Improved system for producing rna-packaged aav particles
AssigneeHuidagene Therapeutics (Singapore) Pte. Ltd.
InventorsWeiya Bai, Zhen Liu, Linyu Shi
Life of this patent
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Abstract
Provided herein are helicase, Rep proteins comprising the helicase, and systems and methods using the Rep proteins for producing RNA-packaged AAV particles.
Claims53
A Rep (e.g., Rep78, Rep68, Rep52, Rep40) protein comprising a helicase domain comprising an amino acid mutation relative to a reference helicase domain (e.g., SEQ ID NO:
- 186).
A polynucleotide encoding a Rep (e.g., Rep78, Rep68, Rep52, Rep40) protein comprising a helicase domain comprising an amino acid mutation relative to a reference helicase domain (e.g., SEQ ID NO:
- 186).
A polynucleotide encoding a Rep78 protein, a Rep68 protein, a Rep52 protein, and a Rep40 protein, wherein the Rep78 protein, the Rep68 protein, the Rep52 protein, and the Rep40 protein share a helicase domain comprising an amino acid mutation relative to a reference helicase domain (e.g., SEQ ID NO:
- 186).
A helicase comprising a helicase domain comprising an amino acid mutation relative to a reference helicase domain (e.g., SEQ ID NO:
- 186).
A polynucleotide encoding a helicase comprising a helicase domain comprising an amino acid mutation relative to a reference helicase domain (e.g., SEQ ID NO:
- 186).
A system for packaging an RNA into an AAV capsid to produce a recombinant RNA-packaged AAV particle (rRAAV particle),
- wherein the RNA comprises:
- (a) an RNA sequence of interest (RSI), e.g., an RNA sequence encoding a protein of interest, and
- (b) an RNA-packaging signal (RPS) capable of interacting, e.g., binding, directly or indirectly, with an RPS-interacting molecule that facilitates packaging of the RNA into the AAV capsid;
- wherein the system comprises:
- (1) one or more capsid proteins (e.g., VP1, VP2, and/or VP3) for assembling the AAV capsid, or one or more coding sequences therefor, or a polynucleotide comprising said coding sequences;
- (2) one or more Rep proteins (e.g., Rep78, Rep68, Rep52, Rep40) comprising a helicase domain comprising an amino acid mutation relative to a reference helicase domain (e.g., SEQ ID NO:
- 186), or one or more coding sequences therefor, or a polynucleotide comprising said coding sequences;
- (3) the RPS-interacting molecule, or a coding sequence therefor, or a polynucleotide comprising said coding sequence;
- (4) the RNA, or a coding sequence therefor, or a polynucleotide comprising said coding sequence, e.g., a transgene vector comprising or encoding the RNA;
- and
- (5) optionally, one or more helper proteins required for AAV packaging (e.g., helper proteins from adenoviral E2a, E4, and/or VA genes), or one or more coding sequences therefor, or a polynucleotide comprising said coding sequences.
A method for the production of a recombinant RNA-packaged AAV particle (rRAAV particle), said method comprising:
- a) culturing for a sufficient time a cell comprising a system for packaging an RNA into a AAV capsid to produce the recombinant RNA-packaged AAV particle (rRAAV particle), and
- b) harvesting the rRAAV particle or a population thereof;
- wherein the RNA comprises:
- (a) an RNA sequence of interest (RSI), e.g., an RNA sequence encoding a protein of interest, and
- (b) an RNA-packaging signal (RPS) capable of interacting, e.g., binding, directly or indirectly, with an RPS-interacting molecule that facilitates packaging of the RNA into the AAV capsid;
- wherein the system comprises:
- (1) one or more capsid proteins (e.g., VP1, VP2, and/or VP3) for assembling the AAV capsid, or one or more coding sequences therefor, or a polynucleotide comprising said coding sequences;
- (2) one or more Rep proteins (e.g., Rep78, Rep68, Rep52, Rep40) comprising a helicase domain comprising an amino acid mutation relative to a reference helicase domain (e.g., SEQ ID NO:
- 186), or one or more coding sequences therefor, or a polynucleotide comprising said coding sequences;
- (3) the RPS-interacting molecule, or a coding sequence therefor, or a polynucleotide comprising said coding sequence;
- (4) the RNA, or a coding sequence therefor, or a polynucleotide comprising said coding sequence, e.g., a transgene vector comprising or encoding the RNA;
- and
- (5) optionally, one or more helper proteins required for AAV packaging (e.g., helper proteins from adenoviral E2a, E4, and/or VA genes), or one or more coding sequences therefor, or a polynucleotide comprising said coding sequences.
