact any therapeutic nucleic acid can be used to express any therapeutic gene expression product. In some cases, the therapeutic gene expression product is a therapeutic protein or peptide (e.g., an antibody, an antigen binding fragment, a peptide or a protein). In one embodiment, the length of the protein encoded by the therapeutic nucleic acid is between 50-2000 amino acids. In some embodiments, the length of the encoded protein is between 50-16000 amino acids. In some embodiments, the length of the encoded protein is between 50-1500 amino acids. In some embodiments, the length of the encoded protein is between 50-1000 amino acids. In some embodiments, the length of the encoded protein is between 50-800 amino acids. In some embodiments, the length of the encoded protein is between 50-600 amino acids. In some embodiments, the length of the encoded protein is between 50-400 amino acids. In some embodiments, the length of the encoded protein is between 50-200 amino acids. In some embodiments, the length of the encoded protein is between 50-100 amino acids. In some embodiments, the length of the encoded peptide is between 4-50 amino acids. In some embodiments, the encoded protein is a tetrapeptide, pentapeptide, hexapeptide, heptapeptide, octapeptide, nonapeptide or decapeptide. In some embodiments, the encoded protein includes a peptide of 2-30 amino acids (e.g., 5-30, 10-30, 2-25, 5-25, 10-25 or 10-20 amino acids). In some embodiments, the encoded protein includes a peptide of at least 11, 12, 13, 14, 15, 17, 20, 25 or 30 amino acids or a peptide of no more than 50 amino acids (e.g., no more than 35, 30, 25, 20, 17, 15, 14, 13, 12, 11 or 10 amino acids).

[0151] Non-limiting examples of therapeutic proteins or peptides include adrenergic agonists, anti-apoptotic factors, apoptosis inhibitors, cytokine receptors, cytokines, cytotoxins, erythropoiesis stimulating agents, glutamate decarboxylase, glycoproteins, growth factors, growth factor receptors, hormones, hormone receptors, interferons, interleukins, interleukin receptors, kinases, kinase inhibitors, nerve growth factors, netrins, neuroactive peptides, neuroactive peptide receptors, neurogenic factors, neurogenic factor receptors, neuropilins, neurotrophic factors, neurotrophins, neurotrophin receptors, N-methyl-D-aspartate antagonists, plexins, proteases, protease inhibitors, protein decarboxylases, protein kinases, protein kinase inhibitors, proteolytic proteins, proteolytic protein inhibitors, semaphoring, semaphoring receptors, serotonin transporters, serotonin uptake inhibitors, serotonin receptors, serine protease inhibitors, serine protease inhibitor receptors, and tumor suppressors. In certain embodiments, the therapeutic protein or peptide is selected from the following: brain-derived neurotrophic factor (BDNF), ciliary neurotrophic factor (CNTF), macrophage colony stimulating factor (CSF), epidermal growth factor (EGF), fibroblast growth factor (FGF), gonadotropin, interferon-γ (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-β (TGF-B), tumor necrosis factor (TNF), vascular endothelial growth factor (VEGF), prolactin, growth hormone, 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.

[0152] The therapeutic gene expression product may include a gene editing component. Non-limiting examples of gene editing components include those required for CRISPR / Cas, artificial site-specific RNA endonucleases (ASREs), zinc finger endonucleases (ZFNs), and transcription factor-like effector nucleases (TALENs). In a non-limiting example, a subject with Huntington's disease is identified. Then, a first amount of rAAV is systemically administered to the subject, which encapsidates a viral vector encoding a ZFN that is engineered to inhibit transcription of the Huntington protein (HTT) gene. The rAAV will contain a modified AAV capsid protein comprising Table 1-2, Figure 1 and the amino acid sequence provided in any one of Formulas I-VII, thereby allowing ZFN to be correctly targeted to the nervous system while reducing expression in off-target organs such as the liver. If necessary, a second or third dose of rAAV is administered to the subject until a therapeutically effective amount of ZFN is expressed in the nervous system of the subject.

[0153] Therapeutic nucleic acids can include non-protein coding genes, for example, sequences encoding antisense RNA, RNAi, shRNA and microRNA (miRNA), miRNA sponges or decoys, for conditional gene deletion recombinase delivery, conditional (recombinase-related) expression including those required for gene editing components described herein. Non-protein coding genes can also encode tRNA, rRNA, tmRNA, piRNA, double-stranded RNA, snRNA, snoRNA and / or long non-coding RNA (IncRNA). In some cases, non-protein coding genes can regulate the expression or activity of target genes or gene expression products. For example, RNA described herein can be used to inhibit gene expression in the cardiovascular system. In some cases, the inhibition of gene expression refers to at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95% and 100% inhibition. In some cases, the protein product of the targeted gene can be inhibited by at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95% and 100%. The gene can be a wild-type gene or a gene with at least one mutation. The targeted protein can be a wild-type protein or a protein with at least one mutation.

[0154] The therapeutic nucleic acid can regulate the expression or activity of a gene or a gene expression product expressed by the gene related to a disease or condition of the heart. For example, in some cases, the therapeutic nucleic acid is a modified version of a gene or the gene described herein. In some cases, the gene or gene expression product is suppressed. In some cases, the gene or gene expression product is enhanced.

[0155] In another example, the therapeutic nucleic acid includes an effector gene expression product, such as a gene editing component that is specific to the gene targeted therein. Non-limiting examples of genes include target genes or gene expression products selected from the following: MYBPC3, MYH7, LMNA, SCN5A, DMD, TAZ, BAG3, DSC2, DSG2, DSP, PKP2, CSRP3, phosphatase inhibitor (1-1) protein, KCNH2, TRPM4, ATP2A2, CACNA1C, DMPK, EPG5, EVC, EVC2, FBN1, NF1, SOS1, NPR1, ERBB4, VIP, MYH7, TNNI3, relaxin, and FXN.

[0156] In some cases, a gene or gene expression product is inhibited. In some cases, a gene or gene expression product is enhanced.

[0157] AAV vectors

[0158] Aspects disclosed herein include plasmid vectors comprising nucleic acid sequences encoding AAV capsids and AAV capsid proteins described herein. The AAV vectors described herein can be used for the assembly of rAAV and viral packaging of heterologous nucleic acids. In addition, the AAV vectors can encode transgenes comprising heterologous nucleic acids.

[0159] AAV vectors can include transgenes that encode heterologous gene expression products (e.g., therapeutic gene expression products, recombinant capsid proteins, etc.) in some cases. Transgenes are cis with two reverse terminal repeats (ITRs) flanking the transgenes. Transgenes can include therapeutic nucleic acids encoding therapeutic gene expression products. Due to the limited packaging capacity of rAAV (about 5kB), in some cases, longer transgenes can be split between two AAV vectors, the first having a 3' splice donor and the second having a 5' splice acceptor. After cell co-infection, concatemers are formed, which are spliced ​​together to express full-length transgenes.

[0160] The transgenic gene is usually inserted so that its expression is driven by an endogenous promoter at the integration site (i.e., a promoter that drives the expression of the endogenous gene into which the transgenic gene is inserted). In some cases, the transgenic gene includes a promoter and / or an enhancer, such as a constitutive promoter or an inducible or tissue / cell specific promoter. As a non-limiting example, the promoter can be a CMV promoter, a CMV-β-actin-intron-β-globin hybrid promoter (CAG), a CBA promoter, a FRDA or FXN promoter, a UBC promoter, a GUSB promoter, a NSE promoter, a synaptophysin promoter, a MeCP2 promoter, a GFAP promoter, a H1 promoter, a U6 promoter, a NFL promoter, a NFH promoter, a SCN8A promoter, or a PGK promoter. As a non-limiting example, the promoter can be a tissue-specific expression element, including but not limited to human elongation factor 1α subunit (EF1α), immediate early cytomegalovirus (CMV), chicken β-actin (CBA) and its derived CAG, β-glucuronidase (GUSB) and ubiquitin C (UBC). Transgenics can include 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), synaptophysin (Syn), 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. Transgenics can include tissue-specific expression elements for astrocytes, such as but not limited to glial fibrillary acidic protein (GFAP) and EAAT2 promoters. The transgene may include tissue-specific expression elements for oligodendrocytes, such as, but not limited to, the myelin basic protein (MBP) promoter.

