阅读说明：本技术 用于生产aav颗粒的载体 (Vectors for production of AAV particles ) 是由 R·卡伍德 A·S·布雷 T·A·佩恩 于 2019-01-18 设计创作，主要内容包括：本发明涉及用于生产腺相关病毒(AAV)颗粒的质粒的生产。特别地,本发明提供包含cap基因和rep基因的核酸分子,其中,cap基因和rep基因都与相同的启动子可操作地相关联。本发明还提供包含本发明的核酸分子的宿主细胞及其使用方法。(The present invention relates to the production of plasmids for the production of adeno-associated virus (AAV) particles. In particular, the invention provides nucleic acid molecules comprising a cap gene and a rep gene, wherein both the cap gene and the rep gene are operably associated with the same promoter. The invention also provides host cells comprising the nucleic acid molecules of the invention and methods of using the same.)

1. A nucleic acid molecule comprising:

(i) a promoter,

(ii) cap gene, and

(iii) the gene for rep is a gene which, when expressed,

in the order 5'→ 3' above, wherein both the cap gene and the rep gene are operably associated with the promoter, and wherein the rep gene is also operably associated with an IRES.

2. The nucleic acid molecule according to claim 1, wherein,

(a) the Rep gene encodes Rep78, Rep68, Rep52 and Rep 40; or

(b) The Rep gene encodes only one, two or three of Rep78, Rep68, Rep52 and Rep 40.

3. The nucleic acid molecule of claim 1, wherein the rep gene:

(a) encodes Rep78 and Rep52, but does not encode Rep68 or Rep 40;

(b) encodes Rep68 and Rep40, but does not encode Rep78 or Rep 52;

(c) encodes Rep68 and Rep52, but does not encode Rep78 or Rep 40; or

(d) Encodes Rep78 and Rep40, but does not encode Rep68 or Rep 52.

4. The nucleic acid molecule according to any one of the preceding claims, wherein Rep52 and/or Rep40 is not transcribed from the promoter.

5. The nucleic acid molecule according to claim 4, wherein Rep52 and/or Rep40 is transcribed from a p19 promoter.

6. The nucleic acid molecule according to any of the preceding claims, wherein the nucleotide sequences of the rep gene and the cap gene are from or derived from an AAV rep gene or an AAV cap gene, preferably from or derived from an AAV serotype 2rep gene or an AAV serotype 2cap gene.

7. The nucleic acid molecule of any one of the preceding claims, wherein the promoter is a cytomegalovirus immediate early (CMV) promoter or a promoter derived from the CMV promoter.

8. The nucleic acid molecule according to any one of claims 1 to 6, wherein the promoter is an inducible promoter.

9. The nucleic acid molecule according to any of the preceding claims, wherein the IRES is a picornavirus IRES (encephalomyocarditis virus, EMCVIRES) or aphtha virus IRES (foot and mouth disease virus, FMDVIRES) or a derivative thereof.

10. An RNA molecule comprising:

(i) cap gene, and

(ii) the gene for rep is a gene which, when expressed,

in the order of 5'→ 3' above, wherein the rep gene is operably associated with an IRES.

11. A plasmid or vector comprising the nucleic acid molecule of any one of claims 1 to 9.

12. A kit, comprising:

(a) the plasmid or vector of claim 11, and one or both of:

(b) an AAV transfer plasmid comprising a transgene flanked by ITRs;

(c) a helper plasmid comprising one or more genes selected from E1A, E1B, E2a, E4 and VA.

13. The kit of claim 12, wherein the helper plasmid does not comprise the E2A gene.

14. A kit, comprising:

(a) the plasmid or vector of claim 11, and one or both of:

(b) an AAV transfer plasmid comprising a transgene flanked by ITRs;

(c) a mammalian host cell (preferably HEK293) comprising one or more viral genes selected from E1A, E1B, E2a, E4 and VA capable of being expressed from the host cell genome.

15. The kit of claim 14, wherein the mammalian host cell does not comprise an E2A gene capable of being expressed from the host cell genome.

16. A mammalian cell comprising a nucleic acid molecule, plasmid or vector according to any one of claims 1 to 9 or 11, preferably wherein the mammalian cell is a human cell, more preferably a HEK293 cell or a derivative thereof.

17. Use of the mammalian cell of claim 16 in the production of an AAV particle.

18. A method for producing an AAV packaging cell, the method comprising the steps of:

(a) stably integrating a nucleic acid molecule according to any one of claims 1 to 9 or a plasmid or vector according to claim 11 into a mammalian cell, thereby producing a mammalian cell expressing a viral rep gene and a viral cap gene.

19. A method for producing AAV, the method comprising the steps of:

(a) introducing a transfer plasmid comprising a transgene flanked by 5 '-AAV ITRs and 3' -AAV ITRs into an AAV packaging cell comprising a nucleic acid molecule according to any one of claims 1 to 9 and sufficient helper genes (preferably selected from one or more of E1A, E1B, E2a, E4 and VA) for packaging the transfer plasmid, the helper genes being present in an episomal helper plasmid within the cell or integrated into the packaging cell genome;

(b) culturing the cell under conditions such that the AAV is assembled and secreted by the cell; and

(c) harvesting the packaged AAV from the supernatant, and optionally purifying the harvested AAV.

20. The method of claim 19, wherein the helper gene does not include the E2A gene.

21. The method of claim 19 or claim 20, wherein the transgene encodes a CRISPR enzyme (preferably Cas9 or Cpf1, or a derivative thereof) or a CRISPR sgRNA.

Technical Field

The present invention relates to the production of plasmids for the production of adeno-associated virus (AAV) particles. In particular, the invention provides nucleic acid molecules comprising a cap gene and a rep gene, wherein both the cap gene and the rep gene are operably associated with the same promoter. The invention also provides host cells comprising the nucleic acid molecules of the invention and methods of using the same.

