Patent Application
20220280655
The present invention relates to new adeno-associated virus (AAV) variants and uses thereof for gene therapy.
Adeno-associated virus (AAV) is a small non-enveloped DNA virus composed by an icosahedral capsid that contains a 4.7 kb linear single-stranded genome.1, 2 AAV genome codes for non-structural proteins (Rep78, 68, 52 and 40), capsid proteins (VP1, VP2, VP3) and the assembly-activating protein (AAP).3, 4 At the extremities, inverse tandem repeats (ITR) are important for the integration in host genomes.5, 6 AAV is a defective virus requiring the presence of a helper virus for an active infection, otherwise it establishes a latent infection through integration into host genome or persistence as circular episomal form.7-9 AAV seroprevalence showed that the infection is endemic in human populations (35-80%) starting during childhood.10-12 Twelve distinct serotypes and more than 100 natural variants have been identified, among which AAV2 is the most frequent type in human.13-16
This small virus has attracted the attention of the gene therapy field because of the lack of identifiable associated disease and the remarkable ability of recombinant AAV (rAAV) vectors to transduce dividing and non-dividing cells with high efficiency, long-term transgene expression, low immunogenicity and specific tissue tropism.17 Although AAV discovery was performed in 1965, many questions regarding AAV infection remain unanswered.2, 18 It is well known that the vector predominantly persist in the nucleus as episomal form with sustained RNA expression raising question about the potential presence of episomal AAV in wild type infection.8 Several helper viruses have been identified but their precise association with wild type liver AAV infection remains unclear. Few data on the specific AAV genotypes in the population and on the frequency of AAV persistence after first infection are available.19
As defined by the claims, the present invention relates to new adeno-associated virus (AAV) variants and uses thereof for gene therapy.
Adeno-associated virus (AAV) is a defective mono-stranded DNA virus, endemic in human population (35-80%). Recurrent clonal AAV2 insertions are associated with the pathogenesis of rare human hepatocellular carcinoma (HCC) developed in normal liver. The aim of the inventors was to characterize the natural history of AAV infection in the liver. Viral DNA was thus quantified in tumor and non-tumor liver tissues of 1461 patients. Presence of episomal form and viral mRNA expression were analyzed using a DNAse/TaqMan based assay and quantitative RT-PCR. In silico analyses of viral capture data identified viral variants and new clonal insertions. AAV DNA was detected in 21% of the patients equally distributed in 2 major viral subtypes: one similar to AAV2, the other hybrid between AAV2 and AAV13 sequences. Thus, the inventors provided an integrated analysis of the wild type AAV infection in the liver with the identification of viral genotypes, molecular forms, helper virus relationship and viral integrations. These findings are important to understand wild type AAV biology. The data are particularly relevant considering the therapeutic potential of this new AAV variants in liver-targeted gene therapy.
As used herein, the term “polynucleotide” or “nucleic acid” refers to a polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides. Thus, this term includes, but is not limited to, single-, double- or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or a polymer comprising purine and pyrimidine bases, or other natural, chemically or biochemically modified, non-natural, or derivatized nucleotide bases. The backbone of the polynucleotide can comprise sugars and phosphate groups (as may typically be found in RNA or DNA), or modified or substituted sugars or phosphate groups. Alternatively, the backbone of the polynucleotide can comprise a polymer of synthetic subunits such as phosphoramidates and thus can be an oligodeoxynucleoside phosphoramidate (P—NH2) or a mixed phosphoramidate-phosphodiester oligomer. In addition, a double-stranded polynucleotide can be obtained from the single stranded polynucleotide product of chemical synthesis either by synthesizing the complementary strand and annealing the strands under appropriate conditions, or by synthesizing the complementary strand de novo using a DNA polymerase with an appropriate primer.
As used herein, the terms “polypeptide” and “protein” are used interchangeably to refer to a polymer of amino acid residues, and are not limited to a minimum length. Such polymers of amino acid residues may contain natural or non-natural amino acid residues, and include, but are not limited to, peptides, oligopeptides, dimers, trimers, and multimers of amino acid residues. Both full-length proteins and fragments thereof are encompassed by the definition. The terms also include post-expression modifications of the polypeptide, for example, glycosylation, sialylation, acetylation, phosphorylation, and the like. Furthermore, for purposes of the present invention, a “polypeptide” refers to a protein which includes modifications, such as deletions, additions, and substitutions (generally conservative in nature), to the native sequence, as long as the protein maintains the desired activity. These modifications may be deliberate, as through site-directed mutagenesis, or may be accidental, such as through mutations of hosts which produce the proteins or errors due to PCR amplification.
As used herein, “expression” refers to the process by which a polynucleotide is transcribed from a DNA template (such as into and mRNA or other RNA transcript) and/or the process by which a transcribed mRNA is subsequently translated into peptides, polypeptides, or proteins. Transcripts and encoded polypeptides may be collectively referred to as “gene product.”
As used herein the term “wild type” or “native” is a term of the art understood by skilled persons and means the typical form of an organism, strain, gene or characteristic as it occurs in nature as distinguished from mutant forms.
As used herein, the term “mutation” has its general meaning in the art and refers to a substitution, deletion or insertion. In particular, the term “substitution” means that a specific amino acid residue at a specific position is removed and another amino acid residue is inserted into the same position.
As used herein, the term “variant” refers to a first composition (e.g., a first molecule), that is related to a second composition (e.g., a second molecule, also termed a “parent” molecule). The variant molecule can be derived from, isolated from, based on or homologous to the parent molecule. A variant molecule can have entire sequence identity with the original parent molecule, or alternatively, can have less than 100% sequence identity with the parent molecule. For example, a variant of a sequence can be a second sequence that is at least 50; 51; 52; 53; 54; 55; 56; 57; 58; 59; 60; 61; 62; 63; 64; 65; 66; 67; 68; 69; 70; 71; 72; 73; 74; 75; 76; 77; 78; 79; 80; 81; 82; 83; 84; 85; 86; 87; 88; 89; 90; 91; 92; 93; 94; 95; 96; 97; 98; 99; 100% identical in sequence compared to the original sequence. Sequence identity is frequently measured in terms of percentage identity (or similarity or homology); the higher the percentage, the more similar are the two sequences. Methods of alignment of sequences for comparison are well known in the art. Various programs and alignment algorithms are described in: Smith and Waterman, Adv. Appl. Math., 2:482, 1981; Needleman and Wunsch, J. Mol. Biol., 48:443, 1970; Pearson and Lipman, Proc. Natl. Acad. Sci. U.S.A., 85:2444, 1988; Higgins and Sharp, Gene, 73:237-244, 1988; Higgins and Sharp, CABIOS, 5:151-153, 1989; Corpet et al. Nuc. Acids Res., 16:10881-10890, 1988; Huang et al., Comp. Appls Biosci., 8:155-165, 1992; and Pearson et al., Meth. Mol. Biol., 24:307-31, 1994). Altschul et al., Nat. Genet., 6:119-129, 1994, presents a detailed consideration of sequence alignment methods and homology calculations. By way of example, the alignment tools ALIGN (Myers and Miller, CABIOS 4:11-17, 1989) or LFASTA (Pearson and Lipman, 1988) may be used to perform sequence comparisons (Internet Program® 1996, W. R. Pearson and the University of Virginia, fasta20u63 version 2.0u63, release date December 1996). ALIGN compares entire sequences against one another, while LFASTA compares regions of local similarity. These alignment tools and their respective tutorials are available on the Internet at the NCSA Website, for instance. Alternatively, for comparisons of amino acid sequences of greater than about 30 amino acids, the Blast 2 sequences function can be employed using the default BLOSUM62 matrix set to default parameters, (gap existence cost of 11, and a per residue gap cost of 1). When aligning short peptides (fewer than around 30 amino acids), the alignment should be performed using the Blast 2 sequences function, employing the PAM30 matrix set to default parameters (open gap 9, extension gap 1 penalties). The BLAST sequence comparison system is available, for instance, from the NCBI web site; see also Altschul et al., J. Mol. Biol., 215:403-410, 1990; Gish. & States, Nature Genet., 3:266-272, 1993; Madden et al. Meth. Enzymol., 266:131-141, 1996; Altschul et al., Nucleic Acids Res., 25:3389-3402, 1997; and Zhang & Madden, Genome Res., 7:649-656, 1997.