[0161] In some embodiments, the promoter is less than 1 kb. The length of the promoter can be 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, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800 or more. The length of the promoter can be between 200-300, between 200-400, between 200-500, between 200-600, between 200-700, between 200-800, between 300-400, between 300-500, between 300-600, between 300-700, between 300-800, between 400-500, between 400-600, between 400-700, between 400-800, between 500-600, between 500-700, between 500-800, between 600-700, between 600-800, or between 700-800. The promoter can provide expression of the therapeutic gene expression product in a targeted tissue, such as, but not limited to, the heart, for a certain period of time.The expression of the therapeutic gene expression product can continue for the following time periods: 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, 17 hours, 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 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, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65 or more years. Expression of the payload may continue 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.

[0162] AAV vectors can include the genome of a helper virus. Assembly of recombinant AAV (rAAV) and packaging of transgenes containing heterologous nucleic acids into rAAV require helper virus proteins. Helper virus genes are adenovirus genes E4, E2a and VA that assist AAV replication when expressed in cells. In some embodiments, the AAV vector includes E2. In some embodiments, the AAV vector includes E4. In some embodiments, the AAV vector includes VA. In some cases, the AAV vector includes one or any combination of helper virus proteins.

[0163] The target gene or gene expression product for transgenesis can be selected from MYBPC3, MYH7, LMNA, SCN5A, DMD, TAZ, BAG3, DSC2, DSG2, DSP, PKP2, CSRP3, phosphatase inhibitor (1-1) protein, KCNH2, TRPM4, ATP2A2, CACNA1C, DMPK, EPG5, EVC, EVC2, FBN1, NF1, SOS1, NPR1, ERBB4, VIP, MYH7, TNNI3, relaxin and FXN.

[0164] The AAV vector may include a viral genome comprising a nucleic acid encoding a recombinant AAV (rAAV) capsid protein described herein. The viral genome may include a replication (Rep) gene encoding a Rep protein and a capsid (Cap) gene encoding an AAP protein in a first open reading frame (ORF1) or a Cap protein in a second open reading frame (ORF2). The Rep protein is selected from Rep78, Rep68, Rep52, and Rep40. In some cases, the Cap gene is modified to encode the modified AAV capsid protein described herein. The wild-type Cap gene encodes three proteins, namely 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 may include a nucleic acid encoding the following: wild-type Rep78, Rep68, Rep52, Rep40, and AAP proteins.

[0165] In some cases, the AAV9 VP1 gene provided in Table 4 is modified to include a gene from Table 1, 2, 3 or Figure 1 Any of the variants listed in . The AAV vectors described herein can be used to produce variant AAV capsids by the methods described herein.

[0166] Table 4. VP1 capsid protein nucleic acid sequence

[0167]

[0168]

[0169] Methods for producing rAAV

[0170] Disclosed herein is a method for producing an AAV capsid comprising an AAV capsid protein and a viral vector encoding a therapeutic nucleic acid. The AAV capsid protein is produced by introducing a first vector, a second vector, and a third vector into a cell (e.g., an immortalized stem cell), wherein the first vector contains a transgenic cassette flanked by an inverted terminal repeat (ITR) sequence from a parental AAV virus (the transgenic cassette has a promoter sequence that drives the transcription of a heterologous nucleic acid in the nucleus of a target cell), the second vector encodes an AAV genome with an AAV capsid protein (encoding the AAV Rep gene and the modified Cap gene of the variant produced), and the third vector encodes auxiliary viral proteins, which are required for assembling the AAV capsid structure and packaging the transgene in the modified AAV capsid structure. The assembled AAV capsid can be isolated and purified from the cell using a suitable method known in the art. Table 1-2 provides DNA sequences for use in the methods described herein.

[0171] Also provided herein is a transgene contained in a recombinant AAV (rAAV) vector and encapsidated by the AAV capsid protein of the present disclosure. The transgene disclosed herein is delivered to a subject for various purposes, such as to treat a disease or condition of the subject. The transgene can be a gene editing component that regulates 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 effectively regulates the activity or expression of itself or another target gene or gene expression product.

[0172] Aspects disclosed herein provide methods for preparing rAAV viruses or viral particles, comprising: (a) introducing into a cell a nucleic acid comprising: (i) a first vector containing a transgene cassette flanked by inverted terminal repeat (ITR) sequences from a parental AAV virus (the transgene cassette having a promoter sequence that drives transcription of a heterologous nucleic acid in the nucleus of a target cell); (ii) a second vector encoding an AAV genome having an AAV capsid protein of the invention; and (iii) a vector encoding helper viral proteins required for assembling an AAV capsid structure and packaging a transgene in a modified AAV capsid structure; (b) expressing the AAV capsid proteins described herein in the cell; (c) assembling AAV particles comprising the AAV capsid proteins disclosed herein; and (d) packaging the AAV particles. In some cases, the cell is mammalian. In some cases, the cell is immortalized. In some cases, the immortalized cell is an embryonic stem cell. In some cases, the embryonic stem cell is a human embryonic stem cell. In some cases, human embryonic stem cells are human embryonic kidney 293 (HEK-293) cells. In some cases, 5'ITR and 3'ITR are derived from AAV2 serotype. In some cases, 5'ITR and 3'ITR are derived from AAV5 serotype. In some cases, 5'ITR and 3'ITR are derived from AAV9 serotype. In some cases, the first nucleic acid sequence and the second nucleic acid sequence are trans. In some cases, the first nucleic acid sequence and the second nucleic acid sequence are cis. In some cases, the first nucleic acid sequence, the second nucleic acid sequence and the third nucleic acid sequence are trans.

[0173] The Cap genes disclosed herein include those from Table 1 or 2 or Figure 1 Any sequence in the sequence.

[0174] In some cases, these methods include packaging a first nucleic acid sequence encoding a therapeutic gene expression product so that it is encapsidated by a modified AAV capsid protein. In some embodiments, rAAV particles are isolated, concentrated and purified using a suitable virus purification method (such as the method described herein).

[0175] In some cases, the rAAV of the present disclosure is produced using the method described in the following: Challis, RC et al., "Natural Experiment Manual (Nat. Protoc.)" 14, 379 (2019). In short, triple transfection of HEK293T cells (ATCC) was performed using polyethyleneimine (PEI), and the virus was collected from both the cell lysate and the culture medium after 120 hours and purified by iodixanol. In a non-limiting example, rAAV is produced by triple transfection of precursor cells (e.g., HEK293T) cells using a standard transfection protocol (e.g., PEI). Viral particles are collected from the culture medium after a certain period of time (e.g., 72 hours after transfection), and viral particles are collected from cells and culture medium at a later time point (e.g., 120 hours after transfection). The virus present in the culture medium is concentrated by precipitation with 8% polyethylene glycol (PEG) and 500mM sodium chloride, and the precipitated virus is added to the lysate prepared from the collected cells. The virus was purified by a stepwise gradient (15%, 25%, 40% and 60%) of iodixanol (Optiprep, Sigma). The virus was concentrated and prepared in PBS. The virus titer was determined by measuring the number of DNase I-resistant vector genome copies (VG) using qPCR and a linearized genomic plasmid as a control.

[0176] The cell can be selected from humans, primates, rodents, cats, dogs, pigs, sheep, cattle, horses, grouper (epine), goats and wolf host cells. In some cases, the cell is a progenitor cell or precursor cell, such as a stem cell. In some cases, the stem cell is a mesenchymal cell, an embryonic stem cell, an induced pluripotent stem cell (iPSC), a fibroblast or other tissue-specific stem cell. The cell can be immortalized. In some cases, the immortalized cell is a HEK293 cell. In some cases, the cell is a differentiated cell. Based on the disclosure provided, it is expected that this system can be used in combination with any transgenic strain, which expresses a recombinase in the target cell type of interest, to develop an AAV capsid that more efficiently transduces the target cell population.

[0177] Treatment

[0178] Disclosed herein are methods for treating a disease or condition of a subject or a symptom of the disease or condition, comprising administering to the subject a therapeutically effective amount of one or more compositions disclosed herein (e.g., rAAV particles, AAV vectors, pharmaceutical compositions). In some embodiments, the composition is an 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 particles encapsidate an AAV vector comprising a transgene (e.g., a therapeutic nucleic acid). In some embodiments, the composition is an rAAV capsid protein conjugated with a therapeutic agent disclosed herein as described herein. In some embodiments, the composition is a pharmaceutical composition comprising rAAV particles and a pharmaceutically acceptable carrier. In some embodiments, one or more compositions are administered to a subject alone (e.g., an independent therapy). In some embodiments, the composition is a first-line therapy for a disease or condition. In some embodiments, the composition is a second-line, third-line, or fourth-line therapy for a disease or condition.