Background

AAV vectors were developed from single-stranded DNA viruses belonging to the parvoviridae family. The virus is capable of infecting a wide range of host cells, including both dividing and non-dividing cells. In addition, the virus is a non-pathogenic virus that produces only a limited immune response in most patients.

The AAV genome comprises two genes that each encode multiple Open Reading Frames (ORFs): the rep gene encodes a non-structural protein required for AAV life cycle and site-specific integration of the viral genome; and the cap gene encodes the structural capsid protein. In addition, these two genes are flanked by Inverted Terminal Repeat (ITR) sequences consisting of 145 bases, which have the ability to form hairpin structures. These hairpin sequences are required for primase-independent synthesis of the second DNA strand and integration of the viral DNA into the host cell genome.

To eliminate any integrating ability of the virus, the recombinant AAV vector removes rep and cap from the DNA of the viral genome. To generate such a vector, the desired transgene is inserted between Inverted Terminal Repeats (ITRs) along with a promoter to drive transcription of the transgene; and the rep gene and the cap gene are supplied in trans in a second plasmid. A third plasmid providing helper genes such as adenovirus E4, E2a, and VA genes is also used. All three plasmids were then transfected into cultured "packaging" cells (such as HEK 293).

Over the past few years, AAV vectors have emerged as a very useful and promising mode of gene delivery. This is due to the following properties of these vectors:

AAV is a small non-enveloped virus, and AAV has only two native genes (rep and cap). Thus, AAV can be easily manipulated to develop vectors for different gene therapies.

AAV particles are not susceptible to shear forces, enzymatic or solvent degradation. This facilitates easy purification and final formulation of these viral vectors.

AAV is non-pathogenic and has low immunogenicity. The use of these vectors further reduces the risk of adverse inflammatory reactions. Unlike other viral vectors (such as lentiviruses, herpes viruses and adenoviruses), AAV is harmless and is not considered to be responsible for any human disease.

AAV vectors can be used to deliver up to 4000bp of genetic sequences to patients.

Although wild-type AAV vectors have been shown to sometimes insert genetic material on human chromosome 19, this property of most AAV vectors is usually eliminated by removing the rep and cap genes from the viral genome. In this case, the virus remains in the host cell in an episomal form. These episomes remain intact in non-dividing cells, while in dividing cells they are lost during cell division.

Disclosure of Invention

However, the inventors of the present invention have recognized that methods for producing AAV vectors can be improved by optimizing the ratio and amount of Rep and Cap proteins present during vector production.

Accordingly, it is an object of the present invention to provide a nucleic acid molecule comprising a cap gene and a rep gene under the control of a single promoter; thus, the Cap polypeptide and the Rep polypeptide are encoded in the same mRNA. Translation of the cap gene will be by attaching methyl guanylic acid cap (m) at the ribosome⁷G) Ligation was initiated at the 5' end of cap mRNA. Translation of the rep gene will be initiated by docking the ribosome at an Internal Ribosome Entry Site (IRES) located upstream of the rep gene.

By using the nucleic acid molecules of the invention, higher viral titers can be obtained.

In some embodiments of the invention, the IRES replaces the wild-type p5 promoter. Another advantage of removing the p5 promoter is that in wild type viruses, the p5 promoter is bound and activated by the E2A DNA Binding Protein (DBP). Thus, removal of the p5 promoter means that the E2A gene (e.g., in a helper plasmid) is not required to produce viral particles.

In one embodiment, the invention provides a nucleic acid molecule comprising:

(i) a promoter,

(ii) cap gene, and

(iii) the gene for rep is a gene which, when expressed,

in the above 5 '-3' order, wherein both the cap gene and the rep gene are operably associated with the promoter, and wherein the rep gene is also operably associated with an IRES.

The present invention also provides a nucleic acid molecule comprising:

(i) cap gene, and

(ii) the gene for rep is a gene which, when expressed,

in the above 5 '-3' order, wherein the rep gene is operably associated with an IRES.

The nucleic acid molecule may be DNA or RNA, preferably DNA. The nucleic acid molecule may be single-stranded or double-stranded, preferably double-stranded.

The nucleic acid molecule of the present invention comprises a rep gene. As used herein, the term "Rep gene" refers to a gene encoding one or more Open Reading Frames (ORFs), wherein each of the ORFs encodes an AAV Rep nonstructural protein or a variant or derivative thereof. These AAV Rep nonstructural proteins (or variants or derivatives thereof) are involved in AAV genome replication and/or AAV genome packaging.

FIG. 1 shows the structure of the wild-type AAV genome, illustrating the organization of the wild-type rep and cap genes.

The wild-type rep gene contains three promoters: p5, p19 and p 40. Two overlapping messenger ribonucleic acids (mrnas) of different lengths can be produced from p5 and p 19. Each of these mrnas contains an intron that may or may not be spliced out using a single splice donor site and two different splice acceptor sites. Six different mrnas can thus be formed, of which only four are functional. Two mrnas that failed to remove introns (one transcribed from p5 and one transcribed from p 19) read through the shared terminator sequence and encode Rep78 and Rep52, respectively. Removal of introns and use of the most 5' splice acceptor site does not result in the production of any functional Rep proteins-the correct Rep68 or Rep40 proteins cannot be produced because the framework of the rest of the sequence is shifted and the correct C-terminus of Rep78 or Rep52 cannot be produced because their terminators are spliced out. In contrast, removal of introns and use of 3' splice acceptors would include the correct C-termini of Rep68 and Rep40, while the terminators of Rep78 and Rep52 are spliced out. Thus, the only functional splicing avoids complete splicing of introns out (to produce Rep78 and Rep52) or the use of 3' splice acceptors (to produce Rep68 and Rep 40). Thus, four different functional Rep proteins with overlapping sequences can be synthesized from these promoters.

In the wild-type rep gene, the p40 promoter is located at the 3' end. In the wild-type AAV genome, transcription of Cap proteins (VP1, VP2, and VP3) is initiated from this promoter.