Use of a Rep protein (e.g., Rep78, Rep68, Rep52, Rep40) comprising a helicase domain comprising an amino acid mutation relative to a reference helicase domain (e.g., SEQ ID NO:
- 186), or a polynucleotide encoding the Rep protein, in the production of a recombinant RNA-packaged AAV particle (rRAAV particle), said production comprising:
- a) culturing for a sufficient time a cell comprising a system for packaging an RNA into a AAV capsid to produce the recombinant RNA-packaged AAV particle (rRAAV particle), and
- b) harvesting the rRAAV particle or a population thereof;
- wherein the RNA comprises:
- (a) an RNA sequence of interest (RSI), e.g., an RNA sequence encoding a protein of interest, and
- (b) an RNA-packaging signal (RPS) capable of interacting, e.g., binding, directly or indirectly, with an RPS-interacting molecule that facilitates packaging of the RNA into the AAV capsid;
- wherein the system comprises:
- (1) one or more capsid proteins (e.g., VP1, VP2, and/or VP3) for assembling the AAV capsid, or one or more coding sequences therefor, or a polynucleotide comprising said coding sequences;
- (2) one or more said Rep proteins (e.g., Rep78, Rep68, Rep52, Rep40), or one or more coding sequences therefor, or a polynucleotide comprising said coding sequences;
- (3) the RPS-interacting molecule, or a coding sequence therefor, or a polynucleotide comprising said coding sequence;
- (4) the RNA, or a coding sequence therefor, or a polynucleotide comprising said coding sequence, e.g., a transgene vector comprising or encoding the RNA;
- and
- (5) optionally, one or more helper proteins required for AAV packaging (e.g., helper proteins from adenoviral E2a, E4, and/or VA genes), or one or more coding sequences therefor, or a polynucleotide comprising said coding sequences.
The Rep protein, the helicase, the polynucleotide, the system, the method, or the use of any preceding claim, wherein the amino acid mutation leads to an increased RNA unwinding property and/or a decreased DNA unwinding property of the helicase or the Rep protein comprising the amino acid mutation.
The Rep protein, the helicase, the polynucleotide, the system, the method, or the use of any preceding claim, wherein the amino acid mutation leads to increased RNA packaging efficiency and/or decreased DNA packaging efficiency.
The Rep protein, the helicase, the polynucleotide, the system, the method, or the use of any preceding claim, wherein the reference helicase domain is the helicase domain of a reference helicase, e.g., a wild type helicase.
The Rep protein, the helicase, the polynucleotide, the system, the method, or the use of any preceding claim, wherein the reference helicase is a superfamily 3 (SF3) helicase.
The Rep protein, the helicase, the polynucleotide, the system, the method, or the use of any preceding claim, wherein the reference helicase is a helicase capable of unwinding DNA.
The Rep protein, the helicase, the polynucleotide, the system, the method, or the use of any preceding claim, wherein the reference helicase is a superfamily 3 (SF3) helicase capable of unwinding DNA.
The Rep protein, the helicase, the polynucleotide, the system, the method, or the use of any preceding claim, wherein the reference helicase domain is the helicase domain of a reference Rep protein, e.g., a wild type Rep protein.
The Rep protein, the helicase, the polynucleotide, the system, the method, or the use of any preceding claim, wherein the reference Rep protein is a reference Rep78 protein, a reference Rep68 protein, a reference Rep52 protein, or a reference Rep40 protein.
The Rep protein, the helicase, the polynucleotide, the system, the method, or the use of any preceding claim, wherein the reference helicase domain is the reference helicase domain shared by a reference Rep78 protein, a reference Rep68 protein, a reference Rep52 protein, and a reference Rep40 protein of a same AAV virus.
The Rep protein, the helicase, the polynucleotide, the system, the method, or the use of any preceding claim, wherein the reference Rep protein, the reference Rep78 protein, the reference Rep68 protein, the reference Rep52 protein, and the reference Rep40 protein are from a wild type AAV virus.
The Rep protein, the helicase, the polynucleotide, the system, the method, or the use of any preceding claim, wherein the wild type AAV virus has a serotype selected from the group consisting of AAV1, AAV2, AAV3A, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAVrh74, AAVrh10, AAV-DJ, AAV.PHP.eB, Anc80L65, Anc80L65AAP, and 7m8.
