Patent Application
20230093183
This application claims priority to Australian Provisional Application No. 2020900529 entitled “Adeno-associated virus capsid polypeptides and vectors”, filed on 25 Feb. 2020, the entire content of which is hereby incorporated herein by reference in its entirety.
The present disclosure relates generally to adeno-associated virus (AAV) capsid polypeptides and encoding nucleic acid molecules. The disclosure also relates to AAV vectors comprising the capsid polypeptides, and nucleic acid vectors (e.g. plasmids) comprising the encoding nucleic acids molecules, as well as to host cells comprising the vectors. The disclosure also relates to methods and uses of the polypeptides, encoding nucleic acids molecules, vectors and host cells.
Gene therapy has most commonly been investigated and achieved using viral vectors, with notable recent advances being based on adeno-associated viral vectors. Adeno-associated virus (AAV) is a replication-deficient parvovirus, the single-stranded DNA genome of which is about 4.7 kb in length. The AAV genome includes inverted terminal repeat (ITRs) at both ends of the molecule, flanking two open reading frames: rep and cap. The cap gene encodes three structural capsid proteins: VP1, VP2 and VP3. The three capsid proteins typically assemble in a ratio of 1:1:8-10 to form the AAV capsid, although AAV capsids containing only VP3, or VP1 and VP3, or VP2 and VP3, have been produced. The cap gene also encodes the assembly activating protein (AAP) from an alternative open reading frame. AAP promotes capsid assembly, acting to target the capsid proteins to the nucleolus and promote capsid formation. The rep gene encodes four known regulatory proteins: Rep78, Rep68, Rep52 and Rep40. These Rep proteins are involved in AAV genome replication, packaging, genomic integration and other processes. More recently, an X gene has been identified in the 3′ end of the AAV2 genome (Cao et al. PLoS One, 2014, 9:e104596). The encoded X protein appears to be involved in the AAV life cycle, including DNA replication.
The ITRs are involved in several functions, in particular integration of the AAV DNA into the host cell genome, as well as genome replication and packaging. When AAV infects a host cell, the viral genome can integrate into the host's chromosomal DNA resulting in latent infection of the cell. Thus, AAV can be exploited to introduce heterologous sequences into cells. In nature, a helper virus (for example, adenovirus or herpesvirus) provides protein factors that allow for replication of AAV virus in the infected cell and packaging of new virions. In the case of adenovirus, genes E1A, E16, E2A, E4 and VA provide helper functions. Upon infection with a helper virus, the AAV provirus is rescued and amplified, and both AAV and the helper virus are produced.
AAV vectors (also referred to as recombinant AAV, rAAV) that contain a genome that lacks some, most or all of the native AAV genome and instead contain one or more heterologous sequences flanked by the ITRs, have been successfully used in gene therapy settings. These AAV vectors are widely used to deliver heterologous nucleic acid to cells of a subject for therapeutic purposes, and in many instances, it is the expression of the heterologous nucleic acid that imparts the therapeutic effect. Although several AAV vectors have now been used in the clinic, there are a limited number that exhibit the required in vivo transduction efficiency of primary human cells/tissues to facilitate adequate expression of the heterologous nucleic acid for therapeutic applications. There is therefore a need to develop alternative AAV vectors that contain capsid proteins that facilitate efficient transduction of host cells in vivo.
The present disclosure is predicated in part on the generation of novel AAV capsid polypeptides. In particular embodiments, the capsid polypeptides facilitate efficient transduction of human cells (such as human hepatocytes) when contained in an AAV vector. Typically, the in vivo transduction of AAV vectors comprising a capsid polypeptide of the present disclosure is improved compared to AAV vectors comprising other AAV capsid polypeptides (e.g. the prototypic AAV2 capsid set forth in SEQ ID NO:1). The capsids polypeptides of the present disclosure are therefore particularly useful in preparing AAV vectors, and in particular, AAV vectors for gene therapy uses. Similarly, AAV vectors comprising a capsid polypeptide of the present disclosure (i.e. having a capsid comprising or consisting of a capsid polypeptide of the present disclosure) are of particular use in gene therapy applications, such as for delivery of heterologous nucleic acids for the treatment of various diseases and conditions.
In one aspect, the disclosure provides a capsid polypeptide, comprising: (i) the sequence of amino acids set forth in any one of SEQ ID Nos:2-20 and 65-79 or a sequence having at least or about 90% or 95% sequence identity thereto; (ii) the sequence of amino acids at positions 138-735 of any one of SEQ ID NOs:2, 6, 7, 9, 10, 12-14, 16-20, 69, 71-74, 76 and 78, positions 138-734 of SEQ ID NO:5, 8 or 11, positions 138-736 of any one of SEQ ID NOs:3, 15, 65, 68, 75, 77 and 79, positions 138-737 of any one of SEQ ID NOs:4, 67 and 70, or positions 138-738 of SEQ ID NO:66; or a sequence having at least or about 90% or 95% sequence identity thereto; and/or (iii) the sequence of amino acids at positions 203-734 of any one of SEQ ID NOs:5, 8 and 11, positions 203-736 of SEQ ID NO:15, positions 204-735 of any one of SEQ ID NOs:2, 6, 7, 9, 10, 12-14, 16-20, 69, 71-74, 76 and 78, positions 204-736 of any one of SEQ ID NOs:3, 65, 68, 75, 77 and 79, positions 204-737 of any one of SEQ ID NOs: 4, 67 and 70, or positions 204-738 of SEQ ID NO:66; or a sequence having at least or about 90% or 95% sequence identity thereto.
In one embodiment, the capsid polypeptide comprises (i) the sequence of amino acids set forth in SEQ ID NO:13 or a sequence having at least or about 90%, 95%, 96%, 97%, 98% or 99% sequence identity thereto; (ii) the sequence of amino acids at positions 138-735 of SEQ ID NO:13 or a sequence having at least or about 90%, 95%, 96%, 97%, 98% or 99% sequence identity thereto; and/or (iii) the sequence of amino acids at positions 204-735 of SEQ ID NO:13 or a sequence having at least or about 90%, 95%, 96%, 97%, 98% or 99% sequence identity thereto.
In a particular examples, the capsid polypeptide comprises one or more of: a) amino acid residues S263, Q264, S265, S268 and H272, with numbering relative to SEQ ID NO:13; b) amino acid residues T546, G547, T549, N550, K551, T552, T553, L554, E555, N556, L558, M559, N561, R566 and P567, with numbering relative to SEQ ID NO:13; c) amino acid residues S580, S581, A585, A586, A590, T592, Q593, V594, and N597, with numbering relative to SEQ ID NO:13; d) amino acid residues D532, S538 and V540, with numbering relative to SEQ ID NO:13; e) amino acid residues S451, Q456, G457, Q460, L462, A466, A469, N470, S472 and A473, with numbering relative to SEQ ID NO:13; f) amino acid residues L493, S494, G505, A506, V518 and V522, with numbering relative to SEQ ID NO:13; g) the sequence of amino acids SQSGASNDNH (SEQ ID NO:58) at positions 263-272, with numbering relative to SEQ ID NO:13; h) the sequence of amino acids TGATNKTTLENVLMTNEEEIRP (SEQ ID NO:59) at positions 546-567, with numbering relative to SEQ ID NO:13; i) the sequence of amino acids SSNLQAANTAAQTQVVNN (SEQ ID NO:60) at positions 582-597, with numbering relative to SEQ ID NO:13; j) the sequence of amino acids DRFFPSSGV (SEQ ID NO:61) at positions 532-540, with numbering relative to SEQ ID NO:13; k) the sequence of amino acids STGGTQGTQQLLFSQAGPANMSA (SEQ ID NO:62) at positions 451-473, with numbering relative to SEQ ID NO:13; and/or l) the sequence of amino acids LSQNNNSNFAWTGATKYHLNGRNSLVNPGV (SEQ ID NO:63) at positions 493-522, with numbering relative to SEQ ID NO:13.
Another aspect of the disclosure relates to a capsid polypeptide, comprising: (i) the sequence of amino acids set forth in SEQ ID NO:13 or a sequence having at least or about 85% sequence identity thereto; (ii) the sequence of amino acids at positions 138-735 of SEQ ID NO:13 or a sequence having at least or about 85% sequence identity thereto; and/or (iii) the sequence of amino acids at positions 204-735 of SEQ ID NO:13 or a sequence having at least or about 85% sequence identity thereto; wherein the capsid polypeptide comprises: a) amino acid residues S263, Q264, S265, S268 and H272, with numbering relative to SEQ ID NO:13; and b) amino acid residues T546, G547, T549, N550, K551, T552, T553, L554, E555, N556, L558, M559, N561, R566 and P567, with numbering relative to SEQ ID NO:13; and/or amino acid residues S580, S581, A585, A586, A590, T592, Q593, V594, and N597, with numbering relative to SEQ ID NO:13.
In some embodiments, the capsid polypeptide comprises a) the sequence of amino acids SQSGASNDNH (SEQ ID NO:58) at positions 263-272, with numbering relative to SEQ ID NO:13; and b) the sequence of amino acids TGATNKTTLENVLMTNEEEIRP (SEQ ID NO:59) at positions 546-567, with numbering relative to SEQ ID NO:13 and/or the sequence of amino acids SSNLQAANTAAQTQVVNN (SEQ ID NO:60) at positions 582-597, with numbering relative to SEQ ID NO:13. In further embodiments, the capsid polypeptide comprises a) the sequence of amino acids ISSQSGASNDNH (SEQ ID NO:80) at positions 261-272, with numbering relative to SEQ ID NO:13; and b) the sequence of amino acids KTGATNKTTLENVLMTNEEEIRP (SEQ ID NO:81) at positions 545-567, with numbering relative to SEQ ID NO:13 and/or the sequence of amino acids SSNLQAANTAAQTQVVNN (SEQ ID NO:60) at positions 582-597, with numbering relative to SEQ ID NO:13.
The capsid polypeptide may comprise amino acid residues D532, S538 and V540, with numbering relative to SEQ ID NO:13. In some embodiments, the capsid polypeptide comprises the sequence of amino acids DRFFPSSGV (SEQ ID NO:61) at positions 532-540, with numbering relative to SEQ ID NO:13. In further embodiments, the capsid polypeptide comprises the sequence of amino acids AMATHKDDEDRFFPSSGV (SEQ ID NO:82) at positions 523-540, with numbering relative to SEQ ID NO:13.
In some examples, the capsid polypeptide comprises amino acid residues S451, Q456, G457, Q460, L462, A466, A469, N470, S472 and A473, with numbering relative to SEQ ID NO:13. In one embodiment, the capsid polypeptide comprises the sequence of amino acids STGGTQGTQQLLFSQAGPANMSA (SEQ ID NO:62) at positions 451-473, with numbering relative to SEQ ID NO:13. In further embodiments, the capsid polypeptide comprises the sequence of amino acids QSTGGTQGTQQLLFSQAGPANMSA (SEQ ID NO:83) at positions 450-473, with numbering relative to SEQ ID NO:13.
In further examples, the capsid polypeptide comprises amino acid residues L493, S494, G505, A506, V518 and V522, with numbering relative to SEQ ID NO:13. In some embodiments, the capsid polypeptide comprises the sequence of amino acids LSQNNNSNFAWTGATKYHLNGRNSLVNPGV (SEQ ID NO:63) at positions 493-522, with numbering relative to SEQ ID NO:13. In further embodiments, the capsid polypeptide comprises the sequence of amino acids RVSTTLSQNNNSNFAWTGATKYHLNGRNSLVNPGV (SEQ ID NO:84) at positions 488-522, with numbering relative to SEQ ID NO:13.
In another aspect, the disclosure provides a capsid polypeptide, comprising: (i) the sequence of amino acids set forth in SEQ ID NO:13 or a sequence having at least or about 85% sequence identity thereto; (ii) the sequence of amino acids at positions 138-735 of SEQ ID NO:13 or a sequence having at least or about 85% sequence identity thereto; and/or (iii) the sequence of amino acids at positions 204-735 of SEQ ID NO:13 or a sequence having at least or about 85% sequence identity thereto; wherein the capsid polypeptide comprises amino acid residues S451, Q456, G457, Q460, L462, A466, A469, N470, S472, A473, L493, S494, G505, A506, V518 V522, D532, S538 V540, T546, G547, T549, N550, K551, T552, T553, L554, E555, N556, L558, M559, N561, R566, P567, S580, S581, A585, A586, A590, T592, Q593, V594, and N597, with numbering relative to SEQ ID NO:13.
In some embodiments, the capsid polypeptide comprises the sequence of amino acids STGGTQGTQQLLFSQAGPANMSA (SEQ ID NO:62) at positions 451-473; the sequence of amino acids LSQNNNSNFAWTGATKYHLNGRNSLVNPGV (SEQ ID NO:63) at positions 493-522; the sequence of amino acids DRFFPSSGV (SEQ ID NO:61) at positions 532-540; the sequence of amino acids TGATNKTTLENVLMTNEEEIRP (SEQ ID NO:59) at positions 546-567; and the sequence of amino acids SSNLQAANTAAQTQVVNN (SEQ ID NO:60) at positions 582-597, with numbering relative to SEQ ID NO:13. In further embodiments, the capsid polypeptide comprises the sequence of amino acids QSTGGTQGTQQLLFSQAGPANMSA (SEQ ID NO:83) at positions 450-473; the sequence of amino acids RVSTTLSQNNNSNFAWTGATKYHLNGRNSLVNPGV (SEQ ID NO:84) at positions 488-522; the sequence of amino acids AMATHKDDEDRFFPSSGV (SEQ ID NO:82) at positions 523-540; the sequence of amino acids KTGATNKTTLENVLMTNEEEIRP (SEQ ID NO:81) at positions 545-567, with numbering relative to SEQ ID NO:13; and the sequence of amino acids SSNLQAANTAAQTQVVNN (SEQ ID NO:60) at positions 582-597, with numbering relative to SEQ ID NO:13. In one example, the capsid polypeptide further comprises a) an insertion of NG after position 262 and residues T263, S264, G265, T268, and T272, with numbering relative to SEQ ID NO:13; or b) an insertion of NG after position 262 and the sequence of amino acids TSGGATNDNT at positions 263-272, with numbering relative to SEQ ID NO:13.
In one embodiment, the capsid polypeptide comprises at least or about 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96% or 97% sequence identity to the sequence of amino acids set forth in SEQ ID NO:13, the sequence of amino acids at positions 138-735 of SEQ ID NO:13, or the sequence of amino acids at positions 204-735 of SEQ ID NO:13.
In another aspect, the disclosure provides an AAV vector, comprising a capsid polypeptide described herein.
In some examples, the vector exhibits increased in vivo transduction efficiency compared to an AAV vector comprising a capsid polypeptide comprising the sequence of amino acids set forth in SEQ ID NO:1. In particular examples, the vector exhibits increased in vivo transduction efficiency of human hepatocytes compared to an AAV vector comprising a capsid polypeptide comprising the sequence of amino acids set forth in SEQ ID NO:1. In one embodiment, transduction efficiency is increased by at least or about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400% or 500%.
In further examples, the AAV vector exhibits increased resistance to neutralization by pooled human immunoglobulins compared to an AAV vector comprising a capsid polypeptide comprising the sequence of amino acids set forth in SEQ ID NO:1. In one embodiment, resistance to neutralization is increased by at least or about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400% or 500%.
The AAV vector of the present disclosure may further include a heterologous coding sequence, such as one that encodes a peptide, polypeptide or polynucleotide. In some examples, the peptide, polypeptide or polynucleotide is a therapeutic peptide, polypeptide or polynucleotide.
In further aspects, provided is an isolated nucleic acid molecule encoding a capsid polypeptide described herein, and a vector comprising the nucleic acid molecule. In some examples, the vector is selected from among a plasmid, cosmid, phage and transposon. A host cell comprising an AAV vector, a nucleic acid molecule or a vector described above and herein is also provided.