[0179] Recombinant adeno-associated virus (rAAV)-mediated gene delivery makes full use of the AAV mechanism of viral transduction for nuclear expression of free heterologous nucleic acids (e.g., transgenic, therapeutic nucleic acids). After delivery to the host's in vivo environment, rAAV will (1) bind to or attach to cell surface receptors on target cells, (2) endocytosis, (3) travel to the nucleus, (4) uncapping the virus to release the encapsidated heterologous nucleic acid, (5) converting the heterologous nucleic acid from single-stranded DNA to double-stranded DNA as a template for transcription in the nucleus, and (6) transcribing the free heterologous nucleic acid in the nucleus of the host cell ("transduction"). rAAV engineered to have increased transduction enrichment (transcription of free heterologous nucleic acids in host cells) is desirable for gene therapy applications.

[0180] 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 a rAAV of the disclosure or a pharmaceutical formulation of the disclosure, wherein the gene product is a therapeutic gene product. In some embodiments, administration is performed intracranially, intraventricularly, intraventricularly, intravenously, intraarterially, intranasally, intrathecally, intracisternal, or subcutaneously.

[0181] Provided herein is a method for treating a disease or condition associated with abnormal expression or activity of a target gene or its gene expression product, the method comprising regulating the expression or activity of the target gene or gene expression product in a subject by administering an rAAV that encapsidates a heterologous nucleic acid disclosed herein. In some cases, the expression or activity of the target gene or gene expression product is reduced relative to the expression or activity in a normal (non-diseased) individual; and administering rAAV to the subject is sufficient to increase the expression of the activity of the target gene or gene expression product. In some cases, the expression or activity of a gene or gene expression product is increased relative to the expression or activity in a normal individual; and administering rAAV to the subject is sufficient to reduce the expression or activity of the target gene or gene expression product. In a non-limiting example, a subject diagnosed with Alzheimer's disease (caused in some cases by gain of function of presenilin 1 and / or presenilin 2 (encoded by genes PSEN1 and PSEN2, respectively)) is administered a rAAV disclosed herein that encapsidates a therapeutic nucleic acid, which is a silencing RNA (siRNA) or other RNAi that has a loss-of-function effect on PSEN1 mRNA.

[0182] Also provided are methods for preventing a disease or condition disclosed herein in a subject, comprising administering to the subject a therapeutically effective amount of a rAAV vector comprising a nucleic acid sequence encoding a therapeutic gene expression product described herein. The rAAV vector may be encapsidated in a modified capsid protein or rAAV virus particle described herein. In some cases, the therapeutic gene expression product effectively regulates the activity or expression of a target gene or gene expression product.

[0183] Disclosed herein are methods of treating a disease or condition in a subject by administering a composition comprising a rAAV disclosed herein. One advantage of the rAAV disclosed herein is that rAAV can be used to treat virtually any disease or condition that would benefit from transgenic therapy, including but not limited to Friedreich's Ataxia, lysosomal storage disorders (including Pompe disease and Danon disease), chronic heart failure, congestive heart failure, cardiomyopathies (including hypertrophic myopathy, dilated myopathy, and arrhythmogenic myopathy), and cardiac arrhythmias.

[0184] In some cases, the disease or condition is localized to a specific in vivo environment of the subject, e.g., the heart. The compositions of the present disclosure are particularly useful in treating the diseases or conditions described herein because these compositions specifically or more effectively target the in vivo environment and deliver therapeutic nucleic acids engineered to modulate the activity or expression of a target gene expression product associated with the pathogenesis or pathology of the disease or condition.

[0185] Provided herein are methods for treating a disease or condition, or a symptom of the disease or condition, in a subject, comprising: (a) diagnosing a subject with a disease or condition that affects a target in vivo environment; and (b) treating the disease or condition by administering to the subject a therapeutically effective amount of a composition disclosed herein (e.g., rAAV particles, AAV vectors, pharmaceutical compositions), wherein the composition is engineered to have increased specificity for the target in vivo environment.

[0186] Disclosed herein are methods of treating a target afflicting disease or condition or a symptom of the disease or condition in a subject, the methods comprising: (a) administering a composition (e.g., rAAV particles, AAV vectors, pharmaceutical compositions) to the subject; and (b) expressing a therapeutic nucleic acid into a target in vivo environment within the subject with increased transduction enrichment.

[0187] In some embodiments, the methods further comprise reducing or eliminating delivery of the heterologous nucleic acid in off-target in vivo environments (such as the liver). In some embodiments, delivery is characterized by an increase in transduction enrichment (eg, of the heterologous nucleic acid) in the heart.

[0188] In some embodiments, a method of treating a disease or condition affecting the heart comprises administering to the heart of a subject a rAAV particle comprising a rAAV capsid protein comprising a protein of Table 1-2, at amino acid positions 588-589 of a parent AAV capsid protein. Figure 1 and about five, six, or seven amino acids of the amino acid sequence provided in Formulas I-VII. In some embodiments, a method of treating a disease or condition affecting the heart comprises administering to the heart of a subject a rAAV particle comprising a rAAV capsid protein comprising an insertion of about five, six, or seven amino acids of the amino acid sequence and in Tables 1-2, Figure 1 and one or more substitutions of the amino acids found at amino acid positions 587-590 [AQAQ] provided in Formulas I-VII. In some embodiments, the parent AAV capsid protein is an AAV9 capsid protein (eg, provided in SEQ ID NO: 1).

[0189] Also provided are methods for regulating target gene expression products, which methods include administering a composition disclosed herein (e.g., rAAV particles, AAV vectors, pharmaceutical compositions) to a subject in need thereof. For example, the methods provided herein include administering to a subject an rAAV having an rAAV capsid protein, which encapsidates a viral vector, which includes a heterologous nucleic acid that regulates the expression or activity of a target gene expression product.

[0190] The term "normal individual" refers to an individual who does not suffer from a disease or condition characterized by an alteration in the expression or activity of a gene or its gene expression product.

[0191] In some instances, a method can be used to treat degenerative muscle diseases or conditions (e.g., familial cardiomyopathy, dilated cardiomyopathy, hypertrophic cardiomyopathy, ischemic cardiomyopathy caused by restrictive cardiomyopathy or coronary artery disease). In some instances, the subject method can be used to treat individuals with heart or cardiovascular disease or conditions, such as cardiovascular disease, aneurysm, angina, arrhythmia, atherosclerosis, cerebrovascular accident (stroke), cerebrovascular disease, congenital heart disease, congestive heart failure, myocarditis, coronary valvular disease, dilated artery disease, diastolic dysfunction, endocarditis, high blood pressure / hypertension, cardiomyopathy, hypertrophic cardiomyopathy, restrictive cardiomyopathy, ischemic cardiomyopathy caused by coronary artery disease, mitral valve prolapse, myocardial infarction (heart attack) or venous thromboembolism. In some instances, the subject suffers from cardiomyopathy or has the risk of suffering from cardiomyopathy.

[0192] In some embodiments, the cardiac disease or condition is selected from Friedreich's Ataxia, lysosomal storage disorders (including Pompe disease and Danon disease), chronic heart failure, congestive heart failure, cardiomyopathies (including hypertrophic myopathy, dilated myopathy, and arrhythmogenic myopathy), and cardiac arrhythmias.

[0193] In some embodiments, the drug formulation comprises a therapeutic nucleic acid encoding a therapeutic gene expression product. In some cases, the therapeutic gene expression product effectively modulates the activity or expression of a target gene or gene expression product selected from the group consisting of MYBPC3, MYH7, LMNA, SCN5A, DMD, TAZ, BAG3, DSC2, DSG2, DSP, PKP2, CSRP3, phosphatase inhibitor (1-1) protein, KCNH2, TRPM4, ATP2A2, CACNA1C, DMPK, EPG5, EVC, EVC2, FBN1, NF1, SOS1, NPR1, ERBB4, VIP, MYH7, TNNI3, relaxin, and FXN.

[0194] In some cases, the therapeutic gene expression product includes a gene editing component. In some cases, the gene editing component is selected from: artificial site-specific RNA endonucleases (ASREs), zinc finger endonucleases (ZFNs), transcription factor-like effector nucleases (TALENs), clustered regularly interspaced short palindromic repeats (CR1SPR) / Cas enzymes and CR1SPR / Cas guide RNAs.

[0195] In some cases, the expression of a gene or the expression or activity of a gene expression product is inhibited by administering a composition to a subject. In some cases, the expression of a gene or the expression or activity of a gene expression product is enhanced by administering a composition to a subject.

