Adenoviral vectors

Abstract

<DP=1>²The present invention relates to an adenoviral vector comprising² a regulatable Major Late Promoter and an exogenous transgene. ²The invention also provides cells comprising such adenoviral vectors, and ²processes using such vectors.²<DP=2>²²

Description

<DP=1>²CA 03070654 2020-01-21²WO 2019 / 020992 PCT / GB2018 / 052083²- 1 -²ADENOVIRAL VECTORS²The present invention relates to an adenoviral vector comprising a regulatable ²Major Late²Promoter and an exogenous transgene. The invention also provides cells ²comprising such²adenoviral vectors, and processes using such vectors.²Adenoviruses are an attractive and versatile tool in biotechnology, having ²well understood ²genetics and the ability to grow to high yields in tissue culture. The ²replication cycle of²Adenoviruses is highly complex, involving both early and late phases. The ²transition from early²to late is considered to occur following DNA replication and the activation of ²the Major Late ²promoter (MLP) in the virus genome. The MLP drives the expression of all virus ²late transcripts, ²and can convert up to 30% of the cells' protein into virus structural ²proteins. Modification to the ²MLP in situ to provide inducible expression has not previously been ²demonstrated, primarily²because the virus DNA polymerase coding sequence is in the opposing DNA ²strand.²Whilst adenoviruses are useful laboratory tools for a number of applications, ²the fact they are so ²productive represents a major problem if they are used in a manufacturing work ²flow, namely ²they generate substantial amounts of virus particles that must be removed ²during downstream²processing. For example, when adenovirus has previously been used for protein ²expression²purposes, or the production of other virus-like particles, or the large scale ²manufacture of ²Adeno-associated virus (AAV), the presence of intact Adenoviral particles at ²the end of the ²production process is highly undesirable.².. The ability to manufacture proteins in mammalian cells is increasingly ²attractive, with many high²value recombinant proteins now being produced in these systems, affording ²optimum protein ²processing, folding and glycosylation. This is frequently achieved using ²transient transfection of ²a plasmid encoding the required transgene under the control of a strong ²promoter, such as the ²Cytomegalovirus (CMV) immediate early promoter. However, compared to some ²viral systems²in other organisms (such as Baculovirus in insect cells), this process is ²relatively inefficient,²often demonstrating variable protein yields that rarely exceed 3% of the total ²cell protein mass.²The extensive characterisation of the Adenovirus genome, coupled with the ²wealth of ²knowledge regarding its gene expression and life cycle, makes this virus an ²ideal candidate²platform on which to significantly improve recombinant protein yields. One ²major benefit is²<DP=2>²CA 03070654 2020-01-21²WO 2019 / 020992 PCT / GB2018 / 052083²- 2 -²demonstrated by the virus' ability to actively hijack the mammalian cell's ²machinery, and to ²inhibit the production of cellular proteins.²However, in addition to inhibiting the production of cellular proteins, ²considerable cellular²resources are used by the virus to produce viral structural proteins. The ²quantity of capsid²proteins produced, for example, is vast and has been calculated to be up to ²30% of total cell ²protein, and removing these proteins and the assembled virus particles after ²production is ²challenging.²Similar to recombinant proteins, the demand for adeno-associated virus ²particles is increasing²significantly, owing to recent clinal successes in the treatment of retinal ²disorders and ²haemophilia. Traditionally, the production of AAV has been achieved through ²two different ²routes. Initially, AAV was generated using wild-type (VVT) Adenovirus serotype ²5 whilst ²transfecting cells with plasmids encoding the Rep and Cap genes and the AAV ²genome. This²allowed the WT adenovirus to provide a number of factors in trans that ²facilitated virus²replication. However, there a number of limitations to this approach: for ²example, each batch of ²AAV must be separated from the Ad5 particles to provide a pure product and ²ensuring that all ²Ad5 has been removed is challenging. Moreover, the fact that during production ²the cell is ²devoting huge resource to the production of Adenoviral particles rather than ²AAV is also² undesirable.²More recently, the adenovirus-based systems have been replaced with plasmids ²encoding the ²sections of the Adenovirus genome required for AAV production. Whilst this has ²solved some of ²the concerns over Adenovirus particles being present in the final virus ²preparation, a number of²issues remain. These include the requirement to pre-manufacture sufficient ²plasmid for²transfection into the production cell line and the inherently inefficient ²process of transfection ²itself. The yields from these systems are also lower than those using Ad5 ²based approaches. ²The inventors have now discovered that transcription of the late adenoviral ²genes can be ²regulated (e.g. inhibited) by the insertion of a repressor element into the ²Major Late Promoter.²By "switching off" expression of the Late genes, the cell's protein-²manufacturing capabilities can²be diverted toward the production of a desired recombinant protein. ²Importantly, the strategic ²silencing of the Major Late promoter in the adenovirus genome in accordance ²with the current ²invention allows the virus to still replicate its DNA in the cell, providing ²thousands of DNA copies ²of the virus genome that can be transcribed for a range of applications.²<DP=3>²CA 03070654 2020-01-21²WO 2019 / 020992 PCT / GB2018 / 052083²- 3 -²This invention provides a range of advantages. For example, it can be used to ²direct a cell's ²protein production capability towards the production of specific recombinant ²proteins at ²increased yields compared to comparable systems wherein the viral Late genes ²are still ²expressed. Furthermore, the ability to "switch off' the production of viral ²structural proteins²means that no or essentially no viral particles are produced during the ²protein-production²process. Consequently, economic savings can be made due to a reduction in the ²need to ²remove virus particles from the purified protein.²The invention also has the advantage of providing a simple, cost-effective, ²way to manufacture²AAV particles where the Rep and Cap proteins of AAV can be encoded within the ²Adenovirus to²provide the high expression levels which are required to make the AAV ²particles by maintaining ²the replication of the Adenoviral genome, but also preventing the production ²Adenovirus ²particles in the final AAV preparation.²Some modifications of the MPL have previously been reported. These include ²making a copy of²the MLP and placing it either in other plasmids for expression level analysis ²(El-Mogy, 2012) or ²within the El region of the Adenovirus genome subject to the insertion of ²sites to regulate its ²expression (Molin, 1998). Other work has included the mutation of the MLP TATA ²box for both ²basic research (Concino etal., 1983; Concino etal., 1984) and in order to ²allow the virus to be²selectively grown only in cells which have a trans-complementing factor in a ²cell. Importantly,²the latter approach provides for a system where the activity of the major late ²promoter is entirely ²dependent on a complementing protein factor being present in a cell, and ²therefore in a cell ²where this factor is absent the MLP is not active. The invention, however, ²provides the converse ²of this, where the MLP maintains it full expression activity level in cells ²where a repressor not²bound, providing high level virus replication with minimal disturbance to the ²virus life cycle.²The virus of the invention described herein is therefore fully active when not ²repressed but is ²capable of being repressed, depending on the presence or absence of a ²repressor. A repressor ²binding site has not previously successfully been inserted into the MLP in ²situ for the regulation²of its expression in an adenovirus genome. The current inventors have further ²improved this²system to place the repressor protein coding sequence under the control of the ²Major Late ²Promoter itself. In this approach, the Major Late Promoter self-represses ²itself because when ²the Major Late Promoter tries to transcribe the structural proteins of the ²virus, it will also ²transcribe a repressor capable of repressing its own activity, thereby ²allowing for a negative²feedback loop that prevents MLP activity and providing tight regulation of MLP ²expression.²<DP=4>²CA 03070654 2020-01-21²WO 2019 / 020992 PCT / GB2018 / 052083²- 4 -²It is therefore an object of the invention to provide an adenoviral vector ²system which provides ²increased yields of recombinant proteins, and can be used to provide non-²adenovirus virus and ²virus-like particles.²It is also an object of the invention to provide a process of producing a ²transgene product in ²adenovirus-infected cells, which reduces the need to remove virus particles ²from the purified ²product which may be a protein, non-adenovirus particle or virus-like ²particle.²In one embodiment, the invention provides an adenoviral vector comprising a ²repressible Major ²Late Promoter (MLP), wherein the MLP comprises one or more repressor elements ²which are ²capable of regulating or controlling transcription of the adenoviral late ²genes, and wherein one ²or more of the repressor elements are inserted downstream of the MLP TATA box.²In another embodiment, the invention provides an adenoviral vector comprising ²a repressible ²Major Late Promoter and an exogenous transgene.²In another embodiment, the invention provides an adenoviral vector comprising: ²²(a) a plurality of adenoviral early genes, and²(b) a plurality of adenoviral late genes under the control of a Major Late ²Promoter (MLP), and ²(c) a transgene,²wherein the MLP comprises one or more repressor elements which are capable of ²regulating or ²controlling transcription of the adenoviral late genes.²Preferably, a gene encoding a repressor protein which is capable of binding to ²the repressor ²element is encoded within the adenoviral genome.²Preferably, the transgene comprises a Tripartite Leader (TPL) in its 5'-UTR.²Preferably, the presence of the repressor element does not affect production ²of the adenoviral ²E2B protein.²Preferably, the repressor protein is transcribed from the MLP.²<DP=5>²CA 03070654 2020-01-21²WO 2019 / 020992 PCT / GB2018 / 052083²- 5 -²Preferably, the one or more repressor elements are inserted between the MLP ²TATA box and ²the +1 position of transcription.²Adenovirus is the most commonly investigated and exploited virus in modern ²scientific research.²It has found utility in the fields of vaccination, gene therapy, virotherapy, ²and also as complex²tools for understanding biological systems (McConnell etal., 2004).²The adenoviridae family of viruses are double stranded DNA viruses which were ²first isolated ²from the adenoids of infected children in 1953 (Rowe etal., 1953). The family ²consists of many²species infecting a broad variety of hosts including humans, birds and ²amphibians and is²divided into four genuses to reflect the wide variety of hosts they infect.²The mastadenoviridae consists of 25 unique adenovirus species infecting ²mammalian hosts. ²Within this genus there are currently seven distinct species of adenovirus ²that infect humans.²They are denoted by a letter as human adenoviruses A-G. VVithin these species ²multiple²serotypes have been identified that are numbered from 1-57 and are all ²recognised as ²independent strains by the International Committee on the Taxonomy of Viruses: ²²Species A: 12, 18,31²Species B: 3,7, 11, 14, 16, 21, 34, 35, 50, 55² Species C: 1,2, 5, 6, 57²Species D: 8, 9, 10, 13, 15, 17, 19, 20, 22, 23, 24, 25, 26, 27, 28, 29, 30, ²32, 33, 36, 37, 38, 39,²42, 43, 44, 45, 46, 47, 48, 49, 51, 53, 54, 56²Species E: 4²Species F: 40, 41² Species G:52²Adenoviruses are small (approximately 90 nnn in diameter) non-enveloped ²viruses with an ²icosahedral capsid with 12 fibres projecting from the icosahedron vertices. ²The virus consists of ²240 hexon trimers, 12 penton pentanners, 60 Illa monomers, 12 fiber timers, 60 ²VII hexanners,²80 IX trimers as well as two terminal repeat proteins and multiple core ²proteins. Many of the²latter are essential for intracellular trafficking and proteolytic cleavage of ²endocytosed viral ²capsids.²The DNA genome of adenovirus varies in length dependent on serotype but is ²typically 34-36²kilobases in length. The adenovirus serotype five genome is usually is 35938 ²base pairs with a²<DP=6>²CA 03070654 2020-01-21²WO 2019 / 020992 PCT / GB2018 / 052083²-6-²103 base pair terminal repeat at each end and a 58% GC content. The genome is ²linear within ²the capsid structure and for the majority of the replication cycle but can ²form pan-handle circular ²structures within the nucleus during genome replication.²The process of cell attachment for adenoviruses varies dependent on the ²serotype. Adenovirus²species C (including serotypes two and five) have been shown to attach to ²cells via their fibre ²domain binding to the cell surface coxsackie and adenovirus receptor (CAR) ²(Tomko etal., ²1997) which is a component of cellular tight junctions (Honda etal., 2000). ²This initial interaction ²is high affinity and is followed by a lower affinity interaction between the ²virus penton base and²cell surface integrins (aV83 & aV85) which are abundant on epithelial cells ²(Wickham etal.,²1993). Following absorption to the cell surface virions are endocytosed and in ²a poorly defined ²step the virus capsid escapes from the endosome into the cytosol. Within the ²cytosol adenoviral ²capsids bind to the microtubule network and are transported via a Dynein ²dependent ²mechanism to the nucleus where the DNA is delivered into the nuclear lumen ²following capsid²interaction with the nuclear pore complex (Trotman etal., 2001; Kelkar etal., ²2004).