As used herein, the term “adeno-associated virus” or “AAV” has its general meaning in the art and refers to a Dependoparvovirus within the Parvoviridae genus of viruses. The term can refer to an AAV derived from a naturally occurring “wild-type” virus, or an AAV derived from a rAAV genome packaged into a capsid derived from capsid proteins encoded by a naturally occurring cap gene. The term “AAV” includes AAV type 1 (AAV-1), AAV type 2 (AAV-2), AAV type 3 (AAV-3), AAV type 4 (AAV-4), AAV type 5 (AAV-5), AAV type 6 (AAV-6), AAV type 7 (AAV-7), AAV type 8 (AAV-8), avian AAV, bovine AAV, canine AAV, porcine AAV, equine AAV, primate AAV, non-primate AAV, and ovine AAV. “Primate AAV” refers to AAV that infect primates, “non-primate AAV” refers to AAV that infect non-primate mammals, “bovine AAV” refers to AAV that infect bovine mammals, etc. The genomic sequences of various serotypes of AAV, as well as the sequences of the native inverted terminal repeats (ITRs), Rep proteins, and capsid subunits are known in the art. Such sequences may be found in the literature or in public databases such as GenBank. See, e.g., GenBank Accession Numbers NC-002077 (AAV-1), AF063497 (AAV-1), NC-001401 (AAV-2), AF043303 (AAV-2), NC-001729 (AAV-3), NC-001829 (AAV-4), U89790 (AAV-4), NC-006152 (AAV-5), AF513851 (AAV-7), AF513852 (AAV-8), and NC-006261 (AAV-8); the disclosures of which are incorporated by reference herein for teaching AAV nucleic acid and amino acid sequences. See also, e.g., Srivistava et al. (1983) J. Virology 45:555; Chiorini et al. (1998) J. Virology 71:6823; Chiorini et al. (1999) J. Virology 73:1309; Bantel-Schaal et al. (1999) J. Virology 73:939; Xiao et al. (1999) J. Virology 73:3994; Muramatsu et al. (1996) Virology 221:208; Shade et al. (1986) J. Virol. 58:921; Gao et al. (2002) Proc. Nat. Acad. Sci. USA 99:11854; Moris et al. (2004) Virology 33:375-383; international patent publications WO 00/28061, WO 99/61601, WO 98/11244; and U.S. Pat. No. 6,156,303. The complete genome of AAV2 is represented by the reference sequence as set forth in SEQ ID NO:1.
As used herein, the term “cap gene” refers to the polynucleotide that encode capsid proteins that form, or contribute to the formation of, the capsid, or protein shell, of the virus. In the case of AAV2, the capsid protein may be VP1, VP2, or VP3. For other parvoviruses, the names and numbers of the capsid proteins can differ. The VP1 capsid protein of AAV2 is represented by the reference amino acid sequence as set forth in SEQ ID NO:2. The VP2 capsid protein of AAV2 is represented by the amino acid sequence that ranges from the amino acid residue at position 138 to the last amino acid residue in SEQ ID NO:2. The VP3 capsid protein of AAV2 is represented by the amino acid sequence that ranges from the amino acid residue at position 203 to the last amino acid residue in SEQ ID NO:2.
As used herein, the term “rep gene” refers to the nucleic acid sequences that encode the non-structural proteins (rep78, rep68, rep52 and rep40) required for the replication and production of virus.
As used herein, the term “packaging” refers to a series of intracellular events that result in the assembly and encapsidation of an AAV virus.
A “helper virus” for AAV refers to a virus that allows AAV (e.g. wild-type AAV) to be replicated and packaged by a mammalian cell. A variety of such helper viruses for AAV are known in the art, including adenoviruses, herpesviruses and poxviruses such as vaccinia. The adenoviruses encompass a number of different subgroups, although Adenovirus type 5 of subgroup C is most commonly used. Numerous adenoviruses of human, non-human mammalian and avian origin are known and available from depositories such as the ATCC. Viruses of the herpes family include, for example, herpes simplex viruses (HSV) and Epstein-Barr viruses (EBV), as well as cytomegaloviruses (CMV) and pseudorabies viruses (PRV); which are also available from depositories such as ATCC. “Helper virus function(s)” refers to function(s) encoded in a helper virus genome which allow AAV replication and packaging (in conjunction with other requirements for replication and packaging described herein). As described herein, “helper virus function” may be provided in a number of ways, including by providing helper virus or providing, for example, polynucleotide sequences encoding the requisite function(s) to a producer cell in trans. For example, a plasmid or other expression vector comprising nucleotide sequences encoding one or more adenoviral proteins is transfected into a producer cell along with nucleotidic sequences enconding for the AAV rep and cap genes and a transgene expression cassette flanked by two ITRs to produce a rAAV
As used herein, the term “virus viral particle” is one that comprises a competently assembled viral capsid and is capable of delivering a polynucleotide component into a cell for which the viral species is tropic. The term does not necessarily imply any replication capacity of the virus. Assays for counting infectious viral particles are described elsewhere in this disclosure and in the art. Viral infectivity can be expressed as the ratio of infectious viral particles to total viral particles. Methods of determining the ratio of infectious viral particle to total viral particle are known in the art. See, e.g., Grainger et al. (2005) Mol. Ther. 11:S337 (describing a TCID50 infectious titer assay); and Zolotukhin et al. (1999) Gene Ther. 6:973. See also the Examples.
As used herein, the term “transgene” refers to a polynucleotide that is introduced into a cell and is capable of being transcribed into RNA and optionally, translated and/or expressed under appropriate conditions. In aspects, it confers a desired property to a cell into which it is introduced, or otherwise leads to a desired therapeutic outcome. In another aspect, it may be transcribed into a molecule that mediates RNA interference, such as miRNA, siRNA, or shRNA.
As used herein, the term “control element” or “control sequence” is a nucleotide sequence involved in an interaction of molecules that contributes to the functional regulation of a polynucleotide, including replication, duplication, transcription, splicing, translation, or degradation of the polynucleotide. The regulation may affect the frequency, speed, or specificity of the process, and may be enhancing or inhibitory in nature. Control elements known in the art include, for example, transcriptional regulatory sequences such as promoters and enhancers. A promoter is a DNA region capable under certain conditions of binding RNA polymerase and initiating transcription of a coding region usually located downstream (in the 3′ direction) from the promoter.
As used herein, the term “operatively linked” or “operably linked” refers to a juxtaposition of genetic elements, wherein the elements are in a relationship permitting them to operate in the expected manner. For instance, a promoter is operatively linked to a coding region if the promoter helps initiate transcription of the coding sequence. There may be intervening residues between the promoter and coding region so long as this functional relationship is maintained.
As used herein, the term “heterologous” means derived from a genotypically distinct entity from that of the rest of the entity to which it is being compared. For example, a polynucleotide introduced by genetic engineering techniques into a plasmid or vector derived from a different species is a heterologous polynucleotide. A promoter removed from its native coding sequence and operatively linked to a coding sequence with which it is not naturally found linked is a heterologous promoter. Thus, for example, an rAAV that includes a heterologous nucleic acid encoding a heterologous gene product is an rAAV that includes a nucleic acid not normally included in a naturally-occurring, wild-type AAV, and the encoded heterologous gene product is a gene product not normally encoded by a naturally-occurring, wild-type AAV.
As used herein, the term “isolated” when referred to a plasmid, nucleic acid, vector, virus, virion, host cell, or other substance refers to a preparation of the substance devoid of at least some of the other components that may also be present where the substance or a similar substance naturally occurs or is initially prepared from. Thus, for example, an isolated substance may be prepared by using a purification technique to enrich it from a source mixture. Enrichment can be measured on an absolute basis, such as weight per volume of solution, or it can be measured in relation to a second, potentially interfering substance present in the source mixture. Increasing enrichments of the embodiments of this disclosure are increasingly more isolated. An isolated plasmid, nucleic acid, vector, virus, host cell, or other substance is in some embodiments purified, e.g., from about 80% to about 90% pure, at least about 90% pure, at least about 95% pure, at least about 98% pure, or at least about 99%, or more, pure.
The term “Pharmaceutically” or “pharmaceutically acceptable” refer to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to a mammal, especially a human, as appropriate.
By a “therapeutically effective amount” is meant a sufficient amount of the rAAV of the present invention to treat the disease at a reasonable benefit/risk ratio. It will be understood that the total daily usage of the rAAV of the present invention will be decided by the attending physician within the scope of sound medical judgment. The specific therapeutically effective dose level for any particular patient will depend upon a variety of factors including the age, body weight, general health, sex and diet of the patient; the time of administration, route of administration, and rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination or coincidental with the specific polypeptide employed; and like factors well known in the medical arts. For example, it is well known within the skill of the art to start doses of the compound at levels lower than those required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved. Thus, the doses of vectors may be adapted depending on the disease condition, the subject (for example, according to his weight, metabolism, etc.), the treatment schedule, etc. A preferred effective dose within the context of this invention is a dose allowing an optimal transduction of the cone photoreceptors. Typically, from 108 to 1010 viral genomes (vg) are administered per dose in mice. Typically, the doses of AAV to be administered in humans may range from 109 to 1012 vg.
Variant AAV2 Capsid Protein:
An object of the present invention relates to a variant VP1 capsid protein that consists of the amino acid sequence as set forth in SEQ ID NO:2 comprising at least one mutation of the table 1.
An object of the present invention relates to a variant VP2 capsid protein that consists of the amino acid sequence that ranges from the amino acid at position 138 to the last amino acid in SEQ ID NO:2 comprising at least one mutation of the table 1.
An object of the present invention relates to a variant VP3 capsid protein that consists of the amino acid sequence that ranges from the amino acid at position 203 to the last amino acid in SEQ ID NO:2 comprising at least one mutation of the table 1.
An object of the present invention relates to a variant VP1 capsid protein that consists of the amino acid sequence as set forth in SEQ ID NO:2 comprising at least one mutation selected from the group consisting of V151A, Q164N; T200S; N201T; M211V; T233Q; M235L; T410Q; S446N; R447K; N449Q; T450S; P451N; T455L; T456Q; Q461L; A467P; S468T; D469S; I470M; R471S; D472L; S474A; R475K; V488L; T491Q; S492A; A493N; E499N; Y500F; S501P; G504A; Q536M; S537H; V539T; S547T; E548N; K549A; T550N; N551D; V552A; I554L; E555D; K556N; S578Y; T581N; R585N; R585S; G586S; R588T; Q589G; A590P; A591T; A593S; A593G; D594T; T597H; V600A; L647M; S658P; T660N; and A663S.