The four wild-type Rep proteins are Rep78, Rep68, Rep52 and Rep 40. Thus, the wild-type Rep gene is a gene encoding four Rep proteins, Rep78, Rep68, Rep52 and Rep 40.

Rep78 and Rep68 can specifically bind to the hairpin formed by the ITRs and can cleave the hairpin at a specific region within the hairpin (i.e., a terminal dissociation site). In wild-type viruses, Rep78 and Rep68 are also required for AAV-specific integration of the AAV genome. Rep78 and Rep68 are transcribed in wild-type virus under the control of the p5 promoter, and the difference between Rep78 and Rep68 reflects the removal (or not) of introns by splicing, so they have different C-terminal protein compositions.

Rep52 and Rep40 are involved in genome packaging. Rep52 and Rep40 are transcribed in wild-type virus under the control of the p19 promoter, and the difference between Rep52 and Rep40 reflects the removal (or not) of introns by splicing, so they have different C-terminal protein compositions.

All four Rep proteins bind ATP and have helicase activity. They up-regulate transcription from the p40 promoter, but down-regulate transcription from the p5 promoter and the p19 promoter.

As used herein, the term "rep gene" encompasses wild-type rep genes and derivatives thereof as well as artificial rep genes with equivalent function.

In one embodiment, the Rep gene encodes a functional Rep78 protein, a functional Rep68 protein, a functional Rep52 protein, and a functional Rep40 protein.

In a preferred example of this embodiment, Rep78 and Rep68 are translated by ribosomes that interface 5' to the Rep78 and Rep68 ATG initiation codons, thereby allowing the production of both proteins. In this example, the Rep78 and Rep68 open reading frames comprise an active p40 promoter that provides for expression of both Rep52 and Rep 40.

In some embodiments of the invention, one or more of the p5 promoter, p19 promoter, and p40 promoter is functionally deleted/defective, e.g., by codon alteration and/or removal of the TATA box, to prevent undesired initiation of transcription from the promoter.

Preferably, the p5 promoter is non-functional (i.e., the p5 promoter cannot be used to initiate transcription). More preferably, the p5 promoter is replaced by an IRES (thus eliminating the function of the p5 promoter). This allows Rep78 or Rep68 to be transcribed in the same mRNA as the cap gene, but the translation of the Rep78 and Rep68 proteins will be under the control of an IRES.

Another advantage of removing the p5 promoter is that in wild type viruses, the p5 promoter is bound and activated by the E2A DNA Binding Protein (DBP). Thus, removal of the p5 promoter means that the E2A gene (e.g., in a helper plasmid) is not required to produce viral particles.

In one embodiment, the rep gene does not have the p5 promoter upstream. In another embodiment, the p5 promoter is not used in AAV packaging.

Preferably, the p19 promoter within the rep gene is functional.

In some embodiments, the function of the p40 promoter is removed/defective within the Rep gene by one or more codon changes.

The cap gene is preferably relocated and its transcription is placed under the control of an alternative promoter (e.g., the CMV immediate early promoter).

There is a degree of redundancy between the functions of the different Rep proteins, and therefore, in some embodiments of the invention, not all of the Rep proteins are necessary.

In some embodiments, the Rep gene encodes only one, two, three or four of Rep78, Rep68, Rep52 and Rep40, preferably one, two or four of Rep78, Rep68, Rep52 and Rep 40.

In some embodiments, the Rep gene does not encode one or more of Rep78, Rep68, Rep52, and Rep 40.

In some embodiments, the Rep gene encodes Rep78 and Rep52, but does not encode Rep68 or Rep 40. In this embodiment, the splice donor site remains in the DNA, but both the 5 'and 3' splice acceptor sites are removed. Thus, introns cannot be removed by splicing, and transcription continues up to the terminator sequences of Rep78 and Rep52 (which are common to both Rep78 and Rep 52). The Rep78 protein is transcribed in the same mRNA as the cap gene (and thus driven by the same promoter), while the translation of Rep78 is driven by an IRES. The transcription of Rep52 is driven by the p19 promoter; thus, transcription of Rep52 forms a separate mRNA and proceeds 5'm on the ribosome⁷G cap dependent docking to translate. Therefore, Rep68 and Rep40 cannot be generated in this embodiment.

In other embodiments, the Rep gene encodes Rep68 and Rep40, but does not encode Rep78 or Rep 52. In this embodiment, the intron sequences between the splice donor and 3' splice acceptor are removed at the DNA level, placing the C-terminus of Rep68 and Rep40 in the same frame as the upstream coding sequence. Thus, Rep68 and Rep40 are generated (but Rep78 and Rep52 are not generated). For clarity, Rep68 is transcribed in the same mRNA as the Cap protein, and Rep68 is translated under the control of an IRES. In contrast, Rep40 is transcribed into a single mRNA by the p19 promoter and on ribosomes by 5' m⁷G cap is docked for translation.

In some embodiments, the Rep gene encodes Rep78 and Rep68, but does not encode Rep52 or Rep 40. This can be achieved by mutating the p19 promoter (e.g., inserting a mutation at the p19 TATA box).

In some embodiments, the Rep gene encodes Rep52 and Rep40, but does not encode Rep78 or Rep 68. This can be achieved by including only the coding sequence of the ATG from Rep 52/40.

As used above, the term "encoding" refers to the functional form of the Rep protein encoded by the Rep gene. Similarly, the term "not encoding" means that the Rep gene does not encode a functional form of the Rep protein.

Without sufficient Rep proteins, the titers (e.g., genomic copies) observed are low (which can be determined by qPCR) since there are fewer ITR plasmids to package and efficient packaging, observations may also include an exaggerated ratio of empty particles to full particles, which can be determined by E L ISA or optical density measurements.

