Method for creating recombinant AAVs

Abstract

This provides a process for producing recombinant adeno-associated virus (AAV) particles. [Solution] The process comprises producing recombinant AAV particles in a first host cell and then producing recombinant AAV particles in a second host cell, wherein the first and second host cells produce first recombinant AAV particles encapsulated in a capsid of a first AAV capsid polypeptide and second recombinant AAV particles encapsulated in a capsid of a second AAV capsid polypeptide. Preferably, the first recombinant AAV particles and the second recombinant AAV particles have different cytotropies toward a producing cell line (for high-efficiency AAV production) and cells associated with a therapeutic indication (for the treatment of said indication).

Description

Technical Field

[0001] The present invention relates to a process for producing recombinant adeno-associated virus (AAV) particles, which is described herein as trans-pseudotyping. The process includes producing recombinant AAV particles in a first host cell and then in a second host cell, wherein in the first host cell and the second host cell, a first AAV cap gene and a second AAV cap gene are expressed, respectively, to produce a first recombinant AAV particle encapsulated in a capsid by a first AAV capsid polypeptide and a second recombinant AAV particle encapsulated in a capsid by a second AAV capsid polypeptide. Each of the first recombinant AAV particle and the second recombinant AAV particle preferably has different cell tropisms for a production cell line (for high-efficiency production of AAV) and a cell related to a therapeutic indication (for treatment of the indication).

[0002] Adeno-associated virus (AAV) is a single-stranded DNA virus belonging to the family Parvoviridae. This virus is a non-pathogenic virus that causes only a limited immune response in most patients. The capsid on the surface of AAV particles defines the cell tropism and tissue tropism of the virus. Some capsids can infect a wide range of host cells, including both dividing and non-dividing cells, while others are quite restricted. Some capsids do not infect standard production cell lines or manufacturing cell lines such as HEK293 cells.

[0003] In recent years, AAV-derived vectors have emerged as a very useful and promising form of gene delivery, due to the following properties of these vectors. AAV is a small, non-enveloped virus with only two endogenous genes (rep and cap). Therefore, it can be easily manipulated to develop vectors for various gene therapies. This is achieved by removing the rep and cap genes from the AAV genome and by replacing these sequences with exogenous sequences (transgenes) that can provide therapeutic effects to patients. -AAV particles are not readily degraded by shear forces, enzymes, or solvents. This facilitates the purification and final formulation of these viral vectors. AAVs are non-pathogenic and have low immunogenicity. Using these vectors further reduces the risk of undesirable inflammatory responses. Unlike other viral vectors such as lentiviruses, herpesviruses, and adenoviruses, AAVs are harmless and are not thought to cause any human disease. - Using AAV vectors, gene sequences of approximately 4500 bp or less can be delivered to patients. - While wild-type AAV vectors have been shown to sometimes insert genetic material into human chromosome 19, this property is generally removed from AAV gene therapy vectors by removing the rep and cap genes from the viral genome. In such cases, the virus remains in episomal form within the host cell. These episomes are preserved in non-dividing cells but are lost during cell division in dividing cells.

[0004] The native AAV genome contains two genes, the rep gene and the cap gene, each encoding multiple open reading frames (ORFs). The rep gene encodes non-structural proteins of the viral genome, necessary for the AAV lifecycle and site-specific integration, while the cap gene encodes a structural capsid protein. Both of these genes also contain terminal inverted repeat sequences (ITRs) consisting of 145 bases, capable of forming hairpin structures. These hairpin sequences are necessary for primase-independent synthesis of the second DNA strand and for the integration of viral DNA into the host cell genome.

[0005] To remove the ability of the virus to integrate, recombinant AAV vectors remove the rep and cap from the viral genome DNA. To produce such a vector, the desired transgene(s), along with a promoter to drive the transcription of the transgene(s), are inserted between terminal inverted repeats (ITRs). The rep and cap genes are supplied trans from a second plasmid or helper virus encoding the rep and / or cap genes. Helper genes such as adenovirus E4, adenovirus E2a, and adenovirus VA genes are also supplied by plasmids or helper viruses. The rep, cap, and helper genes may be supplied on an additional plasmid transfected into the cells, or they may be supplied via a helper virus.

[0006] Traditionally, AAV vector production has been achieved through numerous different pathways.

[0007] Initially, AAV was produced by transfecting cells with plasmids encoding the rep and cap genes and the AAV genome using wild-type (WT) adenovirus serotype 5. This allowed the WT adenovirus to provide numerous factors to the trans that facilitate viral replication. However, this method had several limitations. For example, each batch of AAV had to be isolated from adenovirus (AV) particles after production to provide a pure product, but it was difficult to reliably remove all Ad5. Furthermore, it was undesirable that during production, the cells would allocate considerable resources to producing adenovirus particles rather than AAV.

[0008] Other systems have used stable packaged cell lines that express the rep and cap genes. In such systems, the rep and cap genes are integrated into the cell genome, thus eliminating the need for plasmid-based rep and cap genes. However, these genes are usually integrated infrequently (e.g., 1-2 copies per cell) due to their inherent toxicity. These systems require infection with an adenovirus vector.

[0009] More recently, adenovirus-based systems have been replaced with plasmids encoding the portion of the adenovirus genome necessary for AAV production. This has addressed some concerns regarding adenovirus particles present in the final viral preparation, but numerous problems remain. These include the need to pre-fabricate enough plasmids to transfect the producing cell lines, and the inherent inefficiency of the transfection process itself.

[0010] All current AAV production methods require the AAV genome to be transfused each time, either through plasmid transfection, cell line integration, or helper virus, in order to produce more AAV.

[0011] The inventors have found that by co-infecting cells with an adenovirus expressing the rep gene and the cap gene, and infectious AAV particles produced by any of the above methods, it is possible to amplify AAV particles that have already been produced and contain the AAV genome.

[0012] However, some AAV capsids used on the surface of AAV particles exhibit selectivity for specific target therapeutic tissues (e.g., retinal neurons or hepatocytes). Therefore, the novel approach of continuous AAV proliferation described above is only effective if the produced AAV has the ability to infect the producing cell line (e.g., HEK293 cells). In many cases, AAV is unable to infect, or if it can, it is only very inefficiently (e.g., AAV6 and AAV9 in HEK293 cells).

[0013] In this specification, the inventors describe a method for overcoming this problem using a newly named process called transpsed typing.

[0014] Each AAV particle has a different cell orientation based on the capsid polypeptide encoded by the cap gene. Depending on the cap gene, some capsids can efficiently infect producing cell lines (e.g., the AAV2 capsid in HEK293 cells), while others cannot efficiently infect producing cell lines (e.g., the AAV9 capsid in HEK293 cells). As described above, the inventors have developed a novel method for producing AAV by infecting producing cell lines with AAV and providing the cap gene in trans. Thus, by providing a cap gene that confers the ability to infect producing cell lines to "seed stock" AAV particles (e.g., AAV2 for HEK293 cells), the AAV can initially proliferate continuously. Once sufficient seed stock AAV material encapsulated in a capsid that infects producing cell lines has been produced, in the final production step, another capsid can be provided in trans to produce AAV with the desired new orientation. AAV particles produced with such alternative second cap genes may not infect the producing cell line, but they can be used to infect cells associated with the therapeutic indication. Furthermore, the capsid on their surface provides selectivity and superior specificity for infecting target cells or tissues, but may result in loss or reduced infectivity to the producing cell line. This process is referred to herein as transpsydetyping.

[0015] Accordingly, an object of the present invention is to provide a process for producing recombinant AAV particles, wherein first recombinant AAV particles are produced in first host cells, where the AAV particles are directional to second host cells (e.g., a producing cell line), and the second host cells are then cultured under conditions such that second recombinant AAV particles are produced, and the second recombinant AAV particles are directional to infect cells of a desired therapeutic target tissue or therapeutic target organ.

