Method for preparing adeno-associated virus vector

Abstract

The present invention relates to a nucleic acid molecule encoding at least one AAVRep polypeptide, wherein one or more of the AAV (p5), (p19) and (p40) promoters have been modified to reduce or eliminate the expression of one or more Rep polypeptides, or the nucleic acid molecule does not encode a functional (Rep52) or (Rep40) polypeptide, or the nucleic acid molecule does not encode a functional adenoviral inhibitor sequence. The present invention also relates to a method for producing a recombinant AAV vector by using a 2-adenovirus system, wherein all genes required for AAV replication and packaging (i.e., the AAVrep sequences of the present invention, AAVcap and an AAV transfer vector containing a transgene) can be encoded within two adenoviruses.

Description

[0001] The present invention relates to nucleic acid molecules encoding at least one AAV Rep polypeptide, wherein one or more of the AAV p5, p19 and p40 promoters have been modified to reduce or eliminate expression of one or more Rep polypeptides, or the nucleic acid molecule does not encode a functional Rep52 or Rep40 polypeptide, or the nucleic acid molecule does not encode a functional adenoviral inhibitor sequence.

[0002] The present invention also relates to a method for producing recombinant AAV vectors by using a 2-adenovirus system, wherein all genes required for AAV replication and packaging (i.e., the AAV rep sequence of the present invention, the AAV cap, and the AAV transfer vector containing the transgene) can be encoded within two adenoviruses.

[0003] Adeno-associated virus (AAV) is a single-stranded DNA virus belonging to the Parvoviridae family. This virus is able to infect a wide range of host cells, including both dividing and non-dividing cells. In addition, it is a non-pathogenic virus that produces only a limited immune response in most patients.

[0004] Over the past few years, vectors derived from AAV have emerged as a very useful and promising mode of gene delivery. This is due to the following properties of these vectors:

[0005] -AAVs are small, non-enveloped viruses, and they have only two native genes (rep and cap). Therefore, they can be easily manipulated to develop vectors for different gene therapies. This is achieved by removing the rep and cap genes in the AAV genome and replacing these sequences with foreign sequences (transgenes) that can provide therapeutic benefits to patients.

[0006] -AAV particles are not easily degraded by shear forces, enzymes or solvents. This facilitates the simple purification and final formulation of these viral vectors.

[0007] -AAV is non-pathogenic and has low immunogenicity. The use of these vectors further reduces the risk of adverse inflammatory responses. Unlike other viral vectors such as lentivirus, herpes virus, and adenovirus, AAV is harmless and is not believed to cause any human disease.

[0008] -Gene sequences of up to about 4500 bp can be delivered to patients using AAV vectors.

[0009] -While wild-type AAV vectors have been shown to sometimes insert genetic material into human chromosome 19, most AAV gene therapy vectors generally eliminate this property by removing the rep and cap genes from the viral genome. In this case, the virus remains in an episomal form within the host cell. These episomes remain intact in non-dividing cells, whereas in dividing cells, they are lost during cell division.

[0010] The native AAV genome contains two genes, each encoding multiple open reading frames (ORFs): the rep gene encodes nonstructural proteins required for the AAV life cycle and site-specific integration of the viral genome; and the cap gene encodes the structural capsid protein.

[0011] In addition, the two genes are flanked by 145 bases of inverted terminal repeat (ITR) sequences that have the ability to form hairpin structures. These hairpin sequences are required for the primase-independent synthesis of the second DNA strand and for the integration of the viral DNA into the host cell genome.

[0012] To eliminate any integration ability of the virus, the recombinant AAV vector has rep and cap removed from the DNA of the viral genome. To produce such a vector, the desired transgene is inserted between the inverted terminal repeats (ITRs) along with a promoter that drives transgene transcription; and the rep and cap genes are provided in trans in a second plasmid. Auxiliary genes, such as adenovirus E4, E2a, and VA genes, are also provided. Rep, cap, and auxiliary genes can be provided on additional plasmids that are transfected into cells.

[0013] Traditionally, the production of AAV vectors has been achieved through a number of different approaches.

[0014] Initially, AAV was produced using wild-type (WT) adenovirus serotype 5, and cells were transfected with plasmids encoding the rep and cap genes as well as the AAV genome. This allows the WT adenovirus to provide many trans factors that promote viral replication. However, this approach has many limitations: for example, each batch of AAV must be separated from the adenovirus (AV) particles after manufacturing to provide a pure product, and ensuring the removal of all Ad5 is challenging. In addition, the fact that cells use a lot of resources to produce adenovirus particles instead of AAV during the production process is also undesirable.

[0015] In other systems, stable packaging cell lines expressing rep and cap genes have been used. In such systems, rep and cap genes are integrated into the cell genome, so plasmid-based rep and cap genes are not required. However, due to their inherent toxicity, these genes are usually only integrated at low frequencies (e.g., 1-2 copies per cell). These systems require infection with adenoviral vectors.

[0016] Recently, adenovirus-based systems have been replaced by plasmids encoding the portions of the adenoviral genome required for AAV production. While this has addressed some concerns about the presence of adenoviral particles in the final viral preparation, a number of issues remain. These include the need to pre-manufacture sufficient plasmid for transfection into production cell lines, and the inherently inefficient process of transfection itself. The yields from these systems are also lower than those obtained using Ad5-based approaches.

[0017] In the past, encoding AAV genes (rep and cap) and AAV transfer vectors in adenoviruses has been extensively explored (Fisher, KJ et al., 1996; Liu, XL et al., 1999). Although the AAV genome and AAV Cap DNA sequences are tolerated when inserted into the adenovirus genome, the AAVRep protein and DNA sequences are lethal to adenoviruses and inhibit their replication. Despite the toxicity of AAVRep to adenovirus replication, several publications have reported successful production of adenoviruses encoding AAV Rep polypeptides. However, these adenoviruses are often unstable, coupled with low titers in production, or the rep gene is lost after multiple passages of the virus (Zhang, HG et al., 2001; Zhang, X and Li, CY, Mol. Ther. 2001).

[0018] The inhibition of adenovirus by AAVRep is caused by two main mechanisms. First, the AAVRep protein is an effective inhibitor of adenovirus promoters (including MLP, E2B, E4) (Timpe JM et al., 2006). Secondly, the adenovirus inhibitor sequence is encoded within the AAV rep DNA (located within the p40 promoter, which the virus usually uses to drive the expression of the cap gene). Publications show that by scrambling this "inhibitory" p40 DNA sequence, adenovirus can tolerate the AAV rep gene (Sitaraman, V. et al., 2011; Weger, S. et al., J. Virol. 2016).

[0019] The present invention is based on a combination of steps that enable the AAV rep gene to be stably encoded into an adenoviral vector; the AAV rep gene has never been successfully inserted and maintained in an adenoviral vector before. In particular, in some embodiments, the AAV rep gene promoters p5, p19 and p40 have all been modified or removed. The p5 promoter has been removed to reduce the expression of the Rep 78 and Rep 68 polypeptides, thereby reducing their toxicity; the p19 promoter has been removed to stop the expression of the Rep52 and Rep40 polypeptides; the AV inhibitor sequence within the p40 promoter has been removed or modified, or its transcription has been prevented.

[0020] When using AV to make AAV, preferably only one AV needs to be modified for each desired AAV with different genes. However, the combined molecular size of the rep gene, cap gene and transfer AAV genome will exceed the packaging capacity of the E1 / E3 deleted AV vector.

[0021] The inventors have now discovered that advantages can be gained by using two AVs. Given that genetically distinct AAVs can contain unique capsids and transfer AAV genomes, one aspect of the invention involves contacting cells with two AV vectors: one containing the AAV Rep coding sequence, and the other containing the Cap coding sequence and the transfer AAV genome sequence (the latter defined as a sequence flanked by AAV inverted terminal repeats).

[0022] The inventors have also determined that the presence of the Rep coding sequence and the AAVITR sequence within the same AV is detrimental to AV growth. Although such AVs can be recovered, their yields are generally 5-10 times lower than when the AV contains each sequence independently.

[0023] Thus, in another aspect of the invention, the Rep coding sequence does not contain a promoter driving its expression; and its transcriptional orientation matches that of the E2A, E2B, and E4 transcription units in the AV genome. This ensures that the Rep polypeptide is expressed at low, basal, or minimal levels to reduce toxicity to cells; and that it cannot be transcribed to high levels by transcriptional read-through of the strong E1A promoter embedded in the adenovirus packaging signal element.

[0024] In yet another aspect, the first AV comprising the rep gene may also optionally encode a protein that can transcriptionally activate a promoter driving the expression of the cap gene in the second AV. This allows the transcription of the cap gene to be induced only when the two AVs are present in the same cell, thereby reducing the burden of expressing the AAV cap gene during the AV manufacturing process.

[0025] One object of the present invention is to provide a nucleic acid molecule comprising an AAV rep gene, wherein the rep gene promoter has been modified or eliminated to allow integration of the nucleic acid molecule into an adenoviral vector, and / or its subsequent expression to be less toxic.

[0026] Another object of the present invention is to provide an adenoviral vector and a host cell comprising the nucleic acid molecule of the present invention.

[0027] Another object of the present invention is to provide methods for producing modified host cells and adenoviral vectors comprising the nucleic acid molecules of the present invention; and methods for producing AAV.

[0028] In one embodiment, the present invention provides a nucleic acid molecule, wherein the nucleotide sequence of the nucleic acid molecule encodes at least one AAVRep polypeptide, wherein the Rep polypeptide encoding sequence has two or three of the following characteristics:

[0029] (i) it is not operably associated with a functional AAV p5 promoter;

[0030] (ii) it does not contain a functional AAV p19 promoter or it does not encode functional Rep52 and Rep40 polypeptides;

[0031] (iii) it does not contain a functional AAV p40 promoter or it does not contain a functional adenoviral inhibitor sequence.

[0032] Preferably, the nucleotide sequence encodes functional AAVRep78 and Rep68 polypeptides. Preferably, the nucleic acid molecule is incapable of expressing functional AAVRep52 or Rep40 polypeptides in the absence of an operably associated promoter.

[0033] The nucleic acid molecule of the present invention can be DNA or RNA. It can be single-stranded or double-stranded.

[0034] 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 AAVRep nonstructural protein or a variant or derivative thereof. These AAVRep nonstructural proteins (or their variants or derivatives) are involved in AAV genome replication and / or AAV genome packaging.

[0035] The wild-type rep gene contains three promoters: p5, p19, and p40. Two overlapping messenger RNAs (mRNAs) of different lengths can be produced from p5 and p19. Each of these mRNAs contains introns that can be spliced ​​or unspliced ​​using a single splice donor site and two different splice acceptor sites. Therefore, six different mRNAs can be formed, of which only four are functional. The two mRNAs that fail to remove the introns (one transcribed from p5 and one from p19) read through to a shared terminator sequence and encode Rep78 and Rep52, respectively. Removing the introns and using the 5'-most splice acceptor site does not produce any functional Rep proteins - it cannot produce the correct Rep68 or Rep40 proteins because the frame of the rest of the sequence is frameshifted, and it cannot produce the correct C-terminus of Rep78 or Rep52 because the terminator is spliced. In contrast, removal of the intron and use of the 3' splice acceptor will include the correct C-terminus of Rep68 and Rep40, while the terminators of Rep78 and Rep52 are spliced ​​in. Thus, the only functional splices are either to avoid splicing the intron completely (producing Rep78 and Rep52) or to use the 3' splice acceptor (producing Rep68 and Rep40). Thus, four different functional Rep proteins with overlapping sequences can be synthesized from these promoters.

[0036] In the wild-type rep gene, the p40 promoter is located at the 3' end. Transcription of the Cap proteins (VP1, VP2, and VP3) starts from this promoter in the wild-type AAV genome.

[0037] The four wild-type Rep proteins are Rep78, Rep68, Rep52 and Rep40. Thus, a wild-type rep gene is a gene encoding the four Rep proteins Rep78, Rep68, Rep52 and Rep40.

[0038] As used herein, the term "rep gene" includes wild-type rep genes and their derivatives; and artificial rep genes with equivalent functions. The wild-type rep gene encodes functional Rep78, Rep68, Rep52 and Rep40 polypeptides.

[0039] In a particularly preferred embodiment, the nucleotide sequence encodes functional Rep78 and Rep68 polypeptides.

