Process for making gutless helper-dependent adenoviruses

Abstract

The invention relates to a helper adenovirus genome wherein expression of one or more of the adenoviral early E1 genes, E2 genes including DNA-binding protein (DBP), pre-terminal protein (pTP) and DNA polymerase, or production of functional proteins are regulated. The invention also relates to a process for delivery of the helper-dependent or gutless adenovirus vector genome and modulating replication of the helper virus DNA in the manufacturing cells, in which to allow a sufficient length of time before excision, cutting, or recombination to removal of the viral packaging signal DNA sequence from the helper adenovirus genome prior to induction of viral DNA replication, and expression of helper proteins that are required for adenovirus capsid particle formation and packaging, or encapsidation, of the helper-dependent or gutless adenovirus vector genomes, while preventing the packaging, or encapsidation, of the helper adenoviral genome into preform adenoviral capsids.

Description

[0001] Process for making gutless helper-dependent adenoviruses

[0002] Technical Field

[0003] The present invention relates to a process for production of helper-dependent or gutless adenovirus vectors, an adenovirus vector production system, and use of gutless or helperdependent adenoviruses.

[0004] Field of the invention

[0005] The invention is a new process for manufacture of helper adenoviruses and helper-dependent adenovirus vectors (HDAdVs), high-capacity adenoviral vectors (HCAdVs), or gutless adenoviral vectors, in which the helper virus is eliminated from the helper-dependent adenovirus preparation by a combinatorial method. In the first component of the invention, genomic DNA replication of the Helper virus is inhibited in the packaging cell line. In the second component of the invention, cleavage, recombination and / or splicing of the helper virus DNA within the packaging cell line using a recombinase or endonuclease enzyme prevent packaging of helper virus into capsid particles. In a third component of the invention, genomic DNA replication of the helper virus is enabled to allow expression of helper genes within the packaging cell line. This invention can be used independently or in combination with endonucleases and recombinases that include, but are not limited to, tyrosine and serine recombinases such as FLP, CRE, phiC31, TP901-1, Lambda Red, I-Scel, piggybac transposase, Sleeping beauty transposase, and CRISPR gRNA excision. This invention can be applied in other helper virus-based manufacture systems to eliminate helper virus contamination from the helper-dependent vector.

[0006] Background of the Art

[0007] Gene Therapy

[0008] Gene therapy is defined as the therapeutic approach to deliver nucleic acids into patients’ cells for correction of a genetic disorder. This is usually accomplished by correction, addition, or silencing of a targeted gene and is aimed at treating a variety of human diseases that include both dominant and recessive genetic disorders, cardiovascular and neurodegenerative diseases, and cancer (Lapteva et al. 2020; Mali 2013). Gene delivery into patients’ cells may occur by two routes, ex vivo or in vivo. In the ex vivo route, cells of interest are temporarily isolated from the patient for introduction of the therapeutic construct, before the modified cells are re introduced back into the patient (Gowing, Svendsen, and Svendsen 2017; Anguela and High 2019). While this approach can ensure a stringent quality control process to confirm successful genetic modification and minimise the likelihood of unintentional mutations, ex vivo gene therapy is an expensive and laborious process, and only applicable to cells that are accessible for isolation and genetic manipulation (Naldini 2011; Mejia-Toiber, Castillo, and Giordano 2011). In the in vivo gene delivery approach, which may be local or systemic, vectors carrying the nucleic acids of interest are directly introduced into the patient for targeting cells and tissues at their native anatomical locations (Kumar et al. 2016). In vivo gene delivery remains the gold standard approach to gene therapy as it is the least invasive method, with the theoretical potential to target any cell in the body. Several criteria are critical to achieve success in in vivo gene therapy, including the use of vectors that are safe and efficient, and which can be economically produced in sufficient quantities to meet clinical demands (Macpherson and Rasko 2014; Herzog, Cao, and Srivastava 2010; Goncalves and Paiva 2017; van der Loo and Wright 2016).

[0009] Vectors for Gene Therapy

[0010] Whilst gene therapy using viral vectors has demonstrated significant success in recent years, there remain a number of limitations with the main platforms currently under clinical evaluation. Currently lentiviral vectors are the agent of choice for gene therapy involving proliferating cells, such as stem cells, where integration of the transgene into the host genome is required, while AAV vectors are the agents of choice for post-mitotic targets, such as liver, brain and retina. One of the main issues with AAV is its limited packaging capacity, just 4-5Kb. This restricts gene therapy to relatively small genes (such as Factor IX) or truncated versions of larger genes (such as B-domain-deleted Factor VIII) which often function unpredictably, raising significant safety concerns. In contrast, high-capacity adenoviral vectors (HCAdVs) have a packaging capacity of ~36Kb, making them highly attractive for gene therapy, allowing any human gene to be packaged including combinations of genes to treat complex disorders. The second major issue with AAV is its poor infection efficiency which leads to the use of dangerously high doses that can be fatal. Transduction of cells in vitro typically uses 5000- 100,000 AAV copies / cell compared to about 50 copies / cell of adenovirus. In vivo treatment of haemophilia B in macaques using IxlO11vg / kg of HCAdV produced similar results as 2xl012- 2xl013vg / kg AAV. First generation adenovirus vectors are characterised by short, pro- inflammatory expression profiles and this underpins their use as anticancer agents and recombinant vaccines but limits their utility where long-term transgene expression is required. However the removal of virus genes from adenovirus to produce HCAdV decreases presentation of viral epitopes and endows them with far greater biocompatibility that allows extended transgene expression. For example 100% of treated macaques showed therapeutic levels of Factor IX expression throughout the 1029 day observation period, and 100% of treated baboons showed a-foetoprotein expression for at least 7 years following a single injection. Since both AAV and HCAdV are thought to persist mainly as episomes, their durations of expression are likely to be very similar.

[0011] Adenovirus Structure

[0012] Adenoviruses are small (approximately 90 nm in diameter) non-enveloped viruses with an icosahedral capsid with 12 fibres projecting from the icosahedron vertices. The virus consists of 240 hex on trimers, 12 penton pentamers, 60 IHa monomers, 12 fiber trimers, 60 VII hexamers, 80 IX trimers as well as two terminal repeat proteins and multiple core proteins. Many of the latter are essential for intracellular trafficking and proteolytic cleavage of endocytosed viral capsids.

[0013] The DNA genome of adenovirus varies in length dependent on serotype but is typically 34-36 kilobases in length. The adenovirus serotype five genome is usually is 35938 base pairs with a 103 base pair terminal repeat at each end and a 58% GC content. The genome is linear within the capsid structure and for the majority of the replication cycle but can form pan -handle circular structures within the nucleus during genome replication.

[0014] Adenovirus Infection

[0015] The process of cell attachment for adenoviruses varies dependent on the serotype. Adenovirus species C (including serotypes two and five) have been shown to attach to cells via their fibre domain binding to the cell surface coxsackie and adenovirus receptor (CAR) (Tomko et al., 1997) which is a component of cellular tight junctions (Honda et al., 2000). This initial interaction is high affinity and is followed by a lower affinity interaction between the virus penton base and cell surface integrins (aVp3 & aVp5) which are abundant on epithelial cells (Wickham et al., 1993). Following absorption to the cell surface virions are endocytosed and in a poorly defined step the virus capsid escapes from the endosome into the cytosol. Within the cytosol adenoviral capsids bind to the microtubule network and are transported via a Dynein dependent mechanism to the nucleus where the DNA is delivered into the nuclear lumen following capsid interaction with the nuclear pore complex (Trotman et al., 2001; Kelkar et al., 2004).

[0016] Regulation of Adenovirus Transcription and Translation

[0017] Our understanding of adenovirus genetics, transcription and translation is primarily derived from virological studies using adenovirus species C serotype five. Adenovirus genes are divided into early (El-4) and late (L 1 -5) transcripts, with multiple protein isoforms driven from a range of splicing events. The early regions are divided into El, E2, E3 and E4. El A is the first gene to be expressed after infection, and its mRNA transcripts are alternatively spliced to produce five different proteins ranging from 6-36 kDa (289R, 243R, 217R, 171R, 55R). This initial transcription is driven by a strong constitutively active enhancer element within the El A promoter and allows significant quantities of El A mRNA to be produced. They are one of two sets of proteins in the adenovirus genome which are capable of inducing transformation, with E1B proteins also able to induce cell cycle progression. El A and E1B genes are essential for virus replication. The El A proteins have two major roles within an infected cell. Firstly, they induce the cell to enter the S phase of the cell cycle to allow the efficient replication of the viral genome. Secondly, they induce transcription of the other early promoters within the viral genome through transactivation. These promoters control the production of the E1B, E2, E3 and E4 proteins. El A expression is immediately followed by VARNA and E1B and E3 protein production (Gallimore and Turnell 2001; Radko et al. 2015). These proteins and RNA molecules help to prevent the development of an anti-viral response. These early events in viral replication help to shape the intracellular environment to allow the replication of the viral genome before packaging. The E1B transcription unit, detected shortly after E1A, produces two principal proteins, E1B-19K and E1B-55K, that blocks E1A induced-apoptosis. Later transcription events involve the production of structural proteins and proteins essential for cell lysis which are derived primarily from a single promoter (the major late promoter) which transcribes the late regions Ll-5.

[0018] The E2 region is largely responsible for the replication of the DNA genome, containing the DNA binding protein (E2A), the terminal protein and the DNA polymerase (E2B). E3 contains genes involved in immune regulation of host responses and E4 contains a range of genes involved in regulating cell pathways such as non-homologous end joining (NHEJ) and complexing with E1B-55K to mediate p53 degradation. The sequential process of gene transcription from the adenovirus genome reflects the protein requirements of the virus at each stage of the replication process. The transcription from the adenovirus genome is therefore divided into early and late events dependent on the timing of initiation of transcription from each viral promoter (Figure. 1). Cell lysis is dependent on the E3-11.6K protein (also termed the adenovirus death protein) which despite its labelling as an early gene is only produced late in infection and from the major late promoter (Tollefson et al., 1996).

[0019] The E2 genes are divided into two sections in the adenovirus genome: the E2A and the E2B regions, and both are required for virus replication. E2B contains the DNA polymerase gene which is fundamentally required for the amplification of the virus genomic DNA. Similarly, this region also encodes the pre-terminal protein (pTP) which is required for the initiation of virus genome replication. The terminal protein is covalently attached the end of the virus genomic DNA. The E2A region contains a DNA binding protein (DBP) that is required for DNA replication. All E2 genes are fundamentally required for virus replication. Temperaturesensitive (ts) mutant adenoviruses with mutations mapping to various regions of the E2A and E2B genes, which renders the E2 proteins inactive (or less active) at the non-permissive temperature, have been explored extensively to understand the adenovirus replication mechanisms, improve the safety profile of adenoviral vectors for gene therapy and vaccines as it reduces the activity and toxicity of these viral protein, or as a helper virus for complementing recombinant adeno-associated adenovirus (rAAV) vectors replication. These ts mutants include, but not limited to, tsl 9, tsl07, and tsl25 that mapped to the adenovirus E2A DNA binding protein, ts36, tsl 49, and ts69 that mapped to the E2B region encoding for the virus DNA polymerase, and sublOOr that mapped to the adenovirus E2B pTP (Handa, Shiroki, and Shimojo 1975; Araujo et al., Genet Mol Biol 2022; Carter and Blanton, J Virol. 1978; Carter and Gainsberg, J Virol., 1976; McDonough and Rekosh, Virology, 1982; Roovers et al., Virus Genes, 1990; Freimuth and Ginsberg, 1986). For example, Engelhardt et al., 1994 developed a second-generation adenovirus vector by incorporating a temperature-sensitive mutation into the E2A region (tsl25) of an El-deleted adenoviral vector. At the non-permissive temperature, this virus fails to express late gene products and it also showed sustained in vivo transgene expression in the mouse liver up to 70 days, whereas transgene expression from El -deleted adenovirus vector was observed only up to 14 days (Engelhardt et al., Proc. Nati. Acad. Sci. USA. 1994). The use of ts mutant adenovirus have also been explored to improve the safety profile of helper adenovirus used for the production of rAAV vectors. For example, Farson et al showed that adenoviruses with mutations in the virus E2B DNA polymerase or pTP, rendering them less able to replicate at 37°C, were able to support rAAV production comparable to the wildtype adenovirus. The contaminating helper virus from this approach is expected to be safer compared to the use of replication competent wildtype adenovirus (Farson et al. 2004).

[0020] The E3 genes are primarily involved in regulating cell and host immune responses to virus infection, however, as the majority of viruses used for biotechnology applications are in vitro many, if not all, of these virus genes can be removed without reducing virus replication efficiency. The majority of the E3 genes are not essential for virus replication. However, some genes such as the Adenovirus Death Protein (ADP) are required for efficient replication and virus production.

[0021] The E4 region of the adenovirus genome is similar to the El region in that it is primarily involved in producing proteins that help the virus control and regulate the cell to ensure efficient virus replication and production. The E4 encoded proteins are multifunctional and important for regulating transcription of the cell cycle, cell signalling and DNA repair (Lichtenstein et al. 2004; Evans and Hearing 2003; Reichel et al. 1989). The E4 region includes 6 open reading frames (ORFs) that are able to aid in preventing non-homologous end joining and apoptosis amongst a number of other discrete functions. The relative importance of each E4 transcript to virus replication is variable with some being essential whilst others can be deleted or modified with little to no effect of virus growth kinetics and production. In particular, E4orf6 was shown to enhance viral replication and adenovirus mutants that lack the E4 region were shown to be severely defective for replication of the adenovirus genome or expression of the late viral proteins (Halbert, D. N., J. R. Cutt, and T. Shenk. 1985., J. Virol. 56:250-257).

[0022] Late Gene Expression

[0023] The adenovirus late genes are all transcribed from the same promoter, the Major Late Promoter and all share the same 5' mRNA terminus which contains three exons that collectively form the tri-partite leader sequence. The late genes are expressed by a series of splice events that allow the expression of approximately 13 proteins that either form a part of the virus particle (e.g. Hex on and Fibre) or are involved in its assembly (e.g. 100K protein). The virus late genes are divided into five main transcript families named L1-L5. These transcripts primarily encode proteins that are involved in virus assembly and the structural proteins of the virus. During virus replication they can represent as much as 30-40% of the cells protein content (Gamier, 1994; Ginsberg, 1984). The LI series of transcripts encode for the 13.6K, 52K, and Pllla proteins. These proteins are all involved in virus assembly and particle production. LI genes are required for successful virus assembly but not genomic DNA replication.

[0024] The L2 series of transcripts encode for the penton base, pVII, V, pX proteins. These form structural parts of the virus capsid and are required for the particle to assemble correctly. Penton base contains an RGD motif that is important for virus attachment to the cell surface during infection. L2 genes are required for successful virus assembly but not genomic DNA replication The L3 series of transcripts encode for the pVI, hex on, and protease proteins. The Hexon protein is a major component of the virus capsid and is antigenically diverse between serotypes. The protease protein is involved in cellular entry and the virus capsid maturation. L3 genes are required for successful virus assembly but not genomic DNA replication

[0025] The L4 series of transcripts encode the 100K, 33K, 22K, pVII proteins. These proteins are involved in a range of functions. 100K protein is involved in both aiding virus hexon assembly and nuclear import but may also play a role in shifting cell mRNA translation to capindependent translation. The 22K protein is involved in virus encapsidation. L4 genes are required for successful virus assembly but not genomic DNA replication. However, the 100K protein may aid in shifting cellular protein translation towards those transcripts that contain a tripartite leader (TPL) sequence.

[0026] L5 encodes the Fibre gene. Fibre is a virus structural protein involved in attachment to cell surfaces and in mediating virus cellular infection. The Fibre protein is produced in significant excess of its requirement for virus particle formation. L5 genes are required for successful virus assembly but not genomic DNA replication. Adenovirus derived VA RNA and VA RNAII are important non-coding RNA products that are expressed in the late phase of the virus replication cycle and modulate a range of functions that include regulating translation of late viral genes, antagonising host interferon responses, and interfering with cellular micro (mi)RNA processes (Ma and Mathews 1996; Vachon and Conn 2016).

[0027] Adenovirus Packaging

[0028] The adenovirus packaging sequence involves a series of seven functionally redundant A repeats, referred as A1-A7, of approximately 200 bp and situated between the virus left ITR sequence and the El transcription unit. As the A repeats are functionally redundant, not all are essential for genome packaging into the adenoviral capsid and some A repeats are more efficient than others for directing genome packaging. The repeats Al, A2, A5 and A6 are most important for efficient genome packaging and they act in pairs of A1-A2, A5-A6, and it was suggested that they share a bipartite consensus motif (5’ TTTGN8CG 3’). The packaging signal sequence is extensively characterised for group C adenovirus type 5 (SEQ ID NO: 17) which is located between nucleotides 220 and 400 of the viral genome (Grable and Hearing, 1990, 1992; Tyler et al., 2007). The packaging signal sequence for group A (SEQ ID NO: 15), group B (SEQ ID NO: 16), group D (SED ID 18), group E (SEQ ID NO: 19), group F (SEQ ID NO: 20), and group G (SEQ ID NO: 21) adenoviruses have also been characterised. Sequence alignment of the packaging signal of adenoviruses belonging to different groups show conservation of the bipartite consensus motifs of the packaging repeats, but subtle differences among the A-repeats to reflect variation in the DNA binding specificities of the packaging proteins from different adenovirus species. Packaging of the adenovirus genome occurs in a polar fashion and capsids containing only the left arm portion of the viral genome can be obtained. The packaging signal was shown to maintain efficient function only when positioned within 600bp of either the left or right ITR sequence. Although unclear, this observation may suggest involvement of the ITR sequence and terminal protein in the packaging process (Hearing et al. 1987). While the mechanism of adenovirus packaging remains to be fully established, production of empty capsids from virally infected cells suggests a model of ‘sequential assembly’ to form infectious virions. In this model, particles are first assembled from hexon trimers and penton with incorporation of the minor and non-structural proteins. Subsequently, genome packaging occurs by a process of recognition of the virus packaging element by a packaging complex, that results in insertion of the DNA genome through a portal at an unidentified unique vertex of the capsid. Adenovirus proteins associated with the packaging signal include IVa2, L4 33K, L4 22K, LI 52 / 55K and Illa (Ostapchuk and Hearing 2003; Ahi and Mittal 2016). The intermediate-expressed IVa2 protein is multifunctional and important in stimulating transcription of late viral genes and the genome encapsidation process. The current model of adenovirus packaging suggests IVa2 binds, in a co-dependent complex, with the L4 22K protein to the CG motif within the A repeats of the packaging sequence (Wu, Orozco, and Hearing 2012). This complex further recruits packaging associated proteins Ll- 52 / 55K and Illa, which were shown to contribute to packaging of serotype-specific adenovirus genomes. IVa2 exhibits ATPase function and may serve as a motor protein, in the presence of adenovirus genome and L4 33K, for threading of the genome through the capsid portal (Ahi et al. 2015). Inducible gene regulation systems

[0029] Temporal control of gene expression is fundamental and applicable to almost all aspects of research, including examination of gene and cellular functions, biologies discovery and production, and gene therapy. Numerous approaches have been developed to enable temporal control of gene expression and these can be divided into two classes - endogenous or exogenous control systems. Whereas the former requires environmental or physiological stimuli, such as hypoxia, interferons or metal ions, to regulate gene expression, exogenous control requires external factors, such as a drug molecule, to induce or switch-off gene expression (Naidoo and Young 2012; Ryals et al. 1985; Mayo, Warren, and Palmiter 1982). The most widely explored exogenous gene control approach is the tetracycline regulated gene expression system developed from the TnlO-encoded tetracycline resistance operon in Escherichia coli (E. coli) (Gossen and Bujard 1992). In bacteria, antibiotic resistance to tetracycline is mediated by the TetA protein, which functions as a tetracycline efflux pump located in the cytoplasmic membrane. The tetracycline regulated mechanism is highly evolved, both in sensitivity for detecting intracellular tetracycline and the control response in antibiotic clearance. As overexpression of TetA is lethal to the bacteria, its expression is tightly regulated at the transcription level by a repressor protein TetR. Binding of TetR to the tetracycline operator sites (tetOl and tetO2) within the tet operon repress transcription of both TetR and the TetA resistance gene. Influx of tetracycline results in high affinity binding to the TetR and its disassociation from the tetO sites, resulting in rapid expression of TetA and TetR to remove the intracellular tetracycline, and subsequent gene repression to avoid TetA-induced lethality (Ramos et al. 2005; Das, Tenenbaum, and Berkhout 2016). There are three different variations to this TetR inducible system - Tet-Off, Tet-On and the unmodified TetR system. The unmodified TetR represents the most basic configuration and consists of insertion of tetO sites in the target promoter. Transcriptional repression is mediated by the TetR which interferes with binding of transcription factors and RNA polymerase to the target promoter (Alba, Bosch, and Chillon 2005; Yao et al. 1998). Early studies by Gatz and Quail showed for the first -time repression of a eukaryotic plant promoter in tobacco protoplast using the TetR. Here, the target CaMV 35S promoter was modified with TetO sites flanking the TATA element (Gatz and Quail 1988). Repression of promoters utilising RNA polymerase I, II and III were subsequently demonstrated using the TetR, but highlighted significant variations in the positioning of TetO sites in these different models that was important for efficient gene regulation (Yan, Myler, and Stuart 2001; Frohberg, Heins, and Gatz 1991; Dingermann et al. 1992). In a common configuration to regulate gene expression in mammalian cells, two tetO sites are positioned at approximately 10 bp downstream of the CMV TATA box element to enable TetR-mediated transcriptional repression. Repression can be alleviated by addition of tetracycline, to disrupt binding of the TetR to its operator elements (Wu et al. 2010; Yao et al. 1998). In both Tet-Off and Tet-On approaches, tetO sequences (usually x7 copies, termed tetracycline responsive elements (TRE)) are positioned upstream of the minimal constitutive promoter of a gene target. In the Tet-Off configuration, a transactivator proteins (tTA), consisting of TetR fused to the VP 16 transactivator domain from herpes simplex virus type 1 is introduced to enable automatic expression of the target gene. Addition of tetracycline to cell cultures binds and inhibits activity of the tTA to silence gene expression (Kim et al. 1995; Toniatti et al. 2004). Conversely, the Tet-on configuration utilises a mutant tTA, entitled reverse tTA (rtTA), that binds in the presence of tetracycline to the TetO elements within the target promoter to facilitate gene expression. In this configuration, the absence of tetracycline results in target gene repression (Ramos et al. 2005). TetR inducible expression system offers many advantages as a gene control platform and are often explored in conjunction with viral vectors, such as adenoviruses, AAV and lentiviruses, for both fundamental research and gene therapy application (Goverdhana et al. 2005; Chen et al. 2015). Tetracycline and its analogue doxycycline are antibiotics that are well characterised with a high safety margin in both clinical and preclinical application (Moore, Ling, et al. 2015). However, a major concern of the TetR inducible system is a tendency for leaky gene expression.