[0016] In one embodiment, the present invention provides a process for producing recombinant adeno-associated virus (AAV) particles. The process comprises (a) to (d) below, and optionally (e) below. (a) A step of culturing a first host cell population, wherein each of the first host cells is (i) A nucleic acid molecule encoding a recombinant AAV genome, (ii) A nucleic acid molecule encoding a first AAV cap gene encoding a first AAV capsid polypeptide, wherein the first AAV capsid polypeptide confers a directivity toward a second host cell to the AAV particles containing the polypeptide, (iii) A nucleic acid molecule encoding the AAV rep gene, (iv) A nucleic acid molecule encoding a viral helper gene, The expression of each of (i) to (iv) in the first host cells is a process comprising culturing a population of first host cells in the first host cells under conditions sufficient to produce first recombinant AAV particles containing the recombinant AAV genome, wherein the first AAV particles are encapsulated in a capsid by the first AAV capsid polypeptide; (b) Infecting a second population of host cells with the first recombinant AAV particles produced in step (a); (c) A step of expressing (i) to (iii) in the second host cell population, (i) A nucleic acid molecule encoding a second AAV cap gene encoding a second AAV capsid polypeptide, wherein the second AAV capsid polypeptide confers a directivity toward a third host cell to the AAV particles containing the polypeptide, (ii) Nucleic acid molecule encoding the AAV rep gene, (iii) Nucleic acid molecule encoding a viral helper gene, All of the above (i) to (iii) are either present independently in the second host cell or have been subsequently introduced into the second host cell. In the second host cell, the expression of each of (i) to (iii) is sufficient for the production of a second recombinant AAV particle containing the recombinant AAV genome in the second host cell, and the second recombinant AAV particle is encapsulated in a capsid by the second AAV capsid polypeptide, step; (d) culturing the second host cell in a medium under conditions such that a second recombinant AAV particle containing the recombinant AAV genome is produced, wherein each of the second recombinant AAV particles is encapsulated in a capsid containing the second AAV capsid polypeptide, step; (e) purifying and / or isolating the second recombinant AAV particle from the second host cell or the medium.

[0017] Details of the sequences The following sequences are shown in the sequence listing that forms part of the specification of this application. [Table 1]

[0018] Adeno-associated virus (AAV) is a small (about 20 nm), replication-deficient non-enveloped virus. In some embodiments, AAV is adeno-associated dependoparvovirus A. In other embodiments, AAV is adeno-associated dependoparvovirus B.

[0019] AAV particles are formed from capsid proteins that encapsulate the ssDNA AAV genome in a capsid. The wild-type AAV genome contains two genes, each encoding multiple open reading frames (ORFs). That is, the rep gene encodes non-structural proteins required for the AAV life cycle and site-specific integration of the viral genome, and the cap gene encodes structural capsid proteins. As used herein, the term "recombinant AAV particle" refers to an AAV particle containing a recombinant AAV genome.

[0020] As used herein, the term "recombinant AAV genome" refers to an AAV genome that contains AAV terminal inverted repeat sequences (ITRs) flanking an intervening sequence. The intervening sequence preferably exceeds 100 bp. Further, the intervening sequence does not contain either the AAV rep gene or the AAV cap gene. The intervening sequence preferably contains a transgene. Preferably, the term "recombinant AAV genome" refers to an AAV genome that contains a transgene (instead of the rep gene and the cap gene) flanked by AAV terminal inverted repeat sequences (ITRs).

[0021] As used herein, the terms "AAV genome", "AAV transfer vector", and "transfer plasmid" are used interchangeably. They all refer to a vector that contains 5'- and 3'-viral (preferably, AAV) terminal inverted repeat sequences (ITRs) flanking an intervening sequence.

[0022] The transgene may be a coding sequence or a non-coding sequence, and may be genomic DNA or cDNA. Preferably, the transgene encodes a polypeptide or a fragment thereof.

[0023] Preferably, the transgene is operably associated with one or more transcriptional control elements and / or translational control elements (e.g., enhancer sequences, promoter sequences, terminator sequences, etc.).

[0024] In some embodiments, the transgene encodes a therapeutic polypeptide or a fragment thereof. Preferred therapeutic polypeptides include, for example, antibodies, CAR-T molecules, scFVs, BiTEs, DARPins, and T cell receptors. In some embodiments, the therapeutic polypeptide is a G protein-coupled receptor (GPCR), such as DRD1. In some embodiments, the therapeutic polypeptide is an immunotherapy target, such as CD19, CD40, or CD38. In some embodiments, the therapeutic polypeptide is a functional copy of a gene involved in human visual or retinal function, such as RPE65 or REP. In some embodiments, the therapeutic polypeptide is a functional copy of a gene involved in human hematopoietic production, or a blood component such as factor IX, or one involved in β and α thalassemia or sickle cell anemia. In some embodiments, the therapeutic polypeptide is a functional copy of a gene involved in immune function, such as those in severe combined immunodeficiency (SCID) or adenosine deaminase deficiency (ADA-SCID). In some embodiments, the therapeutic polypeptide is a protein that increases / decreases cell proliferation, such as a growth factor receptor. In some embodiments, the therapeutic polypeptide is an ion channel polypeptide.

[0025] In some embodiments, the therapeutic polypeptide is an immune checkpoint molecule. Preferably, the immune checkpoint molecule is a member of the tumor necrosis factor (TNF) receptor superfamily (e.g., CD27, CD40, OX40, GITR, or CD137) or a member of the B7-CD28 superfamily (e.g., CD28, CTLA4, or ICOS). Preferably, the immune checkpoint molecule is PD1, PDL1, CTLA4, Lag1, or GITR. In some preferred embodiments, the transgene encodes a CRISPR enzyme (e.g., Cas9, dCas9, Cpf1 or its variants or derivatives) or a CRISPR sgRNA.

[0026] Step (a) comprises culturing a first population of host cells, each of which contains (i) a nucleic acid molecule encoding a recombinant AAV genome.

[0027] In this specification, the nucleic acid molecule may be DNA or RNA, and is preferably DNA.

[0028] In one embodiment, the nucleic acid molecule may be in the form of a vector or plasmid containing a nucleic acid molecule encoding a recombinant AAV genome.

[0029] In another embodiment, the nucleic acid molecule may be in the form of a recombinant AAV particle containing a nucleic acid molecule encoding a recombinant AAV genome.

[0030] In this embodiment, the recombinant AAV particles are encapsulated in a capsid by an AAV capsid polypeptide that confers directivity to a first host cell.

[0031] The recombinant AAV particles can be prepared by appropriate methods. General methods for preparing recombinant AAV particles are well known in the art.

[0032] In another embodiment, the nucleic acid molecule may be in the form of a recombinant adenovirus (AV) particle containing a nucleic acid molecule encoding a recombinant AAV genome (as described, for example, in WO2019 / 020922, and further herein).

[0033] In another embodiment, the nucleic acid molecule encoding the recombinant AAV genome may be stably integrated into the genome of the first host cell.

[0034] In some embodiments of the present invention, step (a) further includes a pre-step of introducing a nucleic acid molecule encoding a recombinant AAV genome into the first host cell. The nucleic acid molecule encoding a recombinant AAV genome may be any of the above-described forms.

[0035] In this specification, the term “introduce” includes transformation, and in particular any form of electroporation, conjugation, infection, transduction, or transfection. The term “introduced” is also interpreted similarly, with necessary modifications.

[0036] The first recombinant AAV particle contains a first AAV capsid polypeptide that confers targeting to a second host cell. The second recombinant AAV particle contains a second AAV capsid polypeptide that confers targeting to a third host cell.

[0037] AAV capsid polypeptides are encoded by AAV cap genes. In this specification, the term "cap gene" means a gene that encodes one or more open reading frames (ORFs), each of which encodes an AAV Cap structural protein or a variant or derivative thereof. These AAV Cap structural proteins (or their variants or derivatives) form an AAV capsid.

[0038] The three Cap proteins are VP1, VP2, and VP3, and their sizes are generally 87 kDa, 72 kDa, and 62 kDa, respectively. Therefore, the AAV cap gene is the gene encoding the three Cap proteins VP1, VP2, and VP3. In wild-type AAV, these three proteins are translated from the p40 promoter to form a single mRNA. After this mRNA is synthesized, long or short introns can be cleaved, resulting in the formation of a 2.3 kb or 2.6 kb mRNA. The AAV capsid consists of 60 capsid protein subunits (VP1, VP2, and VP3), which are arranged in an icosahedral symmetry in a 1:1:10 ratio and have an estimated size of 3.9 MDa. In this specification, the term “cap gene” includes the wild-type cap gene and its derivatives, as well as artificial cap genes having equivalent function.