[0040] As used herein, the term Rep78 polypeptide refers to a polypeptide of SEQ ID NO: 22 or a variant thereof, which has at least 80%, 85%, 90%, 95% or 99% sequence identity with a Rep78 polypeptide and encodes a functional Rep78 polypeptide. As used herein, the term Rep68 polypeptide refers to a polypeptide of SEQ ID NO: 23 or a variant thereof, which has at least 80%, 85%, 90%, 95% or 99% sequence identity with a Rep68 polypeptide and encodes a functional Rep68 polypeptide.

[0041] In the production of AAV, in the absence of sufficient functional Rep polypeptide, lower titers (e.g., genome copies) will be observed (this can be determined by qPCR) due to the fact that there are fewer ITR plasmids to be packaged and they cannot be packaged efficiently. Observations may also include an exaggerated empty:full particle ratio; this can be determined by ELISA or optical density measurements.

[0042] The Rep 78 / 68 polypeptide binds ATP and has helicase activity and may be involved in assisting the accumulation of single-stranded genomic precursors and the packaging of newly formed DNA strands into preformed AAV capsids.

[0043] Expression of Rep 78 or Rep 68 polypeptide variants can be determined by Western blot to determine whether these polypeptides are produced at the correct molecular weight.

[0044] The function of these polypeptides can be assayed in purified form using a bioluminescent ATP assay that determines ATP consumption. As both Rep78 and Rep68 have helicase activity, an appropriate helicase assay can also be used.

[0045] In a bioluminescent ATP assay, a test Rep78 polypeptide having an ATP consumption level of at least 80% (preferably at least 90%) of that of a wild-type Rep78 polypeptide (eg, SEQ ID NO: 22) can be considered a functional Rep78 polypeptide.

[0046] In a bioluminescent ATP assay, a test Rep68 polypeptide having an ATP consumption level of at least 80% (preferably at least 90%) of that of a wild-type Rep68 polypeptide (eg, SEQ ID NO: 23) can be considered a functional Rep68 polypeptide.

[0047] A test Rep78 polypeptide having a helicase activity level of at least 80% (preferably at least 90%) of the helicase activity level of a wild-type Rep78 polypeptide (eg, SEQ ID NO: 22) can be considered a functional Rep78 polypeptide.

[0048] A test Rep68 polypeptide having a helicase activity level of at least 80% (preferably at least 90%) of the helicase activity level of a wild-type Rep68 polypeptide (eg, SEQ ID NO: 23) can be considered a functional Rep68 polypeptide.

[0049] The wild-type AAV (serotype 2) rep gene nucleotide sequence is given in SEQ ID NO:1.

[0050] In one embodiment, the term "rep gene" or Rep polypeptide coding sequence refers to a nucleotide sequence having at least 70%, 80%, 85%, 90%, 95%, 99% or 100% sequence identity to SEQ ID NO:1, and which encodes one or more Rep78, Rep68, Rep52 and Rep40 polypeptides, preferably functional Rep78 and 68 polypeptides.

[0051] The Rep52 and Rep40 nucleotide sequences are set forth herein in SEQ ID NOs: 16 and 17. The Rep78 and Rep68 nucleotide sequences are set forth herein in SEQ ID NOs: 20 and 21.

[0052] As used herein, the term "cap gene" refers to a gene encoding one or more open reading frames (ORFs), wherein each of the ORFs encodes an AAV Cap structural protein or a variant or derivative thereof. These AAV Cap structural proteins (or variants or derivatives thereof) form the AAV capsid.

[0053] The three Cap proteins must be functional to produce infectious AAV viral particles that can infect appropriate cells. The three Cap proteins are VP1, VP2, and VP3, and their sizes are usually 87 kDa, 72 kDa, and 62 kDa, respectively. Therefore, the cap gene is the gene that encodes the three Cap proteins VP1, VP2, and VP3.

[0054] 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 excised to form a 2.3 kb or 2.6 kb mRNA.

[0055] The AAV capsid is composed of 60 capsid protein subunits (VP1, VP2, and VP3) arranged in an icosahedral symmetry in a ratio of 1:1:10, with an estimated size of 3.9 MDa.

[0056] As used herein, the term "cap gene" includes wild-type cap genes and their derivatives, as well as artificial cap genes with equivalent functions. The AAV (serotype 2) cap gene nucleotide sequence and the Cap polypeptide sequence are given in SEQ ID NO: 2 and 3, respectively. As used herein, the term "cap gene" preferably refers to a nucleotide sequence having a sequence given in SEQ ID NO: 2 or a nucleotide sequence encoding a polypeptide of SEQ ID NO: 3; or a nucleotide sequence having at least 70%, 80%, 85%, 90%, 95% or 99% sequence identity with SEQ ID NO: 2, or a nucleotide sequence having at least 80%, 90%, 95% or 99% nucleotide sequence identity with a nucleotide sequence encoding a polypeptide of SEQ ID NO: 3 and encoding VP1, VP2 and VP3 polypeptides.

[0057] The rep gene and the cap gene are preferably viral genes or derived from viral genes. More preferably, they are AAV genes or derived from AAV genes. In some embodiments, AAV is adeno-associated dependovirus A. In other embodiments, AAV is adeno-associated dependovirus B.

[0058] Eleven 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. AAV can be from serotype 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11. Preferably, AAV is serotype 1, 2, 5, 6, 7, 8, or 9. Most preferably, AAV serotype is 5 (i.e., AAV5).

[0059] The rep gene and the cap gene (and each protein encoding ORF therein) can be from one or more different viruses (e.g., 2, 3, or 4 different viruses). For example, the rep gene can be from AAV2, and the cap gene can be from AAV5.

[0060] Those skilled in the art recognize that the rep and cap genes of AAV vary from clade to clade and isolate to isolate. The sequences of these genes from all such clades and isolates and their derivatives are encompassed herein.

[0061] As used herein, the term "recombinant AAV genome" refers to an AAV genome comprising a transgene (replacing the rep and cap genes) flanked by AAV inverted terminal repeats (ITRs). As used herein, the terms "AAV genome," "AAV transfer vector," and "transfer plasmid" are used interchangeably herein. They all refer to vectors comprising 5'- and 3'-viral (preferably AAV) inverted terminal repeats (ITRs) flanking a transgene.

[0062] The transgene can be a coding or non-coding sequence. It can be genomic DNA or cDNA. Preferably, the transgene encodes a polypeptide or a fragment thereof. Preferably, the transgene is operably associated with one or more transcriptional and / or translational control elements (e.g., enhancer, promoter, terminator sequence, etc.).

[0063] In some embodiments, the transgene encodes a therapeutic polypeptide or a fragment thereof. Examples of preferred therapeutic polypeptides include antibodies, CAR-T molecules, scFV, BiTEs, designed ankyrin repeats (DARPins), and T cell receptors.

[0064] 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 (such as RPE65 or REP) involved in human vision or retinal function. In some embodiments, the therapeutic polypeptide is a functional copy of a gene involved in human blood production, or a blood component, such as factor IX, or those involved in beta and alpha thalassemia or sickle cell anemia. In some embodiments, the therapeutic polypeptide is a functional copy of a gene involved in immune function, such as in severe combined immunodeficiency disease (SCID) or adenosine deaminase deficiency (ADA-SCID). In some embodiments, the therapeutic polypeptide is a protein that increases / reduces cell proliferation, such as a growth factor receptor. In some embodiments, the therapeutic polypeptide is an ion channel polypeptide.

[0065] In some preferred 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.

[0066] In some preferred embodiments, the transgene encodes a CRISPR enzyme (e.g., Cas9, dCas9, Cpf1, or a variant or derivative thereof) or a CRISPR sgRNA.

[0067] The wild-type AAV p5 promoter promotes the expression of Rep 78 and Rep 68 polypeptides. The p5 promoter is located 5' to the wild-type rep gene.

[0068] The wild-type AAV2 p5 promoter has the nucleotide sequence as given in SEQ ID NO: 4. The core sequence is highlighted in bold.

[0069] As used herein, the term "functional p5 promoter" refers to a nucleotide sequence consisting of the nucleotide sequence of SEQ ID NO: 4 or a variant thereof having at least 80%, 85%, 90%, 95% or 99% sequence identity thereto, or a nucleotide sequence comprising them, and capable of promoting the transcription of an operably associated nucleotide molecule encoding one or more AAVRep polypeptides (preferably Rep 78 and Rep68 polypeptides).

[0070] The level of activity of the p5 promoter can be determined by operably associating the test p5 promoter sequence with an appropriate transgene and measuring the expression level of the transgene.

[0071] When operably associated with the same transgene, expression levels less than 5% (preferably less than 1%) of the wild-type p5 promoter expression level can be considered non-functional.

[0072] In some embodiments, in the nucleic acid molecules of the present invention, (i) the Rep polypeptide coding sequence is not operably associated with a functional AAVp5 promoter. Preferably, the nucleotide sequence is located upstream (5') of the Rep polypeptide coding sequence, more preferably located immediately upstream of the Rep polypeptide coding sequence (i.e., continuously connected to the 5' end of the Rep polypeptide coding sequence). In this way, the expression of Rep78 and Rep68 polypeptides is reduced, thereby reducing the toxicity of these polypeptides to adenovirus.

[0073] In the absence of an operably associated promoter, Rep 78 and / or Rep 68 polypeptides can only be expressed at low, baseline or minimal levels from the nucleic acid molecules of the invention.

[0074] For example, the wild-type AAV p5 promoter sequence (e.g., SEQ ID NO:4) may be rendered non-functional due to the presence of a mutation in the core region (as highlighted above), or it may have a mutation in the TATA element of the promoter such that the TATA element cannot be bound by the TATA binding protein and / or other transcription factors required to initiate transcription.

[0075] Thus, in one embodiment, the Rep polypeptide coding sequence is operably associated with an AAV p5 promoter having one or more mutations in the core region and / or the TATA element. Preferably, these mutations reduce the promoter activity of the AAV p5 promoter compared to the promoter of SEQ ID NO: 4, most preferably rendering it non-functional (as defined above).

[0076] In some preferred embodiments, the Rep polypeptide coding sequence is operably associated with a nucleotide sequence consisting of or comprising a variant of the nucleotide sequence of SEQ ID NO:4, wherein the variant has at least 80%, 85%, 90%, 95% or 99% sequence identity to the nucleotide sequence of SEQ ID NO:4, and wherein, if the variant is operably associated with a transgene, the expression level of the transgene is less than 5% (preferably less than 1%) of the expression level from a promoter having SEQ ID NO:4 operably associated with the transgene.

[0077] In other embodiments, the Rep polypeptide coding sequence is not operably associated with a functional or non-functional AAV p5 promoter.

[0078] In this embodiment, the Rep polypeptide encoding sequence may be operably associated with a nucleotide sequence having less than 99%, 95%, 90% or 85% sequence identity to SEQ ID NO:4 and preferably having no or substantially no promoter activity.

[0079] In some embodiments, the Rep polypeptide coding sequence is not operably associated with an IRES element. In some embodiments, the p5 promoter is not replaced by an IRES.

[0080] In some embodiments of this aspect of the invention, the Rep polypeptide coding sequence is operably linked to SEQ ID NO: 5 (a sequence forming part of the 5'-untranslated region (UTR) of the human β-globin gene).

[0081] The wild-type AAV p19 promoter promotes expression of Rep 52 and Rep 40 polypeptides. The p19 promoter is located within the wild-type rep gene.

[0082] The wild-type AAV2 p19 promoter has the nucleotide sequence given in SEQ ID NO: 6. The highlighted portions are the TATA box and TSS elements.

[0083] As used herein, the term "functional p19 promoter" refers to a nucleotide sequence consisting of the nucleotide sequence of SEQ ID NO: 6 or a variant thereof having at least 80%, 85%, 90%, 95% or 99% sequence identity thereto, or a nucleotide sequence comprising them, and capable of promoting the transcription of an operably associated nucleotide molecule encoding one or more AAVRep polypeptides (preferably Rep 52 and Rep40 polypeptides).

[0084] The activity level of the p19 promoter can be determined by operably associating the test p19 promoter sequence with an appropriate transgene and measuring the expression level of the transgene. Expression levels less than 5% (preferably less than 1%) of the expression level of the wild-type p19 promoter (when operably associated with the same transgene) can be considered non-functional.