SEQ ID NO 1 shows the nucleotide sequence of the wild type AAV (serotype 2) rep gene. SEQ ID NO 2, SEQ ID NO 3, SEQ ID NO 4 and SEQ ID NO 5 give the amino acid sequence of wild type AAV (serotype 2) Rep78, the amino acid sequence of wild type AAV (serotype 2) Rep68, the amino acid sequence of wild type AAV (serotype 2) Rep52 and the amino acid sequence of wild type AAV (serotype 2) Rep40, respectively. The wild type AAV (serotype 2) nucleotide sequence encoding Rep78 is given in SEQ ID NO 6. The nucleotide sequence of the wild type AAV (serotype 2) encoding Rep68 is given in SEQ ID NO. 7. The nucleotide sequence of wild type AAV (serotype 2) encoding Rep52 is given in SEQ ID NO. 8. The nucleotide sequence of a wild-type AAV (serotype 2) encoding Rep40 is given in SEQ ID NO. 9.

In one embodiment, the term "Rep gene" refers to a nucleotide sequence having at least 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID No. 1 and encoding one or more of a Rep78 polypeptide, a Rep68 polypeptide, a Rep52 polypeptide, and a Rep40 polypeptide.

In another embodiment, the term "Rep gene" refers to a nucleotide sequence having at least 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO 6 and encoding a functional Rep78 polypeptide and/or a Rep52 polypeptide (and preferably not encoding a functional Rep68 polypeptide or a Rep40 polypeptide).

In another embodiment, the term "Rep gene" refers to a nucleotide sequence having at least 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID No. 7 and encoding a functional Rep68 polypeptide and/or a Rep40 polypeptide (and preferably not encoding a functional Rep78 polypeptide or Rep52 polypeptide).

In another embodiment, the term "Rep gene" refers to a nucleotide sequence having at least 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO. 8 and encoding a functional Rep52 polypeptide (and preferably not encoding a functional Rep78 polypeptide).

In another embodiment, the term "Rep gene" refers to a nucleotide sequence having at least 70%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO. 9 and encoding a functional Rep40 polypeptide (and preferably not encoding a functional Rep68 polypeptide).

In another embodiment, the term "Rep gene" refers to a nucleotide sequence having at least 90%, 95%, 99%, or 100% sequence identity to the nucleotide sequence encoding SEQ ID NO. 2 and encoding a functional Rep78 polypeptide and/or a Rep52 polypeptide (and preferably not encoding a functional Rep68 polypeptide or a Rep40 polypeptide).

In another embodiment, the term "Rep gene" refers to a nucleotide sequence having at least 90%, 95%, 99%, or 100% sequence identity to the nucleotide sequence encoding SEQ ID NO. 3 and encoding a functional Rep68 polypeptide and/or a Rep40 polypeptide (and preferably not encoding a functional Rep78 polypeptide or a Rep52 polypeptide).

In another embodiment, the term "Rep gene" refers to a nucleotide sequence having at least 90%, 95%, 99%, or 100% sequence identity to the nucleotide sequence encoding SEQ ID NO. 4 and encoding a functional Rep52 polypeptide (and preferably not encoding a functional Rep78 polypeptide).

In another embodiment, the term "Rep gene" refers to a nucleotide sequence having at least 90%, 95%, 99%, or 100% sequence identity to the nucleotide sequence encoding SEQ ID NO. 5 and encoding a functional Rep40 polypeptide (and preferably not encoding a functional Rep68 polypeptide).

In some embodiments, a nucleic acid molecule of the invention does not encode a functional Rep78 polypeptide. In some embodiments, a nucleic acid molecule of the invention does not encode a functional Rep68 polypeptide. In some embodiments, a nucleic acid molecule of the invention does not encode a functional Rep52 polypeptide. In some embodiments, a nucleic acid molecule of the invention does not encode a functional Rep40 polypeptide.

The nucleic acid molecule further comprises a cap gene. As used herein, the term "Cap gene" refers to a gene encoding one or more Open Reading Frames (ORFs), wherein each ORF in the ORF encodes an AAV Cap structural protein or a variant or derivative thereof. These AAV Cap structural proteins (or variants or derivatives thereof) form AAV capsids.

These three Cap proteins must be used to enable the production of infectious AAV viral particles capable of infecting appropriate cells. The three Cap proteins are VP1, VP2 and VP3, respectively, and are typically 87kDa, 72kDa and 62kDa in size, respectively. Thus, the Cap gene is a gene encoding three Cap proteins, VP1, VP2, and VP 3.

In wild-type AAV, these three proteins are translated from the p40 promoter to form a single mRNA. After synthesis of the mRNA, the long or short intron can be excised, resulting in a 2.3kb or 2.6kb mRNA.

Typically, long introns are excised, particularly in the presence of adenovirus. In this form, the first AUG codon (from which VP1 protein synthesis begins) is cleaved, resulting in a reduction in the overall level of VP1 protein synthesis. The first AUG codon that remains is the start codon for VP3 protein. However, upstream of this codon in the same open reading frame is an ACG sequence (encoding threonine) surrounded by an optimal Kozak environment. This contributes to a low level of synthesis of the VP2 protein, whereas the VP2 protein is actually a VP3 protein with additional N-terminal residues, as is VP 1.

If the long intron is spliced out and since the ACG codon is a much weaker translational initiation signal in major splicing, the ratio of AAV structural proteins synthesized in vivo is about 1:1:10, which is the same ratio as in mature virions.A unique fragment at the N-terminus of the VP1 protein has been shown to have phospholipase A2(P L A2) activity, which may be necessary for AAV particle release from late endosomes.

The AAV capsid is composed of 60 capsid protein subunits (VP1, VP2, and VP3) arranged in icosahedral symmetry in a ratio of 1:1:10, with an estimated size of 3.9 MDa.

As used herein, the term "cap gene" encompasses wild-type cap genes and derivatives thereof as well as artificial cap genes with equivalent function. The nucleotide sequence of the Cap gene of AAV (serotype 2) and the sequence of the Cap polypeptide are given in SEQ ID NO:10 and SEQ ID NO:11, respectively.