[0196] Formulations, Dosages and Routes of Administration

[0197] Disclosed herein are methods comprising delivering rAAV particles that encapsidate a heterologous nucleic acid to the heart of a subject, the rAAV particles comprising (i) increased transduction of the heterologous nucleic acid in the heart, wherein the rAAV particles have a rAAV capsid protein that comprises at amino acid positions 588-589 in a parent AAV capsid protein a protein selected from Tables 1-2, Figure 1 and the insertion of five, six or seven amino acids in the amino acid sequence provided in Formula I-VII and as shown in Tables 1-2, Figure 1 and one or more substitutions of the amino acids found at amino acid positions 587-590 [AQAQ] provided in Formulas I-VII.

[0198] Typically, the methods disclosed herein include administering a therapeutic rAAV composition by systemic administration. In some cases, these methods include administering a therapeutic rAAV composition by intravenous ("iv") administration. Therapeutic rAAV compositions can be administered by other routes, such as subcutaneous injection, intramuscular injection, intradermal injection, transcutaneous injection, transdermal administration, intranasal administration, intralymphatic injection, rectal administration, intragastric administration, intraocular administration, intraventricular administration, intrathecal administration, intracisternal administration or any other suitable parenteral administration. The route, dosage, time point and duration of administration of the therapeutic agent can be adjusted. In some embodiments, the therapeutic agent is administered before or after the onset of any one or both of the acute and chronic symptoms of the disease or condition. Other routes of delivery to the heart include, but are not limited to, intracranial administration, lateral ventricular administration and intravascular administration.

[0199] The effective dose and dosage of the pharmaceutical composition for preventing or treating the diseases or conditions disclosed herein are defined by the observed beneficial responses related to the disease or condition or the symptoms of the disease or condition. Beneficial responses include preventing, alleviating, stopping or curing the disease or condition or the symptoms of the disease or condition. In some embodiments, the beneficial response can be measured by detecting the measurable improvement of the presence, level or activity of biomarkers, transcriptome risk profiles or intestinal microbiome in the subject. As used herein, "improvement" refers to the presence, level or activity to the presence, level or activity observed in normal individuals (e.g., individuals without diseases or conditions). In the case where the therapeutic rAAV composition is ineffective or does not fully alleviate the disease or condition or the symptoms of the disease or condition, the dosage and / or route of administration can be changed, or another agent can be administered to the subject together with the therapeutic rAAV composition. In some embodiments, when the patient begins the regimen of the therapeutic rAAV composition, the patient also gradually stops (e.g., the dose is gradually reduced) the second treatment regimen.

[0200] In some cases, a dose of a pharmaceutical composition can include an infectious particle concentration of at least or about 10 7 , 10 8 , 10 9 , 10 10 , 10 11 , 10 12 , 10 13 , 10 14 , 10 15 , 10 16 or 10 17 In some cases, the concentration of infectious particles was 2x10 7 , 2x 10 8 , 2x 10 9 , 2x 10 10 , 2x 10 11 , 2x 10 12 , 2x 10 13 , 2x 10 14 , 2x 10 15 , 2x 10 16 or 2x 10 17 In some cases, the concentration of infectious particles was 3 x 10 7 , 3x 10 8 , 3x 10 9 , 3x 10 10 , 3x 10 11 , 3x 10 12 , 3x10 13 , 3x 10 14 , 3x 1015 , 3x 10 16 or 3x 10 17 In some cases, the concentration of infectious particles was 4 x 10 7 , 4x10 8 , 4x 10 9 , 4x 10 10 , 4x 10 11 , 4x 10 12 , 4x 10 13 , 4x 10 14 , 4x 10 15 , 4x 10 16 or 4x 10 17 In some cases, the concentration of infectious particles was 5 x 10 7 , 5x10 8 , 5x 10 9 , 5x 10 10 , 5x 10 11 , 5x 10 12 , 5x 10 13 , 5x10 14 , 5x 10 15 , 5x 10 16 or 5x 10 17 In some cases, the concentration of infectious particles was 6 x 10 7 , 6x 10 8 , 6x10 9 , 6x 10 10 , 6x 10 11 , 6x 10 12 , 6x 10 13 , 6x 10 14 , 6x 10 15 , 6x 10 16 or 6x 10 17 In some cases, the concentration of infectious particles was 7 x 10 7 , 7x 10 8 , 7x 10 9 , 7x 10 10 , 7x 10 11 , 7x 10 12 , 7x 10 13 , 7x 10 14 , 7x10 15 , 7x 10 16 or 7x 10 17 In some cases, the concentration of infectious particles was 8 x 10 7 , 8x 10 8, 8x 10 9 , 8x10 10 , 8x 10 11 , 8x 10 12 , 8x 10 13 , 8x 10 14 , 8x 10 15 , 8x 10 16 or 8x 10 17 In some cases, the concentration of infectious particles was 9 x 10 7 , 9x 10 8 , 9x 10 9 , 9x 10 10 , 9x 10 11 , 9x 10 12 , 9x 10 13 , 9x 10 14 , 9x 10 15 , 9x 10 16 or 9x 10 17 .

[0201] In some embodiments, disclosed herein are formulations of pharmaceutically acceptable excipients and carrier solutions suitable for delivering rAAV compositions described herein, as well as suitable dosages and treatment regimens for using specific compositions described herein in a variety of treatment regimens. In some embodiments, the amount of therapeutic gene expression product in each therapeutically useful composition can be prepared in such a manner that a suitable dosage will be obtained in any given unit dose of the compound. Those skilled in the art of preparing such pharmaceutical formulations will consider factors such as solubility, bioavailability, biological half-life, route of administration, product shelf life, and other pharmacological factors, and as such, a variety of dosages and treatment regimens may be desired.

[0202] In some embodiments, the pharmaceutical form of the rAAV-based viral composition suitable for injection comprises a sterile aqueous solution or dispersion and a sterile powder for the extemporaneous preparation of a sterile injectable solution or dispersion. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, a polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, etc.), a suitable mixture thereof, and / or a vegetable oil. Suitable fluidity can be maintained, for example, by using a coating such as lecithin, by maintaining the desired particle size in the case of a dispersion, and by using a surfactant. Prevention of the effects of microorganisms can be provided by a variety of antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, thimerosal, etc. In many cases, it will be preferred to include an isotonic agent (e.g., sugar or sodium chloride).

[0203] In some cases, for the use of injectable aqueous solution, if necessary, this injectable aqueous solution can be suitably buffered, and the liquid diluent is first isotonic with enough saline or glucose. These specific aqueous solutions are particularly suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration. Some variations of dosage will inevitably occur according to the symptom of the experimenter being treated. The personnel responsible for use will determine the appropriate dose for individual subjects under any circumstances. In addition, for human use, preparation should meet the sterility, pyrogenicity and general safety and purity standards required by the FDA Office of Biological Products Standards.

[0204] Disclosed herein are sterile injectable solutions comprising rAAV compositions disclosed herein, which are prepared by incorporating the rAAV compositions disclosed herein in the desired amount into an appropriate solvent as needed along with several other ingredients enumerated above, followed by filtration sterilization. Typically, dispersions are prepared by incorporating various sterilized active ingredients into a sterile vehicle containing a basic dispersion medium and other ingredients required from the ingredients enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, preferred preparation methods are vacuum drying and freeze drying techniques, which produce powders of the active ingredient plus any additional desired ingredients from their previous sterile filtered solutions. Injectable solutions can be advantageous for systemic administration (e.g., by intravenous or intrathecal administration).

[0205] The appropriate dose and dosage for administration to a subject is determined by factors including, but not limited to, the particular therapeutic rAAV composition, the disease condition and its severity, the identity of the subject in need of treatment (e.g., weight, sex, age), and may be determined based on the specific circumstances surrounding the case including, for example, the particular agent being administered, the route of administration, the condition being treated, and the subject or host being treated.

[0206] The amount of rAAV composition and the timing of administration of such composition will be within the capabilities of those skilled in the art having the benefit of the present teachings. However, it is likely that administration of a therapeutically effective amount of the disclosed composition can be achieved by a single administration, e.g., a single injection of a sufficient number of infectious particles to provide a therapeutic benefit to a patient receiving such treatment. This is made possible at least in part by the fact that certain target cells (e.g., neurons) do not divide, thereby eliminating the need for multiple or chronic administration.

[0207] In certain embodiments, the data obtained from cell culture assays and animal studies are used to formulate therapeutically effective daily dosage ranges and / or therapeutically effective unit doses for mammals (including humans). In certain embodiments, dosage ranges and / or unit doses vary within this range, depending on the dosage form employed and the route of administration utilized.

[0208] Combination therapy

[0209] Therapeutic rAAV can be used alone or in combination with another therapeutic agent (collectively referred to as a "therapeutic agent"). In some cases, a therapeutic rAAV as used herein is administered alone. The therapeutic agents can be administered together or sequentially in a combination therapy. The combination therapy can be administered on the same day, or can be administered one or more days, weeks, months, or years apart.