[0085] In some embodiments, in the nucleic acid molecules of the invention, (ii) the Rep polypeptide coding sequence does not comprise a functional AAV p19 promoter. In the absence of an operably associated promoter, the Rep 52 and / or Rep 40 polypeptides cannot be expressed from the nucleic acid molecules of the invention. In this way, the expression of the Rep52 and / or Rep40 polypeptides is prevented or inhibited. These polypeptides are not essential for the production of recombinant AAV particles, but they are capable of inhibiting the production of adenovirus. Therefore, in a system in which the adenovirus comprises the nucleic acid molecules of the invention, the prevention or inhibition of the expression of the Rep52 and / or Rep40 polypeptides enhances the production of the adenovirus.

[0086] For example, the wild-type AAV p19 promoter sequence (e.g., SEQ ID NO:6) may be rendered non-functional due to the presence of a mutation in the TSS element, or may have a mutation in the TATA element of the promoter such that the TATA element cannot be bound by TATA binding proteins and / or other transcription factors required for initiation of transcription.

[0087] Thus, in one embodiment, the Rep polypeptide coding sequence is operably associated with an AAV p19 promoter having one or more mutations in the TSS element and / or the TATA element. Preferably, these mutations reduce the promoter activity of the AAV p19 promoter compared to the promoter of SEQ ID NO: 6, most preferably rendering it non-functional (as defined above).

[0088] In some embodiments, the AAV p19 promoter has a deletion comprising some or all of the TATA box. More preferably, the Rep polypeptide coding sequence has a p19 promoter in which the TATA box has been eliminated by replacing one or more (e.g., 1, 2, 3, or 4) nucleotides of the TATA box with alternative nucleotides, such as converting the TATA sequence to TTTT. Preferably, the change is a synonymous mutation or the change retains the box of the coding sequence. In these embodiments, the p19 promoter may still be functional, or non-functional.

[0089] An example of a non-functional p19 sequence is given in SEQ ID NO: 7. The highlighted sequence is a mutated TATA box element which renders the promoter non-functional.

[0090] In some preferred embodiments, the Rep polypeptide coding sequence is operably associated with a nucleotide sequence consisting of or comprising a variant of the nucleotide sequence of SEQ ID NO:6, wherein the variant has at least 80%, 85%, 90%, 95% or 99% sequence identity to the nucleotide sequence of SEQ ID NO:6, and wherein, if the variant is operably associated with a transgene, the expression level of the transgene is less than 5% (preferably less than 1%) of the expression level from a promoter having SEQ ID NO:6 operably associated with the transgene.

[0091] In other embodiments, the Rep encoding polypeptide sequence encodes a functional AAV p19 promoter, but does not encode a functional Rep 52 and / or Rep 40 polypeptide. Preferably, the activity of the Rep78 / 68 polypeptide is not significantly affected.

[0092] For example, the start codon of the Rep52 / 40 polypeptides may be mutated to eliminate expression of these polypeptides. In one example, the nucleotide sequence encoding the start codon (methionine) may be mutated, preferably without significantly affecting expression of the Rep78 / 68 polypeptides.

[0093] Examples of non-functional Rep52 sequences include nucleotide sequences consisting of or comprising variants of the nucleotide sequence of SEQ ID NO: 16 or variants of the nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 18, wherein the variants have at least 80%, 85%, 90%, 95% or 99% sequence identity with the nucleotide sequence and do not encode a functional Rep52 polypeptide sequence.

[0094] Examples of non-functional Rep40 sequences include nucleotide sequences consisting of or comprising variants of the nucleotide sequence of SEQ ID NO: 17 or variants of the nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 19, wherein the variants have at least 80%, 85%, 90%, 95% or 99% sequence identity with the nucleotide sequence and do not encode a functional Rep40 polypeptide sequence.

[0095] The Rep 52 / 40 proteins bind ATP and have helicase activity and may be involved in assisting the accumulation of single-stranded genomic precursors and in assisting the packaging of newly formed DNA strands into preformed AAV capsids. Unlike Rep78 and Rep68, they are generally considered to be disposable for AAV production.

[0096] Expression of Rep 52 or Rep 40 polypeptide variants can be determined by Western blot to determine whether these polypeptides are produced at the correct molecular weight. The function of these polypeptides can be determined in purified form using a bioluminescent ATP assay that determines ATP consumption. Since both Rep52 and Rep40 have helicase activity, an appropriate helicase assay can also be used.

[0097] A test Rep52 polypeptide having a helicase activity level less than 5% (preferably less than 1%) of the helicase activity level of a wild-type Rep52 polypeptide (eg, SEQ ID NO: 18) can be considered a non-functional Rep52 polypeptide.

[0098] A test Rep40 polypeptide having a helicase activity level less than 5% (preferably less than 1%) of the helicase activity level of a wild-type Rep40 polypeptide (eg, SEQ ID NO: 19) can be considered a non-functional Rep40 polypeptide.

[0099] The wild-type AAV p40 promoter promotes the expression of the AAV Cap polypeptide. The p40 promoter is located near the 3' end of the wild-type AAV rep gene.

[0100] The wild-type AAV2 p40 promoter has the nucleotide sequence given in SEQ ID NO: 8. The highlighted element is the TATA element.

[0101] As used herein, the term "functional p40 promoter" refers to a nucleotide sequence consisting of the nucleotide sequence of SEQ ID NO: 8 or a variant thereof having at least 80%, 85%, 90%, 95% or 99% sequence identity thereto, or a nucleotide sequence comprising them, and capable of promoting the transcription of an operably associated nucleotide molecule encoding one or more AAVRep polypeptides (preferably one or more AAV Cap polypeptides).

[0102] The activity level of the p40 promoter can be determined by operably associating the test p40 promoter sequence with an appropriate transgene and measuring the expression level of the transgene. Expression levels less than 5% (preferably less than 1%) of the expression level of the wild-type p40 promoter (when operably associated with the same transgene) can be considered non-functional.

[0103] In some embodiments, in the nucleic acid molecules of the invention, (ii) the Rep polypeptide coding sequence does not comprise a functional AAV p40 promoter. In this way, the adenoviral inhibitor sequence is not transcribed.

[0104] For example, a wild-type AAV p40 promoter sequence (eg, SEQ ID NO: 8) may be rendered non-functional due to the presence of a mutation in the TATA element of the promoter, such that the TATA element cannot be bound by TATA binding proteins and / or other transcription factors required to initiate transcription.

[0105] Thus, in one embodiment, the Rep polypeptide coding sequence is operably associated with an AAV p40 promoter having one or more mutations in the TATA element. Preferably, these mutations reduce the promoter activity of the AAV p40 promoter compared to the promoter of SEQ ID NO: 8, most preferably rendering it non-functional (as defined above).

[0106] In some preferred embodiments, the Rep polypeptide coding sequence is operably associated with a nucleotide sequence consisting of or comprising a variant of the nucleotide sequence of SEQ ID NO:8, wherein the variant has at least 80%, 85%, 90%, 95% or 99% sequence identity to the nucleotide sequence of SEQ ID NO:8, and wherein, when the variant is operably associated with a transgene, the expression level of the transgene is less than 5% (preferably less than 1%) of the expression level from a promoter having SEQ ID NO:8 operably associated with the transgene.

[0107] In other embodiments, the Rep polypeptide coding sequence is not operably associated with a functional or non-functional AAV p40 promoter. In this embodiment, the Rep polypeptide coding sequence can be operably associated with a nucleotide sequence having less than 99%, 95%, 90% or 85% sequence identity to SEQ ID NO: 8 and preferably having no or substantially no promoter activity.

[0108] A preferred non-functional p40 promoter sequence is given in SEQ ID NO: 9. In this sequence, the TATA box element, the transcription start site and the transcription factor binding site are mutated, while the Rep78 and Rep68 polypeptide coding sequences remain unchanged.

[0109] The wild-type AAV p40 promoter sequence contains an adenoviral inhibitor sequence. As used herein, the term "functional adenoviral inhibitor sequence" refers to a nucleotide sequence that, when present in cis to the adenoviral genome, results in significant inhibition of adenoviral replication. The wild-type AAV2 adenoviral inhibitor has the sequence in SEQ ID NO: 10. This sequence forms the p40 promoter and adenoviral inhibitor sequence. The TATA element and the transcription start site form the core of the inhibitor sequence.

[0110] Preferably, a functional adenoviral inhibitor sequence is defined as a sequence having the sequence shown in SEQ ID NO: 10 or a variant thereof having at least 80%, 85%, 90% or 95% sequence identity thereto and capable of inhibiting adenoviral vector replication in a host cell.

[0111] The activity level of the adenoviral inhibitor sequence can be determined by including the adenoviral inhibitor sequence (cis or trans) into the sequence of the AV vector by molecular cloning and then attempting to recover the AV in mammalian cells. Inserting the wild-type adenoviral inhibitor sequence into the AV will completely prevent the recovery and growth of any AV vector. By modifying the sequence of the adenoviral inhibitor sequence, it is possible to successfully recover the AV vector to varying degrees. This can be calculated by measuring the infectious titer of the recovered AV to determine the level of inhibition. Assays that can be used to measure the AV titer include the TCID50 method and the plaque assay.

[0112] Activity levels less than 5% (preferably less than 1%) of the activity level from a wild-type adenoviral inhibitor sequence (under the same conditions) can be considered non-functional.

[0113] The wild-type AAV rep gene contains the p40 promoter sequence that contains the adenoviral suppressor sequence.

[0114] In some embodiments, in the nucleic acid molecules of the invention, (iii) the Rep polypeptide encoding sequence does not comprise a functional adenoviral inhibitor sequence.

[0115] For example, an adenoviral inhibitor sequence can be removed from the Rep polypeptide encoding sequence or can be rendered non-functional (eg, by creating one or more mutations in the adenoviral inhibitor sequence).

[0116] The adenoviral inhibitor sequence may be modified in such a way that transcription of the adenoviral inhibitor sequence in a host cell does not significantly inhibit replication of a wild-type adenovirus in the host cell.

[0117] Preferably, the Rep polypeptide coding sequence encodes functional Rep78 and Rep68 polypeptides, ie, the amino acid coding sequence of Rep78 and Rep68 polypeptides is not affected by removing the adenoviral inhibitor sequence or rendering the adenoviral inhibitor sequence nonfunctional.

[0118] Preferably, the AV inhibitor sequence within the p40 promoter is removed by synonymous codon exchange to retain the Rep encoded amino acid. This reduces the repression of the AV gene.

[0119] In some embodiments, it is not necessary to retain the correct coding sequence for the Rep52 and / or Rep40 polypeptides.

[0120] In one embodiment, the Rep polypeptide coding sequence comprises a functional p40 promoter sequence, ie, removal or rendering non-functional of the adenoviral inhibitor sequence does not affect, significantly reduce, or completely eliminate the function of the p40 promoter.

[0121] In another embodiment, the Rep polypeptide coding sequence does not comprise a functional p40 promoter sequence, ie, removal or rendering non-functional of the adenoviral inhibitor sequence completely eliminates p40 promoter function.

[0122] Preferably, the nucleotide sequence without a functional p40 promoter sequence has the sequence given in SEQ ID NO: 11. In SEQ ID NO: 11, the AV inhibitor sequence within the p40 promoter has been removed by synonymous codon exchange to retain the Rep encoded amino acid to reduce the repression of the AV gene.

[0123] In some embodiments, the nucleic acid molecules of the invention do not comprise a heterologous promoter operably associated with the AAVRep polypeptide encoding sequence.

[0124] In other embodiments, the nucleic acid molecules of the invention do not comprise a promoter adjacent to the AAVRep polypeptide encoding sequence.

[0125] As used herein, in the context of a promoter and a gene, the term "operably-associated" means that the promoter and the gene in question are located within a sufficiently close distance to each other so that the promoter can promote the expression of the gene. In some embodiments, the promoter and the gene are juxtaposed or adjacent.

[0126] Preferably, the Rep polypeptide coding sequence (rep gene) has the sequence given in SEQ ID NO: 12, or a variant thereof having at least 80%, 85%, 90 or 95% nucleotide sequence identity thereto, wherein the p19 promoter is non-functional and the p40 promoter is non-functional and the adenovirus inhibitor sequence is non-functional. In SEQ ID NO: 12, the p19 and p40 promoters are removed, and the Rep78 and Rep68 coding sequences are retained.

[0127] In other preferred embodiments, the Rep polypeptide coding sequence has the following characteristics:

[0128] (i) it is not operably associated with a functional AAV p5 promoter; and

[0129] (iii) it does not contain a functional AAV p40 promoter and it does not contain a functional adenoviral inhibitor sequence.

[0130] More preferably, the Rep polypeptide encoding sequence has a p19 promoter in which the TATA box has been removed, for example by replacing one or more (eg 1, 2, 3 or 4) nucleotides of the TATA box with alternative nucleotides, for example converting the TATA sequence to TTTT.