²Our understanding of adenovirus genetics, transcription and translation is ²primarily derived from ²virological studies using adenovirus species C serotype five. The sequential ²process of gene ²transcription from the adenovirus genome reflects the protein requirements of ²the virus at each²stage of the replication process. The transcription from the adenovirus genome ²is therefore²divided into early and late events dependent on the timing of initiation of ²transcription from each ²viral promoter (Figure 1). The first proteins produced from the virus genome ²are the ElA ²proteins. The El A transcription unit produces multiple mRNA molecules through ²alternative ²splicing which in turn produce multiple proteins ranging from 6-36 kDa. The ²ElA proteins have²two major roles within an infected cell. Firstly, they induce the cell to ²enter the S phase of the²cell cycle to allow the efficient replication of the viral genome. Secondly, ²they induce ²transcription of the other early promoters within the viral genome through ²transactivation. These ²promoters control the production of the El B, E2, E3, E4 and E5 proteins. ElA ²expression is ²immediately followed by VA RNA and El B and E3 protein production. These ²proteins and RNA²molecules help to prevent the development of an anti-viral response. These ²early events in viral²replication help to shape the intracellular environment to allow the ²replication of the viral ²genome before packaging. Later transcription events involve the production of ²structural ²proteins and proteins essential for cell lysis which are derived primarily ²from a single promoter ²(the major late promoter) which transcribes the late regions 1-5. Cell lysis ²is dependent on the²E3-11.6K protein (also termed the adenovirus death protein) which despite its ²labelling as an²<DP=7>²CA 03070654 2020-01-21²WO 2019 / 020992 PCT / GB2018 / 052083²- 7 -²early gene is only produced late in infection and from the major late promoter ²(Tollefson etal., ²1996).²Adenovirus genes are divided into early (El -4) and late (L1-5) transcripts, ²with multiple protein²isoforms driven from a range of splicing events. The early regions are divided ²into El, E2, E3²and E4. El is essential for transitioning the cell into a phase of the cell ²cycle that is conducive to ²virus replication and inhibiting apoptosis and promoting cell division. The E2 ²region is largely ²responsible for the replication of the DNA genome, containing the DNA binding ²protein (E2A), ²the terminal protein and the DNA polymerase (E2B). E3 contains genes involved ²in immune²regulation of host responses and E4 contains a range of genes involved in ²regulating cell²pathways such as non-homologous end joining (NHEJ) and complexing with El B-²55K to ²mediate p53 degradation.²The adenovirus late genes are all transcribed from the same promoter, the ²Major Late Promoter²and all share the same 5' mRNA terminus which contains three exons that ²collectively form the²tri-partite leader sequence. The late genes are expressed by a series of ²splice events that allow ²the expression of approximately 13 proteins that either form a part of the ²virus particle (e.g. ²Hexon and Fibre) or involved in its assembly (e.g. 100K protein).²Adenoviral vectors are vectors which are based on or derived from the genome ²of a virus of the²family Adenoviridae. Preferably, the adenovirus is a human adenovirus from ²group A, B, C, D,²E, F or G. More preferably, the adenovirus is a human adenovirus from group B ²or C or D. ²Even more preferably, the adenovirus is a human adenovirus from groups B or C.²The adenoviral vector comprises a plurality of adenoviral early genes (Figure ²1).²The El A proteins are the translation products of the first gene transcription ²events from the ²adenovirus genome within the nucleus at the ElA region. This initial ²transcription is driven by a ²strong constitutively active enhancer element within the ElA promoter and ²allows significant²quantities of ElA mRNA to be produced. They are one of two sets of proteins in ²the adenovirus²genome which are capable of inducing transformation with El B proteins also ²able to induce cell ²cycle progression. ElA and El B genes are essential for virus replication.²The E2 genes are divided into two sections in the adenovirus genome: the E2A ²and the E2B²regions, and both are required for virus replication. E2B contains the DNA ²polymerase gene²<DP=8>²CA 03070654 2020-01-21²WO 2019 / 020992 PCT / GB2018 / 052083²- 8 -²which is fundamentally required for the amplification of the virus genomic ²DNA. Similarly, this ²region also contains the Terminal protein which is required for the initiation ²of virus genome ²replication. The terminal protein is covalently attached the end of the virus ²genomic DNA. The ²E2A region contains a DNA binding protein that is required for DNA ²replication. All E2 genes² are fundamentally required for virus replication.²The E3 genes are primarily involved in regulating cell and host immune ²responses to virus ²infection, however, as the majority of viruses used for biotechnology ²applications are in vitro ²many, if not all, of these virus genes can be removed without reducing virus ²replication²efficiency. The majority of the E3 genes are not essential for virus ²replication. However, some²genes such as the Adenovirus Death Protein (ADP) are required for efficient ²replication and ²virus production.²The E4 region of the adenovirus genome is similar to the El region in that it ²is primarily involved²in producing proteins that help the virus control and regulate the cell to ²ensure efficient virus²replication and production. The region includes 6 open reading frames (ORFs) ²that are able to ²aid in preventing non-homologous end joining and apoptosis amongst a number of ²other ²discrete functions. The relative importance of each E4 transcript to virus ²replication is variable ²with some being essential whilst others can be deleted or modified with little ²to no effect of virus²growth kinetics and production.²The adenoviral vector of the invention preferably comprises sufficient ²adenoviral early genes in²order for the adenovirus to be capable of replicating the viral genome in the ²nucleus of a cell in²which it is placed.²The adenoviral vector comprises a plurality of adenoviral late genes (Figure ²1).²The virus late genes are divided into five main transcript families named Ll-²L5. These²transcripts primarily encode proteins that are involved in virus assembly and ²the structural²proteins of the virus. During virus replication they can represent as much as ²30-40% of the cells²protein content (Gamier, 1994; Ginsberg, 1984).²The Li series of transcripts encode for the 13.6K, 52K, and PIlla proteins. ²These proteins are all ²involved in virus assembly and particle production. Li genes are required for ²successful virus²assembly but not genomic DNA replication²<DP=9>²CA 03070654 2020-01-21²WO 2019 / 020992 PCT / GB2018 / 052083²- 9 -²The L2 series of transcripts encode for the penton base, pVII, V, pX proteins. ²These form ²structural parts of the virus capsid and are required for the particle to ²assemble correctly. ²Penton base contains an RGD motif that is important for virus attachment to ²the cell surface²during infection. L2 genes are required for successful virus assembly but not ²genomic DNA²replication²The L3 series of transcripts encode for the pVI, hexon, and protease proteins. ²The Hexon ²protein is a major component of the virus capsid and is antigenically diverse ²between serotypes.²The protease protein is involved in cellular entry and the virus capsid ²maturation. L3 genes are²required for successful virus assembly but not genomic DNA replication²The L4 series of transcripts encode the 100K, 33K, 22K, pVII proteins. These ²proteins are ²involved in a range of functions. 100K protein is involved in both aiding ²virus hexon assembly²and nuclear import but may also play a role in shifting cell mRNA translation ²to cap-independent²translation. In one embodiment of the invention, the 100K protein may be ²provided in trans ²within a cell rather than from within the virus genome. The 22K protein is ²involved in virus ²encapsidation. L4 genes are required for successful virus assembly but not ²genomic DNA ²replication. However, the 100K protein may aid in shifting cellular protein ²translation towards² those transcripts that contain a tripartite leader (TPL) sequence.²As such, in one embodiment of the invention, the expression of the 100K ²protein may not be²controlled by the MLP. The 100K protein in one embodiment may be transferred ²to another ²position in the virus genome not under the control of the MLP. In another ²embodiment, the 100K²protein may be transferred to a separate virus. In another embodiment, the ²100K protein may be²transferred to the chromosome of a cell or provide in trans by DNA ²transfection or ²electroporation.²L5 encodes the Fibre gene. Fibre is a virus structural protein involved in ²attachment to cell²surfaces and in mediating virus cellular infection. The Fibre protein is ²produced in significant²excess of its requirement for virus particle formation. An embodiment of the ²invention may ²silence a minimum of one of either Fibre or Hexon (L3) proteins as these are ²some of the most ²abundant proteins produced during virus replication. L5 genes are required for ²successful virus ²assembly but not genomic DNA replication²<DP=10>²CA 03070654 2020-01-21²WO 2019 / 020992 PCT / GB2018 / 052083²- 10 -²The adenoviral vector of the invention comprises sufficient adenoviral late ²genes in order for the ²adenovirus to be capable of being packaged in a cell to produce virus ²particles capable of ²infecting another mammalian cell. The virus genome will contain at least one ²late transcript ²generated from the Major Late promoter. Some proteins may be removed and ²provided in trans² and others may be transferred into a host cell chromosome.²In the wild-type adenoviral genome, a single Major Late Promoter (MLP) is ²responsible for ²promoting the parallel transcription of the adenoviral late transcripts, L1-L5 ²(Figure 1). The ²adenoviral vector of the invention also comprises a MLP. The MLP is situated ²at the 5'-end of²these late transcripts.²Multiple mRNAs are produced from the late transcripts via differential use of ²3'-splice and poly(A) ²sites. All of these transcripts contain the tripartite leader (TPL) sequence.²Figure 2 shows an annotated sequence of the major late promoter. Key features ²in the promoter²include a number of transcription factor binding sites. This includes a CAAT ²box, a UPE box, a ²TATA box, a MAZ / SP1 binding site, a +1 position of transcription, and INR ²element, and an ²enhancer region containing an R1 region, DE1 and DE2 sub-region. These ²features have been ²shown to be important to varying degrees for major late promoter activity. Key ²features that are²known to significantly reduce promoter activity if modified or deleted are the ²TATA box, INR²element, the R region and DE1 and DE2 regions. The TATA box is considered the ²single most ²important feature, with transcription initiation occurring at the 28th base ²pair after the last base of ²the TATA sequence itself.² The nucleotide sequence of the wild-type Ad5 MLP is given below:²cgccctcttcggcatcaaggaaggtgattggtttgtaggtgtaggccacgtgaccgggtgttcctgaaggggggctata²aaagggggt²gggggcgcgttcgtcctca (SEQ ID NO: 1)²The TATA box is underlined in the above sequence and the final base (in bold) ²denotes the²position of transcription initiation (i.e. the +1 position).²In the adenoviral vector of the invention, the MLP comprises one or more ²repressor elements. ²The repressor element(s) are capable of regulating or controlling ²transcription of the adenoviral²<DP=11>²CA 03070654 2020-01-21²WO 2019 / 020992 PCT / GB2018 / 052083²- 11 -²late genes. Preferably, the repressor element(s) are capable of repressing ²transcription of the ²downstream adenoviral late genes.²One or more repressor elements may be present in the MLP. Preferably, there ²are 1, 2, 3 or 4² repressor elements; more preferably 1 0r2.²In some embodiments, one or more (preferably one) repressor elements are ²located²downstream of the MLP's TATA box. In other embodiments, one or more ²(preferably one) ²repressor elements are located upstream of the MLP's TATA box. In other ²embodiments, one²or more (preferably one) repressor elements are located upstream of the MLP's ²TATA box and²one or more (preferably one) repressor elements are located downstream of the ²MLP's TATA ²box.²Preferably, the one or more repressor elements are all placed upstream of the ²+1 transcription²start site. More preferably, the one or more repressor elements are inserted ²between the MLP²TATA box and the +1 position of transcription. Even more preferably, the start ²of one or more ²repressor elements is placed less than 27 nucleotides, more preferably less ²than 20 nucleotides, ²and even more preferably less than 10 nucleotides downstream of the TATA box.²The repressor is a DNA binding protein. The repressor will either bind to DNA ²and directly inhibit²the recruitment of transcription factors required for transcription ²initiation, or the repressor will ²sterically hinder the initiation of transcription by occupying a key footprint ²on the DNA that ²prevents transcription factors that are required for transcription initiation ²from binding. The ²repressor may be regulated by a small molecule that causes a conformational ²shift in the²repressor protein that either enables, or prevents, its DNA binding. The ²repressor is not an²activator.²Promoters regulated by a repressor are typically induced by the presence or ²absence of biotic ²or abiotic factors.²Examples of proteins that could be used as repressors in the context of this ²invention include ²the tetracycline repressor, the lactose repressor, the ecdysone repressor, rat ²glucocorticoid ²receptor, human estrogen receptor, alcohol dehydrogenase I (alcA), ²metallothionein (proteins ²that bind and sequester metal ions), salicylic acid, ethylene and ²benzothiadiazole (BTH).²<DP=12>²CA 03070654 2020-01-21²WO 2019 / 020992 PCT / GB2018 / 052083²- 12 -²Preferably, the repressor is the tetracycline repressor, the lactose repressor ²or the ecdysone ²repressor. Most preferably, the repressor is a Tet repressor protein (TetR).²The TetR binding site may have a wild-type sequence. Preferably, the TetR ²binding site has²been subject to one or more improvements by incorporating minor sequence ²changes. A²preferred version that can be used in an embodiment of the invention has the ²sequence:²tccctatcagtgatagaga (SEQ ID NO: 2)²Alternative versions of the repressor element that bind the TetR protein or ²derivatives of the²TetR protein may also be used in an embodiment of the invention provided that ²the TetR ²repressor protein binds to the TetR binding sequence variant used. Some ²repressor / binding site ²variants will have higher than wild-type affinity for each other; these are ²preferable in an ²embodiment of the invention.² In one embodiment of the invention, the DNA sequence of the TetR protein is:²Atgtcgcgcctggacaaaagcaaagtgattaactcagcgctggaactgttgaatgaggtgggaattgaaggactcacta²ctcgcaag ²ctggcacagaagctgggcgtcgagcagccaacgctgtactggcatgtgaagaataaacgggcgctcctagacgcgcttg²ccatcg²aaatgctggaccgccatcacacccactifigccccctggagggcgaatcctggcaagaifitctgcggaacaatgcaaa²gtcgttccg²gtgcgctctgctgtcccaccgcgatggcgcaaaagtgcacctgggcactcggcccaccgagaaacaatacgaaaccctg²gaaaa ²ccaactggctttectttgccaacagggattttcactggagaatgccctgtacgcactatccgcggtcggccactttacc²ctgggatgcgtc ²ctcgaagatcaggagcaccaagtcgccaaggaggaaagagaaactcctaccactgactcaatgcctccgctcctgagac²aagcc ²atcgagctgttcgaccaccagggtgctgaacctgcatttctgttegggcttgaactgattatctgcggcctggagaaac²agttgaagtgc²gagtcgggatcctag (SEQ ID NO: 3)²or a sequence having at least 80%, more preferably at least 85%, 90% or 95% ²sequence ²identity thereto and which codes for a TetR protein.