The Rep protein, the helicase, the polynucleotide, the system, the method, or the use of any preceding claim, wherein the reference helicase domain (e.g., SEQ ID NO:
- 186) comprises, from N- to C-terminus, Motif A, Motif B, Motif B′, Motif C, and Arginine Finger (R finger).
The Rep protein, the helicase, the polynucleotide, the system, the method, or the use of any preceding claim, wherein the reference Rep78 protein comprises, consists essentially of, or consists an amino acid sequence having a sequence identity of at least about 30% (e.g., at least about 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100%) to the amino acid sequence of any one of SEQ ID NOs:
- 88-109;
- wherein the reference Rep68 protein comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO:
- 284 or a corresponding amino acid sequence comprised in any one of SEQ ID NOs:
- 89-109;
- wherein the reference Rep52 protein comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO:
- 285 or a corresponding amino acid sequence comprised in any one of SEQ ID NOs:
- 89-109;
- or wherein the reference Rep40 protein comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO:
- 286 or a corresponding amino acid sequence comprised in any one of SEQ ID NOs:
- 89-109.
The Rep protein, the helicase, the polynucleotide, the system, the method, or the use of any preceding claim, wherein the reference helicase domain comprises, consists essentially of, or consists an amino acid sequence having a sequence identity of at least about 30% (e.g., at least about 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100%) to the amino acid sequence of any one of SEQ ID NOs:
- 186-207.
The Rep protein, the helicase, the polynucleotide, the system, the method, or the use of any preceding claim, wherein the amino acid mutation is at a position corresponding to one or more positions of position 308 through position 463, optionally position 325 through position 461, of the amino acid sequence of any one of SEQ ID NOs:
- 186-207, wherein the position is numbered according to SEQ ID NO:
- 88.
The Rep protein, the helicase, the polynucleotide, the system, the method, or the use of any preceding claim, wherein the amino acid mutation is at a position corresponding to the position of a conserved amino acid (e.g., a position corresponding to A344 of SEQ ID NO:
- 88) across at least 80%, at least 90%, or 100% of the Rep proteins (e.g., SEQ ID NOs:
- 88-109) of ssDNA viruses.
The Rep protein, the helicase, the polynucleotide, the system, the method, or the use of any preceding claim, wherein the amino acid mutation is at a position corresponding to a position in one or more of Motif A, Motif B, Motif B′, Motif C of the reference helicase domain, a upstream region no more than about 30, 25, 20, 15, 10, or 5 amino acids from the N-terminal of any one of Motif A, Motif B, Motif B′, Motif C, and Arginine Finger (R finger) of the reference helicase domain, and a downstream region no more than about 30, 25, 20, 15, 10, or 5 amino acids from the C-terminal of any one of Motif A, Motif B, Motif B′, Motif C, and Arginine Finger (R finger) of the reference helicase domain.
The Rep protein, the helicase, the polynucleotide, the system, the method, or the use of any preceding claim, wherein the Rep78 protein comprising said amino acid mutation comprises, consists essentially of, or consists an amino acid sequence having a sequence identity of at least about 30% (e.g., at least about 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9%) and less than 100% to the amino acid sequence of any one of SEQ ID NOs:
- 88-109.
The Rep protein, the helicase, the polynucleotide, the system, the method, or the use of any preceding claim, wherein the helicase domain comprising said amino acid mutation comprises, consists essentially of, or consists an amino acid sequence having a sequence identity of at least about 30% (e.g., at least about 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9%) and less than 100% to the amino acid sequence of any one of SEQ ID NOs:
- 186-207.
The Rep protein, the helicase, the polynucleotide, the system, the method, or the use of any preceding claim, wherein the amino acid mutation comprises a mutation at a position corresponding to G325, R327, W331, A336, T337, I343, A344, D371, K372, M373, I375, E378, C405, T419, S420, T422, C425, Q442, D443, M445, K447, E449, L450, T451, L454, D455, H456, D457, F458, and/or V461 of the amino acid sequence of SEQ ID NO:
- 186, wherein the position is numbered according to SEQ ID NO:
- 88.
The Rep protein, the helicase, the polynucleotide, the system, the method, or the use of any preceding claim, wherein the amino acid mutation comprises a mutation at a position corresponding to A336, T337, I343, A344, K372, E378, D443, M445, K447, E449, L450, T451, L454, D455, H456, D457, F458, and/or V461 of the amino acid sequence of SEQ ID NO:
- 186, wherein the position is numbered according to SEQ ID NO:
- 88.