Also provided is a method for introducing a heterologous coding sequence into a host cell, comprising contacting a host cell with an AAV vector of the present disclosure that comprises a heterologous coding sequence. In some examples, the host cell is a hepatocyte. In some embodiments of the method, contacting a host cell with the AAV vector comprises administering the AAV vector to a subject. In other embodiments, the method is in vitro or ex vivo.
In another aspect, provided is a method for producing an AAV vector, comprising culturing a host cell comprising a nucleic acid molecule encoding a capsid polypeptide of the present disclosure, an AAV rep gene, a heterologous coding sequence flanked by AAV inverted terminal repeats, and helper functions for generating a productive AAV infection, under conditions suitable to facilitate assembly of an AAV vector comprising a capsid comprising the capsid polypeptide, wherein the capsid encapsidates the heterologous coding sequence. In some examples, the host cell is a hepatocyte.
In a further aspect, provided is a method for enhancing the in vivo human hepatocyte transduction efficiency of an AAV vector, comprising:
a) identifying a reference capsid polypeptide for transducing human hepatocytes in vivo;
b) modifying the sequence of the reference capsid polypeptide at one or more of positions 263, 264, 265, 268, 272, 546, 547, 549, 550, 551, 552, 553, 554, 555, 556, 558, 559, 561, 566, 567, 580, 581, 585, 586, 590, 592, 593, 594 and 597, with numbering relative to SEQ ID NO:13, to thereby produce a modified capsid polypeptide that comprises: i) amino acid residues S263, Q264, S265, S268 and H272, with numbering relative to SEQ ID NO:13; and ii) amino acid residues T546, G547, T549, N550, K551, T552, T553, L554, E555, N556, L558, M559, N561, R566 and P567, with numbering relative to SEQ ID NO:13; and/or amino acid residues S580, S581, A585, A586, A590, T592, Q593, V594, and N597, with numbering relative to SEQ ID NO:13; and
c) vectorising the modified capsid polypeptide to thereby produce a modified AAV vector.
In some embodiments, the method further comprises modifying the sequence of the reference capsid polypeptide at one or more of positions 532, 538 and 540, with numbering relative to SEQ ID NO:13, wherein the modified capsid polypeptide comprises amino acid residues D532, S538 and V540, with numbering relative to SEQ ID NO:13. In further embodiments, the method further comprises modifying the sequence of the reference capsid polypeptide at one or more of positions 451, 456, 457, 460, 462, 466, 469, 470, 472 and 473, with numbering relative to SEQ ID NO:13, wherein the modified capsid polypeptide comprises amino acid residues S451, Q456, G457, Q460, L462, A466, A469, N470, S472 and A473, with numbering relative to SEQ ID NO:13. In other embodiments, the method further comprises modifying the sequence of the reference capsid polypeptide at one or more of positions 493, 494, 505, 506, 518 and 522, with numbering relative to SEQ ID NO:13, wherein the modified capsid polypeptide comprises amino acid residues L493, S494, G505, A506, V518 and V522, with numbering relative to SEQ ID NO:13.
In another aspect, provided is a method for enhancing the in vivo human hepatocyte transduction efficiency of an AAV vector, comprising:
a) identifying a reference capsid polypeptide for transducing human hepatocytes in vivo;
b) modifying the sequence of the reference capsid polypeptide at one or more of positions 263-272, 546-567 and 582-597 with numbering relative to SEQ ID NO:13, to thereby produce a modified capsid polypeptide that comprises: i) the sequence of amino acids SQSGASNDNH (SEQ ID NO:58) at positions 263-272, with numbering relative to SEQ ID NO:13; and ii) the sequence of amino acids TGATNKTTLENVLMTNEEEIRP (SEQ ID NO:59) at positions 546-567, with numbering relative to SEQ ID NO:13 and/or the sequence of amino acids SSNLQAANTAAQTQVVNN (SEQ ID NO:60) at positions 582-597, with numbering relative to SEQ ID NO:13; and
c) vectorising the modified capsid polypeptide to thereby produce a modified AAV vector.
In some embodiments, the method further comprises modifying the sequence of the reference capsid polypeptide at one or more of positions at positions 532-540, with numbering relative to SEQ ID NO:13, wherein the modified capsid polypeptide comprises the sequence of amino acids DRFFPSSGV (SEQ ID NO:61) at positions 532-540, with numbering relative to SEQ ID NO:13. In further embodiments, the method further comprises modifying the sequence of the reference capsid polypeptide at one or more of positions 451-473, with numbering relative to SEQ ID NO:13, wherein the modified capsid polypeptide comprises the sequence of amino acids STGGTQGTQQLLFSQAGPANMSA (SEQ ID NO:62) at positions 451-473, with numbering relative to SEQ ID NO:1. In other embodiments, the method further comprises modifying the sequence of the reference capsid polypeptide at one or more of positions 493-522, with numbering relative to SEQ ID NO:13, wherein the modified capsid polypeptide comprises the sequence of amino acids LSQNNNSNFAWTGATKYHLNGRNSLVNPGV (SEQ ID NO:63) at positions 493-522, with numbering relative to SEQ ID NO:13.
In some examples of the methods for enhancing the in vivo human hepatocyte transduction efficiency of an AAV vector, the reference capsid polypeptide comprises at least or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence set forth in SEQ ID NO:13. In particular embodiments, the methods further comprise assessing the transduction efficiency of the modified AAV vector in vivo system that utilises human hepatocytes (e.g. an in vivo system that comprises a small animal (e.g. a mouse) with a chimeric liver comprising human hepatocytes, such as the hFRG mouse model. In particular examples, the modified AAV vector produced by the methods has an in vivo transduction efficiency that is enhanced by at least or about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 300% or more compared to a reference AAV vector comprising the reference capsid polypeptide.
Embodiments of the disclosure are described herein, by way of non-limiting example only, with reference to the following drawings.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of skill in the art to which the disclosure belongs. All patents, patent applications, published applications and publications, databases, websites and other published materials referred to throughout the entire disclosure, unless noted otherwise, are incorporated by reference in their entirety. In the event that there is a plurality of definitions for terms, those in this section prevail. Where reference is made to a URL or other such identifier or address, it is understood that such identifiers can change and particular information on the internet can come and go, but equivalent information can be found by searching the internet. Reference to the identifier evidences the availability and public dissemination of such information.
As used herein, the singular forms “a”, “an” and “the” also include plural aspects (i.e. at least one or more than one) unless the context clearly dictates otherwise. Thus, for example, reference to “a polypeptide” includes a single polypeptide, as well as two or more polypeptides.
In the context of this specification, the term “about,” is understood to refer to a range of numbers that a person of skill in the art would consider equivalent to the recited value in the context of achieving the same function or result.
Throughout this specification and the claims that follow, unless the context requires otherwise, the word “comprise”, and variations such as “comprises” and “comprising”, will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.
As used herein, a “vector” includes reference to both polynucleotide vectors and viral vectors, each of which are capable of delivering a transgene contained within the vector into a host cell. Vectors can be episomal, i.e., do not integrate into the genome of a host cell, or can integrate into the host cell genome. The vectors may also be replication competent or replication deficient. Exemplary polynucleotide vectors include, but are not limited to, plasmids, cosmids and transposons. Exemplary viral vectors include, for example, AAV, lentiviral, retroviral, adenoviral, herpes viral and hepatitis viral vectors.
As used herein, “adeno-associated viral vector” or “AAV vector” refers to a vector in which the capsid is derived from an adeno-associated virus, including without limitation, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12 or AAV13, AAV from other clades or isolates, or is derived from synthetic, bioengineered or modified AAV capsid proteins, including chimeric capsid proteins. In particular embodiments, the AAV vector has a capsid comprising a capsid polypeptide of the present disclosure. When referring to AAV vectors, both the source of the genome and the source of the capsid can be identified, where the source of the genome is the first number designated and the source of the capsid is the second number designated. Thus, for example, a vector in which both the capsid and genome are derived from AAV2 is more accurately referred to as AAV2/2. A vector with an AAV6-derived capsid and an AAV2-derived genome is most accurately referred to as AAV2/6. A vector with the bioengineered DJ capsid and an AAV2-derived genome is most accurately referred to as AAV2/DJ. For simplicity, and because most vectors use an AAV2-derived genome, it is understood that reference to an AAV6 vector generally refers to an AAV2/6 vector, reference to an AAV2 vector generally refers to an AAV2/2 vector, etc. An AAV vector may also be referred to herein as “recombinant AAV”, “rAAV”, “recombinant AAV virion”, “rAAV virion”, “AAV variant”, “recombinant AAV variant”, and “rAAV variant” terms which are used interchangeably and refer to a replication-defective virus that includes an AAV capsid shell encapsidating an AAV genome. The AAV vector genome (also referred to as vector genome, recombinant AAV genome or rAAV genome) comprises a transgene flanked on both sides by functional AAV ITRs. Typically, one or more of the wild-type AAV genes have been deleted from the genome in whole or part, preferably the rep and/or cap genes. Functional ITR sequences are necessary for the rescue, replication and packaging of the vector genome into the rAAV virion.
The term “ITR” refers to an inverted terminal repeat at either end of the AAV genome. This sequence can form hairpin structures and is involved in AAV DNA replication and rescue, or excision, from prokaryotic plasmids. ITRs for use in the present disclosure need not be the wild-type nucleotide sequences, and may be altered, e.g., by the insertion, deletion or substitution of nucleotides, as long as the sequences provide for functional rescue, replication and packaging of rAAV.
As used herein, “functional” with reference to a capsid polypeptide means that the polypeptide can self-assemble or assemble with different capsid polypeptides to produce the proteinaceous shell (capsid) of an AAV virion. It is to be understood that not all capsid polypeptides in a given host cell assemble into AAV capsids. Preferably, at least 25%, at least 50%, at least 75%, at least 85%, at least 90%, at least 95% of all AAV capsid polypeptide molecules assemble into AAV capsids. Suitable assays for measuring this biological activity are described e.g. in Smith-Arica and Bartlett (2001), Curr Cardiol Rep 3(1): 43-49.
“AAV helper functions” or “helper functions” refer to functions that allow AAV to be replicated and packaged by a host cell. AAV helper functions can be provided in any of a number of forms, including, but not limited to, as a helper virus or as helper virus genes which aid in AAV replication and packaging. Helper virus genes include, but are not limited to, adenoviral helper genes such as E1A, E1B, E2A, E4 and VA. Helper viruses include, but are not limited to, adenoviruses, herpesviruses, poxviruses such as vaccinia, and baculovirus. The adenoviruses encompass a number of different subgroups, although Adenovirus type 5 of subgroup C (Ad5) is most commonly used. Numerous adenoviruses of human, non-human mammalian and avian origin are known and are available from depositories such as the ATCC. Viruses of the herpes family, which are also available from depositories such as ATCC, include, for example, herpes simplex viruses (HSV), Epstein-Barr viruses (EBV), cytomegaloviruses (CMV) and pseudorabies viruses (PRV). Baculoviruses available from depositories include Autographa californica nuclear polyhedrosis virus.
As used herein, the term “transduction” refers to entry of AAV vector into one or more particular cell types and transferal of the DNA contained within the AAV vector into the cell. Transduction can be assessed by measuring the amount of AAV DNA or RNA expressed from the AAV DNA in a cell or population of cells, and/or by assessing the number of cells in a population that contain AAV DNA or RNA expressed from the DNA. Where the presence or amount of RNA is assessed, the type of transduction assessed is referred to herein as “functional transduction”, i.e. the ability of the AAV to transfer DNA to the cell and have that DNA expressed. The term “transduction efficiency” and grammatical variations thereof refers to the ability of an AAV vector to transduce host cells, and more particularly the efficiency with which an AAV vector transduces host cells. In particular embodiment, the transduction efficiency is in vivo transduction efficiency, and refers to the ability of an AAV vector to transduce host cells in vivo following administration of the vector to the subject. Transduction efficiency can be assessed in a number of ways known to those in the art, including assessing the number of host cells transduced following exposure to, or administration of, a given number of vector particles (e.g. as assessed by expression of a reporter gene from the vector genome, such as GFP or eGFP, using microscopy or flow cytometry techniques); the amount of vector DNA (e.g. number of vector genomes) in a population of host cells following exposure to a given number of vector particles; the amount of vector RNA in population of host cells following exposure to a given number of vector particles; and the level of protein expression from a reporter gene (e.g. GFP or eGFP) in the vector genome in a population of host cells following exposure to, or administration of, a given number of vector particles. The population of host cells can represent a particular number of host cells, a volume or weight of tissue, or an entire organ (e.g. liver). In vivo transduction efficiency can reflect the ability of an AAV vector to access host cells, such as hepatocytes in the liver; the ability of an AAV vector to enter host cells; and/or expression of a heterologous coding sequence contained in the vector genome upon host cell entry.
As used herein, “corresponding nucleotides”, “corresponding amino acid residues” or “corresponding positions” refer to nucleotides, amino acids or positions that occur at aligned loci. The sequences of related or variant polynucleotides or polypeptides are aligned by any method known to those of skill in the art. Such methods typically maximize matches (e.g. identical nucleotides or amino acids at positions), and include methods such as using manual alignments and by using the numerous alignment programs available (for example, BLASTN, BLASTP, ClustlW, ClustlW2, EMBOSS, LALIGN, Kalign, etc) and others known to those of skill in the art. By aligning the sequences of polynucleotides, one skilled in the art can identify corresponding nucleotides. For example, by aligning the prototypic AAV2 capsid polypeptide set forth in SEQ ID NO:1 with another AAV capsid polypeptide (e.g. as shown in
A “heterologous coding sequence” as used herein refers to nucleic acid sequence present in a polynucleotide, vector, or host cell that is not naturally found in the polynucleotide, vector, or host cell or is not naturally found at the position that it is at in the polynucleotide, vector, or host cell, i.e. is non-native. A “heterologous coding sequence” can encode a peptide or polypeptide, or a polynucleotide that itself has a function or activity, such as an antisense or inhibitory oligonucleotide, including antisense DNA and RNA (e.g. miRNA, siRNA, and shRNA). In some examples, the heterologous coding sequence is a stretch of nucleic acids that is essentially homologous to a stretch of nucleic acids in the genomic DNA of an animal, such that when the heterologous coding sequence is introduced into a cell of the animal, homologous recombination between the heterologous sequence and the genomic DNA can occur. In one example, the heterologous coding sequence is a functional copy of a gene for introduction into a cell that has a defective/mutated copy.
As used herein, the term “operably-linked” with reference to a promoter and a coding sequence means that the transcription of the coding sequence is under the control of, or driven by, the promoter.
The term “host cell” refers to a cell, such as a mammalian cell, that has introduced into it the exogenous DNA, such as a vector or other polynucleotide. The term includes the progeny of the original cell into which the exogenous DNA has been introduced. Thus, a “host cell” as used herein generally refers to a cell that has been transfected or transduced with exogenous DNA.
As used herein, “isolated” with reference to a polynucleotide or polypeptide means that the polynucleotide or polypeptide is substantially free of cellular material or other contaminating proteins from the cells from which the polynucleotide or polypeptide is derived, or substantially free from chemical precursors or other chemicals when chemically synthesized.
The term “subject” as used herein refers to an animal, in particular a mammal and more particularly a primate including a lower primate and even more particularly, a human who can benefit from the present invention. A subject, regardless of whether a human or non-human animal or embryo, may be referred to as an individual, subject, animal, patient, host or recipient. The present disclosure has both human and veterinary applications. For convenience, an “animal” specifically includes livestock animals such as cattle, horses, sheep, pigs, camelids, goats and donkeys, as well as domestic animals, such as dogs and cats. With respect to horses, these include horses used in the racing industry as well as those used recreationally or in the livestock industry. Examples of laboratory test animals include mice, rats, rabbits, guinea pigs and hamsters. Rabbits and rodent animals, such as rats and mice, provide a convenient test system or animal model as do primates and lower primates. In some embodiments, the subject is human.