[0030] To improve upon this, attempts have included fusion of the TetR to the Krtippel-associated box (KRAB) repressor domain of human K0X1, use of a minimal promoter from the modified mouse mammary tumor virus (MMTV) for inducible transcription, codon optimisation of the rtTA and identification of novel rtTA by PCR mutagenesis or error-prone reverse transcription using RNA viruses(Deuschle, Meyer, and Thiesen 1995; Hofmann, Nolan, and Blau 1996; Wells et al. 1999; Zhou et al. 2006; Urlinger et al. 2000). A number of other exogenous inducible systems have been explored in mammalian cells and include the cumate gene-switch system, rapamycin-regulated systems, and the TRiP translational repression system (Mullick et al. 2006; Maunder et al. 2017; Naidoo and Young 2012). Of these approaches, the cumate gene-switch is most analogous to the tet-inducible system, in which it was developed from a bacterial repressor, the p-cmt and p-cym operon in Pseudomonas putida (Eaton 1997). Further similarities to the TetR approach include the cumate gene-switch has three configurations where (I) the repressor protein CmyR can be used to block transcription of the target promoter by binding to operator sequences (CuO), or (II) a chimeric transactivator (cTA) composed of CymR fused to the VP 16 activator domain can be used to induce transcription of a minimal promoter encoding six-copies of CuO upstream, or (III) a reverse transactivator (rcTA) can be used to facilitate transcription from the targeted Cu0x6 minimal promoter. While activity of the cTAis inhibited with addition of the cumate drug, function of rcTAis activated by cumate. While the cumate gene-switch system shares some problems with the TetR-inducible approach, such as its propensity to repress highly active promoters, such as CMV, or leaky expression from minimal promotors, it provides a useful alternative in biological systems requiring simultaneous temporal control of multiple transcription units (Mullick et al. 2006).

[0031] Short hairpin RNA (shRNA), antisense RNA, or short-interfering (si) RNA have also been widely used as gene regulation systems via RNA interference. shRNA sequences form short hairpin complexes that can be processed by Dorsha, which is then exported from the nucleus for further processing by Dicer and then loaded into the RNA-induced silencing complex (RISC). shRNA sequences are generally delivered and expressed in cells by plasmid DNA or viral vectors (Moore et al., Methods Mol Biol., 2013). Antisense RNA has also been shown as a potent method to inhibiting adenovirus gene expression and viral replication. Kneidinger et al. showed that siRNA sequences designed to target the adenoviral El A, DNA polymerase, pTP, IVa2, hexon, and protease genes was capable of inhibiting the expression of the target gene and decreasing adenovirus replication. Additionally, siRNAtargeting the adenovirus DNA polymerase mRNA was shown to decrease the copy number of the virus genome in infected cells by several orders of magnitude (Kneidinger et al., Antiviral Res., 2012).

[0032] CRISPR / Cas-based genome editing have also been widely used in regulating and silencing gene expression in various systems including in mammalian cells for controlling viral replication. A common approach is the use of the CRISPR / Cas9 systems to induce doublestranded DNA breaks (DSB) within the cell or virus genomes. In such cases, repair of the DSB by non-homologous end joining results in the addition or deletion of additional nucleotides that interrupt the expression of the functional protein due to base frame shit of the gene coding sequence. Furthermore, CRISPR / dCas interference (CRISPRi) or CRISPR / dCas activation (CRISPRa) systems are able to directly inhibit or activate gene expression. In these systems, a catalytically dead (d) Cas9 protein is fused to a transcriptional activator (CRISPRa) or a transcriptional repressor (CRISPRi), and the guide RNA functions to navigate the protein complex to the genome location, for enhancing or inhibiting gene expression, respectively. High Capacity Adenoviruses

[0033] High Capacity (HCAdV) or "gutless" adenoviruses vectors are particularly attractive for gene therapy applications in vivo because they can package and deliver large DNA payloads (up to 36kb) at high transduction efficiency across a wide range of cell types, and they also show significantly reduced immunogenicity compared to first generation adenoviral vectors. HCAdV vectors are devoid of all viral genes and contain only the adenoviral inverted terminal repeats (ITR) DNA sequence elements at each end of the linear DNA genome and the adenoviral packaging signal ( ) in cis, required for replication and packaging of the HCAdV genome into preformed capsids, respectively (Grable and Hearing, 1992, J. Virol., 66: 723-731; Hearing and Shenk, 1983, Cell, 33: 695-703).

[0034] As HCAdVs are replication defective and devoid of all viral coding genes, their genome replication and manufacture of progeny virions in producer cells requires the co-infection with a second 'helper' adenovirus capable of expressing all of the necessary proteins for replication and encapsidation of the HCAdV genomes. Wildtype (WT) adenoviruses or first-generation El-deleted adenovirus vectors, are both capable of expressing the repertoire of viral genes necessary for replication and packaging of HCAdV vector genomes in capsid particles. The propagation of HCAdV vectors using El -deleted adenovirus helper generally requires the use of the human embryonic kidney (HEK) 293 cell line as they contain stable insertion of the adenovirus E1A and E1B sequence (Graham, et al., 1977, J. Gene. Virol., 36: 59-74) or HEK293-based cell lines, including PerC6 and 911 cells, or amniocytes-based CAP cells, for complementing expression of the adenovirus El and pIX genes (Fallaux et al., 1998, Hum. Gene Th er., 9: 1909-1917, Schiedne and Kochanek, 2000, Hum. Gene Then, 15: 2105-2116).

[0035] Current strategies for decreasing helper virus contamination of HCAdV preparations

[0036] The use of WT adenoviruses or El -deleted adenoviral vectors as helper viruses for propagation of HCAdVs results in the production of unwanted helper virus contaminations in the final HCAdV vector preparations. Various approaches have been developed in attempting to try and reduce or eliminate contamination of the helper virus, in particular by blocking the packaging process of the helper virus. These approaches are usually based on the incorporation of DNA sequence elements within or at close proximity to the packaging signal of the helper virus, in attempts to hinder or eliminate the function of the packaging signal during the HCAdV production phase. To hinder packaging of the helper virus, the Cre-loxP recombination system was explored as a strategy to eliminate the packaging signal of the helper virus (Parks et al. (1996, Proc. Natl. Acad. Sci. USA, 93: 13565-13570). In this platform, the packaging signal of the helper virus is flanked with LoxP sequences, which allows splicing of the packaging signal as the helper virus propagates in Cre-expressing HEK293 cells (Figure 2). Splicing of the packaging signal from the helper virus eliminates its capacity for being packaged into new adenovirus capsids, while expression of the adenovirus genes required for replication of the HCAdV genome is maintained. Attempts to induce inversion of the packaging signal in the helper virus using the Cre-LoxP system has also been explored by Palmer and Ng group (Palmer and Ng (2003, Mol. Then, 8: 846-852). Additionally, use of rare cutting endonucleases, including Seel, has been disclosed in patent application US7135187B2. Here, the inventors reported the insertion of an internal ITR sequences to the right of the Seel recognition site of the helper virus genome. Cleavage of the viral genome in HEK293 cells expressing Seel should facilitate the elimination of the packaging signal, while the internal ITR and right ITR of the viral genome functions to support viral DNA replication. The combination of this method using Seel endonuclease with the Cre / lox system in attempting to enhance reduction of the helper virus was also disclosed in US7135187B2.

[0037] Interestingly, it was suggested (Gonzalez-Aparicio M. et al Biotechnol. J. 2023), that a possible factor limiting the manufacturing yield of high-capacity adenovirus vectors is related to cytotoxicity of the Cre recombinase due to chronic exposure in the packaging cell line. In an attempt to overcome this limitation, the authors developed a split-Cre system wherein the Cre protein is split into two non-functional fragments. Treatment with drugs, such as rapamycin, causes dimerization of the fragments to generate a functional Cre protein. Using this split-Cre system, the authors showed that they were able to reduce helper virus accumulation from 260% to 3% (relative to high-capacity vector) at pre-purification. This significant level of helper virus contamination at the stage of vector harvest (pre-purification) suggested that the purification process by density gradient ultracentrifugation, while challenging to upscale in manufacturing, is essential for eliminating the majority of helper viruses.

[0038] It was also suggested that Cre expression from stable HEK293 cell lines decreases over the course of adenovirus infection, reducing the efficiency for splicing of the loxP -flanked packaging signal from the helper virus. It was shown that up 5% of the helper viral genomes escape Cre-mediated packaging signal excision, with the decrease in Cre efficiency after the onset of viral DNA replication (Ng, P. et al J Virol 2002). The authors postulated that the decrease in Cre expression may be due to the effect of adenovirus-mediated host cell protein shutoff, a viral mechanism for preferential translation of viral proteins. To increase the production Cre for efficient excision of the packaging signal from the helper virus, Gonzalez- Aparicio, M. et al. developed a system wherein the helper virus with the LoxP -flanked packaging signal also encodes a chimeric Mer-Cre-Mer recombinase. Activity of the Mer-Cre- Mer recombinase is modulated using both the Tet-on induction system and 4- hydroxytamoxifen (TAM). While addition of both doxycycline and 4-hydroxytamoxifen enables excision of the packaging signal from the helper virus during production of the HD AdV, it was reported that this system produces helper virus contaminations at approximate 0.1% after purification, which are comparable to levels observed from production using HEK293 cell lines stably expressing Cre (Gonzalez-Aparicio, M. et al. Gene Ther 2011). Alternative approaches using phi C31 recombinase, where attB / attP sites flaking the packaging signal have been reported (Groth et al. Proc. Natl. Acad. Sci. USA, 2000, 97: 5995- 6000). The recombinase C31 specifically recognises the attB / attP sites to excise the packaging signal, but also converts the attB / attP into attR / attL sequences and prevents rerecognition by the C31 recombinase, and therefore reintroduction of the packaging signal into the helper virus. Alba et al. 2007 also reported that insertion of attB / attP sequences to flank the packaging signal of the helper virus results in significantly lengthening its replication cycle to 60h, and therefore reduce contamination of the helper virus, while the HCAdV can be harvested at 36 hours post-production. Further variations to the recombination system for elimination of the packaging signal using FLP / frt recombinase has also been reported. In this model, the FLP recombinase is stably expressed from HEK293 cells to eliminate the frt-flanked packaging signal from the helper virus (Ng et al. (2001, Mol. Then, 3: 809-815). Production of HCAdV from this approach was reported to produce helper virus contaminations of 0.5%, a level comparable to the Cre-LoxP and C31, after purification by caesium chloride ultracentrifugation. Overall, as reviewed in Alba et al. 2005 (Gene Then, 12 Suppl 1 : SI 8-27), the contamination levels of the helper virus, relative to the HCAdV, may differ from 0.02% to 1% at post-purification in the various systems, but these levels are considered too high for potential use in clinical gene therapy.

[0039] Alternative approaches to eliminate the helper virus during the manufacture of the HCAdVs have included systems that imposes a restriction on the packaging size of the adenoviral particles. In one such approach, Sargent et al. 2024 reported that a helper virus with a deletion of the protein IX (pIX) are only able to package up to 35kb of DNA, and helper viruses of 37.5kb can be generated in pIX-complementing HEK293 cell lines. However, high amounts of helper virus contaminations at levels of 500:1 ratio (HCAdV : helper virus) was still observed. Strategies that avoid the use of a helper adenovirus for production of high-capacity adenoviral vectors have also been explored. Kubo et al. 2003 (DOI: 10.1016 / s0006-291x(03)01256-7) reported generation of HCAdVs using an HSV-amplicon. However, it was also reported that low titres of the HSV amplicon resulted in low propagation efficiency of the HCAdV vector, providing significant challenges for scale-up productions. Similarly, a baculovirus-adenovirus hybrid was used for delivery of a packaging-deficient adenovirus genome, flanked by LoxP, to produce HCAdVs in HEK293 cells. However, generation of replication-competent adenovirus and scale-up challenges in production was reported by the authors (Cheskenko et al 2001, DOI: 10.1038 / sj .gt.3301459).

[0040] Packaging cell lines as a strategy to avoid the use of helper virus in production of HCAdV Enormous efforts to stably integrate adenoviral genes to generate stable cell lines for producing HCAdVs have also been explored. However, adenoviral genes are highly cytotoxic and the intricate coordination required for the replication of DNA viruses involves a highly complex sequence of events, which is challenging to reproduce from an episomal DNA or stably integrated cell line (Farley, et al., 2004, J. Virol., 78: 1782-1791). As disclosed in W02000072887A1, a packaging cell line platform encoding an Epstein-Barr virus based episome with the adenoviral genome (El, E3, E4, and E2B pTP and DBP) have been explored for HCAdV production. This system does not rely on a helper virus and the episomal DNA is not expected to be packaged into virions, as it lacks the packaging signal, and its size exceeds the packaging limit of the adenoviral particle. Similarly, Catalucci et al. (2005, J. Virol., 79: 6400-6409) showed an episomal plasmid composed of the origin of replication from the Epstein-Barr virus, and the ITR sequences of the serotype 5 adenovirus (with the genes of the early region (E2) and E4 ORF6) was maintained in a HEK293 cell line stably expressing the nuclear antigen EBNA1 (293EBNA). In this system, the structural genes required for formation of the adenoviral capsid are expressed from the Ad5AEl-4 genome. This platform was limited by several challenges that included difficulties for large-scale productions and the size of the DNA sequences that can be inserted into the vector (~12.5kb). Additionally, the Ad5AEl-4 vector still encoding the adenoviral late genes that poses risk of possible immunogenicity for clinical gene therapy. To improve the safety of the helper adenovirus and reduce unwanted side effects in HCAdV preparations, such as immunogenicity, Zhou et al. 2001 developed a helper adenovirus wherein both the adenovirus El and E2A genes are deleted from the virus genome. HCAdV production is carried out in CreE cells, a HEK293-based cell line expressing both CRE and E2A DNA- binding protein under a tetracycline inducible promoter. In this system, the withdraw of tetracycline from the cell culture enables expression of both CRE and E2A DNA-binding protein for excision of the packaging signal from the helper virus, and adenoviral (helper adenovirus and HCAdV) replication, respectively. While the HCAdV preparations from this approach is expected to exhibit a higher safety profile due to the additional deletion of the E2A gene from the helper virus, the levels of helper virus contamination relative to the HCAdV is comparable, or slightly lower, compared to the use of an E2A-intact helper virus as the decreased helper contamination is dependent on the cell line’s Cre activity. Similarly, a HEK293-based cell line (C7-Cre cells) that expresses the Cre recombinase, and both adenovirus E2B pTP and E2B DNA polymerase have been explored for HCAdV production (Barjot et al. J Gene Med 2002; Reddy et al. Mol Ther. 20002). While production of HCAdV in the C7-Cre cells are complemented with a helper adenovirus deleted of El and the E2B genes, the contamination of helper viruses was observed at levels between 0.3% - <2% relative to the HCAdV genomes. However, the El and E2B-deleted helper virus is expected to also exhibit a higher safety profile due to absence of the viral genes and replication-competent adenoviruses in HCAdV preparations (Sakhuja et al. Mol Ther 2001 D01:https: / / doi.org / 10.1006 / mthe.2001.0363).

[0041] More recently, Lee et al 2019 (DOI: 10.1038 / sl2276-019-0334-z) reported a method for the production of high-capacity adenovirus that does not rely on a helper-virus or stable packaging cell line. In this approach, a helper DNA plasmid deficient of the adenovirus ITRs and packaging signal was used for co-transfection into HEK293 cells alongside plasmids encoding the HCAdV genome and adenovirus pTP gene. While this approach is expected to be free of helper virus contaminations, a significant challenge is the low yield and lack of scalability due to the requirements for manufacturing and transfecting a large quantity of plasmid DNA for HCAdV production.

[0042] These studies have established that there is an important need in the art to achieve a scalable system for the manufacture of HCAdV or gutless adenoviral vectors by exploiting the efficiency of the auxiliary helper virus, but which overcomes the drawback of helper virus contamination. This is particularly important for clinical gene therapy.

[0043] SUMMARY OF THE INVENTION

[0044] The present invention relates to a method for production of HDAdV, HCAdVs, or gutless adenoviral vectors. The invention system described herein is a new method designed to eliminate contamination by helper viruses during production of the HCAdV by inhibiting replication of the helper virus to allow sufficient time for efficient cleavage of DNA sequences within the helper virus using endonucleases or recombinases. Accordingly, this invention provides a useful system for production of HCAdVs, in which the helper virus is eliminated from the HCAdV preparation by a combinatorial process. In the first component of the invention, genomic DNA replication of the helper virus is inhibited in the packaging cell line. In the second component of the invention, endonuclease or recombinase mediate cleavage and / or splicing of the helper virus packaging signal DNA within the packaging cell line, to prevent packaging of helper virus genomes into capsid particles. In a third component of the invention, genomic DNA replication of the packaging signal-deficient helper virus is enabled to allow high level expression of helper genes that are required for adenovirus capsid particle formation and packaging, or encapsidation, of HCAdV genomes within the packaging cell line, while preventing the packaging, or encapsidation, of the helper adenoviral genome into preformed adenoviral capsids.

[0045] This invention can be with endonucleases and recombinases that include, but are not limited to, tyrosine and serine recombinases such as FLP, CRE, phiC31, TP901-1, Lambda Red, I-Scel, piggybac transposase, Sleeping beauty transposase, and CRISPR gRNA excision. This invention can be use in other helper virus production systems to eliminate helper virus contaminations from the helper-dependent vector. Those skilled in the art will appreciate that the recombinases and endonucleases described are merely examples and not meant to be limiting. Other endonucleases and recombinases exist, and new candidates continue to be discovered, which could likewise be employed according to the present invention.

[0046] While some recombinases themselves have an endogenous nuclear localisation signal (NLS), the addition of an exogenous mammalian NLS may enhance their nuclear trafficking, and therefore, the efficiency of packaging signal excision. Sequences encoding a functional NLS are known to those skilled in the art. The present invention accordingly provides a method of producing a helper-dependent adenoviral vector in a population of cells, wherein said cells comprise an adenoviral helper genome which encodes one or more viral genes necessary for replication and / or packaging of the helper-dependent adenoviral vector, wherein replication of the adenoviral helper genome is inhibited while excising a packaging signal sequence therefrom, and wherein replication of the adenoviral helper genome is induced to provide for replication and / or packaging of the genome of the helper-dependent adenoviral vector.

[0047] The present invention further provides a helper-dependent adenoviral vector preparation obtainable according to the method of the invention. The present invention additionally provides a population of cells producing a helper-dependent adenoviral vector obtainable according to the method of the invention.

[0048] The present invention also provides a method of reducing helper virus contamination in a helper-dependent adenoviral vector preparation, comprising producing a helper-dependent adenoviral vector in a population of cells, wherein said cells comprise an adenoviral helper genome which encodes one or more viral genes necessary for replication and / or packaging of the helper-dependent adenoviral vector, wherein replication of the adenoviral helper genome is inhibited while excising a packaging signal sequence therefrom, and wherein replication of the adenoviral helper genome is induced to provide for replication and / or packaging of the genome of the helper-dependent adenoviral vector, optionally wherein the helper-dependent adenoviral vector is produced by a method according to the invention.

[0049] Those skilled in the art will appreciate that no previous reports exist within the prior art that aimed to prevent the DNA replication of the helper adenovirus genome whilst its packaging signal sequence is being excised, recombined, or removed from the viral genome within the cells for the use of HCAdV or gutless adenovirus manufacturing. Furthermore, specific attempts to control the DNA replication of helper adenoviruses using mutations in the El or E2 region, while its packaging signal sequence is being excised, recombined, or removed from the viral genome within the cells have not been reported for HCAdV manufacture.

[0050] The present invention also provides cells which have particularly advantageous properties for adenovirus production, as illustrated in the examples, and related kits and methods. In particular, the invention further provides a cell comprising a nucleic acid sequence encoding a CRE recombinase, a nucleic acid sequence encoding a functional adenovirus E2B DNA polymerase, an adenoviral helper genome which lacks a functional E2B polymerase gene and encodes one or more viral genes necessary for replication and / or packaging of a helperdependent adenoviral vector, a helper-dependent adenoviral genome, wherein the nucleic acid sequence encoding the CRE recombinase and / or the nucleic acid sequence encoding a functional adenovirus E2B DNA polymerase are comprised within an AAV genome or integrated into the genome of the cell, and wherein the CRE recombinase is able to excise a packaging signal from the adenoviral helper genome.

[0051] The invention additionally provides a kit comprising a nucleic acid sequence encoding a CRE recombinase comprised within an AAV genome, an adenoviral helper genome which lacks a functional E2B polymerase gene and encodes one or more viral genes necessary for replication and / or packaging of a helper-dependent adenoviral vector, a helper-dependent adenoviral genome, and a recombinant cell containing a nucleic acid sequence integrated into its genome encoding a functional adenovirus E2B DNA polymerase, wherein the CRE recombinase is able to excise a packaging signal from the adenoviral helper genome.

[0052] The invention additionally provides a kit comprising a recombinant cell containing a nucleic acid sequence encoding a CRE recombinase integrated into its genome, a nucleic acid sequence encoding an adenoviral helper genome which lacks a functional E2B polymerase gene and encodes one or more viral genes necessary for replication and / or packaging of a helperdependent adenoviral vector, a nucleic acid sequence encoding a helper-dependent adenoviral genome, and a nucleic acid sequence encoding a functional adenovirus E2B DNA polymerase comprised within an AAV genome, wherein the CRE recombinase is able to excise a packaging signal from the adenoviral helper genome.

[0053] The invention additionally provides a kit comprising a nucleic acid sequence encoding a CRE recombinase comprised within an AAV genome, a nucleic acid sequence encoding an adenoviral helper genome which lacks a functional E2B polymerase gene and encodes one or more viral genes necessary for replication and / or packaging of a helper-dependent adenoviral vector, a nucleic acid sequence encoding a helper-dependent adenoviral genome, and a nucleic acid sequence encoding a functional adenovirus E2B DNA polymerase comprised within an AAV genome, wherein the CRE recombinase is able to excise a packaging signal from the adenoviral helper genome.

[0054] The invention also provides a method of producing a helper-dependent adenoviral vector in a population of cells, wherein the cells comprise a nucleic acid sequence encoding a CRE recombinase, a nucleic acid sequence encoding a functional adenovirus E2B polymerase, an adenoviral helper genome which lacks a functional E2B polymerase gene and encodes one or more viral genes necessary for replication and / or packaging of a helper-dependent adenoviral vector, a helper-dependent adenoviral genome, wherein the nucleic acid sequence encoding the CRE recombinase and / or the nucleic acid sequence encoding a functional adenovirus E2B DNA polymerase are comprised within an AAV genome or integrated into the genome of said cells, wherein the CRE recombinase excises a packaging signal from the adenoviral helper genome and wherein replication of the adenoviral helper genome provides for replication and / or packaging of the genome of the helper-dependent adenoviral vector.

[0055] Description of the Figures

[0056] Figure 1. Schematic diagram of the adenovirus genome. The adenoviral genome is a linear double- stranded DNA molecule approximately 36 kilobases in length. It is flanked by inverted terminal repeats (ITRs) required for replication and contains a packaging signal (y) near the left end. The genome encodes early (E1-E4) and late (L1-L5) genes involved in replication, host immune evasion, and structural protein production. The El region is essential for initiating transcription and is commonly deleted in recombinant adenoviral vectors to allow transgene insertion and prevent replication in non-complementing cells.

[0057] Figure 2. Schematic diagram of the conventional HCAdV, or gutless adenovirus, manufacture approach using the CRE-loxP recombination system (Adapted from Jager, L. et al 2009).

[0058] The process begins by constructing the HD Ad genome: a plasmid containing the therapeutic or reporter transgene, flanked by adenoviral inverted terminal repeats (ITRs) and the packaging signal (y). This plasmid is linearized and transfected into a packaging cell line, typically HEK293 cells, which supply the adenoviral El proteins in trans. Shortly after transfection, a helper virus is added. This virus contains all necessary adenoviral genes for replication and packaging but has its own packaging signal flanked by loxP sites. The packaging cells typically express Cre recombinase, which excises the helper virus’s packaging signal, preventing its genome from being packaged into viral capsids. The helper virus replicates and provides the viral proteins needed to replicate and package the HD Ad genome. Because the HD Ad genome retains its packaging signal and the helper virus’s signal is excised by the CRE recombinase, ideally only the HD Ad genome is packaged. However, residual contaminating helper virus from unexcised packaging signal is common.