²In one embodiment of the invention, the amino acid sequence of the TetR ²protein is:²MSRLDKSKVINSALELLNEVGIEGLTTRKLAQKLGVEQPTLYWHVKNKRALLDALAIEMLDRHHT ²HFCPLEGESWQDFLRNNAKSFRCALLSHRDGAKVHLGTRPTEKQYETLENQLAFLCQQGFSL ²ENALYALSAVGHFTLGCVLEDQEHQVAKEERETPTTDSMPPLLRQAIELFDHQGAEPAFLFGLE² LIICGLEKQLKCESGS (SEQ ID NO: 4)²<DP=13>²CA 03070654 2020-01-21²WO 2019 / 020992 PCT / GB2018 / 052083²- 13 -²or a sequence having at least 80%, more preferably at least 85%, 90% or 95% ²sequence ²identity thereto and which encodes a TetR protein.²When TetR is bound to the repressor element (e.g. repressor binding site), ²tight suppression of²transcription is obtained. However, in the presence of doxycycline, ²suppression is alleviated,²thus allowing the associated promoter to regain full transcriptional activity. ²Doxycycline is ²preferable over tetracycline.²The repressor element may be introduced into the MLP by modification of the ²MLP sequence.²The repressor element may be introduced by insertion of a repressor element ²into the MLP ²sequence; by substitution of one or more nucleotides in the MLP sequence to ²create a²repressor element sequence; or by a combination of insertion / deletion of a new ²sequence.²In one embodiment of the invention, the distance between the TATA box and the ²+1 position of²DNA transcription of the MLP is maintained (+ / - 5%), but a repressor binding ²site is inserted ²between the two. Within this region, there is a MAX / SP1 binding site. This ²site is not ²fundamental to MLP expression and therefore, in one embodiment of the ²invention, it can be ²replaced. Figure 3 shows an embodiment of the invention where the region ²between the TATA²box and the +1 position of transcription has been replaced with a binding site ²for the tetracycline²repressor protein (TetR).²In some adenoviral vectors, the section of the DNA strand which is opposite to ²that of the MLP ²sequence encodes the early viral protein E2B (which encodes the viral DNA ²polymerase). In²order to maintain the ability of such adenoviral vectors to replicate their ²genomes, it is²necessary to ensure that the modifications which are made to the MLP sequence ²do not ²significantly adversely affect the production, stability or efficacy of the ²E2B protein.²In Adenovirus serotype 5, the MLP resides in the middle of the E2B polymerase ²Exon 2. As²such, in one embodiment of the invention, the insertion of a repressor element ²(e.g. repressor²binding site) into the MLP must maintain the coding frame of the DNA ²polymerase coding ²sequence in the opposing DNA strand or ensure that the modifications which are ²made to the ²MLP sequence do not significantly adversely affect the production, stability ²or efficacy of the ²E2B protein.²<DP=14>²CA 03070654 2020-01-21²WO 2019 / 020992 PCT / GB2018 / 052083²- 14 -²Preferably, some or all of the changes to the MLP sequence are silent changes, ²i.e. they do not ²change the E2B codons. In other embodiments, some or all of the changes to the ²MPL are ²conservative changes, i.e. one or more E2B codons are changed such that they ²encode an ²amino acid having a similar side chain.²Families of amino acid residues having similar side chains have been defined ²in the art. These ²families include amino acids with basic side chains (e.g. lysine, arginine, ²histidine), acidic side ²chains (e.g. aspartic acid, glutamic acid), uncharged polar side chains (e.g. ²glycine, asparagine, ²glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side ²chains (e.g. alanine,²valine, leucine, isoleucine, proline, phenylalanine, nnethionine), beta-²branched side chains (e.g. ²threonine, valine, isoleucine) and aromatic side chains (e.g. tyrosine, ²phenylalanine, tryptophan, ²histidine).²Thus, one or more amino acid residues within the E2B protein can be replaced ²with other amino²acid residues from the same side chain family and the altered E2B protein can ²be tested for any ²significant loss in function or activity.²In one embodiment of the invention, a TetR binding site is positioned into the ²MLP between the ²TATA box and the +1 position. Figure 2 shows the coding sequence of the DNA ²polymerase. In²the region between the MLP TATA box and the +1 position, the amino acid coding ²sequence of ²the DNA polymerase is ENERAPTP (SEQ ID NO: 18). Figure 3 shows that the ²strategic placing ²of a TetR binding site into this region creates a sequence ESLSLIGT (SEQ ID ²NO: 5). This ²sequence maintains the reading frame of the DNA polymerase but also allows for ²more than 60% ²of the wild type amino acids to be exchanged for those with similar side ²chains. Importantly, the²sequence of the TetR binding site contains stop codons in the opposing DNA ²strand if ²positioned incorrectly which would likely create a non-functional DNA ²polymerase.²In one embodiment of the invention, therefore, the sequence on the opposite ²strand of the TetR²binding site is or comprises a sequence encoding ESLSLIGT (SEQ ID NO: 5). ²Preferably, the² DNA polymerase comprises a sequence of SEQ ID NO: 5.²Some loss in amount of E2B mRNA or protein produced may be tolerated, as may ²some ²changes in the amino acid sequence of the E2B protein. The skilled person will ²readily be able ²to assess the effect of changes on the E2B gene sequence on the ability of the ²virus to replicate²simply by assessing the viral titre of a modified adenoviral vector under ²standard conditions in²<DP=15>²CA 03070654 2020-01-21²WO 2019 / 020992 PCT / GB2018 / 052083²- 15 -²comparison to the original unmodified virus. Changes which reduce the viral ²titre by less than ²25%, preferably less than 10% and more preferably less than 1% may be ²considered to be ones ²which do not significantly adversely affect the production, stability or ²efficacy of the E2B protein.²In other adenoviral vectors of the invention, the E2B gene is positioned ²elsewhere in the²adenoviral vector such that modifications in the MLP do not affect the ²production of the E2B ²protein.²An example of a modified MLP which contains one TetR binding site between the ²TATA box² and the +1 position of transcription is given below:²Cgccctcttcggcatcaaggaaggtgattggtttgtaggtgtaggccacgtgaccgggtgttcctgaaggggggctata²aaaggtccc ²tatcagtgatagagactca (SEQ ID NO: 6)² The TATA box is underlined and the TetR binding site is shown in bold.²An example of a modified MLP which contains one TetR binding site between the ²TATA box²and the +1 position of transcription and a second site upstream of the TATA ²box between the²UPE element and the TATA box is shown below:²Cgccctettcggcatcaaggaaggtgattggtttgtaggtgtaggccacgtgactccctatcagtgatagagaactata²aaaggtcc²ctatcagtgatagagactca (SEQ ID NO: 7)²The TATA box is underlined and the TetR binding site is shown in bold.²The repressor which binds to the repressor element(s) may be applied ²exogenously (to the ²cells); it may be encoded within the cellular or mitochondrial genome (or a ²plasmid or vector ²within the cell); or it may be encoded within the adenoviral genome. ²Preferably, the repressor is ²encoded by a gene within the adenoviral genome.²More preferably, the repressor gene is located within one or more of the viral ²early expression ²regions or the late gene expression regions. Even more preferably, the ²repressor gene is ²located in the virus late region. Even more preferably the repressor is ²located in a late transcript ²that is transcribed from an MLP that is regulated by the repressor. The system ²is thereby self-² repressing.²<DP=16>²CA 03070654 2020-01-21²WO 2019 / 020992 PCT / GB2018 / 052083²- 16 -²In one embodiment, the gene encoding the repressor protein is under the ²control of an ²independent promoter (i.e. a promoter which is not the adenoviral major early ²promoter or major ²late promoter). Preferably, the gene encoding the repressor protein is under ²the control of a²strong promoter (e.g. the cytomegalovirus (CMV) promoter). Even more ²preferably, the²repressor protein is under the control of the MLP to which it binds.²Preferably, the mRNA encoding the repressor protein comprises a Tripartite ²Leader (TPL) ²sequence in its 5'-UTR. TPL is a 5' untranslated sequence which is present in ²all of the late, but²none of the early, viral mRNA. TPL facilitates mRNA transport and accumulation ²in the²cytoplasm and is responsible for the selective translation of the late viral ²proteins in preference ²to the cellular proteins.²The Ad5 leader sequence is 201 bp formed by the splicing of three exons during ²the post-² translational modifications.²The adenoviral vector of the invention comprises at least one transgene. A key ²function of the ²adenoviral vector of the invention is to facilitate production of the ²transgene product.²During adenoviral infection up to 90% of the mRNAs can be from the viral ²genome and these²can be preferentially translated to maximise virus output. The selective ²production of virus ²specific mRNA is down to a combination of the amplification of the virus DNA ²polymerase and a ²shift towards the production of late mRNA transcripts. The replication of the ²virus DNA genome ²allows for thousands more copies of the DNA template for transcription to ²occur from. This²activity, coupled with the presence of the tri-partite leader at the 5' end of ²late transcripts, allows²the selective expression of late mRNA. This is because the tripartite leader, ²whilst highly active ²in normal translation processes, is also selectively translated when cap-²independent translation ²is turned on within a cell. Multiple virus proteins are involved in this ²process.²Preferably, the transgene is inserted within one of the adenoviral early ²regions. More preferably,²the transgene is inserted within the adenoviral El region. Even more ²preferably, the El A and ²El B genes are deleted from the Adenovirus genome and the transgene is ²inserted into this ²region. Preferably, the transgene comprises a TPL sequence in its 5'UTR. This ²ensures that ²the transgene is expressed in a cap-independent manner.²<DP=17>²CA 03070654 2020-01-21²WO 2019 / 020992 PCT / GB2018 / 052083²- 17 -²The transgene may be any desired DNA sequence. There may be more than one ²transgene ²(e.g. 2, 3, 4, or 5 transgenes). The DNA sequence of the transgene(s) may be a ²coding or non-²coding sequence. It may be genomic DNA or cDNA. Preferably, the DNA sequence ²encodes a ²polypeptide, more preferably a therapeutic polypeptide. In some cases, the ²transgene will²encode multiple proteins. Preferably, the transgene is operably associated ²with one or more²transcriptional and / or translational control elements (e.g. an enhancer, ²promoter, terminator ²sequence, etc.).²A polypeptide encoded by a transgene could be a human gene or modified form ²thereof or² encode a protein from a virus that infects human cells.²Examples of preferred therapeutic polypeptides include antibodies, CAR-T ²molecules, scFV, ²BiTEs, DARPins and T-cell receptors and antigens from human viral pathogens.²In some embodiments, the therapeutic polypeptide is a G-protein coupled ²receptor (GPCR), e.g.²DRD1. In some embodiments, the therapeutic polypeptide is an immunotherapy ²target, e.g. ²CD19, CD40 or CD38. In some embodiments, the therapeutic polypeptide is a ²functioning copy ²of a gene involved in human vision or retinal function, e.g. RPE65 or REP. In ²some ²embodiments, the therapeutic polypeptide is a functioning copy of a gene ²involved in human²blood production or is a blood component, e.g. Factor IX, or those involved in ²beta and alpha²thalassemia or sickle cell anaemia. In some embodiments, the therapeutic ²polypeptide is a ²functioning copy of a gene involved in immune function such as that in severe ²combined ²immune-deficiency (SCID) or Adenosine deaminase deficiency (ADA-SCID). In some ²²embodiments, the therapeutic polypeptide is a protein which ²increases / decreases proliferation²of cells, e.g. a growth factor receptor. In some embodiments, the therapeutic ²polypeptide is an²ion channel polypeptide. In some preferred embodiments, the therapeutic ²polypeptide is an ²immune checkpoint molecule. Preferably, the immune checkpoint molecule is a ²member of the ²tumour necrosis factor (TNF) receptor superfamily (e.g. CD27, 0D40, 0X40, GITR ²or 0D137) or ²a member of the B7-CD28 superfannily (e.g. 0D28, CTLA4 or ICOS). In some ²embodiments the²polypeptide encoded is an immune checkpoint molecule is PD1, PDL1, CTLA4, Lag1 ²or GITR.²In some embodiments the transgene encodes a virus polypeptide or multiple ²thereof such as ²the antigenically important CMV pentamer.²<DP=18>²CA 03070654 2020-01-21²WO 2019 / 020992 PCT / GB2018 / 052083²- 18 -²In other embodiments, the transgene or transgenes will encode proteins ²involved in the ²replication or structure of viruses other than Adenovirus.²Most vaccines are composed of a collection of viral proteins: either a virus-²like particle (VLP),²inactivated virus particle or a live attenuated virus. VLPs have the advantage ²of typically being²composed entirely of protein and as such have none of the potential ²pathogenicity of the original ²virus. They also have the advantage over inactivated virus particles that ²their structure is the ²same as the original virus and has not been altered by the inactivation ²process. A number of ²VLPs have been shown to be successful vaccines including HPV, Hepatitis B and ²the Malaria²vaccine RTSS. However, production in mammalian systems is often lower than ²required to²make the VLP a commercially-viable product. An example of this is the ²observation that the ²Hepatitis B vaccine VLP is manufactured in Yeast. A system to generate VLPs in ²large ²quantities in mammalian cells is therefore desirable to ensure proper folding ²and glycosylation.²In some embodiments, the transgene of the invention will encode proteins that ²will assemble in,²or outside, of the cell to produce VLPs. These VLPs may make useful vaccine ²products.²In a preferable embodiment, the transgenes will encode proteins from ²Parvoviridae (e.g. adeno-²associated virus), Retroviridae (e.g. HIV), Flaviviridae (e.g. Hepatitis C ²virus) and²Orthomyxoviridae (e.g. Influenza virus), and / or Caliciviridae (e.g. ²Norovirus). In a preferred²embodiment, the transgene will encode Norovirus VP1 or Hepatitis B HBsAG.²Recombinant adeno-associated viruses (rAAV) are in many ways ideal vectors for ²the delivery ²of transgenes to cells for recombinant protein production or gene therapy ²applications. They are²able to infect a wide range of hosts, elicit a robust and sustained transgene ²expression profile,²whilst exerting minimal toxicity to the host cell. AAV is a helper-dependent ²DNA parvovirus that ²requires helper function for completion of its natural life cycle. These ²helper functions may be ²provided by many agents that include co-infection with vaccinia viruses, ²herpesviruses or ²adenoviruses. In the absence of helper agents and functions, AAV infection ²enters a latent²phase wherein the AAV viral genome is integrated into the host cell ²chromosome. Subsequently,²in the presence of a helper virus, the integrated AAV provirus is rescued ²allowing viral ²replication, packaging in preformed protein capsids and production of ²infectious virions.