Recombinant adeno-associated virus comprising truncated CMV promoter sequence and / or truncated polyadenylation sequence

Abstract

[Problem] To provide a new recombinant adeno-associated virus (rAAV) in which fragmentation of vector genome can be reduced. [Solution] An rAAV according to the present invention includes: a truncated CMV promoter having a sequence formed of nucleotides at positions 274-469 in the nucleotide sequence of SEQ ID NO: 1, and the like, a coding sequence operably linked to the sequence of the promotor, and a polyadenylation sequence; or a promoter sequence, a coding sequence operably linked to the promoter sequence, and a truncated polyadenylation sequence including the nucleotide sequence of SEQ ID NO: 3.

Description

Recombinant adeno-associated virus containing a truncated CMV promoter sequence and / or a truncated polyadenylation sequence

[0001] The present invention relates to recombinant adeno-associated viruses (rAAVs) comprising a truncated CMV promoter sequence and / or a truncated polyadenylation sequence.

[0002] rAAV (also referred to as AAV vector) contains a single-stranded DNA genome in which a gene of interest (transgene) is inserted between two inverted terminal repeats (ITRs) in place of the wild-type AAV genome sequence, and is used as a gene transfer vector (Li and Samulski, Nat Rev Genet. 2020; 21(4) 255-272). The sequence inserted between the ITRs and the DNA containing the ITRs are referred to as the vector genome. Depending on the structure of the vector genome, rAAV can be classified as single-stranded AAV (ssAAV) or self-complementary AAV (scAAV). After infecting a target host cell, rAAV is taken up into the cell by endocytosis, translocated from the endosome to the cytoplasm, and then transported into the nucleus. In the case of ssAAV, the single-stranded vector genome released from the capsid in the nucleus is converted into double-stranded DNA and transcribed. This conversion of the single-stranded vector genome into double-stranded DNA requires the synthesis of a complementary strand or the annealing of two complementary single-stranded vector genomes, but these processes are inefficient and limit the rate of transgene expression (McCarty, Mol Ther. 2008; 16(10) 1648-1656).

[0003] scAAV, which has a vector genome consisting of inverted repeat sequences, can avoid the synthesis of complementary strands, which is rate-limiting for transgene expression, because the vector genome forms an intramolecular double-stranded structure, and therefore shows higher expression efficiency than ssAAV (McCarty et al., Gene Ther. 2003; 10(26) 2112-2118; McCarty et al., Gene Ther. 2001; 8(16) 1248-1254; Wang et al., Gene Ther. 2003; 10(26) 2105-2111). scAAV is produced using a construct in which a mutation has been introduced into one of the ITRs that inhibits nick formation by Rep. The vector genome replicated using a construct containing a mutant ITR (mITR) as a template is folded into a double-stranded molecule in which the plus and minus strands of the transgene are linked by the mITR, and is packaged into a capsid.

[0004] The main limitation of AAV vectors is their small packaging capacity; DNA exceeding this capacity (single-stranded DNA of approximately 4.9 kb) is not efficiently packaged, resulting in fragmentation of the vector genome, a decrease in the yield of rAAV containing the full-length vector genome, and a decrease in transduction efficiency (Dong et al., Hum Gene Ther. 1996; 7(17) 2101-2112). In scAAV, which has a double-stranded folded vector genome, the size of the transgene that can be efficiently packaged is approximately half that of ssAAV (Wang et al., Hum Gene Ther Methods. 2012; 23(4) 225-233; Wu et al., Hum Gene Ther. 2007; 18(2) 171-182).

[0005] The small packaging capacity of rAAV limits the use of promoters, polyadenylation sequences, and other gene expression regulatory elements. Therefore, promoters and polyadenylation sequences smaller than commonly used sequences have been developed and used in AAV vectors. For example, the development of AAV vectors using truncated CMV promoters with deleted upstream regions has been reported (Patent Document 1, Non-Patent Document 1). The truncated CMV promoters reported in Patent Document 1 and Non-Patent Document 1 contain a TATA box sequence, a core element of the wild-type CMV promoter, but tend to have lower promoter activity than the full-length CMV promoter, and it has not been shown whether they reduce vector genome fragmentation (Patent Document 1, Non-Patent Document 1). Furthermore, an AAV vector using a truncated human growth hormone polyadenylation sequence has been reported (Patent Document 2).

[0006] International Publication No. WO 2005 / 056762 International Publication No. WO 2007 / 126798

[0007] Ostedgaard et al. , A shortened adeno-associated virus expression cassette for CFTR gene transfer to cystic fibrosis airway epithelia. Proc Natl Acad Sci USA. 2005; 102(8) 2952-2957.

[0008] Under these circumstances, there has been a demand for the development of a new rAAV that can reduce vector genome fragmentation.

[0009] The present invention has been made in consideration of the above circumstances, and provides the following rAAVs, etc.

[0010] [1] A recombinant adeno-associated virus (rAAV) comprising a transgene sequence, the transgene sequence being: (a) a transgene sequence comprising: i) a truncated CMV promoter sequence comprising a sequence consisting of nucleotides 274 to 469 in the nucleotide sequence of SEQ ID NO: 1, or a nucleotide sequence having 90% or more sequence identity to said sequence; ii) a coding sequence operably linked to the truncated CMV promoter sequence; and iii) a polyadenylation sequence; or (b) a transgene sequence comprising: i) a promoter sequence; ii) a coding sequence operably linked to the promoter sequence; and iii) a truncated polyadenylation sequence comprising a sequence consisting of the nucleotide sequence of SEQ ID NO: 3, or a nucleotide sequence having 90% or more sequence identity to said sequence.

[0011] [2] The rAAV according to [1], wherein the transgene sequence comprises: i) a truncated CMV promoter sequence comprising a sequence consisting of nucleotides 274 to 469 in the nucleotide sequence of SEQ ID NO: 1, or a nucleotide sequence having 90% or more sequence identity to said sequence; ii) a coding sequence operably linked to the truncated CMV promoter sequence; and iii) a polyadenylation sequence.

[0012] [3] The rAAV according to [2], wherein the rAAV is a scAAV. [4] The rAAV according to [3], wherein the transgene sequence is 2,115 bases or less in length. [5] The rAAV according to any of [2] to [4], wherein the truncated CMV promoter sequence comprises the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 2, or a nucleotide sequence having 90% or more sequence identity to the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 2. [6] The rAAV according to any of [2] to [5], wherein the polyadenylation sequence is a polyadenylation sequence derived from human growth hormone.

[0013] [7] The rAAV according to any of [2] to [6], wherein the polyadenylation sequence is a truncated polyadenylation sequence derived from human growth hormone. [8] The rAAV according to any of [2] to [7], wherein the polyadenylation sequence is a truncated polyadenylation sequence comprising the nucleotide sequence of SEQ ID NO: 3 or a nucleotide sequence having 90% or more sequence identity to the nucleotide sequence of SEQ ID NO: 3. [9] The rAAV according to any of [2] to [8], wherein the truncated CMV promoter sequence consists of the nucleotide sequence of SEQ ID NO: 1 and the polyadenylation sequence consists of the nucleotide sequence of SEQ ID NO: 3.