[0059] After initial production, the virus-containing supernatant is used to infect fresh packaging cells for amplification. This cycle is repeated several times. The resulting viral mixture contains mostly HD Ad particles and a small amount of helper virus.

[0060] Figure 3. Schematics showing HCAdV production in a HEK293 cell line expressing CRE and inducible E2B DNA polymerase.

[0061] Figure 4: Delaying DNA polymerase-deficient helper adenovirus replication for 24-hours results in enhanced packaging signal excision compared to the simultaneous co-infection of AAV expressing E2B (AAV-E2B) with AAV expressing CRE (AAV -CRE) and the helper adenovirus. The DNA polymerase-deficient helper adenovirus does not replicate in the absence of exogenous E2B. Adenoviral genome copies per well as determined by qPCR for hexon or packaging signal are shown for each condition.

[0062] Figure 5: Delaying DNA polymerase-deficient helper adenovirus replication in monoclonal HEK293 A cells expressing inducible CRE and E2B by withholding doxycycline (dox) for 24- hours results in enhanced packaging signal excision compared to the simultaneous addition of doxycycline with the helper virus. Adenoviral genome copies per well as determined by qPCR for hexon or packaging signal are shown for each condition.

[0063] Figure 6: Schematic of HcAdV production where HEK293 cells are infected with 1) E2B deleted (or mutated) helper adenovirus 2) AAV expressing CRE recombinase and 3) a helperdependent adenoviral vector. The cells are then subsequently infected with AAV-E2B after 24 hours, providing sufficient time for the CRE recombinase to excise the packaging signal from the E2B deleted (or mutated) helper adenovirus prior to adenoviral genome replication.

[0064] Figure 7: Schematic of HcAdV production where HEK293 cells containing an integrated copy of CRE recombinase are infected with 1) E2B deleted (or mutated) helper adenovirus 2) a helper-dependent adenoviral vector. The cells are then subsequently infected with AAV-E2B after 24 hours, providing sufficient time for the CRE recombinase to excise the packaging signal from the E2B deleted (or mutated) helper adenovirus prior to adenoviral genome replication.

[0065] Figure 8: Enhanced excision of helper adenovirus packaging signal in HEK293 cells stably expressing Cre recombinase from a cumate inducible promoter and adenovirus E2B from dual Tet-inducible promoters following a 24-hour delay in the addition of doxycycline (Dox 24 h p.i.) compared to the simultaneous addition of doxycycline with the HAd (Dox TO). Adenoviral genome copies per well as determined by qPCR for hexon or packaging signal are shown for each condition.

[0066] Figure 9: Replication of DNA-polym erase-mutated helper adenovirus in HEK293 cells by coinfection with AAV-E2B results in a comparable genome copy number to a DNA-polymerase intact AE1 / AE3 helper AdV. Adenoviral genome copies per well as determined by qPCR for hexon are shown for each condition.

[0067] Figure 10: HcAdV expressing CMV-GFP can be recovered and amplified by infecting HEK293A cells with a DNA polym erase-mutated helper adenovirus with LoxP sequences flanking the viral packaging sequence as well as a 1 : 1 ratio of AAV expressing CRE recombinase and AAV expressing adenovirus E2B DNA polymerase. Figure 10A shows a representative fluorescent image for GFP expression in infected cells. Figure 10B shows relative fold change of the percentage of GFP-positive cells for P2 and P3 amplifications compared to Pl following infection of HEK293A cells with varying amounts of HcAdV expressing CMV-GFP clarified lysate.

[0068] DETAILED DESCRIPTION

[0069] This patent incorporates, by reference, any publications cited herein to provide a comprehensive description of the relevant state of the art relating to the present invention. All terms, technical and scientific, used throughout are intended to be understood by their common meanings by those skilled in the relevant field. Additionally, the terminology used in this application is not intended to limit the invention. For example, the term "gene" refers to complementary (c) DNAs, RNA, or other polynucleotides that encode gene products and may be operably associated with one or more transcriptional and / or translational control elements (e.g. an enhancer, promoter, terminator sequence, etc.). Furthermore, the term "foreign or exogenous DNA" refers to a nucleic acid or gene derived from a different cell type or organism from the one in which it is expressed, or nucleic acid from the same cell type or organism that has been positioned from its original position within the genome, or nucleic acid (and associated nucleotide sequences) that is not derived from any cell type or organism, but artificially generated and manufactured by de novo DNA synthesis methodologies. Additionally, for terms such as "nucleic acid," "RNA," "DNA," and others, it is not intended to restrict the types of chemical structures that can be utilized in the invention. It well understood in the field that such chemical structures can be substituted for one another, such as that RNA can often substitute for DNA. Therefore, these terms should be interpreted to include such substitutions. In addition, terms such as “expression” of a gene product includes the cellular gene expression, but also transcription and translation of the DNA in any system or context. The term “recombinases”, “nucleases” and “transposase” are used here interchangeably and encompasses enzymes that induce, mediate or facilitate recombination of a nucleic acid sequence, and other nucleic acid modifying enzymes that cause, mediate or facilitate the rearrangement of a nucleic acid sequence, or the excision or insertion of a first nucleic acid sequence from or into a second nucleic acid sequence. The “recognition site” of a “recombinases”, “nucleases” and “transposase” is the region of the nucleic acid sequence that is recognised and / or act upon (e.g. specifically interacts / binds to and facilitate rearrangement, recombination, excision, cut, or insert a second nucleic acid sequence. The terms “induce”, “induction”, “modulate”, “mediate”, “enable”, “repression”, “repress” and “facilitate” in the context of gene expression refers to the increase or decrease of the synthesis, degradation, availability, or activity, of a given gene product(s). The terms “induce”, “induction”, “modulate”, “mediate”, “enable”, “repression”, “repress” and “facilitate” in the context of replication refers to the increase or decrease of the synthesis, degradation, or availability, of viral genomes or DNA molecules with the given cell. The terms “packaging” and “encapsidation” refers to the process wherein viral genetic element, nucleic acid sequences, or DNA molecules are inserted into capsid or viral particles.

[0070] The adenovirus genome is a linear double-stranded DNA genome of about 36 kb and flanked by -100 bp inverted terminal repeats sequences (ITR) that contain the origins of viral DNA replication (Figure 1). Subsequent to the 5' ITR at the 5' terminus, the cis-acting element packaging signal is located which is required for the packaging and encapsidation of newly- formed adenovirus genomes. Terminal proteins (pTP) are covalent attached at the 5’ end of each ITR sequences and are essential for initiating virus DNA replication (Mei et al. 2003; Pronk and van der Vliet 1993). The adenovirus genome is efficiently organised to maximise coding potential by utilising a strategy of overlapping ORFs and encoding of transcriptional units on both strands of the DNA. The genome encodes over 40 proteins that are sequentially and temporally expressed to meet requirements at different phases of the virus replication cycle (Reddy et al. 1998; Saha, Wong, and Parks 2014). Specifically, adenovirus genes are divided into early or late genes depending on the timing of initiation of transcription, that is prior or post DNA replication of the virus genome, respectively. The genome encodes five early transcription units (designated El A, E1B, E2, E3 and E4) and one late transcription unit that is subdivided into LI to L5 (Fessler and Young 1998). The intermediate-expressed proteins (IX and IVa2) and late proteins are expressed after initiation of viral DNA replication (Vales and Darnell 1989; Pardo-Mateos and Young 2004). The adenoviral ITR sequences, which flanks the virus genome, and packaging signal (positioned at approximately 194 - 358 bp) (Grable and Hearing 1992, J. Virol. 64: 2047-2056) are the only cis elements required for DNA replication and packaging of adenoviral genomes into preformed capsid particles. All other essential sequences within the viral genome are thought to be required for expression of viral genes that act in trans to support the virus replication cycle in production of infectious viruses. Therefore, a HCAdV genome (wherein most of the viral genes and DNA sequences have been deleted, except for the in cis ITR and packaging signal, can be packaged in capsid particles if the essential adenoviral genes (including proteins and RNAs) can be provided in trans by a helper virus to aid in the viral replication process.

[0071] As established previously, a significant challenge to overcome in using helper viruses for production of HCAdV for clinical gene therapy is the elimination of helper virus contaminations of the product. It will be appreciated that the significant advancement as disclosed in the present invention has utility in solving the challenge for elimination of helper adenovirus contamination from HCAdV preparations, while employing an auxiliary helper virus to enable an efficient and scalable manufacturing methodology.

[0072] In a first component of the invention, the helper virus, engineered with recombinase and / or endonuclease recognition sequence(s) at close proximity (1 to 5000 bp) to the viral DNA packaging sequence, is added to the manufacturing cells under conditions in which it cannot undergo DNA replication of its genome (Figure 3). Once inside the cell, a sufficient amount of time (at least 6 hours or as long as 96 hours) is allowed for the excision, cutting, or recombination, to ensure removal of the viral packaging signal DNA sequence from the helper adenovirus genome by endonucleases and recombinases that include, but are not limited to, tyrosine and serine recombinases such as FLP, CRE, phiC31, TP901-1, Lambda Red, I-Scel, Piggybac transposase, Sleeping beauty transposase, and CRISPR gRNA excision. In a third component of the invention, the HCAdV genome is introduced into the cells and genomic DNA replication of the helper virus is enabled to allow expression of helper genes within the packaging cell line for adenovirus capsid particle production and formation, and packaging, or encapsidation, of HCAdV genomes, while preventing the packaging, or encapsidation, of the helper adenoviral genome into preformed adenoviral capsids. The efficiency of removal of the packaging signal sequence from the helper virus may be at least 90%, preferably 98%, more preferably 99.5%, before the adenoviral helper genome is induced to replicate its DNA genome. In one embodiment of the invention, the HCAdV genome is engineered with one, preferentially more than one, LoxP sequence(s), or associated sequence variants, at close proximity (1-5000 bp) or flanking the viral packaging DNA sequence. In another embodiment of the invention, the HCAdV genome is engineered with one, preferentially more than one, LoxP2272 sequence(s) within or flanking the viral packaging DNA sequence. In another embodiment of the invention, the HCAdV genome is engineered with one, preferentially more than one, FRT sequence(s) within or flanking the viral packaging DNA sequence. In another embodiment of the invention, the HCAdV genome is engineered with one, preferentially more than one, attB and / or attP sequence(s) within or flanking the viral packaging DNA sequence. In another embodiment of the invention, the HCAdV genome is engineered with one, preferentially more than one, Lambda Red recognition sequence(s) within or flanking the viral packaging DNA sequence. In another embodiment of the invention, the HCAdV genome is engineered with one, preferentially more than one, I-Scel recognition sequence(s) within or flanking the viral packaging DNA sequence. In another embodiment of the invention, the HCAdV genome is engineered with one, preferentially more than one, Piggybac transposon terminal repeat sequence(s) within or flanking the viral packaging DNA sequence. In another embodiment of the invention, the HCAdV genome is engineered with one, preferentially more than one, Sleeping beauty transposon sequence(s) within or flanking the viral packaging DNA sequence. In another embodiment of the invention, the HCAdV genome is engineered with one, preferentially more than one, CRISPR / Cas9 guide (g)RNA recognition sequence(s) within or flanking the viral packaging DNA sequence.

[0073] In one embodiment of the invention, the HCAdV genome is delivered into the cell by DNA transfection using calcium (eg. calcium phosphate), hydrophilic cationic polymer (eg. Linear polyethylenimine) or cationic lipid-based molecules (eg. lipofectamine), or via electroporation or nucleofection. In another embodiment of the invention, the HCAdV genome is delivered into the cell via infection using a delivery vector including, but not limited to, adenovirus, herpes virus, baculovirus, vaccinia virus vectors.

[0074] In another embodiment of the invention, after a defined length of time (at least 6 hours or as long as 96 hours), DNA replication of both the helper virus genomes and helper-dependent viral genomes are induced, by an external induction factor, such that the genes required for adenoviral replication include, but not limited to, the adenoviral E1A, E1B, E2 DNA polymerase, E2 pTP, E2 DBP and E4orf6 are produced, or is active, at sufficient levels as to allow DNA replication of helper genome and HCAdV genomes to proceed. The external induction factors may include the addition or removal of molecules, such as doxycycline, cumate, estrogenic, or tryptophan, or a change in temperature or light in the environment of the cells, or the delivery of nucleic acid into the cells, via transfection or infection using a viral vector or LNP.

[0075] In a preferred embodiment of the invention, HEK293 cells stably expressing the CRE recombinase are engineered to stably express the adenovirus E2B DNA polymerase protein, and wherein expression of the adenovirus E2B DNA polymerase is under inducible transcription control using the Tet-On gene expression system. The CRE recombinase is typically expressed from a promoter (for example, a promoter containing binding sites for a Cumate activator protein followed by a minimal CMV promoter and then an adenovirus UTR, SEQ ID NO: 46) that is activated when the helper virus enters the cell and expresses a gene capable of activating said promoter. In another embodiment of the invention, expression of the Cre recombinase from the HEK293 cells is under an inducible promoter (for example, the cumate inducible system or Tet inducible system) and wherein its activator protein is also expressed in the cell and its activity function can be induced by addition of a small molecule (for example, doxycycline or cumate) to enable gene transcription and expression of the Cre recombinase. In another embodiment of the invention, expression of the Cre recombinase from the HEK293 cells may also be repressed by a transcriptional repressor (for example, TetR, TetR-E37A-P39K, protocatechuic acid repressor (PcaR), or transcription silencer protein (tTS) wherein a TetR is fused to a KRAB domain) or translation repressor (tryptophan RNA-binding attenuation protein (TRAP) from the TRiP system) present within the cell and expression of the Cre recombinase is induced by addition of a molecule that inhibits the function of the repressor protein (for example, doxycycline to inhibit activity of the TetR) or removal of a molecule (for example, removal of L-tryptophan to alleviate repression from the TRAP) from the cell culture media. The Cre recombinase can be introduced into the HEK293 cells by transfection of plasmid DNA encoding the cre recombinase gene expression cassette, or via infection using viral vectors expressing the Cre recombinase such as AAV, adenoviruses vectors, herpes virus vectors, lentiviral vectors, baculovirus vectors and anellovirus vectors etc. The viral vectors may be bocavirus vectors.

[0076] In another embodiment of the invention, expression of adenovirus E2B DNA polymerase from the HEK293 producer cells is under inducible transcription control using the TetR, Tet-Off, cumate-inducible promoters, PcaR inducible system, or a tryptophan-inducible expression (TRiP) system is used. The Tet On, PcaR inducible system may alternatively be used for inducible transcriptional control. Any other inducible translation control system may alternatively be used. Activator proteins (for example, TetR-VP16 or cumate repressor fused to the VP 16 transactivator (CmyR-VP16)) or repressor proteins (for example, TetR, TetR-E37A- P39K, PcaR, or transcription silencer protein (tTS) wherein a TetR is fused to a KRAB domain) can be expressed in the HEK293 cell line by DNA plasmid transfection or infection using viral vectors such as AAV vectors, adenovirus vectors, herpes virus vectors, lentiviral vectors, baculovirus vectors, bocavirus and anellovirus vectors, to induce expression of the E2B DNA polymerase.

[0077] In another embodiment of the invention, expression of adenovirus E2B DNA polymerase from the HEK293 producer cells is under inducible transcription control using the TetR, Tet-On, Tet- Off, cumate-inducible promoters, PcaR inducible system, or an inducible translation control system such as the tryptophan-inducible expression (TRiP) system, and the activator proteins (for example, TetR-VP16 or cumate repressor fused to the VP 16 transactivator (CmyR-VP16)) or repressor proteins (for example, TetR, TetR-E37A-P39K, PcaR, or transcription silencer protein (tTS) wherein a TetR is fused to a KRAB domain) can be stably expressed from the cell’s chromosome and its gene activation or repressor functions can be induced by addition of a small molecules (for example, doxycycline, cumate, or protocatechuic acid) to enable E2B DNA polymerase expression.

[0078] In other embodiments of the invention, the gene encoding the adenovirus E2B DNA polymerase is delivered into the cell by nucleic acid transfection using calcium (calcium phosphate), hydrophilic cationic polymer (polyethylenimine) or cationic lipid-based molecules (lipofectamine), or via electroporation, or via infection using a delivery vector including, but not limited to, adenovirus, adeno-associated virus, herpes virus, baculovirus, or vaccinia virus vectors. In another embodiment of the invention, infection with viral vectors, such as AAV vectors, can be used to deliver and express the adenovirus E2B DNA polymerase, CRE recombinase and the activator protein (for example, TetR-VP16 or CmyR-VP16) in the HEK293 producer cell line. Natural serotypes such as AAV2, AAV1 and AAV6, or specific engineered AAV capsid serotypes (for example, AAV-DJ) are highly permissive for infection of HEK293 cells and may be used for transgene (for example E2B, CRE recombinase, activator protein) delivery and expression. AAV vectors may be used at relatively low multiplicity of infection (MOI), for example of 50-500 vector genome copies per cell.

[0079] Preferentially, an helper adenoviral vector, E1ZE3 -deleted first generation adenovirus vector, further engineered with LoxP sequences flanking its packaging signal DNA sequence, and deletion of the E2B DNA polymerase gene to render it no longer capable of producing adenoviral DNA polymerase proteins in situ, is used for infection of HEK293 cells at low multiplicity of infection (MOI), preferentially an MOI of 1-10. In another embodiment, the deletion in the helper adenovirus vector is the E2B pTP gene and the HEK293 producer cells are engineered to stably express the E2B pTP under inducible transcription control using the Tet-On gene expression system or the E2B pTP is delivered into the cell via nucleic acid transfection, LNPs or infection using a viral vector. In other embodiments of the invention, the cells used production of the HCAdV is not a HEK293 based cell line, but derived of human origins and engineered wherein expression of the adenoviral El proteins are under inducible control such as TetR, Tet-On, Tet-Off inducible systems, PcaR inducible system, cumate inducible promoters, or a tryptophan inducible expression (TRiP) system.

[0080] In other embodiments of the invention, the helper adenovirus vector is genetically modified at the E2 promoter region to enable inducible control of expression of the E2 proteins, including DNA polymerase, DBP and pTP. Preferentially, tetracycline operator sequences (TetO) are inserted in cis into the E2 transcription unit of the helper adenovirus vector, and the gene encoding the TetR-KRAB fusion protein inserted into the El -deleted region of the helper adenovirus vector. In the absence of doxycycline, the E2 genes are repressed to inhibit replication of the helper adenovirus genome, while the presence of doxycycline enables DNA replication to proceed. Helper viruses encoding the TetR-KRAB fusion protein can be used for infection of HEK293 cells expressing the Cre recombinase, and DNA replication of the helper virus genome is inhibited by binding of the TetR-KRAB to the TetO at the E2 transcription unit. After infection of the HEK293 cells with the helper viruses and allowing a sufficient amount of time (at least 6 hours or as long as 96 hours) for excision of viral packaging signal DNA sequence from the helper adenovirus by Cre recombinase, the HCAdV genome is delivered into the HEK293 producer cells via transfection, or infection with a HCAdV seed stock preparation, and E2B DNA polymerase expression from the cell’s chromosome, or in situ from the helper virus, can be induced by doxycycline to enable replication of both the helper adenoviral vector and HCAdV. The resulting HCAdV vectors can be harvested, and the preparation of packaged HCAdV vectors will contain reduced levels, or be free of, helper virus contaminations, simplifying downstream purification.

[0081] Inhibition of replication of the adenoviral helper genome may be due to inactivation of one or more genes encoding for the adenoviral E1A, E1B, E2B DNA polymerase, E2B Preterminal protein, E2A DNA binding proteins and / or E4orf6. The adenovirus El, E2 and / or E4 genes may be inactivated, optionally wherein one or more of the E2 genes are inactivated by one or more mutations selected from ts 19, ts36, ts69, tsl07, ts 125, tsl49 and sublOOr, more preferably ts36, tsl25 or tsl49, most preferably tsl25. Temperature-sensitive (ts) mutant adenoviruses with mutations mapping to various regions of the E2A and E2B genes, which render the E2 proteins inactive (or less active) at the non-permissive temperature, have been explored extensively to understand the adenovirus replication mechanisms, improve the safety profile of adenoviral vectors for gene therapy and vaccines as it reduces the activity and toxicity of these viral protein, or as a helper virus for complementing recombinant adeno-associated adenovirus (rAAV) vectors replication (Handa, Shiroki, and Shimojo 1975; Araujo et al., Genet Mol Biol 2022; Carter and Blanton, J Virol. 1978; Carter and Gainsberg, J Virol., 1976; McDonough and Rekosh, Virology, 1982). Ts mutants described herein include, but are not limited to, ts 19 (Glu to Arg at residue 154), tsl07 (Pro to Ser at residue 413 and Gly to Asp at residue 353), and tsl25 (Pro to Ser at residue 413 and Ala to Pro at residue 347) of the adenovirus E2A DNA binding protein (as described in Roovers et al., Virus Genes 1990; Levine, A .J., Curr Top Microbiol Immunol. 1984), ts36 (Leu to Phe at residue 239), tsl49 (Leu to Phe at residue 411), and ts69 of the E2B region encoding for the virus DNA polymerase (as described in Nicolas et al., PNAS. 1983; Beverly and Williams, J Virol. 1987; Roovers et al., Virus Genes. 1990), and sublOOr (insertion of 3 nucleotides into the pTP ORF at nucleotide position 10,564, upstream of the first AUG) of the adenovirus E2B pTP (as described in Freimuth and Ginsberg, PNAS. 1986). Engelhardt et al., 1994 also developed a second- generation adenovirus vector by incorporating a temperature- sensitive mutation into the E2A region (tsl25) of an El-deleted adenoviral vector. At the non-permissive temperature, this virus fails to express late gene products and it also showed sustained in vivo transgene expression in the mouse liver up to 70 days, whereas transgene expression from El -deleted adenovirus vector was observed only up to 14 days (Engelhardt et al., Proc. Nati. Acad. Sci. USA. 1994).

[0082] The disclosure of the features of the above Ts mutations in the above-mentioned references is incorporated by reference. Ts mutations representing corresponding mutations at corresponding positions to the above Ts mutations in any adenovirus E2A DNA binding protein, E2B DNA polymerase or E2B Preterminal protein are also described herein, including in proteins from other adenovirus serotypes.

[0083] HCAdVs have a packaging capacity of ~36Kb, making them highly attractive for gene therapy, allowing any human gene to be packaged including combinations of genes to treat complex disorders. The total size of foreign DNA may vary from Ikb to 35kb. Preferably, the total size of foreign DNA is from 15kb to 35 kb. There may be more than one transgene (e.g. 2, 3, 4, or 5 transgenes). The DNA sequence of the transgene(s) may be a coding or non-coding sequence. It may be genomic DNA or cDNA. Preferably, the DNA sequence encodes a polypeptide, more preferably a therapeutic polypeptide. In some cases, the transgene will encode multiple proteins. Preferably, the transgene is operably associated with one or more transcriptional and / or translational control elements (e.g. an enhancer, promoter, terminator sequence, etc.).

[0084] A polypeptide encoded by a transgene could be a human gene or modified form thereof or encode a protein from a virus that infects human cells. Examples of preferred therapeutic polypeptides include cytoplasmic proteins, membrane proteins, CRISPR Cas9, antibodies, CAR-T molecules, scFV, BiTEs, DARPins and T-cell receptors and antigens from human viral pathogens.