[0039] In this specification, the AAV cap gene sequences and Cap polypeptide sequences for AAV serotypes 1-9 are shown in SEQ ID NOs: 1-18, respectively. The terms “cap gene” or “Cap polypeptide coding sequence” in this specification preferably include, but are not limited to, the polynucleotide molecules described in (a), (b), and (c) below. (a) A polynucleotide molecule comprising, or consisting of, the nucleotide sequence shown in any one of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, or 17 (preferably SEQ ID NO: 17); (b) Polynucleotide molecules comprising a variant of the nucleotide sequence shown in any one of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, or 17 (preferably SEQ ID NO: 17), or a polypeptide molecule comprising such variant, wherein the variant has at least 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, or 99% (preferably at least 95%) sequence identity with any one of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, or 17 (preferably SEQ ID NO: 17); (c) A polynucleotide molecule having a nucleotide sequence that includes or consists of the nucleotide sequence that encodes (i) or (ii) below: (i) A polypeptide whose amino acid sequence is represented by any one of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, or 18 (preferably SEQ ID NO: 18), (ii) A variant of (i), wherein the variant has at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, or 99% (preferably at least 95%) sequence identity with any one of sequence numbers 2, 4, 6, 8, 10, 12, 14, 16, or 18 (preferably sequence number 18).

[0040] Preferably, the variant is one or more of the VP1 polypeptide, VP2 polypeptide, and VP3 polypeptide, or encodes one or more of these polypeptides.

[0041] The first recombinant AAV particle is encapsulated in a capsid containing a first AAV capsid polypeptide that confers targeting to a second host cell, and optionally, in a capsid containing a first AAV capsid polypeptide that also confers targeting to both the second and first host cells. Thus, the second host cell is a cell that can be infected with AAV particles encapsulated in the first AAV capsid polypeptide.

[0042] The second recombinant AAV particle is encased in a capsid containing a second AAV capsid polypeptide that confers targeting to a third host cell (target cell). Therefore, the third host cell (target cell) is a cell that can be infected with the AAV particle encased in the capsid by the second AAV capsid polypeptide.

[0043] Preferably, the second AAV capsid polypeptide does not confer directivity to the second host cell; that is, the second host cell cannot be infected with the second recombinant AAV particles with high efficiency. In this regard, the term "high efficiency" may be defined as requiring more than 100 viral particles per cell to achieve detectable transgene expression.

[0044] Therefore, the first AAV capsid polypeptide and the second AAV capsid polypeptide will have different amino acid sequences. Preferably, the amino acid sequence identity between the first AAV capsid polypeptide and the second AAV capsid polypeptide is less than 99.5%, for example, less than 99%, less than 98%, less than 97%, less than 96%, less than 95%, or less than 90%, and at least 50%. Preferably, the amino acid sequence identity between the first AAV capsid polypeptide and the second AAV capsid polypeptide is between 80% and 95%. Preferably, the amino acid sequences of the first AAV capsid polypeptide and the second AAV capsid polypeptide differ by at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acids, for example, by 1-5, 5-10, or 10-20 amino acids.

[0045] The first AAV cap gene is not expressed in the second host cell. The second AAV cap gene is not expressed in the first host cell.

[0046] AAV serotypes are determined by the AAV's directivity to host cells. Serotypes are distinct variations within a viral species. These viruses are classified together based on antigens on the cell surface (i.e., the capsid), making epidemiological classification of viruses possible down to the subspecies level.

[0047] The AAV capsid protein contains 12 hypervariable surface regions. Natural capsid polypeptides have different amino acid sequences from one another. The major variant region is recognized as being within the VP3 region of the capsid gene. However, the VP3 region is shared by the VP1 coding sequence and the VP2 coding sequence. Therefore, it is more accurate to say that all capsid coding sequences usually share mutations, since the variable region of the Cap polypeptide is more generally located in the C-terminal half of the Cap gene. For this reason, it is preferable that the first recombinant AAV particles and the second recombinant AAV particles have different serotypes.

[0048] Eleven different AAV serotypes are known. All of the known serotypes can infect cells of a wide variety of host cell types. The serotypes of the first recombinant AAV particles and the second recombinant AAV particles may be selected from the group consisting of serotype 1, serotype 2, serotype 3, serotype 4, serotype 5, serotype 6, serotype 7, serotype 8, serotype 9, serotype 10, and serotype 11, or modified or mutant versions of these serotypes. Preferably, the serotypes of the first recombinant AAV particles and the second recombinant AAV particles are selected from the group consisting of serotype 1, serotype 2, serotype 5, serotype 6, serotype 7, serotype 8, and serotype 9, or modified or mutant versions of these serotypes.

[0049] The first recombinant AAV particle is most preferably serotype 2 (i.e., AAV2). The second recombinant AAV particle is most preferably serotype 9 (i.e., AAV9).

[0050] More preferably, the second recombinant AAV particle is a serotype that exhibits higher directivity to a third host cell (target cell) compared to the directivity of AAV1 to AAV9, or a modified or mutant derivative of that serotype.

[0051] In this specification, the term "rep gene" means a gene that encodes one or more open reading frames (ORFs), each of which encodes an AAV Rep non-structural protein or a variant or derivative thereof. These AAV Rep non-structural proteins (or their variants or derivatives) are involved in AAV genome replication and / or AAV genome packaging.

[0052] The wild-type rep gene contains three promoters: p5, p19, and p40.

[0053] Two overlapping messenger ribonucleic acid (mRNA) sequences of different lengths can be produced from p5 and p19. Each of these mRNAs uses one splice donor site and two different splice acceptor sites, and may or may not contain introns that can be spliced ​​out. Thus, six different mRNAs can be formed, of which only four are functional. The two mRNAs that have not had the introns removed (one transcribed from p5 and the other from p19) are read up to a common terminator sequence and encode Rep78 and Rep52, respectively. The removal of the introns and the use of the 5' most splice acceptor site do not result in the production of any functional Rep proteins—they cannot produce the correct Rep68 or Rep40 proteins because the rest of the sequence frame is shifted, nor can they produce the correct C-terminus of Rep78 or Rep52 because the terminators are spliced ​​out. Conversely, the removal of the intron and the use of the 3' splice acceptor would result in the correct C-terminus of Rep68 and Rep40 being included, and the terminators of Rep78 and Rep52 being spliced ​​out. Therefore, one would either avoid splicing out the intron as a whole with functional splicing alone (producing Rep78 and Rep52), or use the 3' splice acceptor (producing Rep68 and Rep40). As a result, four different functional Rep proteins with duplicate sequences can be synthesized from these promoters.

[0054] In wild-type rep genes, the p40 promoter is located at the 3' end. Transcription of Cap proteins (VP1, VP2, and VP3) begins from this promoter in the wild-type AAV genome.

[0055] The four wild-type Rep proteins are Rep78, Rep68, Rep52, and Rep40. Therefore, the wild-type rep gene is the gene that encodes the four Rep proteins Rep78, Rep68, Rep52, and Rep40.

[0056] In this specification, the term "rep gene" includes wild-type rep genes and their derivatives, as well as artificial rep genes having equivalent function. The wild-type rep genes encode Rep78 polypeptide, Rep68 polypeptide, Rep52 polypeptide, and Rep40 polypeptide.

[0057] The complete nucleotide sequence of the wild-type AAV (serotype 2) rep gene is shown in SEQ ID NO: 19. In this specification, the nucleotide sequences of wild-type AAV (serotype 2) Rep78, Rep68, Rep52, and Rep40 are shown in SEQ ID NOs: 20, 22, 24, and 26, respectively. In this specification, the amino acid sequences of wild-type AAV (serotype 2) Rep78, Rep68, Rep52, and Rep40 are shown in SEQ ID NOs: 21, 23, 25, and 27, respectively.

[0058] The terms “rep gene” or “Rep polypeptide coding sequence” as used herein preferably include, but are not limited to, the polynucleotide molecules described in (a), (b), and (c) below. (a) A polynucleotide molecule comprising, or consisting of, the nucleotide sequence shown in any one of SEQ ID NOs: 19, 20, 22, 24, or 26 (preferably SEQ ID NO: 19); (b) Polynucleotide molecules comprising a variant of the nucleotide sequence shown in any one of SEQ ID NOs: 19, 20, 22, 24, or 26 (preferably SEQ ID NO: 19), or comprising such variant, wherein the variant has at least 80%, 85%, 90%, 95%, or 99% (preferably at least 95%) sequence identity with any one of SEQ ID NOs: 19, 20, 22, 24, or 26 (preferably SEQ ID NO: 19); (c) A polynucleotide molecule having a nucleotide sequence that includes or consists of the nucleotide sequence that encodes (i) or (ii) below: (i) A polypeptide whose amino acid sequence is represented by any one of SEQ ID NOs: 21, 23, 25, or 27 (preferably SEQ ID NO: 21), (ii) A variant of (i), wherein the variant has at least 80%, 85%, 90%, 95%, or 99% (preferably at least 95%) sequence identity with any one of sequence numbers 21, 23, 25, or 27 (preferably sequence number 21).