The Rep protein, the helicase, the polynucleotide, the system, the method, or the use of any preceding claim, wherein the amino acid mutation comprises a substitution corresponding to a substitution selected from the group consisting of G325P, G325I, K326E, K326R, R327P, N328V, W331I, F333H, F333Y, F333K, P335S, A336P, A336S, A336R, T337G, T338G, T341S, N342I, I343T, I343A, I343L, A344T, A344V, A344S, E345N, A346F, H349K, P352T, P365Y, N367D, D368G, C369Y, V370K, D371Q, D371G, D371N, K372Q, K372E, K372N, M373S, M373E, M373A, I375V, W376I, W377M, E378D, E379D, G380L, G380F, C405H, K406R, T419S, T419F, T419I, T419L, S420A, S420M, S420C, N421T, N421S, T422H, T422S, M424N, C425I, Q442K, Q442F, Q442H, Q442R, Q442L, Q442V, D443S, D443R, D443Y, D443N, D443A, M445I, M445R, M445F, F446R, F446I, K447F, K447N, K447H, K447P, K447T, K447A, E449D, E449I, E449R, L450M, L450I, L450V, T451D, T451I, T451E, T451K, T451N, L454F, L454V, D455F, D455K, D455H, D455Y, D455T, D455M, H456F, H456D, H456S, D457E, D457S, D457F, F458Y, F458K, V461L, and a combination thereof, wherein the position is numbered according to SEQ ID NO:
- 88.
The Rep protein, the helicase, the polynucleotide, the system, the method, or the use of any preceding claim, wherein the amino acid mutation comprises a substitution corresponding to a substitution selected from the group consisting of G325P, R327P, W331I, A336P, A336S, A336R, T337G, I343T, I343A, I343L, A344T, A344V, D371Q, K372Q, K372E, K372N, M373S, I375V, E378D, C405H, T419S, S420A, T422H, T422S, C425I, Q442H, Q442R, D443S, D443Y, D443N, D443A, M445I, K447F, K447N, K447T, E449D, L450M, L450I, L450V, T451D, T451E, L454F, D455F, D455Y, D455T, D455M, H456D, H456S, D457E, D457S, D457F, F458Y, V461L, and a combination thereof, wherein the position is numbered according to SEQ ID NO:
- 88.
The Rep protein, the helicase, the polynucleotide, the system, the method, or the use of any preceding claim, wherein the amino acid mutation comprises a substitution corresponding to a substitution selected from the group consisting of A336P, T337G, I343T, A344T, A344V, K372Q, E378D, D443S, M445I, K447F, E449D, L450M, T451D, L454F, D455F, D455T, H456D, D457E, F458Y, V461L, and a combination thereof, wherein the position is numbered according to SEQ ID NO:
- 88.
The Rep protein, the helicase, the polynucleotide, the system, the method, or the use of any preceding claim, wherein the amino acid mutation comprises or consists of a combination substitution corresponding a combination substitution selected from the group consisting of A336P+T337G, K372Q+E378D, D443S+M445I, D443S+L454F+D455F, K447F+E449D+T451D, K447F+L450M, K447F+F458Y, K447F+H456D+F458Y, K447F+V461L, E449D+L450M, L450M+T451D, L454F+D455F, D455T+H456D+D457E+F458Y, H456D+D457E+F458Y, F458Y+V461L, A344T+K372Q, A336P+A344T+K447F, A336P+A344V+K447F, I343T+K447F, I343T+L450M, A344T+K447F, A344V+K447F, A344T+L450M, A344V+L450M, A344T+K447F+E449D+T451D, K372Q+K447F, K372Q+L450M, K372Q++K447F+E449D+T451D, K372Q+V461L, E378D+K447F, E378D+L450M, E378D++K447F+E449D+T451D, A344T+K372Q+K447F, A344V+K372Q+K447F, and a combination thereof, wherein the position is numbered according to SEQ ID NO:
- 88.
The Rep protein, the helicase, the polynucleotide, the system, the method, or the use of any preceding claim, wherein the amino acid mutation comprises or consists of a combination substitution corresponding a combination substitution of A344V and K447F, wherein the position is numbered according to SEQ ID NO:
- 88.
The Rep protein, the helicase, the polynucleotide, the system, the method, or the use of any preceding claim, wherein the helicase domain comprising said amino acid mutation comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO:
- 287.
The Rep protein, the helicase, the polynucleotide, the system, the method, or the use of any preceding claim, wherein the Rep protein comprising said amino acid mutation comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO:
- 288.