[0085] In some embodiments, the therapeutic transgene is the DMD for making a protein called dystrophin. In some embodiments, the therapeutic transgene is the CRISPR Cas9 or a prime editor. In some embodiments, the therapeutic transgene is the MY07A gene for making a protein called myosin VIIA. In some embodiments, the therapeutic transgene is the ABCA4 gene for making the ABCA4 protein. In some embodiments, the therapeutic transgene is the von Willebrand factor gene for making the von Willebrand factor protein. In some embodiments, the therapeutic transgene is the factor VIII gene for making the factor VIII protein. In some embodiments, the therapeutic transgene is the factor IX gene for making the factor IX protein. In some embodiments, the therapeutic transgene is the ALMS1 gene for making the ALMS1 protein. In some embodiments, the therapeutic transgene is the OTOF gene for making the otoferlin protein. In some embodiments, the therapeutic transgene is the GBA1 gene for making the P-Glucocerebrosidase. In some embodiments, the therapeutic transgene is the cadherin-23 gene for making the cadherin-23 protein. In some embodiments, the therapeutic polypeptide is a G-protein coupled receptor (GPCR), e.g. DRD1. In some embodiments, the therapeutic polypeptide is an immunotherapy target, e.g. CD 19, CD40 or CD38. In some embodiments, the therapeutic polypeptide is a functioning copy of a gene involved in human vision or retinal function, e.g. RPE65 or REP. In some embodiments, the therapeutic polypeptide is a functioning copy of a gene involved in human blood production or is a blood component, e.g. Factor IX, or those involved in beta and alpha thalassemia or sickle cell anaemia. In some embodiments, the therapeutic polypeptide is a functioning copy of a gene involved in immune function such as that in severe combined immune-deficiency (SCID) or Adenosine deaminase deficiency (ADA-SCID). In some embodiments, the therapeutic polypeptide is a protein which increases / decreases proliferation of cells, e.g. a growth factor receptor. In some embodiments, the therapeutic polypeptide is an ion channel polypeptide. In some preferred embodiments, the therapeutic polypeptide is an immune checkpoint molecule. Preferably, the immune checkpoint molecule is a member of the tumour necrosis factor (TNF) receptor superfamily (e.g. CD27, CD40, 0X40, GITR or CD137) or a member of the B7-CD28 superfamily (e.g. CD28, CTLA4 or ICOS). In some embodiments the polypeptide encoded is an immune checkpoint molecule is PD1, PDL1, CTLA4, Lagl or GITR. In some embodiments the transgene encodes a virus polypeptide or multiple thereof such as the antigenically important CMV pentamer. In other embodiments, the transgene or transgenes will encode proteins involved in the replication or structure of viruses other than Adenovirus. A gene encoding a polypeptide will also be flanked by regulatory elements known to those in the art to be required for transgene expression, such as a promoter to drive transcription (e.g. the CMV promoter or a promoter derived from the human genome), a 5’ untranslated region, a 3’ untranslated region and a signal for poly adenylation (e.g. the SV40 poly A sequence).

[0086] In some embodiments, the transgene encodes a chimeric antigen receptor. In other embodiments, the transgene encodes a meganuclease, a zinc-finger nuclease or a transcription activator-like effector nuclease. The invention also relates to a cell comprising a nucleic acid sequence encoding a CRE recombinase, , a nucleic acid sequence encoding an adenovirus E2B polymerase, an adenoviral helper genome which lacks a functional E2B polymerase gene and encodes one or more viral genes necessary for replication and / or packaging of a helper-dependent adenoviral vector, a helper-dependent adenoviral genome, wherein the nucleic acid sequence encoding the CRE recombinase and / or the nucleic acid sequence encoding a functional adenovirus E2B DNA polymerase are comprised within an AAV genome or integrated into the genome of the cell, and wherein the CRE recombinase is able to excise a packaging signal from the adenoviral helper genome. The invention further relates to a population of such cells. The CRE recombinase, adenoviral helper genome, helper-dependent adenoviral genome, and adenovirus E2B DNA polymerase may be as described above in relation to a method of producing a helperdependent adenoviral vector of the invention. The cells may be any cell type as described herein, preferably HEK293 cells.

[0087] The nucleic acid sequence encoding a CRE recombinase, the adenoviral helper genome, the helper-dependent adenoviral genome and / or the nucleic acid sequence encoding an adenovirus E2B DNA polymerase may be present in the genome of the cell. The adenoviral helper genome may contain a non-functional E2B gene or may contain an E2B gene deletion. The adenoviral helper genome may be inhibited from replication due to inactivation of E2B polymerase as described herein. The E2B gene may be the only non-functional gene in the adenoviral helper genome.

[0088] In some embodiments, the nucleic acid sequence encoding a CRE recombinase is comprised in an AAV genome, and the nucleic acid sequence encoding an adenovirus E2B DNA polymerase is comprised in an AAV genome. In other embodiments, the cell comprises the nucleic acid sequence encoding a CRE recombinase integrated in its genome, and the nucleic acid sequence encoding an adenovirus E2B DNA polymerase is comprised in an AAV genome. In further embodiments, the cell comprises the nucleic acid sequence encoding a CRE recombinase integrated in its genome and the nucleic acid sequence encoding an adenovirus E2B DNA polymerase integrated in its genome.

[0089] The expression of the CRE recombinase may be inducible by an activator, optionally wherein the activator is a small molecule activator such as doxycycline, or a gene present in the helper adenoviral genome. The expression of the adenoviral E2B DNA polymerase may be inducible by an activator, optionally wherein the activator is a small molecule activator such as doxycycline. The expression of the adenovirus E2B DNA polymerase may be under inducible transcription or translation control. An inducible transcription control system may comprise TetR, Tet-On, Tet-Off, a cumate-inducible promoter, or a PcaR inducible system. An inducible translation control system may comprise the tryptophan-inducible expression (TRiP) system.

[0090] The cell may further comprise a nucleic acid sequence encoding an activator protein or a repressor protein for the inducible transcription or translation control system, optionally wherein the activator protein is TetR-VP16 or cumate repressor fused to the VP 16 transactivator (CmyR-VP16) or the repressor protein is TetR, TetR-E37A-P39K, PcaR, or a transcription silencer protein (tTS) wherein a TetR is fused to a KRAB domain. The nucleic acid sequence encoding an activator protein or a repressor protein for the inducible transcription or translation control system may be introduced into the cell by stable chromosomal integration, DNA plasmid transfection or by infection using viral vectors such as AAV vectors, adenovirus vectors, herpes virus vectors, lentiviral vectors, baculovirus vectors and anellovirus vectors.

[0091] The invention further relates to a kit comprising a nucleic acid sequence encoding a CRE recombinase comprised within an AAV genome, an adenoviral helper genome which lacks a functional E2B polymerase gene and encodes one or more viral genes necessary for replication and / or packaging of a helper-dependent adenoviral vector, a helper-dependent adenoviral genome, and a recombinant cell containing a nucleic acid sequence integrated into its genome encoding a functional adenovirus E2B DNA polymerase, wherein the CRE recombinase is able to excise a packaging signal from the adenoviral helper genome. The invention additionally relates to a kit comprising a recombinant cell containing a nucleic acid sequence encoding a CRE recombinase integrated into its genome, a nucleic acid sequence encoding an adenoviral helper genome which lacks a functional E2B polymerase gene and encodes one or more viral genes necessary for replication and / or packaging of a helper-dependent adenoviral vector, a nucleic acid sequence encoding a helper-dependent adenoviral genome, and a nucleic acid sequence encoding a functional adenovirus E2B DNA polymerase comprised within an AAV genome, wherein the CRE recombinase is able to excise a packaging signal from the adenoviral helper genome. The invention further relates to a kit comprising a nucleic acid sequence encoding a CRE recombinase comprised within an AAV genome, a nucleic acid sequence encoding an adenoviral helper genome which lacks a functional E2B polymerase gene and encodes one or more viral genes necessary for replication and / or packaging of a helperdependent adenoviral vector, a nucleic acid sequence encoding a helper-dependent adenoviral genome, and a nucleic acid sequence encoding a functional adenovirus E2B DNA polymerase comprised within an AAV genome, wherein the CRE recombinase is able to excise a packaging signal from the adenoviral helper genome.

[0092] A kit described above may be suitable for production of a cell as described above. The components of the kit may be as described above in relation to the cell of the invention. The kit may further comprise a nucleic acid sequence encoding an activator protein or a repressor protein for an inducible transcription or translation control system.

[0093] The invention additionally relates to a method of producing a helper-dependent adenoviral vector in a population of cells wherein the cells comprise a nucleic acid sequence encoding a CRE recombinase, a nucleic acid sequence encoding a functional adenovirus E2B polymerase, an adenoviral helper genome which lacks a functional E2B polymerase gene and encodes one or more viral genes necessary for replication and / or packaging of a helper-dependent adenoviral vector, a helper-dependent adenoviral genome, wherein the nucleic acid sequence encoding the CRE recombinase and / or the nucleic acid sequence encoding a functional adenovirus E2B DNA polymerase are comprised within an AAV genome or integrated into the genome of said cells, wherein the CRE recombinase excises a packaging signal from the adenoviral helper genome, and wherein replication of the adenoviral helper genome provides for replication and / or packaging of the genome of the helper-dependent adenoviral vector. The CRE recombinase, adenoviral helper genome, helper-dependent adenoviral genome, and adenovirus E2B DNA polymerase may be as described above in relation to the cell of the invention.

[0094] FURTHER EMBODIMENTS

[0095] The invention also provides:

[0096] 1. A method of producing a helper-dependent adenoviral vector in a population of cells, wherein said cells comprise an adenoviral helper genome which encodes one or more viral genes necessary for replication and / or packaging of the helper-dependent adenoviral vector, wherein replication of the adenoviral helper genome is inhibited while excising a packaging signal sequence therefrom, and wherein replication of the adenoviral helper genome is induced to provide for replication and / or packaging of the genome of the helper-dependent adenoviral vector.

[0097] 2. The method according to embodiment 1, wherein replication of the adenoviral helper genome is induced after excising the packaging signal sequence from the adenoviral helper genome.

[0098] 3. The method according to embodiment 1 or 2, wherein the adenoviral helper genome is introduced into said cells prior to, simultaneously with or subsequently to the helper-dependent adenoviral vector, optionally by transfection, electroporation, a non-viral lipid nanoparticle, or viral infection.

[0099] 4. The method according to any one of the preceding embodiments, wherein the cells are incubated under conditions inhibiting replication of the adenoviral helper genome for a sufficient period to substantially remove the packaging signal sequence from the adenoviral helper genome.

[0100] 5. The method according to embodiment 4, wherein the incubation is for at least 6 hours, preferably at least 12 hours, more preferably at least 24 hours, optionally wherein the incubation is for 12-48 hours.

[0101] 6. The method according to any one of the preceding embodiments, wherein the cells comprise one or more adenovirus early gene regions integrated into their genome, optionally selected from El, E2 and / or E4 regions, optionally wherein the cells further comprise one or more intermediate or late gene regions integrated into their genome.

[0102] 7. The method according to any one of the preceding embodiments, wherein the cells are mammalian cells, optionally HEK293 cells, A549, HeLa or amniocyte -based cells, or derivatives of any thereof, such as PerC6 cells, 911 cells, HEK293-E2 cells, HEK293-E4orf6, HEK293-L4 100K or HEK293-L4 22 / 33K cells.

[0103] 8. The method according to any one of the preceding embodiments, wherein the inhibition of replication of the adenoviral helper genome is due to inactivation of one or more genes encoding for the adenoviral E1A, E1B, E2B DNA polymerase, E2B Preterminal protein, E2A DNA binding proteins and / or E4orf6.

[0104] 9. The method according to embodiment 8, wherein the inactivation of a said gene is by deletion, mutation or insertion of an exogenous nucleic acid sequence, or by prevention of expression, optionally by use of a KRAB-Zinc Finger Protein, an siRNA / shRNA, or CRISPR / dCas9. 10. The method according to embodiment 9, wherein the inactivation of a said gene is due to mutation, deletion, or insertion of an exogenous nucleic acid sequence in the adenovirus El, E2 or E4 promoters.

[0105] 11. The method according to embodiment 10, wherein the adenovirus El, E2 and / or E4 genes are inactivated, optionally wherein one or more of the E2 genes are inactivated by one or more mutations selected from tsl9, ts36, ts69, tsl07, tsl25, tsl49 and sublOOr, more preferably ts36, ts 125 or tsl49, most preferably ts 125.

[0106] 12. The method according to any one of the preceding embodiments, wherein transcription from the El, E2 or E4 promoter is inhibited, optionally wherein the transcription is inducible, such as by use of TetR, Tet-On, Tet-Off, cumate-inducible gene expression, tryptophan- inducible gene expression, IPTG-inducible gene expression, CRISPR-dCas gene induction, TALE / ZINC finger repression, or a Nuclease splicing gene expression system.

[0107] 13. The method according to any one of embodiments 1-12, wherein replication of the adenoviral helper genome is induced by expression of one or more genes necessary for replication of the adenoviral helper genome in the cells, optionally wherein the genes are functional versions of one or more genes inactivated in the adenoviral helper genome, optionally wherein the one or more genes are introduced by transfection, electroporation, a non- viral lipid nanoparticle, or viral infection.

[0108] 14. The method according to embodiment 13, wherein the expression of the one more genes is induced by a small molecule activator, such as tetracycline, doxycycline, cumate, tryptophan, or Isopropyl P- d-1 -thiogalactopyranoside, optionally, wherein the expression of the one more genes is induced by a change in temperature or light conditions.

[0109] 15. The method according to any one of the preceding embodiments, wherein the helper adenoviral genome encodes any or all of the early transcription units, optionally selected from early region 1A (E1A), E1B, E2, E4 and VA RNA, any or all of the Intermediate transcription units, optionally selected from IX, IVa2, and L4 intermediate, and any or all of the late transcription units optionally selected from Hexon, Penton, Fibre, and 100K.

[0110] 16. The method according to any one of the preceding embodiments, wherein the packaging signal sequence comprises any one of SEQ ID NOs 17-23, optionally SEQ ID NO: 18 or 19, preferably SEQ ID NO: 19.

[0111] 17. The method according to any one of the preceding embodiments, wherein said cells express an enzyme capable of excising the packaging signal sequence from the adenoviral helper genome, optionally wherein the packaging signal is flanked by excision sites or recombination recognition sites for the enzyme, optionally wherein expression of the enzyme is induced by an activator encoded in the adenoviral helper genome.

[0112] 18. The method according to embodiment 18, wherein the enzyme is an endonuclease or a recombinase, optionally selected from CRE, FLP, phiC31, TP901-1, Lambda Red, I-Scel, Piggybac transposase, or Sleeping beauty transposase, optionally wherein the enzyme comprises the amino acid sequence of any one of SEQ ID NOs 10-16 or a variant thereof, optionally SEQ ID NO: 10 or SEQ ID NO: 11 or a variant of either thereof, preferably SEQ ID NO: 10 or a variant thereof.

[0113] 19. The method according to any one of the preceding embodiments, wherein the helperdependent adenoviral vector comprises a genome comprising one or more transgenes, wherein the transgenes may comprise non-coding or coding sequences, optionally wherein a said transgene is a human gene, a reporter gene or encodes a therapeutic product, such as an shRNA, CRISPR guide RNA, or siRNA or a polypeptide, preferably wherein the transgene encodes a therapeutic polypeptide, optionally wherein the one or more transgenes comprise a DMD, MY07A, Abca4, COL4A5, von Willebrand factor, factor VIII, factor IX, cadherin-23, ALMS1, OTOF, GBA1, CRISPR guide RNA, or a CRISPR Cas9 transgene, optionally more than one of said transgenes, optionally wherein the one or more transgenes are positioned 3’ to a packaging signal sequence in the genome of the helper-dependent adenoviral vector.

[0114] 20. The method according to any one of the preceding embodiments, wherein the genome of the helper-dependent adenoviral vector comprises inverted terminal repeat sequences, optionally selected from any of SEQ ID NOs 24-30, preferably SEQ ID NO: 25 or 26, most preferably SEQ ID NO: 26.

[0115] 21. The method according to any one of the preceding embodiments, wherein the helper adenoviral and / or helper-dependent adenoviral genome is least lOkb, more preferably at least 20kb, and most preferably at least 30kb in length.

[0116] 22. The method according to any one of the preceding embodiments, wherein the helperdependent adenovirus produced according to the method is used to introduce the helperdependent adenovirus genome into the population of cells.

[0117] 23. A helper-dependent adenoviral vector preparation obtainable according to the method of any one of the preceding embodiments.

[0118] 24. A population of cells producing a helper-dependent adenoviral vector obtainable according to the method of any one of the preceding embodiments.

[0119] 25. A method of reducing helper virus contamination in a helper-dependent adenoviral vector preparation, comprising producing a helper-dependent adenoviral vector in a population of cells, wherein said cells comprise an adenoviral helper genome which encodes one or more viral genes necessary for replication and / or packaging of the helper-dependent adenoviral vector, wherein replication of the adenoviral helper genome is inhibited while excising a packaging signal sequence therefrom, and wherein replication of the adenoviral helper genome is induced to provide for replication and / or packaging of the genome of the helper-dependent adenoviral vector, optionally wherein the helper-dependent adenoviral vector is produced by a method according to any one of embodiments 1-22.

[0120] EXAMPLES

[0121] Example 1: Protocol for production and purification of the helper adenoviruses.

[0122] Production of the helper adenovirus with LoxP sequences flanking the viral packaging sequence is carried out as follows. The sequence fragment encoding the 5' end (nucleotides 1- 197) of an adenovirus type 5 (GenBank: AC_000008), with loxP sequences (SEQ ID NO1) flanking its packaging signal (nucleotides 197-341) is generated by de novo synthesis and cloned into an adenoviral genome plasmid (OGS268, Sigma Aldrich, USA) by molecular cloning and transformed in XLl-Blue competent Escherichia coli (E. coli) (Agilent, USA). Similarly, the helper adenovirus is further modified to incorporate the ts36 temperature sensitive mutation by molecular cloning and Gibson assembly methods as disclosed in Su et al. Sci Rep. 2023; 13: 21670. For bacterial transformation with the plasmid DNA, an aliquot (100 pL) of competent cells is thawed on ice and added to plasmid DNA (10 - 500 ng). Single colonies are picked and grown in LB agar containing 50 g / mL of Kanamycin Sulfate (Sigma- Aldrich, USA) and DNA plasmids extracted from the E. coli using a miniprep or midiprep DNA extraction kit. Positive recombinant plasmids are screened by Sanger sequencing and further propagated in E. coli and plasmid DNA extracted using a maxiprep DNA extraction kit. Adenoviral vectors are recovered in HEK293 cells from plasmid DNA encoding each viral genome. For the viral recovery process, plasmids (20 pg) are linearised with Swal restriction enzyme to release virus ITRs from the bacterial plasmid backbone and purified using genomic Purelink DNA extraction kit (Invitrogen, USA) or DNeasy Blood & Tissue Kit (Qiagen, UK). HEK293 cells can be seeded in T25 tissue culture flasks, at a density of 2E+6 cells per flask, for 24-hours before transfection. Each flask is transfected with 2.5 pg of linearised DNA suspended in 300 pL of OptiMEM (Life Technologies, UK) and complexed using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s protocol. After 4-6 hours, transfection media is exchanged with fresh DMEM containing 2% FBS. Recombinant viruses are harvested from growth media -12-15 days post-transfection upon observation of advanced Cytopathic effect (CPE). To note, for the production of helper adenoviruses harbouring the ts36 temperature sensitive mutation, the HEK293 cells are cultured at 32° C, the permissive temperature, after plasmid transfection to enable function of the adenovirus DNA polymerase in facilitating viral DNA replication. Cells and growth medium are collected and subjected to three rounds of freeze-thaw cycle by transferring between a 37°C incubator and a -80°C freezer. Cellular debris is pelleted by centrifugation at 2500 x g for 20 minutes and supernatant passed through a 0.2 pm fdter and stored at -80°C. Adenoviral vectors are then amplified by a further two rounds in HEK293 cells.

[0123] For large-scale virus amplification, HEK293 cells are seeded in Corning HYPERFlask (SigmaAldrich, MO, USA) for 48-hours so that they are -95% confluent before infection. Growth medium is added to replenish with fresh DMEM containing 2% FBS with viral stocks (purified or crude lysates). Cells are harvested upon display of advanced CPE, usually observed at 48-72 house post infection (hpi), and resuspended in -16 mL of growth medium. Viruses are released by three rounds of freeze-thaw using a 37°C incubator and a -80°C freezer. Lysates are incubated with water- saturated n-butanol (diluted 1:100) for 60 minutes at 4°C and cell debris pelleted by centrifugation at 2500 g for 20 minutes. The supernatant is collected for purification by a caesium chloride (CsCl) gradient. In brief, the CsCl is prepared in ultra-clear centrifuge tubes (Beckman Coulter UK Ltd) and consists of 3 mL of 1 :32 p CsCl (32.0g CsCl, 6.8 mL of 0.5 M Tris-HCl at pH 7.9, 61.2 mL dH2O), underlaid with 2 mL of 1 :45 p CsCl (20.5g CsCl, 2.9 mL 0.5 M Tris-HCl at pH 7.2, 28.5 mL dH2O), and 2 mL of PBS 40% Glycerol can be added on top of the gradient. Approximately 4.5 mL of viral supernatant is added on top of each ultra-clear centrifuge tube. The gradients are centrifuged (30,000 g, 120 minutes, 10°C) using a Thermo Sorvall WX90 plus ultracentrifuge with a TH-641 rotor (ThermoFisher, USA). After centrifugation, two discrete bands are obtained - intact virus particles are presented in the lower band, while the upper band consists of incomplete / empty capsids. Viruses are extracted from the lower band by puncturing the centrifuge tube with a 21 g needle attached to a 6 mL syringe. Virus mixture is injected into a 10K molecular weight cut off Slide-A-Lyzer dialysis cassette (ThermoFisher, UK) and CsCl is removed by four-rounds of dialysis using -500 mL of PBS in each round. Viruses are extracted from the dialysis cassette using a 21 g needle and a syringe and subsequently treated with benzonase 250 U / mL (Sigma- Aldrich, MO, USA), supplemented with magnesium chloride (2 mM), for 1 hour at room temperature. After treatment, the virus mixture is re-injected into a SlideA-Lyzer cassette for three further rounds of dialysis using PBS. The final dialysis step is carried out using PBS containing 10% glycerol and lOmM HEPES buffer (Sigma-Aldrich, USA). Viruses are extracted and stored at -80°C.

[0124] This process is used to generate helper adenovirus wherein the E2B DNA polymerase gene has been deleted by molecular cloning methods. For deletion of the E2B DNA polymerase from helper adenovirus genome (OGS268, Sigma Aldrich, USA), this process is carried out by molecular cloning methods using a series of shuttle plasmids. To carry out such modifications, PCR primers sets are designed to amplify DNA sequences upstream and downstream of the E2B DNA polymerase gene, but omitting the DNA polymerase coding DNA sequences from the amplification. These two PCR-generated fragments (E2B upstream & E2B downstream) are then cloned into the multiple cloning site (MCS) of a standard DNA plasmid (OGS1, Sigma Aldrich, USA) to generate an intermediate E2B shuttle plasmid. Restrictions sites that are recognised by restriction enzymes, such as Bsal or BseRI, that cuts away from the recognition sites are incorporated into the primer design to allow seamless extraction of the E2B fragment from the shuttle plasmid. Sequence integrity of this shuttle plasmid is confirmed by Sanger sequencing or Oxford Nanopore sequencing technology. The E2 region, with deletion of the DNA polymerase, is then extracted from the shuttle plasmid by restriction enzymes, such as Bsal or BseRI, and cloned into the adenovirus genome (OGS268, Sigma Aldrich, USA), via Gibson Assembly, wherein also the unmodified E2 region from the adenoviral genome is extracted using convenient restriction enzymes. Successful deletion of the E2B DNA polymerase from the adenovirus genome plasmid is confirmed using Oxford Nanopore sequencing technology. For production and purification of E2B -deleted helper adenovirus, genome plasmids are transfected into HEK293 cells stably expressing the adenovirus E2B DNA polymerase, and the cells are cultured with doxycycline to enable expression of the DNA polymerase and viral replication.