[0131] In some embodiments, the AAV p19 promoter has a deletion that includes some or all of the TATA box, or one or more TATA bases have been changed from TATA to an alternative nucleotide. Preferably, the change is a synonymous mutation or the change preserves the frame of the coding sequence.

[0132] The p19 promoter may be functional, ie, capable of expressing Rep40 and Rep52 polypeptides therefrom, or may be non-functional.

[0133] In yet another embodiment, the present invention provides an adenoviral vector comprising the nucleic acid molecule of the present invention. The nucleic acid molecule of the present invention is stably integrated into the adenoviral genome.

[0134] In order to accommodate the nucleic acid molecules of the present invention, part or all of one or more adenoviral genes may be deleted (preferably genes that are not adenoviral helper genes).

[0135] For example, the nucleic acid molecules of the invention can 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 can be trans-complemented by a cell line containing the deleted gene (e.g., HEK293 cells containing the adenovirus E1A and E1B regions).

[0136] The nucleic acid molecules of the present invention can be inserted into a region of the adenovirus genome that contains an E1 deletion. In other cases, genes that are not essential to the adenovirus can also be deleted and these sites can be used to insert the nucleic acid molecules of the present invention. For example, the nucleic acid molecules of the present invention can be inserted into the E3 region of the adenovirus, because most E3 genes can be deleted in adenovirus vectors.

[0137] The nucleic acid molecules of the present invention can be inserted into the adenoviral gene in a sense or antisense direction (relative to the transcription direction of the adenoviral gene). A preferred embodiment of the present invention is that the nucleic acid molecules of the present invention are inserted into the E1 region in the same transcription direction as the E4, E2A and E2B expression cassettes. This is to prevent the E1A promoter (usually retained in E1-deleted AV) from acting as a promoter to drive rep gene expression. The E1A promoter cannot be removed because it contains the AV packaging signal.

[0138] A preferred embodiment of the present invention is that the Rep coding sequence does not comprise an upstream promoter.

[0139] In some preferred embodiments, the nucleic acid molecule of the present invention is inserted into the adenovirus E1 gene, preferably wherein a portion of the E1 gene has been deleted. Preferably, the E1 gene is E1A and / or E1B.

[0140] In one embodiment, the invention provides an adenoviral vector comprising a nucleic acid molecule of the invention, wherein the Rep polypeptide (encoded by the nucleic acid molecule of the invention) is expressed at a low, baseline or minimum level. Preferably, the Rep polypeptide coding sequence is not operably associated with any functional promoter. As used herein, the term "low, baseline or minimum level" refers to an expression level of the Rep78 polypeptide from the nucleic acid molecule of the invention that is less than 50%, 40%, 30%, 20% or 10% of the expression level of the wild-type Rep 78 polypeptide operably associated with the wild-type p5 promoter (in the wild-type AAVrep gene). In this way, sufficient Rep polypeptide is provided to enable the production of at least some AAV, but the expression level of the Rep polypeptide is insufficient to completely inhibit adenoviral replication.

[0141] In some embodiments, the nucleic acid molecules of the invention are integrated into the adenoviral genome such that expression of the nucleic acid molecules of the invention is driven by an adenoviral promoter, preferably at a low or minimal level.

[0142] In other embodiments, the nucleic acid molecules of the invention are integrated into the adenoviral genome such that expression of the nucleic acid molecules of the invention is driven by an operably associated heterologous promoter, preferably at a low or minimal level.

[0143] The operably associated promoter can be a constitutive or inducible promoter. In some embodiments, the promoter is a constitutive promoter. In other embodiments, the promoter is inducible or repressible.

[0144] 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-β-globin promoters, or promoters derived therefrom.

[0145] Preferably, the rep gene promoter is the SV40 promoter, or a promoter derived therefrom, or a promoter having equal or reduced strength compared to the SV40 promoter in human cells and human cell lines (eg, HEK-293 cells).

[0146] In some embodiments, the promoter is inducible or inducible by including an inducible or repressible regulatory (promoter) element.

[0147] For example, the promoter may be one that is inducible by doxycycline, tetracycline, IPTG, or lactose.

[0148] In some embodiments of the invention, the nucleic acid molecules of the invention do not comprise and are not operably associated with any functional promoter.

[0149] In some embodiments, the adenoviral vector of the invention further comprises a nucleotide molecule encoding an AAV cap gene.

[0150] Preferably, the AAV cap gene is not juxtaposed with the AAV rep gene or the nucleic acid molecule of the invention. In particular, the AAV cap gene is preferably not operably associated with the rep gene p40 promoter (wild-type AAV p40 promoter or the p40 promoter sequence of the invention).

[0151] For example, the AAV cap gene is preferably not present within 1, 2, 3, 4 or 5 Kb of the AAV rep gene (or the rep gene promoter) or the nucleic acid molecule of the invention in the adenoviral genome.

[0152] Preferably, the AAV cap gene is operably associated with a heterologous promoter.

[0153] Preferably, the AAV cap gene is operably associated with a constitutive or inducible promoter.

[0154] In some embodiments, the AAV cap gene is not operably associated with a promoter or is operably associated with a minimal promoter, such as the minimal promoter region of the CMV, RSV, SV40 promoter, or any composite core promoter region that includes a TATA box and sufficient regulatory binding sites to initiate basal transcription downstream of the TATA box. When the promoter is not used, the TATA box will not be present. This maintains a low, baseline or minimum level of expression of the cap gene.

[0155] As used herein, the term "low, baseline or minimum level" refers to an expression level of a cap gene that is less than 50%, 40%, 30%, 20% or 10% of the expression level of a wild-type cap gene operably associated with a wild-type 40 promoter (e.g., in a wild-type AAV).

[0156] As used herein, the term "heterologous" refers to a genetic element that is not naturally associated with the gene in question.

[0157] In a preferred embodiment of the invention, the AAV cap gene is not juxtaposed to the AAV rep gene or the nucleic acid molecule of the invention.

[0158] Specifically, the AAV cap gene is integrated into the AV under the control of a promoter that is activated by a protein encoded in the AV that encodes the activator protein and the rep gene.

[0159] In yet another embodiment of the invention, the adenoviral vector comprising the nucleic acid molecule of the invention (the first adenoviral vector) further encodes a polypeptide capable of transcriptionally activating a (remote) promoter (e.g., a promoter present in the second adenoviral vector). Preferably, the promoter in the second adenoviral vector is a promoter that is operably associated with (i.e., drives expression of) the AAV cap gene.

[0160] In some embodiments, the adenoviral vector encodes a polypeptide capable of transcriptionally activating a promoter that is not present in the adenoviral vector.

[0161] Examples of such polypeptides include the VP16 transcriptional activator from herpes simplex virus and the transactivator domain from the p53 protein. These sequences can be linked to a DNA binding domain, such as those that bind to a cumate binding site or a tetracycline binding site.

[0162] This allows induction of cap gene transcription only when both the first and second adenoviral vectors are present in the same cell, thereby reducing the burden of expressing the AAV cap gene during adenoviral manufacturing.

[0163] The present invention also provides a host cell comprising the nucleic acid molecule of the present invention freely located in the host cell. The host cell can be an isolated cell, for example, they are not present in a living animal or mammal. Preferably, the host cell is a mammalian cell.

[0164] Examples of mammalian cells include cells from any organ or tissue of humans, mice, rats, hamsters, monkeys, rabbits, donkeys, horses, sheep, cattle and apes. Preferably, the cells are human cells. The cells may be primary cells or immortalized cells. Preferred cells include HEK-293, HEK 293T, HEK-293E, HEK-293FT, HEK-293S, HEK-293SG, HEK-293FTM, HEK-293SGGD, HEK-293A, MDCK, C127, A549, HeLa, CHO, mouse myeloma, PerC6, 911 and Vero cell lines. HEK-293 cells have been modified to include E1A and E1B proteins, which eliminates the need to provide these proteins on a helper plasmid. Similarly, PerC6 and 911 cells include similar modifications and may also be used.

[0165] Most preferably, the human cell is HEK293, HEK293T, HEK293A, PerC6, 911 or HeLaRC32. Other preferred cells include Hela, CHO and VERO cells.

[0166] The inventors have found that the presence of the Rep coding sequence and the AAVITR sequence within the same adenovirus is detrimental to the growth of the adenovirus. Although such adenovirus can be recovered, the yield is generally 5-10 times lower than when the AV contains each sequence independently. Therefore, advantages can be obtained by separating the Rep coding sequence and the AAVITR sequence, i.e., by placing them in two separate adenoviral vectors.

[0167] In yet another embodiment, the present invention provides a kit comprising:

[0168] (A) a first adenoviral vector comprising a nucleic acid molecule of the present invention, and

[0169] (B) a second adenoviral vector comprising

[0170] (i) a nucleic acid molecule encoding an AAV Cap polypeptide, and / or

[0171] (ii) A nucleic acid molecule encoding a recombinant AAV genome.

[0172] Preferably, the first adenoviral vector further encodes a polypeptide capable of transcriptionally activating a promoter present in the second adenoviral vector. Preferably, the promoter in the second adenoviral vector is a promoter operably associated with (ie, driving expression of) the AAV cap gene.

[0173] WO2019 / 020992 discloses that the transcription of late adenoviral genes can be regulated (e.g., inhibited) by inserting a repressor element into the major late promoter. By "turning off" the expression of adenoviral late genes, the protein manufacturing capacity of the cell can be diverted to the production of desired recombinant proteins or AAV particles. Preferably, the adenoviral vector of the present invention comprises a repressible major late promoter (MLP), more preferably, wherein the MLP comprises one or more repressor elements capable of regulating or controlling the transcription of adenoviral late genes, and wherein the one or more repressor elements are inserted downstream of the MLP TATA box.

[0174] Preferred features for producing viral (preferably AAV) particles include the following:

[0175] - wherein one or more repressor elements are inserted between the MLP TATA box and the +1 position of transcription.

[0176] - wherein the repressor element is an element capable of being bound by a repressor protein.

[0177] - wherein a gene encoding a repressor protein capable of binding to the repressor element is encoded within the adenoviral genome.

[0178] -wherein the repressor protein is transcribed under the control of MLP.

[0179] - wherein the repressor protein is tetracycline repressor, lactose repressor or ecdysone repressor, preferably tetracycline repressor (TetR).

[0180] - wherein the repressor element is a tetracycline repressor binding site comprising or consisting of the sequence shown in SEQ ID NO: 13.

[0181] - wherein the nucleotide sequence of MLP comprises or consists of the sequence shown in SEQ ID NO: 14 or 15.

[0182] - wherein the presence of the repressor element does not affect the production of adenoviral E2B protein.

[0183] - wherein the adenoviral vector encodes an adenoviral L4100K protein and wherein the L4100K protein is not under the control of MLP.

[0184] - wherein the transgene is inserted into one of the adenoviral early regions, preferably into the adenoviral E1 region and not into a transfer plasmid.

[0185] - wherein the transgene comprises a tripartite leader (TPL) in its 5'-UTR.

[0186] - wherein the transgene encodes a therapeutic polypeptide.

[0187] - wherein the transgene encodes a viral protein, preferably a protein that can be assembled intracellularly or extracellularly to produce virus-like particles, preferably wherein the transgene encodes Norovirus VP1 or Hepatitis B HBsAG.

[0188] In yet another embodiment, the present invention also provides a method for producing a modified host cell, the method comprising the steps of:

[0189] (a) introducing the nucleic acid molecule of the present invention into a host cell such that the nucleic acid molecule:

[0190] (i) stably integrated into the host cell genome, or

[0191] (ii) Existing freely in the host cell.

[0192] Preferably, the nucleic acid molecule of the present invention is present free in the host cell.

[0193] In some embodiments of the invention, the nucleic acid molecule of the invention in the cell does not comprise any functional promoter and is not operably associated with any functional promoter.

[0194] In some embodiments, the host cell is a cell that expresses or is capable of expressing a Cap polypeptide and / or an AAV genome.

[0195] For example, the host cell may be a cell in which one or more DNA molecules comprising nucleotide sequences encoding Cap polypeptides and / or AAV genomes are stably integrated. The nucleotide sequences encoding Cap polypeptides and / or AAV genomes are preferably operably associated with suitable regulatory elements (e.g., inducible or constitutive promoters).