[0014]

[10] The rAAV of [1], wherein the transgene sequence comprises: i) a promoter sequence; ii) a coding sequence operably linked to the promoter sequence; and iii) a truncated polyadenylation sequence comprising a sequence consisting of the nucleotide sequence of SEQ ID NO: 3, or a nucleotide sequence having 90% or more sequence identity to said sequence.

[0015]

[11] The rAAV according to

[10] , wherein the truncated polyadenylation sequence comprises a sequence consisting of the nucleotide sequence of SEQ ID NO: 3.

[12] The rAAV according to

[10] or

[11] , wherein the promoter sequence is a CMV promoter sequence.

[13] The rAAV according to any of

[10] to

[12] , wherein the rAAV is a single-stranded AAV (ssAAV).

[0016] The rAAV of the present invention can reduce vector genome fragmentation due to oversize during the production process, and has the activity of expressing a transgene protein, and can be used to prevent and / or treat various diseases.

[0017] Figure 1 shows a schematic diagram of the plasmid construct and the size of the transgene sequence. Figure 2 shows the results of alkaline agarose gel electrophoresis analysis of the vector genome of an scAAV having an AAV8 capsid prepared using the plasmid construct shown in Figure 1. The size of the full-length vector genome as single-stranded DNA is shown at the bottom of the figure. Figure 3 shows the results of Western blotting analysis of hTFEB (human transcription factor EB: hTFEB) expression in HEK293 cells one day after transfection with the plasmid construct shown in Figure 1 (n = 2). The expression level of the transgene protein hTFEB was corrected using the β-actin expression level in the same sample as the total hTFEB expression level, including endogenous expression, and then expressed as a percentage relative to KA072. Figure 4 shows the results of Western blot analysis of hTFEB expression in HEK293 cells 1 day after transfection with the plasmid construct shown in Figure 1 (n = 3). The expression level of the transgene protein hTFEB was corrected using the β-actin expression level in the same sample as the total hTFEB expression level, including endogenous expression, and then expressed as a percentage relative to KA072.

[0018] The present invention will be described in detail below. The scope of the present invention is not limited to these descriptions, and other than the following examples, modifications can be made as appropriate without departing from the spirit of the present invention. This specification encompasses the entire specification of Japanese Patent Application No. 2023-143041 (filed September 4, 2023), which is the basis for the priority claim of this application. All publications cited in this specification, such as prior art documents, published patent applications, patent publications, and other patent documents, are incorporated herein by reference.

[0019] <1. Definition> Adeno-associated virus (AAV) is a small, linear, single-stranded DNA virus belonging to the Parvoviridae family. The AAV genome is a linear, single-stranded DNA of approximately 4.7 kb, with approximately half being positive or negative strand. Hairpin structures called ITRs are present at both ends of the genome. The sequence flanked by the ITRs contains the Rep gene, which encodes nonstructural proteins, and the Cap gene, which encodes capsid protein. AAV serotypes vary depending on the type of capsid protein, and each serotype exhibits different host tropism. Known AAV serotypes include AAV1 to 11. The ITR sequence differs depending on the AAV serotype.

[0020] As used herein, the term "coding sequence" refers to a sequence containing nucleotides that encode a protein to be expressed in a host cell (hereinafter referred to as a "transgene protein"). The transgene protein used in the present invention is not particularly limited, as long as it can be expressed in a host cell.

[0021] As used herein, the term "transgene sequence" refers to a nucleotide sequence that includes a coding sequence and elements necessary for the expression of a transgene protein. When a transgene sequence is referred to herein by a specific sequence, the term "transgene sequence" encompasses both the plus and minus strands of the transgene sequence.

[0022] As used herein, "vector genome" refers to a single-stranded DNA containing a transgene sequence and ITRs that is thought to be packaged into rAAV. In the case of ssAAV, a single-stranded DNA containing the plus strand of the transgene sequence and ITRs may be packaged into the capsid, or a single-stranded DNA containing the minus strand of the transgene sequence and ITRs may be packaged into the capsid. In the case of scAAV, a single-stranded DNA containing the plus and minus strands of the transgene sequence, as well as ITRs and mITRs, is folded into a double strand and packaged into the capsid. In this specification, these single-stranded DNAs are collectively referred to as vector genomes. However, as used herein, "vector genome" may also refer to a fragmented vector genome.

[0023] As used herein, a "recombinant adeno-associated virus (rAAV)" refers to an AAV having a vector genome. As used herein, a rAAV may also be referred to as an "AAV vector."

[0024] Examples of rAAV include single-stranded AAV (ssAAV) and self-complementary AAV (scAAV). ssAAV is an rAAV whose vector genome consists of single-stranded DNA containing the plus or minus strand of a transgene sequence, and double-stranded DNA is formed in a host cell by synthesis of a strand complementary to the plus or minus strand of the transgene sequence or by annealing of two complementary single-stranded DNAs. scAAV is an rAAV whose vector genome is designed so that the plus and minus strands of the transgene sequence are linked by an ITR (mITR) into which a mutation (including base substitution or deletion) that inhibits nick formation by Rep is introduced, and the resulting vector genome is folded to form a double-stranded DNA structure. Given that the packaging capacity of rAAV is approximately 4.9 kb for single-stranded DNA, the size of the transgene sequence is thought to be limited to approximately 4.6 kb for ssAAV and approximately half the size (approximately 2.2 kb) for scAAV, which has a self-complementary single-stranded DNA genome.

[0025] As used herein, the term "truncated CMV promoter sequence" refers to a sequence that contains a TATA box sequence derived from a human cytomegalovirus (CMV) promoter sequence (hereinafter also referred to as a "wild-type CMV promoter sequence"), has promoter activity, and is shorter than the wild-type CMV promoter sequence. The extent to which the truncated CMV promoter sequence of the present invention is shorter than the wild-type CMV promoter sequence is not particularly limited, but examples of the truncated CMV promoter sequence that is shorter than the wild-type CMV promoter sequence include those that are at least 54 bases, 85 bases, or 397 bases shorter than the wild-type CMV promoter sequence. Wild-type CMV promoter sequences derived from multiple human CMV substrains have been reported, and an example of such a sequence is the wild-type CMV promoter sequence shown in SEQ ID NO: 4. In one embodiment of the rAAV of the present invention, the truncated CMV promoter sequence of the present invention is, for example, a promoter sequence that contains a sequence consisting of nucleotides 274 to 469 of the nucleotide sequence of SEQ ID NO: 1, has promoter activity, and is shorter than the wild-type CMV promoter sequence.

[0026] As used herein, "operably linked" means that the truncated CMV promoter sequence is linked to a coding sequence so that the protein encoded by the coding sequence can be expressed in a host cell.