[0125] Example 2: Protocol for creating stable cell lines containing a DNA encoding an adenovirus inducible E2B DNA polymerase

[0126] In order to establish a stable cell line expressing DNA molecules encoding the adenovirus E2B DNA polymerase under control of a Tet-On inducible promoter, HEK293 cells are seeded in Dulbecco's Modified Eagle (DMEM) media (10% FCS, 1% penicillin / streptomycin) into T25 flasks (10 cm or 6 cm dishes may also be used) 24 hr hours prior to transfection so as to be at 80% confluent at time of plasmid transfection. Cells are then transfected using the PEI method. Briefly, the transfection mixture consists of 15 pg plasmid DNA in a 1:3 ratio with Branched PEI (25 KDa) respectively, which is then added to two vials of 150 ul of DMEM (2% fetal calf serum (FCS)) Optimem media. Other media may be used. Preferably the media used is Optimem to complex the DNA and is free of both FCS and Penicillin and / or Streptomycin. The media / DNA mix and the media / PEI mix are then combined and incubated for 20 minutes at room temperature to allow complex formation. At the time of transfection, the pre-existing media in which the cells have been seeded is removed by aspiration and changed for fresh DMEM media containing 10% FCS. Transfection mixtures are then added drop-wise into the flasks and gently swirled to evenly distribute the transfection complexes in the media. 24 hours post-transfection, the media and transfection mixtures in each flask is removed by aspiration and replaced with DMEM (10% FCS, 1% penicillin / streptomycin) media containing puromycin which is then added to the flasks at varying concentrations to determine the optimal antibiotic concentration required. For each DNA plasmid being tested, one flask is maintained at each of the following concentrations: 0 pg / ml, 0.5 pg / ml, 1.5 pg / ml, 3 pg / ml, 5 pg / ml, 7.5 pg / ml and 10 pg / ml puromycin per ml of growth media.

[0127] Over the next 4 weeks, media in the flasks are changed every 3-4 days, maintaining the same concentrations of puromycin relevant to each flask, and the flasks are continuously evaluated for cell death and formation of cell foci / colonies. Formation of foci from single surviving cells is typically observed in the flasks maintained at either 3 pg / ml and 5 pg / ml puromycin in flasks transfected with plasmids containing the DNA molecules encoding E2B DNA polymerase and selection gene. After 4 weeks, two alternative approaches are taken. The contents of each of flask are processed as follows: 1. Gently trypsinized and passaged into larger T75 flasks, to make mixed population cell lines, maintaining the same puromycin concentration for each line (this is then be followed by single cell isolation by limited dilution or FACS to identify single monogenic cell lines); or 2. Single colonies are encircled by a polycarbonate ring fixed temporarily to the flasks surface with sterile grease, followed by trypsinisation and colony isolation from within the ring and transfer to a 6-well plate containing the same media containing puromycin. FACS is the more preferable method of clonal cell line isolation. After cells derived from colonies reach sufficient confluence, they are passaged with a 5 -fold dilution; the remaining cells are tested for the integration of the DNA by QPCR and expression of the adenovirus DNA polymerase gene by the addition of doxycycline. Measurement of the adenovirus DNA polymerase gene expression is carried out by detecting mRNA expression by reverse-transcription (RT) qPCR or protein expression by Western blotting analysis. The cell lines are maintained from this point onwards at the same concentration of puromycin as originally selected in, or the puromycin can be withdrawn. Cell banks are created and stored at -170° C. using the cells remaining from each passage. In some instances, it is possible to increase the expression of the E2B DNA polymerase by increasing the puromycin concentration in 1-2 pg / ml increments, selecting for only the cells expressing the highest quantity puromycin resistance. Cell lines are created in the same way as described above but also using transposons. In this method, the approach is the same as described above, except that the DNA encoding the Tet-inducible adenovirus E2B DNA polymerase expression cassette is flanked by the transposon ITRs and a plasmid encoding a transposase that binds the ITRs is simultaneously introduced into the cell line. Cell lines are also created in the same way as described above but using a lentiviral vector to introduce the Tet-inducible adenovirus E2B DNA polymerase expression cassette. Using a lentiviral vector containing the DNA encoding the adenovirus E2B DNA polymerase means that the cells are infected with the lentiviral vector rather than transfection. After 24 hours post infection, cells are selected in the same way as described above provided that the lentiviral vector contains a selection gene such as puromycin resistance. Furthermore, using these above described processes, the Tet-inducible adenovirus E2B DNA polymerase expression cassette is also introduced into HEK293 cells stably expressing the Cre recombinase (HEK293 / Cre stable cell line, FenicsBio CL- 1586), or FLP recombinase (Gentarget Inc, Cat# SC006) to generate cell lines expressing both the Adenovirus DNA polymerase and CRE or FLP recombinase, respectively.

[0128] Example 3: Infection of a cell line expressing the adenovirus E2B DNA polymerase

[0129] Cells containing a stably-integrated E2B DNA polymerase that enables replication of a DNA polymerase-deficient helper adenovirus (Pol-A AdV helper) are achieved by introduction of the Pol-A AdV helper into cells via a suitable delivery method. This could be by transfection, electroporation or infection via viral vectors and non-viral lipid nanoparticles. HEK293 cells stably containing the E2B DNA polymerase are seeded in suspension at 1.5E+6 cells / mL in a shake flask of appropriate size e.g. a 250 mL unbaffled polypropylene shake flask, using an appropriate media e.g. BalanCD or CD293 media. Cells are then transfected or infected with a Pol-A AdV helper. Wherein expression of the E2B DNA polymerase is inducible by the Tet- On system, cell culture media is supplemented with doxycycline or tetracycline at a concentration of 1 pg / mL to induce expression of the DNA polymerase. The production of the Pol-A AdV helper is then monitored by testing the cell supernatant or cell lysate by either qPCR.

[0130] Example 4: Quantification of total adenoviral genomes

[0131] For quantification of total adenovirus genomes in HEK293 cells, total DNA is extracted from culture media and cellular lysates using Purelink genomic DNA miniprep kit (Invitrogen, CA, USA). Five microlitres of DNA eluent is used in qPCR reactions using TaqMan Fast Advanced Master Mix (Applied Biosystems, CA, USA) in a StepOnePlus Real-Time PCR System (Applied Biosystems, CA, USA).

[0132] Example 5: Quantification of encapsulated adenoviral genomes

[0133] For quantification of genome encapsulated adenovirus particles, 2 pL of viral samples, is harvested from culture medium or cell lysates, and treated with 1U of TURBO DNase (ThermoFisher Scientific, MA, USA) in a 20 pL reaction for 2-hours at 37° C. TURBO DNase is heat-inactivated at 75° C for 10-minutes. Five microlitres of samples are diluted at 1:200 using nuclease-free water and used in the PCR reaction to quantify encapsulated Ad5 using Ad5 hexon primers and probe. Standard curves for qPCR analyses are generated using a gBlock gene fragment suspended in nuclease-free water (Integrated DNA Technologies, IA, USA) and CT values of PCR reaction are used to calculate DNA copy number by extrapolation to the standard curves (a qPCR standard of 3E+8 - 3E+10 copies / well).

[0134] Example 6: TCID50 assay for determining infectious adenovirus

[0135] HEK293 cells are seeded in 96-well tissue culture plates at a density of 1E+4 cells per well for 24 hours. Eight 10-fold serial dilutions of each adenovirus stock are made in DMEM containing 2% FBS at a total volume of 1.2 mL. Ten replicates of each diluted sample (1 x 10-5 to 1 x 10-12) are added at a volume of 100 pL per well on each plate. 100 pL of DMEM containing 2% FBS is added to the final two columns as a negative control. Plates are monitored over 12 days for the presence for viral plaques or EGFP expression under a brightfield microscope and fluorescence microscope, respectively. Positive infection events in different wells are also determined by qPCR detection using primers and probes directed against the adenovirus genomes or transgene. Infectious adenovirus are determined as TCID50 per mL using the KARBER-SPEARMAN statistical method.

[0136] Example 7: Adenovirus cell internalisation assay

[0137] HEK293 cells are seeded in 48-well tissue culture plates at 7.5E+4 for 24 hours. Cells are counted before infection and are infected with adenoviral vectors or HCAdV at various multiplicity of infection (MOI) doses (MOI 1 - 100). At 6 hours post infection, cells are trypsinised and extensively washed for three rounds with PBS before extraction of total DNA using a DNeasy Blood & Tissue Kits (Qiagen, Venlo, Netherlands) and viral genomes are quantified by qPCR.

[0138] Example 8: Production of HCAdV vectors from HEK293 cells expressing CRE and inducible E2B DNA polymerase

[0139] For production of HCAdV vectors, plasmid DNA encoding the HCAdV genome and transgene expression cassette, such as a HCAdV expressing the EGFP reporter (Vector ID: VB010000- 9299hac, VectorBuilder, USA), are linearized with PacI restriction enzymes to release the bacterial plasmid backbone and purified using genomic Purelink DNA extraction kit (Invitrogen, USA) or DNeasy Blood & Tissue Kit (Qiagen, UK) to use for transfection. HCAdV genomes plasmids encoding various transgenes such as DMD, MY07A, Abca4, COL4A5, or a CRISPR Cas9 gRNA expression cassette are constructed by de novo DNA synthesis of the transgene DNA, and associated regulatory elements, and inserted into the HCAdV genome by Gibson Assembly or standard molecular cloning methods using restriction enzymes. HEK293 cells stably expressing the CRE recombinase and Tet-inducible E2B DNA polymerase (HEK293-CRE-Tet-E2BPol), are seeded in T25 tissue culture flasks, at a density of 2E+6 cells per flask, for 24-hours before transfection. Each flask is infected with the Pol-A adenovirus helper at a MOI of 3, and transfected with 2.5 pg of linearised HCAdV genome suspended in 300 pL of OptiMEM (Life Technologies, UK) and complexed using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s protocol. After 24 hours, transfection / infection media is exchanged with fresh DMEM containing 2% FBS and supplemented with doxycycline (1 pg / mL) to induce expression of the E2B DNA polymerase from the cell to facilitate replication of the Pol-A adenovirus helper, after CRE-mediated excision of the packaging signal, and to aid replication and packaging of the HCAdV genomes. Recombinant viruses are harvested from growth media at 3-5 days post-transfection upon observation of advanced CPE. HCAdV and helper adenovirus genomes are quantified as described in Example 4-6, to determine the quantity of total, encapsidated genomes, and TCID50 infectious titre, respectively. HCAdV is further propagated to a high titre in the HEK293-CRE-Tet-E2BPol cells using a similar process as described above, except that the HCAdV genomes are delivered by infection. In this amplification process, HEK293-CRE-Tet- E2BPol are co-infected with both the HCAdV and the Pol-A adenovirus helper, each at an MOI of 3. After 24 hours, the infection media is exchanged with fresh DMEM containing 2% FBS and supplemented with doxycycline (1 pg / mL) to induce expression of the E2B DNA polymerase for replication of the Pol-A adenovirus helper and HCAdV genomes. Using this process the HCAdV is sequentially amplified and purified by CsCl gradient ultracentrifugation to high titre and concentration as described in Example 1.

[0140] Example 9: HCAdV purification by ion-exchange chromatography

[0141] HCAdV vectors are also effectively purified by anion-exchange chromatography, which is performed in AKTA Basic low-pressure liquid chromatography system. Clarification of the cell lysate is carried out using an ULTRA PRIME GF 5 pm 6” (KGF-A-0506GG, GE Healthcare Life Sciences) and ULTA PRIME CG 4” (KMP-CG9204GG, GE Healthcare Life Sciences) at a flux of 600 LMH. Viral vectors can be concentrated by tangential flow filtration (TFF) using a 750 kDa hollow fiber with a membrane area of 290 cm2 (UFP-750-C-3X2MA, GE Healthcare Life Sciences). Purification is carried out using a Hiscale XK50 / 20 column (28988952, GE Healthcare Life Sciences) packed with 120 mL of Capto Q impRes resin (17547301, GE Healthcare Life Sciences). To equilibrate the column, 50 mM Hepes, 150 mM NaCl, pH 6.5 is used. Viral vector elution is performed with 50 mM Hepes, IM NaCl, pH 6.5, and the fractions can be pooled and diluted using 20 mM Tris, 25 mM NaCl, pH 8. For polishing, an XK50 / 60 column (28988951, GE Healthcare Life Sciences) packed with 300 mL of Sepharose 4 FF (17014901, GE Life Sciences) is used. The run is performed at a flow rate of 12 cm / h and a using buffer containing 20 mM Tris, 25 mM NaCl, pH 8. The vector materials are pooled and formulated with 20 mM Tris, 25 mM NaCl, 25% glycerol (v / v), pH 8, to a final glycerol concentration of 2.5% (v / v), and sterile filtered.

[0142] Example 10: AAV production via triple transfection HEK293A cells were used to produce AAV expressing CRE recombinase (AAV-CRE) (SEQ ID NO: 32) or adenovirus E2B (AAV-E2B) (SEQ ID NO: 33), where either gene is regulated by the CMV promoter (SEQ ID NO: 34). HEK293 cells were seeded in two T175 tissue culture flasks, at a density of 1E+7 cells per flask and left to adhere overnight. The following day, cells were transfected with an equimolar ratio of a plasmid encoding AAV rep and cap genes (pTrans), an AAV transfer plasmid containing AAV ITRs flanking a GOI of interest along with sequences required for the GOI expression (pCis) and a plasmid encoding adenoviral helper genes required for AAV replication (pHelper) in 5% DMEM. A 1:3 molar ratio of DNA:PEI (PEI Pro) was used for the transfection according to the manufacturer’s (Polyplus) protocol. Following a 72-hour incubation, the supernatant was discarded, and the cells were collected in AAV lysis buffer (150 mM NaCl, 50 mM Tris-HCl pH 8.5, and 2 mM MgCh in PBS). Following three freeze-thaw cycles, the supernatant was clarified, aliquoted, and the titre of the AAV was determined by qPCR using primers specific to the inverted terminal repeat of the AAV genome sequence.

[0143] The data in Table 1 below shows the average titres of AAV expressing CMV-Cre and AAV expressing CMV-E2B.

[0144] Table 1

[0145] Example 11: Excision of helper adenovirus packaging signal in HEK293 cells by viral vector co-infection using E2B mutated helper adenovirus, AAV expressing CRE (AAV- CRE) and AAV expressing adenovirus E2B (AAV-E2B).

[0146] HEK293A cells were used to produce AAV expressing CRE recombinase (AAV-CRE) (SEQ ID NO: 32) or adenoviral E2B (AAV-E2B) (SEQ ID NO: 33) under control of the CMV promoter (SEQ ID NO: 34) within the AAV ITRs (SEQ ID NO: 31) via triple transfection. An E2B polymerase-mutated helper adenovirus (Pol-A AdV helper) (SEQ ID NO: 36) incapable of replicating its own genome and engineered with LoxP sequences flanking the viral packaging sequence (SEQ ID NO: 35) (HAd) was recovered and propagated in HEK293A cells stably expressing the adenovirus E2B (SEQ ID NO: 42). The titre was determined via qPCR with primers and a probe targeting the adenovirus hexon gene. To determine excision of the the packaging signal from the helper adenovirus vector, HEK293A cells were seeded in 10% DMEM at a density of 7.5E+4 cells in a 48-well plate and left to adhere overnight. The following day, they were co-infected with the HAd and AAV-CRE at varying MOIs in 2% DMEM. AAV-E2B was added simultaneously (TO) for co-infection with the (HAd and AAV- CRE, or was added to the HEK293 A cells 24 hours later 24-hours (T24). Cells were incubated for either 2-, 72-, or 96-hours post-infection with the HAd followed by total DNA extraction. qPCR with primers and a probe targeting either the hexon gene or the adenoviral packaging signal was used to determine the number of intact vs CRE recombined adenoviral genome (i.e adenoviral genomes wherein the packaging signal was removed / spliced by Cre) copies per well for each condition.

[0147] The data in Figure 4 shows enhanced excision of the packaging signal (i.e reduced packaging of Pol-A AdV helper into adenoviral capsids) from the HAd following a 24-hour delay in the addition of AAV-E2B (T24) compared to co-infection of the AAV-E2B with the HAd and AAV-CRE (TO). The data also demonstrate that the HAd does not replicate in the absence of exogenous E2B, and that despite the 24-hour delay in the addition of the polymerase, the amount of HAd genome replication, as determined by hexon qPCR, was comparable to the TO condition.

[0148] Example 12: Excision of helper adenovirus packaging signal in HEK293 cells stably expressing Cre recombinase and adenovirus E2B from inducible promoters.

[0149] A DNA polymerase-mutated helper adenovirus (Pol-A AdV helper) (SEQ ID NO: 36) with LoxP sequences flanking the viral packaging sequence (SEQ ID NO: 35) and encoding a CmyR-VP16 (SEQ ID NO: 37)-IRES (SEQ ID NO: 38)-tTS (SEQ ID NO: 39)-P2A (SEQ ID NO: 40)-rtTA (SEQ ID NO: 41) expressed from the PGK promoter (HAd) was produced in HEK293A cells stably expressing adenovirus E2B (SEQ ID NO: 42). The titre was determined via qPCR with primers and a probe targeting the hexon gene. Specifically, the CmyR-VP16 was added to the virus to induce the expression of CRE from the HEK293 cell line, and the tTS-P2A-rtTA (Tet transactivator protein) to induce expression of the integrated E2B from the inducible promoter via addition of doxycycline.

[0150] A clonal HEK293A cell line stably encoding ere recombinase (SEQ ID NO: 10) with the adenovirus tripartite leader (TPL) sequence (SEQ ID NO: 45) position under a minimal CMV promoter with cumate operator / activator (CuO) (SEQ ID NO: 44) binding sites upstream of the TATA box element, and adenovirus E2B under a Tet-inducible promoter encoding tetracycline operator sites (TetO) (SEQ ID NO: 43) was generated using the piggyBac transposon system. The clonal HEK293 cells were seeded in 10% DMEM at a density of 7.5E+4 cells in a 48-well plate and left to adhere overnight. The following day, the cells were infected with the HAd at varying MOIs in 2% DMEM. The culture media was supplemented with doxycycline (1 pg / mL) either 24-hours pre-infection with the HAd (24 h pre), alongside HAd addition (TO), or 24-hours post-infection with the HAd (24 h p.i.) to induce expression of the integrated adenovirus E2B from the Tet-inducible promoter and initiate adenoviral genome replication. The cells were left to incubate until the experiment was terminated at either 2-, 72- , or 96-hours post-HAd infection followed by DNA extraction using a lysis buffer. qPCR with primers and a probe targeting either the hexon gene or the adenoviral packaging signal was used to determine the number of genome copies per well for each condition.

[0151] The data in Figure 5 shows enhanced excision of the packaging signal (i.e reduced packaging of HAd into adenoviral capsids) following a 24-hour delay in the addition of doxycycline (Dox 24 h p.i.) compared to the simultaneous addition of doxycycline with the HAd (Dox TO) or preinfection with the HAd (24 h pre).

[0152] Example 13: Excision of helper adenovirus packaging signal in HEK293 cells stably expressing Cre recombinase from a cumate inducible promoter and adenovirus E2B from a Tet-inducible promoter

[0153] A DNA polymerase-mutated helper adenovirus (Pol- A AdV helper) (SEQ ID NO: 36) with LoxP sequences flanking the viral packaging sequence (SEQ ID NO: 35) and encoding a CmyR-VP16 (SEQ ID NO: 37)-IRES (SEQ ID NO: 38)-tTS (SEQ ID NO: 39)-P2A (SEQ ID NO: 40)-rtTA (SEQ ID NO: 41) expressed from the PGK promoter (HAd) was produced in HEK293A cells stably expressing adenovirus E2B (SEQ ID NO: 42). The titre was determined via qPCR with primers and a probe targeting the hexon gene. Specifically, the CmyR-VP16 was added to the virus to induce the expression of CRE from the HEK293 cell line, and the tTS-P2A-rtTA (Tet transactivator protein) to induce expression of the integrated E2B from the inducible promoter via addition of doxycycline.

[0154] A clonal HEK293A cell line was generated using the piggyBac transposon system, stably expressing TetR-E37A-P39K from the EFS promoter, and encoding ere recombinase (SEQ ID NO: 10) with the adenovirus tripartite leader (TPL) sequence (SEQ ID NO: 45) positioned under an inducible promoter consisting of the minimal CMV promoter with cumate operator / activator (CuO) (SEQ ID NO: 44) binding sites upstream of the TATA box element, and an adenovirus E2B DNA polymerase positioned under a dual Tet-inducible promoter, consisting of tetracycline operator sites (TetO) (SEQ ID NO: 43) inserted upstream a minimal CMV promoter and TetO-4C5G sites (a variant of TetO that is repressed by TetR-E37A-P39K) positioned downstream of the TATA box element of the minimal CMV. This stable HEK293 cell line was seeded in 10% DMEM at a density of 7.5E+4 cells in a 48-well plate and left to adhere overnight. The following day, the cells were infected with 7.52E+8 genome copies of the HAd in 2% DMEM. The culture media was supplemented with doxycycline (1 pg / mL) either alongside HAd addition (TO), or 24-hours post-infection with the HAd (24 h p.i.) to induce expression of the integrated adenovirus E2B from the dual Tet-inducible promoter and initiate adenoviral genome replication. The cells were left to incubate until the experiment was terminated at either 2-, 72-hours post- HAd infection followed by DNA extraction using a lysis buffer. qPCR with primers and a probe targeting either the hexon gene or the adenoviral packaging signal was used to determine the number of genome copies per well for each condition.

[0155] The data in Figure 8 shows significant inhibition of HAd genome replication in the absence of doxycycline at 72 hours post infection, and high levels of genome replication with doxycycline treatment. Additionally, the data shows enhanced excision of the packaging signal (i.e reduced packaging of HAd into adenoviral capsids) following a 24-hour delay in the addition of doxycycline (Dox 24 h p.i.) compared to the simultaneous addition of doxycycline with the HAd (Dox TO).

[0156] Example 14: Replication of DNA-polymerase-mutated helper adenovirus in HEK293 cells by co-infection with AAV-E2B AE1 / AE3 or AEl / AE3-LoxP-PS-LoxP or AEl / AE2B / AE3-LoxP-PS-LoxP helper adenoviruses were produced in either HEK293A cells or HEK293A cells stably expressing constitutive E2B (for the AE2B deleted variant). The titre for each virus was determined via qPCR with primers and a probe targeting the hexon gene following turboDNAse and Proteinase K treatment.

[0157] HEK293A cells were seeded in 10% DMEM at a density of 7.5E+4 cells in a 48-well plate and left to adhere overnight. The following day, the cells were infected with the three helper AdV variants (AE1 / AE3 or AEl / AE3-LoxP-PS-LoxP or AEl / AE2B / AE3-LoxP-PS-LoxP) at a MOI of 20 in 2% DMEM in the absence or presence of AAV expressing CMV-E2B. The cells were left to incubate until the experiment was terminated at 72-hours post-infection followed by DNA extraction using a lysis buffer. qPCR with primers and a probe targeting the hexon gene was used to determine the number of genome copies per well for each condition.

[0158] The data in Figure 9 demonstrates comparable genome copies per well following a 72-hour infection with three helper AdV variants (AE1 / AE3 or AEl / AE3-LoxP-PS-LoxP or AEl / AE2B / AE3-LoxP-PS-LoxP) indicating that LoxP site addition or DNA polymerase deletion does not affect viral titres when supplemented with AAV expressing CMV-E2B.