[0059] Preferably, the variant is one or more of the Rep78 polypeptide, Rep68 polypeptide, Rep52 polypeptide, and Rep40 polypeptide, or encodes one or more of these polypeptides.

[0060] The rep gene and the cap gene (and each of the protein-coding ORFs within them) may originate from one or more different viruses (e.g., two, three, or four different viruses). For example, the rep gene may originate from AAV2, while the cap gene may originate from AAV5.

[0061] It is known to those skilled in the art that the rep and cap genes of AAV differ among strains and isolates. Sequences of these genes derived from all such strains and isolates are included herein, as are their derivatives.

[0062] Nucleic acid molecules encoding viral helper genes are also expressed in host cells. Helper genes are necessary to promote the replication of the AAV genome and the production of infectious AAV particles. Suitable viral helper genes include one or more or all of the E1A, E1B, E4, and VA RNAs, and optionally the E2A gene. The helper genes are preferably viral helper genes derived from adenovirus, herpesvirus, or poxvirus. Most preferably, the helper genes are adenovirus helper genes. Since the genomes of some host cells (e.g., HEK293 cells) stably incorporate the E1A and E1B genes, it is not necessary to introduce further copies of these latter genes into such host cells.

[0063] To produce recombinant AAV particles containing the recombinant AAV genome, the AAV genome must be present in the host cell, and the Rep polypeptide and Cap polypeptide must be produced within the host cell or provided trans. Furthermore, sufficient viral helper genes (e.g., adenovirus E4, E1A, E1B, and VA RNA, and optionally the E2A gene) are required.

[0064] In this specification, the term “introduce” includes transformation, and in particular any form of electroporation, conjugation, infection, transduction, or transfection. The term “introduced” is also interpreted similarly, with necessary modifications.

[0065] Nucleic acid molecules encoding the AAV cap gene, the AAV rep gene, and the viral helper gene may be present independently in the first host cell and / or the second host cell in one or more of the following forms (i) to (v), or may be introduced separately into the first host cell and / or the second host cell. (i) It is stably integrated into the genome of the host cell; (ii) It exists as an episome within the host cell; (iii) Present within the recombinant adenovirus in the host cell; (iv) Introduced into host cells by recombinant adenovirus; or (v) It is introduced into host cells via a vector or plasmid.

[0066] The morphologies of the nucleic acid molecules encoding the AAV cap gene, AAV rep gene, and viral helper gene in the first host cell and the second host cell may be the same or different.

[0067] In the embodiment in which (iv) or (v) above is introduced into host cells in step (a), this introduction may be performed before or after the introduction of the nucleic acid molecule encoding the recombinant AAV genome.

[0068] For both the first and second host cells, the nucleic acid molecule encoding the AAV cap gene is preferably in the form of a vector or plasmid, or present in the recombinant adenovirus.

[0069] Preferably, the vector, plasmid, or recombinant adenovirus is introduced into the host cell before or after the introduction of the nucleic acid molecule encoding the recombinant AAV genome, or before or after the transfection of recombinant AAV particles.

[0070] Most preferably, the nucleic acid molecule encoding the first AAV cap gene is introduced into a first host cell in the form of a plasmid or as an AV recombinant genome (for example, as described in WO2019 / 020922, and further herein).

[0071] Most preferably, the nucleic acid molecule encoding the second AAV cap gene is introduced into a second host cell via an AV recombinant genome (as described, for example, in WO2019 / 020922, and further herein).

[0072] Preferably, the nucleic acid molecule encoding the AAV rep gene is introduced into a first host cell in the form of a plasmid or as an AV recombinant genome (for example, as described in WO2019 / 020922, and further herein). Preferably, the nucleic acid molecule encoding the AAV rep gene is introduced into a second host cell as an AV recombinant genome (for example, as described in WO2019 / 020922, and further herein).

[0073] The nucleic acid molecule encoding the viral helper gene is preferably in the form of a helper adenovirus. The AV E1a and AV E1b genes are preferably integrated into the host cell genome (e.g., HEK293 cells).

[0074] Most preferably, components (i) to (iv) of step (a) will be delivered simultaneously.

[0075] As described above, the AAV rep gene and / or AAV cap gene may be introduced (e.g., transfected) into a host cell separately by one or more recombinant adenoviruses, or they may be present in one or more recombinant adenoviruses already present in the host cell.

[0076] To adapt the AAV rep gene and / or AAV cap gene, one or more adenovirus genes may be partially or entirely deleted. These may include helper genes such as E1a and E1b, which are integrated into the host cell genome (e.g., HEK293 cells).

[0077] For example, the AAV rep gene and / or AAV cap gene may be inserted into one of the adenovirus early genes, or into a site where an early gene has been deleted from the adenovirus. In the latter example, the deleted early gene may be trans-complemented by a cell line containing the deleted gene, such as HEK293 cells containing the adenovirus E1A and E1B regions.

[0078] The AAV rep gene and / or AAV cap gene may be inserted into a region of the adenovirus genome containing an E1 deletion. In other examples, genes not essential to the adenovirus can also be deleted, and these sites can be used for insertion of the nucleic acid molecules of the present invention. For example, since most E3 genes can be deleted in adenovirus vectors, the AAV rep gene and / or AAV cap gene may be inserted into the E3 region of the adenovirus.

[0079] The AAV rep gene and / or AAV cap gene may be inserted into the adenovirus gene in a sense orientation or an antisense orientation (relative to the transcription direction of the adenovirus gene). A preferred embodiment of the present invention would be that, when the AAV rep gene and / or AAV cap gene are inserted into the E1 region, they are in the same transcription direction as the E4, E2A, and E2B expression cassettes. This is to prevent the E1A promoter (often retained in E1 deletion AVs) from acting as a promoter driving rep gene expression. The E1A promoter cannot be removed because it contains AV packaging signals.

[0080] A preferred embodiment of the present invention would be that the Rep-coding sequence does not include an upstream promoter.

[0081] In some preferred embodiments, the AAV rep gene and / or AAV cap gene are inserted into the adenovirus E1 gene, preferably with a portion of the E1 gene deleted. Preferably, the E1 gene is E1A and / or E1B.

[0082] The rep gene, the cap gene, and the viral helper gene may, independently, be operably related to a promoter, such as a constitutive promoter, an inducible promoter, an inhibitory promoter, or a minimal promoter, or they may not be operably related to a promoter. In this specification, the term “operably related” in the context of promoters and genes means that the promoter and gene in question are located close enough to each other that the promoter can promote the transcription of the gene. In some embodiments, the promoter and the gene are juxtaposed or in close proximity.

[0083] The promoter operably associated with the AAV cap gene is preferably a constitutive promoter or an inductive promoter, and more preferably a constitutive promoter such as a minimal CMV promoter. In some embodiments, the first AAV cap gene and / or the second AAV cap gene are either not operably associated with a promoter or are operably associated with a minimal promoter.

[0084] Preferably, when an AAV cap gene and a promoter operably associated with it are encoded within recombinant AV, the promoter used will be selected based on the toxicity of the cap gene to adenovirus and the required expression level. We have found that some cap genes can be expressed under a CMV promoter and have little to no effect on AV replication (e.g., AAV9). Conversely, some cap genes can only be inserted into AV when driven by a minimal CMV promoter with relatively low expression (e.g., AAV6). Therefore, the promoter driving the cap gene will preferably be based on experimental testing of low-expression and high-expression promoters. In this case, the high-expression promoter is the CMV promoter, and the low-expression promoter is the minimal promoter region of the same promoter.