[0027] As used herein, the term "polyadenylation sequence" refers to a DNA sequence located 3' of a coding sequence that enables the addition of a polyadenosine (polyA) tail to the 3' end of mRNA transcribed from a promoter sequence (also referred to as polyadenylation), and includes a DNA motif (also referred to as a PAS motif) corresponding to a polyadenylation signal (PAS). A PAS is generally a conserved 6-base sequence present at the 3' end of an mRNA, and is necessary for mRNA cleavage and the addition of a polyA tail. Known examples of PAS include AAUAAA (AATAAA as the DNA motif) and AUUAAA (ATAAA as the DNA motif). In one embodiment of the rAAV of the present invention, the polyadenylation sequence of the present invention includes AATAAA or ATTAAA. The polyadenylation sequence of the present invention is a DNA sequence that enables polyadenylation, includes a PAS motif, and is shorter than a wild-type polyadenylation sequence (hereinafter referred to as a "truncated polyadenylation sequence"). The extent to which the truncated polyadenylation sequence of the present invention is shorter than a wild-type polyadenylation sequence is not particularly limited, but, for example, in the case of a polyadenylation sequence derived from human growth hormone, it is preferably at least 382 bases or 478 bases shorter than the wild-type polyadenylation sequence. In one embodiment of the rAAV of the present invention, the truncated polyadenylation sequence of the present invention is a DNA sequence that enables polyadenylation, includes a PAS motif, and is a promoter sequence that is shorter than a wild-type polyadenylation sequence, for example, including a sequence consisting of the nucleotides of SEQ ID NO: 3.

[0028] As used herein, "sequence identity" refers to the value "Identity" calculated by the blastn program (Altschul et al., J. Mol. Biol. 1990; 215(3) 403-410, available from https: / / blast.ncbi.nlm.nih.gov / ). The main parameters used in blastn are as follows: Word size = 11, Match / Mismatch Scores = 2, -3, Gapcosts = 5, 2

[0029] 2. rAAV of the Present Invention The present invention provides the following rAAV (hereinafter also referred to as "rAAV of the present invention"): A rAAV comprising a transgene sequence, wherein the transgene sequence is: (a) a transgene sequence comprising: i) a truncated CMV promoter sequence comprising a sequence consisting of nucleotides 274 to 469 of the nucleotide sequence of SEQ ID NO: 1, or a nucleotide sequence having 90% or more sequence identity to said sequence, ii) a coding sequence operably linked to the truncated CMV promoter sequence, and iii) a polyadenylation sequence; or (b) a transgene sequence comprising: i) a promoter sequence, ii) a coding sequence operably linked to the promoter sequence, and iii) a truncated polyadenylation sequence comprising a sequence consisting of the nucleotide sequence of SEQ ID NO: 3, or a nucleotide sequence having 90% or more sequence identity to said sequence.

[0030] In one embodiment of the rAAV of the present invention, the following rAAV is provided: An rAAV comprising a transgene sequence, the transgene sequence comprising: i) a truncated CMV promoter sequence comprising a sequence consisting of nucleotides 274 to 469 of the nucleotide sequence of SEQ ID NO: 1, or a nucleotide sequence having 90% or more sequence identity to said sequence; ii) a coding sequence operably linked to said truncated CMV promoter sequence; and iii) a polyadenylation sequence.

[0031] In one embodiment of the rAAV of the present invention, the rAAV is a ssAAV or a scAAV. In another aspect, the rAAV is a scAAV.

[0032] In one embodiment, the rAAV is a ssAAV.

[0033] In one embodiment, the rAAV is a scAAV.

[0034] In one embodiment of the rAAV of the present invention, the rAAV is an scAAV, and the transgene sequence is 2115 bases or less in length, or 2084 bases or less in length.

[0035] More specifically, the truncated CMV promoter sequence used in the present invention is not particularly limited as long as it comprises: (a) a sequence consisting of nucleotides 274 to 469 in the nucleotide sequence of SEQ ID NO: 1 (hereinafter referred to as sequence a); (b) a nucleotide sequence having 90% or more sequence identity to sequence a (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more); or (c) a nucleotide sequence in which 1 to 60, 1 to 10, 1 to 7, 1 to 5, 1 to 3, or 1 to 2 bases have been deleted, substituted, inserted, and / or added to sequence a; the truncated CMV promoter sequence has promoter activity; and is shorter than the wild-type CMV promoter sequence. Furthermore, the truncated CMV promoter sequence used in the present invention is not limited to (A) the entire promoter sequence containing the above sequence a (hereinafter referred to as sequence A), but also includes (B) a nucleotide sequence having 90% or more (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to the above sequence A, or (C) a promoter sequence that contains a nucleotide sequence in which 1 to 60, 1 to 10, 1 to 7, 1 to 5, 1 to 3, or 1 to 2 bases have been deleted, substituted, inserted, and / or added to the above sequence A, and that has promoter activity and is shorter than the wild-type CMV promoter sequence. The examples of truncated CMV promoter sequences in this and the following paragraphs are applicable throughout this specification.

[0036] In one embodiment of the rAAV of the present invention, the truncated CMV promoter sequence used in the present invention comprises: (a) the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 2; (b) a nucleotide sequence in which 1 to 60, 1 to 10, 1 to 7, 1 to 5, 1 to 3, or 1 to 2 bases have been deleted, substituted, inserted, and / or added relative to the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 2, and which has promoter activity; or (c) a nucleotide sequence having 90% or more (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 2, and which has promoter activity. In another aspect, the truncated CMV promoter sequence used in the present invention consists of (d) the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 2, (e) a nucleotide sequence in which 1 to 60, 1 to 10, 1 to 7, 1 to 5, 1 to 3, or 1 to 2 bases have been deleted, substituted, inserted, and / or added to the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 2, and which has promoter activity, or (f) a nucleotide sequence having 90% or more sequence identity (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) to the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 2, and which has promoter activity. In one embodiment of the rAAV of the present invention, the truncated CMV promoter sequence of the present invention may further comprise a transcription start site (TSS).