[0196] For example, the host cell can be a host cell comprising one or more DNA plasmids or vectors, the DNA plasmids or vectors comprising nucleotide sequences encoding Cap polypeptides and / or AAV genomes. The nucleotide sequences encoding Cap polypeptides and / or AAV genomes are preferably operably associated with suitable regulatory elements (e.g., inducible or constitutive promoters).

[0197] The host cell may be an AAV packaging cell or an AAV producer cell.

[0198] 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.

[0199] In yet another embodiment, the present invention provides a method for producing a modified adenoviral vector, the method comprising the steps of:

[0200] (a) Introducing the nucleic acid molecule of the present invention into an adenoviral vector.

[0201] Preferably, the nucleic acid molecule is stably integrated into the adenoviral vector genome.

[0202] In some embodiments of the invention, the nucleic acid molecule of the invention in the adenoviral vector genome does not comprise any functional promoter and is not operably associated with any functional promoter.

[0203] In yet another embodiment, the invention provides a method for producing AAV particles, the method comprising the steps of:

[0204] (a) infecting a mammalian host cell with the first adenoviral vector of the present invention;

[0205] (b) infecting the host cell with a second adenoviral vector,

[0206] wherein the second adenoviral vector comprises a recombinant AAV genome comprising a transgene, wherein at least one of the first and second adenoviral vectors comprises an AAV cap gene;

[0207] (c) culturing the mammalian host cell in a culture medium under conditions to produce AAV particles comprising the transgene; and

[0208] (d) Isolating or purifying AAV particles from cells or cell culture medium.

[0209] At least one adenoviral vector also contains sufficient accessory genes (eg, E4, E1, E2a, and VA) for packaging the AAV genome.

[0210] In yet another embodiment, the invention provides a method for producing AAV particles, the method comprising the steps of:

[0211] (a) infecting a mammalian host cell with an adenoviral vector, the vector comprising

[0212] (i) a recombinant AAV genome containing a transgene, and

[0213] (ii) AAV cap gene,

[0214] wherein the mammalian host cell comprises the nucleic acid molecule of the present invention either episomally located within the host cell or stably integrated into the host cell genome;

[0215] (b) culturing the mammalian host cell in a culture medium under conditions to produce AAV particles comprising the transgene; and

[0216] (c) Isolating or purifying AAV particles from host cells or cell culture medium.

[0217] The adenoviral vector also contains sufficient accessory genes for packaging the AAV genome (eg, E4, E2a, and VA, including the E2A gene).

[0218] In yet another embodiment, the invention provides a method for producing AAV particles, the method comprising the steps of:

[0219] (a) infecting a mammalian host cell with the first adenoviral vector of the present invention,

[0220] wherein the mammalian host cell comprises a recombinant AAV genome stably integrated into the host cell genome, wherein the recombinant AAV genome comprises a transgene, and wherein:

[0221] (i) the adenoviral vector further comprises an AAV cap gene, or

[0222] (ii) the AAV cap gene is stably integrated into the mammalian host cell genome, or

[0223] (iii) infecting the cells with a second adenoviral vector comprising the AAV cap gene;

[0224] (b) culturing the mammalian host cell in a culture medium under conditions to produce AAV particles comprising the transgene; and

[0225] (c) Isolating or purifying AAV particles from cells or cell culture medium.

[0226] The at least one adenoviral vector present in the cell also comprises sufficient accessory genes for packaging the AAV genome (eg, E4, E1A, E1B and VA, and optionally the E2A gene).

[0227] In yet another embodiment, the present invention provides a method for producing a recombinant AAV particle, the method comprising the steps of:

[0228] (a) infecting a mammalian host cell with a first adenoviral vector of the present invention, wherein:

[0229] (i) the first adenoviral vector further comprises an AAV cap gene, or

[0230] (ii) infecting the cells with a second adenoviral vector comprising the AAV cap gene;

[0231] (b) infecting a mammalian host cell with a recombinant AAV comprising a transgene;

[0232] (c) culturing the mammalian host cell in a culture medium under conditions to produce AAV particles comprising the transgene; and

[0233] (d) isolating and / or purifying AAV particles from cells or cell culture medium.

[0234] The at least one adenoviral vector introduced (infected) into the cell also contains sufficient accessory genes for packaging the AAV genome (eg, E4, E1A, E1B and VA, and optionally the E2A gene).

[0235] The steps in steps (a) and (b) may be performed in any order. The initial recombinant AAV comprising the transgene may be prepared initially by any suitable means.

[0236] In the above embodiments, the initial recombinant AAV comprising the transgene and the adenoviral vector can then be reintroduced (e.g., reinfected) into another host cell to repeat the process. Where the major late promoter of the adenovirus is regulated, primarily AAV will be produced. Thus, in addition to the AAV (e.g., a portion) produced from the above embodiments, the process can be repeated by adding more adenoviral vectors to the host cells, thereby passage the AAV by adding more adenoviral vectors. Thus, steps (a) and / or (b) can be repeated as needed.

[0237] There are many established algorithms that can be used for comparing two amino acid or nucleotide sequences.Usually, a sequence serves as a reference sequence, and a test sequence can be compared thereto.The sequence comparison algorithm calculates the sequence identity percentage of the test sequence relative to the reference sequence based on the specified program parameters.The comparison for the amino acid or nucleotide sequence compared can be performed, for example, by an algorithm (such as GAP, BESTFIT, FASTA or TFASTA) or BLAST and BLAST 2.0 algorithms performed by a computer.

[0238] The percentage of amino acid sequence identity and nucleotide sequence identity can be obtained using the BLAST alignment 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 alignment parameters are used.

[0239] Standard protein-protein BLAST (blastp) can be used to find similar sequences in protein databases. Like other BLAST programs, blastp is designed to find local regions of similarity. When sequence similarity spans the entire sequence, blastp will also report a global alignment, which is the preferred result for protein identification purposes. It is preferred to use standard or default alignment parameters. In some cases, the "low complexity filter" can be disabled.

[0240] BLAST protein searches can also be performed using the BLASTX program, score = 50, word length = 3. To obtain gapped alignments for comparison purposes, Gapped BLAST (in BLAST 2.0) as described in Altschul et al. (1997) Nucleic Acids Res. 25:3389 can be used. Alternatively, PSI-BLAST (in BLAST 2.0) can be used to perform iterated searches to detect long-range relationships between molecules. (See Altschul et al. (1997) supra). When using BLAST, Gapped BLAST, PSI-BLAST, the default parameters of each program can be used.

[0241] About nucleotide sequence comparison, MEGABLAST, discontinuous-megablast and blastn can be used to achieve this goal.Preferably use standard or default comparison parameters.MEGABLAST is designed to be specially used for effectively finding long comparisons between very similar sequences.Discontinuous MEGABLAST can be used to find nucleotide sequences similar but not identical to nucleic acid of the present invention.

[0242] The BLAST nucleotide algorithm finds similar sequences by breaking down into short subsequences called words. The program first identifies exact matches (word hits) with the query word. The BLAST program then expands these word hits in multiple steps to generate a final gap comparison. In some embodiments, a BLAST nucleotide search can be performed with the BLASTN program, score = 100, word length = 12.

[0243] One of the important parameters that controls the sensitivity of a BLAST search is the word length. The most important reason that blastn is more sensitive than MEGABLAST is that it uses a shorter default word length (11). Because of this, blastn is superior to MEGABLAST in finding alignments with related nucleotide sequences from other organisms. The word length is adjustable in blastn and can be reduced from the default value to a minimum of 7 to increase search sensitivity.

[0244] By using the newly introduced discontinuous megablast page (www.ncbi.nlm.nih.gov / Web / Newsltr / FallWinter02 / blastlab.html), a more sensitive search can be achieved. The algorithm used in this page is similar to the algorithm reported by Ma et al. (Bioinformatics.2002Mar; 18(3):440-5). Discontinuous megablast does not require exact word matches as seeds for alignment extension, but uses discontinuous words within a longer window of the template. In encoding mode, the third base swing is considered by focusing on finding matches at the first and second codon positions and ignoring mismatches at the third position. Searching in discontinuous MEGABLAST using the same word length is more sensitive and effective than using the standard blastn of the same word length. The unique parameters of discontinuous megablast are: word length: 11 or 12; template: 16, 18 or 21; template type: encoding (0), non-coding (1) or both (2).

[0245] In some embodiments, the BLASTP 2.5.0+ algorithm (eg, available from NCBI) can be used using default parameters.

[0246] In other embodiments, the BLAST global alignment program (eg, available from NCBI) can be used using a Needleman-Wunsch alignment of two protein sequences with the following gap costs: presence 11 and extension 1.

[0247] The disclosure of each reference set forth herein is specifically incorporated by reference in its entirety. BRIEF DESCRIPTION OF THE DRAWINGS

[0248] Figure 1 : This shows the structure of the wild-type AAV genome, illustrating the various AAV promoters within the rep gene.

[0249] Figure 2: AAVRep protein can inhibit basal transcription of the adenovirus major late promoter.

[0250] FIG. 3 : AAVRep DNA stably integrates and replicates along with the adenoviral genome.

[0251] Figure 4: Co-infection with TERA vector significantly increased AAV production in HEK293 cells compared to the helper-free transfection method.

[0252] Figure 5: Coinfection with TERA vector encoding the AAV transfer genome and AAV2 cap and TERA encoding AAV Rep78-68 significantly increased AAV production in HEK293 cells compared to the helper-free plasmid transfection method.

[0253] Figure 6 : AAV was produced by co-infection of HEK293 cells with TERA-AAV and TERA-RepCap or without helper plasmid transfection. Results are shown as the ratio of total AAV infectious particles and transducing particles relative to the total genome containing AAV particles. Quantification was performed by a modified TCID50 method, in which serial dilutions of AAV were added to HEK293 cells and GFP-positive cells were counted at the lowest dilution. AAV titers were then reverse calculated according to the TCID50 method.

[0254] Figure 7 : Demonstrates that the present invention does not interfere with the suppressible adenoviral system as disclosed in WO2019 / 020992. Total infectious adenoviral particles are shown, indicating that this approach can still be used to prevent adenoviral contamination of AAV preparations when AAVRep and Cap genes are integrated into adenoviral vectors. Quantification was performed by QPCR targeting adenoviral hexon sequences.

[0255] Figure 8 : Demonstration that integration of AAVRep, Cap, and genome into an adenoviral vector allows plasmid-free AAV production and significantly improves yield compared to plasmid-based production methods. Quantification was performed using QPCR targeting the EGFP expression cassette in the AAV vector.

[0256] Fig. 9 : Serial passaging of adenoviral vectors encoding the AAV rep and AAV Cap genes. QPCR quantification of these sequences relative to the adenoviral hexon sequence indicates that these genes have been stably inserted and can be propagated over long periods of time.

[0257] Fig.10 : Western blot showing the expression of AAVRep from TERA-RepCap showing the expression of all major isoforms of AAVRep.

[0258] Fig.11 : Western blot of cells infected with adenoviral vectors encoding AAVRep (TERA-RepCap, TERA2.0, or TERA2.0+Dox) or AAV2 reference material.

[0259] Fig.12: Demonstrated that AAV yields were higher when cells were infected with two adenoviral vectors (TERA2.0), one encoding Rep and Cap from AAV serotype 5 and one encoding the AAV genome, compared to the unhelper approach.

[0260] Fig.13 : Demonstrated that AAV yields were higher when cells were infected with two adenoviral vectors (TERA2.0), one encoding Rep and Cap from AAV serotype 6 and one encoding the AAV genome, compared to the unhelper approach.

[0261] Fig.14 : Demonstrated that AAV yields were higher when cells were infected with two adenoviral vectors (TERA2.0), one encoding Rep and Cap from AAV serotype 9 and one encoding the AAV genome, compared to the unhelper approach.

[0262] Figure 15A shows that integration of the Rep coding sequence into an adenoviral vector significantly increased AAV production compared to a plasmid encoding Rep or a triple plasmid / no helper approach. Figure 15B shows that AAV productivity integrated into an adenoviral vector was significantly superior even when the Rep promoter was driven by the strong CMV promoter. Example

[0263] The present invention is further illustrated by the following examples, wherein parts and percentages are by weight, and degrees are in degrees Celsius, unless otherwise stated. It should be understood that these examples, although showing preferred embodiments of the present invention, are given only by way of illustration. Through the above discussion and these examples, those skilled in the art can determine the essential characteristics of the present invention, and without departing from its spirit and scope, the present invention can be subjected to various changes and modifications to adapt it to various uses and conditions. Therefore, according to the foregoing description, in addition to those shown and described herein, various modifications of the present invention will be apparent to those skilled in the art. Such modifications are also intended to fall within the scope of the appended claims.