As used herein, the term "cap gene" preferably refers to a nucleotide sequence having the sequence given in SEQ ID NO. 10 or a nucleotide sequence encoding SEQ ID NO. 11; or a nucleotide sequence having at least 70%, 80%, 85%, 90%, 95% or 99% sequence identity to SEQ ID NO. 10 or at least 80%, 90%, 95% or 99% sequence identity to the nucleotide sequence encoding SEQ ID NO. 11 and encoding a VP1 polypeptide, a VP2 polypeptide and a VP3 polypeptide.

The rep gene and cap gene are preferably viral genes or derived from viral genes. More preferably, they are or are derived from AAV genes. In some embodiments, the AAV is adeno-associated dependent parvovirus a. In other embodiments, the AAV is adeno-associated dependent parvovirus B.

11 different AAV serotypes are known. All known serotypes can infect cells from a variety of different tissue types. Tissue specificity is determined by the capsid serotype. The AAV may be from serotype 1, serotype 2, serotype 3, serotype 4, serotype 5, serotype 6, serotype 7, serotype 8, serotype 9, serotype 10, or serotype 11. Preferably, the AAV is serotype 1, serotype 2, serotype 5, serotype 6, serotype 7, or serotype 8. Most preferably, the AAV serotype is 2 (i.e., AAV 2).

The rep and cap genes (and each protein-encoding ORF therein) may be from one or more different viruses (e.g., 2, 3, or 4 different viruses). For example, the rep gene may be from AAV2, while the cap gene may be from AAV 5. It is recognized by those skilled in the art that the rep and cap genes of AAV vary with clades and isolates. Sequences of these genes and their derivatives from all of these clades and isolates are encompassed herein.

The cap gene and the rep gene are present in the nucleic acid in the order of 5'→ 3'. However, since Rep52 and/or Rep40 may be transcribed from their own p19 promoter, the position of the coding sequence encoding Rep52 and/or Rep40 may be altered. For example, the coding sequences encoding Rep52 and/or Rep40 may be placed upstream or downstream of the cap gene and Rep gene encoding Rep 78/68; or indeed on the reverse strand of the nucleic acid of the invention or on a different nucleic acid.

Both the cap gene and the rep gene are operably associated with the same promoter. The promoter is preferably 5' (i.e., upstream) of the cap gene and rep gene. In some embodiments, the promoter is a constitutive promoter. In other embodiments, the promoter is inducible or repressible.

Examples of constitutive promoters include CMV, SV40, PGK (human or mouse), HSV TK, SFFV, ubiquitin, elongation factor α, CHEF-1, FerH, Grp78, RSV, adenovirus E1A, CAG or CMV- β -globin promoters, or promoters derived therefrom.

In some embodiments, the promoter is inducible or repressible by virtue of comprising an inducible or repressible regulatory (promoter) element. For example, the promoter may be one induced with doxycycline, tetracycline, IPTG or lactose.

Preferably, the inducible promoter element comprises a plurality of Tet operator sequences to which the Tet repressor protein (TetR) is capable of binding. In the bound state, a strict inhibition of transcription is obtained. However, in the presence of doxycycline (or, less preferably, tetracycline), repression is mitigated, thereby allowing the promoter to obtain full transcriptional activity. Such inducible promoter elements are preferably placed downstream of another promoter, for example downstream of the CMV promoter.

The TetR binding site may have a wild-type sequence, many of which are known in the art. Preferably, the TetR binding site is one or more improved by the incorporation of minor sequence changes. A preferred form that can be used in embodiments of the invention has the sequence tccctatcagtgatagaga (SEQ ID NO: 12).

Alternative forms of repressor elements that bind to a TetR protein or a derivative of a TetR protein may also be used in embodiments of the invention, provided that the TetR repressor protein binds to the TetR binding sequence variant used. Some of the repression/binding site variants will have a higher affinity for each other than wild-type; these are preferred in the embodiments of the present invention.

The TetR gene is typically integrated into the chromosome of a human (host) cell. The gene may or may not be integrated adjacent to or in conjunction with the cap gene or rep gene. In some embodiments, the TetR gene is co-expressed with the cap gene or the rep gene.

In one embodiment of the invention, the nucleotide sequence of the TetR protein is as given in SEQ ID NO 13 or a nucleotide sequence having at least 80%, more preferably at least 85%, 90% or 95% sequence identity to SEQ ID NO 13 and encoding a TetR protein.

In another embodiment of the invention the amino acid sequence of the TetR protein is as given in SEQ ID NO 14 or an amino acid sequence having at least 80%, more preferably at least 85%, 90% or 95% sequence identity to SEQ ID NO 14 and encoding a TetR protein.

Preferably, the promoter operably associated with the cap gene and the rep gene is the CMV immediate early promoter or a derivative thereof. In some particularly preferred embodiments, the promoter is a promoter as defined in WO2017/149292 (more preferably, a promoter as defined therein as "p 565"). Preferably, the promoter operably associated with the cap gene and the rep gene is not an AAV promoter, e.g., it is not an AAV p5 promoter, p19 promoter, or p40 promoter.

Translation of the cap Gene is preferably from the canonical 5'm at the 5' end of the mRNA⁷G-cap is started.

The rep gene is also operably associated with an Internal Ribosome Entry Site (IRES).

IRES regulates the translation of rep mRNA. IRES are distinct regions of a nucleic acid molecule that are capable of recruiting eukaryotic ribosomes to mRNA in a process known as cap-independent translation. IRES is usually located in the 5' -UTR of RNA viruses. They facilitate translation of viral RNA in a cap-independent manner.

Examples of viral IRES include picornavirus IRES (encephalomyocarditis virus, EMCV IRES), aphtha virus IRES (foot and mouth disease virus, FMDV IRES), kaposi's sarcoma-associated herpesvirus IRES, hepatitis a, hepatitis c IRES, pestivirus IRES, cricket paralysis virus Internal Ribosome Entry Site (IRES), gloomy virus Internal Ribosome Entry Site (IRES), and 5' -leader IRES and intercistron IRES in the 1.8-kb family of immediate early transcripts (IRES) 1.