[0037] The polyadenylation sequence used in the present invention is not particularly limited, as long as it is a sequence that allows polyadenylation. In one embodiment of the rAAV of the present invention, the polyadenylation sequence used in the present invention includes the polyadenylation sequence derived from Simian virus 40 (SV40), the polyadenylation sequence derived from human beta globin, the polyadenylation sequence derived from rabbit beta globin, the polyadenylation sequence derived from bovine growth hormone, or the polyadenylation sequence derived from human growth hormone. In another embodiment, the polyadenylation sequence used in the present invention is the polyadenylation sequence derived from human growth hormone. As used herein, the term "shortened polyadenylation sequence derived from human growth hormone" refers to a sequence that includes a sequence derived from the polyadenylation sequence derived from human growth hormone shown in SEQ ID NO: 5 (hereinafter also referred to as "wild-type human growth hormone polyadenylation sequence"), enables polyadenylation, and is shorter than the wild-type human growth hormone polyadenylation sequence. In another aspect, the truncated polyadenylation sequence derived from human growth hormone of the present invention comprises: (A) the nucleotide sequence of SEQ ID NO: 3; (B) a nucleotide sequence in which 1 to 10, 1 to 7, 1 to 5, 1 to 3, or 1 to 2 bases have been deleted, substituted, inserted, and / or added in the nucleotide sequence of SEQ ID NO: 3, and which enables polyadenylation; or (C) a nucleotide sequence having 90% or more (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to the nucleotide sequence of SEQ ID NO: 3, and which enables polyadenylation.In another embodiment, the truncated polyadenylation sequence derived from human growth hormone of the present invention comprises (D) the nucleotide sequence of SEQ ID NO: 3, (E) a nucleotide sequence in which 1 to 10, 1 to 7, 1 to 5, 1 to 3, or 1 to 2 bases have been deleted, substituted, inserted, and / or added in the nucleotide sequence of SEQ ID NO: 3, and which enables polyadenylation, or (F) a nucleotide sequence which has 90% or more sequence identity (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) to the nucleotide sequence of SEQ ID NO: 3, and which enables polyadenylation. The exemplary explanations of polyadenylation sequences and truncated polyadenylation sequences in this paragraph are applicable throughout the present specification.

[0038] In one aspect of the present invention, the following rAAV is provided: an rAAV comprising a transgene sequence, the transgene sequence comprising: i) a truncated CMV promoter sequence comprising a sequence consisting of nucleotides 274 to 469 of the nucleotide sequence of SEQ ID NO: 1, or a nucleotide sequence having 90% or more sequence identity to said sequence; ii) a coding sequence operably linked to said truncated CMV promoter sequence; and iii) a truncated polyadenylation sequence derived from human growth hormone.

[0039] In one aspect of the present invention, the following rAAV is provided: an rAAV comprising a transgene sequence, the transgene sequence comprising: i) a truncated CMV promoter sequence comprising a sequence consisting of nucleotides 274 to 469 of the nucleotide sequence of SEQ ID NO: 1, or a nucleotide sequence having 90% or more sequence identity to said sequence; ii) a coding sequence operably linked to said truncated CMV promoter sequence; and iii) a truncated polyadenylation sequence comprising the nucleotide sequence of SEQ ID NO: 3.

[0040] In one aspect of the present invention, the following rAAV is provided: an rAAV comprising a transgene sequence, the transgene sequence comprising: i) a truncated CMV promoter sequence comprising a sequence consisting of nucleotides 274 to 469 of the nucleotide sequence of SEQ ID NO: 1, or a nucleotide sequence having 90% or more sequence identity to said sequence; ii) a coding sequence operably linked to said truncated CMV promoter sequence; and iii) a sequence consisting of the nucleotide sequence of SEQ ID NO: 3.

[0041] In one aspect of the present invention, there is provided an rAAV comprising a transgene sequence comprising: i) a truncated CMV promoter sequence comprising the nucleotide sequence of SEQ ID NO: 1; ii) a coding sequence operably linked to the truncated CMV promoter sequence; and iii) a truncated polyadenylation sequence derived from human growth hormone.

[0042] In one aspect of the present invention, the following rAAV is provided: an rAAV comprising a transgene sequence, the transgene sequence comprising: i) a truncated CMV promoter sequence comprising the nucleotide sequence of SEQ ID NO: 1; ii) a coding sequence operably linked to the truncated CMV promoter sequence; and iii) a truncated polyadenylation sequence comprising the nucleotide sequence of SEQ ID NO: 3.

[0043] In one aspect of the present invention, there is provided an rAAV comprising a transgene sequence, the transgene sequence comprising: i) a truncated CMV promoter sequence comprising the nucleotide sequence of SEQ ID NO: 1; ii) a coding sequence operably linked to the truncated CMV promoter sequence; and iii) a sequence consisting of the nucleotide sequence of SEQ ID NO: 3.

[0044] In one aspect of the present invention, there is provided an rAAV comprising a transgene sequence comprising: i) a truncated CMV promoter sequence consisting of the nucleotide sequence of SEQ ID NO: 1; ii) a coding sequence operably linked to the truncated CMV promoter sequence; and iii) a truncated polyadenylation sequence derived from human growth hormone.

[0045] In one aspect of the present invention, there is provided an rAAV comprising a transgene sequence comprising: i) a truncated CMV promoter sequence consisting of the nucleotide sequence of SEQ ID NO: 1; ii) a coding sequence operably linked to the truncated CMV promoter sequence; and iii) a truncated polyadenylation sequence comprising the nucleotide sequence of SEQ ID NO: 3.

[0046] In one aspect of the present invention, there is provided an rAAV comprising a transgene sequence, the transgene sequence comprising: i) a truncated CMV promoter sequence consisting of the nucleotide sequence of SEQ ID NO: 1; ii) a coding sequence operably linked to the truncated CMV promoter sequence; and iii) a sequence consisting of the nucleotide sequence of SEQ ID NO: 3.

[0047] In one aspect of the present invention, the following rAAV is provided: an rAAV comprising a transgene sequence, the transgene sequence comprising: i) a truncated CMV promoter sequence comprising the nucleotide sequence of SEQ ID NO: 2; ii) a coding sequence operably linked to the truncated CMV promoter sequence; and iii) a truncated polyadenylation sequence derived from human growth hormone.

[0048] In one aspect of the present invention, the following rAAV is provided: an rAAV comprising a transgene sequence, the transgene sequence comprising: i) a truncated CMV promoter sequence comprising the nucleotide sequence of SEQ ID NO:2; ii) a coding sequence operably linked to the truncated CMV promoter sequence; and iii) a truncated polyadenylation sequence comprising the nucleotide sequence of SEQ ID NO:3.

[0049] In one aspect of the present invention, there is provided an rAAV comprising a transgene sequence, the transgene sequence comprising: i) a truncated CMV promoter sequence comprising the nucleotide sequence of SEQ ID NO:2; ii) a coding sequence operably linked to the truncated CMV promoter sequence; and iii) a sequence consisting of the nucleotide sequence of SEQ ID NO:3.

[0050] In one aspect of the present invention, there is provided an rAAV comprising a transgene sequence comprising: i) a truncated CMV promoter sequence consisting of the nucleotide sequence of SEQ ID NO: 2; ii) a coding sequence operably linked to the truncated CMV promoter sequence; and iii) a truncated polyadenylation sequence derived from human growth hormone.

[0051] In one aspect of the present invention, the following rAAV is provided: an rAAV comprising a transgene sequence, the transgene sequence comprising: i) a truncated CMV promoter sequence consisting of the nucleotide sequence of SEQ ID NO:2; ii) a coding sequence operably linked to the truncated CMV promoter sequence; and iii) a truncated polyadenylation sequence comprising the nucleotide sequence of SEQ ID NO:3.