[0159] Example 15: Production of HC dV vectors from HEK293 cells infected with AAV expressing CRE and adenovirus E2B DNA polymerase

[0160] For production of HCAdV vectors, plasmid DNA encoding the HCAdV genome and CMV- eGFP as a proxy for a transgene expression cassette, was linearized with Pmel restriction enzyme to release the bacterial plasmid backbone and was purified using a genomic DNA extraction kit to use for transfection.

[0161] A T25 flask of HEK293A cells were seeded at a density of 2E+6 cells per flask and left to adhere overnight in 10% DMEM. The following day, cells were transfected with 5 pg of linearized HcAdV plasmid DNA and simultaneously co-infected with a DNA polymerase- mutated helper adenovirus (Pol- A AdV helper) (SEQ ID NO: 36) with LoxP sequences flanking the viral packaging sequence (SEQ ID NO: 35) as well as a 1:1 ratio of AAV expressing CRE recombinase and AAV expressing adenovirus E2B DNA polymerase. Recombinant viruses were harvested from growth media at 72-hours post-infection (PO) and were propagated a further three times (P1-P3) following recovery using a similar process as described above in HEK293A cells. For propagation, the HCAdV genomes were delivered by infection with 1 mL of the viral lysate for the Pl amplification and 2 mL for P2 and P3 amplifications.

[0162] To confirm and evaluate the HcAdV recovery and amplification process, either 100 pL, 250 pL, or 500 pL of the clarified viral lysate was added to 6E+5 HEK293A cells per well in a 6- well plate. Following a 72-hour incubation with the clarified HcAdV lysate, a Celigo imaging cytometer and flow cytometry were used to image, and quantify GFP expression in infected cells, respectively.

[0163] Figure 10A shows that functional HcAdV expressing CMV-GFP were recovered using the dual AAV method as described above.

[0164] Figure 10B demonstrates an increase in the relative fold change of the percentage of GFP- positive cells for P2 and P3 amplifications compared to Pl following infection of HEK293A cells with varying amounts of HcAdV expressing CMV-GFP clarified lysate.

[0165] Description of sequences

[0166] SEQ ID NOs 1-9 are nucleotide sequences of excision sites or recombinase recombination sites.

[0167] SEQ ID NOs 10-16 are amino acid sequences of recombinases or endonucleases.

[0168] SEQ ID NOs 17-23 are nucleotide sequences of packaging signal sequences.

[0169] SEQ ID NOs 24-31 are nucleotide sequences of inverted terminal repeat sequences.

[0170] SEQ ID NOs 32-34 are nucleotide sequences or fragments thereof of AAVs comprising CMV-E2B or CMV-CRE.

[0171] SEQ ID NOs 35-41 are nucleotide sequences of excision / recombinase-recombination site- flanked packaging signal sequences, genes, or regulatory elements used in the helper adenovirus.

[0172] SEQ ID NOs 42-46 are nucleotide sequences of genes, regulatory elements, or combinations thereof used in cell lines.

[0173] SEQ ID NO: 1: Ataacttcgtatagcatacattatacgaagttat (LoxP)

[0174] SEQ ID NO: 2: Gaagttcctattctctagaaagtataggaacttc (FRT)

[0175] SEQ ID NO: 3: TAGGGATAACAGGGTAAT (I-SecI)

[0176] SEQ ID NO: 4: ttaaccctagaaagatagtctgcgtaaaattgacgcatgcattcttgaaatattgctctctctttctaaatagcgcgaatccgtcgctgtgca tttaggacatctcagtcgccgcttggagctcccgtgaggcgtgcttgtcaatgcggtaagtgtcactgattttgaactataacgaccgcgt gagtcaaaatgacgcatgattatcttttacgtgacttttaagatttaactcatacgataattatattgttatttcatgttctacttacgtgataactt attatatatatattttcttgttatagatatc (piggybac 5’TR)

[0177] SEQ ID NO: 5: aaaagttttgttactttatagaagaaattttgagtttttgtttttttttaataaataaataaacataaataaattgtttgttgaatttattattagtatgt aagtgtaaatataataaaacttaatatctattcaaattaataaataaacctcgatatacagaccgataaaacacatgcgtcaattttacgcat gattatctttaacgtacgtcacaatatgattatctttctagggttaa (piggybac 3’TR)

[0178] SEQ ID NO: 6: gtgccagggcgtgcccttgggctccccgggcgcg (attB) SEQ ID NO: 7:

[0179] CCAGATCCCTATACAGTTGAAGTCGGAAGTTTACATACACTTAAGTTGGAGTCAT

[0180] TAAAACTCGTTTTTCAACTACTCCACAAATTTCTTGTTAACAAACAATAGTTTTGG

[0181] CAAGTCAGTTAGGACATCTACTTTGTGCATGACACAAGTCATTTTTCCAACAATT

[0182] GTTTACAGACAGATTATTTCACTTATAATTCACTGTATCACAATTCCAGTGGGTCA

[0183] GAAGTTTACATACACT (Sleeping Beauty TR 5')

[0184] SEQ ID NO: 8:

[0185] AGTGTATGTAAACTTCTGACCCACTGGGAATGTGATGAAAGAAATAAAAGCTGA

[0186] AATGAATCATTCTCTCTACTATTATTCTGATATTTCACATTCTTAAAATAAAGTGG

[0187] TGATCCTAACTGACCTAAGACAGGGAATTTTTACTAGGATTAAATGTCAGGAATT

[0188] GTGAAAAAGTGAGTTTAAATGTATTTGGCTAAGGTGTATGTAAACTTCCGACTTC

[0189] AACTGTATAGGGATCTGGT (Sleeping Beauty TR 3')

[0190] SEQ ID NO: 9:

[0191] CCCCAACTGGGGTAACCTTTGAGTTCTCTCAGTTGGGG (attp)

[0192] SEQ ID NO: 10:

[0193] MSNLLTVHQNLPALPVDATSDEVRKNLMDMFRDRQAFSEHTWKMLLSVCRSWAA

[0194] WCKLNNRKWFPAEPEDVRDYLLYLQARGLAVKTIQQHLGQLNMLHRRSGLPRPSD

[0195] SNAVSLVMRRIRKENVDAGERAKQALAFERTDFDQVRSLMENSDRCQDIRNLAFLGI

[0196] AYNTLLRIAEIARIRVKDISRTDGGRMLIHIGRTKTLVSTAGVEKALSLGVTKLVERWI

[0197] SVSGVADDPNNYLFCRVRKNGVAAPSATSQLSTRALEGIFEATHRLIYGAKDDSGQR YLAWSGHSARVGAARDMARAGVSIPEIMQAGGWTNVNIVMNYIRNLDSETGAMVR LLEDGD (CRE)

[0198] SEQ ID NO: 11:

[0199] HMMPQFDILCKTPPKVLVRQFVERFERPSGEKIALCAAELTYLCWMITHNGTAIKRA

[0200] TFMSYNTIISNSLSFDIVNKSLQFKYKTQKATILEASLKKLIPAWEFTIIPYYGQKHQSD

[0201] ITDIVSSLQLQFESSEEADKGNSHSKKMLKALLSEGESIWEITEKILNSFEYTSRFTKTK TLYQFLFLATFINCGRFSDIKNVDPKSFKLVQNKYLGVIIQCLVTETKTSVSRHIYFFS ARGRIDPLVYLDEFLRNSEPVLKRVNRTGNSSSNKQEYQLLKDNLVRSYNKALKKN APYSIFAIKNGPKSHIGRHLMTSFLSMKGLTELTNVVGNWSDKRASAVARTTYTHQI

[0202] TAIPDHYFALVSRYYAYDPISKEMIALKDETNPIEEWQHIEQLKGSAEGSIRYPAWNG

[0203] IISQEVLDYLSSYINRRI (FLP)

[0204] SEQ ID NO: 12:

[0205] MGSSLDDEHILSALLQSDDELVGEDSDSEISDHVSEDDVQSDTEEAFIDEVHEVQPTS SGSEILDEQNVIEQPGSSLASNKILTLPQRTIRGKNKHCWSTSKSTRRSRVSALNIVRS QRGPTRMCRNIYDPLLCFKLFFTDEIISEIVKWTNAEISLKRRESMTGATFRDTNEDEI YAFFGILVMTAVRKDNHMSTDDLFDRSLSMVYVSVMSRDRFDFLIRCLRMDDKSIR

[0206] PTLRENDVFTPVRKIWDLFIHQCIQNYTPGAHLTIDEQLLGFRGRCPFRMYIPNKPSK YGIKILMMCDSGTKYMINGMPYLGRGTQTNGVPLGEYYVKELSKPVHGSCRNITCD NWFTSIPLAKNLLQEPYKLTIVGTVRSNKREIPEVLKNSRSRPVGTSMFCFDGPLTLVS YKPKPAKMVYLLSSCDEDASINESTGKPQMVMYYNQTKGGVDTLDQMCSVMTCSR

[0207] KTNRWPMALLYGMINIACINSFIIYSHNVSSKGEKVQSRKKFMRNLYMSLTSSFMRK RLEAPTLKRYLRDNISNILPNEVPGTSDDSTEEPVTKKRTYCTYCPSKIRRKANASCK KCKKVICREHNIDMCQSCF (Piggybac transposase)

[0208] SEQ ID NO: 13:

[0209] MGKSKEISQDLRKKIVDLHKSGSSLGAISKRLKVPRSSVQTIVRKYKHHGTTQPSYRS GRRRVLSPRDERTLVRKVQINPRTTAKDLVKMLEETGTKVSISTVKRVLYRHNLKGR SARKKPLLQNRHKKARLRFARAHGDKDRTFWRNVLWSDETKIELFGHNDHRYVWR KKGEACKPKNTIPTVKHGGGSIMLWGCFAAGGTGALHKIDGIMRKENYVDILKQHL

[0210] KTSVRKLKLGRKWVFQQDNDPKHTSKHVRKWLKDNKVKVLEWPSQSPDLNPIENL WAELKKRVRARRPTNLTQLHQLCQEEWAKIHPTYCGKLVEGYPKRLTQVKQFKGN ATKY (Sleeping beauty transposase)

[0211] SEQ ID NO: 14:

[0212] MDTYAGAYDRQSRERENSSAASPATQRSANEDKAADLQREVERDGGRFRFVGHFSE APGTSAFGTAERPEFERILNECRAGRLNMIIVYDVSRFSRLKVMDAIPIVSELLALGVT IVSTQEGVFRQGNVMDLIHLIMRLDASHKESSLKSAKILDTKNLQRELGGYVGGKAP YGFELVSETKEITRNGRMVNVVINKLAHSTTPLTGPFEFEPDVIRWWWREIKTHKHL

[0213] PFKPGSQAAIHPGSITGLCKRMDADAVPTRGETIGKKTASSAWDPATVMRILRDPRIA GFAAEVIYKKKPDGTPTTKIEGYRIQRDPITLRPVELDCGPIIEPAEWYELQAWLDGR GRGKGLSRGQAILSAMDKLYCECGAVMTSKRGEESIKDSYRCRRRKVVDPSAPGQH EGTCNVSMAALDKFVAERIFNKIRHAEGDEETLALLWEAARRFGKLTEAPEKSGERA NLVAERADALNALEELYEDRAAGAYDGPVGRKHFRKQQAALTLRQQGAEERLAEL EAAEAPKLPLDQWFPEDADADPTGPKSWWGRASVDDKRVFVGLFVDKIVVTKSTT GRGQGTPIEKRASITWAKPPTDDDEDDAQDGTEDVAA (phiC31)

[0214] SEQ ID NO: 15:

[0215] MGSGRSMKNIKKNQVMNLGPNSKLLKEYKSQLIELNIEQFEAGIGLILGDAYIRSRDE

[0216] GKTYCMQFEWKNKAYMDHVCLLYDQWVLSPPHKKERVNHLGNLVITWGAQTFKH QAFNKLANLFIVNNKKTIPNNLVENYLTPMSLAYWFMDDGGKWDYNKNSTNKSIV LNTQSFTFEEVEYLVKGLRNKFQLNCYVKINKNKPIIYIDSMSYLIFYNLIKPYLIPQM MYKLPNTISSETFLK (I-SecI)

[0217] SEQ ID 16:

[0218] MTAPKKKRKVMTKKVAIYTRVSTTNQAEEGFSIDEQIDRLTKYAEAMGWQVSDTYT

[0219] DAGFSGAKLERPAMQRLINDIENKAFDTVLVYKLDRLSRSVRDTLYLVKDVFTKNKI DFISLNESIDTSSAMGSLFLTILSAINEFERENIKERMTMGKLGRAKSGKSMMWTKTA FGYYHNRKTGILEIVPLQATIVEQIFTDYLSGISLTKLRDKLNESGHIGKDIPWSYRTL RQTLDNPVYCGYIKFKDSLFEGMHKPIIPYETYLKVQKELEERQQQTYERNNNPRPF

[0220] QAKYMLSGMARCGYCGAPLKIVLGHKRKDGSRTMKYHCANRFPRKTKGITVYNDN KKCDSGTYDLSNLENTVIDNLIGFQENNDSLLKIINGNNQPILDTSSFKKQISQIDKKIQ KNSDLYLNDFITMDELKDRTDSLQAEKKLLKAKISENKFNDSTDVFELVKTQLGSIPI NELSYDNKKKIVNNLVSKVDVTADNVDIIFKFQLA (TP901 )

[0221] SEQ ID NO: 17:

[0222] Gaaatgatgttttttgggcgttgtttgtgcaaattttgtgttttaggcgcgaaaactgaaatgcggaagtgaaaattgatgacggcaatttta ttataggcgcggaatatttaccgagggcagagtgaactctgagcctctacgtgtgggtttcgatacgtgagcgacggggaaactccac gttggcgctcaaagggcgcgtttattgttctgtcagctgatcgtttggg (Adl2 group A)

[0223] SEQ ID NO: 18: gcttcttttctcacggaactacttagttttcccacggtatttaacaggaaatgaggtagttttgaccggatgcaagtgaaaattgctgattttc gcgcgaaaactgaatgaggaagtgtttttctgaataatgtggtatttatggcagggtggagtatttgttcagggccaggtagactttgacc cattacgtggaggtttcgattaccgtgttttttacctgaatttccgcgtaccgtgtcaaagtcttctgtttttacgtaggt (Ad35 group B) SEQ ID NO: 19: ggaagtgacaattttcgcgcggttttaggcggatgttgtagtaaatttgggcgtaaccgagtaagatttggccattttcgcgggaaaactg aataagaggaagtgaaatctgaataattttgtgttactcatagcgcgtaatatttgtctagggccgcggggactttgaccgtttacgtgga gactcgcccaggtgtttttctcaggtgttttccgcgttccgggtcaaagttggcgttttattattata (Ad5 group C)

[0224] SEQ ID NO: 20: gactttaaacccggaaacggccgattttcccacggccacgcccggatatgaggtaattttgggcggttgcaaataaaattaggacatgg tggcgccaaaactgaatgaggaagtgaaaagcgaaaaataccggctcccgcccaggggcggaatatttaccgagggccaacagac tttgaccgattacgtggggtttcgattacggtgttttttttctcgccaatttccgcgtccgtgttaaatccggtgtttatgtta (Ad8 group D)

[0225] SEQ ID NO: 21: ggaagtagacagttttcccacgcttactgacaggatatgaggtagttttgggtggatgcaagtgaaaattctccattttcgcgcgaaaact gaatgaggaagtgaatgtctgagtcattttgcggttatgacagggtggagtatttgccgagggccgagtagactttgaccgtttccgtgg aggtttcgattaccgtgtttttacctaaatttccgcgtacggtgtcaaagttcggtgtttttacgtaggtgt (Ad4 group E)

[0226] SEQ ID NO: 22: gtgtattgggcgggtttttgtaactttttggttattttggcgcgaaaactgagtaatgcggaagttgaacgaactctggactttttatggctag ggagggaaaactgctgatcattgctgaactttggggctttgacgtggcggtttccctacgtggcactgccacgcgaatgctcaaagtcc ttattttattgtgtgttcagcccttttgagggtatttaaacacagccagaacgtcaagaggccactcttgagtgccagcgagtagagttttct cctc (Ad41 group F)

[0227] SEQ ID NO: 23: gcttagcttttacgtatgcggaaggaggttttatgccggaagttgggtaatttgggcgtatacttgtaagttttgtgtaaattggcgcgaaaa ctgggtaatgaggaagttgaggttaatatgtactttttatgactgggcggaatttctgctgatcagcagtgaactttgggcgctgacgggg aggtttcgctacgtggcagtaccacgagaaggctcaaaggtcccatttattgtactcctcagcgttttcgctg (Ad52 group G)

[0228] SEQ ID NO:

[0229] 24: cctatctaataatataccttatactggactagtgccaatattaaaatgaagtgggcgtagtgtgtaatttgattgggtggaggtgtggc tttggcgtgcttgtaagtttgggcggatgaggaagtggggcgcggcgtgggagccgggcgcgccggatgtgacgt (Ad 12 group A) SEQ ID NO: 25

[0230] : catcatcaataatataccttatagatggaatggtgccaatatgtaaatgaggtgattttaaaaagtgtgggccgtgtggtgattggctgtg gggttaacggttaaaaggggcggcgcggccgtgggaaaatgacgtt (Ad35 group B)

[0231] SEQ ID NO: 26: catcatcaataatataccttattttggattgaagccaatatgataatgagggggtggagtttgtgacgtggcgcggggcgtgggaacgg ggcgggtgacgtag (Ad5 group C)

[0232] SEQ ID NO: 27: ctatctatataatataccccacaaagttaatatgcaaatgagcttttaaattttaacggttttagggcagggccaacgttaagtggttaacaa gcggtaatgcagttgacgtcaagacg (Ad8 group D)

[0233] SEQ ID NO: 28: catcatcaataatataccttatagatggaatggtgccaatatgtaaatgaggtgatttaaaaaagtgcgcgctgtgtggtgattggccgtg gagtaaatggctaaaaggggtggggtaatgctgggaggtgacgtaacttatgtgggaggagttatgttgcaagttattgtgttaaatgtg acgtaaaaccaggtgtggtttaaacac (Ad4 group E)

[0234] SEQ ID NO: 29: catcatcaataatataccttaaagctggaaacgagccaatatgataatgagtgaggaggggctaggggtggtgcgaagtgacgtatag gtaggcggggtgggaaagggtggaggtgaatgacgttggggtcgaaggttggggagtggcgcggcgga (Ad41 group F)

[0235] SEQ ID NO: 30: catcatcaataatataccttaaaactggaaacgtgccaatatgataatgagcggggaggagcgaggcggggccggggtgacgt

[0236] (Ad52 group G)

[0237] SEQ ID NO: 31: tgcaggcagctgcgcgctcgctcgctcactgaggccgcccgggcaaagcccgggcgtcgggcgacctttggtcgcccggcctcag tgagcgagcgagcgcgcagagagggagtggccaactccatcactaggggttcct (AAV ITR)

[0238] SEQ ID NO: 32: tgcaggcagctgcgcgctcgctcgctcactgaggccgcccgggcaaagcccgggcgtcgggcgacctttggtcgcccggcctcag tgagcgagcgagcgcgcagagagggagtggccaactccatcactaggggttcctgattacagatctggctacactcgatcagcagtt agataataaaatcgctatccatcgaagatggatgtgtgttggttttttgtgtgtgtaacgcaacgattgatagcataaccccttggggcctct aaacgggtcttgaggggttttttgaattgttgacattgattattgactagttattaatagtaatcaattacggggtcattagttcatagcccata tatggagttccgcgttacataacttacggtaaatggcccgcctggctgaccgcccaacgacccccgcccattgacgtcaataatgacgt atgttcccatagtaacgccaatagggactttccattgacgtcaatgggtggagtatttacggtaaactgcccacttggcagtacatcaagt gtatcatatgccaagtacgccccctattgacgtcaatgacggtaaatggcccgcctggcattatgcccagtacatgaccttatgggacttt cctacttggcagtacatctacgtattagtcatcgctattaccatggtgatgcggttttggcagtacatcaatgggcgtggatagcggtttga ctcacggggatttccaagtctccaccccattgacgtcaatgggagtttgttttggcaccaaaatcaacgggactttccaaaatgtcgtaac aactccgccccattgacgcaaatgggcggtaggcgtgtacggtgggaggtctatataagcagagctcactctcttccgcatcgctgtct gcgagggccagctgttgggctcgcggttgaggacaaactcttcgcggtctttccagtactcttggatcggaaacccgtcggcctccga acggtactccgccgccgagggacctgagcgagtccgcatcgaccggatcggaaaacctctcgagaaaggcgtctaaccagtcaca gtcgcaagaattcaggaggtacccaccatgtccaatttactgaccgtacaccaaaatttgcctgcattacccgtcgatgcaacgagtgat gaggttcgcaagaacctgatggatatgttcagggatcgccaggcgttttctgagcatacctggaaaatgcttctgtccgtttgccggtcgt gggcggcatggtgcaagttgaataaccggaaatggtttcccgcagaacctgaagatgttcgcgattatcttctatatcttcaggcccgcg gtctggcagtaaaaactatccagcaacatttgggccagctaaacatgcttcatcgtcggtccgggctgccacgaccaagtgacagcaa tgctgtttcactggttatgcggcgcatccgaaaagaaaacgtggatgccggtgaacgtgcaaaacaggctctagcgtttgaacgcact gatttcgaccaggttcgttcactcatggaaaatagcgaccgctgccaggatatacgtaatctggcatttctggggattgcttacaacaccc tgttacgtatagccgaaattgccaggatcagggttaaagacatctcacgtactgacggtgggagaatgttaatccatattggcagaacg aaaacgctggttagcaccgcaggtgtagagaaggcacttagcctgggggtaactaaactggtcgagcgatggatttccgtctctggtg tagctgatgatccgaataactacctgttttgccgggtcagaaaaaatggtgttgccgcgccatctgccaccagccagctatcaactcgc gccctggaagggatttttgaagcaactcaccgattgatttacggcgctaaggatgactctggtcagagatacctggcctggtctggaca cagtgcccgtgtcggagccgcgcgagatatggcccgcgctggagtttcaataccggagatcatgcaagctggtggctggaccaatgt aaatattgtgatgaactatatccgtaacctggatagtgaaacaggggcaatggtgcgcctgctggaagatggcgactagaagttgtctc ctcctgcactgactgactgatacaatcgatttctggatccgcaggcctctgctagcttgactgactgagatacagcgtaccttcagctcac agacatgataagatacattgatgagtttggacaaaccacaactagaatgcagtgaaaaaaatgctttatttgtgaaatttgtgatgctattg ctttatttgtaaccattataagctgcaataaacaagttaacaacaacaattgcattcattttatgtttcaggttcagggggaggtgtgggagg ttttttctatcctgcaggcgatctctaggaacccctagtgatggagttggccactccctctctgcgcgctcgctcgctcactgaggccggg cgaccaaaggtcgcccgacgcccgggctttgcccgggcggcctcagtgagcgagcgagcgcgcagctgcctgca (AAV- CMV-CRE)