The invention also includes non-natural derivatives of the above IRES that retain the ability to recruit eukaryotic ribosomes to the mRNA. In some preferred embodiments, the IRES is an encephalomyocarditis virus (EMCV) IRES. In one embodiment of the invention, the nucleotide sequence of the EMCV IRES is as given in SEQ ID No. 15 or a nucleotide sequence having at least 80%, more preferably at least 85%, 90% or 95% sequence identity to SEQ ID No. 15 and encoding an IRES.

In other embodiments, the IRES is a Foot and Mouth Disease Virus (FMDV) IRES. In one embodiment of the invention, the nucleotide sequence of the FMDV IRES is as given in SEQ ID No. 16 or a nucleotide sequence having at least 80%, more preferably at least 85%, 90% or 95% sequence identity to SEQ ID No. 16 and encoding the IRES.

The rep gene is operably associated with an IRES. Preferably, the IRES is located downstream of the cap gene and upstream of the translation initiation site of Rep 78/68.

The generation of stable cell lines in mammalian culture typically requires selection methods to promote the growth of cells containing any exogenously added DNA.

Preferably, the nucleic acid molecule of the invention additionally comprises a selection gene or an antibiotic resistance gene. For this reason, a series of genes are known which provide resistance to specific compounds when the DNA encoding them is inserted into the genome of mammalian cells.

Preferably, the selection gene is puromycin N-acetyltransferase (Puro), hygromycin phosphotransferase (Hygro), blasticidin S deaminase (Blast), neomycin phosphotransferase (Neo), glutathione S-transferase (GS), bleomycin resistance gene (Sh ble), or dihydrofolate reductase (DHFR). Each of these genes provides resistance to small molecules known to be toxic to mammalian cells, or in the case of GS, provides a method for the production of glutathione by cells in the absence of glutathione in the growth medium.

In a preferred embodiment of the invention, the resistance gene is Puro. This gene is particularly effective because many cell lines used in common tissue culture are not resistant to Puro; this is not the case for Neo, and many HEK293 derivatives in particular have been Neo resistant (e.g., HEK293T cells) due to previous genetic manipulation by researchers. Puro selection also has the advantage of being toxic within a short time window (<72 hours), and therefore Puro selection allows for rapid testing of variables and for rapid removal from culture systems of cells that do not carry exogenous DNA to be inserted into the genome. This is not the case for some other selection methods, such as Hygro, which has a much slower onset of toxicity.

The development of stable cell lines using selection genes (e.g. Puro) requires the expression of resistance genes in the cells. This can be achieved by a variety of methods, including but not limited to Internal Ribosome Entry Sites (IRES), 2A cleavage systems, alternative splicing and dedicated promoters.

In a preferred embodiment of the invention, the selection gene will be expressed from a dedicated promoter. The promoter will preferably transcribe in human cells at a lower level than the specialized promoter driving the rep or cap genes.

Each of the genes encoding a polypeptide or RNA in a nucleic acid molecule will preferably be operably associated with one or more regulatory elements. This ensures that the polypeptide or RNA is expressed at the desired level and at the desired time. As used herein, the term "regulatory element" includes one or more of an enhancer, promoter, intron, polyA, insulator, or terminator.

The genes used in the AAV plasmids or vectors disclosed herein are preferably separated by polyA signals and/or insulators in order to keep transcriptional readthrough to other genes to a minimum.

Although some advantages may be obtained by using copies of the same regulatory element (e.g., promoter sequence) with more than one polypeptide or RNA encoding nucleotide sequence (in terms of coordinated expression thereof), in the context of the present invention, it is highly desirable to use different regulatory elements with each polypeptide or RNA encoding nucleotide sequence.

Thus, preferably the rep gene and the cap gene are operably associated with different regulatory elements, such as different promoters, different introns, different polyas, different insulators and/or different terminator sequences. More preferably, the degree of nucleotide sequence identity between the rep promoter and the cap promoter is less than 95% or less than 90%, more preferably less than 85%, 80%, 70% or 60%. More preferably, the degree of nucleotide sequence identity between the rep terminator and the cap terminator is less than 95% or less than 90%, more preferably less than 85%, 80%, 70% or 60%. In this way, the risk of homologous recombination between these regulatory elements is reduced.

In most embodiments, the nucleic acid molecule of the invention will be a plasmid or vector for production of AAV. Thus, in most embodiments, a nucleic acid molecule of the invention (or a vector or plasmid comprising the same) will not comprise an Inverted Terminal Repeat (ITR).

In some embodiments, a nucleic acid molecule of the invention (or a vector or plasmid comprising the same) will not comprise one or more genes selected from adenovirus E1A, adenovirus E1B, adenovirus E4, adenovirus E2A, or adenovirus VA. In some preferred embodiments, the nucleic acid molecule of the invention (or a vector or plasmid system comprising the same) does not comprise an adenoviral E2A gene. As used herein, the term "E2A" or "E2A gene" refers to the viral E2A gene or variants or derivatives thereof. Preferably, the E2A gene is derived or derived from a human adenovirus such as Ad 5. In one embodiment of the invention, the nucleotide sequence of the adenovirus E2A gene is as given in SEQ ID NO 17 or in a nucleotide sequence having at least 80%, more preferably at least 85%, 90% or 95% sequence identity to SEQ ID NO 17 and encoding an extended DNA binding protein that facilitates replication of viral DNA.

In another embodiment, a plasmid or vector comprising a nucleic acid molecule of the invention is provided.