[0085] In some embodiments, the Rep polypeptide is expressed at low, baseline, or minimal levels. In some embodiments, the AAV rep gene is not operably associated with any functional promoter. In this specification, the terms “low, baseline, or minimal levels” refer to Rep78 polypeptide expression levels that are less than 50%, less than 40%, less than 30%, less than 20%, or less than 10% of the expression level of the wild-type Rep78 polypeptide operably associated with the wild-type p5 promoter (of the wild-type AAV rep gene). Thus, sufficient Rep polypeptide is provided to enable the production of at least some AAV, but the Rep polypeptide expression level is insufficient to completely inhibit adenovirus replication.

[0086] The promoter operably associated with the helper gene is preferably a constitutive promoter or an inducible promoter, and more preferably a constitutive promoter.

[0087] Examples of constitutive promoters include CMV, SV40, PGK (human or mouse), HSV TK, SFFV, ubiquitin, elongation factor alpha, CHEF-1, FerH, Grp78, RSV, adenovirus E1A, CAG or CMV-beta-globulin promoter, or promoters derived from them.

[0088] Preferably, the rep gene promoter is the SV40 promoter or a promoter derived therefrom, or a promoter with the same or lower strength compared to the SV40 promoter in human cells and human cell lines (e.g., HEK-293 cells).

[0089] In some embodiments, the promoter is inducible or repressive by including an inducible or repressible regulatory (promoter) element. For example, the promoter may be inducible with doxycycline, tetracycline, IPTG, or lactose.

[0090] The rep gene and the cap gene may each be independently operably associated with a terminator such as the SV40 polyadenylation signal.

[0091] In embodiments of the present invention, when the nucleic acid molecule encoding the recombinant AAV genome is in the form of recombinant AAV particles, the recombinant AAV particles are encapsulated in a capsid by an AAV capsid polypeptide that confers targeting to a first host cell. The second host cell is a cell that can infect the AAV particles encapsulated in the first AAV capsid polypeptide. The third host cell (target cell) is a cell that can infect the AAV particles encapsulated in the second AAV capsid polypeptide.

[0092] The objective of step (a) is to produce a large quantity of recombinant AAV particles to be used in step (b). Therefore, the first host cells are preferably cells capable of producing high-titer first recombinant AAV particles. In some embodiments, the first host cells are derived from an AAV-producing cell line or AAV manufacturing cell line, i.e., a cell line containing all the polypeptides necessary for the production of AAV capsids and the maturation of AAV. The first host cells may comprise one or more different types of cells (for example, a mixture of HEK293 cells and PerC6 cells). Preferably, the first host cells are all of the same type.

[0093] The first host cell is preferably a mammalian cell. Examples of mammalian cells include those derived from any organ or tissue of humans, mice, rats, hamsters, monkeys, rabbits, donkeys, horses, sheep, cattle, and great apes. The cell is preferably a human cell. The cell may be a primary cell or an immortalized cell.

[0094] Preferred first host cell lines include HEK-293 cell lines, HEK-293T cell lines, HEK-293E cell lines, HEK-293 FT cell lines, HEK-293S cell lines, HEK-293SG cell lines, HEK-293 FTM cell lines, HEK-293SGGD cell lines, HEK-293A cell lines, MDCK cell lines, C127 cell lines, A549 cell lines, HeLa cell lines, CHO cell lines, mouse myeloma cell lines, PerC6 cell lines, 911 cell lines, and Vero cell lines. HEK-293 cells have been modified to contain E1A and E1B proteins so that these proteins do not need to be supplied to the helper plasmid. Similarly, PerC6 and 911 cells can also be used with less modification. Most preferably, the human cells are HEK293, HEK293T, HEK293A, PerC6, 911, or HeLaRC32. Other preferred cells include HeLa cells, CHO cells, and VERO cells. Most preferably, the first host cells are HEK293 cells, HEK293T cells, HEK293A cells, PerC6 cells, or 911 cells.

[0095] The objective of step (a) is to produce a large quantity of the first recombinant AAV particles to be used in step (b). Step (b) involves infecting a second population of host cells with the first recombinant AAV particles produced in step (a).

[0096] In this process, recombinant AAV particles are brought into contact with a second group of host cells to infect the second host cells with the first recombinant AAV particles. The reason these second host cells can be infected with the first recombinant AAV particles is likely because the AAV capsid surrounding the first recombinant AAV particles provides them with targeting for the second host cells.

[0097] The infection step in step (b) is preferably carried out using a composition containing purified AAV particles. That is, the composition contains no first host cells, and preferably, no cells.

[0098] The objective of step (b) is to produce a large quantity of recombinant AAV particles for subsequent treatment. Therefore, the second host cell is preferably a cell capable of producing a second recombinant AAV particle with a high titer.

[0099] The second host cell may be any of the first host cell types, or a mixture thereof. The second host cell is most preferably HEK293 cells, HEK293T cells, HEK293A cells, PerC6 cells, or 911 cells.

[0100] The type of the first host cell may be the same as or different from the type of the second host cell.

[0101] The following table shows preferred combinations of a first host cell / second host cell and specific serotype AAV particles that are directional to these host cells. [Table 2]

[0102] The first host cell and / or the second host cell may be a recombinant host cell.

[0103] Step (c) is, (c) A step of expressing (i) to (iii) in the plurality of second host cells, (i) A nucleic acid molecule encoding a second AAV cap gene encoding a second AAV capsid polypeptide, wherein the second AAV capsid polypeptide confers a directivity toward a third host cell to the AAV particles containing the polypeptide, (ii) Nucleic acid molecule encoding the AAV rep gene, (iii) Nucleic acid molecule encoding a viral helper gene, All of the above (i) to (iii) are either present independently in the second host cell or have been subsequently introduced into the second host cell. The expression of each of (i) to (iii) in the second host cell is sufficient to produce a second recombinant AAV particle containing the recombinant AAV genome in the second host cell. The process involves the second recombinant AAV particle being encapsulated in a capsid by the second AAV capsid polypeptide.

[0104] In step (c), a second (i.e., different) AAV cap gene is expressed together with the AAV rep gene and viral helper gene (compared to step (a)) to promote the replication of the AAV genome and produce a second recombinant AAV particle encapsulated by a second AAV capsid polypeptide. This produces recombinant AAV particles with targeting to a third host cell.

[0105] The second AAV cap gene is expressed in the second host cell. The second AAV cap gene is different from the first AAV cap gene. The second AAV cap gene is defined as described above. The first AAV cap gene is not expressed in the second host cell.

[0106] The nucleic acid molecule encoding the AAV rep gene is as defined above. The nucleic acid molecules encoding the AAV rep gene used in steps (a) and (c) may be the same or different.

[0107] The nucleic acid molecule encoding the viral helper gene is as defined above. The nucleic acid molecules encoding the viral helper gene used in steps (a) and (c) may be the same or different.

[0108] Step (d) includes culturing the second host cells in a culture medium under conditions such that a second recombinant AAV particle containing the recombinant AAV genome is produced, the second recombinant AAV particle being encapsulated in a capsid containing the second AAV capsid polypeptide. Host cell culture conditions for AAV production are well known in the art. During the culture step, the Rep polypeptide and the second Cap polypeptide will be produced, as will the viral helper polypeptide. The first Cap polypeptide will not be produced (because the second host cells do not express the first AAV cap gene). The recombinant AAV genome will be replicated and encapsulated in a capsid by the second Cap (capsid) polypeptide to produce the second recombinant AAV particle.

[0109] Step (e) is optional. Step (e) includes purifying and / or isolating the second recombinant AAV particles from the second host cells or the culture medium. Methods for purifying and / or isolating recombinant AAV particles from host cells and culture medium are well known in the art.

[0110] The second AAV capsid polypeptide confers tropism to the second recombinant AAV particles toward a third host cell (target cell). The third host cell is preferably a cell that does not possess the same AAV receptor as the highly efficient producing / manufacturing cell line (HEK293, PerC6, 911, etc.). The first AAV capsid polypeptide does not confer effective tropism toward the third host cell to the AAV particles encapsulated by the polypeptide. The second recombinant AAV particles will exhibit stronger tropism toward the third host cell than toward the first or second host cell. The third host cell is preferably not of the same type as the first or second host cell.