In some embodiments, the variant VP1 capsid protein of the present invention has an amino acid sequence selected from the group consisting of SEQ ID NO: 3 to SEQ ID NO:61.
A further object of the present invention relates to a variant VP1 capsid protein of the present invention has an amino acid sequence selected from the group consisting of SEQ ID NO:26 (i.e. capsid #3013), SEQ ID NO:9 (i.e. capsid #2087), SEQ ID NO: 20 (i.e. capsid #1449), SEQ ID NO: 10 (i.e. capsid #2206), SEQ ID NO: 12 (i.e. capsid #1534), SEQ ID NO: 56 (i.e. capsid #163), SEQ ID NO: 23 (i.e. capsid #1343), SEQ ID NO: 53 (i.e. capsid #790), SEQ ID NO: 6 (i.e. capsid #1017), SEQ ID NO: 11 (i.e. capsid #877), SEQ ID NO:8 (i.e. capsid #1273), SEQ ID NO: 3 (i.e. capsid #2497), SEQ ID NO: 60 (i.e. capsid #1055), SEQ ID NO:14 (i.e. capsid #1919), SEQ ID NO: 4, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:13, SEQ ID NO:15 to SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:27 to SEQ ID NO:55, SEQ ID NO:57 to SEQ ID NO:59, SEQ ID NO:61.
A further object of the present invention relates to a variant VP2 capsid protein that consists of the amino acid sequence that ranges from the amino acid at position 138 to the last amino acid in SEQ ID NO:2 comprising at least one mutation selected from the group consisting of V151A, Q164N; T200S; N201T; M211V; T233Q; M235L; T410Q; S446N; R447K; N449Q; T450S; P451N; T455L; T456Q; Q461L; A467P; S468T; D469S; I470M; R471S; D472L; S474A; R475K; V488L; T491Q; S492A; A493N; E499N; Y500F; S501P; G504A; Q536M; S537H; V539T; S547T; E548N; K549A; T550N; N551D; V552A; I554L; E555D; K556N; S578Y; T581N; R585N; R585S; G586S; R588T; Q589G; A590P; A591T; A593S; A593G; D594T; T597H; V600A; L647M; S658P; T660N; and A663S
In some embodiments, the variant VP2 capsid protein of the present invention has an amino acid sequence that ranges from the amino acid at position 138 to the last amino acid in SEQ ID NO:3 to SEQ ID NO:61.
A further object of the present invention relates to a variant VP2 capsid protein of the present invention has an amino acid sequence that ranges from the amino acid at position 138 to the last amino acid in SEQ ID NO:26 (i.e. capsid #3013), SEQ ID NO:9 (i.e. capsid #2087), SEQ ID NO: 20 (i.e. capsid #1449), SEQ ID NO: 10 (i.e. capsid #2206), SEQ ID NO: 12 (i.e. capsid #1534), SEQ ID NO: 56 (i.e. capsid #163), SEQ ID NO: 23 (i.e. capsid #1343), SEQ ID NO: 53 (i.e. capsid #790), SEQ ID NO: 6 (i.e. capsid #1017), SEQ ID NO: 11 (i.e. capsid #877), SEQ ID NO:8 (i.e. capsid #1273), SEQ ID NO: 3 (i.e. capsid #2497), SEQ ID NO: 60 (i.e. capsid #1055), SEQ ID NO:14 (i.e. capsid #1919), SEQ ID NO: 4, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:13, SEQ ID NO:15 to SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:27 to SEQ ID NO:55, SEQ ID NO:57 to SEQ ID NO:59, SEQ ID NO:61.
A further object of the present invention relates to a variant VP3 capsid protein that consists of the amino acid sequence that ranges from the amino acid at position 203 to the last amino acid in SEQ ID NO:2 comprising at least one mutation selected from the group consisting of M211V; T233Q; M235L; T410Q; S446N; R447K; N449Q; T450S; P451N; T455L; T456Q; Q461L; A467P; S468T; D469S; I470M; R471S; D472L; S474A; R475K; V488L; T491Q; S492A; A493N; E499N; Y500F; S501P; G504A; Q536M; S537H; V539T; S547T; E548N; K549A; T550N; N551D; V552A; I554L; E555D; K556N; S578Y; T581N; R585N; R585S; G586S; R588T; Q589G; A590P; A591T; A593S; A593G; D594T; T597H; V600A; L647M; S658P; T660N; and A663S
In some embodiments, the variant VP3 capsid protein of the present invention has an amino acid sequence that ranges from the amino acid at position 203 to the last amino acid in SEQ ID NO:3 to SEQ ID NO:61.
A further object of the present invention relates to a variant VP3 capsid protein of the present invention has an amino acid sequence that ranges from the amino acid at position 203 to the last amino acid in SEQ ID NO:26 (i.e. capsid #3013), SEQ ID NO:9 (i.e. capsid #2087), SEQ ID NO: 20 (i.e. capsid #1449), SEQ ID NO: 10 (i.e. capsid #2206), SEQ ID NO: 12 (i.e. capsid #1534), SEQ ID NO: 56 (i.e. capsid #163), SEQ ID NO: 23 (i.e. capsid #1343), SEQ ID NO: 53 (i.e. capsid #790), SEQ ID NO: 6 (i.e. capsid #1017), SEQ ID NO: 11 (i.e. capsid #877), SEQ ID NO:8 (i.e. capsid #1273), SEQ ID NO: 3 (i.e. capsid #2497), SEQ ID NO: 60 (i.e. capsid #1055), SEQ ID NO:14 (i.e. capsid #1919), SEQ ID NO: 4, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:13, SEQ ID NO:15 to SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:27 to SEQ ID NO:55, SEQ ID NO:57 to SEQ ID NO:59, SEQ ID NO:61.
Polynucleotides that Encodes for the Variant Capsid Proteins:
A further object of the present invention relates to a polynucleotide that encodes for a variant capsid protein of the present invention.
In some embodiments, the polynucleotide of the present invention that encodes for a variant VP1 capsid of the present invention consists of the nucleic sequence that ranges from the nucleotide at position 2203 to the nucleotide at position 4410 in SEQ ID NO:1 and that comprises at least one mutation selected from Table 1.
In some embodiments, the polynucleotide of the present invention that encodes for a variant VP1 capsid of the present invention consists of the nucleic sequence that ranges from the nucleotide at position 2203 to the nucleotide at position 4410 in SEQ ID NO:1 and that comprises at least one mutation selected from the group consisting of 2433A>G; 2511C>T; 2654T>C; 2685G>A; 2692CAG>AAC; 2721T>C; 2745A>C; 2794C>T; 2800A>T; 2804A>C; 2808G->A; 2833A>G; 2853C>T; 2859C>T; 2877G>A; 2899AC>CA; 2905A>C; 2946C>T; 2967C>T; 2970C>G; 2976A>G; 2979A>G; 2982T>C; 3003G>A; 3012T>C; 3036T>C; 3123A>G; 3129G>A; 3165A>G; 3297C>T; 3303A>G; 3318G>C; 3360A>G; 3375A>C; 3411G>T; 3417C>T; 3430ACC>CAG; 3444T>C; 3450G>A; 3490C>T; 3495C>T; 3498T>G; 3501C>G; 3513C>G; 3534C>T; 3535T>C; 3539G>A; 3542GA>AG; 3547AAC>CAA; 3550ACT>TCA; 3553CCA>AAT; 3558T>C; 3564C>T; 3565ACC>CTT; 3568AC>CA; 3576A>C; 3576A>T; 3577A>C; 3582T>A; 3584A>T; 3589TC>AG; 3594G>A; 3597C>T3601GCG>CCC 3605GT>CC 3607GA>AG 3612T>G 3613CGG>TCT 3616GAC>CTT 3621G>A 3622T>G 3626GG>AA 3636T>G 3645C>T 3648T>C 3652CGC>AGA 3663A>T 3664GTA>CTG 3672ACA>CAG 3676TCT>GCA 3679GCG>AAC 3684T>C 3696T>C 3697GAA>AAC 3701AC>TT 3703TCG>CCC 3713GA>CG 3720C>A 3726C>T 3729C>T 3732C>A 3739AGA>CGG 3747T>G 3748C>T 3753G>T 3759G>A 3762C>A 3765G>A 3768C>T 3774A>C 3783G>A 3804T>C 3807T>C 3808CA>AT 3811AGC>CAT 3816G>A 3817GTT>ACC 3822C>G 3825C>A 3831G>A 3834G>A 3840C>A 3841T>A 3844GAG>AAT 3847AAA>GCT 3851CA>AC 3853AAT>GAC 3857T>C 3861C>T 3862ATT>TTG 3867A>C 3870>T 3885C>T 3891G>A 3898AGG>CGC 3903A>C 3930T>C 3933T>A 3935C>A 3939A>G 3942T>A 3944CC>AT 3948C>T 3949CTC>TTG 3954G>A 3956GA>AC 3957A>C 3958GGC>TCA 3963C>T 3965G>C 3967CAA>GGT 3970G>C 3973G>A 3978C>T 3979G>T 3980C>G 3982GA>AC 3990C>T 3991ACA>CAC 3999C>A 4001TT>CG 4003CTT>TTA 4008A>T 4011C>T 4017C>G 4026C>T 4027A>C 4032T>C 4041T>G 4047G>A 4053C>T 4059A>C 4068A>T 4074G>C 4077C>T 4083T>C 4089C>T 4092C>T 4098C>A 4101C>G 4107T>C 4110A>T 4113C>T 4116A>G 4119T>G 4128T>G 4134A>G 4140T>C 4141CTC>ATG 4149G>A 4155C>T 4158G>C 4161A>T 4164T>A 4167G>C 4174TCG>CCC 4179C>A 4181C>A 4189GCG>TCT 4194A>C 4206C>T 4221C>T 4230A>G 4269A>G 4287T>C 4293A>G 4296T>C 4320G>A and 4356C>T.