Examples of preferred embodiments of the invention include nucleic acid molecules comprising the following elements in the following order:

CMV promoter-AAV 2cap gene-FMDV IRES-rep gene

p565 promoter-AAV 2cap gene-EMCV IRES-rep gene

CMV promoter-AAV 2cap Gene-EMCV IRES-rep Gene

In some preferred embodiments, the "Rep gene" refers to a gene encoding a Rep78 polypeptide, a Rep52 polypeptide, a Rep68 polypeptide, and a Rep40 polypeptide. In other preferred embodiments, the term "Rep gene" refers to a gene that encodes a Rep78 polypeptide and a Rep52 polypeptide (but preferably does not encode a functional Rep68 polypeptide or a Rep40 polypeptide).

In general, one sequence serves as a reference sequence to which a test sequence may be compared.A sequence comparison algorithm calculates the percent sequence identity of the test sequence relative to the reference sequence based on specified program parameters.an alignment of amino acid or nucleic acid sequences for comparison may be performed, for example, by a computer-implemented algorithm (e.g., GAP, BESTFIT, FASTA, or TFASTA) or the B L AST algorithm and the B L AST2.0 algorithm.

Amino acid sequence identity and percentage of nucleotide sequence identity may be obtained using B L AST alignment methods (Altschul et al (1997), "Gapped B L AST and PSI-B L AST: a new generation of protein induced abase search programs," Nucleic Acids Res.25:3389 and 3402; and http:// www.ncbi.nlm.nih.gov/B L AST.) preferably, standard or default alignment parameters are used.

The standard protein, protein B L AST (blastp), may be used to find similar sequences in a protein database as other B L AST programs blastp is designed to find similar local regions.

B L AST protein search may also be performed using the B L ASTX program, score 50, word length 3 in order to obtain a gap alignment for comparison purposes, as described in Altschul et al (1997) Nucleic Acids res.25:3389, gap B L AST (Gapped B L AST) (in B L AST 2.0) or PSI-B L AST (in B L AST 2.0) may be used to perform an iterative search to detect the distance relationship between molecules (see Altschul et al (1997), supra.) when B L AST, Gapped B L AST, PSI-B L AST are used, the default parameters of the respective programs may be used.

For nucleotide sequence comparison, MEGAB L AST, discontinuous megablast (discontinuous-megablast) and blastn may be used to accomplish this.

The B L AST nucleotide algorithm finds similar sequences by dividing the query into short subsequences called words the program first identifies exact matches to the query words (word hits) then the B L AST program expands these word hits in multiple steps to generate the final gap alignment.

One of the important parameters that determine the sensitivity of the B L AST search is the word size (word size). the most important reason that blastn is more sensitive than MEGAB L AST is that blastn uses a shorter default word size (11). for this reason, blastn outperforms MEGAB L AST in finding alignments of related nucleotide sequences from other organisms.

Html, a more sensitive search can be achieved using a newly introduced discontinuous megablast page (www.ncbi.nlm.nih.gov/Web/newswtr/fallwenter 02/blastlab. html) that uses an algorithm similar to that reported by Ma et al (bioinformatics.2002, 3/18 (3): 440-5). discontinuous megablast uses non-overlapping words within a longer window of the template, rather than requiring exact word matching as a seed for comparative expansion.in the coding mode, the third base wobble is considered by focusing on finding a match at the first and second codon positions while ignoring the mismatch at the third position.a discontinuous MEGAB L AST of the same word length is used to search more and efficiently than a standard blastn search of the same word length. the unique parameters of discontinuous megablast are word length: 11 or 12; template: 16, 18 or 21; type: coding (0), non-coded (1), or both (2).

In some embodiments, the B L ASTP 2.5.0+ algorithm (such as available from NCBI) may be used using default parameters in other embodiments, the B L AST global alignment program (such as available from NCBI) may be used by using a Needleman-Wunsch alignment of two protein sequences with a gap penalty of initial 11 and extension 1.

One method for producing recombinant AAV is based on transient transfection of all elements required for AAV production into a host cell (such as HEK293 cells). This typically involves co-transfection of AAV producer cells with 3 plasmids:

(a) a plasmid containing an AAV ITR, which carries a gene of interest;

(b) a plasmid carrying an AAV rep-cap gene; and

(c) plasmids of essential helper genes isolated from adenoviruses are provided.

In some cases, plasmid (c) is not required because the helper genes are stably integrated into (and can be expressed from) the host cell genome.

Accordingly, the present invention provides a kit comprising:

(a) a plasmid or vector comprising a nucleic acid molecule of the invention and one or more of

(b) An AAV transfer plasmid comprising a transgene flanked by ITRs;

(c) a helper plasmid comprising one or more genes selected from adenovirus E1A, adenovirus E1B, adenovirus E4 and adenovirus VA.

In some embodiments of the invention, the helper plasmid additionally comprises the E2A gene. In other embodiments, the helper plasmid does not comprise the E2A gene. In the latter case, deletion of the E2A gene significantly reduces the amount of DNA required in the helper plasmid.

The present invention also provides a kit comprising:

(a) a plasmid or vector comprising a nucleic acid molecule of the invention and one or more of

(b) An AAV transfer plasmid comprising a transgene flanked by ITRs;

(c) a mammalian host cell (e.g. HEK293) comprising one or more viral genes selected from E1A, E1B, E4 and VA capable of being expressed from the host cell genome.

In some embodiments of the invention, the mammalian host cell further comprises an E2A gene capable of being expressed from the host cell genome. In other embodiments, the mammalian host cell does not comprise an adenovirus E2A gene.

The kit also includes materials for purifying AAV particles, such as those associated with density banding and purification of viral particles, e.g., one or more of centrifuge tubes, iodixanol, dialysis buffer, and dialysis cassettes.

The invention also provides a mammalian cell comprising a nucleic acid molecule, plasmid or vector of the invention. The nucleic acid molecules of the invention can be stably integrated into the nuclear genome of a mammalian cell or can be present within a vector or plasmid (e.g., episome) within the cell.

Preferably, the nucleic acid molecule of the invention is stably integrated into the nuclear genome of a mammalian cell (and wherein the rep gene and the cap gene are capable of being expressed therefrom).