As used herein, the term “conservative sequence modifications” or “conservative substitution” refers to amino acid modifications that do not significantly affect or alter the characteristics of a vector containing the amino acid sequence. Such conservative modifications include amino acid substitutions, additions and deletions. Modifications can be introduced into a vector that are compatible with various embodiments by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions are ones in which an amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, one or more amino acid residues within a capsid can be replaced with other amino acid residues from the same side chain family and the altered capsid can be tested for tropism and/or the ability to deliver a payload using the functional assays described herein.
It will be appreciated that the above described terms and associated definitions are used for the purpose of explanation only and are not intended to be limiting.
The present disclosure is predicated in part on the identification of novel AAV capsid polypeptides. Typically, the capsid polypeptides, when present in the capsid of an AAV vector, facilitate efficient transduction of human cells (such as human hepatocytes). The in vivo transduction of cells by AAV vectors having a capsid comprising a capsid polypeptide of the present disclosure is generally increased or enhanced compared to AAV vectors comprising a reference AAV capsid polypeptide (e.g. the prototypic AAV2 capsid set forth in SEQ ID NO:1). Transduction or transduction efficiency of AAV vectors can be increased by at least or about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000% or more, e.g. an AAV vector comprising a capsid polypeptide of the present disclosure can be at least or about 1.2×, 1.5×, 2×, 3×, 4×, 5×, 6×, 7×, 8×, 9×, 10×, 11×, 12×, 13×, 14×, 15×, 16×, 17×, 18×, 19×, 20×, 30×, 40×, 50×, 60×, 70×, 80×, 90×, 100× or more efficient at transducing cells in vivo compared to a reference AAV capsid polypeptide (e.g. one set forth in SEQ ID NO:1). In particular examples, the increased transduction or transduction efficiency is observed in human liver tissue or human hepatocytes.
AAV vectors comprising a capsid of the present disclosure may also exhibit enhanced or increased resistance to neutralization by pooled human immunoglobulins (also referred to as intravenous immunoglobulin or IVIg). The resistance to IVIg neutralization can be observed in vivo or in vitro using well-known assays, such as those described in the Examples below. The resistance to IVIg neutralization can be increased by at least or about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000% or more, e.g. the resistance to IVIg neutralization of the AAV vector comprising a capsid polypeptide of the present disclosure can be at least or about 1.2×, 1.5×, 2×, 3×, 4×, 5×, 6×, 7×, 8×, 9×, 10×, 11×, 12×, 13×, 14×, 15×, 16×, 17×, 18×, 19×, 20×, 30×, 40×, 50×, 60×, 70×, 80×, 90×, 100× or more than the resistance to IVIg neutralization of an AAV vector comprising a reference AAV capsid polypeptide (e.g. one set forth in SEQ ID NO:1).
The capsid polypeptides of the present disclosure are therefore particularly useful in preparing AAV vectors, and in particular AAV vectors for gene therapy uses. In exemplary embodiments, the capsid polypeptides of the present disclosure are particularly useful in preparing AAV vectors that transduce hepatocytes, and in particular, human hepatocytes, and are thus useful for gene therapy applications targeting the liver.
Provided herein are polypeptides, including isolated polypeptides, comprising all or a portion of an AAV capsid polypeptide set forth in any one of SEQ ID Nos: 2-20 and 65-79, including all or a portion of the VP1 protein (comprising amino acid residues corresponding to those at positions 1-735 of SEQ ID NO:1), VP2 protein (comprising amino acid residues corresponding to those at positions 138-735 of SEQ ID NO:1) and/or the VP3 protein (comprising amino acid residues corresponding to those at positions 203-735 of SEQ ID NO:1), and variants thereof, including variants comprising at least or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the VP1, VP2 or VP3 proteins described herein.
Capsid polypeptides of the disclosure include those comprising all or a portion of the VP1 protein set forth in SEQ ID NO:2 (also referred to as AAVC11.01) or a polypeptide having at least or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. Thus, also included in the present disclosure are capsid polypeptides comprising all or a portion of the VP2 protein set forth as amino acids 138-735 of SEQ ID NO:2 or comprising a sequence having at least or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the VP2 protein set forth as amino acids 138-735 of SEQ ID NO:2 or a functional fragment thereof; and capsid polypeptides comprising all or a portion of the VP3 protein set forth as amino acids 204-735 of SEQ ID NO:2 or comprising a sequence having at least or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the VP3 protein set forth as amino acids 204-735 of SEQ ID NO:2 or a functional fragment thereof.
Capsid polypeptides of the disclosure also include those comprising all or a portion of the VP1 protein set forth in SEQ ID NO:3 (also referred to as AAVC11.02) or a polypeptide having at least or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. Thus, also included in the present disclosure are capsid polypeptides comprising all or a portion of the VP2 protein set forth as amino acids 138-736 of SEQ ID NO:3 or comprising a sequence having at least or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the VP2 protein set forth as amino acids 138-736 of SEQ ID NO:3 or a functional fragment thereof; and capsid polypeptides comprising all or a portion of the VP3 protein set forth as amino acids 204-736 of SEQ ID NO:3 or comprising a sequence having at least or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the VP3 protein set forth as amino acids 204-736 of SEQ ID NO:3 or a functional fragment thereof.
Exemplary capsid polypeptides of the disclosure also include those comprising all or a portion of the VP1 protein set forth in SEQ ID NO:4 (also referred to as AAVC11.03) or a polypeptide having at least or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. Thus, also included in the present disclosure are capsid polypeptides comprising all or a portion of the VP2 protein set forth as amino acids 138-737 of SEQ ID NO:4 or comprising a sequence having at least or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the VP2 protein set forth as amino acids 138-737 of SEQ ID NO:4 or a functional fragment thereof; and capsid polypeptides comprising all or a portion of the VP3 protein set forth as amino acids 204-737 of SEQ ID NO:4 or comprising a sequence having at least or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the VP3 protein set forth as amino acids 204-737 of SEQ ID NO:4 or a functional fragment thereof.
Also provided herein are capsid polypeptides comprising all or a portion of the VP1 protein set forth in SEQ ID NO:5 (also referred to as AAVC11.04) or a polypeptide having at least or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. Thus, also included in the present disclosure are capsid polypeptides comprising all or a portion of the VP2 protein set forth as amino acids 138-734 of SEQ ID NO:5 or comprising a sequence having at least or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the VP2 protein set forth as amino acids 138-734 of SEQ ID NO:5 or a functional fragment thereof; and capsid polypeptides comprising all or a portion of the VP3 protein set forth as amino acids 203-734 of SEQ ID NO:5 or comprising a sequence having at least or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the VP3 protein set forth as amino acids 203-734 of SEQ ID NO:5 or a functional fragment thereof.
Capsid polypeptides of the disclosure also include those comprising all or a portion of the VP1 protein set forth in SEQ ID NO:6 (also referred to as AAVC11.05) or a polypeptide having at least or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. Thus, also included in the present disclosure are capsid polypeptides comprising all or a portion of the VP2 protein set forth as amino acids 138-735 of SEQ ID NO:6 or comprising a sequence having at least or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the VP2 protein set forth as amino acids 138-735 of SEQ ID NO:6 or a functional fragment thereof; and capsid polypeptides comprising all or a portion of the VP3 protein set forth as amino acids 204-735 of SEQ ID NO:6 or comprising a sequence having at least or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the VP3 protein set forth as amino acids 204-735 of SEQ ID NO:6 or a functional fragment thereof.
Capsid polypeptides of the disclosure also include those comprising all or a portion of the VP1 protein set forth in SEQ ID NO:7 (also referred to AAVC11.06) or a polypeptide having at least or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. Thus, also included in the present disclosure are capsid polypeptides comprising all or a portion of the VP2 protein set forth as amino acids 138-735 of SEQ ID NO:7 or comprising a sequence having at least or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the VP2 protein set forth as amino acids 138-735 of SEQ ID NO:7 or a functional fragment thereof; and capsid polypeptides comprising all or a portion of the VP3 protein set forth as amino acids 204-735 of SEQ ID NO:7 or comprising a sequence having at least or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the VP3 protein set forth as amino acids 204-735 of SEQ ID NO:7 or a functional fragment thereof.
Other exemplary capsid polypeptides of the disclosure include those comprising all or a portion of the VP1 protein set forth in SEQ ID NO:8 (also referred to as AAVC11.07) or a polypeptide having at least or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. Thus, also included in the present disclosure are capsid polypeptides comprising all or a portion of the VP2 protein set forth as amino acids 138-734 of SEQ ID NO:8 or comprising a sequence having at least or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the VP2 protein set forth as amino acids 138-734 of SEQ ID NO:8 or a functional fragment thereof; and capsid polypeptides comprising all or a portion of the VP3 protein set forth as amino acids 203-734 of SEQ ID NO:8 or comprising a sequence having at least or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the VP3 protein set forth as amino acids 203-734 of SEQ ID NO:8 or a functional fragment thereof.
Further exemplary capsid polypeptides of the disclosure include those comprising all or a portion of the VP1 protein set forth in SEQ ID NO:9 (also referred to as AAVC11.08) or a polypeptide having at least or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. Thus, also included in the present disclosure are capsid polypeptides comprising all or a portion of the VP2 protein set forth as amino acids 138-735 of SEQ ID NO:9 or comprising a sequence having at least or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the VP2 protein set forth as amino acids 138-735 of SEQ ID NO:9 or a functional fragment thereof; and capsid polypeptides comprising all or a portion of the VP3 protein set forth as amino acids 204-735 of SEQ ID NO:9 or comprising a sequence having at least or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the VP3 protein set forth as amino acids 204-735 of SEQ ID NO:9 or a functional fragment thereof.
Capsid polypeptides of the present disclosure also include those comprising all or a portion of the VP1 protein set forth in SEQ ID NO:10 (also referred to as AAVC11.09) or a polypeptide having at least or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. Thus, also included in the present disclosure are capsid polypeptides comprising all or a portion of the VP2 protein set forth as amino acids 138-735 of SEQ ID NO:10 or comprising a sequence having at least or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the VP2 protein set forth as amino acids 138-735 of SEQ ID NO:10 or a functional fragment thereof; and capsid polypeptides comprising all or a portion of the VP3 protein set forth as amino acids 204-735 of SEQ ID NO:10 or comprising a sequence having at least or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the VP3 protein set forth as amino acids 204-735 of SEQ ID NO:10 or a functional fragment thereof.
Capsid polypeptides of the present disclosure also include those comprising all or a portion of the VP1 protein set forth in SEQ ID NO:11 (also referred to as AAVC11.10) or a polypeptide having at least or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. Thus, also included in the present disclosure are capsid polypeptides comprising all or a portion of the VP2 protein set forth as amino acids 138-734 of SEQ ID NO:11 or comprising a sequence having at least or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the VP2 protein set forth as amino acids 138-734 of SEQ ID NO:11 or a functional fragment thereof; and capsid polypeptides comprising all or a portion of the VP3 protein set forth as amino acids 203-734 of SEQ ID NO:11 or comprising a sequence having at least or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the VP3 protein set forth as amino acids 203-734 of SEQ ID NO:11 or a functional fragment thereof.
Exemplary capsid polypeptides of the present disclosure also include those comprising all or a portion of the VP1 protein set forth in SEQ ID NO:12 (also referred to as AAVC11.11) or a polypeptide having at least or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. Thus, also included in the present disclosure are capsid polypeptides comprising all or a portion of the VP2 protein set forth as amino acids 138-735 of SEQ ID NO:12 or comprising a sequence having at least or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the VP2 protein set forth as amino acids 138-735 of SEQ ID NO:12 or a functional fragment thereof; and capsid polypeptides comprising all or a portion of the VP3 protein set forth as amino acids 204-735 of SEQ ID NO:12 or comprising a sequence having at least or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the VP3 protein set forth as amino acids 204-735 of SEQ ID NO:12 or a functional fragment thereof.
Further exemplary capsid polypeptides of the present disclosure include those comprising all or a portion of the VP1 protein set forth in SEQ ID NO:13 (also referred to as AAVC11.12) or a polypeptide having at least or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. Thus, also included in the present disclosure are capsid polypeptides comprising all or a portion of the VP2 protein set forth as amino acids 138-735 of SEQ ID NO:13 or comprising a sequence having at least or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the VP2 protein set forth as amino acids 138-735 of SEQ ID NO:13 or a functional fragment thereof; and capsid polypeptides comprising all or a portion of the VP3 protein set forth as amino acids 204-735 of SEQ ID NO:13 or comprising a sequence having at least or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the VP3 protein set forth as amino acids 204-735 of SEQ ID NO:13 or a functional fragment thereof.
Also provided are capsid polypeptides that comprise all or a portion of the VP1 protein set forth in SEQ ID NO:14 (also referred to as AAVC11.13) or a polypeptide having at least or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. Thus, also included in the present disclosure are capsid polypeptides comprising all or a portion of the VP2 protein set forth as amino acids 138-735 of SEQ ID NO:14 or comprising a sequence having at least or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the VP2 protein set forth as amino acids 138-735 of SEQ ID NO:14 or a functional fragment thereof; and capsid polypeptides comprising all or a portion of the VP3 protein set forth as amino acids 204-735 of SEQ ID NO:14 or comprising a sequence having at least or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the VP3 protein set forth as amino acids 204-735 of SEQ ID NO:14 or a functional fragment thereof.
Capsid polypeptides of the present disclosure also include those that comprise all or a portion of the VP1 protein set forth in SEQ ID NO:15 (also referred to as AAVC11.14) or a polypeptide having at least or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. Thus, also included in the present disclosure are capsid polypeptides comprising all or a portion of the VP2 protein set forth as amino acids 138-736 of SEQ ID NO:15 or comprising a sequence having at least or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the VP2 protein set forth as amino acids 138-736 of SEQ ID NO:15 or a functional fragment thereof; and capsid polypeptides comprising all or a portion of the VP3 protein set forth as amino acids 203-736 of SEQ ID NO:15 or comprising a sequence having at least or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the VP3 protein set forth as amino acids 203-736 of SEQ ID NO:15 or a functional fragment thereof.
Capsid polypeptides of the present disclosure also include those that comprise all or a portion of the VP1 protein set forth in SEQ ID NO:16 (also referred to as AAVC11.15) or a polypeptide having at least or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. Thus, also included in the present disclosure are capsid polypeptides comprising all or a portion of the VP2 protein set forth as amino acids 138-735 of SEQ ID NO:16 or comprising a sequence having at least or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the VP2 protein set forth as amino acids 138-735 of SEQ ID NO:16 or a functional fragment thereof; and capsid polypeptides comprising all or a portion of the VP3 protein set forth as amino acids 204-735 of SEQ ID NO:16 or comprising a sequence having at least or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the VP3 protein set forth as amino acids 204-735 of SEQ ID NO:16 or a functional fragment thereof.
Exemplary capsid polypeptides of the present disclosure also include those that comprise all or a portion of the VP1 protein set forth in SEQ ID NO:17 (also referred to as AAVC11.16) or a polypeptide having at least or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. Thus, also included in the present disclosure are capsid polypeptides comprising all or a portion of the VP2 protein set forth as amino acids 138-735 of SEQ ID NO:17 or comprising a sequence having at least or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the VP2 protein set forth as amino acids 138-735 of SEQ ID NO:17 or a functional fragment thereof; and capsid polypeptides comprising all or a portion of the VP3 protein set forth as amino acids 204-735 of SEQ ID NO:17 or comprising a sequence having at least or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the VP3 protein set forth as amino acids 204-735 of SEQ ID NO:17 or a functional fragment thereof.