In some embodiments, the polynucleotide of the present invention that encodes for a variant VP1 capsid of the present invention consists the nucleic sequence that ranges from the nucleotide at position 2203 to the nucleotide at position 4410 in SEQ ID NO: 62; 63; 64; 65; 66; 67; 68; 69; 70; 71; 72; 73; 74; 75; 76; 77; 78; 79; 80; 81; 82; 83; 84; 85; 86; 87; 88; 89; 90; 91; 92; 93; 94; 95; 96; 97; 98; 99; 100; 101; 102; 103; 104; 105; 106; 107; 108; 109; 110; 111; 112; 113; 114; 115; 116; 117; or 118.
In some embodiments, the polynucleotide of the present invention that encodes for a variant VP1 capsid of the present invention consists the nucleic sequence that ranges from the nucleotide at position 2203 to the nucleotide at position 4410 in SEQ ID NO: 85 (i.e. capsid #3013), SEQ ID NO: 68 (i.e. capsid #2087), SEQ ID NO: 79 (i.e. capsid #1449), SEQ ID NO: 69 (i.e. capsid #2206), SEQ ID NO: 71 (i.e. capsid #1534), SEQ ID NO: 115 (i.e. capsid #163), SEQ ID NO: 82 (i.e. capsid #1343), SEQ ID NO: 112 (i.e. capsid #790), SEQ ID NO:65 (i.e. capsid #1017), SEQ ID NO: 70 (i.e. capsid #877), SEQ ID NO: 67 (i.e. capsid #1273), SEQ ID NO: 62 (i.e. capsid #2497); SEQ ID NO: 119 (i.e. capsid #1055); SEQ ID NO: 73 (i.e. capsid #1919), SEQ ID NO:63, SEQ ID NO:64; SEQ ID NO:66, SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO:80, SEQ ID NO:81, SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:86, SEQ ID NO:87; SEQ ID NO:88; SEQ ID NO:89; SEQ ID NO:90; SEQ ID NO: 91; SEQ ID NO:92; SEQ ID NO:93; SEQ ID NO:94; SEQ ID NO:95; SEQ ID NO:96; SEQ ID NO:97; SEQ ID NO:98, SEQ ID NO: 99; SEQ ID NO:100; SEQ ID NO:101; SEQ ID NO:102; SEQ ID NO:103, SEQ ID NO:104; SEQ ID NO:105, SEQ ID NO: 106; SEQ ID NO:107, SEQ ID NO:108, SEQ ID NO:109, SEQ ID NO: 110; SEQ ID NO:111, SEQ ID NO:113; SEQ ID NO:114; SEQ ID NO:116, SEQ ID NO: 117; SEQ ID NO:118 or SEQ ID NO:120.
In some embodiments, the polynucleotide of the present invention that encodes for a variant VP2 capsid of the present invention consists of the nucleic sequence that ranges from the nucleotide at position 2614 to the nucleotide at position 4410 in SEQ ID NO:1 and that comprises at least one mutation selected from Table 1.
In some embodiments, the polynucleotide of the present invention that encodes for a variant VP2 capsid of the present invention consists of the nucleic sequence that ranges from the nucleotide at position 2614 to the nucleotide at position 4410 in SEQ ID NO:1 and that comprises at least one mutation selected from the group consisting of 2654T>C; 2685G>A; 2692CAG>AAC; 2721T>C; 2745A>C; 2794C>T; 2800A>T; 2804A>C; 2808G->A; 2833A>G; 2853C>T; 2859C>T; 2877G>A; 2899AC>CA; 2905A>C; 2946C>T; 2967C>T; 2970C>G; 2976A>G; 2979A>G; 2982T>C; 3003G>A; 3012T>C; 3036T>C; 3123A>G; 3129G>A; 3165A>G; 3297C>T; 3303A>G; 3318G>C; 3360A>G; 3375A>C; 3411G>T; 3417C>T; 3430ACC>CAG; 3444T>C; 3450G>A; 3490C>T; 3495C>T; 3498T>G; 3501C>G; 3513C>G; 3534C>T; 3535T>C; 3539G>A; 3542GA>AG; 3547AAC>CAA; 3550ACT>TCA; 3553CCA>AAT; 3558T>C; 3564C>T; 3565ACC>CTT; 3568AC>CA; 3576A>C; 3576A>T; 3577A>C; 3582T>A; 3584A>T; 3589TC>AG; 3594G>A; 3597C>T3601GCG>CCC 3605GT>CC 3607GA>AG 3612T>G 3613CGG>TCT 3616GAC>CTT 3621G>A 3622T>G 3626GG>AA 3636T>G 3645C>T 3648T>C 3652CGC>AGA 3663A>T 3664GTA>CTG 3672ACA>CAG 3676TCT>GCA 3679GCG>AAC 3684T>C 3696T>C 3697GAA>AAC 3701AC>TT 3703TCG>CCC 3713GA>CG 3720C>A 3726C>T 3729C>T 3732C>A 3739AGA>CGG 3747T>G 3748C>T 3753G>T 3759G>A 3762C>A 3765G>A 3768C>T 3774A>C 3783G>A 3804T>C 3807T>C 3808CA>AT 3811AGC>CAT 3816G>A 3817GTT>ACC 3822C>G 3825C>A 3831G>A 3834G>A 3840C>A 3841T>A 3844GAG>AAT 3847AAA>GCT 3851CA>AC 3853AAT>GAC 3857T>C 3861C>T 3862ATT>TTG 3867A>C 3870>T 3885C>T 3891G>A 3898AGG>CGC 3903A>C 3930T>C 3933T>A 3935C>A 3939A>G 3942T>A 3944CC>AT 3948C>T 3949CTC>TTG 3954G>A 3956GA>AC 3957A>C 3958GGC>TCA 3963C>T 3965G>C 3967CAA>GGT 3970G>C 3973G>A 3978C>T 3979G>T 3980C>G 3982GA>AC 3990C>T 3991ACA>CAC 3999C>A 4001TT>CG 4003CTT>TTA 4008A>T 4011C>T 4017C>G 4026C>T 4027A>C 4032T>C 4041T>G 4047G>A 4053C>T 4059A>C 4068A>T 4074G>C 4077C>T 4083T>C 4089C>T 4092C>T 4098C>A 4101C>G 4107T>C 4110A>T 4113C>T 4116A>G 4119T>G 4128T>G 4134A>G 4140T>C 4141CTC>ATG 4149G>A 4155C>T 4158G>C 4161A>T 4164T>A 4167G>C 4174TCG>CCC 4179C>A 4181C>A 4189GCG>TCT 4194A>C 4206C>T 4221C>T 4230A>G 4269A>G 4287T>C 4293A>G 4296T>C 4320G>A and 4356C>T.
In some embodiments, the polynucleotide of the present invention that encodes for a variant VP2 capsid of the present invention consists of the nucleic sequence that ranges from the nucleotide at position 2614 to the nucleotide at position 4410 in SEQ ID NO: 62; 63; 64; 65; 66; 67; 68; 69; 70; 71; 72; 73; 74; 75; 76; 77; 78; 79; 80; 81; 82; 83; 84; 85; 86; 87; 88; 89; 90; 91; 92; 93; 94; 95; 96; 97; 98; 99; 100; 101; 102; 103; 104; 105; 106; 107; 108; 109; 110; 111; 112; 113; 114; 115; 116; 117; or 118.
In some embodiments, the polynucleotide of the present invention that encodes for a variant VP2 capsid of the present invention consists of the nucleic sequence that ranges from the nucleotide at position 2614 to the nucleotide at position 4410 in SEQ ID NO: 85 (i.e. capsid #3013), SEQ ID NO: 68 (i.e. capsid #2087), SEQ ID NO: 79 (i.e. capsid #1449), SEQ ID NO: 69 (i.e. capsid #2206), SEQ ID NO: 71 (i.e. capsid #1534), SEQ ID NO: 115 (i.e. capsid #163), SEQ ID NO: 82 (i.e. capsid #1343), SEQ ID NO: 112 (i.e. capsid #790), SEQ ID NO:65 (i.e. capsid #1017), SEQ ID NO: 70 (i.e. capsid #877), SEQ ID NO: 67 (i.e. capsid #1273), SEQ ID NO: 62 (i.e. capsid #2497); SEQ ID NO: 119 (i.e. capsid #1055); SEQ ID NO: 73 (i.e. capsid #1919), SEQ ID NO:63, SEQ ID NO:64; SEQ ID NO:66, SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO:80, SEQ ID NO:81, SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:86, SEQ ID NO:87; SEQ ID NO:88; SEQ ID NO:89; SEQ ID NO:90; SEQ ID NO: 91; SEQ ID NO:92; SEQ ID NO:93; SEQ ID NO:94; SEQ ID NO:95; SEQ ID NO:96; SEQ ID NO:97; SEQ ID NO:98, SEQ ID NO: 99; SEQ ID NO:100; SEQ ID NO:101; SEQ ID NO:102; SEQ ID NO:103, SEQ ID NO:104; SEQ ID NO:105, SEQ ID NO: 106; SEQ ID NO:107, SEQ ID NO:108, SEQ ID NO:109, SEQ ID NO: 110; SEQ ID NO:111, SEQ ID NO:113; SEQ ID NO:114; SEQ ID NO:116, SEQ ID NO: 117; SEQ ID NO:118 or SEQ ID NO:120
In some embodiments, the polynucleotide of the present invention that encodes for a variant VP3 capsid of the present invention consists of the nucleic sequence that ranges from the nucleotide at position 2809 to the nucleotide at position 4410 in SEQ ID NO:1 and that comprises at least one mutation selected from Table 1.