[0264] In the following examples, reference to "TERA vector" refers to an adenoviral vector in which the transcription of late adenoviral genes from the major late promoter is regulated, as described in WO2019 / 020992. WO2019 / 020992 discloses that the transcription of late adenoviral genes can be regulated (e.g., inhibited) by inserting a repressor element into the major late promoter. By "turning off" the expression of adenoviral late genes, the protein manufacturing capacity of the cell can be diverted to the production of desired recombinant proteins or AAV particles.

[0265] WO2019 / 020992 discloses that an adenovirus vector comprising a repressor element in the major late promoter may also encode a TetR protein downstream and under the transcriptional control of the major late promoter. In the absence of doxycycline, the TetR protein will bind to the major late promoter repressor element and prevent the activity of the promoter. In the presence of doxycycline, the TetR protein cannot bind to the repressor element in the major late promoter. Therefore, in the presence of doxycycline, the major late promoter of the adenovirus is active and the structural genes of the adenovirus are expressed and the virus can replicate.

[0266] The vector "TERA-AAV" encodes the AAV transfer genome.

[0267] The vector "TERA-RepCap" encodes AAV Rep and Cap. In this construct, there is no functional Rep p5 or p40 promoter; the Rep adenovirus inhibitor sequence is not present; and the Rep p19 promoter has been modified to delete the TATA box, although the promoter is still functional. The rep gene is inserted in the E1 region of the adenovirus. Cap is expressed from the CMV promoter. The cap gene is inserted in the E1 region of the adenovirus. The transcription direction of the CMV promoter is not toward the Rep coding sequence.

[0268] The TERA2.0 method refers to the process of adding TERA-AAV and TERA-RepCap to cells.

[0269] The vectors TERA-RepCap5, TERA-RepCap6, and TERA-RepCap9 encode the AAV Rep and Cap genes from AAV5, AAV6, or AAV9, respectively. In TERA-RepCap5, the Cap gene is driven by the minimal CMV promoter region. In TERA-RepCap6 and TERA-RepCap9, the Cap gene is driven by the full-length CMV promoter. In these constructs, there is no functional Rep p5 or p40 promoter; there is no Rep adenovirus inhibitor sequence; and the Rep p19 promoter has been modified to eliminate the TATA box, although the promoter is still functional. The rep gene is inserted in the E1 region of the adenovirus. The Cap gene expression cassette is inserted in the E1 region of the adenovirus. The transcription direction of the minimal CMV promoter or the full-length CMV promoter is not toward the Rep coding sequence.

[0270] The triple plasmid transfection or helper-free approach refers to the transfection of an adenoviral helper plasmid (as opposed to the use of adenovirus), a plasmid encoding the AAV Rep and Cap found in nature, and a plasmid containing the AAV genome with a CMV-driven EGFP expression cassette inserted between the AAV ITRs.

[0271] Plasmids pRepCap2, pRepCap5, pRepCap6, and pRepCap9 encode the AAV Rep and Cap genes from AAV2, AAV5, AAV6, or AAV9, respectively. In these constructs, the AAV Rep and Cap genes are configured as they are found in nature.

[0272] Plasmid pAAV-EGFP encodes the left and right ITRs from the AAV genome. A CMV promoter driving the expression of the EGFP reporter gene is inserted between the ITRs, followed by a polyadenylation signal.

[0273] Plasmid pHelper contains the adenoviral sequences required for AAV helper. This includes the E4 region, the E2A region, and the VA gene. This plasmid does not encode the adenoviral E1 region as it is present in the HEK293 cells used in the studies described herein.

[0274] Example 1: AAVRep protein inhibits transcription of adenovirus major late promoter

[0275] To determine the effect of AAVRep protein on transcription from the adenoviral major late promoter (MLP), wild-type MLP or TetR-repressible MLP sequences were inserted into a plasmid expressing the EGFP reporter protein by molecular cloning methods. The MLP promoter plasmid was co-transfected into HEK293 cells with a control CMV plasmid or a plasmid construct expressing AAVRep78, Rep68, Rep52, or Rep40 under the control of the CMV promoter. EGFP reporter gene expression was quantified by flow cytometry 48 hours after transfection.

[0276] The results are as follows Figure 2A and 2B These results indicate that high expression of the Rep protein inhibits the activity of the adenovirus major late promoter or a modified form of the major late promoter containing a TetR binding site.

[0277] Example 2: AAVRep DNA is stably integrated and replicated with the adenoviral genome

[0278] To construct an adenoviral vector encoding AAVRep78 and Rep68 polypeptides, the p5 and p19 promoters (involved in the transcription of AAVRep52 and AAVRep40) and the p40 adenoviral inhibitor sequence were scrambled (while retaining AAVRep78 and Rep68 polypeptides) and inserted into the E1 deletion region of the adenoviral vector by molecular cloning methods.

[0279] This resulted in Figure 3B The vector shown in , wherein the adenoviral vector encodes the Rep coding sequence and the AAV genome expressing EGFP.

[0280] The adenoviral vector genome was transfected into HEK293 cells and the viral vector was harvested approximately 15 days after transfection after complete cytopathic effect was observed. The vector was subsequently passaged in HEK293 cells (>5) and the adenoviral particles were treated with DNase to degrade any encapsulated DNA. The presence of the AAV Rep gene and adenoviral hexon gene sequences was determined by QPCR. The results are shown in Figure 3A As shown, and the vector is Figure 3B shown.

[0281] Figure 3A The results showed that the frequencies of AAVRep coding DNA and adenovirus hexon DNA were equal, proving that Rep DNA inserted into the adenovirus genome was stable.

[0282] Example 3: Co-infection with TERA vector significantly increased AAV production in HEK293 cells compared to the non-helper plasmid transfection method.

[0283] HEK293 cells were seeded at 9e4 cells / well in 48-well tissue culture plates for 24 hours. Cells were triple transfected with no helper plasmids or co-transfected with the following in the presence of 0.5ug / ml doxycycline or DMSO: A) TERA encoding an AAV transfer genome with an EGFP expression cassette and the AAV2 Cap2 gene driven by a CMV promoter (TERA-AAV-Cap) at an MOI of 100, and B) TERA encoding an AAV transfer genome with an EGFP expression cassette and AAV Rep78-68 (TERA-AAV-Rep) at an MOI of 50. AAV vectors were harvested 96 hours after transduction, and encapsulated AAV particles and contaminating adenoviral particles were quantified by QPCR.

[0284] The results are as follows Figure 4A As shown, and the vector is Figure 4B These results indicate that AAV can be produced at high titers using a combination of TERA-AAV-Cap and TERA-AAV-Rep. Figure 4A The titers achieved were shown to be significantly higher than those achieved using triple transfection or the unassisted approach. Figure 4B A schematic diagram of the adenoviral genome comprising the AAV components of TERA-AAV-Rep and TERA-AAV-Cap is shown.

[0285] Example 4: Co-infection with TERA vector encoding the AAV transfer genome and AAV2 cap and TERA encoding AAV Rep78-68 significantly increased AAV production in HEK293 cells compared to the helper-free transfection method.

[0286] HEK293 cells were seeded at 9e4 cells / well in 48-well tissue culture plates for 24 hours. Cells were triple transfected with no helper plasmids or co-transfected with the following (at the indicated MOIs) in the presence of 0.5ug / ml doxycycline or DMSO: A) TERA-AAV-Cap, B) TERA-AAV-Rep, or C) TERA encoding AAV Rep78-68 (TERA-Rep78-68). AAV vectors were harvested 96 hours after transduction, and encapsulated AAV particles and contaminating adenovirus particles were quantified by QPCR.

[0287] The results are as follows Figure 5 These results suggest that two viruses containing all AAV components provide improved AAV titers that are much higher than those achieved with the no-helper / triple plasmid transfection approach. Figure 5 As shown, the flexibility and versatility of this method are demonstrated. In each production run, at least one viral vector comprises a Rep construct of the present invention.

[0288] Example 5: Production of AAV2 vectors in HEK293 cells using the TERA2.0 system, which consists of two TERA vectors, one encoding the AAV transfer genome and the second encoding AAV rep and cap.

[0289] Until the development of the invention described herein, it was not possible to encode both AAV Rep and Cap into a single adenoviral vector. In order to simplify the AAV manufacturing process, a new adenoviral vector (TERA-RepCap) encoding both AAV Rep and AAV capsid from serotype 2 was constructed. In this example, AAV production using this new adenoviral vector (TERA-RepCap) in combination with TERA-AAV was tested, and this method was called TERA-2.0. This method was compared with the triple plasmid transfection / no helper method.

[0290] HEK293 cells were infected with TERA-AAV and TERA-RepCap to embody the TERA2.0 method, each at an MOI of 25.

[0291] In addition, HEK293 cells were triple transfected with helper-free plasmids (pAAV-EGFP; pRepCap2; pHelper). Cells were cultured for 4 days before vector harvesting. Samples were treated with DNase for 2 hours at 37°C and quantified by qPCR using primers and probes for the EGFP transgene. AAV vectors generated from these two methods were used to infect fresh HEK293 cells and transduction units were quantified by TCID50 assay.

[0292] The results are as follows Figure 6 The ratio of genome copies (GC) to transducing units (TU) shown on the right axis is determined by dividing the intact AAV particles determined by qPCR by the transducing units measured from the TCID50 assay. The results of this example show that infection of cells with TERA2.0 (coinfection with TERA-AAV and TERA-RepCap) can increase the production of transducing active AAV vector by 1000-fold compared to preparations generated by triple plasmid transfection.

[0293] Example 6: Determination of contaminating adenovirus levels in the AAV2 production process using the TERA2.0 method.

[0294] AAV production was performed in HEK293 cells infected with TERA2.0 method (co-infection with TERA-AAV and TERA-RepCap, each with an MOI of 25) and cultured for 4 days with 0.5ug / ml doxycycline or DMSO (-DOX). In the absence of doxycycline (DMSO group), adenoviral late genes were inhibited and the viral replication cycle was truncated. In addition, the presence of doxycycline in the growth medium enabled the expression of late adenoviral genes from TERA vectors and the production of adenovirus. The vectors were harvested by three rounds of freeze-thaw and contaminated adenovirus was detected by TCID50 assay by infecting fresh HEK293 cells and culturing with 0.5ug / ml doxycycline.

[0295] This example shows that the production of AAV vectors using two TERA vectors (one encoding the AAV transfer genome and the other encoding AAVRep and Cap) in the absence of doxycycline significantly inhibited adenoviral replication and produced clean AAV preparations. A reduction of more than 9 logs of contaminating adenovirus was observed compared to the production of AAV in the presence of doxycycline.

[0296] Example 7: Production of AAV2 vectors in HEK293 cells using the TERA2.0 system compared to a triple plasmid transfection method in a 1 L stirred tank bioreactor.

[0297] HEK293 cell suspension was infected using the TERA2.0 method (coinfection with TERA-AAV and TERA-RepCap) at an MOI of 25 TCID50 units / cell for each.

[0298] In addition, HEK293 cells were triple transfected with helper-free plasmids (pAAV-EGFP; pRepCap2; pHelper). Cells were cultured for 4 days before vector harvesting. Samples were treated with DNase for 2 hours at 37°C and quantified by qPCR using primers and probes for the EGFP transgene to determine the titer of complete particles (viral genome (VG)).

[0299] This example demonstrates that AAV2 vectors can be produced in suspension-adapted HEK293 cells using co-infection of TERA vectors encoding the AAV transfer genome as well as AAV Rep and Cap. The AAV2 vector titers obtained by this method increased intact particles by more than 100-fold.

[0300] Example 8: Evaluation of genetic stability of TERA vectors encoding AAV rep and cap by qPCR.

[0301] Plasmid DNA encoding TERA-RepCap was transfected into HEK293 cells for vector recovery and further amplified by serial passages. HEK293 cells were infected in HYPERFlask cell culture vessels (MOI-15). The vector was harvested 3 days later and purified by cesium chloride ultracentrifugation to produce serial passage 4. This process was repeated to produce vector passages 5, 6, and 7. DNA was extracted from the purified TERA-RepCap vector of each passage, and AAV2 rep and cap and Ad5 hexon genes were quantified by qPCR. Fig. 9 The data in show the copy number of AAV2 rep and cap genes relative to Ad5 hexon and compared to a DNA plasmid encoding TERA-RepCap (no significant difference, two-way ANOVA).