[0239] SEQ ID NO: 33: tgcaggcagctgcgcgctcgctcgctcactgaggccgcccgggcaaagcccgggcgtcgggcgacctttggtcgcccggcctcag tgagcgagcgagcgcgcagagagggagtggccaactccatcactaggggttcctgatatcatcaactttgtatagaaaagttggcgat cgcatcgatgttgacattgattattgactagttattaatagtaatcaattacggggtcattagttcatagcccatatatggagttccgcgttac ataacttacggtaaatggcccgcctggctgaccgcccaacgacccccgcccattgacgtcaataatgacgtatgttcccatagtaacgc caatagggactttccattgacgtcaatgggtggagtatttacggtaaactgcccacttggcagtacatcaagtgtatcatatgccaagtac gccccctattgacgtcaatgacggtaaatggcccgcctggcattatgcccagtacatgaccttatgggactttcctacttggcagtacatc tacgtattagtcatcgctattaccatggtgatgcggttttggcagtacatcaatgggcgtggatagcggtttgactcacggggatttccaa gtctccaccccattgacgtcaatgggagtttgttttggcaccaaaatcaacgggactttccaaaatgtcgtaacaactccgccccattgac gcaaatgggcggtaggcgtgtacggtgggaggtctatataagcagagctcgctaccatttggagacggtatcctagcggccgccaag tttgtacaaaaaagcaggctgccaccatggccctcgtgcaagcccatagagcccggagactgcacgctgaagcccctgacagcgga gatcagcctcctcgtagaagagtgcggcaacagcccactagagccgctcctgcccctgctagagccagacgtagacgggcacctgc acccagccctggcggtagcggcgcccctcctaccagcggcggctccccagcctctccccttctggacgcttcttctaaggacacacc ggctgctcacagaccccctcgcggaaccgtggtggcccctagaggatgtggactgctgcaggccatcgacgctgccaccaaccag cctctcgagatcagataccaccttgacctggcccgagccctgacaagactgtgcgaggtgaatctgcaggagctgccccccgacctg acacccagagaactgcagacaatggacagctcccatctcagagacgtggtcatcaagctgagaccacccagagccgacatctggac actgggaagcagaggcgtggtcgtgcgcagcaccgtgacacctctggaacagcctgatggccagggccaagccgccgaagtgga ggatcaccagcctaacccccctggcgagggcctgaaattccccctgtgcttcctcgtgcggggaagacaggttaacctggtccaaga cgtgcagcccgtgcacagatgccagtactgcgcccggttctacaagagccagcacgagtgtagcgccagacggagagatttttactt ccaccacatcaactcccacagcagcaactggtggagagaaattcagttcttccccatcggctcccacccgagaacagagagactcttc gtgacctacgacgtggagacatacacatggatgggcgccttcggcaagcagctggtgcctttcatgctggtgatgaaattcggcggcg acgagcctctggtgaccgccgcgagagatctggccgccaacctgggctgggacagatgggaacaggatcctctgacattctactgta tcacccccgagaagatggccatcggcagacagtttcggaccttccgggatcacctgcagatgctgatggcccgggatctgtggagct ccttcgtagccagtaaccctcacctggccgattgggccctgagcgaacacggcctcagctcccctgaggagctgacctatgaagagtt aaagaaactgcctagcatcaagggcatcccccgtttcctggagctctacatcgtgggccacaacatcaacggcttcgatgagatcgtg ctggccgctcaagtgatcaacaacagaagcgaggtgccagggccttttcggatcacccggaatttcatgcctcgggccggcaaaatc ctgttcaacgatgtgacatttgccttgcctaatcctagaagcaagaaaagaaccgatttcctgctgtgggagcaaggcggatgcgacga caccgacttcaagtaccagtacctgaaggtgatggtcagagacacattcgccctgacacacaccagcctgagaaaggccgcccagg cttacgccctgcctgtggaaaagggctgctgcgcctatcaggccgtgaaccagttctacatgctgggctcttatagaagcgaggccga cggcttccctatccaggagtactggaaggaccgggaagagttcgtgctgaatagagagctctggaagaagaagggccaagacaagt acgacatcatcaaggaaacactggactactgtgccctggacgtgcaggtgacagccgaactggtgaacaaactgagggacagctac gccagttttgtgagagatgccgtgggcctgaccgatgcctcctttaacgtgttccagagaccgaccatcagctctaatagccacgccatt ttcagacagatcgtgtttcgggccgagcaacctgctagaagcaacctcggcccagatctgctggcccctagccacgagctgtacgact acgtgcgggcgtctatccggggcggaagatgctaccccacctacctgggtatcctgagagagcccctgtacgtgtacgacatctgtg gaatgtacgcctctgctctgacccacccaatgccttggggccctcctctgaacccttacgagcgggccctggcagccagagcttggca gcaggccctggatctgcagggctgcaaaatcgactacttcgacgcccggctgctgcctggcgtgttcaccgtggatgctgatcctcct gacgagacccagctggatcctctgcctccattctgcagccgcaagggcggccggctgtgttggacaaacgagaggctgcgaggcg aggtggcgacctccgtggacctggtgaccctccacaaccggggctggcgggtgcaccttgtgcctgatgaaagaaccaccgtgttcc ccgaatggcggtgcgtggcccgggaatacgtgcagctgaacattgccgccaaggaaagagccgacagagacaagaaccagaccc tgcggagcatcgccaagctgctgtctaacgccctgtacggcagcttcgccacaaagctggacaacaaaaagatcgtgttcagcgatc agatggacgccgcgaccctgaagggcattacagccggccaggtgaatatcaagtcttcgagcttcctggaaaccgacaacctgagc gccgaagtcatgcctgcttttcagagagagtactccccacagcagctggccctggccgactctgacgccgaggagagcgaggacga gcgagcccctacccctttctacagcccaccatctggcacccctgggcatgtggcctacacctataagcccatcacctttctggacgccg aggaaggcgacatgtgcctgcataccctggaacgggttgaccccctggtggacaatgacagataccctagccacctggccagcttcg tgctggcttggacaagagctttcgtcagcgagtggtccgagttcctgtacgaagaagatagaggcacccccctggaggatagacccct caaaagcgtctacggcgatacagattctctttttgtgaccgaaagaggccacagactgatggaaaccagaggaaagaaacggatcaa gaagcacggcggcaacctggtgtttgacccagagagaccagagctgacctggctggttgaatgtgaaacagtgtgcggcgcatgcg gcgccgacgcctacagccctgagagcgtgttcctggctcctaagctgtatgctctgaaaagcctgcactgcccctcatgcggagcctc cagcaagggcaagctgagagctaagggtcatgccgctgagggcctggactacgacacaatggtgaagtgctacctggccgacgcc cagggcgaggaccggcagcggttcagcaccagcagaacaagcctgaagcggacattggcttctgcccaacctggagctcacccttt caccgtgactcagacaacactgaccaggaccctgcgcccctggaaggacatgaccctcgccaggctagatgagcaccgtctcctgc cctacagcgagtcccggcctaaccccagaaacgaggaaatctgctggatcgagatgccgtgagctagcttgactgactgagatacag cgtaccttcagctcacagacatgataagatacattgatgagtttggacaaaccacaactagaatgcagtgaaaaaaatgctttatttgtga aatttgtgatgctattgctttatttgtaaccattataagctgcaataaacaagttaacaacaacaattgcattcattttatgtttcaggttcaggg ggaggtgtgggaggttttttctatcctgcaggcgatctctaggaacccctagtgatggagttggccactccctctctgcgcgctcgctcg ctcactgaggccgggcgaccaaaggtcgcccgacgcccgggctttgcccgggcggcctcagtgagcgagcgagcgcgcagctgc ctgca (AAV-CMV-E2B)

[0240] SEQ ID NO: 34: gttgacattgattattgactagttattaatagtaatcaattacggggtcattagttcatagcccatatatggagttccgcgttacataacttacg gtaaatggcccgcctggctgaccgcccaacgacccccgcccattgacgtcaataatgacgtatgttcccatagtaacgccaataggga ctttccattgacgtcaatgggtggagtatttacggtaaactgcccacttggcagtacatcaagtgtatcatatgccaagtacgccccctatt gacgtcaatgacggtaaatggcccgcctggcattatgcccagtacatgaccttatgggactttcctacttggcagtacatctacgtattag tcatcgctattaccatggtgatgcggttttggcagtacatcaatgggcgtggatagcggtttgactcacggggatttccaagtctccaccc cattgacgtcaatgggagtttgttttggcaccaaaatcaacgggactttccaaaatgtcgtaacaactccgccccattgacgcaaatggg cggtaggcgtgtacggtgggaggtctatataagcagagctc (CMV promoter)

[0241] SEQ ID NO: 35:

[0242] Tagtgtggcggaagtgtgatgttgcaagtgtggcggaacacatgtaagcgacggatgtggcaaaagtgacgtttttggtgtgcgcata acttcgtatagcatacattatacgaagttatcggtgtacacaggaagtgacaattttcgcgcggttttaggcggatgttgtagtaaatttgg gcgtaaccgagtaagatttggccattttcgcgggaaaactgaataagaggaagtgaaatctgaataattttgtgttactcatagcgcgtaa tatttgtctagggccgcggggactttgaccgtttacgtggagactcgcccaggtgtttttctcaggtgttttccgcgttccgggtcaaagtt ggcgttttattattatagtcagctgacggcgatcgcataacttcgtatagcatacattatacgaagttatgcgatcgctggggcggccgcc atcgatggg (LoxP-Ad5 packaging signal-LoxP) SEQ ID NO: 36: ttttcggccagcgcgtttacgatcgccagcaccttctcgtgcgtggggtttgcgcgcgccgggaccaccgcttccagaattgcggaga gccggttggcctgcggctgctgccggaacgcgtcaggattgcgcgcagtcagcgacatgatgcggtccatgacctggcgccagtcg tccgtggagttaaggccggacggctggctctgcagcgccgcccgcaccgccgggtccgttgcgtcttgcatcatctgatcagaaacat caccgcttagtactcgccgtcctctggctcgtactcatcgtcctcgtcatattcctccacgccgccgacgttgccagcgcgcgcgggtg ccaccgccagcccaggtccggccccagctgcctccagggcgcgtcggcttggggcccagcgcaggtcagcgcccgcgtcaaagt aggactcggcctctctatcgccgctgcccgtgccagccagggccctttgcaggctgtgcatcagctcgcggtcgctgagctcgcgcc gccggctcacgctcacggccttgtggatgcgctcgttgcgataaacgcccaggtcgtcgctcaaggtaagcaccttcagcgccatgc gcatgtagaacccctcgatctttacctccttgtctatgggaacgtaaggggtatggtatatcttgcgggcgtaaaacttgcccaggctaag catggaatagttgatggcggccaccttgtcagccaggctcaagctgcgctcctgcaccactatgctctgcaggatgtttatcaaatcgag cagccagcggccctcgggctctactatgtttagcagcgcatccctgaatgcctcgttgtccctgctgtgctgcactataaggaacagctg cgccatgagcggcttgctatttgggttttgctccagcgcgcttacaaagtcccacagatgcatcagtcctatagccacctcctcgcgcgc cacaagcgtacgcacgtggttgttaaagcttttttgaaagttaatctcctggttcaccgtctgctcgtatgcggttaccaggtcggcggcc gccacgtgtgcgcgcgcgggactaatcccggttcgcgcgtcgggctcaaagtcctcctcgcgcagcaaccgctcgcgattcaggcc atgccgcagctcgcgccctgcgtggaactttcgatcccgcatctcctcgggctcctctccctcgcggtcgcgaaacaggttctgccgc ggcacgtacgcctcacgcgtatcacgcttcagctgcacccttgggtgccgctcaggagagggcgctcctagccgcgccaggccctc gccctcctccaagtccaggtagtgccgggcccggcgccgcgggggttcgtaatcaccatctgctgccgcgtcaaccgcggatgtcg cccctcctgacgcggtaggaggaggggagggtgccctgcatgtctgccgctgctcttgctcttgccgctgctgaggaggggggcgc atctgccgcagcaccggatgcatctgggaaaagcaaaaaaggggctcgtccctgtttccggaggaatttgcaagcggggtcttgcat gacggggaggcaaacccccgttcgccgcagtccggccggtccgagactcgaaccgggggtcccgcgactcaacccttggaaaata accctccggctacagggagcgagccacttaatgctttcgctttccagcctaaccgcttacgctgcgcgcggccagtggccaaaaaag ctagcgcagcagccgccgcgcctggaaggaagccaaaaggagcactcccccgttgtctgacgtcgcacacctgggttcgacacgc gggcggtaaccgcatggatcacggcggacggccggatacggggctcgaaccccggtcgtccgccatgatacccttgcgaatttatc caccagaccacggaagagtgcccgcttacaggctctccttttgcacggtctagagcgtcaacgattgcgcgcgcctgaccggccaga gcgtcccgaccatggagcactttttgccgctgcgcaacatctggaaccgcgtccgcgactttccgcgcgcctccaccaccgccgccg gcatcacctggatgtccaggtacatctacggatatcatcgccttatgttggaagatctcgcccccggagccccggccaccctacgctgg cccctctaccgccagccgccgccgcactttttggtgggataccagtacctggtgcggacttgcaacgactacgtatttgactcgagggc ttactcgcgtctcaggtacaccgagctctcgcagccgggtcaccagaccgttaactggtccgttatggccaactgcacttacaccatca acacgggcgcataccaccgctttgtggacatggatgacttccagtctaccctcacgcaggtgcagcaggccatattagccgagcgcgt tgtcgccgacctagccctgcttcagccgatgaggggcttcggggtcacacgcatgggaggaagagggcgccacctacggccaaac tccgccgccgccgcagcgatagatgcaagagatgcaggacaagaggaaggagaagaagaagtgccggtagaaaggctcatgcaa gactactacaaagacctgcgccgatgtcaaaacgaagcctggggcatggccgaccgcctgcgcattcagcaggccggacccaagg acatggtgcttctgtcgaccatccgccgtctcaagaccgcctactttaattacatcatcagcagcacctccgccagaaacaaccccgac cgccgcccgctgccgcccgccacggtgctcagcctaccttgcgactgtgactggttagacgcctttctcgagaggttttccgatccggt cgatgcggactcgctcaggtccctcggcggcggagtacctacacaacaattgttgagatgcatcgttagcgccgtatccctgccgcat ggcagccccccgccaacccataaccgggacatgacgggcggcgtcttccaactgcgcccccgcgagaacggccgcgccgtcacc gagaccatgcgccgtcgccgcggggagatgatcgagcgctttgtcgaccgcctcccggtgcgccgtcgtcgccgccgtgtcccccc tcccccaccgccgccagaagaagaagaaggggaggcccttatggaagaggagattgaagaagaagaagaggcccctgtagccttt gagcgcgaggtgcgcgacactgtcgccgagctcatccgtcttctggaggaggagttaaccgtgtcggcgcgcaactcccagtttttca acttcgccgtggacttctacgaggccatggagcgccttgaggccttgggggatatcaacgaatccacgttgcgacgctgggttatgtac ttcttcgtggcagaacacaccgccaccaccctcaactacctctttcagcgcctgcgaaactacgccgtcttcgcccggcacgtggagct caatctcgcgcaggtggtcatgcgcgcccgcgatgccgaagggggcgtggtctacagccgcgtctggaacgagggaggcctcaac gccttctcgcagctcatggcccgcatttccaacgacctcgccgccaccgtggagcgagccggacgcggagatctccaggaggaag agatcgagcagttcatggccgagatcgcctatcaagacaactcaggagacgtgcaggagattttgcgccaggccgccgtcaacgac accgaaattgattctgtcgaactctctttcaggttcaagctcaccgggcccgtcgtcttcacgcagaggcgccagattcaggagatcaa ccgccgcgtcgtcgcgttcgccagcaacctacgcgcgcagcaccagctcctgcccgcgcgcggcgccgacgtgcccctgccccct ctcccggcgggtccggagccccccctacctccgggggctcgcccgcgtcaccgcttttagatgcatcatccaaggacacccccgcg gcccaccgcccgccgcgcggtaccgtagtcgcgccgcggggatagggcctcttgcaagccatctgagccgccaccaaccagccc ctataaattaggtatcacctggatctagcccgcgccctgacccgtctatgcgaggtaaacctgcaggagctcccgcctgacctgacgc cgcgggagctccagaccatggacagctcccatctgcgcgatgttgtcatcaagctccgaccgccgcgcgcggacatctggactttgg gctcgcgcggcgtggtggtccgatccaccgtaactcccctcgagcagccagacggtcaaggacaagcagccgaagtagaagacca ccagccaaacccgccaggcgaggggctcaaattcccactctgcttccttgtgcgcggtcgtcaggtcaacctcgtgcaggatgtacag cccgtgcaccgctgccagtactgcgcacgtttttacaaaagccagcacgagtgttcggcccgtcgcagggacttctactttcaccacat caatagccactcctccaattggtggcgggagatccagttcttcccgatcggctcgcatcctcgcaccgagcgtctctttgtcacctacga tgtagagacctatacttggatgggggcctttgggaagcagctcgtgcccttcatgctggtcatgaagttcggcggagatgagcctctag tgactgccgcgcgagacctagccgcgaaccttggatgggaccgctgggaacaagacccgcttaccttctactgcatcaccccagaaa aaatggccataggtcgccagtttaggacctttcgcgaccacctgcaaatgctaatggcccgtgacctgtggagctcattcgtcgcttcca accctcatcttgcagactgggccctttcagagcacgggctcagctcccctgaagagctcacctacgaggaacttaaaaaattgccttcc atcaagggcatcccgcgcttcttggaactttacattgtgggccacaacatcaacggctttgacgagatcgtgctcgccgcccaggtaatt aacaaccgttccgaggtgccgggacccttccgcatcacacgcaactttatgcctcgcgcgggaaagatactcttcaacgatgtcacctt cgccctgccaaatccgcgttccaaaaagcgcacggactttttgctctgggagcagggcggatgcgacgacactgacttcaaatacca gtacctcaaagtcatggtcagggacacctttgcgctcacccacacctcgctccggaaggccgcgcaggcatacgcgctacccgtaga aaagggatgctgcgcctaccaggccgtcaaccagttctacatgctaggctcttaccgttcggaggccgacgggtttccgatccaagag tactggaaagaccgcgaagagtttgtcctcaaccgcgagctgtggaaaaaaaagggacaggataagtatgacatcatcaaggaaacc ctggactactgcgccctagacgtgcaggtcaccgccgagctggtcaacaagctgcgcgactcctacgcctccttcgtgcgtgacgcg gtaggtctcacagacgccagcttcaacgtcttccagcgtccaaccatatcatccaactcacatgccatcttcaggcagatagtcttccga gcagagcagcccgcccgtagcaacctcggtcccgacctcctcgctccctcgcacgaactatacgattacgtgcgcgccagcatccgc ggtggaagatgctaccctacatatcttggaatactcagagagcccctctacgtttacgacatttgcggcatgtacgcctccgcgctcacc caccccatgccatggggtcccccactcaacccatacgagcgcgcgcttgccgcccgcgcatggcagcaggcgctagacttgcaag gatgcaagatagactacttcgacgcgcgcctgctgcccggggtctttaccgtggacgcagaccccccggacgagacgcagctagac cccctaccgccattctgctcgcgcaagggcggccgcctctgctggaccaacgagcgcctacgcggagaggtagccaccagcgttg accttgtcaccctgcacaaccgcggttggcgcgtgcacctggtgcccgacgagcgcaccaccgtctttcccgaatggcggtgcgttg cgcgcgaatacgtgcagctaaacatcgcggccaaggagcgcgccgatcgcgacaaaaaccaaaccctgcgctccatcgccaagtt gctgtccaacgccctctacgggtcgtttgccaccaagcttgacaacaaaaagattgtcttttctgaccagatggatgcggccaccctcaa aggcatcaccgcgggccaggtgaatatcaaatcctcctcgtttttggaaactgacaatcttagcgcagaagtcatgcccgcttttcagag ggagtactcaccccaacagctggccctcgcagacagcgatgcggaagagagtgaggacgaacgcgcccccacccccttttatagc cccccttcaggaacacccggtcacgtggcctacacctacaaaccaatcaccttccttgatgccgaagagggcgacatgtgtcttcaca ccctggagcgagtggaccccctagtggacaacgaccgctacccctcccacttagcctccttcgtgctggcctggacgcgagcctttgt ctcagagtggtccgagtttctatacgaggaggaccgcggaacaccgctcgaggacaggcctctcaagtctgtatacggggacacgg acagccttttcgtcaccgagcgtggacaccggctcatggaaaccagaggtaagaaacgcatcaaaaagcatgggggaaacctggttt ttgaccccgaacggccagagctcacctggctcgtggaatgcgagaccgtctgcggggcctgcggcgcggatgcctactccccgga atcggtatttctcgcgcccaagctctacgccctcaaaagtctgcactgcccctcgtgcggcgcctcctccaagggcaagctgcgcgcc aagggccacgccgcggaggggctggactatgacaccatggtcaaatgctacctggccgacgcgcagggcgaagaccggcagcg cttcagcaccagcaggaccagcctcaagcgcaccctggccagcgcgcagcccggagcgcaccccttcaccgtgacccagactacg ctgacgaggaccctgcgcccgtggaaagacatgaccctggcccgtctggacgagcaccgactactgccgtacagcgaaagccgcc ccaacccgcgaaacgaggagatatgctggatcgagatgccgtag (E2B mutant)

[0243] SEQ ID NO: 37: atgccaaagagaaggacccaggccgaaagggcaatggagacacagggcaaactgatcgccgccgccctgggcgtgctgagaga gaagggctacgccggctttaggatagccgacgtgcccggcgccgccggagtgtcccggggtgcacagtcccaccatttccccacca agctggagctgctgctggccacctttgagtggctgtatgagcagattaccgagaggagcagggccagactggctaaactgaagcca gaagatgatgtgatccagcagatgctggacgatgccgccgaatttttcctggatgacgacttctcaatcagtctggatctgatcgtggcc gccgaccgggaccccgccctgcgggagggcatccagagaacagtggagcggaacagattcgtggtggaggacatgtggctgggc gtgctggtgtcccgggggctgtccagagacgatgccgaggacattctgtggctgatttttaatagcgtgagagggctggccgtgcggt cactgtggcagaaggacaaggaacggtttgagagagtgagaaacagtaccctggaaatcgccagggagcgctacgccaaatttaag aggtatcccaccgacgccctggacgacttcgacctggatatgctgccagccgacgccctggacgacttcgatctggacatgctgccc gccgacgcactggacgacttcgacctggacatgctgcccggctag (CmyR- VP 16)

[0244] SEQ ID NO: 38: acgttactggccgaagccgcttggaataaggccggtgtgcgtttgtctatatgttattttccaccatattgccgtcttttggcaatgtgaggg cccggaaacctggccctgtcttcttgacgagcattcctaggggtctttcccctctcgccaaaggaatgcaaggtctgttgaatgtcgtga aggaagcagttcctctggaagcttcttgaagacaaacaacgtctgtagcgaccctttgcaggcagcggaaccccccacctggcgaca ggtgcctctgcggccaaaagccacgtgtataagatacacctgcaaaggcggcacaaccccagtgccacgttgtgagttggatagttgt ggaaagagtcaaatggctcccctcaagcgtattcaacaaggggctgaaggatgcccagaaggtaccccattgtatgggatctgatctg gggcctcggtgcacatgcttttcatgtgtttagtcgaggttaaaaaacgtctaggccccccgaaccacggggacgtggttttcctttgaa aaacacgatgataata (IRES)

[0245] SEQ ID NO: 39: atgtctagattagataaaagtaaagtgattaacagcgcattagagctgcttaatgaggtcggaatcgaaggtttaacaacccgtaaactc gcccagaagctaggtgtagagcagcctacattgtattggcacgtgcgcaacaagcagactcttatgaacatgctttcagaggcaatact ggcgaagcatcacacccgttcagcaccgttaccgactgagagttggcagcagtttctccaggaaaatgctctgagtttccgtaaagcat tactggtccatcgtgatggagcccgattgcatatagggacctctcctacgcccccccagtttgaacaagcagaggcgcaactacgctgt ctatgcgatgcagggttttcggtcgaggaggctcttttcattctgcaatctatcagccattttacgttgggtgcagtattagaggagcaagc aacaaaccagatagaaaataatcatgtgatagacgctgcaccaccattattacaagaggcatttaatattcaggcgagaacctctgctga aatggccttccatttcgggctgaaatcattaatatttggattttctgcacagttagatgaaaaaaagcatacacccattgaggatggtaata aaccaaaaaagaagagaaagctagcagtgtcagtgacatttgaagatgtggctgtgctctttactcgggacgagtggaagaagctgga tctgtctcagagaagcctgtaccgtgaggtgatgctggagaattacagcaacctggcctccatggcaggattcctgtttaccaaaccaa aggtgatctccctgttgcagcaaggagaggatccctgg (tTS)