The cells may be isolated cells, for example, they are not in a living animal or mammal. Examples of mammalian cells include cells from any organ or tissue of humans, mice, rats, hamsters, monkeys, rabbits, donkeys, horses, sheep, cows, and apes. Preferably, the cell is a human cell. The cell may be a primary cell or an immortalized cell.

Preferred cells include HEK-293, HEK293T, HEK-293E, HEK-293FT, HEK-293S, HEK-293SG, HEK-293FTM, HEK-293SGGD, HEK-293A, MDCK, C127, A549, He L a, CHO, mouse myeloma, PerC6, 911, and Vero cell lines HEK-293 cells have been modified to contain the E1A protein and the E1B protein and this eliminates the need to provide these proteins on helper plasmids.

Preferably, the cell of the invention is capable of inducing the expression of the rep gene and the cap gene.

The invention also provides an AAV packaging cell (preferably a mammalian cell, more preferably a human cell) comprising one or both of (a) a nucleic acid molecule of the invention and optionally (b) an AAV transfer plasmid comprising a transgene flanked by ITRs and (c) a helper plasmid comprising one or more genes selected from E1A, E1B, E4 and VA. In some embodiments of the invention, the helper plasmid additionally comprises the E2A gene. In other embodiments, the helper plasmid does not comprise the E2A gene. In the latter case, deletion of the E2A gene significantly reduces the amount of DNA required in the helper plasmid.

The nucleic acid molecules, plasmids and vectors of the invention may be prepared by any suitable technique. Recombinant methods for producing the nucleic acid molecules and packaging cells of the invention are well known in the art (e.g. "molecular cloning: A laboratory Manual" (fourth edition), Green, MR and Sambrook, J., (2014 updates)).

The expression of the rep and cap genes from the nucleic acid molecules of the invention can be determined in any suitable assay, for example by qPCR to determine the number of genomic copies per ml (as described in the examples herein).

In another embodiment, a method for producing an AAV packaging cell is provided, the method comprising the steps of:

(a) the nucleic acid molecules of the invention are stably integrated into mammalian cells, thereby producing mammalian cells that express viral rep and cap genes.

The invention also provides a use of the AAV packaging cell of the invention in the production of an AAV particle.

The present invention also provides a method for producing AAV, comprising the steps of:

(a) introducing a transfer plasmid comprising a transgene flanked by 5 '-AAV ITRs and 3' -AAV ITRs into an AAV packaging cell comprising a nucleic acid molecule of the invention and sufficient helper genes (preferably selected from one or more of E1A, E1B, E4 and VA) for packaging the transfer plasmid, either in an episomal helper plasmid within the cell or integrated into the packaging cell genome;

(b) culturing the cell under conditions such that AAV is assembled and secreted by the cell; and

(c) packaged AAV was harvested from the supernatant.

In some embodiments of the invention, the helper gene additionally comprises the E2A gene. In other embodiments, the helper gene does not include the E2A gene.

Preferably, the harvested AAV is subsequently purified.

As used herein, the term "introducing" one or more plasmids or vectors into a cell includes transformation, as well as any form of electroporation, conjugation, infection, transduction, or transfection, and the like.

In some preferred embodiments, the transgene encodes a CRISPR enzyme (e.g., Cas9, Cpf1) or crisprrsgrna.

Methods for such introduction are well known in the art (e.g., Proc. Natl. Acad. Sci. USA.1995, 8/1; 92 (16): 7297-.

The disclosure of each reference set forth herein is specifically incorporated by reference in its entirety.

Drawings

FIG. 1 shows the organization of Rep protein genes and Cap protein genes in the wild-type AAV genome.

Fig. 2A, 2B and 2C show three embodiments of the nucleic acid molecule of the invention. In fig. 2B, "OXGP 3" refers to a CMV promoter variant with two Tet operator sites.

FIGS. 3 to 4 show the results of determination of the number of copies of AAV genomes produced per ml in cells transfected with various rep-cap plasmids. OxG-a standard RepCap configuration as found in wild-type viruses, including a distally located p5 promoter; CMV-configuration in which both the Rep and Cap sequences are placed under the CMV promoter in a5 '-3' order CMV-Cap-CMV-Rep; a configuration in which a Rep sequence and a Cap sequence are placed under a PGK promoter and a CMV promoter, respectively, in a5 '-3' order CMV-Cap-PGK-Rep; CMV-EMCV — a configuration in which a Cap sequence is placed under the CMV promoter and a Rep sequence is placed under the control of an IRES EMCV in a5 '-3' order CMV-Cap-EMCV-Rep. In fig. 4, virus-containing cell lysates were diluted 500-fold and quantified using qPCR. This demonstrates physical potency.

Fig. 5 shows the results after flow cytometry analysis of HEK293T cells 72 hours after infection with AAV particles. Data are given as the percentage of GFP positive cells in P1. P1 corresponds to viable cells in the sample.

Fig. 6 shows the transduction units per ml of infected virus sample, as calculated from the results of fig. 5 and the number of infected cells. This demonstrates the infectious titer.

FIG. 7 shows the results of determination of the number of copies of AAV genomes per ml produced in cells transfected with various rep-cap plasmids. For details of the plasmids, see FIGS. 3-4 above. Clontech refers to the 3-plasmid system (pAAV-CMV-EGFP; pHelper; pRepCap-miR342) supplied by Clontech.

Figure 8 shows the titers (GC, genomic copies) obtained from the viruses produced: a) a 3-plasmid AAV system of the invention; b) the system of a), wherein the pSF-helper plasmid is replaced by a plasmid comprising only the CMV-E4orf6 (coding sequence); c) the system of a), wherein the pSF-helper plasmid is replaced with pSF-E4orf 6-VAI; and d) the system of a), wherein the pSF-helper plasmid is removed and replaced by filler DNA (control).

Detailed Description