[0302] This example demonstrates that the AAVRep and Cap genes are stably propagated within the TERA vector during serial passaging in HEK293 cells.

[0303] Example 9: Evaluation of AAV Rep expression from TERA vectors encoding AAV rep and cap Western blot detection of Rep protein from AAV production process using TERA2.0 method.

[0304] HEK293 cells were co-infected with TERA vectors encoding the AAV transfer genome (TERA-AAV) and AAV Rep and Cap (TERA-RepCap) (MOI-25 each). Samples were harvested 24 and 48 hours after infection, and total cell extracts (25uL) were probed with AAV2 Rep antibodies by Western blotting. Data are presented as duplicate biological replicates.

[0305] The results are as follows Fig.10 This example shows that the TERA vector encoding AAV Rep and Cap expressed high levels of AAV Rep 52 and 40, and also expressed low levels of Rep78 and Rep68.

[0306] Example 10: Evaluation of AAV2 capsid proteins from particles produced using TERA vectors encoding AAV rep and cap.

[0307] HEK293 cells were co-infected with TERA-AAV and TERE-RepCap (in the presence or absence of 0.5 μg / mL doxycycline) (each MOI-25), or co-transfected with 50 GC / cell of AAV2 particles (generated by TERA2.0) and TERA-RepCap (MOI-25). Samples were harvested 96 hours after infection and cell lysates (25uL) were probed with anti-AAV2 VP1 / 2 / 3 antibodies.

[0308] The results are as follows Fig.11 Standard AAV2 (ATCC VR-1616) is also shown. This result shows that the relative composition of AAV2 capsid subunits VP1, VP2 and VP3 is the same as that of the AAV2 vector from the reference standard and the material produced from the triple plasmid transfection method.

[0309] Example 11: Production of AAV5 vectors in HEK293 cells using the TERA2.0 system consisting of two TERA vectors, one encoding the AAV transfer genome and the second encoding AAV rep and cap 5. AAV production was compared with a triple plasmid transfection method.

[0310] HEK293 cells were infected using the TERA2.0 method described previously, but in which the AAV2 capsid was replaced with a capsid from AAV serotype 5 (TERA-AAV and TERA-RepCap5, in which Rep is not expressed from a heterologous promoter, and Cap5 is expressed from a minimal CMV promoter). TERA-AAV was used at an MOI of 25, and TERA-RepCap5 was used at 75 genome copies (GC) / cell. Alternatively, HEK293 cells were triple transfected with no helper plasmid (pAAV-EGFP; pRepCap5; pHelper). Cells were cultured for 4 days before vector harvesting. Samples were treated with DNase for 2 hours at 37°C and quantified by qPCR using primers and probes for the EGFP transgene.

[0311] The results are as follows Fig.12The results of this example demonstrate that co-infection of two TERA vectors encoding the AAV transfer genome as well as the AAV rep and cap5 genes can increase intact AAV5 particles by more than 16-fold compared to the triple plasmid transfection / no helper approach.

[0312] Example 12: Production of AAV6 vectors in HEK293 cells using the TERA2.0 system consisting of two TERA vectors, one encoding the AAV transfer genome and the second encoding AAVRep and Cap 6. AAV production was compared with a triple plasmid transfection method.

[0313] HEK293 cells were infected using the TERA2.0 method described previously, but the AAV2 capsid was replaced with a capsid from AAV serotype 6 (TERA-AAV and TERA-RepCap6). TERA-AAV was used at an MOI of 25, and TERA-RepCap6 was used at 75 genome copies (GC) / cell. In addition, HEK293 cells were triple transfected with no helper plasmid (pAAV-EGFP; pRepCap6; pHelper). Cells were cultured for 4 days before vector harvesting. Samples were treated with DNase for 2 hours at 37°C and quantified by qPCR using primers and probes for the EGFP transgene.

[0314] The results are as follows Fig.13 The results of this example demonstrate that co-infection of two TERA vectors encoding the AAV transfer genome as well as the AAV Rep and Cap6 genes can increase intact AAV6 particles by more than 18-fold compared to the triple plasmid transfection / no helper approach.

[0315] Example 13: Production of AAV9 vectors in HEK293 cells using the TERA2.0 system consisting of two TERA vectors, one encoding the AAV transfer genome and the second encoding AAVRep and Cap 9. AAV production was compared with a triple plasmid transfection method.

[0316] HEK293 cells were infected using the TERA2.0 method described previously, but the AAV2 capsid was replaced with a capsid from AAV serotype 9 (TERA-AAV and TERA-RepCap9). TERA-AAV was used at an MOI of 25, and TERA-RepCap9 was used at 50 genome copies (GC) / cell. In addition, HEK293 cells were triple transfected with no helper plasmid (pAAV-EGFP; pRepCap9; pHelper). Cells were cultured for 4 days before vector harvesting. Samples were treated with DNase for 2 hours at 37°C and quantified by qPCR using primers and probes for the EGFP transgene.

[0317] The results are as follows Fig.14 The results of this example demonstrate that co-infection of two TERA vectors encoding the AAV transfer genome as well as the AAV rep and Cap9 genes can increase intact AAV9 particles by more than 8.5-fold compared to the triple plasmid transfection / no helper approach.

[0318] Example 14: AAV2 vectors were generated in HEK293 cells using two TERA vectors, I) encoding AAVRep (TERA-Rep) and II) encoding AAV-EGFP and AAV Cap2 expression cassettes driven by the CMV promoter (ERA-AAV-EGFP-Cap2), compared to a helper plasmid-free approach or replacing TERA-Rep with a plasmid encoding the Rep coding sequence driven by the native AAV p5 promoter.

[0319] To generate AAV2-EGFP vectors, HEK293 cells were co-infected with TERA-Rep (MOI 5, 10 or 50) and TERA-AAV-EGFP-Cap2 (MOI 100). This was compared to HEK293 cells transfected with plasmid p5-Rep (in which the Rep78 / 68 polypeptide is expressed by its native p5 promoter) and infected with TERA-AAV-EGFP-Cap2 (MOI 100). The cells were cultured for 4 days before vector harvesting. Samples were treated with DNase for 2 hours at 37°C and quantified by qPCR using primers and probes for the EGFP transgene. The productivity of the complete AAV2-EGFP was compared with a non-assisted transfection method (HF), in which HEK293 cells were transfected with an AAV transfer genome plasmid (pAAV-EGFP), plasmid pRepCap2 and plasmid pHelper. Control samples were transfected with filler DNA (pUC19) instead of pRepCap2 to control for the efficiency of DNase treatment.

[0320] The results of Figure 15A show that co-transfection of two TERA vectors, TERA-Rep and TERA-AAV-EGFP-Cap2, produced significantly greater amounts of intact AAV2 vectors compared to transfections in which Rep was expressed from the native p5 AAV promoter and transfections without a helper plasmid to achieve the Rep expression plasmid.

[0321] The results in Figure 15B show the same experiment as in Figure 15A, but in which the p5 promoter driving Rep expression was replaced with the strong CMV promoter; this did not increase AAV productivity relative to the use of TERA-Rep.

[0322] References

[0323] Fisher,KJet al(1996)A novel adenovirus-adeno-associated virushybrid vector that displays efficient rescue and delivery of theAAV genome.Humgene ther.7:2079-2087.

[0324] Liu,

[0325] Sitaraman,V.et al(2011)Computationally designed adeno-associatedvirus(AAV)rep 78is efficiently maintainedwithin an adenovirusvector.Proc.Natl.Acad.Sci.108:14294-14299.

[0326] Timpe,JMet al(2006)Effect of adeno-associated virus on adenovirusreplication and gene expression during coinfection.J.Virol.16:7807-7815.

[0327] Weger, S. et al (2016) A regulatory element near the 3’end of adeno-associated virus rep gene inhibits adenovirus replication in cis by means of p40 promoter-associated short transcripts. J. Virol. 90:3981-3993.

[0328] Zhang, H-G. et al (2001) Recombinant adenovirus expressing adeno-associated virus cap and rep proteins supports production of high-titre recombinant adeno-associated virus. Gene ther. 8:704-712.

[0329] Zhang, X. and Li, C-Y. (2001) Generation of recombinant adeno-associated virus vectors by a complete adenovirus-mediated approach. Mol. Ther. 3:787-693.

[0330] Sequence

[0331] SEQ ID NO:1

[0332] Rep nucleotide sequence (AAV serotype 2)

[0333]

[0334] SEQ ID NO:2

[0335] Cap nucleotide sequence (AAV serotype 2)

[0336]

[0337] SEQ ID NO:3

[0338] Cap amino acid sequence (AAV serotype 2)

[0339] MAADGYLPDWLEDTLSEGIRQWWKLKPGPPPPKPAERHKDDSRGLVLPGYKYLGPFNGLDKGEPVNEADAAALEHDKAYDRQLDSGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVFQAKKRVLEPLGLVEEPVKTAPGKKRPVEHSPVEPDSSSGTGKAGQQPARKRLNFGQTGDADSVPDPQPLGQPPAAPSGLGTNTMATGSGAPMADNNEGADGVGNSSGNWHCDSTWMGDRVITTSTRTWALPTYNNHLYKQISSQSGASNDNHYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTQNDGTTTIANNLTSTVQVFTDSEYQLPYVLGSAHQGCLPPFPADVFMVPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFTFSYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTNTPSGTTTQSRLQFSQAGASDIRDQSRNWLPGPCYRQQRVSKTSADNNNSEYSWTGATKYHLNGRDSLVNPGPAMASHKDDEEKFFPQSGVLIFGKQGSEKTNVDIEKVMITDEEEIRTTNPVATEQYGSVSTNLQRGNRQAATADVNTQGVLPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKHPPPQILIKNTPVPANPSTTFSAAKFASFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYNKSVNVDFTVDTNGVYSEPRPIGTRYLTRNL*

[0340] SEQ ID NO:4

[0341] gtcctgtattagaggtcacgtgagtgttttgcgacattttgcgacaccatgtggtcacgctgggtatttaagcccgagtgagcacgcagggtctccattttgaagcgggaggtttgaacgcgcagccgcc

[0342] SEQ ID NO:5

[0343] agatctttgtcgatcctaccatccactcgacacacccgccagcggccgctgccaagcttccgagctctcgaattc

[0344] SEQ ID NO:6

[0345] gtcacaaagaccagaaatggcgccggaggcgggaacaaggtggtggatgagtgctacatccccaattacttgctccccaaaacccagcctgagctccagtgggcgtggactaatatggaacagtatttaagcgcctgtttgaatctcacggagcgtaaacggttggtggcgcagcatctgacgcacgtgtcgcagacgcaggagcagaacaaagagaatcagaatcccaattctgatgcgccggtgatcagatcaaaaacttcagccaggtac

[0346] SEQ ID NO:7

[0347] gtcacaaagaccagaaatggcgccggaggcgggaacaaggtggtggatgagtgctacatccccaattacttgctccccaaaacccagcctgagctccagtgggcgtggactaatatggaacagtacctcagcgcctgtttgaatctcacggagcgtaaacggttggtggcgcagcatctgacgcacgtgtcgcagacgcaggagcagaacaaagagaatcagaatcccaattctgatgcgccggtgatcagatcaaaaacttcagccaggtac

[0348] SEQ ID NO:8

[0349] Ggtcaccaagcaggaagtcaaagactttttccggtgggcaaaggatcacgtggttgaggtggagcatgaattctacgtcaaaaagggtggagccaagaaaagacccgcccccagtgacgcagatataagtgagcccaaacgggtgcgcgagtcagttgcgcagccatcgacgtcagacgcggaagcttcgatcaactacgcagacaggt

[0350] SEQ ID NO:9

[0351] gtgacaaaacaagaggtgaaggacttctttcgttgggccaaagaccatgtggtcgaggtcgaacacgagttctatgtgaagaaaggaggcgcgaagaagcgcccagcgccatcggacgctgacatctccgaaccgaagcgcgtgagagagagcgtggcacaaccatcaacctcggatgccgaggcatccatcaattatgcggacaggt

[0352] SEQ ID NO:10

[0353] Gtcaccaagcaggaagtcaaagactttttccggtgggcaaaggatcacgtggttgaggtggagcatgaattctacgtcaaaaagggtggagccaagaaaagacccgcccccagtgacgcagatataagtgagcccaaacgggtgcgcgagtcagttgcgcagccatcgacgtcagacgcggaagcttcgatcaactacgcagacaggt