[0246] SEQ ID NO: 40: gagggcaggggaagtcttctaacatgcggggacgtggaggaaaatcccggcccc (T2A)

[0247] SEQ ID NO: 41: atgtctagactggacaagagcaaagtcataaacggcgctctggaattactcaatggagtcggtatcgaaggcctgacgacaaggaaa ctcgctcaaaagctgggagttgagcagcctaccctgtactggcacgtgaagaacaagcgggccctgctcgatgccctgccaatcgag atgctggacaggcatcatacccacttctgccccctggaaggcgagtcatggcaagactttctgcggaacaacgccaagtcattccgct gtgctctcctctcacatcgcgacggggctaaagtgcatctcggcacccgcccaacagagaaacagtacgaaaccctggaaaatcagc tcgcgttcctgtgtcagcaaggcttctccctggagaacgcactgtacgctctgtccgccgtgggccactttacactgggctgcgtattgg aggaacaggagcatcaagtagcaaaagaggaaagagagacacctaccaccgattctatgcccccacttctgagacaagcaattgag ctgttcgaccggcagggagccgaacctgccttccttttcggcctggaactaatcatatgtggcctggagaaacagctaaagtgcgaaa gcggcgggccggccgacgcccttgacgattttgacttagacatgctcccagccgatgcccttgacgactttgaccttgatatgctgcct gctgacgctcttgacgattttgaccttgacatgctccccgggtaa (rtTA)

[0248] SEQ ID NO: 42: atggccctcgtgcaagcccatagagcccggagactgcacgctgaagcccctgacagcggagatcagcctcctcgtagaagagtgc ggcaacagcccactagagccgctcctgcccctgctagagccagacgtagacgggcacctgcacccagccctggcggtagcggcg cccctcctaccagcggcggctccccagcctctccccttctggacgcttcttctaaggacacaccggctgctcacagaccccctcgcgg aaccgtggtggcccctagaggatgtggactgctgcaggccatcgacgctgccaccaaccagcctctcgagatcagataccaccttga cctggcccgagccctgacaagactgtgcgaggtgaatctgcaggagctgccccccgacctgacacccagagaactgcagacaatg gacagctcccatctcagagacgtggtcatcaagctgagaccacccagagccgacatctggacactgggaagcagaggcgtggtcgt gcgcagcaccgtgacacctctggaacagcctgatggccagggccaagccgccgaagtggaggatcaccagcctaacccccctgg cgagggcctgaaattccccctgtgcttcctcgtgcggggaagacaggttaacctggtccaagacgtgcagcccgtgcacagatgcca gtactgcgcccggttctacaagagccagcacgagtgtagcgccagacggagagatttttacttccaccacatcaactcccacagcagc aactggtggagagaaattcagttcttccccatcggctcccacccgagaacagagagactcttcgtgacctacgacgtggagacataca catggatgggcgccttcggcaagcagctggtgcctttcatgctggtgatgaaattcggcggcgacgagcctctggtgaccgccgcga gagatctggccgccaacctgggctgggacagatgggaacaggatcctctgacattctactgtatcacccccgagaagatggccatcg gcagacagtttcggaccttccgggatcacctgcagatgctgatggcccgggatctgtggagctccttcgtagccagtaaccctcacctg gccgattgggccctgagcgaacacggcctcagctcccctgaggagctgacctatgaagagttaaagaaactgcctagcatcaaggg catcccccgtttcctggagctctacatcgtgggccacaacatcaacggcttcgatgagatcgtgctggccgctcaagtgatcaacaaca gaagcgaggtgccagggccttttcggatcacccggaatttcatgcctcgggccggcaaaatcctgttcaacgatgtgacatttgccttg cctaatcctagaagcaagaaaagaaccgatttcctgctgtgggagcaaggcggatgcgacgacaccgacttcaagtaccagtacctg aaggtgatggtcagagacacattcgccctgacacacaccagcctgagaaaggccgcccaggcttacgccctgcctgtggaaaaggg ctgctgcgcctatcaggccgtgaaccagttctacatgctgggctcttatagaagcgaggccgacggcttccctatccaggagtactgga aggaccgggaagagttcgtgctgaatagagagctctggaagaagaagggccaagacaagtacgacatcatcaaggaaacactgga ctactgtgccctggacgtgcaggtgacagccgaactggtgaacaaactgagggacagctacgccagttttgtgagagatgccgtggg cctgaccgatgcctcctttaacgtgttccagagaccgaccatcagctctaatagccacgccattttcagacagatcgtgtttcgggccga gcaacctgctagaagcaacctcggcccagatctgctggcccctagccacgagctgtacgactacgtgcgggcgtctatccggggcg gaagatgctaccccacctacctgggtatcctgagagagcccctgtacgtgtacgacatctgtggaatgtacgcctctgctctgacccac ccaatgccttggggccctcctctgaacccttacgagcgggccctggcagccagagcttggcagcaggccctggatctgcagggctg caaaatcgactacttcgacgcccggctgctgcctggcgtgttcaccgtggatgctgatcctcctgacgagacccagctggatcctctgc ctccattctgcagccgcaagggcggccggctgtgttggacaaacgagaggctgcgaggcgaggtggcgacctccgtggacctggt gaccctccacaaccggggctggcgggtgcaccttgtgcctgatgaaagaaccaccgtgttccccgaatggcggtgcgtggcccggg aatacgtgcagctgaacattgccgccaaggaaagagccgacagagacaagaaccagaccctgcggagcatcgccaagctgctgtc taacgccctgtacggcagcttcgccacaaagctggacaacaaaaagatcgtgttcagcgatcagatggacgccgcgaccctgaagg gcattacagccggccaggtgaatatcaagtcttcgagcttcctggaaaccgacaacctgagcgccgaagtcatgcctgcttttcagaga gagtactccccacagcagctggccctggccgactctgacgccgaggagagcgaggacgagcgagcccctacccctttctacagcc caccatctggcacccctgggcatgtggcctacacctataagcccatcacctttctggacgccgaggaaggcgacatgtgcctgcatac cctggaacgggttgaccccctggtggacaatgacagataccctagccacctggccagcttcgtgctggcttggacaagagctttcgtc agcgagtggtccgagttcctgtacgaagaagatagaggcacccccctggaggatagacccctcaaaagcgtctacggcgatacaga ttctctttttgtgaccgaaagaggccacagactgatggaaaccagaggaaagaaacggatcaagaagcacggcggcaacctggtgtt tgacccagagagaccagagctgacctggctggttgaatgtgaaacagtgtgcggcgcatgcggcgccgacgcctacagccctgag agcgtgttcctggctcctaagctgtatgctctgaaaagcctgcactgcccctcatgcggagcctccagcaagggcaagctgagagcta agggtcatgccgctgagggcctggactacgacacaatggtgaagtgctacctggccgacgcccagggcgaggaccggcagcggtt cagcaccagcagaacaagcctgaagcggacattggcttctgcccaacctggagctcaccctttcaccgtgactcagacaacactgac caggaccctgcgcccctggaaggacatgaccctcgccaggctagatgagcaccgtctcctgccctacagcgagtcccggcctaacc ccagaaacgaggaaatctgctggatcgagatgccgtg (E2B polymerase)

[0249] SEQ ID NO: 43: ggtaccgagctcgactttcacttttctctatcactgatagggagtggtaaactcgactttcacttttctctatcactgatagggagtggtaaa ctcgactttcacttttctctatcactgatagggagtggtaaactcgactttcacttttctctatcactgatagggagtggtaaactcgactttca cttttctctatcactgatagggagtggtaaactcgactttcacttttctctatcactgatagggagtggtaaactcgactttcacttttctctatc actgatagggagtggtaaactcgacctatataagcagagctcgtttagtgaaccgtcagatcgcctggagacgccatccacgctgtttt gacctccatagaagacaccgggaccgatccagcctccgcggccccgaattg (TRE2G)

[0250] SEQ ID NO: 44: gatctgtcgaccaactagtaccccgggaactcgtcacgacgaaagaaacaaaccaacctgtctgtattatcaaagaaacaaaccaacc tgtctgtattatcaaagaaacaaaccaacctgtctgtattatcaaagaaacaaaccaacctgtctgtattatcaaagaaacaaaccaacct gtctgtattatcaaagaaacaaaccaacctgtctgtattatccgaaggtaggcgtgtacggtgggaggtctatataagcagagctggttta gtgaaccg (6 x CuO-minimal CMV promoter)

[0251] SEQ ID NO: 45: actctcttccgcatcgctgtctgcgagggccagctgttgggctcgcggttgaggacaaactcttcgcggtctttccagtactcttggatcg gaaacccgtcggcctccgaacggtactccgccgccgagggacctgagcgagtccgcatcgaccggatcggaaaacctctcgagaa aggcgtctaaccagtcacagtcgcaag (Adenovirus TPL UTR)

[0252] SEQ ID NO: 46: gatctgtcgaccaactagtaccccgggaactcgtcacgacgaaagaaacaaaccaacctgtctgtattatcaaagaaacaaaccaacc tgtctgtattatcaaagaaacaaaccaacctgtctgtattatcaaagaaacaaaccaacctgtctgtattatcaaagaaacaaaccaacct gtctgtattatcaaagaaacaaaccaacctgtctgtattatccgaaggtaggcgtgtacggtgggaggtctatataagcagagctggttta gtgaaccgactctcttccgcatcgctgtctgcgagggccagctgttgggctcgcggttgaggacaaactcttcgcggtctttccagtact cttggatcggaaacccgtcggcctccgaacggtactccgccgccgagggacctgagcgagtccgcatcgaccggatcggaaaacc tctcgagaaaggcgtctaaccagtcacagtcgcaag (6 x CuO-minimal CMV promoter-adenovirus TPL)

Claims

CLAIMS1. A method of producing a helper-dependent adenoviral vector in a population of cells, wherein said cells comprise an adenoviral helper genome which encodes one or more viral genes necessary for replication and / or packaging of the helper-dependent adenoviral vector, wherein replication of the adenoviral helper genome is inhibited while excising a packaging signal sequence therefrom, and wherein replication of the adenoviral helper genome is induced to provide for replication and / or packaging of the genome of the helper-dependent adenoviral vector.

2. The method according to claim 1, wherein replication of the adenoviral helper genome is induced after excising the packaging signal sequence from the adenoviral helper genome.

3. The method according to claim 1 or 2, wherein the adenoviral helper genome is introduced into said cells prior to, simultaneously with or subsequently to the helperdependent adenoviral vector, optionally by transfection, electroporation, a non-viral lipid nanoparticle, or viral infection.

4. The method according to any one of the preceding claims, wherein the cells are incubated under conditions inhibiting replication of the adenoviral helper genome for a sufficient period to substantially remove the packaging signal sequence from the adenoviral helper genome.

5. The method according to claim 4, wherein the incubation is for at least 6 hours, preferably at least 12 hours, more preferably at least 24 hours, optionally wherein the incubation is for 12-48 hours.

6. The method according to any one of the preceding claims, wherein the cells comprise one or more adenovirus early gene regions integrated into their genome, optionally selected from El, E2 and / or E4 regions, optionally wherein the cells further comprise one or more intermediate or late gene regions integrated into their genome.

7. The method according to any one of the preceding claims, wherein the cells are mammalian cells, optionally HEK293 cells, A549, HeLa or amniocyte-based cells, orderivatives of any thereof, such as PerC6 cells, 911 cells, HEK293-E2 cells, HEK293-E4orf6,HEK293-L4 100K or HEK293-L4 22 / 33K cells.

8. The method according to any one of the preceding claims, wherein the inhibition of replication of the adenoviral helper genome is due to inactivation of one or more genes encoding for the adenoviral E1A, E1B, E2B DNA polymerase, E2B Preterminal protein, E2A DNA binding proteins and / or E4orf6.

9. The method according to claim 8, wherein the inactivation of a said gene is by deletion, mutation or insertion of an exogenous nucleic acid sequence, or by prevention of expression, optionally by use of a KRAB-Zinc Finger Protein, an siRNA / shRNA, or CRISPR / dCas9.

10. The method according to claim 9, wherein the inactivation of a said gene is due to mutation, deletion, or insertion of an exogenous nucleic acid sequence in the adenovirus El, E2 or E4 promoters.

11. The method according to claim 10, wherein the adenovirus El, E2 and / or E4 genes are inactivated, optionally wherein one or more of the E2 genes are inactivated by one or more mutations selected from ts 19, ts36, ts69, tsl07, ts 125, tsl49 and sublOOr, more preferably ts36, ts 125 or tsl49, most preferably ts 125.

12. The method according to any one of the preceding claims, wherein transcription from the El, E2 or E4 promoter is inhibited, optionally wherein the transcription is inducible, such as by use of TetR, Tet-On, Tet-Off, cumate-inducible gene expression, tryptophan-inducible gene expression, IPTG-inducible gene expression, CRISPR-dCas gene induction, TALE / ZINC finger repression, or a Nuclease splicing gene expression system.

13. The method according to any one of claims 1-12, wherein replication of the adenoviral helper genome is induced by expression of one or more genes necessary for replication of the adenoviral helper genome in the cells, optionally wherein the genes are functional versions of one or more genes inactivated in the adenoviral helper genome, optionally wherein the one or more genes are introduced by transfection, electroporation, a non- viral lipid nanoparticle, or viral infection.

14. The method according to claim 13, wherein the expression of the one or more genes is induced by a small molecule activator, such as tetracycline, doxycycline, cumate, tryptophan, or Isopropyl P- d-1 -thiogalactopyranoside, optionally, wherein the expression of the one or more genes is induced by a change in temperature or light conditions.

15. The method according to any one of the preceding claims, wherein the helper adenoviral genome encodes any or all of the early transcription units, optionally selected from early region 1A (El A), E1B, E2, E4 and VARNA, any or all of the Intermediate transcription units, optionally selected from IX, IVa2, and L4 intermediate, and any or all of the late transcription units optionally selected from Hexon, Penton, Fibre, and 100K.

16. The method according to any one of the preceding claims, wherein the packaging signal sequence comprises any one of SEQ ID NOs 17-23, optionally SEQ ID NO: 18 or 19, preferably SEQ ID NO: 19.

17. The method according to any one of the preceding claims, wherein said cells express an enzyme capable of excising the packaging signal sequence from the adenoviral helper genome, optionally wherein the packaging signal is flanked by excision sites or recombination recognition sites for the enzyme, optionally wherein expression of the enzyme is induced by an activator encoded in the adenoviral helper genome.

18. The method according to claim 17, wherein the enzyme is an endonuclease or a recombinase, optionally selected from CRE, FLP, phiC31, TP901-1, Lambda Red, I-Scel, Piggybac transposase, or Sleeping beauty transposase, optionally wherein the enzyme comprises the amino acid sequence of any one of SEQ ID NOs 10-16 or a variant thereof, optionally SEQ ID NO: 10 or SEQ ID NO: 11 or a variant of either thereof, preferably SEQ ID NO: 10 or a variant thereof.

19. The method according to any one of the preceding claims, wherein the helperdependent adenoviral vector comprises a genome comprising one or more transgenes, wherein the transgenes may comprise non-coding or coding sequences, optionally wherein a said transgene is a human gene, a reporter gene or encodes a therapeutic product, such as an shRNA, CRISPR guide RNA, or siRNA or a polypeptide, preferably wherein the transgene encodes a therapeutic polypeptide, optionally wherein the one or more transgenes comprise aDMD, MYO7A, Abca4, COL4A5, von Willebrand factor, factor VIII, factor IX, cadherin-23, ALMS1, OTOF, GBA1, CRISPR guide RNA, or a CRISPR Cas9 transgene optionally more than one of said transgenes, optionally wherein the one or more transgenes are positioned 3’ to a packaging signal sequence in the genome of the helper-dependent adenoviral vector.

20. The method according to any one of the preceding claims, wherein the genome of the helper-dependent adenoviral vector comprises inverted terminal repeat sequences, optionally selected from any of SEQ ID NOs 24-30, preferably SEQ ID NO: 25 or 26, most preferably SEQ ID NO: 26.

21. The method according to any one of the preceding claims, wherein the helper adenoviral and / or helper-dependent adenoviral genome is least lOkb, more preferably at least 20kb, and most preferably at least 30kb in length.

22. The method according to any one of the preceding claims, wherein the helperdependent adenovirus produced according to the method is used to introduce the helperdependent adenovirus genome into the population of cells.

23. The method according to any one of the preceding claims, wherein the inhibition of replication of the adenoviral helper genome is due to inactivation of E2B DNA polymerase.

24. The method according to any one of the preceding claims, wherein the replication of the adenoviral helper genome is induced by expression of a functional E2B DNA polymerase gene, optionally wherein the functional E2B DNA polymerase gene is introduced by viral infection, preferably wherein the gene encoding the functional E2B DNA polymerase gene is comprised within an AAV genome.

25. The method according to any one of claims 17 to 24, wherein the enzyme capable of excising the packaging signal sequence from the adenoviral helper genome is a CRE recombinase, optionally SEQ ID NO: 10 or a variant thereof, optionally wherein a gene encoding the CRE recombinase is introduced by viral infection, preferably wherein the gene encoding the CRE recombinase is comprised within an AAV genome.

26. The method according to any one of claims 4 to 25, wherein the incubation under conditions inhibiting replication of the adenoviral helper genome is for at least about 20hours, more preferably at least about 24 hours, optionally about 20 to about 30 hours or about 24 to about 30 hours.

27. A helper-dependent adenoviral vector preparation obtainable according to the method of any one of the preceding claims.

28. A population of cells producing a helper-dependent adenoviral vector obtainable according to the method of any one of the preceding claims.

29. A method of reducing helper virus contamination in a helper-dependent adenoviral vector preparation, comprising producing a helper-dependent adenoviral vector in a population of cells, wherein said cells comprise an adenoviral helper genome which encodes one or more viral genes necessary for replication and / or packaging of the helper-dependent adenoviral vector, wherein replication of the adenoviral helper genome is inhibited while excising a packaging signal sequence therefrom, and wherein replication of the adenoviral helper genome is induced to provide for replication and / or packaging of the genome of the helper-dependent adenoviral vector, optionally wherein the helper-dependent adenoviral vector is produced by a method according to any one of claims 1-26.

30. A cell comprising a nucleic acid sequence encoding a CRE recombinase, a nucleic acid sequence encoding a functional adenovirus E2B DNA polymerase, an adenoviral helper genome which lacks a functional E2B polymerase gene and encodes one or more viral genes necessary for replication and / or packaging of a helper-dependent adenoviral vector, a helperdependent adenoviral genome, wherein the nucleic acid sequence encoding the CRE recombinase and / or the nucleic acid sequence encoding a functional adenovirus E2B DNA polymerase are comprised within an AAV genome or integrated into the genome of the cell, and wherein the CRE recombinase is able to excise a packaging signal from the adenoviral helper genome.

31. A kit comprising a nucleic acid sequence encoding a CRE recombinase comprised within an AAV genome, an adenoviral helper genome which lacks a functional E2B polymerase gene and encodes one or more viral genes necessary for replication and / or packaging of a helperdependent adenoviral vector, a helper-dependent adenoviral genome, and a recombinant cell containing a nucleic acid sequence integrated into its genome encoding a functional adenovirusE2B DNA polymerase, wherein the CRE recombinase is able to excise a packaging signal from the adenoviral helper genome.

32. A kit comprising a recombinant cell containing a nucleic acid sequence encoding a CRE recombinase integrated into its genome, a nucleic acid sequence encoding an adenoviral helper genome which lacks a functional E2B polymerase gene and encodes one or more viral genes necessary for replication and / or packaging of a helper-dependent adenoviral vector, a nucleic acid sequence encoding a helper-dependent adenoviral genome, and a nucleic acid sequence encoding a functional adenovirus E2B DNA polymerase comprised within an AAV genome, wherein the CRE recombinase is able to excise a packaging signal from the adenoviral helper genome.

33. A kit comprising a nucleic acid sequence encoding a CRE recombinase comprised within an AAV genome, a nucleic acid sequence encoding an adenoviral helper genome which lacks a functional E2B polymerase gene and encodes one or more viral genes necessary for replication and / or packaging of a helper-dependent adenoviral vector, a nucleic acid sequence encoding a helper-dependent adenoviral genome, and a nucleic acid sequence encoding a functional adenovirus E2B DNA polymerase comprised within an AAV genome, wherein the CRE recombinase is able to excise a packaging signal from the adenoviral helper genome.

34. A method of producing a helper-dependent adenoviral vector in a population of cells according to claim 30, wherein the CRE recombinase excises a packaging signal from the adenoviral helper genome and wherein replication of the adenoviral helper genome provides for replication and / or packaging of the genome of the helper-dependent adenoviral vector.

35. The cell of claim 30 or the method of claim 34, wherein the nucleic acid sequence encoding a CRE recombinase is comprised in an AAV genome, and the nucleic acid sequence encoding a functional adenovirus E2B DNA polymerase is comprised in an AAV genome36. The cell of claim 30 or the method of claim 34, wherein the cell comprises the nucleic acid sequence encoding a CRE recombinase integrated in its genome, and the nucleic acid sequence encoding a functional adenovirus E2B DNA polymerase is comprised in an AAV genome.

37. The cell of claim 30 or the method of claim 34, wherein the cell comprises the nucleic acid sequence encoding a CRE recombinase integrated in its genome and the nucleic acid sequence encoding a functional adenovirus E2B DNA polymerase integrated in its genome.

38. The cell, kit or method according to any one of claims 30-37, wherein expression of the CRE recombinase is inducible by an activator, optionally wherein the activator is a small molecule activator such as doxycycline, or a gene present in the helper adenoviral genome.

39. The cell, kit or method according to any one of claims 30-38, wherein expression of the functional adenoviral E2B DNA polymerase is inducible by an activator, optionally wherein the activator is a small molecule activator such as doxycycline.

40. A cell, kit or method according to any one of claims 30-39 wherein expression of the functional adenovirus E2B DNA polymerase is under inducible transcription or translation control.

41. The cell, kit or method according to claim 40, wherein the inducible transcription control comprises a TetR, Tet-On, Tet-Off, or cumate-inducible promoter, a PcaR inducible system, and / or the inducible translation control system comprises the tryptophan-inducible expression (TRiP) system.

42. The cell, kit or method according to claim 41 comprising a nucleic acid sequence encoding an activator protein or a repressor protein for the inducible transcription or translation control system, optionally wherein the activator protein is TetR-VP16 or cumate repressor fused to the VP16 transactivator (CmyR-VP16)) or the repressor protein is TetR, TetR-E37A-P39K, PcaR, or a transcription silencer protein (tTS) wherein a TetR is fused to a KRAB domain.

43. The cell or method according to claim 42, wherein the nucleic acid sequence encoding an activator protein or a repressor protein for the inducible transcription or translation control system is introduced into the cell by stable chromosomal integration, DNA plasmid transfection or by infection using viral vectors such as AAV vectors, adenovirus vectors, herpes virus vectors, lentiviral vectors, baculovirus vectors, bocavirus vectors and anellovirus vectors.

44. The cell, kit or method according to any one of claims 30-43, wherein the cell is a mammalian cell, optionally encoding an adenoviral El region.

45. The cell, kit or method according to claim 44, wherein the mammalian cell is selected from HEK293 cells, A549, HeLa or amniocyte-based cells, or derivatives of any thereof, such as PerC6 cells, 911 cells, HEK293-E2 cells, HEK293-E4orf6, HEK293-L4 100K or HEK293-L422 / 33K cells, preferably HEK293 cell.