[0111] Preferred third host cells (target cells) include, but are not limited to, neurons (preferably retinal neurons), hepatocytes, muscle cells, stem cells (e.g., hematopoietic stem cells, mesenchymal stem cells, embryonic stem cells, adipose-derived stem cells and induced pluripotent stem cells and their derivatives), immune cells (including B lymphocytes, T lymphocytes, natural killer cells, monocytes, macrophages and granulocytes), endothelial cells, cardiovascular cells, epithelial cells, mesenchymal cells, pancreatic α cells, pancreatic β cells, cardiomyocytes, spleen cells, adipocytes, glial cells, fibroblasts, Kupffer cells and cancer cells (e.g., leukemia cells, lymphoma cells, myeloma cells, carcinoma cells, sarcoma cells and melanoma cells).

[0112] In some embodiments, the process further includes step (f) of infecting a third host cell with the second recombinant AAV particles of the present invention. The second recombinant AAV particles are encapsulated in a capsid by the second AAV capsid polypeptide, thereby conferring directivity to the third host cell. This facilitates the infection of the third host cell by the second recombinant AAV particles.

[0113] It is preferable that each step of the above process be performed in a specified order.

[0114] The present invention also provides a second recombinant AAV particle that can be obtained or obtained by the process of the present invention.

[0115] The present invention also provides a process for pseudotyping recombinant AAV particles to alter their range of host tropism. The process comprises steps (a) to (d) of the present invention, and optionally step (e).

[0116] WO2019 / 020992 discloses that the transcription of late adenovirus genes can be regulated (e.g., inhibited) by inserting a repressor element into the major late promoter. By "switching off" the expression of late adenovirus genes, the cell's protein production capacity can be redirected to the production of desired recombinant proteins or AAV particles. The entire contents of WO2019 / 020992 are incorporated herein by reference.

[0117] Therefore, in some embodiments, the recombinant adenovirus (i.e., adenovirus vector) comprises a repressible major late promoter (MLP) and a plurality of adenovirus late genes, wherein the MLP comprises one or more repressor elements that can regulate or control the transcription of the adenovirus late genes, and one or more of the repressor elements are inserted downstream of the MLP TATA box.

[0118] In other embodiments, the recombinant adenovirus (i.e., adenovirus vector) comprises (a) a plurality of adenovirus early genes, (b) a plurality of adenovirus late genes under the control of a major late promoter (MLP), and (c) a transgene (e.g., including an AAV rep gene and an AAV cap gene), wherein the MLP comprises one or more repressor elements capable of regulating or controlling the transcription of the adenovirus late genes, and one or more of the repressor elements are inserted downstream of the MLP TATA box.

[0119] Preferred characteristics for producing viral (preferably AAV) particles include the following: - One or more repressor elements are inserted between the MLP TATA box and the transfer +1 position. - The repressor element can bind to a repressor protein. - The gene encoding the repressor protein that can bind to the repressor element is encoded within the adenovirus genome. - The repressor protein is transcribed under the control of the MLP. - The repressor protein is a tetracycline repressor, a lactose repressor, or an ecdysone repressor, and is preferably a tetracycline repressor (TetR). - The repressor element is a tetracycline repressor binding site comprising the sequence shown in Sequence ID No. 28, or consisting of the sequence shown. - The nucleotide sequence of the MLP includes or consists of the sequence shown in SEQ ID NO: 29 or SEQ ID NO: 30. - The presence of the repressor element does not affect the production of adenovirus E2B protein. - The adenovirus vector encodes the adenovirus L4 100K protein, which is not under the control of the MLP. - The introduced gene is inserted into one of the early regions of the adenovirus, preferably into the adenovirus E1 region instead of the transfer plasmid. - The aforementioned transgene contains a triplicate leader (TPL) in its 5'-UTR. - The introduced gene encodes a therapeutic polypeptide. - The introduced gene encodes a viral protein, preferably a protein that can gather inside or outside a cell to produce virus-like particles, and preferably the introduced gene encodes norovirus VP1 or hepatitis B HBsAG.

[0120] Preferably, one or more repressor elements are inserted downstream of the MLP TATA box. Preferably, the transgene includes an AAV rep gene and an AAV cap gene.

[0121] Numerous algorithms have been established for aligning two amino acid sequences or nucleic acid sequences. Typically, one sequence serves as a reference sequence, and the test sequence is compared against the reference sequence. Sequence comparison algorithms calculate the percentage sequence identity of the test sequence to the reference sequence based on specified program parameters. Alignment of amino acid sequences or nucleic acid sequences for comparison may be performed, for example, by computer-run algorithms (e.g., GAP, BESTFIT, FASTA, or TFASTA), or by algorithms such as BLAST and BLAST 2.0.

[0122] Percentage amino acid sequence identity and percentage nucleotide sequence identity may be determined using the BLAST matching method (Altschul et al. (1997), "Gapped BLAST and PSI-BLAST: a new generation of protein database search programs", Nucleic Acids Res. 25:3389-3402; and http: / / www.ncbi.nlm.nih.gov / BLAST). Preferably, standard or default matching parameters are used.

[0123] Standard protein-protein BLAST (blastp) may be used to find similar sequences within a protein database. Like other BLAST programs, blastp is designed to find similar local regions. If the sequence similarity extends to the entire sequence, blastp also reports global consistency, which is a desirable result for protein identification purposes. Preferably, standard or default consistency parameters are used. In some cases, the "low complexity filter" may be removed.

[0124] BLAST protein searches may be performed using the BLASTX program, with a score of 50 and a word length of 3. To obtain gap matching for comparison purposes, gap BLAST (included in BLAST2.0) can be used as described in Altschul et al. (1997) Nucleic Acids Res. 25: 3389. Alternatively, sequential searches to detect distant relationships between molecules can be performed using PSI-BLAST (included in BLAST2.0) (see Altschul et al. (1997) above). When using BLAST, gap BLAST, or PSI-BLAST, the default parameters of each program may be used.

[0125] For the comparison of nucleotide sequences, this goal may be achieved using MEGABLAST, discontinuous MEGABLAST, and blastn. Preferably, standard or default matching parameters are used. MEGABLAST is specifically designed to efficiently find long matches between very similar sequences. Discontinuous MEGABLAST can be used to find nucleotide sequences that are similar to, but not identical to, the nucleic acids of the present invention.

[0126] The BLAST nucleotide algorithm finds similar sequences by breaking down the query into short subsequences called words. The program first identifies exact matches with the query words (word hits). The BLAST program then expands these word hits in multiple steps to generate final gap matches. In some embodiments, BLAST nucleotide searching can be performed with the BLASTN program, score = 100, word length = 12.

[0127] One of the key parameters governing the accuracy of BLAST searches is word size. The most important reason why blastn is more sensitive than MEGABLAST is that it uses a shorter default word size (11). For this reason, blastn is superior to MEGABLAST in finding matching sequences for related nucleotide sequences originating from other organisms. In blastn, the word size can be adjusted, and it can be shortened from the default value to a minimum of 7 to increase search sensitivity.

[0128] The newly introduced discontinuous megablast page allows for more sensitive searches (www.ncbi.nlm.nih.gov / Web / Newsltr / FallWinter02 / blastlab.html). This page uses an algorithm similar to that reported by Ma et al. (Bioinformatics. 2002 Mar; 18(3): 440-5). Discontinuous megablast uses discontinuous words within a longer window of the template, rather than requiring a perfect word match as a seed for consistent extension. In coding mode, it takes into account third base variation by focusing on finding matches at the first and second codon positions while ignoring third-position mismatches. Searches in discontinuous megablast with the same word size are more sensitive and efficient than standard blastn with the same word size. Parameters specific to discontinuous megablast are word size: 11 or 12; template: 16, 18, or 21; template type: coding (0), non-coding (1), or both (2).

[0129] In some embodiments, the BLASTP2.5.0+ algorithm (such as one available from NCBI) can be used with default parameters.

[0130] In other embodiments, the BLAST Global Alignment program using gap cost:Existence 11 and Extension 1 for Needleman-Wunsch alignment of two protein sequences may be used (e.g., available from NCBI).

[0131] The nucleic acid molecules, plasmids, and vectors of the present invention can be prepared by appropriate techniques. Recombination methods for producing the nucleic acid molecules and producing cell lines of the present invention are well known in the art (for example, "Molecular Cloning: A Laboratory Manual" (Fourth Edition), Green, MR and Sambrook, J., (updated 2014)).