In some embodiments, the polynucleotide of the present invention that encodes for a variant VP3 capsid of the present invention consists of the nucleic sequence that ranges from the nucleotide at position 2809 to the nucleotide at position 4410 in SEQ ID NO:1 and that comprises at least one mutation selected from the group consisting of 2833A>G; 2853C>T; 2859C>T; 2877G>A; 2899AC>CA; 2905A>C; 2946C>T; 2967C>T; 2970C>G; 2976A>G; 2979A>G; 2982T>C; 3003G>A; 3012T>C; 3036T>C; 3123A>G; 3129G>A; 3165A>G; 3297C>T; 3303A>G; 3318G>C; 3360A>G; 3375A>C; 3411G>T; 3417C>T; 3430ACC>CAG; 3444T>C; 3450G>A; 3490C>T; 3495C>T; 3498T>G; 3501C>G; 3513C>G; 3534C>T; 3535T>C; 3539G>A; 3542GA>AG; 3547AAC>CAA; 3550ACT>TCA; 3553CCA>AAT; 3558T>C; 3564C>T; 3565ACC>CTT; 3568AC>CA; 3576A>C; 3576A>T; 3577A>C; 3582T>A; 3584A>T; 3589TC>AG; 3594G>A; 3597C>T3601GCG>CCC 3605GT>CC 3607GA>AG 3612T>G 3613CGG>TCT 3616GAC>CTT 3621G>A 3622T>G 3626GG>AA 3636T>G 3645C>T 3648T>C 3652CGC>AGA 3663A>T 3664GTA>CTG 3672ACA>CAG 3676TCT>GCA 3679GCG>AAC 3684T>C 3696T>C 3697GAA>AAC 3701AC>TT 3703TCG>CCC 3713GA>CG 3720C>A 3726C>T 3729C>T 3732C>A 3739AGA>CGG 3747T>G 3748C>T 3753G>T 3759G>A 3762C>A 3765G>A 3768C>T 3774A>C 3783G>A 3804T>C 3807T>C 3808CA>AT 3811AGC>CAT 3816G>A 3817GTT>ACC 3822C>G 3825C>A 3831G>A 3834G>A 3840C>A 3841T>A 3844GAG>AAT 3847AAA>GCT 3851CA>AC 3853AAT>GAC 3857T>C 3861C>T 3862ATT>TTG 3867A>C 3870>T 3885C>T 3891G>A 3898AGG>CGC 3903A>C 3930T>C 3933T>A 3935C>A 3939A>G 3942T>A 3944CC>AT 3948C>T 3949CTC>TTG 3954G>A 3956GA>AC 3957A>C 3958GGC>TCA 3963C>T 3965G>C 3967CAA>GGT 3970G>C 3973G>A 3978C>T 3979G>T 3980C>G 3982GA>AC 3990C>T 3991ACA>CAC 3999C>A 4001TT>CG 4003CTT>TTA 4008A>T 4011C>T 4017C>G 4026C>T 4027A>C 4032T>C 4041T>G 4047G>A 4053C>T 4059A>C 4068A>T 4074G>C 4077C>T 4083T>C 4089C>T 4092C>T 4098C>A 4101C>G 4107T>C 4110A>T 4113C>T 4116A>G 4119T>G 4128T>G 4134A>G 4140T>C 4141CTC>ATG 4149G>A 4155C>T 4158G>C 4161A>T 4164T>A 4167G>C 4174TCG>CCC 4179C>A 4181C>A 4189GCG>TCT 4194A>C 4206C>T 4221C>T 4230A>G 4269A>G 4287T>C 4293A>G 4296T>C 4320G>A and 4356C>T.
In some embodiments, the polynucleotide of the present invention that encodes for a variant VP3 capsid of the present invention consists of the nucleic sequence that ranges from the nucleotide at position 2809 to the nucleotide at position 4410 in SEQ ID NO: 62; 63; 64; 65; 66; 67; 68; 69; 70; 71; 72; 73; 74; 75; 76; 77; 78; 79; 80; 81; 82; 83; 84; 85; 86; 87; 88; 89; 90; 91; 92; 93; 94; 95; 96; 97; 98; 99; 100; 101; 102; 103; 104; 105; 106; 107; 108; 109; 110; 111; 112; 113; 114; 115; 116; 117; or 118.
In some embodiments, the polynucleotide of the present invention that encodes for a variant VP3 capsid of the present invention consists of the nucleic sequence that ranges from the nucleotide at position 2809 to the nucleotide at position 4410 in SEQ ID NO: 112, SEQ ID NO: 115, SEQ ID NO: 101, SEQ ID NO:105, SEQ ID NO: 111, SEQ ID NO:98, SEQ ID NO:107, SEQ ID NO:109, SEQ ID NO:108, SEQ ID NO:103, SEQ ID NO:116, SEQ ID NO: 62; SEQ ID NO:63; SEQ ID NO:64; SEQ ID NO:65; SEQ ID NO:66; SEQ ID NO:67; SEQ ID NO:68; SEQ ID NO:69; SEQ ID NO:70; SEQ ID NO:71; SEQ ID NO:72; SEQ ID NO:73; SEQ ID NO:74; SEQ ID NO:75; SEQ ID NO:76; SEQ ID NO:77; SEQ ID NO:78; SEQ ID NO:79; SEQ ID NO:80; SEQ ID NO:81; SEQ ID NO:82; SEQ ID NO:83; SEQ ID NO:84; SEQ ID NO:85; SEQ ID NO:86; SEQ ID NO:87; SEQ ID NO:88; SEQ ID NO:89; SEQ ID NO:90; SEQ ID NO: 91; SEQ ID NO:92; SEQ ID NO:93; SEQ ID NO:94; SEQ ID NO:95; SEQ ID NO:96; SEQ ID NO:97; SEQ ID NO: 99; SEQ ID NO:100; SEQ ID NO:101; SEQ ID NO:102; SEQ ID NO:104; SEQ ID NO: 106; SEQ ID NO: 110; SEQ ID NO:113; SEQ ID NO:114; SEQ ID NO: 117; SEQ ID NO:118, SEQ ID NO:119 or SEQ ID NO:120 SEQ ID NO: 85 (i.e. capsid #3013), SEQ ID NO: 68 (i.e. capsid #2087), SEQ ID NO: 79 (i.e. capsid #1449), SEQ ID NO: 69 (i.e. capsid #2206), SEQ ID NO: 71 (i.e. capsid #1534), SEQ ID NO: 115 (i.e. capsid #163), SEQ ID NO: 82 (i.e. capsid #1343), SEQ ID NO: 112 (i.e. capsid #790), SEQ ID NO:65 (i.e. capsid #1017), SEQ ID NO: 70 (i.e. capsid #877), SEQ ID NO: 67 (i.e. capsid #1273), SEQ ID NO: 62 (i.e. capsid #2497); SEQ ID NO: 119 (i.e. capsid #1055); SEQ ID NO: 73 (i.e. capsid #1919), SEQ ID NO:63, SEQ ID NO:64; SEQ ID NO:66, SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO:80, SEQ ID NO:81, SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:86, SEQ ID NO:87; SEQ ID NO:88; SEQ ID NO:89; SEQ ID NO:90; SEQ ID NO: 91; SEQ ID NO:92; SEQ ID NO:93; SEQ ID NO:94; SEQ ID NO:95; SEQ ID NO:96; SEQ ID NO:97; SEQ ID NO:98, SEQ ID NO: 99; SEQ ID NO:100; SEQ ID NO:101; SEQ ID NO:102; SEQ ID NO:103, SEQ ID NO:104; SEQ ID NO:105, SEQ ID NO: 106; SEQ ID NO:107, SEQ ID NO:108, SEQ ID NO:109, SEQ ID NO: 110; SEQ ID NO:111, SEQ ID NO:113; SEQ ID NO:114; SEQ ID NO:116, SEQ ID NO: 117; SEQ ID NO:118 or SEQ ID NO:120.
AAV Comprising a Capsid Protein of the Present Invention:
A further object of the present invention relates to an adeno-associated virus (AAV) that comprises at least one capsid protein of the present invention.
In some embodiments, the AAV is a wildtype AAV. In some embodiments, the AAV comprises a genome sequence as set forth in SEQ ID NO: 62-120.