²In current approaches to using AAV as a biotechnology tool for DNA delivery, ²AAV is²engineered to replace the viral rep and cap coding sequence region with the ²transgene of²<DP=19>²CA 03070654 2020-01-21²WO 2019 / 020992 PCT / GB2018 / 052083²- 19 -²interest for delivery into target cells. The rep and cap gene sequences ²required for replication, ²integration and expression of viral capsids are supplied in trans by DNA ²expression plasmids, ²viral vectors or cell lines which are engineered to stably express these genes ²from the host ²genome.²A traditional method of rAAV production involves co-transfection of two DNA ²vectors: 1) a ²plasmid containing the rAAV sequences; and 2) a plasmid containing AAV rep and ²cap ²sequences. Subsequently, helper functions can be provided in trans by ²infection with an ²adenovirus or herpes virus. The amount of rep proteins required for efficient ²rAAV production is²unclear. While it was reported that attenuation of the translation initiation ²codon for Rep78 / 68 ²led to high levels of rAAV production (Li etal., 1997 J. Virol. 71:5236-5243), ²others have ²reported the exchange of the p5 promoter responsible for transcription of AAV ²rep with strong ²viral promoters, such as the human immunodeficiency virus long terminal repeat ²(HIV LTR) ²have resulted in the production of high levels of rAAV (see US 5,658,776). ²Since the discussed²methods required the co-transfection of two plasmids and co-infection with a ²helper virus e.g. ²adenovirus, to produce rAAV, reproducibility and batch variation are ²significantly problematic. ²Additionally, rAAV are required to be purified from helper agents (e.g. ²adenoviruses) for ²downstream and industrial application. The final preparation also has to be ²free from the helper ²virus proteins, particularly those that are antigens from the helper virus as ²this could affect the² human response to such an AAV product.²A second method required for production of rAAV involves the co-transfection ²of three DNA ²plasmid vectors into producer cell lines. The cis plasmids contain: the 1) ²rAAV ITRs and ²transgene; 2) a trans DNA plasmid encoding the AAV rep and cap; and 3) a trans ²DNA plasmid²encoding sequences from a helper virus (e.g. adenovirus). While this method ²has the advantage ²of being free of contamination from helper virus particles, co-transfection of ²three DNA plasmids ²with significantly large sizes results in poor reproducibility and substantial ²drop in rAAV viral titre ²yield.²A third method involves the construction of rAAV packaging cell lines where ²the rep and cap ²gene sequences are integrated into host cell genome. Typically, rAAV is ²produced from this ²method by transfection of the DNA cis plasmid encoding the AAV ITRs and ²transgene, followed ²by introduction of the helper agent (e.g. infection with helper adenoviruses). ²Alternatively, the ²helper viruses carrying the cis DNA AAV ITRS and transgenes (e.g. hybrid ²Ad / AAV) can be²introduced into producer cell lines for production of rAAV (see US 5,856,152).²<DP=20>²CA 03070654 2020-01-21²WO 2019 / 020992 PCT / GB2018 / 052083²- 20 -²The disadvantage of this method is that it requires complex engineering of ²producer cell lines ²wherein the AAV rep and cap are stably integrated and expressed to a ²sufficient amount.²Additionally, use of helper viruses for delivery of the cis DNA sequence (e.g. ²AAV ITRS and²transgenes) results in contamination from helper viruses and requires ²downstream purification.²Furthermore, toxicity of the AAV rep protein in cell lines have been reported ²and constitutive ²expression results in anti-proliferation and cell death (Yang etal., 1994 J. ²Virol. 68(8), 4847-²4856). Additionally, the Rep protein was also reported to induce a negative ²feedback loop for ²repression of its own transcription (Beaton etal., 1989 J. Virol. 63,450-²4454). Other attempts to²circumvent AAV rep toxicity by modification of packaging cell lines to ²expressed Rep have used²an inducible transcription system (see US 5,837,484 and Ogasawara etal., 1999 ²J. Virol. 80, ²2477-2480). Typically, these stable systems have failed to yield the required ²level of AAV to ²make them commercially scalable systems.²A fourth method of producing rAAV involves the use of helper agents (e.g. ²helper adenoviruses)²for delivery of the AAV rep and cap genes. In this method, the recombinant ²adenoviruses ²comprise rep and cap genes which are inserted into the genome of the helper ²virus (typically, in ²the El region of a recombinant adenovirus). These are subsequently used for ²infection into ²packaging cell lines that may contain the cis AAV DNA sequence (e.g. AAV ITRs ²and transgene)²or the cis DNA plasmids can be introduced by methods of transfection (Zhang et ²al., 2001 Gene.²Ther. 8(9), 704-712). The significant disadvantages of this method are similar ²to those of the ²traditional method of rAAV production, where difficult and costly purification ²steps are required ²for removal of contamination from the helper virus and any proteins ²(particularly structural ²proteins) from the final product.²In one embodiment of the invention, one or more transgenes in the adenoviral ²vector of the ²invention encodes an AAV Rep-Cap polypeptide. In another embodiment of the ²invention, one ²or more of the transgenes encodes an AAV genome. Preferably, the AAV genome ²comprises ²or consists of a 5' and 3' ITR sequence from AAV with intervening sequences. ²In another²embodiment of the invention, one or more of the transgenes encodes a Rep-Cap ²polypeptide²from an AAV and an AAV genome with ITRs flanking a sequence of interest. Rep ²or Cap or ²sub-transcripts or regions thereof may be encoded individually in an ²embodiment of the ²invention.²<DP=21>²CA 03070654 2020-01-21²WO 2019 / 020992 PCT / GB2018 / 052083²- 21 -²The adenoviral viral vector may additionally comprise, but not limited to, one ²or more other ²elements, selected from the group consisting of reporter genes, restriction ²enzyme sites, ²promoters, poly-adenylation signals, un-translated regions, enhancers and ²insulators.²In some embodiments, the adenoviral vector additionally comprises one or more ²multiple-²restriction enzyme sites. These sites will most preferably be Type II and ²either 6 or 8 base pairs ²in length.²The invention also provides a composition comprising an adenovirus particle ²comprising an²adenoviral vector of the invention, together with one or more physiologically-²acceptable carriers,²excipients or diluents. Examples of suitable physiologically-acceptable ²carriers, excipients or ²diluents for use with virus particles are well known in the art. Preferably, ²the composition ²comprises an adenovirus particle comprising an adenoviral vector of the ²invention in an ²aqueous buffer solution. Preferably, the aqueous buffer solution comprises ²MgCl2 and / or² glycerol.²The invention also provides a kit comprising an adenoviral vector of the ²invention, wherein the ²kit additionally comprises one or more additional components selected from the ²group ²consisting of (i) a cell line that allows the viral vector to infect the cells ²and replicate its genome,²wherein the cell line will repress the viral vector's MLP and (ii) one or more ²DNA plasmids for²aiding in the construction of the viral vector.²Preferably, the kit will comprise a plasmid containing the viral vector that ²contains sites for ²insertion of a transgene. More preferably there will be two plasmids: the ²viral vector plasmid²and a shuttle plasmid to allow the easy manipulation of the viral vector. The ²shuttle plasmid will²contain either regions of homology to the viral vector to allow homologous ²recombination to ²create a final viral vector, or restriction sites that are compatible with the ²viral vector to allow ²shuttling of DNA from the shuttle plasmid to the viral vector plasmid.²The kit may also contain materials for the purification of the viral particles ²such as those²involved in the density banding and purification of viral particles, e.g. one ²or more of centrifuge ²tubes, benzonase, dialysis buffers, dialysis cassettes.²In other embodiments, the invention provides a viral vector (preferably an ²adenoviral vector) or²a nucleic acid molecule comprising the nucleotide sequence:²<DP=22>²CA 03070654 2020-01-21²WO 2019 / 020992 PCT / GB2018 / 052083²- 22 -²Aggccagcacgaaggaggctaagtgggaggggtagcggtcgttgtccactagggggtccactcgctccagggtgtgaag²acacat ²gtcgccctcttcggcatcaaggaaggtgattggifigtaggtgtaggccacgtgaccgggtgttectgaaggggggcta²taaaaggtcc ²ctatcagtgatagagactcactctcttccgcatcgctgtctgcgagggccagctgttggggtgagtactccctctgaaa²agogggcat²gacttctgcgctaagattgtcagtttccaaaaacgaggaggatttgatattcacctggcccgcggtgatgcctttgagg²gtggccgcatc²catctggtcagaaaagacaatctttttgttgtcaagcttggtggcaaacgacccgtagagggcg (SEQ ID ²NO: 8)²or nucleotide sequence having at least 80%, more preferably at least 85%, 90% ²or 95% ²sequence identity thereto.²In some embodiments, the above-mentioned nucleotide sequence or sequence ²having identity ²thereto comprises the sequence tccctatcagtgatagaga (SEQ ID NO: 2)(i.e. the ²repressor element ²is not altered).²In other embodiments, the above-mentioned nucleotide sequence or sequence ²having identity²thereto comprises a different repressor element to that shown in bold above.²The invention also provides a mammalian cell comprising an adenoviral vector ²of the invention. ²The cells may be isolated cells, e.g. they are not present in a living animal. ²Examples of²mammalian cells include those from any organ or tissue from humans, mice, ²rats, hamsters,²monkeys, rabbits, donkeys, horses, sheep, cows and apes. Preferably, the cells ²are human ²cells. The cells may be primary or immortalised cells. Preferred human cells ²include HEK293, ²HEK293T, HEK293A, PerC6, 911, HeLa and COS cells.²HEK-293 cells have been modified to contain the El A and El B proteins and ²this allows the²creation of viruses that have a deletion of the ElA and El B regions to be ²grown in this cell line ²by trans-complementation. Similarly, PerC6 and 911 cells contain a similar ²modification and can ²also be used. Most preferably, the human cells are HEK293, HEK293T, HEK293A, ²PerC6 or ²911. Other preferred cells include CHO and VERO cells.²Adenoviral vectors may be produced by several methods, the most common of ²which involves ²homologous recombination of adenovirus plasmids in either mammalian cells or ²microorganisms, including bacteria and yeast. Two plasmids, termed a shuttle ²plasnnid and an ²adenoviral (also called backbone) plasmid, are recombined into a DNA molecule ²that²<DP=23>²CA 03070654 2020-01-21²WO 2019 / 020992 PCT / GB2018 / 052083²- 23 -²incorporates sequences from both plasmids. This DNA molecule can then be ²transfected into ²mammalian packaging cell lines to generate adenovirus particles.²In an embodiment of the invention wherein the MLP is regulated by a TetR ²protein, the virus is²recovered in the presence of doxycycline which will inhibit the repressor ²protein and thereby²prevent it from binding to the repressor element / site in the MLP promoter. ²This should allow for ²production of the adenovirus at high titres.²For protein production or the expression of virus proteins, infecting with the ²virus into any cell²line that can be infected by adenovirus (e.g. HEK-293 cells), in the absence ²of doxycycline, will²allow the expressed TetR repressor protein to bind to the MLP repressor ²element and thereby ²shutting down the production of virus late gene expression and preventing ²virus particle ²production.²The TetR protein can be provided within the cell line infected, either ²transiently or stably, or²encoded within the virus itself. If under the control of the MLP then with ²each genome replication, ²activation of the MLP will provide a feedback loop, further shutting down any ²potential new virus ²late gene expression.²In a further embodiment, the invention provides a process for producing a ²modified mammalian²cell, the process comprising the step:²(a) infecting a mammalian cell with an adenoviral vector of the ²invention, ²whereby the mammalian cell then comprises the adenoviral vector.²In yet a further embodiment, the invention provides a process for producing a ²transgene product,²the process comprising the steps:²(a) infecting mammalian cells with an adenoviral vector of the invention;²(b) culturing the infected mammalian cells in a culture medium under ²conditions such ²that the transgene is expressed; and²(c) isolating or purifying transgene product from the cells or from the cell ²culture medium.²Suitable conditions for the culturing of infected mammalian cells are well ²known in the art.²<DP=24>²- 24 -²Transgene product expression levels may be improved by the co-expression of ²the 100kDa²L4 / eIF48 protein. Furthermore, the repressor protein needs to be active as a ²repressor (e.g.²doxycycline is not present if the repressor protein is TetR).²The processes of the invention are preferably carried out in vitro or ex vivo.²BRIEF DESCRIPTION OF THE FIGURES²Figure 1 shows a schematic diagram of the adenovirus genome and shows the main ²early and²late expression regions. The MLP is also shown. Splice patterns are denoted by ²dotted lines.²Figure 2 shows annotated sequences (SEQ ID NOs: 12-14) of the major late ²promoter region of² wild type adenovirus serotype 5.²Figure 3 shows annotated sequences (SEQ ID NOs: 15-17) of the modified major ²late promoter ²region of adenovirus serotype 5.²Figure 4 shows schematic diagrams of plasmid vectors constructed for ²expression of the GFP ²reporter from the Ad5 repressor mutant Major late promoters.²Figure 5 shows plasmid vectors expressing the GFP reporter gene from MLP ²promoters.²Figure 6 shows transcriptional repression of the repressor mutant MLP by the ²TETR protein.²Figure 7 shows examples of MLP repressor Ad5 in repression in HEK293 T-Rex Flp ²cells, a cell ²line stably expressing the TETR protein.²Figure 8 shows examples of MLP repressor Ad5 in repression in 293Ad cells ²transfected with a ²TETR expression plasmid (pTETR), under the control of the constitutive CMV ²(cytomegalovirus) ²promoter.²Date Recue / Date Received 2020-11-17²<DP=25>²CA 03070654 2020-01-21²WO 2019 / 020992 PCT / GB2018 / 052083²- 25 -²Figure 9 shows viral genome replication of repressed MLP mutant Ad5 in 293 T-²Rex Flp cells, a ²cell line stably expressing the TETR protein.²Figure 10 shows Ad5 with TET1b modified MLP variants infection of HEK293 cells ²at low MOI.²A) HEK293 cells were transduced with E1 / E3 Ad5 or Ad5 TET1b MLP expressing ²EGFP under²control of the CMV promoter at MOI 0.1 and in the presence doxycycline ²0.2pg / mL or DMSO. ²Image of day-6 post infection by fluorescence microscopy. HEK293 cells were ²also harvested ²for flow-cytometry analysis to determine the percentage of EGFP positive cell ²within the gated ²population (B). Data as mean SD of triple biological repetition.