[0052] In one aspect of the present invention, there is provided an rAAV comprising a transgene sequence, the transgene sequence comprising: i) a truncated CMV promoter sequence consisting of the nucleotide sequence of SEQ ID NO:2; ii) a coding sequence operably linked to the truncated CMV promoter sequence; and iii) a sequence consisting of the nucleotide sequence of SEQ ID NO:3.

[0053] In one aspect of the present invention, the following rAAV is provided: an rAAV comprising a transgene sequence, the transgene sequence comprising: i) a truncated CMV promoter sequence comprising the nucleotide sequence of SEQ ID NO: 2; ii) a coding sequence operably linked to the truncated CMV promoter sequence; and iii) a truncated polyadenylation sequence comprising the nucleotide sequence of SEQ ID NO: 3, wherein the rAAV is a scAAV and the transgene sequence is 2115 bases or less in length.

[0054] In one aspect of the present invention, the following rAAV is provided: an rAAV comprising a transgene sequence, the transgene sequence comprising: i) a truncated CMV promoter sequence comprising the nucleotide sequence of SEQ ID NO: 2; ii) a coding sequence operably linked to the truncated CMV promoter sequence; and iii) a truncated polyadenylation sequence comprising the nucleotide sequence of SEQ ID NO: 3, wherein the rAAV is a scAAV and the transgene sequence is 2084 bases or less in length.

[0055] In one aspect of the present invention, there is provided the following rAAV: an rAAV comprising a transgene sequence, the transgene sequence comprising: i) a truncated CMV promoter sequence consisting of the nucleotide sequence of SEQ ID NO: 2; ii) a coding sequence operably linked to the truncated CMV promoter sequence; and iii) a sequence consisting of the nucleotide sequence of SEQ ID NO: 3, wherein the rAAV is a scAAV and the transgene sequence is 2115 bases or less in length.

[0056] In one aspect of the present invention, there is provided the following rAAV: an rAAV comprising a transgene sequence, the transgene sequence comprising: i) a truncated CMV promoter sequence consisting of the nucleotide sequence of SEQ ID NO: 2; ii) a coding sequence operably linked to the truncated CMV promoter sequence; and iii) a sequence consisting of the nucleotide sequence of SEQ ID NO: 3, wherein the rAAV is a scAAV and the transgene sequence is 2084 bases or less in length.

[0057] In one aspect of the present invention, the following rAAV is provided: an rAAV comprising a transgene sequence, the transgene sequence comprising: i) a truncated CMV promoter sequence comprising the nucleotide sequence of SEQ ID NO: 1; ii) a coding sequence operably linked to the truncated CMV promoter sequence; and iii) a truncated polyadenylation sequence comprising the nucleotide sequence of SEQ ID NO: 3, wherein the rAAV is a scAAV and the transgene sequence is 2115 bases or less in length.

[0058] In one aspect of the present invention, the following rAAV is provided: an rAAV comprising a transgene sequence, the transgene sequence comprising: i) a truncated CMV promoter sequence comprising the nucleotide sequence of SEQ ID NO: 1; ii) a coding sequence operably linked to the truncated CMV promoter sequence; and iii) a truncated polyadenylation sequence comprising the nucleotide sequence of SEQ ID NO: 3, wherein the rAAV is a scAAV and the transgene sequence is 2084 bases or less in length.

[0059] In one aspect of the present invention, there is provided the following rAAV: an rAAV comprising a transgene sequence, the transgene sequence comprising: i) a truncated CMV promoter sequence consisting of the nucleotide sequence of SEQ ID NO: 1; ii) a coding sequence operably linked to the truncated CMV promoter sequence; and iii) a sequence consisting of the nucleotide sequence of SEQ ID NO: 3, wherein the rAAV is a scAAV and the transgene sequence is 2115 bases or less in length.

[0060] In one aspect of the present invention, there is provided the following rAAV: an rAAV comprising a transgene sequence, the transgene sequence comprising: i) a truncated CMV promoter sequence consisting of the nucleotide sequence of SEQ ID NO: 1; ii) a coding sequence operably linked to the truncated CMV promoter sequence; and iii) a sequence consisting of the nucleotide sequence of SEQ ID NO: 3, wherein the rAAV is a scAAV and the transgene sequence is 2084 bases or less in length.

[0061] In one embodiment of the rAAV of the present invention, the following rAAV is provided: An rAAV comprising a transgene sequence, wherein the transgene sequence comprises: i) a promoter sequence; ii) a coding sequence operably linked to the promoter sequence; and iii) a polyadenylation sequence comprising a sequence consisting of the nucleotide sequence of SEQ ID NO: 3, or a nucleotide sequence having 90% or more sequence identity to the sequence.

[0062] In one embodiment, the rAAV is a ssAAV.

[0063] In one embodiment, the rAAV is a scAAV.

[0064] In one embodiment, the rAAV is an scAAV, and the transgene sequence is 2187 bases or less in length.

[0065] In one embodiment, the rAAV is an scAAV, and the transgene sequence is 2115 bases or less in length.

[0066] In one embodiment, the rAAV is an scAAV, and the transgene sequence is 2084 bases or less in length.

[0067] The truncated polyadenylation sequence used in the present invention may be any of the polyadenylation sequences described in this section.

[0068] The promoter sequence is not particularly limited, and examples that can be used include a CMV promoter, a truncated CMV promoter, a CMV early enhancer / chicken beta actin gene (CAG, CBh, CBA) promoter, a phosphoglycerate kinase gene (PGK) promoter, a synapsin 1 gene (SYN1) promoter, a gamma synuclein gene (SNCG) promoter, a rhodopsin kinase gene (GRK1) promoter, a cone rod homeobox gene (CRX) promoter, a retinal homeobox gene (RX) promoter, and an RPE65 gene promoter.

[0069] In one aspect of the present invention, there is provided an rAAV comprising a transgene sequence, the transgene sequence comprising: i) a CMV promoter sequence; ii) a coding sequence operably linked to the CMV promoter sequence; and iii) a polyadenylation sequence comprising a sequence consisting of the nucleotide sequence of SEQ ID NO: 3, or a nucleotide sequence having 90% or more sequence identity to the sequence.

[0070] In one aspect of the present invention, the following rAAV is provided: An rAAV comprising a transgene sequence, the transgene sequence comprising: i) a CMV promoter sequence; ii) a coding sequence operably linked to the CMV promoter sequence; and iii) a polyadenylation sequence comprising a sequence consisting of the nucleotide sequence of SEQ ID NO:3.

[0071] In one embodiment of the rAAV of the present invention, the rAAV of the present invention can use, as the AAV serotype, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, or an artificially modified serotype derived from any of these AAVs.

[0072] In one embodiment of the rAAV of the present invention, the ITRs contained in the rAAV of the present invention can be ITRs derived from AAV2 or AAV5. In another aspect, an scAAV, which is an rAAV of the present invention, can be produced by using an ITR derived from AAV2 or AAV5 and an ITR (mITR) into which a mutation (including base substitution or deletion) that inhibits nick formation by Rep has been introduced.