The Rep protein, the helicase, the polynucleotide, the system, the method, or the use of any preceding claim, wherein the Rep protein comprise a mutation partially or substantially abolishing the endonuclease activity of the Rep protein, e.g., in the Original Binding Domain (OBD) of the Rep protein, optionally, the mutation comprises or consists of a mutation corresponding to a Y156F mutation, a K146A+D149A+E150A mutation (KDE-mu), or an E83A+K84A+E86A mutation (EKE-mu), wherein the position is numbered according to SEQ ID NO:
- 88.
The Rep protein, the helicase, the polynucleotide, the system, the method, or the use of any preceding claim, wherein Rep protein comprises a combination substitution comprising or consisting of Y156F, A344V, and K447F, wherein the position is numbered according to SEQ ID NO:
- 88.
The Rep protein, the helicase, the polynucleotide, the system, the method, or the use of any preceding claim, wherein the Rep protein comprise the amino acid sequence of SEQ ID NO:
- 289.
The Rep protein, the helicase, the polynucleotide, the system, the method, or the use of any preceding claim, wherein the RPS-interacting molecule is said Rep protein.
The Rep protein, the helicase, the polynucleotide, the system, the method, or the use of any preceding claim, wherein the RPS-interacting molecule comprises an RPS-binding protein (RPSBP) capable of binding directly or indirectly to the RNA packaging signal (RPS).
The Rep protein, the helicase, the polynucleotide, the system, the method, or the use of any preceding claim, wherein the Rep protein is (e.g., N-terminally, C-terminally, internally) fused with the RPSBP, optionally, via a peptide linker.
The Rep protein, the helicase, the polynucleotide, the system, the method, or the use of any preceding claim, wherein the RPS is located at or near the 5′ end of the RSI, at or near the 3′ end of the RSI, or internal to the RSI.
The Rep protein, the helicase, the polynucleotide, the system, the method, or the use of any preceding claim, wherein the RNA comprises one, two, or three copies of the RPS.
The Rep protein, the helicase, the polynucleotide, the system, the method, or the use of any preceding claim, wherein the RPS comprises an MS2 sequence (e.g., SEQ ID NO:
- 54), an PP7 binding site (e.g., SEQ ID NO:
- 56), and/or a Com binding site (e.g., SEQ ID NO:
- 58).
The Rep protein, the helicase, the polynucleotide, the system, the method, or the use of any preceding claim, wherein (a) the RPS comprises an MS2 sequence (e.g., SEQ ID NO:
- 54), and the RPSBP comprises a bacteriophage-derived MS2 coat protein (MCP) (e.g., SEQ ID NO:
- 49);
- (b) the RPS comprises an PP7 binding site (e.g., SEQ ID NO:
- 56), and the RPSBP comprises a PP7 bacteriophage coat protein (PCP) (e.g., SEQ ID NO:
- 51), or (c) the RPS comprises a Com binding site (e.g., SEQ ID NO:
- 58), and the RPSBP comprises a phage COM protein (COM) (e.g., SEQ ID NO:
- 53).
The Rep protein, the helicase, the polynucleotide, the system, the method, or the use of any preceding claim, wherein the RNA, or a coding sequence therefor, or a polynucleotide comprising said coding sequence, e.g., a transgene vector comprising or encoding the RNA, lacks a functional DNA packaging signal, e.g., an AAV ITR (such as, 5′ AAV2 ITR and/or 3′ AAV2 ITR), or a coding sequence therefor.
The Rep protein, the helicase, the polynucleotide, the system, the method, or the use of any preceding claim, wherein the RNA is transcribed from a polynucleotide (e.g., a transgene plasmid) lacking a functional DNA packaging signal, e.g., an AAV ITR (such as, 5′ AAV2 ITR and/or 3′ AAV2 ITR), or a coding sequence therefor.
The Rep protein, the helicase, the polynucleotide, the system, the method, or the use of any preceding claim, wherein the AAV capsid comprises a capsid from an AAV having a serotype selected from the group consisting of AAV1, AAV2, AAV3A, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAVrh74, AAVrh10, AAV-DJ, AAV.PHP.eB. Anc80L65, Anc80L65AAP, and 7m8.
The Rep protein, the helicase, the polynucleotide, the system, the method, or the use of any preceding claim, wherein the RNA is not bound to the AAV capsid.
A vector comprising the polynucleotide of any preceding claim;
- optionally, wherein the vector is a plasmid.
A (isolated) (host) cell comprising the Rep protein, the helicase, the polynucleotide, the system, or the vector of any preceding claim.
A recombinant RNA-packaged AAV particle (rRAAV particle) or a population thereof produced by the method of any preceding claim.
Dependencies are inferred from the claim text.