[0210] Additional therapeutic agents may include small molecules. Additional therapeutic agents may include antibodies or antigen binding fragments. Additional therapeutic agents may include lipid nanoparticle-based therapies, antisense oligonucleotide therapies, and other viral therapies.

[0211] Additional therapeutic agents may include cell-based therapies. Exemplary cell-based therapies include, but are not limited to, 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. Any one of NK cells or CAR-NK cells or a combination of NK cells and CAR-NK cells can be used in combination with the methods disclosed herein. In some embodiments, NK cells and CAR-NK cells are derived from human induced pluripotent stem cells (iPSC), cord blood, or cell lines. NK cells and CAR-NK cells may include cytokine receptors and suicide genes. Cell-based therapies may include stem cell therapy. Stem cell therapy may be embryonic stem cells or somatic stem cells. Stem cells may be separated from donors (allogeneic) or separated from subjects (autologous). Stem cells may be adipose-derived stem cells (eASC), hematopoietic stem cells (HSC), mesenchymal stem (stromal) cells (MSC), or induced pluripotent stem cells (iPSC) derived from cells of subjects.

[0212] Reagent test kit

[0213] Kits comprising the compositions disclosed herein are disclosed herein. Kits for treating or preventing a disease or condition of the heart are also disclosed herein. In some cases, the disease or condition is cancer, a pathogen infection, a lung disease or condition, a nervous system disease, a muscle disease, or an immune disorder, such as a disease or condition described herein.

[0214] In one embodiment, the kit may include a therapeutic or prophylactic composition containing an effective amount of rAAV particles and a composition of a recombinant AAV (rAAV) capsid protein disclosed herein, which rAAV particles encapsidate a recombinant AAV vector encoding a therapeutic nucleic acid (e.g., a therapeutic nucleic acid). In another embodiment, the kit may include a therapeutic or prophylactic composition containing an effective amount of cells modified by rAAV described herein in a unit dosage form expressing a therapeutic nucleic acid ("modified cells"). In some embodiments, the kit includes a sterile container that may contain a therapeutic composition; such containers may be boxes, ampoules, bottles, vials, tubes, bags, pouches, blister packs, or other suitable container forms known in the art. Such containers may be made of plastic, glass, laminated paper, metal foil, or other materials suitable for containing drugs.

[0215] In some cases, the test kit further includes cells. In some cases, the cells are mammalian. In some cases, the cells are immortalized. In some cases, the immortalized cells are embryonic stem cells. In some cases, the embryonic stem cells are human embryonic stem cells. In some cases, the human embryonic stem cells are human embryonic kidney 293 (HEK-293) cells. In some cases, the test kit further includes an AAV vector, which includes a heterologous nucleic acid encoding a therapeutic gene expression product. In some cases, the AAV vector is an episome.

[0216] In some cases, the rAAV is provided with instructions for administering the rAAV to a subject having a disease or condition (e.g., a disease of the heart) or at risk of developing the disease or condition. The instructions can generally include information about the use of the composition for treating or preventing a disease or condition.

[0217] In some cases, the instructions include at least one of the following: a description of the therapeutic rAAV composition; dosage tables and administration for treating or preventing a disease or condition disclosed herein; precautions; warnings; indications; contraindications; overdose information; adverse reactions; animal pharmacology; clinical studies; and / or references. These instructions can be printed directly on the container (if present), or as a label applied to the container or as a separate sheet, brochure, card, or folder provided with or applied to the container. In some cases, the instructions provide procedures for administering rAAV alone to a subject. In some cases, these instructions provide that rAAV is formulated for systemic delivery.

[0218] definition

[0219] The terms used herein are for the purpose of describing a particular situation only and are 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. In addition, where the terms "including / includes," "having / has / with," or variations thereof, are used in the detailed description and / or claims, such terms are intended to be inclusive in a manner similar to the term "comprising."

[0220] The term "about" or "approximately" means within an acceptable error range for a 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, for example, limitations of the measurement system. For example, depending on the practice of a given value, "about" can mean within 1 or more than 1 standard deviation. Where specific values ​​are described in the application and claims, the term "about" should be assumed to mean an acceptable error range for that specific value unless otherwise stated.

[0221] As used herein, "consisting essentially of" when used to define compositions and methods shall be meant to exclude other elements having any significance to the combination for the stated purpose. Thus, a composition consisting essentially of elements as defined herein would not exclude other materials or steps that do not materially affect the basic and novel characteristics of the claimed disclosure, such as a composition for treating skin conditions such as acne, eczema, psoriasis, and rosacea.

[0222] The terms "homologous", "homology" or "homology percentage" as used herein generally refer to an amino acid sequence or nucleic acid sequence that has the same or similar sequence as a reference sequence. As of the date of filing of this application, the latest version of BLAST can be used to determine the homology percentage of a sequence.

[0223] The term "increase" is used herein to generally mean an increase in a statistically significant amount. In some embodiments, the term "increase" means an increase of at least 10% compared to a reference level, such as 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 an increase of 100%, or any increase between 10%-100%. Other examples of "increase" include an increase of at least 2 times, at least 5 times, at least 10 times, at least 20 times, at least 50 times, at least 100 times, at least 1000 times compared to a reference level.

[0224] The term "reduce (decreased / decrease)" is generally used in this article to mean a statistically significant amount of reduction. In certain embodiments, "reduce (decreased / decrease)" means to reduce at least 10% compared with a reference level, for example, reduce 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 at most and comprising 100% reduction (for example, non-existent level or undetectable level compared with a reference level) or any reduction between 10-100%. In the context of markers or symptoms, these terms mean a statistically significant reduction of such levels. Reduction can be, for example, at least 10%, at least 20%, at least 30%, at least 40% or more, and is preferably reduced to the level accepted in the normal range as an individual not suffering from a given disease.

[0225] The term "subject" is any organism. In some cases, the organism is a mammal. Non-limiting examples of mammals include any member of the mammal class: humans, non-human primates, such as chimpanzees and other apes and monkey species; farm animals, such as cattle, horses, sheep, goats, pigs, etc.; domestic animals, such as rabbits, dogs and cats, etc.; laboratory animals, including rodents, such as rats, mice and guinea pigs, etc. On the one hand, mammals are humans. As used herein, the term "animal" includes humans and non-human animals. In one embodiment, a "non-human animal" is a mammal, such as a rodent (such as a rat or mouse). In one embodiment, a "non-human primate" is a mammal, such as a monkey. In some cases, a subject is a patient, which, as used herein, may refer to a subject diagnosed with a specific disease or condition.

[0226] As used herein, the term "gene" refers to a nucleic acid fragment (also referred to as a "coding sequence" or "coding region") that encodes a single protein or RNA, optionally together with associated regulatory regions (such as promoters, operators, terminators, etc.), which may be located upstream or downstream of the coding sequence.

[0227] As used herein, the term "adeno-associated virus" or "AAV" refers to an adeno-associated virus or a derivative thereof. Non-limiting examples of AAV include type 1 AAV (AAV1), type 2 AAV (AAV2), type 3 AAV (AAV3), type 4 AAV (AAV4), type 5 AAV (AAV5), type 6 AAV (AAV6), type 7 AAV (AAV7), type 8 AAV (AAV8), type 9 AAV (AAV9), type 10 AAV (AAV10), type 11 AAV (AAV11), type 12 AAV (AAV12), avian AAV, cattle AAV, canine AAV, horse AAV, primate AAV, non-primate AAV and sheep AAV. In some cases, AAV is described as "primate AAV", which refers to the AAV that infects primates. Similarly, AAV can infect cattle animals (e.g., "cattle AAV" etc.). In some cases, AAV is wild-type or naturally occurring. In some cases, the AAV is recombinant.

[0228] As used herein, the term "AAV capsid" refers to a capsid protein or peptide of an adeno-associated virus. In some cases, the AAV capsid protein is configured to encapsidate genetic information (e.g., transgenic, therapeutic nucleic acid, viral genome). In some cases, the AAV capsid of the present disclosure is a modified AAV capsid relative to the corresponding parent AAV capsid protein.

[0229] As used herein, the term "tropism" refers to a quality or property of an AAV capsid, which quality or property may include specificity for expressing encapsidated genetic information into an in vivo environment relative to a second in vivo environment, and / or increased or decreased enrichment for expressing encapsidated genetic information into an in vivo environment relative to a second in vivo environment. In some cases, the in vivo environment is a cell type. In some cases, the in vivo environment is an organ or organ system.