[0073] In one embodiment of the rAAV of the present invention, the transgene sequence contained in the rAAV of the present invention may further include post-transcriptional regulatory elements, various expression regulatory elements (see, for example, Goeddel, Gene Expression Technology, Methods in Enzymology, 1990. 185. Academic Press, San Diego), translation initiation codons, translation termination codons, Kozak sequences, splicing junctions, etc. As used herein, the term "post-transcriptional regulatory element" refers to a sequence that regulates gene expression by post-transcriptional control.

[0074] The rAAV of the present invention can be used as a pharmaceutical composition for preventing and / or treating various diseases by incorporating a transgene sequence containing an appropriate coding sequence and, if necessary, formulating it according to standard methods. The present invention also provides a method for preventing and / or treating various diseases, comprising administering the rAAV of the present invention or the pharmaceutical composition to a subject (patient) in need of such prevention and / or treatment. Furthermore, the present invention may also provide the use of the rAAV of the present invention for preventing and / or treating various diseases, the use of the rAAV of the present invention for producing a medicament or pharmaceutical composition for preventing and / or treating various diseases, and the rAAV of the present invention for use in preventing and / or treating various diseases.

[0075] 3. Methods for Producing the rAAV of the Present Invention The rAAV of the present invention can be produced using, for example, known methods such as (1) a method using a plasmid, (2) a method using a baculovirus, (3) a method using herpes simplex virus, (4) a method using an adenovirus, or (5) a method using yeast (see, e.g., Appl Microbiol Biotechnol. 2018; 102(3): 1045-1054). For example, in the method using a plasmid (1), known scAAV production plasmids or ssAAV production plasmids can be used to insert the transgene sequence of the rAAV of the present invention. The transgene sequence and ITR sequence of the rAAV of the present invention can be chemically synthesized using known DNA synthesis methods. Since the produced rAAV is present in the cell culture medium and / or intracellularly, the host cells are disrupted by freezing and thawing, treating with a surfactant, ultrasonication, or the like, and then the virus is recovered from the cell culture medium, etc., and the virus fraction is separated and purified by ion exchange chromatography and / or hydrophobic interaction chromatography, cesium chloride density gradient centrifugation, sucrose density gradient centrifugation, iodixanol density gradient centrifugation, ultrafiltration, diafiltration, affinity chromatography, polyethylene glycol precipitation, ammonium sulfate precipitation, or the like, to prepare the desired rAAV.

[0076] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples. When commercially available kits or reagents were used, the experiments were carried out according to the attached protocols unless otherwise specified.

[0077] Example 1: Preparation of plasmid construct Human TFEB (hTFEB) cDNA (SEQ ID NO: 7; see GenBank Accession no. NM_007162) prepared by gene synthesis was inserted into a plasmid construct for producing scAAV, pscAAV-CMV-hTFEB-hGHpA (KA060; SEQ ID NO: 6), which contains a wild-type CMV promoter (SEQ ID NO: 4; see the 566th to 1157th bases of the 1848-base nucleotide sequence registered in GenBank Accession no. X03922) and a wild-type human growth hormone polyadenylation sequence (hGHpA) (SEQ ID NO: 5), to obtain a plasmid construct pscAAV-CMV-hTFEB-hGHpA (referred to as KA072; SEQ ID NO: 8).

[0078] Furthermore, the wild-type hGHpA was removed from KA060, and a truncated polyadenylation sequence derived from human growth hormone (hGHpA(SS); SEQ ID NO: 3) consisting of hTFEB cDNA and a sequence containing a PAS motif was inserted to obtain the plasmid construct pscAAV-CMV-hTFEB-hGHpA(SS)-1 (designated KA074; SEQ ID NO: 9). Furthermore, an unnecessary restriction enzyme site contained downstream of the wild-type CMV promoter of KA074 was removed to prepare the plasmid construct pscAAV-CMV-hTFEB-hGHpA(SS)-2 (designated KA093; SEQ ID NO: 10).

[0079] Next, plasmid constructs pscAAV-CMV(S2)-hTFEB-hGHpA(SS) (KA094; SEQ ID NO: 11) and pscAAV-CMV(S3)-hTFEB-hGHpA(SS) (KA095; SEQ ID NO: 12) containing truncated CMV promoters (CMV(S2); SEQ ID NO: 2 and CMV(S3); SEQ ID NO: 1, respectively) were prepared by deleting 54 and 85 bases from the 5' end of the wild-type CMV promoter of KA074. Furthermore, a plasmid construct pscAAV-CMV(173)-hTFEB-hGHpA(SS) (designated KA152; SEQ ID NO: 13) was prepared in place of the wild-type CMV promoter of KA074, which contains a truncated CMV promoter region corresponding to the truncated CMV promoter consisting of the nucleotide sequence shown as SEQ ID NO: 1 in the aforementioned Patent Document 1. In addition, a plasmid construct pscAAV-CMV(S9)-hTFEB-hGHpA(SS) (designated KA101; SEQ ID NO: 14) was prepared in which the CMV proximal enhancer region of KA095 was further deleted, leaving the distal enhancer. Furthermore, a known truncated hGHpA (referred to as S-hGHpA; a natural human growth hormone polyadenylation sequence, corresponding to the nucleotide sequence shown as SEQ ID NO: 16 in Table 3 of Patent Document 2 (GenBank Accession no. NC_000017.11:c63917140-63917250)) was inserted into KA072 hGHpA to prepare pscAAV-CMV-hTFEB-short hGHpA (referred to as KE001; SEQ ID NO: 17).

[0080] A schematic representation of these plasmid constructs is shown in FIG.

[0081] Example 2: rAAV Production HEK293T cells (ATCC, catalog number CRL-3216) were cultured at 3 × 10 ３ cells / cm ２ The cells were seeded at a density of 1000 x g and cultured in 10% fetal bovine serum (FBS, Hyclone). ＴＭThe cells were statically cultured for 4 days in Dulbecco's Modified Eagle Medium (DMEM, Sigma-Aldrich, Catalog No. D6429) containing Triton X-10 (GE Healthcare Life Sciences, Catalog No. SH30070.03). KA072 or KA074 obtained in Example 1 was introduced into the cells together with two helper plasmids, pRC_AAV8 (SEQ ID NO: 15) and pHelper (SEQ ID NO: 16), using a transfection reagent (Polysciences, Catalog No. 24765-2, PEI MAX (registered trademark)). The cells were statically cultured in DMEM containing 2% FBS. Three days after transfection, the culture medium was diluted with Triton X-10 to a final concentration of 0.2%. ＴＭ X-100 was added, and the mixture was allowed to stand at room temperature for 1 hour, and then filtered through a 0.22 μm bottle-top filter to obtain a lysate.