[0354] SEQ ID NO:11gtgacaaaacaagaggtgaaggacttctttcgttgggccaaagaccatgtggtcgaggtcgaacacgagttctatgtgaagaaaggaggcgcgaagaagcgcccagcgccatcggacgctgacatctccgaaccgaagcgcgtgagagagagcgtggcacaaccatcaacctcggatgccgaggcatccatcaattatgcggacaggt

[0355] SEQ ID NO:12

[0356]

[0357] SEQ ID NO:13

[0358] TetR binding site

[0359] tccctatcag tgatagaga

[0360] SEQ ID NO:14

[0361] Modified MLP

[0362] cgccctcttc ggcatcaagg aaggtgattg gtttgtaggt gtaggccacg tgaccgggtg

[0363] ttcctgaagg ggggctataa aaggtcccta tcagtgatag agactca

[0364] SEQ ID NO:15

[0365] Modified MLP

[0366] cgccctcttc ggcatcaagg aaggtgattg gtttgtaggt gtaggccacg tgactcccta

[0367] tcagtgatag agaactataa aaggtcccta tcagtgatag agactca

[0368] SEQ ID NO:16

[0369] Rep52 nucleotide sequence

[0370]

[0371] SEQ ID NO:17

[0372] Rep40 nucleotide sequence

[0373] ATGGAGCTGGTCGGGTGGctcgtggacaaggggattacctcggagaagcagtggatccaggaggaccaggcctcatacatctccttcaatgcggcctccaactcgcggtcccaaatcaaggctgccttggacaatgcgggaaagattatgagcctgactaaaaccgcccccgactacctggtgggccagcagcccgtggaggacatttccagcaatcggatttataaaattttggaactaaacgggtacgatccccaatatgcggcttccgtctttctgggatgggccacgaaaaagttcggcaagaggaacaccatctggctgtttgggcctgcaactaccgggaagaccaacatcgcggaggccatagcccacactgtgcccttctacgggtgcgtaaactggaccaatgagaactttcccttcaacgactgtgtcgacaagatggtgatctggtgggaggaggggaagatgaccgccaaggtcgtggagtcggccaaagccattctcggaggaagcaaggtgcgcgtggaccagaaatgcaagtcctcggcccagatagacccgactcccgtgatcgtcacctccaacaccaacatgtgcgccgtgattgacgggaactcaacgaccttcgaacaccagcagccgttgcaagaccggatgttcaaatttgaactcacccgccgtctggatcatgactttgggaaggtcaccaagcaggaagtcaaagactttttccggtgggcaaaggatcacgtggttgaggtggagcatgaattctacgtcaaaaagggtggagccaagaaaagacccgcccccagtgacgcagatataagtgagcccaaacgggtgcgcgagtcagttgcgcagccatcgacgtcagacgcggaagcttcgatcaactacgcagacagattggctcgaggacactctctcTAG

[0374] SEQ ID NO:18

[0375] Rep52 Amino Acid Sequence (AAV Serotype 2)

[0376] MELVGWLVDKGITSEKQWIQEDQASYISFNAASNSRSQIKAALDNAGKIMSLTKTAPDYLVGQQPVEDISSNRIYKILELNGYDPQYAASVFLGWATKKFGKRNTIWLFGPATTGKTNIAEAIAHTVPFYGCVNWTNENFPFNDCVDKMVIWWEEGKMTAKVVESAKAILGGSKVRVDQKCKSSAQIDPTPVIVTSNTNMCAVIDGNSTTFEHQQPLQDRMFKFELTRRLDHDFGKVTKQEVKDFFRWAKDHVVEVEHEFYVKKGGAKKRPAPSDADISEPKRVRESVAQPSTSDAEASINYADRYQNKCSRHVGMNLMLFPCRQCERMNQNSNICFTHGQKDCLECFPVSESQPVSVVKKAYQKLCYIHHIMGKVPDACTACDLVNVDLDDCIFEQ*

[0377] SEQ ID NO:19

[0378] Rep40 Amino Acid Sequence (AAV Serotype 2)

[0379] MELVGWLVDKGITSEKQWIQEDQASYISFNAASNSRSQIKAALDNAGKIMSLTKTAPDYLVGQQPVEDISSNRIYKILELNGYDPQYAASVFLGWATKKFGKRNTIWLFGPATTGKTNIAEAIAHTVPFYGCVNWTNENFPFNDCVDKMVIWWEEGKMTAKVVESAKAILGGSKVRVDQKCKSSAQIDPTPVIVTSNTNMCAVIDGNSTTFEHQQPLQDRMFKFELTRRLDHDFGKVTKQEVKDFFRWAKDHVVEVEHEFYVKKGGAKKRPAPSDADISEPKRVRESVAQPSTSDAEASINYADRLARGHSL*

[0380] SEQ ID NO:20

[0381] Rep78 Nucleotide Sequence (AAV Serotype 2)

[0382]

[0383] SEQ ID NO:21

[0384] Rep68 nucleotide sequence (AAV serotype 2)

[0385]

[0386] SEQ ID NO:22

[0387] Rep78 Amino Acid Sequence (AAV Serotype 2)

[0388] MPGFYEIVIKVPSDLDGHLPGISDSFVNWVAEKEWELPPDSDMDLNLIEQAPLTVAEKLQRDFLTEWRRVSKAPEALFFVQFEKGESYFHMHVLVETTGVKSMVLGRFLSQIREKLIQRIYRGIEPTLPNWFAVTKTRNGAGGGNKVVDECYIPNYLLPKTQPELQWAWTNMEQYLSACLNLTERKRLVAQHLTHVSQTQEQNKENQNPNSDAPVIRSKTSARYMELVGWLVDKGITSEKQWIQEDQASYISFNAASNSRSQIKAALDNAGKIMSLTKTAPDYLVGQQPVEDISSNRIYKILELNGYDPQYAASVFLGWATKKFGKRNTIWLFGPATTGKTNIAEAIAHTVPFYGCVNWTNENFPFNDCVDKMVIWWEEGKMTAKVVESAKAILGGSKVRVDQKCKSSAQIDPTPVIVTSNTNMCAVIDGNSTTFEHQQPLQDRMFKFELTRRLDHDFGKVTKQEVKDFFRWAKDHVVEVEHEFYVKKGGAKKRPAPSDADISEPKRVRESVAQPSTSDAEASINYADRYQNKCSRHVGMNLMLFPCRQCERMNQNSNICFTHGQKDCLECFPVSESQPVSVVKKAYQKLCYIHHIMGKVPDACTACDLVNVDLDDCIFEQ*

[0389] SEQ ID NO:23

[0390] Rep68 Amino Acid Sequence (AAV Serotype 2)

[0391] *

[0392] Sequence Listing Free Text

[0393] <210> 1

[0394] <213> Rep nucleotide sequence (adeno-associated virus 2)

[0395] <210> 2

[0396] <213> Cap nucleotide sequence (adeno-associated virus 2)

[0397] <210> 3

[0398] <213> Cap amino acid sequence (adeno-associated virus 2)

[0399] <210> 4

[0400] <213> Wild-type adeno-associated virus 2p5 promoter

[0401] <210> 5

[0402] <213> Sequence forming part of the 5'-untranslated region (UTR) of the Homo sapiens β-globin gene

[0403] <210> 6

[0404] <213> Wild-type adeno-associated virus 2p19 promoter

[0405] <210> 7

[0406] <223> Non-functional p19 sequence

[0407] <210> 8

[0408] <213> Wild-type adeno-associated virus 2p40 promoter

[0409] <210> 9

[0410] <223> Non-functional p40 promoter sequence

[0411] <210> 10

[0412] <213> Wild-type adeno-associated virus 2 adenovirus inhibitor

[0413] <210> 11

[0414] <223> Nucleotide sequence without functional p40 promoter sequence

[0415] <210> 12

[0416] <223> The p19 and p40 promoters of the rep gene sequence were eliminated, and the Rep78 and Rep68 coding sequences were retained.

[0417] <210> 13

[0418] <223> TetR binding site

[0419] <210> 14

[0420] <223> Modified MLP

[0421] <210> 15

[0422] <223> Modified MLP

[0423] <210> 16

[0424] <223> Rep52 nucleotide sequence

[0425] <210> 17

[0426] <223> Rep40 nucleotide sequence

[0427] <210> 18

[0428] <213> Rep52 amino acid sequence (adeno-associated virus 2)

[0429] <210> 19

[0430] <213> Rep40 amino acid sequence (adeno-associated virus 2)

[0431] <210> 20

[0432] <213> Rep78 nucleotide sequence (adeno-associated virus 2)

[0433] <210> twenty one

[0434] <213> Rep68 nucleotide sequence (adeno-associated virus 2)

[0435] <210> twenty two

[0436] <213> Rep78 amino acid sequence (adeno-associated virus 2)

[0437] <210> twenty three

[0438] <213> Rep68 amino acid sequence (adeno-associated virus 2)

Claims

1. An adenoviral vector comprising a nucleic acid molecule, wherein the nucleotide sequence of the nucleic acid molecule encodes at least one AAV Rep polypeptide, wherein the Rep polypeptide encoding sequence has the following characteristics: (i) it is not operably associated with a functional AAV p5 promoter; (ii) it does not contain a functional AAV p19 promoter; and (iii) it does not contain a functional adenovirus inhibitor sequence, wherein the functional adenovirus inhibitor sequence is as shown in SEQ ID NO:

10.

2. The adenoviral vector of claim 1, wherein the nucleotide sequence encodes functional AAV Rep78 and Rep68 polypeptides.

3. The adenoviral vector of claim 1, wherein the nucleic acid molecule does not comprise a p5 promoter.

4. The adenoviral vector of claim 1, wherein the Rep polypeptide encoding sequence is not operably associated with an IRES element.

5. The adenoviral vector of claim 1, wherein the adenoviral inhibitor sequence cannot be transcribed.

6. The adenoviral vector of claim 1, wherein the nucleic acid molecule is located in one of the adenoviral early genes.

7. The adenoviral vector of claim 1, wherein the nucleic acid molecule is located in the E1 region in the same transcriptional orientation as the E4, E2A and E2B expression cassettes.

8. The adenoviral vector of claim 1, wherein the adenoviral vector additionally comprises an AAV cap gene.

9. The adenoviral vector of claim 1, wherein the adenoviral vector comprises a repressible major late promoter (MLP).

10. A kit comprising: (A) the adenoviral vector according to any one of claims 1 to 9, and (B) Another adenoviral vector comprising (i) a nucleic acid molecule encoding an AAV Cap polypeptide, and / or (ii) A nucleic acid molecule encoding a recombinant AAV genome.

11. The kit of claim 10, wherein the adenoviral vector additionally encodes a polypeptide capable of transcriptionally activating a promoter present in the additional adenoviral vector.

12. A method for producing AAV particles, the method comprising the steps of: (a) infecting a mammalian host cell with the adenoviral vector according to any one of claims 1 to 9; (b) infecting the host cell with another adenoviral vector, wherein the other adenoviral vector comprises a recombinant AAV genome comprising a transgene, wherein at least one of the adenoviral vectors comprises an AAV cap gene; (c) culturing the mammalian host cell in a culture medium under conditions to produce AAV particles comprising the transgene; and (d) isolating or purifying AAV particles from the host cells or from the culture medium.

13. A method for producing AAV particles, the method comprising the steps of: (a) infecting a mammalian host cell with an adenoviral vector as claimed in any one of claims 1 to 9, wherein the mammalian host cell comprises a recombinant AAV genome stably integrated into the host cell genome, wherein the recombinant AAV genome comprises a transgene, and wherein: (i) the adenoviral vector additionally comprises an AAV cap gene, or (ii) the AAV cap gene is stably integrated into the mammalian host cell genome, or (iii) infecting the cells with an additional adenoviral vector comprising an AAV cap gene; (b) culturing the mammalian host cell in a culture medium under conditions to produce AAV particles comprising the transgene; and (c) isolating or purifying AAV particles from the cells or from the culture medium.

14. A method for producing recombinant AAV particles, the method comprising the steps of: (a) infecting a mammalian host cell with the adenoviral vector according to any one of claims 1 to 9, wherein: (i) the adenoviral vector additionally comprises an AAV cap gene, or (ii) infecting the cells with an additional adenoviral vector comprising an AAV cap gene; (b) infecting the mammalian host cell with a recombinant AAV comprising a transgene; (c) culturing the mammalian host cell in a culture medium under conditions to produce AAV particles comprising the transgene; and (d) isolating and / or purifying AAV particles from the cells or from the culture medium.

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