[0132] The disclosures of each reference document described herein are incorporated herein by reference in their entirety. [Brief explanation of the drawing]

[0133] [Figure 1] Figure 1 shows the production of recombinant AAV2 vectors in HEK293 cells upon co-infection with AAV2, AAV5, AAV6, or AAV9 and a tetracycline-activating suppressed adenovirus (TERA-RepCap2) encoding the AAV Rep and Cap2 genes. [Figure 2] Figure 2 shows the production of recombinant AAV9 vectors in HEK293 cells upon co-infection with either recombinant AAV2 or recombinant AAV9 and a tetracycline-activating suppressor adenovirus (TERA-RepCap9) encoding the AAV Rep and Cap9 genes. [Examples]

[0134] The present invention is further illustrated by the following embodiments, where parts and proportions are by weight unless otherwise noted, and temperatures are in Celsius. It should be understood that these embodiments are presented for illustrative purposes only, while illustrating preferred embodiments of the present invention. Based on the above considerations and these embodiments, those skilled in the art can confirm the essential characteristics of the present invention and make various modifications and alterations to the invention to suit various uses and conditions without departing from its spirit and scope. Therefore, in addition to those presented and described herein, various alterations of the present invention will be apparent to those skilled in the art from prior descriptions. Such alterations are also intended to fall within the scope of the appended claims.

[0135] General method Recovery of adenovirus vectors containing AAV components All recombinant adenovirus vectors are initially recovered from plasmid DNA encoding each viral genome within HEK293 cells. Plasmid (20 μg) was linearized with Swar restriction enzyme to release the viral ITR from the bacterial plasmid backbone and purified using the PureLink DNA extraction kit for genomes (Invitrogen, California, USA). HEK293 cells were transferred to multiple T25 tissue culture flasks at a rate of 2 × 10⁶ cells per flask, 24 hours prior to transfection. 6Cells were seeded at a specified density. Each flask was transfected with 2.5 μg of linearized DNA using Lipofectamine 2000 (Invitrogen) according to the manufacturer's protocol. After 4 hours, the transfection medium was replaced with fresh 2% FBS-containing DMEM (with 0.5 μg / mL doxycycline or DMSO added). Recombinant viruses were collected from the growth medium within 12 days of transfection, upon observation of complete CPE. Single clones were isolated by two consecutive dilutions and further grown in HEK293 cells (and cultured in 0.5 μg / mL doxycycline or DMSO). Viruses were collected on day 3 post-infection after three freeze-thaw cycles. Cell debris was pelleted by centrifugation, and the supernatant was filtered through a 0.2 μm filter. For large-scale viral amplification and purification, HEK293 cells were seeded on Corning® HYPERFlask® (Sigma-Aldrich, Missouri, USA) for 48 hours and cultured to 95% confluence before infection. For adenovirus vectors containing rep and cap genes, the respective HYPERFlasks were infected with the viral stock solution at an MOI of 3 for 3 days. For adenovirus vectors containing a modified major late promoter regulated by the tetR protein, cell culture was performed with the addition of doxycycline 0.5 μg / mL. Once complete CPE appeared, cells were harvested and the virus was released by three freeze-thaw cycles. The virus was purified by two CsCl gradient band formations. During this process, benzonase 250 U / mL (Sigma-Aldrich, Missouri, USA) was added after the first banding to degrade the free DNA.

[0136] AAV Transduction Assay Transducible AAVs can be measured using a modified TCID50 assay. HEK293 cells were placed in a 96-well tissue culture plate, with 1 × 10⁶ cells per well. 4Seed the cells at the specified density for 24 hours. Then, dilute the stock solution of each crude AAV lysate 10-fold in eight steps using 1.2 mL of 2% FBS-containing DMEM. Next, each diluted sample (1 × 10⁻⁶) -2 ~1 × 10 -10 Ten copies of the ) were added to each plate at a volume of 100 μL per well. As a negative control, 100 μL of 2% FBS-containing DMEM was added to the last two rows. Furthermore, the plates were observed using a fluorescence microscope (EVOS FL imaging system, Thermo Fisher Scientific, Massachusetts, USA) to confirm the presence of EGFP-expressing cells in each well at 96 hpi (96 hours post-infection). Transduction units per 1 mL could be calculated using the KARBER-SPEARMAN statistical method.

[0137] Quantification of viral genome and gene expression using qPCR To quantify the entire adenovirus genome in HEK293 cells, total DNA was extracted from the culture medium and cell lysates using the Purelink genomic DNA miniprep kit (Invitrogen, California, USA). 5 μL of DNA eluate was used for a qPCR reaction using the StepOnePlus Real-Time PCR System (Applied Biosystems, California, USA) with TaqMan Fast Advanced Master Mix (Applied Biosystems, California, USA). To quantify genome-encapsulated AAV particles and adenovirus particles, 2 μL of viral sample collected from culture medium or cell lysates was treated with 1 U of TURBO DNase (Thermo Fisher Scientific, Massachusetts, USA) in 20 μL of reaction solution at 37°C for 2 hours. The TURBO DNase was heat-inactivated at 75°C for 10 minutes. Encapsulated Ad5 was quantified using Ad5 hexone primers and probes, and encapsulated AAV genomes were quantified using EGFP primers and probes, by using 5 μL of sample diluted 1:200 with nuclease-free water in the PCR reaction. The total titer of the AAV vector produced using helper adenovirus was determined by subtracting the adenovirus encapsulating the genome. A standard curve for qPCR analysis was prepared using gBLOCK gene fragments suspended in nuclease-free water (Integrated DNA Technologies, Iowa, USA). Furthermore, the DNA copy number was calculated by extrapolating the CT value of the PCR reaction to the standard curve (qPCR standard is 3 × 10⁻⁶). 8 -3 × 10 1 Copy / Well).

[0138] Quantification of AAV capsids by ELISA The assembled AAV2 capsid can be detected using the AAV2 titration ELISA kit (Progen, Heidelberg, Germany) according to the manufacturer's instructions.

[0139] Example 1: Production of AAV in a first host cell by either plasmid or adenovirus method. The production of helper-free AAV2 and helper-free AAV9 was performed according to the Takara AAVpro Helper Free System (Clontech, California, USA). When producing AAV2 and AAV9 in a 6-well tissue culture treatment plate, HEK293 cells were used at a rate of 7.5 × 10⁶ per well. 4Cells were seeded at cell density 24 hours prior to transfection. To produce without helpers, each well was transfected with 2.5 μg of plasmid DNA diluted with Opti-MEM (Gibco, Massachusetts, USA) containing pHelper, pAAV-CMV-EGFP, and pRepCap2 or pRepCap9 in a DNA mass ratio of 1:1:1. A complex with a DNA-to-PEI mass ratio of 1:3 was formed using linear PEI 25 kDA (Polysciences). When producing AAV using an adenovirus vector containing the AAV rep gene and AAV cap gene, HEK293 cells are placed in a 48-well tissue culture plate at a rate of 7.5 × 10⁶ cells per well. 4 Cells were seeded at the specified cell density 24 hours prior to production. Each well was co-infected with AAV containing the AAV genome encoding EGFP and adenovirus containing both the rep and cap genes. Four hours after treatment, the infection medium was replaced with fresh 2% FBS-containing DMEM (with doxycycline 0.5 μg / mL or DMSO added). To determine the efficiency of the DNase reaction in the degradation of free DNA, each experiment included a helper-free production control, using the stuffer plasmid pUC19 instead of pRepCap2 or pRepCap9. The AAV vector was harvested by three freeze-thaw cycles of cells suspended in growth medium. Cells were pelleted by centrifugation at 3000 g for 20 minutes, and the supernatant was filtered through a 0.2 μm filter.

[0140] Example 2: Infection of a second host cell using a different AAV serotype to produce AAV2. Figure 1 shows the production of recombinant AAV2 vectors in HEK293 cells upon co-infection with AAV2, AAV5, AAV6, or AAV9 and tetracycline-activating suppressed adenovirus (TERA-RepCap2) encoding the Rep and Cap2 genes of AAV. The data demonstrate that using different serotypes of AAV at the same concentration can significantly affect the amount of AAV subsequently produced from a second host cell. AAV2 and AAV6 are known to efficiently infect HEK293 cells, while AAV9 and AAV5 are known to be less efficient infecting HEK293 cells. This infection efficiency directly affects the production levels of each serotype. These production levels are directly compared to those obtained using helper-free plasmid transfection. In this example, HEK293 cells were seeded at a rate of 9e4 cells / well in a 48-well tissue culture plate format for 24 hours. HEK293 cells were triple-transfected or co-infected with helper-free plasmids using 50 genome copies per cell of recombinant AAV2, AAV5, AA6, or AAV9 encoding AAV Rep and Cap2, as well as a tetracycline-activating suppressor adenovirus encoding an EGFP reporter. AAV vectors were collected 96 hours after transduction, and encapsulated AAV particles were quantified by QPCR.