²Figure 11 shows EGFP expression from Ad5 with TETO1b modified MLP. A) Plasmid ²expression of EGFP encoding Ad5 TPL from the CMV promoter. HEK293 cells ²transfected with ²plasmid DNA expressing EGFP from a promoterless plasmid or under control of ²the CMV ²promoter with and without the Ad5 TPL. EGFP expression determined by flow ²cytometry 48h²post-transfection. B) MLP TETO1b modified Ad5 expressing EGFP (with and ²without Ad5 TPL)²under control of the CMV promoter in T-Rex Flp cells treated with DMSO or ²doxycycline ²0.2ug / mL. Cells analysis by flow cytometry 24h, 48h and 72h post-infection. C) ²EGFP ²expression under control of the CMV promoter from plasmid DNA or MLP modified ²Ad5 in ²HEK293. HEK293 cells were transfected with plasmid expressing TETR prior to ²transduction²with the CMV promoter plasmid expressing EGFP or MLP modified Ad5 expressing ²EGFP (with²and without the Ad5 TPL) under control of the CMV promoter, in the presence of ²doxycycline or ²DMSO. Cells were harvested for flow cytometry 48h post-transduction. D) ²Fluorescent ²microscopy image of pTETR transfected HEK293 cells transduced with MLP ²modified Ad5 ²expressing EGFP encoding Ad5 TPL, in the presence of DMSO or doxycycline ²0.2pg / mL. Image² 48h post-infection. MFI (median fluorescent intensity). Data as mean SD.²Figure 12 shows Western blot detection of adenovirus structural proteins from ²TERA-CMV-²EGFP or E1 / E3 Ad5-CMV-EGFP infection in HEK293 cells. A) TERA or E1 / E3 Ad5 was ²used to²transduce HEK293 at MOI 100 or 1000. Virus were harvested from cell lysate ²(left blot) or²growth medium (right blot). B) Western blot analysis of growth media from ²HEK293 cells²cultured in a dose escalation of doxycycline and transduced with TERA or E1 / E3 ²deleted Ad5 at ²MOI 10 or MOI 100 (C). All virus samples harvested at 72h post-infection and ²detected using ²anti-Ad5 antibodies with automated Western machine (Wes Simple Western).²<DP=26>²CA 03070654 2020-01-21²WO 2019 / 020992 PCT / GB2018 / 052083²- 26 -²Figure 13 shows DNA and virus replication of TERA in HEK293 cells. A) HEK293 ²cells were ²infected with TERA or E1 / E3 Ad5, encoding the CMV EGFP expression cassette, at ²MOI 1, 10 ²or 100, and total DNA was harvested at the indicated timepoint post-infection ²for QPCR analysis. ²B) TERA or Ei / E3 Ad5, encoding the CMV EGFP expression cassette, was used to ²infect²HEK293 at MOI 10 or 100, in the presence of doxycycline 0.5pg / mL or DMSO. ²Total DNA was²harvested at the indicated timepoint post-infection for QPCR analysis.²Figure 14 shows reporter EGFP expression from TERA in HEK293 cells. A) HEK293 ²cells were ²transduced with TERA (M0110) expressing EGFP from the CMV promoter (with and ²without²Ad5 TPL), and E1 / E3 Ad5 (M0110) or plasmid DNA (0.75ug per ¨7.0x 104 cell ²using Branch²PEI) expressing the EGFP also under control of the CMV promoter. HEK293 cells ²were ²harvested at 24h and 48h post transduction for flow cytometry analysis. B) ²HEK293 cells, in the ²presence of DMSO or doxycycline 0.5ug / mL, infected at MOI 100 with TERA ²expressing EGFP ²under the CMV promoter. Virus harvested from cell lysate 72h post transduction ²and detected²using anti-Ad5 and Anti-EGFP antibody by automated Western blot machine (Wes ²Simple²Protein).²Figure 15 shows TERA expression of BiTE in HEK293 cells. A) HEK293 cells were ²transduced ²with Ei / E3 Ad5 or TERA expressing EGFP under control of the CMV (with and ²without the Ad5²TPL) at an MOI 10. Culture media harvested at 48h post-transduction and (5pL) ²probed with²anti-6x His antibodies for BiTE expression using automated Western (Wes Simple ²Protein). B) ²Chemiluminescence signals from each sample were further quantified and plotted ²to give ²relative expression intensity based on the detected peak and area under curve. ²C) Total DNA ²were harvested timepoint Oh, 24h, 48h, 72h from the virus infected HEK293 ²cells and genome²quantified by QPCR. D) Detection of adenovirus protein from HEK293 cells ²transduced with²E1 / E3 Ad5 or TERA engineered to express BiTE. Culture media harvested at 48h ²post-²transduction and (5pL) probed with anti-Ad5 antibodies for Ad5 major ²structural proteins using ²automated Western (Wes Simple Protein). E) HEK293 cells were transduced with ²plasmid DNA ²(0.75pg per ¨7.0x 104 cell using Branch PEI) or TERA expressing BiTE encoding ²Ad5 TPL and²under control of the CMV promoter at MOI 10. Growth media from transduced ²cells were²harvested at the specified timepoint 24h, 48h, and 72h post-transduction and ²(5pL) probed for ²BiTE expression using anti-6x His antibody.²Figure 16 shows rAAV vector production in HEK293 cells with TERA. A) HEK293 ²cells, in the²presence of DMSO or doxycycline 0.5pg / mL, were transfected with plasmid ²expressing AAV²<DP=27>²CA 03070654 2020-01-21²WO 2019 / 020992 PCT / GB2018 / 052083²- 27 -²Rep and Cap genes prior to transduction with MOI 100 or 500 of E1 / E3 Ad5 or ²TERA encoding ²the rAAV genome, a CMV-EGFP expression cassette flanked by AAV ITRs, or Helper-²free ²production via triple plasmids transfection. Total recombinant viruses, from ²cell lysate and ²growth media, were extracted 72h post-transduction and DNase I resistant ²genomes were²quantified by QPCR primer and probe sets directed against the Fiber sequence ²of the²adenovirus genome or the CMV promoter sequence, shared by both rAAV and ²adenovirus. ²Total genome encapsulated adenovirus per well is plotted alongside total ²genome encapsulated ²rAAV per well determined by subtraction of genome encapsulated adenovirus from ²each sample. ²B) Culture media (5pL) from each specified rAAV production method were probed ²for²adenovirus capsid proteins with anti-Ad5 antibodies using automated Western ²blot machine²(Wes Simple Protein). C) rAAV capsids or viral particles (VP) produced by ²Helper-free ²transfection method or TERA-AAV transduction at MOI 500, in the presence of ²DMSO, were ²determined by ELISA. Data presented as viral particles produced per cell at ²point of²transduction and plotted alongside total encapsulated rAAV genomes determined ²by QPCR²analysis. E) Percentage of genome encapsulated rAAV determined from rAAV ²capsids are²shown. F) Infectious culture media harvested from rAAV production using TERA-²AAV ²transduction of HEK293 cells at MOI 500, in the presence of DMSO or ²doxycycline 0.5pg / mL, ²was used for infection of fresh HEK293, in the presence of doxycycline ²0.5pg / mL, at 1:100 ²dilution. Cell monolayer imaged by fluorescence microscopy 24h and 96h post ²infection. G)²Contaminating infectious adenoviruses were determined from rAA samples ²produced by method²of Helper-free transfection, or E1 / E3 Ad5 or TERA transduction at MOI 100 and ²500, in the ²presence of DMSO, by Tissue Culture Infectious Dose 50 (TCID50) assay. ²Contaminating ²adenovirus from use of TERA in AAV production, at MOI 100 or 500, is presented ²as ²percentage infectious adenovirus determined relative to E1 / E3 Ad5 control.² EXAMPLES²The present invention is further illustrated by the following Examples, in ²which parts and²percentages are by weight and degrees are Celsius, unless otherwise stated. It ²should be ²understood that these Examples, while indicating preferred embodiments of the ²invention, are²given by way of illustration only. From the above discussion and these ²Examples, one skilled in²the art can ascertain the essential characteristics of this invention, and ²without departing from ²the spirit and scope thereof, can make various changes and modifications of ²the invention to ²adapt it to various usages and conditions. Thus, various modifications of the ²invention in ²addition to those shown and described herein will be apparent to those skilled ²in the art from the²<DP=28>²CA 03070654 2020-01-21²WO 2019 / 020992 PCT / GB2018 / 052083²- 28 -²foregoing description. Such modifications are also intended to fall within the ²scope of the ²appended claims.²Materials and Methods²The methods used for cloning and production of the adenoviral vectors were as ²follows.²Plasmid amplification in prokaryotic systems ²Transformation of Escherichia coli (E. co / i) allowed amplification of plasmids ²and subsequent²screening for desired final DNA clones. DNA transformation was performed by ²thawing E. coli²cells on ice, followed by the addition of 5-100 ng of DNA per transformation. ²Cells were²incubated in ice for 20 minutes followed by 3 minutes heat-shock at 37 C. LB ²medium (0.9 mL ²per sample) was added to each tube and incubated for a further 15 minutes at ²37 C. Samples ²were either poured into nutrient agar plates containing selection antibiotic ²or 100 pL of each ²sample was streaked out to allow individual colony isolation.²Successfully transformed clones were amplified and selected using antibiotic ²resistance using ²the appropriate antibiotic in growth media. Bacterial culture broth and plates ²containing either ²Kanamycin (50 pg / mL, Kanamycin Sulfate, Invitrogen, UK) or Ampicillin (50 ²pg / mL, Ampicillin ²Sodium salt, Sigma Aldrich) were prepared using Lennox LB Broth Base (20 g / L) ²and Lennox²LB Agar (32 g / L) (Invitrogen, UK), respectively. Single bacterial colonies ²obtained on culture²plates after transformation were picked and grown overnight in broth (5 mL ²mini-prep or 250 mL ²maxi-prep) at 37 C with agitation (200 rpm) using a Sanyo orbital incubator ²(SANYO Electric ²Biomedical Co. Ltd, USA).²Plasmids were purified by mini-prep using the QIAprep Spin Miniprep Kit ²(Qiagen, UK) or maxi-²prep using the Qiagen High Speed Maxi-prep kit (Qiagen, UK). Plasmid clones ²were selected ²and confirmed by diagnostic restriction digestion and DNA sequencing.²Bacterial cells ²For the majority of small (<10 Kb) DNA constructs, DH5a E. coli bacteria ²(Invitrogen) were used²to amplify plasmids. For larger (>10 Kb) constructs, XL10 Gold Ultraconnpetent ²E. coli bacteria ²(Stratagene) were used to amplify DNA. These cells have been engineered in ²order to allow the ²efficient delivery of large DNA plasmids via heat-shock treatment and reduce ²bias towards ²selecting for small plasmids libraries. For growing DNA which was free of DAM ²and DMC²methylation at key restriction sites, JM110 E. coli (Stratagene) were used. ²These cells are²<DP=29>²CA 03070654 2020-01-21²WO 2019 / 020992 PCT / GB2018 / 052083²- 29 -²deficient in methyl transferase enzymes and allow high purity methyl deficient ²DNA to be ²amplified. For recombination of DNA molecules, BJ5183 E. coli cells were used. ²These cells ²allow the recombination between two DNA molecules due to both the mutation of ²Endonuclease ²I and the expression of a RecET homologue which allows the efficient repair of ²double-strand²breaks at homologous regions. All strains were grown in Lennox LB media or on ²LB agar plates²at 37 C.²DNA restriction digests (preparative and qualitative) ²Restriction endonucleases were used to generate diagnostic digestion patterns ²or to produce²DNA molecules with sticky (non-blunt) ends or blunt ends, allowing subsequent ²ligation of DNA²fragments with other DNA molecules with either blunt or compatible overhangs. ²All digestions ²were performed according to conditions specified by the manufacturer (enzymes ²were ²purchased from New England Biolabs, UK or Promega, UK). Where digestions were ²performed ²for molecular engineering purposes, DNA fragments of correct sizes were ²visualised by gel²electrophoresis and purified either from excised gel bands using the QIAEX II ²Gel Extraction Kit²for large fragments (>8 Kb) (Qiagen, UK) and the MinElute Gel Extraction Kit ²for small ²fragments (Qiagen, UK), or, where it was unnecessary to resolve fragments of ²different sizes, ²purified directly from the digestion reaction (QIAquick PCR Purification Kit). ²HyperLadderTM I ²(Bioline Ltd, UK) was used as a molecular weight marker for fragments ranging ²from 1-10 Kb.²For larger DNA fragments a Lambda Hind3 digest ladder was used (New England ²Biolabs, UK).²For fragments less than 1 Kb a 100 base pair ladder was used (New England ²Biolabs, UK).²DNA ligation ²Donor (insert) and recipient (vector) DNA were digested with single or double ²(for directional²cloning) restriction enzyme(s) generating compatible overhangs for ²complementary base-pairing.²Digested insert was gel-purified or cleaned up, while the vector was de-²phosphorylated by ²treatment with 2 pL of calf intestinal alkaline phosphatase (37 C, 1 h) ²(Invitrogen, UK) to reduce ²background contamination resulting from vector re-ligation. Vectors were then ²purified using the ²MinElute Gel Extraction Kit (Qiagen, UK) or the QIAquick PCR Purification Kit ²(Qiagen, UK)²using the manufacturer's protocols. All purified DNA was eluted in nuclease-²free water. Ligation²reactions were prepared using T4 DNA Ligase (New England Biolabs, UK) ²according to the ²manufacturer's protocol.²<DP=30>²CA 03070654 2020-01-21²WO 2019 / 020992 PCT / GB2018 / 052083²- 30 -²Heat shock competent cells²Heat Shock DNA competent E. co / / cells were produced by inoculating a single ²colony of E. coli ²into 5 mL 2xYT broth media (16 g / L Bacto Tryptone, 10g / L Yeast Extract, 5g / L ²NaCI, pH 7.0 ²with 5M NaOH) which was shaken at 37 C for 4 hours. This was transferred to ²200 mL pre-²warmed 2xYT media. When the culture reached OD 480 the cells were pelleted at ²3000 rpm in²a swing out centrifuge at 4 C for 10 minutes. Cells were re-suspended in a ²total of 80 mL cold ²TFBI1 (30 mM KC2H302 (Potassium acetate), 100 mM RbCI, 10 mM CaC12.2H20, 50 mM ²²MnC12.4H20, 15% v / v glycerol, adjusted to pH 5.8 using 0.2 M CH3COOH, filter ²sterile). Cells ²were spun as above and re-suspended in 8mL of TFBI2 (10 mM MOPS, 10 mM RbCI, ²75 mM²CaC12.2H20, 15% v / v glycerol, adjusted to pH 6.6 using 5 M KOH, filter ²sterile). 100 pl of cells²were pipette into pre-chilled 1.5 mL microfuge tubes and frozen on dry ice. ²Tubes were then ²transferred to -80 C for long term storage.²Polymerase chain reaction (cloning) ²Polymerase chain reactions (PCR) were performed using oligonucleotide primers ²designed²according to the requirements for molecular manipulation, sequencing or ²screening procedures ²(purchased from Sigma-Genosys, UK). Primer sequences employed for vector ²engineering and ²molecular characterisation purposes are described below.²For sub-cloning of DNA fragments generated by PCR amplification from DNA ²templates, high²fidelity AccuPrime TM Pfx proofreading DNA polymerase (Invitrogen, UK) was ²used. For routine ²screening for the presence of specific DNA sequences by PCR, PCR SuperMix ²(Invitrogen, UK) ²was used. Primers were dissolved in de-ionised, nuclease-free water at 10 pM; ²0.8-1 pL of ²each of the forward and reverse primers were used per PCR reaction. Reactions ²were carried²out according to recommended protocols provided by the suppliers of enzymes ²and reagents.²Where necessary, primer concentrations were varied and a gradient of annealing ²temperatures ²was run to determine optimal reaction conditions. PCRs were performed using a ²PTC-225 ²Peltier Thermal Cycler DNA Engine Tetrad (MJ Research, USA).