Exemplary capsid polypeptides also include those comprising all or a portion of the VP1 protein set forth in SEQ ID NO:18 (also referred to as AAVC11.17) or a polypeptide having at least or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. Thus, also included in the present disclosure are capsid polypeptides comprising all or a portion of the VP2 protein set forth as amino acids 138-735 of SEQ ID NO:18 or comprising a sequence having at least or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the VP2 protein set forth as amino acids 138-735 of SEQ ID NO:18 or a functional fragment thereof; and capsid polypeptides comprising all or a portion of the VP3 protein set forth as amino acids 204-735 of SEQ ID NO:18 or comprising a sequence having at least or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the VP3 protein set forth as amino acids 204-735 of SEQ ID NO:18 or a functional fragment thereof.
Further exemplary capsid polypeptides of the present disclosure include those comprising all or a portion of the VP1 protein set forth in SEQ ID NO:19 (also referred to as AAVC11.18) or a polypeptide having at least or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. Thus, also included in the present disclosure are capsid polypeptides comprising all or a portion of the VP2 protein set forth as amino acids 138-735 of SEQ ID NO:19 or comprising a sequence having at least or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the VP2 protein set forth as amino acids 138-735 of SEQ ID NO:19 or a functional fragment thereof; and capsid polypeptides comprising all or a portion of the VP3 protein set forth as amino acids 204-735 of SEQ ID NO:19 or comprising a sequence having at least or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the VP3 protein set forth as amino acids 204-735 of SEQ ID NO:19 or a functional fragment thereof.
Capsid polypeptides of the present disclosure also include those comprising all or a portion of the VP1 protein set forth in SEQ ID NO:20 (also referred to as AAVC11.19) or a polypeptide having at least or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. Thus, also included in the present disclosure are capsid polypeptides comprising all or a portion of the VP2 protein set forth as amino acids 138-735 of SEQ ID NO:20 or comprising a sequence having at least or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the VP2 protein set forth as amino acids 138-735 of SEQ ID NO:20 or a functional fragment thereof; and capsid polypeptides comprising all or a portion of the VP3 protein set forth as amino acids 204-735 of SEQ ID NO:20 or comprising a sequence having at least or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the VP3 protein set forth as amino acids 204-735 of SEQ ID NO:20 or a functional fragment thereof.
Capsid polypeptides of the present disclosure also include those comprising all or a portion of the VP1 protein set forth in any one of SEQ ID NOs:65-79 or a polypeptide having at least or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. Thus, also included in the present disclosure are capsid polypeptides comprising all or a portion of the VP2 protein set forth as amino acids 138-735 of any one of SEQ ID NOs: 69, 71-74, 76 and 78, amino acids 138-736 of any one of SEQ ID NOs: 65, 68, 75, 77 and 79, amino acids 138-737 of SEQ ID NOs: 67 or 70, or amino acids 138-738 of SEQ ID NO:66; or comprising a sequence having at least or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the aforementioned VP2 protein or a functional fragment thereof. Also included in the present disclosure are capsid polypeptides comprising all or a portion of the VP3 protein set forth as amino acids 204-735 of any one of SEQ ID NOs: 69, 71-74, 76 and 78, amino acids 204-736 of any one of SEQ ID NOs: 65, 68, 75, 77 and 79, amino acids 204-737 of SEQ ID NO: 67 or 70, or amino acids 204-738 of SEQ ID NO:66; or comprising a sequence having at least or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the aforementioned VP3 protein or a functional fragment thereof.
In some examples, the capsid polypeptides described above and herein comprise all or a portion of one or more variable regions having a sequence that is the same as the sequence of the corresponding variable region present in the AAVC11.12 polypeptide (SEQ ID NO:13). The variable regions of AAV capsid polypeptides have been described (see e.g. Drouin and Agbandje-McKenna, 2013, Future Virol. 8(12): 1183-1199) and include VR-I, spanning positions 260-267; VR-II, spanning positions 326-330; VR-III, spanning positions 380-384; VR-IV, spanning positions 449-467; VR-V, spanning positions 487-504; VR-VI, spanning positions 522-538; VR-VII, spanning positions 544-557; VR-VIII, spanning positions 580-592; and VR-IX, spanning positions 703-711 with numbering relative to AAV2. The AAVC11.12 polypeptide, which was generated from a DNA shuffled library, contains a VR-I of AAV2 origin, VR-IV and VR-V of AAV10 origin, and VR-VI, VR-VII, and VR-VIII of AAV7 origin (when using the VR regions as defined above and in Drouin and Agbandje-McKenna, 2013, the VR-I spans positions 261-268; the VR-IV spans positions 450-468; the VR-V spans positions 488-505; the VR-VI spans positions 523-539; the VR-VII spans positions 545-557; and the VR-VIII spans positions 580-592 of the AAVC11.12 polypeptide set forth in SEQ ID NO:13). Thus, in some examples, the capsid polypeptides of the present disclosure comprise all or a portion of one or more of the VR-I, VR-IV, VR-V, VR-VI, VR-VII and VR-VIII of the AAVC11.12 polypeptide. In some embodiments, capsid polypeptides have at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to all or a portion of one or more of the VR-I, VR-IV, VR-V, VR-VI, VR-VII and VR-VIII of the AAVC11.12 polypeptide
In one example, the capsid polypeptides of the present disclosure (e.g. a capsid polypeptide comprising a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the VP1, VP2 or VP3 protein of any one of SEQ ID NOs: 2-20 or 65-79) comprise amino acid residues S263, Q264, S265, S268 and H272 (i.e. including residues in or near the VR-I of AAVC11.12); amino acid residues S451, Q456, G457, Q460, L462, A466, A469, N470, S472 and A473 (i.e. including residues in and/or near the VR-IV of AAVC11.12); amino acid residues L493, S494, G505, A506, V518 and V522 (i.e. including residues in or near the VR-V of AAVC11.12); amino acid residues D532, S538 and V540 (i.e. including residues in or near the VR-VI of AAVC11.12); amino acid residues T546, G547, T549, N550, K551, T552, T553, L554, E555, N556, L558, M559, N561, R566 and P567 (i.e. including residues in or near the VR-VII of AAVC11.12); and/or amino acid residues S580, S581, A585, A586, A590, T592, Q593, V594, and N597 (i.e. including residues in or near the VR-VIII of AAVC11.12); with numbering relative to SEQ ID NO:13.
In further examples, the capsid polypeptides comprise the sequence of amino acids SQSGASNDNH (SEQ ID NO:58) at positions 263-272; the sequence of amino acids ISSQSGASNDNH (SEQ ID NO:80) at positions 261-272; the sequence of amino acids STGGTQGTQQLLFSQAGPANMSA (SEQ ID NO:62) at positions 451-473; the sequence of amino acids QSTGGTQGTQQLLFSQAGPANMSA (SEQ ID NO:83) at positions 450-473; the sequence of amino acids LSQNNNSNFAWTGATKYHLNGRNSLVNPGV (SEQ ID NO:63) at positions 493-522; the sequence of amino acids RVSTTLSQNNNSNFAWTGATKYHLNGRNSLVNPGV (SEQ ID NO:84) at positions 488-522; the sequence of amino acids DRFFPSSGV (SEQ ID NO:61) at positions 532-540; the sequence of amino acids AMATHKDDEDRFFPSSGV (SEQ ID NO:82) at positions 523-540; the sequence of amino acids TGATNKTTLENVLMTNEEEIRP (SEQ ID NO:59) at positions 546-567; the sequence of amino acids KTGATNKTTLENVLMTNEEEIRP (SEQ ID NO:81) at positions 545-567; and/or the sequence of amino acids SSNLQAANTAAQTQVVNN (SEQ ID NO:60) at positions 582-597; with numbering relative to SEQ ID NO:13.
In a particular example, the capsid polypeptides of the present disclosure (e.g. a capsid polypeptide comprising a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the VP1, VP2 or VP3 protein of any one of SEQ ID NOs: 2-20 or 65-79) comprise all or a portion of the VR-I of AAVC11.12, and all or a portion of the VR-VII and/or VR-VIII of AAVC11.12. Thus, in one example, the polypeptides comprise a) amino acid residues S263, Q264, S265, S268 and H272; and b) amino acid residues T546, G547, T549, N550, K551, T552, T553, L554, E555, N556, L558, M559, N561, R566 and P567; and/or amino acid residues S580, S581, A585, A586, A590, T592, Q593, V594, and N597, with numbering relative to SEQ ID NO:13. In further examples, the capsid polypeptides comprise a) the sequence of amino acids SQSGASNDNH (SEQ ID NO:58) at positions 263-272; and b) the sequence of amino acids TGATNKTTLENVLMTNEEEIRP (SEQ ID NO:59) at positions 546-567 and/or the sequence of amino acids SSNLQAANTAAQTQVVNN (SEQ ID NO:60) at positions 582-597, with numbering relative to SEQ ID NO:13. In other examples, the capsid polypeptides comprise the sequence of amino acids ISSQSGASNDNH (SEQ ID NO:80) at positions 261-272; and b) the sequence of amino acids KTGATNKTTLENVLMTNEEEIRP (SEQ ID NO:81) at positions 545-567 and/or the sequence of amino acids SSNLQAANTAAQTQVVNN (SEQ ID NO:60) at positions 582-597, with numbering relative to SEQ ID NO:13. Such capsid polypeptides can further include all or a portion of the VR-VI of AAVC11.12 (e.g. amino acid residues D532, S538 and V540; the sequence of amino acids DRFFPSSGV (SEQ ID NO:61) at positions 532-540; and/or the sequence of amino acids AMATHKDDEDRFFPSSGV (SEQ ID NO:82) at positions 523-540), all or a portion of the VR-IV of AAVC11.12 (e.g. comprising amino acid residues S451, Q456, G457, Q460, L462, A466, A469, N470, S472 and A473; the sequence of amino acids STGGTQGTQQLLFSQAGPANMSA (SEQ ID NO:62) at positions 451-473, and/or the sequence of amino acids QSTGGTQGTQQLLFSQAGPANMSA (SEQ ID NO:83) at positions 450-473), and/or all or a portion of the VR-V of AAVC11.12 (e.g. comprising amino acid residues L493, S494, G505, A506, V518 and V522, the sequence of amino acids LSQNNNSNFAWTGATKYHLNGRNSLVNPGV (SEQ ID NO:63) at positions 493-522, and/or the sequence of amino acids RVSTTLSQNNNSNFAWTGATKYHLNGRNSLVNPGV (SEQ ID NO:84) at positions 488-522), with numbering relative to SEQ ID NO:13.
In some embodiments, capsid polypeptides of the present disclosure comprise a sequence of amino acids having at least about 50%, 60%, 70%, 80%, or 90% sequence identity to SEQ ID NO: 58 and include at least one substitution at any of positions 264-272 (e.g., at least one conservative substitution, e.g., at least two, three, four, or five substitutions). In some embodiments, capsid polypeptides of the present disclosure comprise a sequence of amino acids having at least about 50%, 60%, 70%, 80%, or 90% sequence identity to SEQ ID NO: 58 (e.g., at least one conservative substitution, e.g., at least two, three, four, or five substitutions) and include at least one substitution at any of positions 266, 267, 269, 270, and 271. In some embodiments, capsid polypeptides of the present disclosure comprise a sequence of amino acids having at least about 50%, 60%, 70%, 80%, or 90% sequence identity to SEQ ID NO: 58 and include at least one deletion or insertion. In some embodiments, capsid polypeptides may comprise S at position 263, or a conservative substitution thereof. In some embodiments, capsid polypeptides may comprise Q at position 264, or a conservative substitution thereof. In some embodiments, capsid polypeptides may comprise S at position 265, or a conservative substitution thereof. In some embodiments, capsid polypeptides may comprise S at position 268, or a conservative substitution thereof. In some embodiments, capsid polypeptides may comprise H at position 272, or a conservative substitution thereof.
In some embodiments, capsid polypeptides of the present disclosure comprise a sequence of amino acids having at least about 65%, 70%, 75%, 80%, 85%, 90%, or 95% sequence identity to SEQ ID NO: 59 and include at least one substitution at any of positions 545-567 (e.g., at least one conservative substitution, e.g., at least two, three, four, five, six, or seven substitutions). In some embodiments, capsid polypeptides of the present disclosure comprise a sequence of amino acids having at least about 65%, 70%, 75%, 80%, 85%, 90%, or 95% sequence identity to SEQ ID NO: 59 (e.g., at least one conservative substitution, e.g., at least two, three, four, five, six, or seven substitutions) and include at least one substitution at any of positions 545, 548, 557, 560, 562, 563, 564, or 565. In some embodiments, capsid polypeptides of the present disclosure comprise a sequence of amino acids having at least about 65%, 70%, 75%, 80%, 85%, 90%, or 95% sequence identity to SEQ ID NO: 59 and include at least one deletion or insertion. In some embodiments, capsid polypeptides may comprise T at position 546, or a conservative substitution thereof. In some embodiments, capsid polypeptides may comprise G at position 547, or a conservative substitution thereof. In some embodiments, capsid polypeptides may comprise T at position 549, or a conservative substitution thereof. In some embodiments, capsid polypeptides may comprise N at position 550, or a conservative substitution thereof. In some embodiments, capsid polypeptides may comprise K at position 551, or a conservative substitution thereof. In some embodiments, capsid polypeptides may comprise T at position 552, or a conservative substitution thereof. In some embodiments, capsid polypeptides may comprise T at position 553, or a conservative substitution thereof. In some embodiments, capsid polypeptides may comprise L at position 554, or a conservative substitution thereof. In some embodiments, capsid polypeptides may comprise E at position 555, or a conservative substitution thereof. In some embodiments, capsid polypeptides may comprise N at position 556, or a conservative substitution thereof. In some embodiments, capsid polypeptides may comprise L at position 558, or a conservative substitution thereof. In some embodiments, capsid polypeptides may comprise M at position 559, or a conservative substitution thereof. In some embodiments, capsid polypeptides may comprise N at position 561, or a conservative substitution thereof. In some embodiments, capsid polypeptides may comprise R at position 566, or a conservative substitution thereof. In some embodiments, capsid polypeptides may comprise P at position 567, or a conservative substitution thereof.
In some embodiments, capsid polypeptides of the present disclosure comprise a sequence of amino acids having at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% sequence identity to SEQ ID NO: 60 (e.g., at least one conservative substitution, e.g., at least two, three, four, five, six, seven, eight, or nine substitutions) and include at least one substitution at any of positions 581-597. In some embodiments, capsid polypeptides of the present disclosure comprise a sequence of amino acids having at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% sequence identity to SEQ ID NO: 60 (e.g., at least one conservative substitution, e.g., at least two, three, four, five, six, seven, eight, or nine substitutions) and include at least one substitution at any of positions 582, 583, 584, 587, 588, 589, 591, 595, or 596. In some embodiments, capsid polypeptides of the present disclosure comprise a sequence of amino acids having at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% sequence identity to SEQ ID NO: 60 and include at least one deletion or insertion. In some embodiments, capsid polypeptides may comprise S at position 580, or a conservative substitution thereof. In some embodiments, capsid polypeptides may comprise S at position 581, or a conservative substitution thereof. In some embodiments, capsid polypeptides may comprise A at position 585, or a conservative substitution thereof. In some embodiments, capsid polypeptides may comprise A at position 586, or a conservative substitution thereof. In some embodiments, capsid polypeptides may comprise A at position 590, or a conservative substitution thereof. In some embodiments, capsid polypeptides may comprise T at position 592, or a conservative substitution thereof. In some embodiments, capsid polypeptides may comprise O at position 593, or a conservative substitution thereof. In some embodiments, capsid polypeptides may comprise V at position 594, or a conservative substitution thereof. In some embodiments, capsid polypeptides may comprise N at position 597, or a conservative substitution thereof.