[0141] Example 3: Infection of a second host cell with AAV2 and AAV9 to produce AAV9 Figure 2 shows the production of recombinant AAV9 vectors in HEK293 cells by co-infection with either recombinant AAV2 or recombinant AAV9 and tetracycline-activating suppressed adenovirus (TERA-RepCap9) encoding the Rep and Cap9 genes of AAV. Recombinant AAV production is compared to helper-free plasmid transfection. The above data shows that infecting HEK293 cells with AAV2 at MOI 50 and TERA-RepCap9 at MOI 75 produces 4.69 times more AAV9 than using AAV9 at MOI 750 and TERA-RepCap9 at MOI 75. Therefore, per molecule of AAV, producing AAV9 using AAV2 is approximately 70 times more efficient in HEK293 cells than producing AAV9 using AAV9. This represents a basic example of the present invention. In this example, HEK293 cells were seeded at 9e4 cells / well in a 48-well tissue culture plate format for 24 hours. HEK293 cells were triple-transfected with helper-free plasmids or co-infected with TERA-RepCap9 virus encoding AAV Rep and Cap9, or recombinant AAV2 vector or recombinant AAV9 vector encoding an EGFP reporter, at the indicated MOI. The AAV vector was recovered 96 hours after transduction, and encapsulated AAV particles were quantified by QPCR.

[0142] Example 4: Infection of target cells AAV is transduced into target cells from a second host cell. This is achieved by various methods appropriate to the disease state during treatment. Delivery to a third host cell is done via direct in vivo delivery by injection into the patient. As a result, AAV is either injected directly into the therapeutic target tissue or delivered intravenously, but it is possible to infect the target cells with the second capsid polypeptide. By applying AAV to cells isolated from patients, the cells can be infected ex vivo. These infected cells can then be reintroduced to the patients.

[0143] SEQUENCE LISTING FREE TEXT <210> 1 <223> AAV1 - Capsid Nucleotide Sequence (AF063497.1) <210> 2 <223> AAV1 - Capsid Protein Sequence <210> 9 <223> AAV5 - Capsid Nucleotide Sequence (AF085716.1) <210> 10 <223> AAV5 - Capsid Protein Sequence <210> 11 <223> AAV6 - Capsid Nucleotide Sequence (AF028704.1) <210> 12 <223> AAV6 - Capsid Protein Sequence <210> 13 <223> AAV7 - Capsid Nucleotide Sequence (AF513851.1) <210> 14 <223> AAV7 - Capsid Protein Sequence <210> 15 <223> AAV8 - Capsid Nucleotide Sequence (AF513852.1) <210> 16 <223> AAV8 - Capsid Protein Sequence <210> 17 <223> AAV9 - Capsid Nucleotide Sequence (AY530556.1) <210> 18 <223> AAV9 - Capsid Protein Sequence <223> 28 <223> TetR binding site <223> 29 <223> Modified MLP <223> 30 <223> Modified MLP

Claims

1. A process for producing recombinant adeno-associated virus (AAV) particles, the process comprising (a) to (d) below, and optionally (e) below. (a) A step of culturing a first host cell population, wherein each of the first host cells is (i) A nucleic acid molecule encoding a recombinant AAV genome, (ii) A nucleic acid molecule encoding a first AAV cap gene encoding a first AAV capsid polypeptide, wherein the first AAV capsid polypeptide confers a directivity toward a second host cell to the AAV particles containing the polypeptide, (iii) A nucleic acid molecule encoding the AAV rep gene, (iv) A nucleic acid molecule encoding the viral helper gene, The expression of each of (i) to (iv) in the first host cells is a step comprising culturing a population of first host cells under conditions sufficient to produce first recombinant AAV particles containing the recombinant AAV genome in the first host cells, wherein the first recombinant AAV particles are encapsulated in a capsid by the first AAV capsid polypeptide; (b) Infecting a second group of host cells with the first recombinant AAV particles produced in step (a); (c) A step of expressing (i) to (iii) in the second host cell population, (i) A nucleic acid molecule encoding a second AAV cap gene encoding a second AAV capsid polypeptide, wherein the second AAV capsid polypeptide confers a directivity toward a third host cell to the AAV particles containing the polypeptide, (ii) Nucleic acid molecule encoding the AAV rep gene, (iii) Nucleic acid molecule that represents a viral helper gene, All of the above (i) to (iii) are either present independently in the second host cell or have been subsequently introduced into the second host cell. The expression of each of (i) to (iii) in the second host cell is sufficient to produce a second recombinant AAV particle containing the recombinant AAV genome in the second host cell, wherein the second recombinant AAV particle is capsid-encapsulated by the second AAV capsid polypeptide; (d) A step of culturing the second host cells in a culture medium under conditions such that a second recombinant AAV particle containing the recombinant AAV genome is produced, wherein each of the second recombinant AAV particles is enclosed in a capsid containing the second AAV capsid polypeptide; (e) A step of purifying and / or isolating the second recombinant AAV particles from the second host cells or the culture medium.

2. The process according to claim 1, wherein step (a) further comprises a pre-step of introducing the nucleic acid molecule encoding the recombinant AAV genome into the first host cell.

3. The process according to claim 1 or 2, wherein the first host cell and / or the second host cell is a HEK293 cell, a HEK293T cell, a HEK293A cell, a PerC6 cell, or a 911 cell.

4. The process according to any one of the claims, wherein the nucleic acid molecule encoding the recombinant AAV genome, which is introduced into the first host cell, is in the form of a plasmid, a recombinant adenovirus, or is incorporated into the genome of a cell or a recombinant AAV particle.

5. The process according to any one of the claims, wherein the nucleic acid molecule encoding the first AAV cap gene and / or the nucleic acid molecule encoding the rep gene are introduced into the first host cell in the form of a plasmid or by an AV recombinant genome.

6. The process according to any one of the claims, wherein the nucleic acid molecule encoding the second AAV cap gene and / or the nucleic acid molecule encoding the rep gene are introduced into the second host cell in the form of a plasmid or by an AV recombinant genome.

7. The process according to any one of the claims, wherein the amino acid sequence identity between the first AAV capsid polypeptide and the second AAV capsid polypeptide is less than 99.5%, preferably less than 99%, less than 98%, less than 97%, less than 96%, less than 95%, or less than 90%, and at least 50%.

8. The process according to any one of the claims, wherein the first recombinant AAV particle is serotype 2 (AAV2).

9. The process according to any one of the claims, wherein the second recombinant AAV particle is serotype 9 (AAV9) or a modified or mutant derivative of serotype 9.

10. The process according to any one of the claims, wherein the third host cell is selected from the group consisting of neurons (e.g., retinal neurons), hepatocytes, muscle cells, stem cells (preferably hematopoietic stem cells, mesenchymal stem cells, embryonic stem cells, adipose stem cells and induced pluripotent stem cells and their derivatives), immune cells (including B lymphocytes, T lymphocytes, natural killer cells, monocytes, macrophages and granulocytes), endothelial cells, cardiovascular cells, epithelial cells, mesenchymal cells, pancreatic a cells or pancreatic b cells, cardiomyocytes, spleen cells, adipocytes, glial cells, fibroblasts, Kupffer cells and cancer cells (preferably leukemia cells, lymphoma cells, myeloma cells, carcinoma cells, sarcoma cells or melanoma cells).

11. The process according to any one of the claims, wherein the nucleic acid molecule encoding the first cap gene and / or the second cap gene and / or the rep gene is present in a recombinant adenovirus (AV), the recombinant adenovirus comprising a repressible major late promoter (MLP) and a plurality of adenovirus late genes, the MLP comprising one or more repressor elements capable of regulating or controlling the transcription of the adenovirus late genes, and one or more of the repressor elements being inserted downstream of an MLP TATA box.

12. A second recombinant AAV particle obtained or obtainable by the process described in any one of the preceding claims.

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