² Agarose gel electrophoresis²Size fractionation of DNA on 1% (w / v) agarose (Invitrogen, UK) gels containing ²ethidium ²bromide (Sigma-Aldrich, UK) (0.5 pg / mL) in 0.5 x TAE buffer (4.84 g Tris-base, ²1.09 g glacial ²acetic acid, 0.29 g EDTA in 1 L) enabled visualisation of DNA and size ²estimation, by relative ²comparison to commercially available molecular weight markers described above. ²DNA²molecules from purified plasmids or fragments from PCR reactions or ²restriction digestions were²<DP=31>²CA 03070654 2020-01-21²WO 2019 / 020992 PCT / GB2018 / 052083²- 31 -²subjected to gel electrophoresis (10 volts per cm gel length, 30-120 minutes) ²in horizontal DNA ²electrophoresis gel tanks (Bio-Rad Laboratories Ltd, UK). DNA bands were ²visualised using a ²gel and fluorescent imaging system (AlphaImager, Alpha lnnotech Corporation, ²USA) under ²ultraviolet light.² Double stranded deoxyribonucleic acid quantitation ²DNA was quantified using a Nanodrop (Thermo Scientific, UK) spectrophotometer. ²Prior to ²analysis equipment was blanked using distilled water and then background ²readings were ²performed using the solution in which the DNA was dissolved (TE, EB or H20). ²Samples were²tested in triplicate and data was interpreted using the ND-1000v 3.1.0 ²software according to the ²manufacturer's instructions. The final quantity of DNA was expressed as ²nanograms / microlitre.²Calcium phosphate transfection for adenovirus recovery ²Swal-linearised adenoviral plasmids containing either recombinant adenoviral ²genomic DNA or²the wild-type adenovirus genome were transfected into HEK-293 cells using ²Lipofectamine 2000 ²transfection. HEK-293 cells were seeded in wells of a 6-well plate 24 h prior ²to transfection so ²that they were 70-80% confluent at the time of transfection. For each well, ²lipofectamine 2000 ²suspended in 100u1Opti-MEM Reduced Serum Media (Thermo Fisher Scientific) was ²mixed ²with DNA solution (2.5 pg in 100 pL Opti-MEM Reduced Serum Media) at a 1:2 ²ratio of total²DNA mass to lipofectamine 2000 and incubated at room temperature before being ²added drop-²wise to adherent HEK-293 which were growing in DMEM with 10% Fetal Bovine ²Serum (FBS) ²from Gibco (Thermo Fisher Scientific UK).²The culture media containing the lipofectamine 2000 transfection complex was ²removed at 4h²post-transfection. Fresh DMEM media containing 2% FCS was added to each well. ²Cytopathic ²effects (CPE) were observed in wells containing successfully transfected cells ²between 12 and ²15 days post-transfection. Virus stocks were serially 10-fold diluted into 96 ²well plates. Single ²clones were picked and amplified in 10 cm dishes from which infectious ²supernatants were ²collected and stored as seed stock for further amplification and virus ²production.² Virus production and purification by banding on Caesium Chloride gradients ²Single virus clones were amplified in HEK-293 cells cultured in DMEM media ²containing 5% ²FCS, using approximately 15-25 confluent 175 cm2 monolayers for each ²purification. HEK-293 ²monolayers containing virus-packed cells were harvested (72 hours post-²infection, when CPE²was observable but infected cells were not lysed) by gentle agitation and ²pelleted cells were re-²<DP=32>²CA 03070654 2020-01-21²WO 2019 / 020992 PCT / GB2018 / 052083²- 32 -²suspended in infectious supernatant (16 mL, volume accommodated in three ²banding columns), ²then lysed by three freeze-thaw cycles to release virus particles. The mixture ²containing lysed ²cells and free virus particles was centrifuged at 245 g (10 min, 4 C). The ²mixture was incubated ²on ice for 60 min, followed by centrifugation at 800 g (10 min, 4 C). The ²pellet was discarded²and the supernatant (containing virus particles) was loaded onto centrifuge ²tubes (Ultra-clearTM²Beckman Centrifuge tubes, Beckman Coulter UK Ltd) containing a caesium ²chloride (CsCI) ²gradient. The gradients were centrifuged (25,000 rpm, 10 C, 120 min without ²deceleration ²using a Beckman L8-70M Ultracentrifuge with rotor type SW40 TI) (Beckman ²Coulter, Inc, USA). ²Two discrete bands were obtained after centrifugation: a faint band higher in ²the column²containing 'empty' viral particles, and a thicker, opaque lower band ²containing intact infectious²viral particles. The virus was harvested by puncturing the tube below the ²level of the virus band ²with an 18-gauge needle and extracting the desired band into a syringe.²CsCI was subsequently removed by consecutive dialyses in buffers (500 mL, 4 ²C) containing²50 mM HEPES, lx PBS, 0.1 g / L CaCl2, 0.2 g / L (initial dialysis) or 0.1 g / L ²(final dialysis) MgC12²and 10% glycerol at pH 7.8, using a 3-15 mL dialysis cassette for the initial ²dialysis (Pierce ²Slide-A-Lyzer0 Dialysis Cassette, Pierce Biotechnology, Inc., USA) after which ²the virus was ²recovered and treated with Benzonase DNA nuclease (6 pL / mL, Novagen, UK) for ²30 minutes ²at room temperature. The virus was subsequently re-banded by CsCI gradient ²centrifugation²and dialysed overnight in the final dialysis buffer using a 0.5-3 mL cassette ²(Pierce Slide-A-²Lyzer0 Dialysis Cassette, Pierce Biotechnology, Inc., USA). For each dialysis, ²the buffer was ²changed and replaced with fresh buffer after 1 hour and 2 hours, followed by ²an overnight ²dialysis. Virus obtained after the final dialysis was aliquoted and stored ²immediately at -80 C.² Double-stranded DNA measurement for adenovirus quantitation ²Adenoviral DNA concentrations were determined using the PicoGreen assay (Quant-²iTTm ²PicoGreen dsDNA Reagent, Molecular Probes, Invitrogen). The assay contains a ²fluorescent ²nucleic acid probe for double-stranded DNA (dsDNA), allowing quantification of ²adenoviral ²genome content. Appropriate dilutions of virus stock solutions (10 to 100-²fold) were made and²were transferred (15 pL) to a solution containing 1 x TE buffer (255 pL) and ²0.5 % sodium²dodecyl sulphate (SDS) in 1 x TE (30 pL). Samples were incubated at 56 C (30 ²min) to disrupt ²virus particles. Six four-fold serial dilutions of known concentrations of the ²bacteriophage ²lambda DNA provided in the Quant-iTTm PicoGreen Kit were carried out (highest ²concentration ²at 1 pg / mL), allowing the construction of a standard curve (last standard ²blank) from which the²DNA content in unknown samples were calculated. The PicoGreen reagent was ²diluted 200-²<DP=33>²CA 03070654 2020-01-21²WO 2019 / 020992 PCT / GB2018 / 052083²- 33 -²fold in 1 x TE buffer. 100 pL of the diluted reagent was placed in wells of a ²black 96-well plate ²(Corning, UK) for each standard or unknown sample. 50 pL of appropriately ²diluted standards ²and samples were added to the wells in duplicate. The plate was read in a ²Wallac 1420 Victor2 ²multi-label counter, using the 'Fluorescein 485 / 535 nm, 1 second' program for ²analysis. The²number of viral particles present in each sample was calculated on the basis ²that 1 pg of DNA is²approximately equal to 2.7 x 1010 adenoviral particles.²Calculation of plaque forming units of adenoviral preparations using the ²tissue culture infectious ²dose 50 (TCID50) method ²The tissue culture infectious dose 50 (TCID50) method (Karber, 1931) was used ²to estimate the²number of infectious virus particles, or plaque forming units (pfu), and is ²based on the ²development of cytopathic effects (CPE) in HEK-293 cells after infection with ²10-fold serially ²diluted samples of virus preparation. This method is described in full in the ²manual for the ²Ad Easy non-replicating virus platform available from QBiogene (France).² Adenovirus preparations ²All adenoviruses were grown in HEK-293 cells, purified by double banding in ²CsCI gradients as ²described above. Viral particle (vp) number was determined by measuring DNA ²content using a²modified version of the PicoGreen assay (lnvitrogen, Paisley, UK) (Mittereder ²et al. 1996).²Infectivity was calculated using the TCID50 system with the KARBER statistical ²method (Karber,²1931) and was used to estimate the adenovirus titer (T0ID50 units / mL) and ²corrected to ²determine plaque forming units / mL (pfu / mL).²Maintenance of cell lines²HEK293 human embryonic kidney cells were obtained from the European Collection ²of Cell²Cultures (Porton Down, UK), and maintained in DMEM media with 10 % FCS (FAA ²Laboratories, ²Yeovil, UK) including penicillin (25 U / nnL) and streptomycin (10 nng / mL) at 37 ²0C in 5 % CO2 in a ²humidified incubator.² Real time (quantitative) PCR (Q¨PCR) for Ad5²The Q-PCR methodology for measurement of adenoviral particles has been ²previously ²described (Green etal., 2004). Briefly, viral DNA from infected cells or ²tissue samples was ²extracted using a mammalian genomic Genelute DNA extraction kit (Sigma). ²Reactions were ²performed using Applied Biosystems master mix following the manufacturer's ²protocol. The²cycles were as follows: 94 degrees Celsius 10 min, then 40 times at 94 ²Celsius 30 s, 60²<DP=34>²CA 03070654 2020-01-21²WO 2019 / 020992 PCT / GB2018 / 052083²- 34 -²Celsius 1 min. Primers sequences for targeting Ad5 fiber were: Forward primer ²¨ 5' TGG CTG ²TTA AAG GCA GTT TGG 3' (SEQ ID NO: 9) (Ad5 32350-32370 nucleotides) and ²reverse ²primer -5' GCA CTC CAT ITT CGT CAA ATC TT 3' (SEQ ID NO: 10) (Ad5 32433-32411 ²nt) ²and the TaqMan probe- 5' TCC AAT ATC TGG AAC AGT TCA AAG TGC TCA TCT 3' (SEQ ²ID².. NO: 11) (Ad5 32372-32404 nt), dual labelled at the 5' end with 6-²carboxyfluorescein and the 3'²end with 6-carboxytetramethylrhodamine. The results were analyzed with the ²Sequence ²Detection System software (Applied Biosystems). Standard curves for tissues ²and cells were ²prepared by spiking samples of cell lysate or tissue homogenate with serial ²dilutions of known ²concentrations of virus particles followed by extraction and analysis of each ²sample separately² by Q-PCR as described above.²Viral genomic DNA extraction ²Where determination of adenoviral genome numbers was necessary, viral DNA was ²extracted²from culture supernatants or cell lysates prepared in either lysis solution C ²(Sigma, UK) or lysis².. solution from the luciferase assay system (Promega, UK). Samples were ²treated with proteinase²K (1 mg / mL) and incubated at 56 degrees Celsius for 20 minutes to degrade ²viral capsid ²proteins, followed by a 20 minute incubation at 70 degrees Celsius. Total ²cellular DNA ²extraction was then performed using the Genelute mammalian DNA extraction kit ²(Sigma-²Aldrich, UK) according to the manufacturer's instructions.² Measuring MLP Activity by Luminometry ²Cells were seeded in triplicate in 12 well plates. After 24 h plasmid DNA (0.5 ²pg) was added to ²50 pL of HBS buffer and mixed with 2.5 pL DOTAP reagent (Roche) also in 50 pL ²sterile HBS. ²The complex was incubated at room temperature for 30 min. 100 pL of ²transfection mixture was²added to each well and incubated at 37 0C for 4 h. Cells were washed with PBS ²and incubated²with DMEM containing 2 % fetal calf serum (FCS) (PAA Laboratories, Yeovil, ²UK). 24 h²following transfection media was removed and 150 pL of reporter cell lysis ²buffer (Promega) ²was added to the cells. Cells were then frozen at -80 C for 1 h before ²thawing. Luciferin (25 pL) ²(Promega, Southampton, UK) was added to 25 pL aliquots of cell lysate and ²relative².. luminescence was measured by luminometry (Lumat LB9507, Berthold ²Technologies, Redbourn,²UK).²Virus Infections ²Cells are cultured using the conditions described above, or conditions ²favourable for any such²cell line, in which a transgene within the virus of the invention is to be ²expressed. Virus is added²<DP=35>²CA 03070654 2020-01-21²WO 2019 / 020992 PCT / GB2018 / 052083²- 35 -²to the culture supernatant of the cells at an MOI from 1-1000, more preferably ²an MOI of 1-100 ²and more preferably still an MOI of 10-100.²Measuring protein expression ²Transgene expression level or the level of expression of late proteins within ²a cell is measured²via Western blot or an Enzyme-linked Imnnunosorbant Assay (ELISA). The method ²taken ²depends on whether the product being measured is intracellular or secreted ²outside of the cell. ²For the former, the level of protein is measured by lysing the cells to create ²a cell lysate followed ²by western blot or ELISA. For secreted material, the supernatant is analysed ²immediately by² western blot or ELISA.²If a western blot protocol is used, the following approach is taken. 30 pg of ²protein is loaded ²onto a 10% polyacrylamide gel after quantitation using a Quanti Pro BCA assay ²(Sigma Aldrich, ²cat: QPBCA-1KT). The gel is run at 160 V for 1 h and then protein is blotted ²onto to a²nitrocellulose membrane overnight at 4 degrees Celsius at 30V. Nitrocellulose ²membrane is²stained with Ponceau solution (described below) to confirm equal loading and ²transfer of ²samples and then blocked using 5 % milk powder (Fluke, Sigma Aldrich) for 2 h ²(E1A) or ²overnight (Aldolase A). Membranes are then washed twice with PBS, 0.1 % Tween ²20, and then ²once with PBS. For detecting repression levels of adenovirus late proteins, an ²anti-Hexon²western blot is used. A primary antibody recognising Hexon (AbCam, Cambridge, ²UK, cat²number: Ab8249) is added at 1:500 dilution in 2.5 % milk powder in PBS for 1 ²hr. Membrane is ²washed as above. Secondary anti-rabbit HRP labelled antibody is added at ²1:1000 dilution in ²2.5 % milk powder for 1 h. The membranes are washed as above and then bathed ²in ECL ²western blotting detection reagent (Amersham, GE Healthcare) at 0.125 ²mL / cm2for 1 minute.²Blot is visualised in an Alpha Innotech gel documentation system for 1, 5 and ²10 minutes using²chemi-luminescence detection. Molecular weights are calculated against a dual ²colour ²molecular weight ladder (Bio-Rad).²Ponceau staining²To confirm successful transfer of protein from poly-acrylamide gels onto ²nitrocellulose²membranes and to ensure equal loading and transfer, membranes are stained for ²2 minutes ²with Ponceau S solution (0.1 % w / v Ponceau S in 5 % v / v acetic acid made up ²with de-ionised ²distilled H20) prior to blocking. The stain is then poured off and membranes ²washed briefly in ²PBS twice before imaging. Membranes are then washed twice with PBS 0.1 % Tween ²20 and² then once with PBS to remove all residual stain prior to blocking.²<DP=36>²CA 03070654 2020-01-21²WO 2019 / 020992 PCT / GB2018 / 052083²- 36 -²Rather than assessing protein levels by western blot, an ELISA may be used. To ²do this, a ²sample to be studied is first diluted in coating buffer (cat. 28382 ²Thermofisher). Typically, ²samples are coated onto the plates at different concentrations such as 1:10; ²1:100; 1:500 to²determine optimal detection range. A standard curve is created by using a pre-²made quantity of²the protein to be detected and coating wells the highest standard point of ²4pg / nnl by dilution ²followed by 2-fold serial dilution into adjacent wells. For example, a ²standard protein at ²0.2mg / nril requires 4p1 with 196p1 of non-transfected culture media and 1.8m1 ²of coating buffer. ²This is used to generate an 8-point standard curve by performing 2-fold serial ²dilution by adding²lml of reconstituted standard and lml of coating buffer in 1.5 ml tubes. 