In some embodiments, capsid polypeptides of the present disclosure comprise a sequence of amino acids having at least about 30%, 40%, 50%, 60%, 70%, 80%, or 90% sequence identity to SEQ ID NO: 61 (e.g., at least one conservative substitution, e.g., at least two, three, four, five, or six substitutions) and include at least one substitution at any of positions 532-540. In some embodiments, capsid polypeptides of the present disclosure comprise a sequence of amino acids having at least about 30%, 40%, 50%, 60%, 70%, 80%, or 90% sequence identity to SEQ ID NO: 61 (e.g., at least one conservative substitution, e.g., at least two, three, four, five, or six substitutions) and include at least one substitution at any of positions 533, 534, 535, 536, 537, or 539. In some embodiments, capsid polypeptides of the present disclosure comprise a sequence of amino acids having at least about 30%, 40%, 50%, 60%, 70%, 80%, or 90% sequence identity to SEQ ID NO: 61 and include at least one deletion or insertion. In some embodiments, capsid polypeptides may comprise D at position 532, or a conservative substitution thereof. In some embodiments, capsid polypeptides may comprise S at position 538, or a conservative substitution thereof. In some embodiments, capsid polypeptides may comprise V at position 540, or a conservative substitution thereof.
In some embodiments, capsid polypeptides of the present disclosure comprise a sequence of amino acids having at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% sequence identity to SEQ ID NO: 62 (e.g., at least one conservative substitution, e.g., at least two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, or thirteen substitutions) and include at least one substitution at any of positions 451-473. In some embodiments, capsid polypeptides of the present disclosure comprise a sequence of amino acids having at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% sequence identity to SEQ ID NO: 62 (e.g., at least one conservative substitution, e.g., at least two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, or thirteen substitutions) and include at least one substitution at any of positions 452. 453. 454. 455. 458, 459, 461, 463, 464, 465, 467, 468, or 471. In some embodiments, capsid polypeptides of the present disclosure comprise a sequence of amino acids having at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% sequence identity to SEQ ID NO: 62 and include at least one deletion or insertion. In some embodiments, capsid polypeptides may comprise S at position 451, or a conservative substitution thereof. In some embodiments, capsid polypeptides may comprise Q at position 456, or a conservative substitution thereof. In some embodiments, capsid polypeptides may comprise G at position 457, or a conservative substitution thereof. In some embodiments, capsid polypeptides may comprise Q at position 460, or a conservative substitution thereof. In some embodiments, capsid polypeptides may comprise L at position 462, or a conservative substitution thereof. In some embodiments, capsid polypeptides may comprise A at position 466, or a conservative substitution thereof. In some embodiments, capsid polypeptides may comprise A at position 469, or a conservative substitution thereof. In some embodiments, capsid polypeptides may comprise N at position 470, or a conservative substitution thereof. In some embodiments, capsid polypeptides may comprise S at position 472, or a conservative substitution thereof. In some embodiments, capsid polypeptides may comprise A at position 473, or a conservative substitution thereof.
In some embodiments, capsid polypeptides of the present disclosure comprise a sequence of amino acids having at least about 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% sequence identity to SEQ ID NO: 63 (e.g., at least one conservative substitution, e.g., at least two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, twenty one, twenty two, twenty three, or twenty four substitutions) and include at least one substitution at any of positions 493-522. In some embodiments, capsid polypeptides of the present disclosure comprise a sequence of amino acids having at least about 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% sequence identity to SEQ ID NO: 63 (e.g., at least one conservative substitution, e.g., at least two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, twenty one, twenty two, twenty three, or twenty four substitutions) and include at least one substitution at any of positions 495, 496, 497, 498, 499, 500, 501, 502, 503, 504, 507, 508, 509, 510, 511, 512, 513, 514, 515, 516, 517, 519, 520, or 521. In some embodiments, capsid polypeptides of the present disclosure comprise a sequence of amino acids having at least about 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% sequence identity to SEQ ID NO: 63 and include at least one deletion or insertion. In some embodiments, capsid polypeptides may comprise L at position 493, or a conservative substitution thereof. In some embodiments, capsid polypeptides may comprise S at position 494, or a conservative substitution thereof. In some embodiments, capsid polypeptides may comprise G at position 505, or a conservative substitution thereof. In some embodiments, capsid polypeptides may comprise A at position 506, or a conservative substitution thereof. In some embodiments, capsid polypeptides may comprise V at position 518, or a conservative substitution thereof. In some embodiments, capsid polypeptides may comprise V at position 522, or a conservative substitution thereof.
In a particular example, the capsid polypeptides of the present disclosure (e.g. a capsid polypeptide comprising a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the VP1, VP2 or VP3 protein of any one of SEQ ID NOs: 2-20 or 65-79) comprise all or a portion of the VR-IV, VR-V, VR-VI, VR-VII and VR-VIII of AAVC11.12. Thus, in one example, the polypeptides comprise amino acid residues S451, Q456, G457, Q460, L462, A466, A469, N470, S472, A473, L493, S494, G505, A506, V518 V522, D532, S538 V540, T546, G547, T549, N550, K551, T552, T553, L554, E555, N556, L558, M559, N561, R566, P567, S580, S581, A585, A586, A590, T592, Q593, V594, and N597, with numbering relative to SEQ ID NO:13. In particular examples, the capsid polypeptides comprise the sequence of amino acids STGGTQGTQQLLFSQAGPANMSA (SEQ ID NO:62) at positions 451-473; the sequence of amino acids LSQNNNSNFAWTGATKYHLNGRNSLVNPGV (SEQ ID NO:63) at positions 493-522; the sequence of amino acids DRFFPSSGV (SEQ ID NO:61) at positions 532-540; the sequence of amino acids TGATNKTTLENVLMTNEEEIRP (SEQ ID NO:59) at positions 546-567; and the sequence of amino acids SSNLQAANTAAQTQVVNN (SEQ ID NO:60) at positions 582-597, with numbering relative to SEQ ID NO:13. In still further examples, the polypeptides comprise the sequence of amino acids QSTGGTQGTQQLLFSQAGPANMSA (SEQ ID NO:83) at positions 450-473; the sequence of amino acids RVSTTLSQNNNSNFAWTGATKYHLNGRNSLVNPGV (SEQ ID NO:84) at positions 488-522; the sequence of amino acids AMATHKDDEDRFFPSSGV (SEQ ID NO:82) at positions 523-540; the sequence of amino acids KTGATNKTTLENVLMTNEEEIRP (SEQ ID NO:81) at positions 545-567, with numbering relative to SEQ ID NO:13; and the sequence of amino acids SSNLQAANTAAQTQVVNN (SEQ ID NO:60) at positions 582-597, with numbering relative to SEQ ID NO:13. Typically, such polypeptides do not have the VR-I from AAVC11.12 (i.e. do not have the AAV2 VR-I). These polypeptides may have a VR-I from AAV8. For example, the polypeptides may have an insertion of NG after position 262, and contain residues T263, S264, G265, T268, and T272, with numbering relative to SEQ ID NO:13. In particular examples, the polypeptide contains an insertion of NG after position 262 and the sequence of amino acids TSGGATNDNT at positions 263-272, with numbering relative to SEQ ID NO:13.
Also provided are nucleic acid molecules, including isolated nucleic acid molecules, encoding a capsid polypeptide described herein. Thus, for example, amongst the nucleic acid molecules provided herein are those encoding the VP1, VP2 and/or VP3 of any one of the capsid polypeptides described herein. Non-limiting examples of nucleic acid molecules therefore include those set forth in SEQ ID NOs:21-39 and 85-99, those having at least or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, and those that hybridize with medium or high stringency to nucleic acid molecules comprising a sequence set forth in any one of SEQ ID NOs:21-39 and 85-99.
The present disclosure also provides vectors comprising a nucleic acid molecule that encodes a capsid polypeptide described herein, and vectors comprising a capsid polypeptide described herein. The vectors include nucleic acid vectors that comprise a nucleic acid molecule that encodes a capsid polypeptide described herein, and AAV vectors that have a capsid comprising a capsid polypeptide described herein.
Vectors of the present disclosure include nucleic acid vectors that comprise a polynucleotide that encodes all or a portion of a capsid polypeptide described herein, e.g. that encodes a polypeptide comprising an amino acid sequence set forth in any one of SEQ ID NOs:2-20 or an amino acid sequence having at least or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to a sequence set forth in any one of SEQ ID NOs:2-20, or a fragment thereof (e.g. all or a portion of the VP2 or VP3 protein), as described above. The vectors can be episomal vectors (i.e., that do not integrate into the genome of a host cell) or can be vectors that integrate into the host cell genome. Exemplary vectors that comprise a nucleic acid molecule encoding a capsid polypeptide include, but are not limited to, plasmids, cosmids, transposons and artificial chromosomes. In particular examples, the vectors are plasmids.
Vectors, such as plasmids, suitable for use in bacterial, insect and mammalian cells are widely described and well-known in the art. Those skilled in the art would appreciate that vectors of the present disclosure may also contain additional sequences and elements useful for the replication of the vector in prokaryotic and/or eukaryotic cells, selection of the vector and the expression of a heterologous sequence in a variety of host cells. For example, the vectors of the present disclosure can include a prokaryotic replicon (that is, a sequence having the ability to direct autonomous replication and maintenance of the vector extra-chromosomally in a prokaryotic host cell, such as a bacterial host cell. Such replicons are well known in the art. In some embodiments, the vectors can include a shuttle element that makes the vectors suitable for replication and integration in both prokaryotes and eukaryotes. In addition, vectors may also include a gene whose expression confers a detectable marker such as a drug resistance gene, which allows for selection and maintenance of the host cells. Vectors may also have a reportable marker, such as gene encoding a fluorescent or other detectable protein. The nucleic acid vectors will likely also comprise other elements, including any one or more of those described below. Most typically, the vectors will comprise a promoter operably linked to the nucleic acid encoding the capsid protein.
The nucleic acid vectors of the present disclosure can be constructed using known techniques, including, without limitation, the standard techniques of restriction endonuclease digestion, ligation, transformation, plasmid purification, in vitro or chemical synthesis of DNA, and DNA sequencing. The vectors of the present disclosure may be introduced into a host cell using any method known in the art. Accordingly, the present disclosure is also directed to host cells comprising a vector or nucleic acid described herein.
Provided herein are AAV vectors comprising a capsid polypeptide described herein, such as a polypeptide comprising all or a portion of an AAV capsid protein (e.g. a polypeptide comprising the amino acid sequence set forth in any one of SEQ ID NOs:2-20 or an amino acid sequence having at least or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to a sequence set forth in any one of SEQ ID NOs:2-20, or a fragment thereof (e.g. all or a portion of the VP2 or VP3 protein).
Methods for vectorizing a capsid protein are well known in the art and any suitable method can be employed for the purposes of the present disclosure. For example, the cap gene can be recovered (e.g. by PCR or digest with enzymes that cut upstream and downstream of cap) and cloned into a packaging construct containing rep. Any AAV rep gene may be used, including, for example, a rep gene is from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12 or AAV13 and any variants thereof. Typically, the cap gene is cloned downstream of rep so the rep p40 promoter can drive cap expression. This construct does not contain ITRs. This construct is then introduced into a packaging cell line with a second construct containing ITRs, typically flanking a heterologous coding sequence. Helper function or a helper virus are also introduced, and recombinant AAV comprising a capsid generated from capsid proteins expressed from the cap gene, and encapsidating a genome comprising the transgene flanked by the ITRs, is recovered from the supernatant of the packaging cell line. Various types of cells can be used as the packaging cell line. For example, packaging cell lines that can be used include, but are not limited to, HEK293 cells, HeLa cells, and Vero cells, for example as disclosed in US20110201088. The helper functions may be provided by one or more helper plasmids or helper viruses comprising adenoviral helper genes. Non-limiting examples of the adenoviral helper genes include E1A, E1B, E2A, E4 and VA, which can provide helper functions to AAV packaging. Helper viruses of AAV are known in the art and include, for example, viruses from the family Adenoviridae and the family Herpesviridae. Examples of helper viruses of AAV include, but are not limited to, SAdV-13 helper virus and SAdV-13-like helper virus described in US20110201088, helper vectors pHELP (Applied Viromics). A skilled artisan will appreciate that any helper virus or helper plasmid of AAV that can provide adequate helper function to AAV can be used herein.
In some instances, rAAV virions are produced using a cell line that stably expresses some of the necessary components for AAV virion production. For example, a plasmid (or multiple plasmids) comprising the nucleic acid containing a cap gene identified as described herein and a rep gene, and a selectable marker, such as a neomycin resistance gene, can be integrated into the genome of a cell (the packaging cells). The packaging cell line can then be transfected with an AAV vector and a helper plasmid or transfected with an AAV vector and co-infected with a helper virus (e.g., adenovirus providing the helper functions). The advantages of this method are that the cells are selectable and are suitable for large-scale production of the recombinant AAV. As another non-limiting example, adenovirus or baculovirus rather than plasmids can be used to introduce the nucleic acid encoding the capsid polypeptide, and optionally the rep gene, into packaging cells. As yet another non-limiting example, the AAV vector is also stably integrated into the DNA of producer cells, and the helper functions can be provided by a wild-type adenovirus to produce the recombinant AAV.
In still further instances, the AAV vectors are produced synthetically, by synthesising AAV capsid proteins and assembling and packaging the capsids in vitro.
Typically, the AAV vectors of the present disclosure also comprise a heterologous coding sequence. The heterologous coding sequence may be operably linked to a promoter to facilitate expression of the sequence. The heterologous coding sequence can encode a peptide or polypeptide, such as a therapeutic peptide or polypeptide, or can encode a polynucleotide or transcript that itself has a function or activity, such as an antisense or inhibitory oligonucleotide, including antisense DNA and RNA (e.g. miRNA, siRNA, and shRNA). In some examples, the heterologous coding sequence is a stretch of nucleic acids that is essentially homologous to a stretch of nucleic acids in the genomic DNA of an animal, such that when the heterologous coding sequence is introduced into a cell of the animal, homologous recombination between the heterologous coding sequence and the genomic DNA can occur. As would be appreciated, the nature of the heterologous coding sequence is not essential to the present disclosure. In particular embodiments, the vectors comprising the heterologous coding sequence(s) will be used in gene therapy.
In particular examples, the heterologous coding sequence encodes a peptide or polypeptide, or polynucleotide, whose expression is of therapeutic use, such as, for example, for the treatment of a disease or disorder. For example, expression of a therapeutic peptide or polypeptide may serve to restore or replace the function of the endogenous form of the peptide or polypeptide that is defective (i.e. gene replacement therapy). In other examples, expression of a therapeutic peptide or polypeptide, or polynucleotide, from the heterologous sequence serves to alter the levels and/or activity of one or more other peptides, polypeptides or polynucleotides in the host cell. Thus, according to particular embodiments, the expression of a heterologous coding sequence introduced by a vector described herein into a host cell can be used to provide a therapeutic amount of a peptide, polypeptide or polynucleotide to ameliorate the symptoms of a disease or disorder. In other instance, the heterologous coding sequence is a stretch of nucleic acids that is essentially homologous to a stretch of nucleic acids in the genomic DNA of an animal, such that when the heterologous sequence is introduced into a cell of the animal, homologous recombination between the heterologous coding sequence and the genomic DNA can occur. Accordingly, the introduction of a heterologous sequence by an AAV vector described herein into a host cell can be used to correct mutations in genomic DNA, which in turn can ameliorate the symptoms of a disease or disorder.