[0082] Next, 2 mM magnesium chloride and 100 U / mL Benzonase® (Merck, catalog number 1.01656.0001) (final concentrations) were added to the lysate, and the mixture was incubated overnight at 4°C. After incubation, the mixture was transferred to an affinity support (Thermo Fisher Scientific, catalog number A36743, POROS ＴＭ CaptureSelect ＴＭ rAAV particles were purified using AAVX Affinity Resin. The rAAV particles were eluted from the carrier with 100 mM glycine, 150 mM NaCl, and 0.01% polysorbate 20 (pH 2.7) and neutralized by adding 3% (v / v) of the eluate to 2 M Tris-HCl (pH 9.0). The rAAV produced using KA072 is referred to as AAV-KA072, and the rAAV produced using KA074 is referred to as AAV-KA074.

[0083] Furthermore, rAAVs were produced in a similar manner using KA093, KA094, KA095, and KA101 and purified by affinity chromatography. The resulting rAAVs are designated AAV-KA093, AAV-KA094, AAV-KA095, and AAV-KA101, respectively.

[0084] Example 3: Analysis of rAAV genomic DNA The rAAV prepared in Example 2 was treated with DNase I and then with Proteinase K, and the rAAV genomic DNA was purified using a DNA purification column (QIAGEN, Catalog No. 28104, QIAquick® PCR Purification Kit). ＴＭ The single-stranded DNA concentration was calculated using the chromatogram, and 100 to 200 ng of DNA was analyzed by alkaline agarose gel electrophoresis containing 50 mM sodium hydroxide (NaOH). The rAAV genomic DNA and DNA size markers were denatured in the presence of 50 mM NaOH / 0.3% SDS at 95°C for 5 minutes before electrophoresis.

[0085] After electrophoresis, the gel was stained with DNA staining reagent (Biotium, Cat. No. 41003, GelRed ＴＭ ) and then visualized with a UV transilluminator (GE Healthcare, Amersham, ＴＭ Imager 680) or ChemiDoc ＴＭ The vectors were analyzed using a Touch Imaging System (BIO-RAD) (Figure 2). The estimated sizes of the full-length vector genomes are shown at the bottom of Figure 2 and in Table 1. As shown in Figure 2, AAV-KA072 did not contain a full-length vector genome, but contained heterogeneous DNA smaller than the full-length vector genome, which is likely a fragmented vector genome. On the other hand, AAV-KA074 contained a full-length vector genome, and the proportion of fragmented vector genomes was significantly reduced. In AAV-KA093, the proportion of full-length vector genomes further increased, while AAV-KA094, AAV-KA095, and AAV-KA101 mostly contained full-length vector genomes.

[0086]

[0087] Example 4: Transgene protein expression analysis using plasmid constructs To evaluate the activity of the truncated CMV promoter sequence and truncated human growth hormone polyadenylation sequence to increase the expression of a transgene protein, transgene protein expression analysis was performed using a plasmid construct. One day before the transfection experiment, HEK293 cells (ATCC, catalog number CRL-1573) were seeded on a collagen I-coated 24-well microplate (Iwaki, catalog number 4820-010) and incubated with 10% FBS (Hyclone). ＴＭ The cells were cultured in DMEM (Sigma-Aldrich, catalog number D6429) containing 1% PBS (GE Healthcare Life Sciences, catalog number SH30084.03) and 1% penicillin-streptomycin (Thermo Fisher Scientific, catalog number 15070-063) at 37°C and 5% CO ２ One day after cell seeding, in order to introduce the plasmids into the cells, equal amounts of the plasmid constructs (KA072, KA074, KA093, KA094, KA095, KA101, and KA152) prepared in Example 1 were added to the cells in a transfection reagent (Lipofectamine ＴＭ 2000, Thermo Fisher Scientific, Catalog No. 11668-027), low serum medium (Opti-MEM ＴＭ The mixture was mixed with 100% ethanol (100% ethanol, Thermo Fisher Scientific, Cat. No. 31985-062) and incubated at room temperature for 5 minutes, then added dropwise to the cells. ２ After static culture for 1 day under these conditions, the cells were washed with D-PBS(-) (Fujifilm Wako Pure Chemical Industries, Ltd., catalog number 045-29795), and a cell lysis buffer was added to recover the lysate, which was then stored at -80°C. ＴＭ RIPA Buffer (Thermo Fisher Scientific, Cat. No. 89901) was diluted with 1 / 1000 volume of Benzonase® Nuclease (Merck Millipore, Cat. No. 70664) and 1 / 100 volume of Halt. ＴＭProtease and Phosphatase Inhibitor Cocktail (Thermo Fisher Scientific, Catalog No. 78441) was added to prepare the lysate. After thawing on ice, the lysate was centrifuged at 4°C and 12,000 rpm for 5 minutes using a centrifuge (Hitachi), and the supernatant was used for subsequent tests. The protein concentration of each sample was determined by Pierce. ＴＭ The BCA Protein Assay Kit (Thermo Fisher Scientific, Catalog No. 23227) was used, and absorbance at 562 nm was measured using a microplate reader (SpectraMax® Plus 384, Molecular Devices). hTFEB expression in HEK293 cells was confirmed by Western blotting using equal amounts of protein per sample. The following antibodies were used for detection: The primary antibody used for hTFEB detection was Mouse anti-TFEB antibody (SANTA CRUZ Biotechnology, catalog number sc-166736), and the secondary antibody used for β-actin detection was Rabbit anti-β-actin antibody (Cell Signaling Technology, catalog number 4967S). ＴＭ Anti-mouse IgG, HRP-Linked Whole Ab from Sheep (GE Healthcare Life Sciences, Catalog No. NA931V) was used for detecting β-actin using ECL. ＴＭ Anti-rabbit IgG, HRP-linked F(ab')2 fragment (from Donkey) (GE Healthcare Life Sciences, Catalog No. NA9340V) were used. Western blot detection was performed using ECL. ＴＭ Prime Western Blotting Detection Reagents (Cytiva, Cat. No. RPN2232) were used, and ChemiDoc ＴＭImages were taken using a Touch Imaging System (BIO-RAD) and quantified using Image Lab software (BIO-RAD). Analysis of transgene protein expression levels was performed by correcting using the β-actin expression level in the same sample, and the total hTFEB expression level, including endogenous hTFEB expression, was calculated relative to the other constructs, with KA072 set at 100 and the background value set at 0. The plasmid constructs (KA072, KE001, and KA074) prepared in Example 1 were also evaluated for their activity of increasing transgene protein expression using the same method.