A further object of the present invention relates to an AAV wherein the amino acid sequence of VP1 capsid protein is encoded by the nucleotides 2203-4410 of in SEQ ID NO: 85 (i.e. capsid #3013), SEQ ID NO: 68 (i.e. capsid #2087), SEQ ID NO: 79 (i.e. capsid #1449), SEQ ID NO: 69 (i.e. capsid #2206), SEQ ID NO: 71 (i.e. capsid #1534), SEQ ID NO: 115 (i.e. capsid #163), SEQ ID NO: 82 (i.e. capsid #1343), SEQ ID NO: 112 (i.e. capsid #790), SEQ ID NO:65 (i.e. capsid #1017), SEQ ID NO: 70 (i.e. capsid #877), SEQ ID NO: 67 (i.e. capsid #1273), SEQ ID NO: 62 (i.e. capsid #2497); SEQ ID NO: 119 (i.e. capsid #1055); SEQ ID NO: 73 (i.e. capsid #1919), SEQ ID NO:63, SEQ ID NO:64; SEQ ID NO:66, SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO:80, SEQ ID NO:81, SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:86, SEQ ID NO:87; SEQ ID NO:88; SEQ ID NO:89; SEQ ID NO:90; SEQ ID NO: 91; SEQ ID NO:92; SEQ ID NO:93; SEQ ID NO:94; SEQ ID NO:95; SEQ ID NO:96; SEQ ID NO:97; SEQ ID NO:98, SEQ ID NO: 99; SEQ ID NO:100; SEQ ID NO:101; SEQ ID NO:102; SEQ ID NO:103, SEQ ID NO:104; SEQ ID NO:105, SEQ ID NO: 106; SEQ ID NO:107, SEQ ID NO:108, SEQ ID NO:109, SEQ ID NO: 110; SEQ ID NO:111, SEQ ID NO:113; SEQ ID NO:114; SEQ ID NO:116, SEQ ID NO: 117; SEQ ID NO:118 or SEQ ID NO:120; or, the amino acid sequence of VP2 capsid protein is encoded by nucleotides 2614-4410 of in SEQ ID NO: 85 (i.e. capsid #3013), SEQ ID NO: 68 (i.e. capsid #2087), SEQ ID NO: 79 (i.e. capsid #1449), SEQ ID NO: 69 (i.e. capsid #2206), SEQ ID NO: 71 (i.e. capsid #1534), SEQ ID NO: 115 (i.e. capsid #163), SEQ ID NO: 82 (i.e. capsid #1343), SEQ ID NO: 112 (i.e. capsid #790), SEQ ID NO:65 (i.e. capsid #1017), SEQ ID NO: 70 (i.e. capsid #877), SEQ ID NO: 67 (i.e. capsid #1273), SEQ ID NO: 62 (i.e. capsid #2497); SEQ ID NO: 119 (i.e. capsid #1055); SEQ ID NO: 73 (i.e. capsid #1919), SEQ ID NO:63, SEQ ID NO:64; SEQ ID NO:66, SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO:80, SEQ ID NO:81, SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:86, SEQ ID NO:87; SEQ ID NO:88; SEQ ID NO:89; SEQ ID NO:90; SEQ ID NO: 91; SEQ ID NO:92; SEQ ID NO:93; SEQ ID NO:94; SEQ ID NO:95; SEQ ID NO:96; SEQ ID NO:97; SEQ ID NO:98, SEQ ID NO: 99; SEQ ID NO:100; SEQ ID NO:101; SEQ ID NO:102; SEQ ID NO:103, SEQ ID NO:104; SEQ ID NO:105, SEQ ID NO: 106; SEQ ID NO:107, SEQ ID NO:108, SEQ ID NO:109, SEQ ID NO: 110; SEQ ID NO:111, SEQ ID NO:113; SEQ ID NO:114; SEQ ID NO:116, SEQ ID NO: 117; SEQ ID NO:118 or SEQ ID NO:120 and the amino acid sequence of VP3 capsid protein is encoded by nucleotides 2809-4410 of in SEQ ID NO: 85 (i.e. capsid #3013), SEQ ID NO: 68 (i.e. capsid #2087), SEQ ID NO: 79 (i.e. capsid #1449), SEQ ID NO: 69 (i.e. capsid #2206), SEQ ID NO: 71 (i.e. capsid #1534), SEQ ID NO: 115 (i.e. capsid #163), SEQ ID NO: 82 (i.e. capsid #1343), SEQ ID NO: 112 (i.e. capsid #790), SEQ ID NO:65 (i.e. capsid #1017), SEQ ID NO: 70 (i.e. capsid #877), SEQ ID NO: 67 (i.e. capsid #1273), SEQ ID NO: 62 (i.e. capsid #2497); SEQ ID NO: 119 (i.e. capsid #1055); SEQ ID NO: 73 (i.e. capsid #1919), SEQ ID NO:63, SEQ ID NO:64; SEQ ID NO:66, SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO:80, SEQ ID NO:81, SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:86, SEQ ID NO:87; SEQ ID NO:88; SEQ ID NO:89; SEQ ID NO:90; SEQ ID NO: 91; SEQ ID NO:92; SEQ ID NO:93; SEQ ID NO:94; SEQ ID NO:95; SEQ ID NO:96; SEQ ID NO:97; SEQ ID NO:98, SEQ ID NO: 99; SEQ ID NO:100; SEQ ID NO:101; SEQ ID NO:102; SEQ ID NO:103, SEQ ID NO:104; SEQ ID NO:105, SEQ ID NO: 106; SEQ ID NO:107, SEQ ID NO:108, SEQ ID NO:109, SEQ ID NO: 110; SEQ ID NO:111, SEQ ID NO:113; SEQ ID NO:114; SEQ ID NO:116, SEQ ID NO: 117; SEQ ID NO:118 or SEQ ID NO:120.
In some embodiments, the AAV of the present invention is a rAAV that comprises a heterologous nucleic acid comprising a transgene.
In some embodiments, the transgene encodes for a therapeutic protein. The term “therapeutic protein” refers to a protein which is defective or missing from the subject in question, thus resulting in a disease state or disorder in the subject, or to a protein which confers a benefit to the subject in question, such as an antiviral, antibacterial or antitumor function. A therapeutic protein can also be one which modifies any one of a wide variety of biological functions, such as endocrine, immunological and metabolic functions. Typically, the transgene may encode for a polypeptide involved in immune response, hematopoiesis, inflammation, cell growth and proliferation, cell lineage differentiation, or stress response.
In some embodiments, the transgene encodes for a site-specific endonuclease that provides for site-specific knock-down of gene function, e.g., where the endonuclease knocks out an allele associated with a disease. For example, where a dominant allele encodes a defective copy of a gene that, a site-specific endonuclease can be targeted to the defective allele and knock out the defective allele. In some embodiments, the rAAV of the present invention comprises a polynucleotide that encodes a site-specific endonuclease; and a polynucleotide that encodes a functional copy of a defective allele, where the functional copy encodes a functional protein. Site-specific endonucleases that are suitable for use include, e.g., zinc finger nucleases (ZFNs); transcription activator-like effector nucleases (TALENs), and CRISPR-associated endonuclease. As used herein, the term “CRISPR-associated endonuclease” has its general meaning in the art and refers to clustered regularly interspaced short palindromic repeats associated which are the segments of prokaryotic DNA containing short repetitions of base sequences. In particular, the CRISPR-associated endonuclease is Cas9 or a derivative thereof. The Cas9 nuclease can have a nucleotide sequence identical to the wild type Streptococcus pyrogenes sequence. Alternatively, the wild type Streptococcus pyrogenes Cas9 sequence can be modified. For instance, the Cas9 nuclease sequence can be for example, the sequence contained within a commercially available vector such as PX330 or PX260 from Addgene (Cambridge, Mass.). In some embodiments, the Cas9 endonuclease can have an amino acid sequence that is a variant or a fragment of any of the Cas9 endonuclease sequences of Genbank accession numbers KM099231.1 GL669193757; KM099232.1; GL669193761; or KM099233.1 GL669193765 or Cas9 amino acid sequence of PX330 or PX260 (Addgene, Cambridge, Mass.). The Cas9 nucleotide sequence can be modified to encode biologically active variants of Cas9, and these variants can have or can include, for example, an amino acid sequence that differs from a wild type Cas9 by virtue of containing one or more mutations (e.g., an addition, deletion, or substitution mutation or a combination of such mutations). For example the Cas9 nuclease can be mutated in the conserved FiNH and RuvC domains, which are involved in strand specific cleavage. For example, an aspartate-to-alanine (D10A) mutation in the RuvC catalytic domain allows the Cas9 nickase mutant (Cas9n) to nick rather than cleave DNA to yield single-stranded breaks, and the subsequent preferential repair through HDR can potentially decrease the frequency of unwanted indel mutations from off-target double-stranded breaks. In some embodiments, the rAAV of the present invention comprises one or more guide RNA. As used herein, the term “one or more guide RNA” refers to the RNAs that guide the insertion or deletion of residues. In the context of the invention, the guide RNA is used for recruiting Cas9 to specific genomic loci. In some embodiments, the guide RNA can be a sequence complementary to a coding or a non-coding sequence. In some embodiments, the subject is administered with a combination of at least one vectors comprising one polynucleotide encoding for a Cas9 endonuclease and at least one vector comprising the guide RNA.
In some embodiments, the transgene encodes for an interfering RNA (RNAi), in particular a siRNA. A “small interfering” or “short interfering RNA” or siRNA is a RNA duplex of nucleotides that is targeted to a gene interest (a “target gene”). An “RNA duplex” refers to the structure formed by the complementary pairing between two regions of a RNA molecule. siRNA is “targeted” to a gene in that the nucleotide sequence of the duplex portion of the siRNA is complementary to a nucleotide sequence of the targeted gene. In some embodiments, the length of the duplex of siRNAs is less than 30 nucleotides.