In non-limiting examples, the heterologous coding sequence encodes an expression product that, when delivered to a subject, and in particular the liver of a subject, treats a liver-associated disease or condition. In illustrative embodiments, the liver-associated disease or condition is selected from among a urea cycle disorder (UCD; including N-acetylglutamate synthase deficiency (NAGSD), carbamylphosphate synthetase 1 deficiency (CPS1D), ornithine transcarbamylase deficiency (OTCD), argininosuccinate synthetase deficiency (ASSD), argininosuccinate lyase (ASLD), arginase 1 deficiency (ARG1D), citrin or aspartate/glutamate carrier deficiency and the mitochondrial ornithine transporter 1 deficiency causing hyperornithinemia-hyperammonemia-homocitrullinuria syndrome (HHH syndrome)), organic acidopathy (or organic academia, including methylmalonic acidemia, propionic acidemia, isovaleric acidemia, and maple syrup urine disease), aminoacidopathy, glycogenoses (Types I, III and IV), Wilson's disease, Progressive Familial Intrahepatic Cholestasis, primary hyperoxaluria, complementopathy, coagulopathy (e.g. hemophilia A, hemophilia B, von Willebrand disease (VWD)), Crigler Najjar syndrome, familial hypercholesterolaemia, α-1-antitrypsin deficiency, mitochondria respiratory chain hepatopathy, and citrin deficiency. Those skilled in the art would readily be able to select an appropriate heterologous coding sequence useful for treating such diseases. In some examples, the heterologous coding sequence comprises all or a part of a gene that is associated with the disease, such as all or a part of a gene set forth in Table 2. Introduction of such a sequence to the liver can be used for gene replacement or gene editing/correction, e.g. using CRISPR-Cas9. In particular examples, the heterologous coding sequence encodes a protein encoded by a gene that is associated with the disease, such as a gene set forth in Table 2.
The heterologous coding sequence in the AAV vector is flanked by 3′ and 5′ AAV ITRs. AAV ITRs used in the vectors of the disclosure need not have a wild-type nucleotide sequence, and may be altered, e.g., by the insertion, deletion or substitution of nucleotides. Additionally, AAV ITRs may be derived from any of several AAV serotypes, including without limitation, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12 or AAV13. Such ITRs are well known in the art.
As will be appreciated by a skilled artisan, any method suitable for purifying AAV can be used in the embodiments described herein to purify the AAV vectors, and such methods are well known in the art. For example, the AAV vectors can be isolated and purified from packaging cells and/or the supernatant of the packaging cells. In some embodiments, the AAV is purified by separation method using a CsCl or iodixanol gradient centrifugation. In other embodiments, AAV is purified as described in US20020136710 using a solid support that includes a matrix to which an artificial receptor or receptor-like molecule that mediates AAV attachment is immobilized.
The vectors of the present disclosure can comprise promoters. In instances where the vector is a nucleic acid vector comprising nucleic acid encoding the capsid polypeptide, the promoter may facilitate expression of the nucleic acid encoding the capsid polypeptide. In instances where the vector is an AAV vector, the promoter may facilitate expression of a heterologous coding sequence, as described above.
In some examples, the promoters are AAV promoters, such as the p5, p19 or p40 promoter. In other examples, the promoters are derived from other sources. Examples of constitutive promoters include, without limitation, the retroviral Rous sarcoma virus (RSV) LTR promoter (optionally with the RSV enhancer), the cytomegalovirus (CMV) promoter (optionally with the CMV enhancer), the SV40 promoter, the dihydrofolate reductase promoter, the 8-actin promoter, the phosphoglycerol kinase (PGK) promoter, and the EF1α promoter. Inducible promoters allow regulation of gene expression and can be regulated by exogenously supplied compounds, environmental factors such as temperature, or the presence of a specific physiological state, e.g., acute phase, a particular differentiation state of the cell, or in replicating cells only. Non-limiting examples of inducible promoters regulated by exogenously supplied promoters include the zinc-inducible sheep metallothionine (MT) promoter, the dexamethasone (Dex)-inducible mouse mammary tumor virus (MMTV) promoter, the T7 polymerase promoter system; the ecdysone insect promoter, the tetracycline-repressible system, the tetracycline-inducible system, the RU486-inducible system and the rapamycin-inducible system. Still other types of inducible promoters which may be useful in this context are those which are regulated by a specific physiological state, e.g., temperature, acute phase, a particular differentiation state of the cell, or in replicating cells only. In some embodiments, tissue specific promoters are used. Non-limiting examples of such promoters include the liver-specific thyroxin binding globulin (TBG) promoter, insulin promoter, glucagon promoter, somatostatin promoter, pancreatic polypeptide (PPY) promoter, synapsin-1 (Syn) promoter, creatine kinase (MCK) promoter, mammalian desmin (DES) promoter, a α-myosin heavy chain (a-MHC) promoter, a cardiac Troponin T (cTnT) promoter, beta-actin promoter, and hepatitis B virus core promoter. The selection of an appropriate promoter is well within the ability of one of ordinary skill in the art.
The vectors can also include transcriptional enhancers, translational signals, and transcriptional and translational termination signals. Examples of transcriptional termination signals include, but are not limited to, polyadenylation signal sequences, such as bovine growth hormone (BGH) poly(A), SV40 late poly(A), rabbit beta-globin (RBG) poly(A), thymidine kinase (TK) poly(A) sequences, and any variants thereof. In some embodiments, the transcriptional termination region is located downstream of the posttranscriptional regulatory element. In some embodiments, the transcriptional termination region is a polyadenylation signal sequence.
The vectors can include various posttranscriptional regulatory elements. In some embodiments, the posttranscriptional regulatory element can be a viral posttranscriptional regulatory element. Non-limiting examples of viral posttranscriptional regulatory element include woodchuck hepatitis virus posttranscriptional regulatory element (WPRE), hepatitis B virus posttranscriptional regulatory element (HBVPRE), RNA transport element, and any variants thereof. The RTE can be a rev response element (RRE), for example, a lentiviral RRE. A non-limiting example is bovine immunodeficiency virus rev response element (RRE). In some embodiments, the RTE is a constitutive transport element (CTE). Examples of CTE include, but are not limited to, Mason-Pfizer Monkey Virus CTE and Avian Leukemia Virus CTE.
A signal peptide sequence can also be included in the vector to provide for secretion of a polypeptide from a mammalian cell. Examples of signal peptides include, but are not limited to, the endogenous signal peptide for HGH and variants thereof; the endogenous signal peptide for interferons and variants thereof, including the signal peptide of type I, II and III interferons and variants thereof; and the endogenous signal peptides for known cytokines and variants thereof, such as the signal peptide of erythropoietin (EPO), insulin, TGF-β1, TNF, IL1-α, and IL1-β, and variants thereof. Typically, the nucleotide sequence of the signal peptide is located immediately upstream of the heterologous sequence (e.g., fused at the 5′ of the coding region of the protein of interest) in the vector.
In further examples, the vectors can contain a regulatory sequence that allows, for example, the translation of multiple proteins from a single mRNA. Non-limiting examples of such regulatory sequences include internal ribosome entry site (IRES) and 2A self-processing sequence, such as a 2A peptide site from foot-and-mouth disease virus (F2A sequence).
Also provided herein are host cells comprising a nucleic acid molecule or vector or of the present disclosure. In some instances, the host cells are used to amplify, replicate, package and/or purify a polynucleotide or vector. In other examples, the host cells are used to express a heterologous sequence, such as one packaged within AAV vector. Exemplary host cells include prokaryotic and eukaryotic cells. In some instances, the host cell is a mammalian host cell. It is well within the skill of a skilled artisan to select an appropriate host cell for the expression, amplification, replication, packaging and/or purification of a polynucleotide, vector or rAAV virion of the present disclosure. Exemplary mammalian host cells include, but are not limited to, HEK293 cells, HeLa cells, Vero cells, HuH-7 cells, and HepG2 cells. In particular examples, the host cell is a hepatocyte or cell-line derived from a hepatocyte.
Also provided are compositions comprising the nucleic acid molecules, polypeptides and/or vectors of the present disclosure. In particular examples, provided are pharmaceutical compositions comprising the AAV vectors disclosed herein and a pharmaceutically acceptable carrier. The compositions can also comprise additional ingredients such as diluents, stabilizers, excipients, and adjuvants.
The carriers, diluents and adjuvants can include buffers such as phosphate, citrate, or other organic acids; antioxidants such as ascorbic acid; low molecular weight polypeptides (e.g., less than about 10 residues); proteins such as serum aAAVC.umin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and/or nonionic surfactants such as Tween™, Pluronics™ or polyethylene glycol (PEG). In some embodiments, the physiologically acceptable carrier is an aqueous pH buffered solution.
The AAV vectors of the present disclosure, and compositions containing the AAV vectors, may be used in methods for the introduction of a heterologous coding sequence into a host cell. Such methods involve contacting the host cell with the AAV vector. This may be performed in vitro, ex vivo or in vivo. In particular embodiments, the host cell is a hepatocyte (e.g. a human hepatocyte).
When the methods are performed ex vivo or in vivo, typically the introduction of the heterologous sequence into the host cell is for therapeutic purposes, whereby expression of the heterologous sequence results in the treatment of a disease or condition. Thus, the AAV vectors disclosed herein can be administered to a subject (e.g., a human) in need thereof, such as subject with a disease or condition amendable to treatment with a protein, peptide or polynucleotide encoded by a heterologous sequence described herein.
When used in vivo, titers of AAV vectors to be administered to a subject will vary depending on, for example, the particular recombinant virus, the disease or disorder to be treated, the mode of administration, the treatment goal, the individual to be treated, and the cell type(s) being targeted, and can be determined by methods well known to those skilled in the art. Although the exact dosage will be determined on an individual basis, in most cases, typically, recombinant viruses of the present disclosure can be administered to a subject at a dose of between 1×1010 genome copies of the recombinant virus per kg of the subject and 1×1014 genome copies per kg. In other examples, less than 1×1010 genome copies may be sufficient for a therapeutic effect. In other examples, more than 1×1014 genome copies may be required for a therapeutic effect.
The route of the administration is not particularly limited. For example, a therapeutically effective amount of the AAV vector can be administered to the subject via, for example, intramuscular, intravaginal, intravenous, intraperitoneal, subcutaneous, epicutaneous, intradermal, rectal, intraocular, pulmonary, intracranial, intraosseous, oral, buccal, or nasal routes. The AAV vector can be administrated as a single dose or multiple doses, and at varying intervals.
Also provided are methods for producing an AAV vector described above and herein, i.e. one comprising a capsid polypeptide of the present disclosure. Such methods comprise culturing a host cell comprising a nucleic acid molecule encoding a capsid polypeptide the present disclosure, an AAV rep gene, a heterologous coding sequence flanked by AAV inverted terminal repeats, and helper functions for generating a productive AAV infection, under conditions suitable to facilitate assembly of an AAV vector comprising a capsid polypeptide of the present disclosure, wherein the capsid encapsidates the heterologous coding sequence.
In further aspects, provided are methods for enhancing the in vivo human hepatocyte transduction efficiency of an AAV vector. As demonstrated herein, some variable regions, and combinations of capsid variable regions, are important for efficient transduction of human hepatocytes by an AAV vector. In particular, the presence of all or a part of VR-VII and/or VR-VIII from AAV7 in a capsid polypeptide imparts enhanced transduction by AAV vectors of a human hepatocyte in vivo. VR-I from AAV2 can also enhance the transduction by AAV vectors of a human hepatocyte in vivo.
Thus, provided herein are methods for enhancing the in vivo human hepatocyte transduction efficiency of an AAV vector (or producing an AAV vector with enhanced in vivo human hepatocyte transduction efficiency), which include the steps of modifying the sequence of a reference capsid polypeptide at one or more of positions 263, 264, 265, 268, 272, 546, 547, 549, 550, 551, 552, 553, 554, 555, 556, 558, 559, 561, 566, 567, 580, 581, 585, 586, 590, 592, 593, 594 and 597, with numbering relative to SEQ ID NO:13, to thereby produce a modified capsid polypeptide that comprises: i) amino acid residues S263, Q264, S265, S268 and H272, with numbering relative to SEQ ID NO:13; and ii) amino acid residues T546, G547, T549, N550, K551, T552, T553, L554, E555, N556, L558, M559, N561, R566 and P567, with numbering relative to SEQ ID NO:13; and/or amino acid residues S580, S581, A585, A586, A590, T592, Q593, V594, and N597, with numbering relative to SEQ ID NO:13. Additional modifications can optionally be made at or adjacent to one or more other variable regions, such as VR-IV, VR-V and VR-VI. For example, modifications can be made at one or more of positions 532, 538 and 540, with numbering relative to SEQ ID NO:13, wherein the modified capsid polypeptide comprises amino acid residues D532, S538 and V540, with numbering relative to SEQ ID NO:13. In another example, modifications can be at one or more of positions 451, 456, 457, 460, 462, 466, 469, 470, 472 and 473, with numbering relative to SEQ ID NO:13, wherein the modified capsid polypeptide comprises amino acid residues S451, Q456, G457, Q460, L462, A466, A469, N470, S472 and A473, with numbering relative to SEQ ID NO:13. In a further example, modifications can be made at one or more of positions 493, 494, 505, 506, 518 and 522, with numbering relative to SEQ ID NO:13, wherein the modified capsid polypeptide comprises amino acid residues L493, S494, G505, A506, V518 and V522, with numbering relative to SEQ ID NO:13.
Methods for enhancing the in vivo human hepatocyte transduction efficiency of an AAV vector (or producing an AAV vector with enhanced in vivo human hepatocyte transduction efficiency) also include those methods that include the steps of modifying the sequence of a reference capsid polypeptide at one or more of positions 263-272, 546-567 and 582-597 with numbering relative to SEQ ID NO:13, to thereby produce a modified capsid polypeptide that comprises: i) the sequence of amino acids SQSGASNDNH (SEQ ID NO:58) at positions 263-272, with numbering relative to SEQ ID NO:13; and ii) the sequence of amino acids TGATNKTTLENVLMTNEEEIRP (SEQ ID NO:59) at positions 546-567, with numbering relative to SEQ ID NO:13 and/or the sequence of amino acids SSNLQAANTAAQTQVVNN (SEQ ID NO:60) at positions 582-597, with numbering relative to SEQ ID NO:13.
Methods for enhancing the in vivo human hepatocyte transduction efficiency of an AAV vector (or producing an AAV vector with enhanced in vivo human hepatocyte transduction efficiency) also include those methods that include the steps of modifying the sequence of a reference capsid polypeptide at one or more of positions 261-272, 545-567 and 582-597 with numbering relative to SEQ ID NO:13, to thereby produce a modified capsid polypeptide that comprises: i) the sequence of amino acids ISSQSGASNDNH (SEQ ID NO:80) at positions 261-272, with numbering relative to SEQ ID NO:13; and ii) the sequence of amino acids KTGATNKTTLENVLMTNEEEIRP (SEQ ID NO:81) at positions 545-567, with numbering relative to SEQ ID NO:13 and/or the sequence of amino acids SSNLQAANTAAQTQVVNN (SEQ ID NO:60) at positions 582-597, with numbering relative to SEQ ID NO:13.
Additional modifications can optionally be made at or adjacent to one or more other variable regions, such as VR-IV, VR-V and VR-VI. For example, modifications can be made at one or more of positions 532-540, with numbering relative to SEQ ID NO:13, wherein the modified capsid polypeptide comprises the sequence of amino acids DRFFPSSGV (SEQ ID NO:61) at positions 532-540, with numbering relative to SEQ ID NO:13; at one or more of positions 523-540, with numbering relative to SEQ ID NO:13, wherein the modified capsid polypeptide comprises the sequence of amino acids AMATHKDDEDRFFPSSGV (SEQ ID NO:82) at positions 523-540, with numbering relative to SEQ ID NO:13; at one or more of positions 451-473, with numbering relative to SEQ ID NO:13, wherein the modified capsid polypeptide comprises the sequence of amino acids STGGTQGTQQLLFSQAGPANMSA (SEQ ID NO:62) at positions 451-473, with numbering relative to SEQ ID NO:1; at one or more of positions 450-473, with numbering relative to SEQ ID NO:13, wherein the modified capsid polypeptide comprises the sequence of amino acids QSTGGTQGTQQLLFSQAGPANMSA (SEQ ID NO:83) at positions 450-473, with numbering relative to SEQ ID NO:1; at one or more of positions 493-522, with numbering relative to SEQ ID NO:13, wherein the modified capsid polypeptide comprises the sequence of amino acids LSQNNNSNFAWTGATKYHLNGRNSLVNPGV (SEQ ID NO:63) at positions 493-522, with numbering relative to SEQ ID NO:13; and/or at one or more of positions 488-522, with numbering relative to SEQ ID NO:13, wherein the modified capsid polypeptide comprises the sequence of amino acids RVSTTLSQNNNSNFAWTGATKYHLNGRNSLVNPGV (SEQ ID NO:84) at positions 488-522, with numbering relative to SEQ ID NO:13.