[0230] The term "AAV vector" as used herein refers to a nucleic acid polymer encoding genetic information associated with a virus. The AAV vector can be a recombinant AAV vector (rAAV), which refers to an AAV vector produced using a recombinant genetics method. In some cases, the rAAV vector includes at least one heterologous polynucleotide (e.g., a polynucleotide other than a wild-type or naturally occurring AAV genome, such as a transgene).

[0231] As used herein, the term "AAV particle" refers to an AAV virus, a virion, an AAV capsid protein, or a component thereof. In some cases, the AAV particle is modified relative to a parent AAV particle.

[0232] The term "gene product" of "gene expression product" refers to the expression product of a polynucleotide sequence, such as a polypeptide, peptide, protein or RNA (including interfering RNA (e.g., siRNA, miRNA, shRNA) and messenger RNA (mRNA)).

[0233] As used herein, the term "heterologous" refers to a genetic element (eg, coding region) or gene expression product (eg, RNA, protein) derived from an entity that is genotypically different from the rest of the entity to which it is being compared.

[0234] As used herein, the term "endogenous" refers to a genetic element (eg, coding region) or gene expression product (eg, RNA, protein) that is naturally present in or associated with an organism or a specific cell within an organism.

[0235] As used herein, the term "treat / treating / treatment" refers to the alleviation or elimination of a disorder, disease or condition; or one or more of the symptoms associated with a disorder, disease or condition; or the alleviation or elimination of the cause of the disorder, disease or condition itself. The desired therapeutic effect may include, but is not limited to, preventing the occurrence or recurrence of the disease, alleviating symptoms, reducing any direct or indirect pathological consequences of the disease, preventing metastasis, reducing the rate of disease progression, improving or alleviating the disease state, and alleviating or improving prognosis.

[0236] The term "therapeutically effective amount" refers to that amount of a compound or therapy which, when administered, is sufficient to prevent the development of, or alleviate to some extent, one or more of the symptoms of a disorder, disease, or condition of a disease; or an amount of a compound sufficient to elicit the biological or medical response of a cell, tissue, system, animal or human that is being sought by a researcher, veterinarian, medical doctor or clinician.

[0237] The terms "pharmaceutically acceptable carrier", "pharmaceutically acceptable excipient", "physiologically acceptable carrier", or "physiologically acceptable excipient" refer to a pharmaceutically acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, excipient, solvent, or encapsulating material. A component may be "pharmaceutically acceptable" in the sense that it is compatible with the other ingredients of a pharmaceutical formulation. It may also be suitable for contact with human and animal tissues or organs at a reasonable benefit / risk ratio without producing excessive toxicity, irritation, allergic response, immunogenicity, or other problems or complications. See Remington: The Science and Practice of Pharmacy, 21st ed., Lippincott Williams & Wilkins: Philadelphia, PA, 2005; Handbook of Pharmaceutical Excipients, 5th ed., Rowe et al., eds., The Pharmaceutical Press and the American Pharmaceutical Association: 2005; and Handbook of Pharmaceutical Additives, 3rd ed., Ash and Ash, eds., Gower Publishing Company: 2007; Pharmaceutical Preformulation and Formulation, Gibson, ed., CRC Press LLC: Boca Raton, FL, 2004.

[0238] The term "pharmaceutical composition" refers to a mixture of a compound disclosed herein and other chemical components such as a diluent or carrier. A pharmaceutical composition can facilitate administration of a compound to an organism. There are a variety of techniques for administering a compound in the art, including but not limited to systemic administration.

[0239] Non-limiting examples of "samples" 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 extracts, cheek swabs, cells or other body fluids or tissues, including but not limited to tissues obtained by surgical biopsy or surgical resection. Alternatively, the sample can be obtained by a primary patient-derived cell line, or can be an archived patient sample in the form of a stored sample or a fresh frozen sample.

[0240] The term "in vivo" is used to describe events that occur within the body of a subject.

[0241] The term "in vitro" is used to describe events that occur in a container used to hold laboratory reagents, such that the material is separated from the biological source from which the material was obtained. In vitro assays may encompass cell-based assays, in which living or dead cells are employed. In vitro assays may encompass cell-free assays, in which intact cells are not employed.

[0242] The terms "cardiac" or "cardiovascular system" mean tissue selected from the group consisting of the heart, veins and aorta.

[0243] The term "systemic delivery" is defined as a route of administration in which a drug or other substance enters the circulatory system so that the entire body is affected. Administration can be by enteral administration (drugs are absorbed through the gastrointestinal tract) or parenteral administration (usually injection, infusion or implantation). The "circulatory system" includes the blood circulatory system. Examples of systemic administration include intra-arterial, intravenous or intrathecal injections. The terms "systemic administration" and "systemic delivery" are used interchangeably.

[0244] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0245] Examples

[0246] Example 1

[0247] Methods for Identifying Modified Capsid Proteins in Cynomolgus Macaques

[0248] The therapeutic applicability of engineered adeno-associated viruses (AAVs) is primarily concerned with how their transduction profiles translate into human applications. Although previous engineering efforts have focused on in vitro or in vivo rodent screening platforms because of their ease of use and flexibility, screening efforts conducted directly in non-human primates (NHPs) are more likely to identify viruses that translate. Cynomolgus monkeys (an old world NHP) were selected for engineering efforts. Engineering efforts were focused on the region on the surface of the AAV9 capsid at amino acid position 588, which is one of the most exposed loops on the capsid surface, which is a variable region between natural AAV serotypes and plays a role in receptor binding. A library of viral capsids was created by randomly inserting 7 amino acids at this site within AAV9, and the novel tropism to the NHP heart was analyzed.

[0249] Plasmids . The first round of viral DNA libraries was generated by amplifying 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 changes. The resulting library insert was then introduced into the rAAV-ΔCap-in-rev-RNA plasmid by Gibson assembly as previously described. The resulting capsid DNA library rAAV-Cap-Cag-GFP11 contains a diversity of approximately 1.28 billion variants at the amino acid level. The second round of viral DNA libraries was generated in a similar manner to the first round, but instead of inserting the NNK degenerate primer at 588, a synthetic oligomer pool (Twist Bioscience) was used to generate only selected variants in UBC-Cap-DNA and CAG-Cap-DNA constructs with CAP. This second round DNA library contained a diversity of 20,000 variants at the amino acid level, with 3 codons replicated for each variant. The AAV2 / 9 REP-AAP-ΔCAP plasmid transfected into HEK293T cells to provide the Rep gene for library virus production prevented the production of wild-type AAV9 capsids during viral library production following plausible recombination events between this plasmid and the library plasmid containing the library insert at each stage of co-transfection.

[0250] Virus production. Recombinant AAV was produced according to established protocols. Briefly, immortalized HEK293T cells (ATCC) were quadruple transfected with four vectors using polyethyleneimine (PEI). The first vector was a rAAV-Cap-in-cis-Lox library flanked by inverted terminal repeat (ITR) sequences from the parental AAV virus. The second vector was an AAV2 / 9REP-AAP-ΔCAP plasmid. The third vector contained nucleic acids encoding auxiliary viral proteins required for viral assembly and packaging of heterologous nucleic acids into modified capsid structures. The fourth was a pUC-18 plasmid, which contained the correct PEI / DNA ratio for optimal transfection enrichment. Only 10 ng of rAAV-Cap-in-cis-Lox library DNA (per 150 mm plate) was transfected to reduce the possibility of multiple library DNAs entering the same cell. Viral particles were harvested from cells and culture medium 60 hours after transfection. The virus present in the culture medium is concentrated by precipitation with 8% polyethylene glycol and 500mM sodium chloride, and the precipitated virus is added to the lysate prepared by the collected cells. Virus is purified by iodixanol (Optiprep, Sigma) stepwise gradient (15%, 25%, 40% and 60%). The virus is concentrated and deployed in PBS. The virus titer is determined by measuring the number of DNA enzyme I resistance vector genome copies (VG) using qPCR and the linearized genomic plasmid as a control.

[0251] animal . The cynomolgus monkey procedure was approved by the IACUC committee of Envol Biomedical. Cynomolgus monkeys were born and raised in the Envol Biomedical colony and were housed in family groups under standard conditions. They were fed ad libitum and were enriched as part of the primate enrichment program for NHPs at Envol Biomedical. For AAV infusion, animals were screened for endogenous neutralizing antibodies (Nab). None of the screened animals showed any detectable blocking reaction when the serum was diluted 1:10. The group was then housed alone for several days and adapted to the new room before injection. A total of four young monkeys were used for screening, two young monkeys for the first round of libraries, and two young monkeys for the second round of libraries. The animals were restrained and the test articles were administered by intravenous infusion for 10 minutes. During life, activity and behavior were monitored daily.