In some embodiments, the transgene encodes for an antisense oligonucleotide. As used herein, the term “antisense oligonucleotide” is understood to refer to a nucleotide sequence which is substantially complementary to a target nucleotide sequence in a pre-mRNA molecule, hnRNA (heterogenous nuclear RNA) or mRNA molecule. The degree of complementarity (or substantial complementarity) of the antisense sequence is preferably such that a molecule comprising the antisense sequence can form a stable hybrid with the target nucleotide sequence in the RNA molecule under physiological conditions.
Other transgenes of interest include, without limitation, synthetic long non-coding RNAs (SINEUPs; Carrieri et al., 2012, Nature 491: 454-7; Zucchelli et al., 2015, RNA Biol 12(8): 771-9; Indrieri et al., 2016, Sci Rep 6: 27315) and artificial microRNAs. Other specific transgene of interest useful in the practice of the present invention are described below.
In some embodiments, the transgene is operably linked to a constitutive promoter. In some embodiments the transgene is operably linked to an inducible promoter. In some embodiments, the transgene is operably linked to a tissue-specific or cell type-specific regulatory element.
Numerous methods are known in the art for production of rAAV vectors, including transfection, stable cell line production, and infectious hybrid virus production systems which include adenovirus-AAV hybrids, herpesvirus-AAV hybrids (Conway, J E et al., (1997) J. Virology 71(11):8780-8789) and baculovirus-AAV hybrids. rAAV production cultures for the production of rAAV virus particles all require; 1) suitable host cells, including, for example, human-derived cell lines such as HeLa, A549, or 293 cells, or insect-derived cell lines such as SF-9, in the case of baculovirus production systems; 2) suitable helper virus function, provided by wild-type or mutant adenovirus (such as temperature sensitive adenovirus), herpes virus, baculovirus, or a plasmid construct providing helper functions; 3) AAV rep and cap genes and gene products; 4) a transgene (such as a therapeutic transgene) flanked by at least one AAV ITR sequences; and 5) suitable media and media components to support rAAV production. Suitable media known in the art may be used for the production of rAAV vectors. These media include, without limitation, media produced by Hyclone Laboratories and JRH including Modified Eagle Medium (MEM), Dulbecco's Modified Eagle Medium (DMEM), custom formulations such as those described in U.S. Pat. No. 6,566,118, and Sf-900 II SFM media as described in U.S. Pat. No. 6,723,551, each of which is incorporated herein by reference in its entirety, particularly with respect to custom media formulations for use in production of recombinant AAV vectors. The rAAV particles can be produced using methods known in the art. See, e.g., U.S. Pat. Nos. 6,566,118; 6,989,264; and 6,995,006. In practicing the invention, host cells for producing rAAV particles include mammalian cells, insect cells, plant cells, microorganisms and yeast. Host cells can also be packaging cells in which the AAV rep and cap genes are stably maintained in the host cell or producer cells in which the AAV vector genome is stably maintained. Exemplary packaging and producer cells are derived from 293, A549 or HeLa cells. AAV vectors are purified and formulated using standard techniques known in the art.
In some embodiments, rAAV particles may be produced by a triple transfection method, such as the exemplary triple transfection method provided infra. Briefly, a plasmid containing a rep gene and a capsid gene, along with a helper adenoviral plasmid, may be transfected (e.g., using the calcium phosphate method) into a cell line (e.g., HEK-293 cells), and virus may be collected and optionally purified.
In some embodiments, rAAV particles may be produced by a producer cell line method, such as the exemplary producer cell line method provided infra (see also (referenced in Martin et al., (2013) Human Gene Therapy Methods 24:253-269). Briefly, a cell line (e.g., a HeLa cell line) may be stably transfected with a plasmid containing a rep gene, a capsid gene, and a promoter-transgene sequence. Cell lines may be screened to select a lead clone for rAAV production, which may then be expanded to a production bioreactor and infected with an adenovirus (e.g., a wild-type adenovirus) as helper to initiate rAAV production. Virus may subsequently be harvested, adenovirus may be inactivated (e.g., by heat) and/or removed, and the rAAV particles may be purified.
In some embodiments, a method is provided for producing any rAAV particle as disclosed herein comprising (a) culturing a host cell under a condition that rAAV particles are produced, wherein the host cell comprises (i) one or more AAV package genes, wherein each said AAV packaging gene encodes an AAV replication and/or encapsidation protein; (ii) a rAAV pro-vector comprising a transgene flanked by at least one AAV ITR, and (iii) an AAV helper function; and (b) recovering the rAAV particles produced by the host cell. rAAV vector particles of the invention may be harvested from rAAV production cultures by lysis of the host cells of the production culture or by harvest of the spent media from the production culture, provided the cells are cultured under conditions known in the art to cause release of rAAV particles into the media from intact cells, as described more fully in U.S. Pat. No. 6,566,118). Suitable methods of lysing cells are also known in the art and include for example multiple freeze/thaw cycles, sonication, microfluidization, and treatment with chemicals, such as detergents and/or proteases.
In some embodiments, the rAAV particles are purified. The term “purified” as used herein includes a preparation of rAAV particles devoid of at least some of the other components that may also be present where the rAAV particles naturally occur or are initially prepared from. Thus, for example, isolated rAAV particles may be prepared using a purification technique to enrich it from a source mixture, such as a culture lysate or production culture supernatant. Enrichment can be measured in a variety of ways, such as, for example, by the proportion of DNase-resistant particles (DRPs) or genome copies (gc) present in a solution, or by infectivity, or it can be measured in relation to a second, potentially interfering substance present in the source mixture, such as contaminants, including production culture contaminants or in-process contaminants, including helper virus, media components, and the like. rAAV particles may be isolated or purified using one or more of the following purification steps: equilibrium centrifugation; flow-through anionic exchange filtration; tangential flow filtration (TFF) for concentrating the rAAV particles; rAAV capture by apatite chromatography; heat inactivation of helper virus; rAAV capture by hydrophobic interaction chromatography; buffer exchange by size exclusion chromatography (SEC); nanofiltration; and rAAV capture by anionic exchange chromatography, cationic exchange chromatography, or affinity chromatography. These steps may be used alone, in various combinations, or in different orders. In some embodiments, the method comprises all the steps in the order as described below. Methods to purify rAAV particles are found, for example, in Xiao et al., (1998) Journal of Virology 72:2224-2232; U.S. Pat. Nos. 6,989,264 and 8,137,948; and WO 2010/148143.
Therapeutic Uses and Pharmaceutical Compositions:
The rAAV of the present invention are particularly suitable for delivering the transgene to a cell of interest. Accordingly, a further object of the present invention relates to a method of delivering a transgene to a cell comprising contacting the cell with an amount of a rAAV of the present invention.
In particular, the rAAV of the present invention are particularly suitable for gene therapy. Gene therapy consists in introducing in tissue of interest a functional copy of a transgene (e.g. a gene) that is deficient therein (gene replacement therapy), or by delivering to the tissue a transgene which will have a beneficial effect on the disease to be treated (symptomatic therapy). In some embodiments, the rAAV of the present invention is particularly suitable for delivering a transgene in liver.
Thus a further object of the present invention relates to a method for delivering a transgene to a tissue in a subject, the method comprising administering to the subject: an effective amount of a rAAV of the present invention.
More specifically, the present invention provides a method of therapy in a patient in need thereof, comprising administering to the patient a therapeutically effective amount of a rAAV of the present invention.
A further object of the present invention relates to a pharmaceutical composition comprising the rAAV of the present invention with pharmaceutically acceptable excipients, and optionally sustained-release matrices, such as biodegradable polymers. The pharmaceutical composition of the present invention thus comprises a pharmaceutically acceptable excipient, carrier, buffer, stabiliser or other materials well known to those skilled in the art. A pharmaceutically acceptable carrier or excipient refers to a non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type. Such materials should be non-toxic and should not interfere with the efficacy of the active ingredient (i.e. the rAAV of the present invention of the invention). The precise nature of the carrier or other material may be determined by the skilled person according to the route of administration, i.e. here subretinal injection. The pharmaceutical composition is typically in liquid form. Liquid pharmaceutical compositions generally include a liquid carrier such as water, petroleum, animal or vegetable oils, mineral oil or synthetic oil. Physiological saline solution, magnesium chloride, dextrose or other saccharide solution or glycols such as ethylene glycol, propylene glycol or polyethylene glycol may be included. For injection, the active ingredient will be in the form of an aqueous solution, which is pyrogen-free and has suitable pH, isotonicity and stability. Those of relevant skill in the art are well able to prepare suitable solutions using, for example, isotonic vehicles such as Sodium Chloride Injection, Ringer's Injection, Lactated Ringer's Injection. Preservatives, stabilisers, buffers, antioxidants and/or other additives may be included, as required. For delayed release, the rAAV of the present invention may be included in a pharmaceutical composition, which is formulated for slow release, such as in microcapsules formed from biocompatible polymers or in liposomal carrier systems according to methods known in the art. Typically, the pharmaceutical composition of the present invention is supplied in a prefilled syringe. A “ready-to-use syringe” or “prefilled syringe” is a syringe which is supplied in a filled state, i.e. the pharmaceutical composition to be administered is already present in the syringe and ready for administration. Prefilled syringes have many benefits compared to separately provided syringe and vial, such as improved convenience, affordability, accuracy, sterility, and safety. The use of prefilled syringes results in greater dose precision, in a reduction of the potential for needle sticks injuries that can occur while drawing medication from vials, in pre-measured dosage reducing dosing errors due to the need to reconstituting and/or drawing medication into a syringe, and in less overfilling of the syringe helping to reduce costs by minimising drug waste. In some embodiments the pH of the liquid pharmaceutical composition of the present invention is in the range of 5.0 to 7.0, 5.1 to 6.9, 5.2 to 6.8, 5.3 to 6.7 or 5.4 to 6.6.