It will be understood that any modification or combination of modifications, e.g. amino acid replacement or substitution, amino acid deletion and/or amino acid insertion, will result in a change of amino acid sequence in the modified capsid polypeptide compared to the reference capsid polypeptide. Thus, for example, reference to modification does not include within its scope amino acid substitutions where one amino acid residue is substituted with the same amino acid residue, or modifications when an amino acid deletion is accompanied by an insertion of that deleted amino acid, such that there is no difference in the amino acid sequence of the modified capsid polypeptide compared to the reference capsid polypeptide sequence, i.e. the amino acid sequence of the modified capsid polypeptide can not be the same as (or must be different to) the amino acid sequence of the reference capsid polypeptide sequence.
Typically, the methods include an initial step of first identifying a reference capsid polypeptide for transducing human hepatocytes in vivo. The reference capsid polypeptide may be any AAV polypeptide, such as an AAV1, AAV2, AAV3, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12 or AAV13 capsid polypeptide, or a synthetic or chimeric capsid polypeptide. In illustrative embodiments, the reference polypeptide comprises at least or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence set forth in SEQ ID NO:13. Reference capsid polypeptides include those comprising all or a portion of the VP1 protein, VP2 protein or VP3 protein. Thus, in some embodiments, the reference capsid polypeptide comprises all or a portion of a VP1 protein having at least or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence set forth in SEQ ID NO:13 (also referred to as AAVC11.12); all or a portion of a VP2 protein having at least or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the VP2 protein set forth as amino acids 138-735 of SEQ ID NO:13; and all or a portion of a VP3 protein having at least or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the VP3 protein set forth as amino acids 204-735 of SEQ ID NO:13.
Methods for modifying the sequence of a reference capsid polypeptide or polynucleotide so as to produce a modified capsid polypeptide or polynucleotide are well known in the art, and any such method can be utilised so as to perform the methods of the present disclosure. For example, the modification of the sequence of the reference capsid polynucleotide to produce a modified capsid polynucleotide can be performed using any method known in the art, including recombinant and synthetic methods, performed (either in part or in whole) in silico and/or in vitro. In a particular example, the modification of the sequence is performed in silico, followed by de novo synthesis of the modified capsid polynucleotide having the modified sequence (e.g. by gene synthesis methods such as those involving the chemical synthesis of overlapping oligonucleotides following by gene assembly).
The modified capsid polynucleotides may be contained in nucleic acid vector, such as a plasmid, for subsequent expression, replication, amplification and/or manipulation. Vectors suitable for use in bacterial, insect and mammalian cells are widely described and well-known in the art. Those skilled in the art would appreciate that the vectors may also contain additional sequences and elements useful for the replication of the vector in prokaryotic and/or eukaryotic cells, selection of the vector and the expression of a heterologous sequence in a variety of host cells. For example, the vectors can include a prokaryotic replicon, which is a sequence having the ability to direct autonomous replication and maintenance of the vector extrachromosomally in a prokaryotic host cell, such as a bacterial host cell. Such replicons are well known in the art. In some embodiments, the vectors can include a shuttle element that makes the vectors suitable for replication and integration in both prokaryotes and eukaryotes. In addition, vectors may also include a gene whose expression confers a detectable marker such as a drug resistance gene, which allows for selection and maintenance of the host cells. Vectors may also have a reportable marker, such as gene encoding a fluorescent or other detectable protein. The nucleic acid vectors will likely also comprise other elements, including any one or more of those described below. Most typically, the vectors will comprise a promoter operably linked to the nucleic acid encoding the capsid protein.
The nucleic acid vectors can be constructed using known techniques, including, without limitation, the standard techniques of restriction endonuclease digestion, ligation, transformation, plasmid purification, in vitro or chemical synthesis of DNA, and DNA sequencing. The vectors comprising a modified capsid polynucleotide may be introduced into a host cell using any method known in the art.
Following modification, the modified capsid are then vectorised. Methods for vectorising a capsid polypeptide are well known in the art and non-limiting examples are described above.
The AAV vector produced by these methods typically has an in vivo transduction efficiency that is enhanced compared to a reference AAV vector having a capsid comprising the reference capsid polypeptide. The transduction efficiency can be enhanced by at least or about, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900% 1000%, or more, e.g. the transduction efficiency of the AAV vector can be at least or about 2×, 3×, 4×, 5×, 6×, 7×, 8×, 9×, 10×, 12×, 13×, 14×, 15×, 16×, 17×, 18×, 19×, 20×, 30×, 40×, 50×, 60×, 70×, 80×, 90×, 100× or more efficient at transducing cells in vivo.
Thus, also provided are AAV vectors produced by the methods of the present disclosure.
In order that the invention may be readily understood and put into practical effect, particular preferred embodiments will now be described by way of the following non-limiting examples.
The reference in this specification to any prior publication (or information derived from it), or to any matter which is known, is not, and should not be taken as an acknowledgment or admission or any form of suggestion that that prior publication (or information derived from it) or known matter forms part of the common general knowledge in the field of endeavour to which this specification relates.
Parental AAV cap genes (AAV1 through 12, AAV-mAAV1 (WO2019227168) and AAV-EVE1 (WO2017192699) were cloned into the plasmid p-RescueVector (pRV 1-12), a construct based on the pGEM-T Easy Vector System (catalog [Cat] #A1360; Promega) modified to harbor trimethoprim resistance and randomized ends flanking the capsids, for optimal Gibson Assembly (GA). Individual clones were Sanger sequenced (Garvan Molecular Genetics). Capsid genes (serotypes 1-12) were excised using SwaI and NsiI (NEB), mixed at 1:1 molar ratio, and digested with 1:10 prediluted DNaseI (Cat #M030S; NEB) for 2-5 min. The pool of fragments was separated on a 1% (w/v) agarose gel and fragments ranging from 200 to 1,000 bp were recovered using the Zymoclean Gel DNA Recovery Kit (Cat #D4001T; Zymogen). For each primer-less PCR reassembly reaction, 500 ng of gel-extracted fragments was used, and fully reassembled capsids were amplified in a second PCR with primers (Shuffling Rescue-F/R, Table 3) binding the cap gene and carrying overlapping ends to pRV plasmids. A GA reaction was performed by mixing an equal volume of 2 GA Master Mix (Cat #E2611L; NEB) with 1 pmoL PCR-amplified and DpnI-treated pRV (BB GAR-F/R, Table 3) and 1 pmol of the recovered shuffled capsids, at 50° C. for 30 min. DNA was ethanol precipitated and electroporated into SS320 electrocompetent E. coli (Cat #60512-2; Lucigen). The total number of transformants was calculated by preparing and plating five 10-fold serial dilutions of the electroporated bacteria. The pool of transformants was grown overnight in 250 mL of Luria-Bertani media supplemented with trimethoprim (10 mg/mL). Total pRV library plasmids were purified with an EndoFree Maxiprep Kit (Cat #12362; QIAGEN). pRV-based libraries were then digested overnight with SwaI and NsiI, and 1.4 μg of insert was ligated at 16° C. with T4 DNA ligase (Cat #M0202; NEB) for 16 hr into 1 μg of a replication-competent AAV2-based plasmid platform (p-Replication-Competent [p-RC]) containing ITR-2 and rep2, and unique SwaI and NsiI sites flanking a 1-kb randomized stuffer [ITR2-rep2-(SwaI)-stuffer-(NsiI)-ITR2]. Ligation reactions were concentrated by using ethanol precipitation, electroporated into SS320 electro-competent bacteria, and grown as described above. Total pRC library plasmids were purified with an EndoFreeMaxiprep Kit (Cat #12362; QIAGEN).
A humanised FRG (hFRG) mouse was injected with 1×1011 vg of replication-competent RC-AAVC11 by i.v. tail vein administration. 5×109 PFUs of wild-type human adenovirus-5 (ATCC, VR-5, Lot #70010153) were administered intraperitoneally (i.p.) 24 hr later. The xenograft liver was harvested 72 hr after hAd5 administration, homogenised and snap frozen in liquid nitrogen. To extract AAV particles, approximately 0.3 g fragment of liver was subjected to three freeze-thaw cycles and mechanical homogenisation in the presence of 2× w/v of PBS. Sample was subsequently centrifuged for 30 min at 4° C. at top speed in a table-top centrifuge to separate the virus-containing supernatant from cellular debris. To inactivate wtAd5, the virus-containing supernatant was incubated at 65° C. for 30 min. Following titration by qPCR, 200 μL of the virus-containing supernatant was administrated i.p. into hFRG mouse for subsequent round of selection. A total of 5 rounds of selection were performed for this selection.
After round five of selection, AAV capsid sequences were recovered from the supernatant by PCR using primers flanking the capsid region (CapRescue-F/R, Table 3). PCR-amplified cap genes were cloned by GA in-frame downstream of the rep2 gene in a recipient pHelper packaging plasmid opened by PCR amplification using the following primers (pHelper-F/R) and DpnI treated. Individual clones containing full-length cap candidates were then Sanger sequenced.
AAV constructs were packaged into AAV capsids using HEK293 cells and a helper-virus-free system as previously described (Xiao et al, 1998 J Virol, 1998. 72(3): 2224-32). Genomes were packaged in capsid serotypes AAV2, AAV8, LK03 and NP59 using packaging plasmid constructs pAAV2, pAAV8, pLK03 and pAAVNP59, respectively. Replication-competent (RC) library AAVC11 was packaged by co-transfection of a corresponding plasmid containing the full-length AAV genome (ITR2-rep2-cap-ITR2) and pAd5 into HEK-293T cells.
All vector/virus were purified using iodixanol gradient ultracentrifugation as previously described (Khan et al. 2011. Nat Protoc, 2011. 6(4): p. 482-501). AAV preparations were titred using real-time quantitative PCR (qPCR) using eGFP-specific qPCR primers GFP-qPCR-For/Rev or AAV2-rep-specific qPCR primers Rep-qPCR-For/Rev (Table 3). For in vivo testing of capsid candidates (Example 2), n=4 independent barcoded transgenes were packaged per capsid using two different concentrations (n=2 barcoded transgenes at high dose: 10 μg/transgene per preparation, and n=2 barcoded transgenes at low dose: 1 μg/transgene per preparation). The presence of the two distinct populations was confirmed by next-generation sequencing of the pre-injection mix. For further comparisons, n=5 barcoded transgenes were packaged at increasing concentration by co-transfecting 2, 4, 8, 12 and 16 μg per barcode per preparation. NGS analysis of vector mix confirmed presence of the five barcoded populations per capsid.
All animal care and experimental procedures were approved by the joint Children's Medical Research Institute (CMRI) and The Children's Hospital at Westmead Animal Care and Ethics Committee. CMRI's established Fah−/−/Rag2−/−/Il2rg−/− (FRG) mouse colony was used to breed recipient animals. FRG mice were housed in individually ventilated cages with 2-(2-nitro-4-trifluoro-methyAAVC.enzoyl)-1,3-cyclohexanedione (NTBC)-supplemented in drinking water. FRG mice, 6 to 8 weeks old, were engrafted with human hepatocytes (Lonza Group Ltd., Basel, Switzerland) as described previously (Azuma et al., 2007, Nat Biotechnol. 25(8):903-10). Humanised FRG (hFRG) mice were placed on 10% NTBC 1 week prior to transduction with vectors and were maintained on 10% NTBC until harvest.
The vector for injection was made up to a final volume of 150 μL using saline. Mice were randomly selected and transduced by intravenous injection (lateral tail vein) with the indicated vectors at a dose of 1×1010 vg/vector for NGS comparison, and at a dose of 2×1011 vg/vector for immunohistochemistry. For in vivo IVIg screening, 5 mg or 20 mg of IVIg (Intragam 10, CSL Behring) were injected into hFRG (i.v.) 24h prior to vector injection. Mice were euthanized by CO2 inhalation 2 weeks after transduction for immunohistochemistry and 1 week after transduction for barcoded Next-Generation Sequencing (NGS) analysis. Hepatocytes for flow cytometry analysis were obtained by collagenase perfusion of the liver (see below).
To perfuse mouse liver and obtain single-cell suspension, the inferior vena cava (IVC) was cannulated, and the solutions were pumped with an osmotic minipump (Gilson Minipuls 3) in the following order: 25 mL of Hank's balanced salt solution (−/−) (−/−) (cat #H9394; Sigma), 25 mL of HBSS (−/−) supplemented with 0.5 mM EDTA, 25 ml HBSS (−/−), and 25 mL of HBSS (−/−) supplemented with 5 mM CaCl2), 0.05% wt/vol collagenase IV (Sigma) and 0.01% wt/vol DNase I (Sigma).
Following perfusion, the liver was carefully removed and placed in a Petri dish containing 25 ml of DuAAVC.ecco's modified Eagle's medium (DMEM) supplemented with 10% foetal bovine serum (FBS). The blunt end of a scalpel blade was used to break the liver capsule to release the cells into the medium. After collection, the cells were spun down at 50× g for 3 min at 4° C. The pellet was resuspended in 21 mL of DMEM and passed through a 100-μm nylon cell strainer. Isotonic Percoll (9 mL) (1 part of 10×PBS (−/−) with 9 parts of Percoll; GE Healthcare) was added to the cell suspension to separate live and dead cells. Live cells were pelleted at 650× g for 10 min at 4° C. and the pellet was resuspended in FACS buffer (PBS (−/−) with 5% FBS and 5 mM EDTA). To delineate between mouse liver cells and human hepatocytes, cells were labelled with phycoerythrin (PE)-conjugated anti-human-HLA-ABC (clone W6/32; Invitrogen 12-9983-42; 1:20), biotin-conjugated anti-mouse-H2Kb (done AF6-88.5, BD Pharmigen 553568; 1:100) and allophycocyanin (APC)-conjugated streptavidin (eBioscience 17-4317-82; 1:500). GFP-positive labelled samples were sorted to a minimal 95% purity using a BD Influx cell sorter. Sorting of the GFP-positive population was included to enrich for murine hepatocytes among non-parenchymal cells, given the hepatocyte-restricted expression of the pLSP1-GFP-WPRE-BGHpA AAV construct. Flow cytometry was performed in the Flow Cytometry Facility, Westmead Institute for Medical Research, Westmead, NSW, Australia. The data were analysed using FlowJo 7.6.1 (Flow®), LLC).
Human AAAVC.umin ELISA
Levels of human cell engraftment in chimeric mice were assessed by measuring presence of human aAAVC.umin on peripheral blood, using the Human AAAVC.umin ELISA Quantitation Kit (Bethyl, cat #E80-129) as previously reported (Azuma et al., 2007, Nat Biotechnol. 25(8):903-10).
AAV transgene constructs were cloned using standard molecular biological techniques. All of the vectors used in the study contain AAV2 ITR sequences. The AAV construct pLSP1-eGFP-WPRE-BGHpA, which encodes eGFP under the transcriptional control of a heterologous promoter containing one copy of the SERPINA1 (hAAT) promoter and two copies of the APOE enhancer element, has been previously reported (Dane et al., 2009, Mol Ther, 2009. 17(9): 1548-54). Eighty four (n=84) versions of the pLSP1-eGFP-BC-WPRE-BGHpA construct were produced by cloning n=84 unique 6-nucleotide-long barcodes (BC) downstream of eGFP.