[0252] DNA / RNA recovery and sequencing The first and second round virus libraries were cultured at 2.5 x 10 13vg / kg animal was injected into cynomolgus monkeys, and the rAAV genome was recovered two weeks after injection. The animals were euthanized and recovered, snap frozen, and placed at -80°C for long-term storage, as well as other peripheral tissues such as heart, spleen, adrenal glands, kidneys and limbs. For round 1, the brain was divided into eleven brain regions, 100 mg per brain region, and for round 2, the brain was divided into thirteen brain regions. MagMAXDNA ULTRA (A25597) and Bead Ruptor 96 (OMNI, INC) were used to homogenize 100 mg of each heart and liver in buffer, and viral DNA was isolated according to the manufacturer's recommended protocol. The recovered viral DNA was treated with RNase and purified with a Zymo DNA cleaning and concentrator kit (D4033). Using 50% of the total extracted viral DNA as a template, the viral genome was enriched by 25 cycles of PCR amplification, where the primers flanked the 588-589 insertion site in the capsid genome. After Zymo DNA purification, samples were diluted 1:10 to 1:1000 depending on tissue type, and each dilution was further amplified around the variable region of the library with 10 cycles of PCR. Subsequently, the samples were further amplified for 10 cycles using custom primers with Illumina indexes. The amplified products were run on 2% low melting point agarose gel (Thermo Fisher Scientific, 16520050) to better separate and recover the 210 bp band.

[0253] For the second round of libraries only, packaged viral library DNA was isolated from the injected viral library by digesting the viral capsid and purifying the contained ssDNA. These viral genomes were amplified by two PCR amplification steps, as was viral DNA extracted from tissue to add adapters and indexes for Illumina next-generation sequencing, and purified after gel electrophoresis. This viral library DNA, along with viral DNA extracted from tissue, was deeply sequenced using Illumina's NextSeq 2000 system.

[0254] NGS data alignment and processing . Raw fastq files from NGS runs were processed using a custom script (CapsidaCapSeq tool). For the first round of libraries, the pipeline used to process these datasets involved filtering to remove low-quality reads, utilizing a quality score for each sequence and removing bias caused by PCR-induced mutations or high GC content. The filtered datasets were then aligned using a perfect string matching algorithm and adjustments were made to improve alignment quality. Read counts for each sequence were extracted and presented by tissue, at which point all sequences found in the heart were compiled to form the second round of libraries.

[0255] For the second round of libraries, the read counts by tissue were similarly tabulated. Then, a read count of 1 was added to each sequence to remove zero values, all cardiac regions for each sequence were summed, and the read sequences for each codon copy of a given 7-mer amino acid sequence were summed to obtain a single value for each peptide insertion. Finally, the data were normalized to log10 counts per million (Cpm). Enrichment values ​​were calculated using normalized cardiac cpm relative to viral cpm and converted to log10.

[0256] Although preferred embodiments of the present invention have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. Without departing from the present invention, those skilled in the art will now appreciate that many variations, changes and substitutions are possible. It should be understood that each alternative of the embodiments of the present invention described herein can be used to practice the present invention. The appended claims are intended to define the scope of the present invention, and thus cover methods and structures within the scope of these claims and their equivalents.

[0257] 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.

Claims

1. An AAV capsid protein comprising a sequence as provided in Tables 1-2 or Figure 1.

2. An AAV capsid comprising an insert sequence of Formula I: X 1 -G-G-X 2 -X 3 -X 4 -X 5 (I) Where X 1 is an amino acid selected from G, I, K, L and V; X 2 is an amino acid selected from the group consisting of E, G, H, L, N, S and Q; X 3 is an amino acid selected from the group consisting of A, F, I, L, P, T and Y; X 4 is an amino acid selected from the group consisting of F, L, S and Y; and X 5 is an amino acid selected from the group consisting of E, H, I, K, P, S and T.

3. The AAV capsid protein according to claim 2, wherein X 1 It is I, K or L.

4. The AAV capsid protein according to claim 2, wherein X 1 It's K.

5. The AAV capsid protein according to claim 2, wherein X 1 It's I.

6. The AAV capsid protein according to claim 2, wherein X 2 It is L, H or Q.

7. The AAV capsid protein according to claim 2, wherein X 2 It's L.

8. The AAV capsid protein according to claim 2, wherein X 2 It's H.

9. The AAV capsid protein according to claim 2, wherein X 3 It is I, P or Y.

10. The AAV capsid protein according to claim 2, wherein X 4 It is F or L.

11. The AAV capsid protein according to claim 2, wherein X 5 It is I or H.

12. The AAV capsid protein of claim 2, wherein the sequence is selected from KGGLPFI, IGGHYLH, LGGQILI, LGGGFSE, IGGNLLK, KGGETSS, VGGSTYP, and GGGLAST.

13. An AAV capsid protein comprising an insertion sequence of formula II: RSX 6 -X 7 -X 8 -X 9 -X 10 (II) Where X 6 is an amino acid selected from the group consisting of E, G, I, N, Q and T; X 7 is an amino acid selected from L, H, Q, S and T; X 8 is an amino acid selected from D, E, H, P and S; X 9 is an amino acid selected from the group consisting of A, F, L, R, S and T; and X 10 is an amino acid selected from the group consisting of E, I, M, S and V.

14. The AAV capsid protein according to claim 13, wherein X 10 It is I, V or M.

15. The AAV capsid protein according to claim 13, wherein X 10 It’s M.

16. The AAV capsid protein according to claim 13, wherein X 9 It is F or L.

17. The AAV capsid protein according to claim 13, wherein X 8 It is P or H.

18. The AAV capsid protein according to claim 13, wherein X 7 It is H or L.

19. The AAV capsid protein according to claim 13, wherein X 6 It is E or G.

20. The AAV capsid protein of claim 13, wherein the insertion sequence is selected from RSGHPFV, RSGLHLM, RSELHFI, RSQTSTE, RSISDRV, RSTTDAS, and RSNQESV.

21. An AAV capsid protein comprising an insertion sequence of Formula III: R-X 11 -G-X 12 -X 13 -X 14 -X 15 (III) Where X 11 is an amino acid selected from the group consisting of A, D, E, I, N, V ​​and S; X 12 is an amino acid selected from E, H, L, M, S and V; X 13 is an amino acid selected from the group consisting of F, H, L, P, V and Y; X 14 is an amino acid selected from F, L and T; and X 15 is an amino acid selected from the group consisting of D, H, K, L, M, Q, S and V.

22. The AAV capsid protein according to claim 21, wherein X 11 is an amino acid selected from the group consisting of A, D, N and S.

23. The AAV capsid protein according to claim 21, wherein X 12 is an amino acid selected from the group consisting of E, H, L and V.

24. The AAV capsid protein according to claim 21, wherein X 13 is an amino acid selected from the group consisting of F, H, P and Y.

25. The AAV capsid protein according to claim 21, wherein X 14 It is F or L.

26. The AAV capsid protein according to claim 21, wherein X 15 is an amino acid selected from the group consisting of K, L, M and V.

27. The AAV capsid protein of claim 21, wherein the insertion sequence is selected from RSGHPFV, RNGEHFK, RDGHYLL, RNGEFFH, REGMPFL, RAGVHFV, RSGLHLM, RIGVHTS, RIGSVTQ, and RVGSLTD.

28. An AAV capsid protein comprising an insertion sequence of Formula IV: X 16 -X 17 -GHX 18 -X 19 -X 20 (IV) Where X 16 is an amino acid selected from I, R and L; X 17 is an amino acid selected from D, G, Q and S; X 18 is an amino acid selected from I, P and Y; X 19 is an amino acid selected from F or L; and X 20 is an amino acid selected from the group consisting of H, L and V.

29. The AAV capsid protein according to claim 28, wherein X 16 It is L or R.

30. The AAV capsid protein according to claim 28, wherein X 17 is an amino acid selected from the group consisting of P, Q and S.

31. The AAV capsid protein according to claim 28, wherein X 19 It is F or L.

32. The AAV capsid protein according to claim 28, wherein X 20 It's L.

33. The AAV capsid protein according to claim 28, wherein X 20 It's V.

34. The AAV capsid protein according to claim 28, wherein X 18 It is I or Y.

35. The AAV capsid protein of claim 28, wherein the insertion sequence as described in Figure 1 is selected from RSGHPFV, RDGHYLL, LQGHILL and IGGHYLH.