The invention will be further illustrated by the following figures and examples. However, these examples and figures should not be interpreted in any way as limiting the scope of the present invention.
Luciferase activity of controls and new capsids capsids. Each column represents the average of activity in at least 3 mice expressed as fold change versus AAV2. Standard deviations are displayed. On the right, the phylogenetic tree of the VP1 amino acid sequences was constructed using the Neighbor-Joining method. The evolutionary distances were computed using the Poisson correction method and are in the units of the number of amino acid substitutions per site.
A-C). Luciferase activity of controls and new capsids. Each column represents the average of activity in at least 3 mice expressed as fold change versus AAV2. Standard deviations are displayed. Statistical analysis on fold change was performed using one-way ANOVA. Dunnett's multiple comparison test was used to compare the mean of each capsid with the mean of the controls (* versus PBS, # versus AAV2 and $ versus AAV*). *, #, $=p<0.05; **, ##, $$=p<0.01; ***, ###, $$$=p<0.001.
Results:
Identification of Two Major AAV Genotypes in the Liver
We screened frozen liver tissues from 1,319 patients for the presence of AAV viral DNA with 6 Taqman probes distributed along the genome that collectively recognize all known AAV genotypes 1 to 13. We identified AAV DNA in 18% of non-tumor liver tissues (n=233), 82 of which were further analyzed by viral capture with 120-mer oligonucleotide probes covering the entire genome of all known AAV genotypes (350 probes/genotype). After sequencing, a complete coverage of the viral genome was obtained in 57 samples, which allowed to reconstruct full-length AAV sequences (
AAV Infection and Episomal Form
In the 233 liver samples positive for AAV DNA, quantification of AAV followed a bimodal distribution with 97% of the tissues showing a low number of copy/cell (ranging from 4.6e-05 to 0.04) and only 8 patients with higher quantity of AAV ranging from 0.07 to 0.18 copy/cell (data not shown). Relationship between AAV detection, clinical and histological features of the patients showed that AAV was significantly enriched in female (p<0.001), young patients (p=0.016) and occurred more frequently in a background of non-fibrotic liver (p<0.001; data not shown).
In 64/233 (27.5%) of the tissues positive for AAV, all the genomic AAV regions were amplified suggesting the presence of the entire viral genome. In order to experimentally search for episomal AAV form, we designed a DNAse/TaqMan based assay (data not shown) which allowed to detected episomal AAV in 60 patients, corresponding to 26% of AAV positive samples and 4.5% of all patients. Using in silico analyses of the AAV capture sequencing, among the 57 cases with a complete reconstructed AAV genomic sequence, we identified 14 cases with 3′ITR-5′ITR junctions without clonal insertion. The 3′ITR-5′ITR junctions showed various sequences presenting a double-D ITR structure, in flip or flop configuration, with a 125 bp deletion confirmed by Sanger sequencing (data not shown).
AAV Transcription is Associated with Episomal Form
Then, we screened for AAV RNA expression in 101 non-tumor liver tissues positive for AAV by qRT-PCR. AAV transcript was identified in 64% of the tested liver tissues. We showed that either AAV REP or CAP expression were enriched in liver tissues with episomal form (p<0.001) and both transcripts were more frequently associated in presence of episomal than not-episomal AAV form (p=0.022), defining a population of patients with an “episomal-expressed AAV” (data not shown). A higher AAV copies per cell was identified in liver tissues with episomal-expressed AAV, supporting the hypothesis of a viral active infection in these liver samples (data not shown). Episomal AAV were also more frequent in female (p<0.001) and non-cirrhotic patients (p<0.001; data not shown). Analysis of AAV positivity in function of age showed a peak of frequency at 25% in the 30-40 years class. AAV episomal form was more frequent in young patients (<40 years old) reaching the highest frequency level in the twenties (data not shown). These results suggest that AAV active infection is more frequent in the second and third decade during life, while inactive not-episomal forms subsist after the primary infection.
Discussion:
In this study, we provided a comprehensive description at large scale of wild type AAV infection in the liver and of its oncogenic consequences in human. Despite the large number of studies available on recombinant AAV vectors (rAAV), the natural history of wild type AAV infection is still poorly understood. Here, we provided an extensive characterization of the different AAV viral forms in the largest series of tumor and non-tumor liver tissues to better understand the AAV infection in human.
The prevalence of AAV was observed in 21% of patients in non-tumor and/or tumor liver in agreement with the seroprevalence of antibody against AAV identified in 40 to 80% of the general population.10-12 Our result showed that half of the patients demonstrate persistent AAV DNA in the liver during life, mainly in the population of young and female patients without liver fibrosis. However, since most of our liver tissues were sampled from patients with liver diseases, the exact prevalence of AAV DNA in the liver of healthy individuals remains to be evaluated.
Two major AAV genotypes, AAV2 and hybrids AAV2/13, were identified in our cohort. The classification of AAV viruses is currently based on their serological response and on the amino acid sequence of the VP1 region defined by Gao and colleagues.14 However, this classification in viral clades does not include all the AAV serotypes and the most recently isolated viral clones from non-human primates.16, 46 Moreover, most of the sequences isolated from human tissues were restricted to VP1 region, in particular only one full-length AAV sequence from clade C was publicly available.43 Here, we provided the whole AAV genome sequences of 57 human clones classified with half of them belonging to the reference AAV2 sequence, whereas the others were hybrids between AAV2 in the 5′part and AAV13 in the 3′ corresponding to the previous clade C of the VP1 classification. Our work dramatically increased the number human AAV full-length sequences publicly available enlightening the genotypes and genomic variants associated with an efficient natural AAV infection of the liver.
In conclusion, these findings are important to understand wild type AAV biology. Our data are particularly relevant considering the large usage of AAV vector in liver-targeted gene therapy.
Material and Methods
Plasmid Construction for New Serotypes
To construct the plasmid containing AAV2 Rep sequence and the new Cap genes, the capsid sequences were synthesized (GENEWIZ). The fragment was inserted in the plasmid pAAV2 which contains AAV2 Rep and AAV2 Cap in order to replace the AAV2 Cap with the corresponding new Cap sequence.
AAV Production
HEK293T cells were grown in suspension in 50 mL of serum-free medium. The cells were transfected with 3 plasmids: i) a transgene plasmid, containing AAV2 ITRs flanking an expression cassette ii) the helper plasmid pXX6, containing adenoviral sequences necessary for AAV production, and iii) a plasmid containing AAV Rep and Cap genes, defining the serotype of AAV. Two days after transfection, the cells were lysed to release the AAV particles.
The viral lysate was purified by affinity chromatography. Viral genomes were quantified by a TaqMan real-time PCR assay using primers and probes corresponding to the ITRs of the AAV vector genome (Rohr et al., J. Virol. Methods, 2002, 106, 81-88).
In Vivo Studies
All mouse studies were performed according to the French and European legislation on animal care and experimentation (2010/63/EU) and approved by the local institutional ethical committee (protocol no. 2016-002C). AAV vectors were administered intravenously via the tail vein to 6 weeks old male C57B16/J mice. PBS-injected littermates were used as controls. 15 days after vector injections, tissues were harvested and homogenized in DNAse/RNAse free water using Fastprep tubes (6.5 m/s; 60 seconds).
Luciferase Activity
Luciferase assay was used to measure the expression of the reporter gene used as transgene. Tissue lysates were centrifuged at 10000 rpm for 10 min, the supernatant was diluted in lysis buffer in a white opaque 96-well plate. Luciferase activity was measured using EnSpire (PerkinElmer) through sequential injections of assay buffer containing ATP and luciferine.
Protein quantification was performed on the samples using BCA assay in order to normalize the RLU (relative luminescence unit) on the quantity protein. The final results were expressed as RLU/mg of protein and normalized as fold change versus AAV2 control.
Results:
Recombinant AAV vectors were produced by cloning the Cap genes identified in EXAMPLE 1 in a plasmid suitable for vector production. A transgene expression cassette flanked by AAV2 ITRs and expressing a luciferase reporter gene was encapsidated in the so derived AAV vectors. Triple transfection of HEK293 cells was used to produce the vectors followed by immunoaffinity column purification. The capsid sequences that were not efficiently produced as rAAV vectors were excluded. The vectors were tested in wild-type C57Bl6/J mice through intravenous injection of the different vectors at the dose of 1×1011 vg/mice. Fifteen days post-injection, animals were sacrificed and the levels of expression of the transgene were measured in isolated tissues. All new capsids detargeted the liver (
Throughout this application, various references describe the state of the art to which this invention pertains. The disclosures of these references are hereby incorporated by reference into the present disclosure.
| Number | Date | Country | Kind |
|---|---|---|---|
| 19305521.7 | Apr 2019 | EP | regional |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2020/061380 | 4/23/2020 | WO |