To extract DNA from sorted cells, the cells were resuspended in 200 μL lysis buffer (100 mM Tris-HCl pH 8.5 (Astral Scientific, BioSD8141-450ML), 5 mM EDTA (ThermoFisher), 0.2% (w/v) sodium dodecyl sulphate (Sigma-Aldrich), 200 mM NaCl (Sigma-Aldrich) containing 50 μg/mL of proteinase K (Bioline). Samples were incubated overnight at 56° C. degrees. DNA was extracted using a standard phenol:chloroform protocol using phenol:chloroform:isoamyl alcohol (25:24:1) (Sigma-Aldrich), followed by DNA ethanol precipitation.
RNA from sorted cells was extracted using the Direct-Zol kit (Zymogen Cat #R2062) and treated with TURBO DNase (ThermoFsher, Cat #AM2238). cDNA was synthesised using the SuperScript IV First-Strand Synthesis System, following manufacturer's instructions (ThermoFisher, Cat #18091050).
HEK293 cells were validated and provided by ATCC. HuH-7 cells were provided by Dr Jerome Laurence (The University of Sydney). All cells were cultured in DuAAVC.ecco's Modified Eagle Medium (DMEM) (Gibco, 11965-092) supplemented with 10% FBS (Sigma Aldrich, F9423-500 mL, Lot #16K598), 100 Units/mL Penicillin, 100 μg/mL Streptomycin (Sigma Aldrich, P4458) and passaged using TrypLE Express Enzyme (Gibco, 12604-21). For HuH-7 cultures, media were supplemented also with non-essential amino acids (Gibco, 11140-050). AH cells were tested for mycoplasma and were mycoplasma-free. For transduction studies, cells were plated into 24-well plates in complete DMEM at 2×105 cells per well and incubated overnight in a tissue-culture incubator at 37° C./5% COD. 16 hrs later, the vector stock was diluted in 1 ml of complete DMEM and added to cells (at the indicated vector genome copies per cell (vac/cell). When indicated, serial 2-fold dilutions of intravenous immunoglobulin (IVIg) (Intragam 10, CSL Behring) were mixed with vectors for 1h at 37° C. prior to cell transduction.
After a 72-h incubation, the cells were harvested using TrypLE Express (Gibco) and analysed for GFP using BD LSRFortessa cell analyser. The data were analysed using FlowJo 7.6.1.
The 150 base pair region surrounding the 6-mer barcode was amplified with Q5 High-Fidelity DNA Polymerase (NEB, Cat #M0491L) using BC_F and BC_R primers (Table 3). Next-generation sequencing library preparations and sequencing using a 2×150 paired-end (PE) configuration were performed by Genewiz (Suzhou, China) using an Illumina MiSeq instrument. A workflow was written in Snakemake (5.6) (Koster et al. 2012 Bioinformatics 28:2520-2522) to process reads and count barcodes. Paired reads were merged using BBMerge and then filtered for reads of the expected length in a second pass through BBDuk, both from BBTools 38.68. The merged, filtered fastq files were passed to a Perl (5.26) script that identified barcodes corresponding to AAV variants.
Mouse livers were fixed with 4% (w/v) paraformaldehyde, cryo-protected in 10-30% (w/v) sucrose before freezing in O.C.T. (Tissue-Tek; Sakura Finetek USA, Torrance, Calif.). Frozen liver sections (5 μm) were permeabilised in −20° C. methanol, then room temperature 0.1% Triton X-100, and then reacted with anti-human GAPDH antibody (Abcam, Cat #ab215227, Clone AF674), and DAPI (Invitrogen, D1306) at 0.08 ng/mL. After immunolabelling, the images were captured and analysed on a Zeiss Axio Imager.M1 using ZEN 2 software. The percentage of transduced human hepatocytes per field of view was determined by counting total human GAPDH-positive cells and eGFP/human GAPDH double-positive cells.
When specified, clones were Sanger-sequenced at the Garvan Molecular Genetics facility of the Garvan Institute of Medical Research (Darlinghurst, NSW, Australia) with External_Seq_F/R primers (Table 3).
Vector copy numbers were measured with primers GFP-qPCR-For/Rev using Droplet Digital (dd)PCR (Bio-Rad, Berkeley, US) with QX200 ddPCR EvaGreen Supermix (Bio-Rad, Cat #1864034) and following manufacturer's instructions. Vector genomes were normalised to human aAAVC.umin copy number using primers human_AAAVC._F/R_ddPCR.
A shuffled DNA library was generated as described in Example 1. Replication-competent virus produced with the library were produced and injected into a hFRG mouse, and 5 rounds of selection were performed as described above to identify sixteen AAV capsid polypeptides: AAVC11.01 (SEQ ID NO:2), AAVC11.02 (SEQ ID NO:3), AAVC11.03 (SEQ ID NO:4), AAVC11.04 (SEQ ID NO:5), AAVC11.05 (SEQ ID NO:6), AAVC11.06 (SEQ ID NO:7), AAVC11.07 (SEQ ID NO:8), AAVC11.8 (SEQ ID NO:9), AAVC11.09 (SEQ ID NO:10), AAVC11.10 (SEQ ID NO:11), AAVC11.11 (SEQ ID NO:12), AAVC11.12 (SEQ ID NO:13), AAVC11.13 (SEQ ID NO:14), AAVC11.14 (SEQ ID NO:15), AAVC11.15 (SEQ ID NO:16), and AAVC11.16 (SEQ ID NO:17) (Table 4).
Four barcoded AAV transgenes (Liver Specific Promoter (LSP)-GFP-Barcode-WPRE-BGHpA) were packaged into each capsid (AAVC11.01- AAVC11.16 capsid, AAV2, AAV8, LK03 and NP59) to produce vectors. As the yield from AAVC11.03, AAVC11.10 and AAVC11.16 vectors was lower than that of AAV2, these were excluded from further testing. The remaining vectors were co-injected (1×1010 vg/capsid; a total of 1.8×1011 vg/capsid) into a hFRG mouse for comparison of function. One week after injection the chimeric liver from the mouse was perfused and human and murine hepatocytes were single cell sorted. DNA and RNA were recovered from the mouse and human populations of hepatocytes and NGS of the barcoded transgene was performed on the DNA and RNA (cDNA).
As shown in
AAVC11.01, AAVC11.04, AAVC11.05, AAVC11.06, AAVC11.07, AAVC11.09, AAVC11.11, AAVC11.12, AAVC11.13 and AAVC11.15, as well as AAV2, AAV8, LK03 and NP59, were re-packaged with 5× barcoded transgene/capsid at increasing barcode concentration with the aim of studying the ratio of DNA to RNA conversion. The AAV-DJ vector was also included as a titer control. For each capsid, 5×15 cm HEK293T plates (˜20M cells−15 mL media) were independently transfected, processed and titered.
The vectors (excluding AAV-DJ) were then mixed at equal ratio (1×1010 vg/capsid) and injected into a single hFRG mouse. Human and murine hepatocytes were isolated and sorted after one week. DNA and RNA were extracted and NGS performed on the DNA and cDNA. NGS of the pre-injection mix was also performed for validation, and the DNA and RNA (cDNA) reads from hepatocytes were normalized to pre-injection reads. This normalization is expressed as ‘Human Entry Index’ (HEI), which is a constant for each capsid on a determined experiment and expresses how efficient a given capsid is at physically transducing human hepatocytes in relation to the other capsids included in the experiment. It was observed that regardless of initial barcode concentration, the HEI for each capsid remained constant (data not shown).
cDNA reads were then normalized to DNA reads. This normalization is expressed as ‘Human Expression Index’ (HEXI), which is a constant for each capsid on a determined experiment and indicated how efficient a given capsid is at functionally transducing human hepatocytes, i.e. converting DNA reads into RNA reads. This is an important property, as some AAV capsids (e.g. AAV2) are relatively efficient at entering the hepatocytes but relatively deficient at functional transduction (i.e. transgene expression).
The HEI and HEXI were converted into a normalized percentage read to analyze the overall functional transduction power of the tested capsids. This data is shown in
It has been observed that the rate of DNA to RNA conversion follows a linear trend, with a slope corresponding to each specific HEXI (RNA/DNA). Non-normalized DNA reads vs non-normalized RNA reads were plotted, where the x-axis extension gives an estimate of how efficient a capsid is at human entry, and the slope gives the approximate ratio of DNA to RNA conversion. When doing such an analysis, it becomes apparent that AAV2 is relatively better than AAV8 at human entry, but AAV8 is relatively better than AAV2 at expression (functional transduction) (data not shown). This analysis was performed with NP59 and AAVC11.04, AAVC11.06, AAVC11.11, AAVC11.12 and AAVC11.13, and demonstrated that each of AAVC11.04, AAVC11.06, AAVC11.11, AAVC11.12 and AAVC11.13 is comparable to NP59, a highly efficient capsid described previously (Paulk et al., 2018, Mol Ther 26:289-303).
Having identified the most functional AAVC11 variants, their relative in vivo performance in human hepatocytes in the presence of pooled human immunoglobulins was investigated. To do so, following a method recently reported (Cabanes-Creus et al. 2020, Mol Ther Methods Clin Dev, 17:1139-1154), five barcoded AAV-LSP1-eGFP cassettes were packaged at increasing concentrations in the selected AAV variant capsids. AAV2, AAV8, AAV-LK03, and AAV-NP59 were included as controls. Three hFRG animals were passively immunized by intravenous administration of increasing doses of pooled human IgGs 24h before AAV administration (1×1010 vgs/capsid). A control hFRG animal that received no IVIg was also included (the same animal as used for the study shown in
The relative performance of the individual AAV variants in the human hepatocytes harvested from hFRG #1 (the no-IVIg control) was then analysed. As shown in
As a final validation, the top three performers (AAVC11.06, AAVC11.11, and AAVC11. 12) were injected into individual humanised FRG mice, using AAV-NP59 as a control (2×1011 vgs/hFRG). As shown in
AAVC11.12 and AAVC11.13 were injected into individual hFRG mice at 2×1011 vg/mouse. Livers were harvested two weeks after injection and processed for immunohistochemistry. DAPI (blue) was used to stain all cells (murine/human) and an antibody against human GAPDH (hGAPDH, red) was used to stain only human cells. eGFP (green) expressed from the AAV indicated cells that were functionally-transduced with rAAV. It was observed that AAVC11.12 and AAVC11.13 preferentially transduced human hepatocytes (data not shown).
The inventors then investigated whether relative transduction efficiency among AAV variants is dependent on the origin of the engrafted human hepatocytes. To do so, an equimolar mix was produced of barcoded AAVs that, in addition to AAVC11.12, contained prototypical variants (AAV2, AAV3b, AAV5, AAV8), bioengineered variants (AAV-LK03, AAV-NP59, AAV2-N496D (Cabanes-Creus et al. 2020, Mol Ther Methods Clin Dev, 17:1139-1154), AAV2-RC01 as well as the naturally occurring human variant AAV-hu.Lvr02 (Australian provisional patent no. 2020904687 and Cabanes-Creus et al. 2020, Sci Transl Med, 12(560):eaba3312). FRG mice were engrafted with hepatocytes from seventeen different human donors, varying in age, gender, and ethnicity (n=2 hFRGs per donor, n=1 for donor 13 and 16). The level of liver repopulation was assessed by measuring the concentration of human albumin in the blood, with the aim of performing the barcoded NGS-based comparison at mid-levels of engraftment (average of 3.6 mg human albumin/mL blood, which corresponds to a 20-60% level of human engraftment). Although there was an evident variability in the engraftment rate between donors, a positive correlation between the concentration of human albumin and the percentage of human hepatocytes in harvested livers was observed (data not shown). Each animal was injected i.v. with 1×1011 vg, which corresponds to a dose of 1×1010 vg per capsid variant. One-week post-injection, the chimeric livers were perfused, human GFP positive hepatocytes were sorted, and the vector copy number per cell and the barcode composition for each sample was analysed. It was observed that the AAV vector mix transduces human hepatocytes more efficiently than murine cells, as estimated by the respective GFP positive population in live cells (
It was observed that the three top capsids based on RNA reads (AAVC11.06, AAVC11.12, AAVC11.13) were part of a phylogenetic cluster. Four additional clones from the same selection that clustered with AAVC11.06, AAVC11.12 and AAVC11.13 were sequenced and named AAVC11.17 (SEQ ID NO:18), AAVC11.18 (SEQ ID NO:19), and AAVC11.19 (SEQ ID NO:20) (Table 5).
Phylogenetic analysis and analysis of the parental contribution was performed. As shown in
Given the substantially superior performance of AAVC11.12 when compared to other liver-tropic vectors, studies to investigate which capsid regions were the main determinants of human hepatocyte tropism in the hFRG model were performed. Due to the fact that AAVC11.12 was selected from a DNA-family shuffled library, it harbours regions of multiple parental variants (AAV1/AAV6, AAV2, AAV3b, AAV7, AAV10, and AAV12) as depicted in detail in
Given the differential performance of AAVC11.12 (SEQ ID NO:13) and AAV8 (SEQ ID NO:64) in human and murine cells and so as to understand which functional capsid domains are responsible for the superior function of AAVC11.12, a series of domain swaps between the two AAV was generated. As schematically shown in
Two independent barcoded-AAV NGS comparisons among these variants were then performed. In the first experiment (N=2 hFRGs, hFRG #1 and #2), AAVC11.12 and AAV8 were included as controls, as well as AAV8-Swaps1-7. As shown in
In the second comparison (N=2 hFRGs, hFRGs #3 and #4,
To validate these results, a multiplexed immunofluorescence comparison of AAV8+Swap5 and AAV8+Swap6 was performed in two independent hFRGs. Briefly, to allow visualisation of transduction patterns of two AAVs in the same animal, two AAV cassettes expressing the Cerulean or the Venus fluorescent reporters under the control of a liver-specific promoter were cloned. 1×1011 vg of AAV8-Cerulean with Swap5-Venus was mixed with AAV8-Cerulean with Swap6-Venus and injected into two independent hFRG mice. The immunofluorescence experiments confirmed the NGS results, with Swap5 transducing human hepatocytes substantially better than AAV8, and Swap6 displaying poor cell entry and strong expression in both human and murine hepatocytes (data not shown).
In a further validation of the results, the same barcoded mix from the first experiment (i.e. AAVC11.12 and AAV8, as well as AAV8-Swaps1-7) was injected in two highly engrafted mice. The highly engrafted mice had an average of 11 mg human albumin per mL blood, compared to the “low engraftment” mice from the previous experiments, which had an average of 1.8 mg human albumin per mL blood. The relative NGS reads mapped to each capsid were analyzed as previously for DNA and cDNA populations. As shown in
In summary, it appears that VR-VII (in particular) and VR-VIII, both from AAV7, alone or in combination, are important for efficient transduction of human hepatocytes (as evidenced by the reduction in transduction for Swap11 and Swap12 compared to Swap7). Conversely, it appears that VR-VI (also from AAV7) is dispensable for improving AAV8 performance in humans (see Swap5 compared to Swap10). VR-I, which is from AAV2, may be important for entry of human hepatocytes, such that the combination of the AAVC11.12 VR-I and VR-VII and/or VR-VIII appears to impart good entry of human hepatocytes and also good expression. In contrast, the combination present in Swap6, i.e. VR-I from AAV8, VR-IV and V from AAV10, and VR-VI, VR-VII and VR-VIII from AAV7, appears to impart much poorer entry into human hepatocytes but strong expression nonetheless, a phenotype that may have some advantages in the context of gene therapy (e.g. comparable expression with less physical transduction, potentially lessening concerns around DNA integration).
| Number | Date | Country | Kind |
|---|---|---|---|
| 2020900529 | Feb 2020 | AU | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/AU2021/050158 | 2/25/2021 | WO |