36. An AAV capsid protein comprising an AAV capsid protein comprising an insertion sequence of Formula V: R-X 21 -X 22 -X 23 -H-F-X 24 (V) Where X 21 is an amino acid selected from A, N and S; X 22 is an amino acid selected from D, E and G; X 23 is an amino acid selected from E, L and V; and X 24 is an amino acid selected from the group consisting of I, K, Q and V.

37. The AAV capsid protein according to claim 36, wherein X 21 It is A.

38. The AAV capsid protein according to claim 36, wherein X 22 It is D.

39. The AAV capsid protein according to claim 36, wherein X 23 It's E.

40. The AAV capsid protein according to claim 36, wherein X 23 It's L.

41. The AAV capsid protein according to claim 36, wherein X 24 It's Q.

42. The AAV capsid protein of claim 36, wherein the insertion sequence is selected from RADLHFQ, RAGVHFV, RNGEHFK, and RSELHFI.

43. An AAV capsid protein comprising an AAV capsid protein comprising an insertion sequence of Formula VI: X 25 -X 26 -X 27 -X 28 -X 29 -LL(VI) Where X 25 is an amino acid selected from A, L, R and Y; X 26 is an amino acid selected from the group consisting of Q, E, D, T, K and F; X 27 is an amino acid selected from G, Q and A; X 28 is an amino acid selected from the group consisting of H, G, T, V and D; and X 29 is an amino acid selected from the group consisting of I, S, T and Y.

44. The AAV capsid protein according to claim 43, wherein X 25 It is L or R.

45. The AAV capsid protein according to claim 43, wherein X 26 is an amino acid selected from the group consisting of Q, E and D.

46. ​​The AAV capsid protein according to claim 43, wherein X 27 It is G or Q.

47. The AAV capsid protein according to claim 43, wherein X 28 It is H or G.

48. The AAV capsid protein of claim 43, wherein X 29 It is I or Y.

49. The AAV capsid protein of claim 43, wherein the insertion sequence is selected from the group consisting of LQGHILL, REQGYLL, RDGHYLL, YTGTSLL, AKAVTLL, and AFGDSLL.

50. An AAV capsid protein comprising an insertion sequence of Formula VII: X 30 -X 31 -X 32 -X 33 -X 34 -S-S(VII) Where X 30 is an amino acid selected from the group consisting of A, E, F, H, I, K, L, M, N, P, Q, S, T, V and Y; X 31 is an amino acid selected from the group consisting of F, G, H, I, K, L, M, P, Q, R, S, T and V; X 32 is an amino acid selected from the group consisting of A, G, H, L, M, P, Q, S and V; X 33 is an amino acid selected from the group consisting of E, G, H, I, P, Q, R, S, T and V; and X 34 is an amino acid selected from the group consisting of D, G, I, L, M, N, R, S, T and V.

51. The AAV capsid protein according to claim 50, wherein X 30 It is V, Q or H.

52. The AAV capsid protein of claim 50, wherein X 31 It is F, L or M.

53. The AAV capsid protein according to claim 50, wherein X 32 It is S, A or Q.

54. The AAV capsid protein according to claim 50, wherein X 33 It is G, H or S.

55. The AAV capsid protein according to claim 50, wherein X 34 It is G, V or M.

56. The AAV capsid protein of claim 50, wherein the sequence is selected from the group consisting of VFSGVSS, QLQHGSS, HMASMSS, SSLTLSS, SVPSSSS, ITPSVSS, MTVQVSS, SHLSVSS, YPHQTSS, PQGGSSS, FRSSLSS, ISVSISS, ETQRGSS, IHPPSSS, TSAITSS, FKSQSSS, ASHSTSS, AVMRDSS, KGGETSS, VVSVTSS, LISIRSS, NSLGLSS, and HLAQNSS.

57. An AAV capsid protein comprising an insertion sequence of Formula VIII: DLX 35 -X 36 -X 37 -X 38 -X 39 (VIII) Where X 35 is an amino acid selected from G, H, Q, S and T; X 36 is an amino acid selected from K, N, P, R and S; X 37 is an amino acid selected from H, L, R and S; X 38 is an amino acid selected from the group consisting of D, K, R, S, T and V; and X 39 is an amino acid selected from the group consisting of A, D, E, L and V.

58. The AAV capsid protein of claim 57, wherein X 35 It is H or S.

59. The AAV capsid protein of claim 57, wherein X 36 It is K or R.

60. The AAV capsid protein of claim 57, wherein X 37 It is L or S.

61. The AAV capsid protein of claim 57, wherein X 38 It is S or V.

62. The AAV capsid protein of claim 57, wherein X 39 It is A or L.

63. The AAV capsid protein of claim 57, wherein the sequence is selected from DLSRLSL, DLHKSVA, DLGNLKV, DLTPHKD, DLQRSDL, and DLGSRRE.

64. The AAV capsid protein of any one of claims 1 to 63, wherein the AAV is AAV9.

65. The AAV capsid protein of any one of claims 1 to 64, wherein the AAV is provided in SEQ ID NO:

1.

66. The AAV capsid protein of any one of claims 1 to 65, wherein the insertion is between amino acid 588 and amino acid 589.

67. The AAV capsid protein of any one of claims 1 to 66, wherein 60 copies of the AAV capsid protein are assembled into an AAV capsid.

68. The AAV capsid protein of any one of claims 1 to 67, wherein the AAV capsid protein is present in VP1, VP2, and VP3 of the AAV capsid.

69. The AAV capsid protein of any one of claims 1 to 68, wherein the AAV capsid protein is characterized by at least one of: increased transduction enrichment when measured in the cardiovascular system of a subject when delivered systemically to the subject.

70. The AAV capsid protein of any one of claims 1 to 69, wherein the AAV capsid protein further comprises an amino acid substitution comprising A589N or Q590P.

71. An AAV capsid comprising the AAV capsid protein of any one of claims 1 to 70.

72. The AAV capsid of claim 71, wherein the AAV capsid is chimeric.

73. The AAV capsid of any one of claims 71 to 72, which is isolated and purified.

74. The AAV capsid of any one of claims 71 to 73, formulated as a pharmaceutical formulation for systemic administration to treat a disease or condition of the heart, the pharmaceutical formulation further comprising a pharmaceutically acceptable carrier.

75. The AAV capsid protein of any one of claims 1 to 70, wherein the capsid protein is expressed in the cardiovascular system.

76. The AAV capsid protein of claim 75, wherein the cardiovascular system comprises cardiomyocytes.

77. The AAV capsid of any one of claims 75 to 76, wherein the cardiovascular system comprises a tissue selected from the heart.

78. A recombinant vector comprising a nucleic acid encoding the AAV capsid protein according to any one of claims 1 to 70.

79. A kit comprising: a) a first vector, the first vector comprising the recombinant vector according to claim 78; b) a second vector encoding a helper virus protein; and c) a third vector, said third vector comprising a therapeutic nucleic acid encoding a therapeutic gene expression product.

80. A method of treating a disease or condition in a subject, the method comprising administering a therapeutically effective amount of a pharmaceutical formulation comprising an AAV capsid protein according to any one of claims 1 to 70.

81. The method of claim 80, wherein the disease or the condition is a disease or condition of the subject's heart.

82. A method for preparing recombinant AAV particles from the AAV capsids of any one of claims 1 to 70, the method comprising: a. introducing into the 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, the recombinant viral genome comprising a capsid (Cap) gene, the Cap gene being modified to express the AAV capsid according to any one of claims 1 to 70; as well as 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 that encapsidates the first nucleic acid.

83. The AAV capsid protein of any one of claims 1 to 70, wherein the AAV capsid protein is characterized by increased transduction enrichment when measured in a tissue of a subject when delivered systemically to the subject.

84. The AAV capsid protein of any one of claims 1 to 70, wherein the tissue is heart or myocardial tissue.

85. An AAV particle comprising the AAV capsid protein of any one of claims 1 to 70 and a viral genome.

86. A peptide comprising an amino acid sequence selected from the group consisting of DLSRLSL, KLDPLLM, TPKHLLI, PDQRRTD, RHDPLVM, SHFSGRN, VSAVRDR, VGVPVVL, LLPSNYL, QLQITKE, LQVTSVL and VFSGVSS.

87. A recombinant adeno-associated virus (rAAV) comprising a capsid comprising any one of: KGGLPFI, DLSRLSL, RSGHPFV, RADLHFQ, LQGHILL, KLDPLLM, IGGHYLH, RAGVHFV, LGGQILI, TPKHLLI, RNGEHFK, KDSIPFR, PDQRRTD, RHDPLVM, DNYTRHT, RSGLHLM and TNSVRVV.

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