100 ²microliters of each²standard is then added in duplicate to the ELISA plate. 100 microlitres of ²each sample is added ²to a well of the immuno-microplate. 100 microlitres of coating buffer is added ²to one well as a ²no-antigen control. 100 microlitres of a positive control sample is included, ²if available. The plate ²is incubated at 4 degrees Celsius overnight. The plate is washed with PBS-T ²(0.1% Tween)²three times. 200pL of blocking buffer is added (the original coating buffer ²with 2% BSA) per well²and the plate is incubated at 37 C for lhr. The plate is washed with PBS-T ²(0.1% tween) three ²times). The Primary antibody (1*) is added to the diluent solution at the ²required dilution and 50 ²pL is added to each well. The plate is incubated at room temperature for 1-2 ²hours. The plate is ²washed with PBS-T (0.1% tween) three times. A HRP-labelled secondary antibody ²is added to²the diluent solution at the required dilution. 50 microlitres is added to each ²well. The plate is²incubated at room temperature for 1 hour. The plate is washed with PBS-T (0.1% ²tween) three ²times. The plate is washed with PBS once. 50 microlitres of 3,3' 5,5'-²Tetramethylbenzidine ²(TMB) is added to each well. It is then necessary to wait for the colour to ²develop and to record ²the time taken. 50 microlitres of the stop solution (1 M HCI) is added. Data ²acquisition is² achieved by reading the plates absorbance in a plate reader at 450nm.²Measuring VLP production ²The levels of VLP production from a cell Transgene expression level or the ²level of expression²of late proteins are measured via Western blot or ELISA as described above but ²using² antibodies recognising an epitope on the VLP surface.²Example 1: Construction of plasmid vectors for expression of the GFP reporter ²from the²Ad5 repressor mutant Major late promoters²Five expression constructs were created wherein the GFP reporter gene was ²transcribed by the² wildtype Ad5 MLP (pMLPwt-GFP), repressor mutant MLPs (pMLP-TET01a-GFP; pMLP-²<DP=37>²CA 03070654 2020-01-21²WO 2019 / 020992 PCT / GB2018 / 052083²- 37 -²TETO1b-GFP; pMLP-TET02-GFP) or the control construct in which no internal ²promoter was ²present (pMCS-GFP) for determining low level baseline transcription. The ²vectors are shown in ²Figure 4.² Example 2: Expression of the GFP reporter gene from MLP promoters²Five expression constructs were created wherein the GFP reporter gene was ²transcribed by the ²wildtype Ad5 MLP (pMLPwt-GFP), repressor mutant MLPs (pMLP-TETO1a-GFP; pMLP-²TETO1b-GFP; pMLP-TET02-GFP) or the control construct in which no internal ²promoter was ²present (pMCS-GFP) for determining low level baseline transcription. HEK293 ²cells were²seeded in tissue culture treated 48-well plates at a density of 3e4 ²cells / well, 24-hours before²transfection. HEK293 cells were transfected with the plasmids pMLPwt-GFP; pMLP-²TETO1a-²GFP; pMLP-TETO1b-GFP; pMLP-TET02-GFP; pMCS-GFP using branched PEI (25kDA) at a ²²1:3 ratio of total DNA mass to PEI. Transfection was carried out in ²triplicates and cells were ²harvested at 24 hours, 48 hours and 72 hours post transfection for analysis by ²flow cytometry.²Data is presented in Figure 5 as MF1 (mean fluorescent intensity) of GFP ²positive cells. Error²bars indicate SD of triple biological replicates. Data NS p>0.05 by Student's ²t-test.²Example 3: Transcriptional repression of the repressor mutant MLP by the TETR ²protein ²Five expression constructs were created wherein the GFP reporter gene was ²transcribed by the²wildtype Ad5 MLP (pMLPwt-GFP), repressor mutant MLP (pMLP-TETO1a-GFP; pMLP-²TETO1b-²GFP; pMLP-TET02-GFP) or the control construct in which no internal promoter ²was present ²(pMCS-GFP) for determining low level baseline transcription. Wildtype Ad5 MLP ²(pMLPwt-GFP), ²repressor mutant [VIP (pMLP-TETO1a-GFP; pMLP-TETO1b-GFP; pMLP-TET02-GFP) or ²the ²control construct expressing the GFP reporter was co-transfected with a TETR ²expression²plasmid (pTETR), under the control of the constitutive CMV (cytomegalovirus) ²promoter, at a 1:1²ratio of total DNA mass, in HEK293 cells treated with doxycycline 0.2pg / m1 or ²DMSO. HEK293 ²cells were seeded in tissue culture treated 48-well plates at a density of 3e4 ²cells / well, 24-hours ²before transfection. Transfection was carried out in triplicates using ²branched PEI (25kDA) at a ²1:3 ratio of total DNA mass to PEI and cells were analysed by flow cytonnetry ²at 24, 48 and 72²hours post transfection. Data is presented in Figure 6 as percentage ²repression of MF1 (mean²fluorescent intensity) in GFP positive cells compared against the activity of ²the Ad5 MLP ²wildtype (pMLPwt-GFP) and normalised against the promoterless control ²construct for ²background expression signal. Data as mean SD NS p>0.05; *p.-0.05; **p-0.01; ²²0.001; ****P0.0001; unpaired, two tailed Student's.²<DP=38>²CA 03070654 2020-01-21²WO 2019 / 020992 PCT / GB2018 / 052083²- 38 -²Example 4: Ad5 MLP repressor in repression in HEK293 T-Rex Flp cells²Adenoviruses engineered by molecular cloning methods to incorporate the ²repressor sequences ²were recovered in HEK293 cells. Ad5 genome with the MLP repressor sequence MLP-²TET01a,²MLP-TETO1b, TET02 or wild-type MLP were excised away from bacterial plasmid ²DNA by²restriction digest with Swal restriction enzymes to release the 5' and 3' ²flanking Ad5 ITR²sequences. HEK293 cells were seeded in 10-cm tissue culture plates at a ²density of 1e5 ²cells / plate for 24-hours and transfected with 10pg of DNA containing MLP ²repressor Ad5 or Ad5 ²MLPwt genomes, using lipofectamine 2000 at a 1:2 ratio of total DNA mass to ²lipofectamine ²2000. Viruses were harvested following observation of viral infection and ²cytoplasmic effect,².. typically, 10-15 days post transfection. 293 T-Rex Flp cells were seeded in ²48-well tissue culture²treated plates at a density of 4e4 cells / well for 24-hours and transduced with ²adenoviruses Ad5-²MLP-TET01a, Ad5-MLP-TET01b, Ad5-MLP-TET02 or standard E1-E3 deleted Ad5 (Ad5-²MLPwt) ²in the presence of doxycycline 0.2pg / m1 or DMSO and imaged by light miscopy 10-²days post ²infection. Data shown in Figure 7 is representative of triplicate biological ²replicates. Bar =² 250pm.²Example 5: Ad5 MLP repressor in repression in 293Ad cells transfected with a ²TETR ²expression plasmid (pTETR), under the control of the constitutive CMV ²(cytomegalovirus) ²promoter²Adenoviruses engineered by molecular cloning methods to incorporate the ²repressor sequences²MLP-TETO1b were recovered in HEK293 cells. Ad5 genome with the MLP repressor ²sequence ²MLP-TETO1b were excised away from bacterial plasmid DNA by restriction digest ²with Swal ²restriction enzymes to release the 5' and 3' flanking Ad5 ITR sequences. ²HEK293 cells were ²seeded in 10-cm tissue culture plates at a density of 1e5 cells / plate for 24-²hours and transfected²with bug of DNA containing MLP repressor Ad5, using lipofectamine 2000 at a ²1:2 ratio of total²DNA mass to lipofectamine 2000. Viruses were harvested following observation ²of viral infection ²and cytoplasmic effect, typically, 10-15 days post transfection. HEK293 cells ²were seeded in 10-²cm tissue culture plates were transfected with pTETR (5pg) or stuffer DNA, ²using lipofectamine ²2000 at a 1:2 ratio of total DNA mass to lipofectamine 2000, prior to ²infection with adenoviruses²Ad5-MLP-TET01b. The results are shown in Figure 8. Data imaged by light ²microscopy 24, 48,²72 and 96 hours post-infection.²Example 6: Viral genome replication of repressed MLP mutant Ad5 in 293 T-Rex ²Flp cells ²Adenoviruses engineered by molecular cloning methods to incorporate the ²repressor sequences²were recovered in HEK293 cells. Ad5 genome with the MLP repressor sequence MLP-²TET01a,²<DP=39>²CA 03070654 2020-01-21²WO 2019 / 020992 PCT / GB2018 / 052083²- 39 -²MLP-TET01b, TET02 or wildtype MLP were excised away from bacterial plasmid DNA ²by ²restriction digest with Swal restriction enzymes to release the 5' and 3' ²flanking Ad5 ITR ²sequences. 293 T-Rex Flp cells were seeded in 10-cm tissue culture plates at a ²density of 1e5 ²cells / plate for 24-hours and transfected with 10pg of DNA containing MLP ²repressor Ad5 or Ad5²MLPwt genomes, using lipofectamine 2000 at a 1:2 ratio of total DNA mass to ²lipofectamine²2000. Viruses were harvested following observation of viral infection and ²cytoplasmic effect, ²typically 10-15 days post transfection. 293 T-Rex Flp cells were seeded in 48-²well tissue culture ²treated plates at a density of 4e4 cells / well for 24-hours and transduced with ²adenoviruses Ad5-²MLP-TETO1a, Ad5-MLP-TET01b, Ad5-MLP-TET02 or standard E1-E3 deleted Ad5 (Ad5-²MLPwt)²in the presence of doxycycline 0.2ug / nril or DMSO 293 T-Rex Flp cells were ²infected with MLP²mutant or MLP-wt Ad5 in the presence of doxycycline or DMSO. Total viral and ²cellular DNA ²was harvested 10-days post infection. Viral genome copy was quantified by QPCR ²using ²TaqMan0 probes. Ad5 of known titres were used for generating the standard ²curve and data is ²presented (Figure 9) as increase in genome copy above the initial viral load ²used for infection.²Data representative of triplicate biological replicates and presented as mean ² SEM **10.01;²unpaired, two tailed Student's.²Example 7: MLP repression blocks adenovirus spread across HEK293 cell ²monolayer ²To determine the effect of MLP repression on inhibiting adenovirus production ²and virus²spreading, adenovirus with wild-type MLP or TETOlb modification was engineered ²to express²the EGFP reporter, with and without Ad5 TPL fused to the EGFP initiation ²codon. EGFP ²reporter was expressed from the virus El deleted region and under control of ²the CMV promoter. ²Adenoviruses expressing EGFP were used to infect a monolayer of Flp-In T-REx ²293 cells ²treated with doxycycline or DMSO at MOI 1 and EGFP expression was monitored at ²6-days by²fluorescence microscopy and flow cytometry. While viral spreading i.e EGFP ²expression across²the cell monolayer was not inhibited in the control adenovirus with wild-type ²MLP, virus ²spreading of adenoviruses with MLP TETOlb was blocked in Flp-In T-REx 293 ²cells in the ²absence of doxycycline, indicating repression of virion replication (Figure ²10A).²Following 6-days post infection, cells were harvested for flow cytometry to ²determine the relative²proportion of EGFP expressing cells within the gated population. In line with ²results from ²fluorescent microscopy, the proportion of cells expressing EGFP was comparable ²in Flp-In T-²R Ex 293 cells treated with doxycycline or DMSO following infection with the ²control adenovirus ²(Figure 10B). However, whilst the population of EGFP positive cells were lower ²from infection²with MLP TETOlb modified adenoviruses in cells treated with doxycycline ²compared to the²<DP=40>²CA 03070654 2020-01-21²WO 2019 / 020992 PCT / GB2018 / 052083²- 40 -²control virus, presumably due to variation in infectivity and replication, ²cells treated with DMSO²showed a significantly lower popular of EGFP expressing cells, indicating that ²EGFP is only ²expressed from cells transduced by viruses from the first-round infection.²Example 8: MLP repression enhanced expression of EGFP containing Ad5 TPL exon ²1-2-²3²To determine whether fusion of Ad5 TPL exons to the initiation codon of EGFP ²affected gene ²expression, plasnnids engineered to expression EGFP, with and without the ²leader sequence, ²under the control of the CMV promoter were transfected into HEK293 cells and ²EGFP²expression determined by flow cytometry at 48 hours post-transfection. The ²results are shown²in Figure 11A. Whilst the promoter-less control plasmid showed background ²levels of EGFP, ²there were no significant differences in the levels of EGFP expression from ²plasmid containing ²the TPL sequence.²To assess whether protein expression is increased following MLP repression, ²MLP TETO1b²modified adenoviruses expressing the EGFP reporter from the CMV promoter, with ²and without ²Ad5 TPL, were infected into Flp-In T-REx 293 cells treated with doxycycline or ²DMSO at an MOI ²10, and EGFP expression was measured by flow cytometry 24, 48 and 72-hours ²post-infection. ²The results are shown in Figure 11B. While MLP TETO1b adenovirus expressing ²EGFP²containing the TPL showed an increase in median fluorescence intensity (MFI) ²compared EGFP²lacking the TPL sequence, repression of the adenovirus MLP from infection in ²the DMSO ²treated cells resulted in a further ¨2-fold increase in EGFP expression ²compared to virus ²infection into cells treated with doxycycline, which enables virion ²production.²Similarly, a ¨3-fold increase in EGFP expression was observed in HEK293 cells ²when²adenovirus MLP was repressed by transient expression of the repressor TETR ²(see Figures ²11C-11D). Plasmid expressing TETR from the CMV promoter was transfected into ²HEK293 ²cells treated with doxycycline or DMSO. Subsequently, cells were infected with ²adenovirus ²containing MLP TETO1b modification and expressing EGFP (with and without the ²TPL exons)²from the CMV promoter or transfected using Lipofectamine with a CMV expression ²plasmid that²transcribed EGFP. EGFP expression was determined by flow cytometry 48-hours ²post ²transduction with the virus and plasmid. Results showed comparable levels of ²EGFP expression, ²as determined by MFI, from HEK293 cells transduced with the EGFP expressing ²adenoviruses ²or plasmid DNA. However, ¨3-fold increase in EGFP expression was only observed ²from²infection with the MLP TET01b modified adenovirus expression EGFP with the TPL ²exons in²<DP=41>²CA 03070654 2020-01-21²WO 2019 / 020992 PCT / GB2018 / 052083²- 41 -²cells treated with DMSO, which allowed repression of Ad MLP TETO1b by TETR, ²indicating ²enhance protein production by cap-independent translation as this effect was ²not observed in ²the EGFP coding sequence without the TPL exons.²Example 9: MLP self-repression by Tetracycline-Enabled Repression Adenovirus ²(TERA)²inhibits production of adenoviral structural proteins²Adenoviruses with MLP TETO1b modification was further engineered to express ²the repressor ²TETR from a splice acceptor sequence under direct control of the virus ²internal MLP, position at²the E3-deleted region of the virus, to construct TERA. The results are shown ²in Figure 12. ²To assess repression of viral structural proteins from TERA, control ²adenovirus E1 / E3 deleted ²or TERA was used for infection of HEK293 cells in the presence of doxycycline ²or DMSO, at an ²MOI 100 or 1000. Adenovirus capsid proteins from cellular lysate and growth ²medium were ²probed using anti-Ad5 antibodies by Western blot 72-hours post infection. ²Comparable levels of²adenovirus structural proteins were detected from TERA and the control virus ²when doxycycline²was present in the growth medium, from both cell lysates and culture media. ²However, in the ²treated DMSO group, structural proteins from TERA were undetectable from the ²blot. ²Presumably, in the absence of doxycycline, TERA infection into HEK293 cells ²enabled ²production of viral MLP transcribed TETR to self-repression of its own MLP and ²a blockade in² the production of capsid proteins.