[0088] Cells transfected with KA074, which contains a truncated human growth hormone polyadenylation sequence, showed similar levels of hTFEB expression upregulation as KA072 without the truncated modification. The upregulation of hTFEB expression was also maintained in cells transfected with KA093, which contains a truncated CMV promoter with an unnecessary restriction enzyme site removed downstream of the CMV promoter in addition to the truncated human growth hormone polyadenylation sequence, and KA094 and KA095, which contain truncated CMV promoters with a 5' deletion. On the other hand, the upregulation of hTFEB expression was limited in cells transfected with KA101, which contains a truncated CMV promoter with an additional deletion of the CMV proximal enhancer region, and KA152, which contains a truncated CMV promoter with an additional deletion of the 5' end of the CMV promoter (Figure 3). These results suggest that the truncated human growth hormone-derived polyadenylation sequence (hGHpA(SS)) shown in SEQ ID NO: 3 and the truncated CMV promoter CMV(S3) shown in SEQ ID NO: 1 have activity equivalent to that of the wild-type human growth hormone polyadenylation sequence and the wild-type CMV promoter, respectively. Furthermore, it is suggested that the sequence (nucleotides 274 to 440 in the nucleotide sequence of SEQ ID NO: 1) that is located upstream of the core element TATA box (nucleotides 463 to 469 in SEQ ID NO: 1) and contained in CMV(S2) and CMV(S3) (a sequence consisting of nucleotides 274 to 469 in the nucleotide sequence of SEQ ID NO: 1) and that is deleted in CMV(S9) is important for achieving activity equivalent to that of the wild-type CMV promoter. rAAV constructed using these plasmid constructs are considered to have the potential to increase expression of transgene proteins, as shown in Figure 3 .

[0089] Furthermore, although hGHpA(SS) (SEQ ID NO: 3) is a shorter sequence than S-hGH pA (SEQ ID NO: 16 of Patent Document 2), the activity of increasing hTFEB expression in cells transfected with KA074 containing hGHpA(SS) was comparable to the activity of increasing hTFEB expression in cells transfected with KA072 containing the wild-type human growth hormone polyadenylation sequence or KE001 containing a known truncated human growth hormone polyadenylation sequence (Figure 4). rAAV containing a truncated human growth hormone polyadenylation sequence (hGHpA(SS); SEQ ID NO: 3), which is even shorter than the known truncated human growth hormone polyadenylation sequence (S-hGH pA; SEQ ID NO: 16 of Patent Document 2), is considered to potentially have activity equivalent to that of rAAV containing the wild-type human growth hormone polyadenylation sequence.

[0090] The rAAV of the present invention is expected to reduce vector genome fragmentation due to oversize during the production process and to have the activity of expressing a transgene protein, and is useful, for example, for research, development, and commercial production of rAAV for preventing and / or treating various diseases.

[0091] SEQ ID NO: 1: Synthetic construct (CMV promoter (S3)) SEQ ID NO: 2: Synthetic construct (CMV promoter (S2)) SEQ ID NO: 3: Synthetic construct (hGHpA(SS)) SEQ ID NO: 6: Synthetic construct (pscAAV-CMV-hGHpA(KA060)) SEQ ID NO: 8: Synthetic construct (pscAAV-CMV-hTFEB-hGHpA(KA072)) SEQ ID NO: 9: Synthetic construct (pscAAV-CMV-hTFEB-hGHpA(SS)-1(KA074)) SEQ ID NO: 10: Synthetic construct (pscAAV-CMV-hTFEB-hGHpA(SS)-2(KA093)) SEQ ID NO: 11: Synthetic construct (pscAAV-CMV(S2)-hTFEB-hGHpA(SS)(KA094)) SEQ ID NO: 12: Synthetic construct (pscAAV-CMV(S3)-hTFEB-hGHpA(SS)(KA095)) SEQ ID NO: 13: Synthetic construct (pscAAV-CMV(173)-hTFEB-hGHpA(SS)(KA152)) SEQ ID NO: 14: Synthetic construct (pscAAV-CMV(S9)-hTFEB-hGHpA(SS)(KA101)) SEQ ID NO: 15: Synthetic construct (helper plasmid pRC_AAV8) SEQ ID NO: 16: Synthetic construct (helper plasmid pHelper) SEQ ID NO: 17: Synthetic construct (pscAAV-CMV-hTFEB-short hGHpA(KE001))

Claims

1. A recombinant adeno-associated virus (rAAV) comprising a transgene sequence, the transgene sequence being: (a) a transgene sequence comprising: i) a truncated CMV promoter sequence comprising a sequence consisting of nucleotides 274 to 469 of the nucleotide sequence of SEQ ID NO:1, or a nucleotide sequence having 90% or more sequence identity to said sequence; ii) a coding sequence operably linked to said truncated CMV promoter sequence; and iii) a polyadenylation sequence; or (b) a transgene sequence comprising: i) a promoter sequence; ii) a coding sequence operably linked to said promoter sequence; and iii) a truncated polyadenylation sequence comprising a sequence consisting of the nucleotide sequence of SEQ ID NO:3, or a nucleotide sequence having 90% or more sequence identity to said sequence.

2. The rAAV of claim 1, wherein the transgene sequence comprises: i) a truncated CMV promoter sequence comprising a sequence consisting of nucleotides 274 to 469 of the nucleotide sequence of SEQ ID NO:1, or a nucleotide sequence having 90% or greater sequence identity thereto; ii) a coding sequence operably linked to the truncated CMV promoter sequence; and iii) a polyadenylation sequence.

3. The rAAV of claim 2, wherein the rAAV is a self-complementary AAV (scAAV).

4. The rAAV of claim 3, wherein the transgene sequence is 2115 bases or less in length.

5. The rAAV of any one of claims 2 to 4, wherein the truncated CMV promoter sequence comprises the nucleotide sequence of SEQ ID NO:1 or SEQ ID NO:2, or a nucleotide sequence having 90% or greater sequence identity to the nucleotide sequence of SEQ ID NO:1 or SEQ ID NO:

2.

6. The rAAV of any one of claims 2 to 4, wherein the polyadenylation sequence is a polyadenylation sequence derived from human growth hormone.

7. The rAAV of any one of claims 2 to 4, wherein the polyadenylation sequence is a truncated polyadenylation sequence derived from human growth hormone.

8. The rAAV of any one of claims 2 to 4, wherein the polyadenylation sequence is a truncated polyadenylation sequence comprising the nucleotide sequence of SEQ ID NO:3 or a nucleotide sequence having 90% or more sequence identity to the nucleotide sequence of SEQ ID NO:

3.

9. The rAAV of any one of claims 2 to 4, wherein the truncated CMV promoter sequence consists of the nucleotide sequence of SEQ ID NO:1 and the polyadenylation sequence consists of the nucleotide sequence of SEQ ID NO:

3.

10. The rAAV of claim 1, wherein the transgene sequence comprises: i) a promoter sequence; ii) a coding sequence operably linked to the promoter sequence; and iii) a truncated polyadenylation sequence comprising a sequence consisting of the nucleotide sequence of SEQ ID NO:3, or a nucleotide sequence having 90% or greater sequence identity thereto.

11. The rAAV of claim 10, wherein the truncated polyadenylation sequence comprises the nucleotide sequence of SEQ ID NO:

3.

12. The rAAV of claim 10, wherein the promoter sequence is a CMV promoter sequence.

13. The rAAV of any one of claims 10 to 12, wherein the rAAV is a single-stranded AAV (ssAAV).

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