²In a doxycycline dose-escalation study to assess production of TERA in HEK293 ²cells, control ²adenovirus with the wildtype MLP or TERA was used for infection of HEK293 ²cells at MOI 10 ²and 100, treated with DMSO or doxycycline dose of 0.1, 0.2, 0.5 and 1.0 pg / mL. ²The results are².. shown in Figures 12B-12C. Comparable levels of Ad5 major structural ²proteins were detected²for the control adenovirus infection in cells treated with DMSO or ²doxycycline, while capsid ²proteins expressed from TERA increased with increasing with increasing doses ²of doxycycline ²up to 0.5 pg / mL, consistent for both MOI assessed.².. Example 10: TERA viral replication is comparable to E1 / E3 deleted ²adenovirus and MLP²repression enhanced DNA replication²To assess virus replication of TERA, control E1 / E3 deleted adenovirus or TERA ²was used to ²infect a nnonolayer of HEK293 cells at MOI 1, 10 and 100, in the presence of ²doxycycline, and ²total genomic DNA was extracted from growth media and cellular lysate at time-²point 0, 24, 48²<DP=42>²CA 03070654 2020-01-21²WO 2019 / 020992 PCT / GB2018 / 052083²- 42 -²and 72-hours post viral infection. Abundance of adenovirus genomic DNA was ²determined by ²QPCR. The results are shown in Figure 13.²For all three infection MOI assessed, virus replication of TERA was comparable ²to the control²adenovirus, where the relative abundance of viral genome increased sharply at ²24h, with >4-log²increases in total viral genomes at 72h post-infection.²Genome replication of TERA during MLP repression was further assessed by QPCR. ²The virus ²TERA or control E1 / E3 deleted adenovirus was used for infecting HEK293 cells ²in the presence²of doxycycline or DMSO at an MOI of 10 and 100 and total genonnic DNA was ²harvested at²time-point 0, 24, 48, 72, and 96-hours post infection viral genome ²quantification by QPCR. ²Similarly, while virus replication of TERA was comparable to the control ²virus, DNA replication ²from TERA significantly increased after 24-hours post infection in HEK293 ²cells treated with the ²DMSO control group compared to infection into cells cultured with doxycycline. ²Presumably,²repression of the virus MLP inhibited TERA from completion of its replicative ²life-cycle, thereby²enabling exponential viral genome amplification.²Example 11: TERA enhanced expression of the EGFP reporter²To assess EGFP reporter expression from TERA under control of the CMV ²promoter, TERA²engineered to express EGFP, with and without the Ad5 TPL exons, were used to ²infect HEK293²cells and EGFP expression is compared to expression from E1 / E3 control ²adenovirus and ²method of transient transfection with a CMV expression plasmid.²The results are shown in Figure 14.²While E1 / E3 adenovirus and TERA exhibited increase EGFP expression compared to²expression from a CMV plasmid vector, TERA expressing EGFP containing Ad5 TPL ²exons ²enhanced production above levels observed from both E1 / E3 control vector and ²TERA without ²the TPL exons. Presumably, self-repression of TERA MLP by the virally encoded ²TETR resulted ²in repression of Ad5 late nnRNA transcripts and enabling cap-independent ²translation of the² prevailing TPL EGFP mRNAs.²Additionally, Western blot confirmed that EGFP expression using the control ²E1 / E3 adenovirus ²and TERA, in the presence of doxycycline, was accompanied with contaminating ²adenovirus ²proteins. In contrast, the absence of doxycycline enabled repression of Ad5 ²structural proteins²in TERA while EGFP expression was maintained.²<DP=43>²CA 03070654 2020-01-21²WO 2019 / 020992 PCT / GB2018 / 052083²- 43 -²Example 12: TERA enhanced production of a Bi-specific T-cell Engager (BiTE)²To compare production of a model secretory protein using TERA against a method ²of transient²expression from plasmid DNA, TERA and plasmid vector (pCMV-BiTE) were ²engineered to ²express a Bi-Specific T-Cell Engager (BiTE) against tumour specific antigen ²EpCAM under the² control of the CMV promoter. The results are shown in Figure 15.²Virus TERA expressing BiTE containing the Ad5 TPL exons or plasmid pCMV-BiTE ²was used to ²transduce HEK293 cells at MO1 10 or 0.75pg DNA per well, respectively, and ²protein harvested ²from growth media at time-point 24, 48 and 72-hours post-transduction and ²probed using anti-6x²His antibodies. Western blot showed significant increase in BiTE expressing ²from TERA²compared to production from using the CMV expression plasmid in all three time-²point of ²harvest.²Figure 15D shows Western blot detection of BiTE protein expressed from TERA ²compared to²El / E3 deleted adenovirus. HEK293 cells, cultured in the absence of ²doxycycline, were infected²with adenoviruses E1 / E3-deleted or TERA-expressing BiTE under control of the ²CMV promoter ²(with and without Ad5 TPL exons) at M0110 and BiTE protein expression was ²measured at 48-²hours post-infection from growth medium. TERA exhibited increase production of ²BiTE ²compared to control adenovirus; however, -2-fold increase in BiTE production ²was observed²from infection with TERA expressing BiTE containing the Ad5 TPL exons. ²Additionally, while²viral structural proteins were detected from infection with the El / E3 deleted ²virus control, virus ²capsid proteins were undetectable following infection by TERA. Amplification ²of the viral ²genomic DNA was confirmed to be maintained and comparable to the control virus ²by QPCR ²analysis.²Example 13: TERA enhances production of rAAV free of Ad5 particle contaminants ²²To evaluate the use of TERA for delivery of helper-functions and rAAV DNA for ²the production ²rAAV viral vectors, E1 / E3 deleted control adenovirus and TERA was modified in ²the virus El ²deleted region to encode an rAAV genonne, AAV2 ITRs flanking an EGFP ²expression cassette²under control of the CMV promoter. The results are shown in Figure 16.²rAAV encoding EGFP transgene was produced by an established Helper-free triple ²transfection ²method, or via infection with control E1 / E3 deleted adenovirus or TERA. HEK293 ²cells were ²triple-transfected with plasmid encoding 1) rAAV genome encoding EGFP, 2) AAV2 ²Rep and²Cap, and 3) Ad5 Helper, or transduced with plasmid encoding AAV2 Rep and Cap, ²and control²<DP=44>²CA 03070654 2020-01-21²WO 2019 / 020992 PCT / GB2018 / 052083²- 44 -²E1 / E3 deleted adenovirus or TERA, in the presence of doxycycline or DMSO. ²Recombinant ²viruses were harvested from cellular lysates and growth media 72-hours post ²transduction and ²viral genomes, from encapsulated adenovirus and rAAV, were quantified by QPCR ²using primer ²and probe sets directed against adenovirus DNA encoding Fibre, or CMV promoter ²encoded by² both rAAV and adenoviruses.²Figure 16 C shows quantification of encapsulated adenovirus genomes from ²cellular lysate and ²growth medium. While significant levels of adenoviruses were detected ²following infection with ²the control adenovirus and TERA in HEK293 cells treated with DMSO and ²doxycycline,²respectively, presence of adenoviruses were below the lowest standard and ²detection range of²the assay from HEK293 cells, absent of doxycycline, when transduced with TERA ²or Helper-²free production method. In contrast, significant levels of recombinant viruses ²from all samples ²were detected by QPCR assay using primer and probe sets directed against the ²encoded CMV ²promoter, present in both adenoviruses and rAAVs (Figure 16D).²Since virus quantification used QPCR primers and probe directed against the ²encoded CMV ²promoter detected both rAAV and the adenoviral delivery vectors, total rAAV ²from each ²production method and conditions were determined by subtraction of overall ²adenovirus ²particles contamination detected (Figure 16A). E1 / E3 deleted adenovirus ²produced rAAV that²were comparable or above the Helper-free production system by triple ²transfection, which was²also accompanied with significant levels of adenovirus contamination. In ²contrast, HEK293 cells ²infected with TERA-AAV, in the absence of doxycycline, yielded -5-fold ²increase in rAAV ²compared to Helper-free plasmid transfections with adenovirus contamination ²beyond the limit ²of the QPCR detection. (Figure 16B) Adenovirus particles contamination in ²growth medium was²further confirmed by Western blot using antibodies directed against Ad5 capsid ²protein. In line²with results from QPCR analysis, adenovirus structural proteins were ²undetectable from Helper-²free production method or infection with TERA (M01100 and 500) in HEK293 cells ²absent of ²doxycycline, while adenovirus proteins were readily detected from TERA ²infection in the ²presence of doxycycline, enabling active MLP, and from the E1 / E3 control ²virus.²To determine rAAV particles with an encapsulated rAAV genome, rAAV capsids ²produced from ²methods of Helper-free plasmid transfection, or via TERA infection into ²plasmid Rep and Cap ²transfected HEK293 cells, were quantified. Figure 16E shows total rAAV2 ²capsids and DNase-I ²resistant genome encapsulated particles determined by ELISA directed toward ²formed-AAV2²viral capsids and quantification of rAAV genome by QPCR, respectively. Figure ²16F shows per²<DP=45>²CA 03070654 2020-01-21²WO 2019 / 020992 PCT / GB2018 / 052083²- 45 -²cell basis, TERA MOI 500 infection produced significantly higher amounts of ²rAAV2 capsids and ²DNase-I resistant genome encapsulated rAAVs, compared to Helper-free ²production method. ²Overall, Helper-free rAAV2 production and TERA produced genome encapsulated ²particles ²accounting to -30% and -50% of total rAAV2 capsid particles, respectively.²Contaminating adenovirus particles from the use of TERA in the production rAAV ²in HEK293 ²cells, cultured in the absence of doxycycline, were undetectable via Western ²blot and QPCR. ²Figure 16H shows contaminating infectious adenovirus following production of ²rAAV by method ²of Helper-free, and infection by control E1 / E3 adenovirus or TERA was ²determine by Tissue²Culture Infectious Dose 50 assay (TCID50) in HEK293 cells. As shown in Figure ²16G, ²transduction of HEK293 cells with crude rAAV produced by method of TERA ²infection in the ²presence of doxycycline results in early EGFP expression and adenovirus CPE ²cell morphology. ²In stark contrast, the absence of doxycycline enables MLP and adenovirus self-²repression as ²seen from the delayed expression of EGFP reporter and an intact cell ²monolayer, presumably²expressed from rAAV and the time required for second-strand DNA synthesis of ²rAAV genome. ²Additionally, while infectious adenoviruses were undetected by rAAV2 Helper-²free production ²method lacking adenovirus, rAAV production by TERA infection at MOI 100 and ²500, exhibit ²contaminating infectious adenovirus at levels of 0.0003% (>2e6 fold ²repression) and 0.007% ²compared to rAAV2 production using the control E1 / E3 adenovirus, respectively. ²Overall, in the²absence of doxycycline to enable MLP self-repression, TERA infection in HEK293 ²cells at MOI ²100 and 500 reduced contaminating adenoviruses by -3 x 106 and -1.5 x 104 ²folds, respectively, ²compared to infection with E1 / E3 deleted control adenovirus.²REFERENCES²Babiss LE, Ginsberg HS. Adenovirus type 5 early region lb gene product is ²required for efficient ²shutoff of host protein synthesis. J Virol. 1984 Apr;50(1):202-12.²Beaton, A., Palumbo, P., Berns, K.I. Expression from the adeno-associated ²virus p5 and p19 ²promoters is negatively regulated in trans by the rep protein. J Virol. 1989. ²63(10):4450-4454.²Concino M, Goldman RA, Caruthers MH, Weinnnann R. Point mutations of the ²adenovirus major ²late promoter with different transcriptional efficiencies in vitro. J Biol ²Chem. 1983 Jul ²10;258(13):8493-6.²<DP=46>²CA 03070654 2020-01-21²WO 2019 / 020992 PCT / GB2018 / 052083²- 46 -²Concino MF, Lee RF, Merryweather JP, Weinmann R. The adenovirus major late ²promoter ²TATA box and initiation site are both necessary for transcription in vitro. ²Nucleic Acids Res. ²1984 Oct 11;12(19):7423-33.²El-Mogy, M and Haj-Ahmad, Y. Transgene Expression under the Adenoviral Major ²Late²Promoter, Tripartite Leader Sequence and El Genes in Absence and Presence of ²Adenovirus ²Infection. Journal of Molecular Biology Research; 2012, Vol. 2, No. 1²Gamier, A, Johanne, C. Nadeau, I. Kamen, A. and Massie. B. Scale-up of the ²adenovirus²expression system for the production of recombinant protein in human 293S ²cells.²Cytotechnology 15: 145-155, 1994. 145²Green, N. K., C. W. Herbert, et al. (2004). "Extended plasma circulation time ²and decreased²toxicity of polymer-coated adenovirus." Gene Therapy 11(16): 1256-1263.²Honda, T., H. Saitoh, et al. (2000). "The coxsackievirus-adenovirus receptor ²protein as a cell²adhesion molecule in the developing mouse brain." Molecular Brain Research ²77(1): 19-28.²Karber, G. (1931). "50% end-point calculation." Arch Exp Pathol Pharmak 162: ²480-483.²Kelkar, S. A., K. K. Pfister, et al. (2004). "Cytoplasmic dynein mediates ²adenovirus binding to²microtubules." 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"Isolation of a Cytopathogenic ²Agent from Human²Adenoids Undergoing Spontaneous Degeneration in Tissue Culture." Proceedings ²of the ²Society for Experimental Biology and Medicine 84(3): 570-573.²Tollefson, A. E., J. S. Ryerse, et al. (1996). "The E3-11.6-kDa adenovirus ²death protein (ADP) is²required for efficient cell death: Characterization of cells infected with adp ²mutants." Virology²220(1): 152-162.²Tomko, R. P., R. L. Xu, et al. (1997). "HCAR and MCAR: The human and mouse ²cellular ²receptors for subgroup C adenoviruses and group B coxsackieviruses." ²Proceedings of the²National Academy of Sciences of the United States of America 94(7): 3352-3356.²Trotman, L. C., N. Mosberger, et al. (2001). "Import of adenovirus DNA ²involves the nuclear ²pore complex receptor CAN / Nup214 and histone Hi." Nature Cell Biology 3(12): ²1092-1100.²Wickham, T. J., P. Mathias, et al. (1993). "Integrin-Alpha-V-Beta-3 and ²Integrin-Alpha-V-Beta-5²Promote Adenovirus Internalization but Not Virus Attachment." Cell 73(2): 309-²319.²Yang, Q., Chen, F., Trempe, J.P. Characterization of cell lines that inducibly ²express the adeno-²associated virus rep proteins. J. Virol. 1994. 68(8): 4847-4856.²Zhang, H-G., Wang, Y.M., Xie, J.F., Liang, X., Hsu, H-C., Zhang, X., Douglas, ²J., Cruiel, D.T., ²Mountz, J.D. Recombinant adenovirus expressing adeno-associated virus cap and ²rep proteins ²supports production of high-titer recombinant adeno-associated virus. Gene. ²Ther. 2001. 8(9): ²704-712.²

Claims

<DP=1>²48²CLAIMS²1. An adenoviral vector comprising a repressible Major Late Promoter (MLP) ²and a plurality ²of adenoviral late genes, wherein the MLP comprises one or more repressor ²elements ²which are capable of regulating or controlling transcription of the adenoviral ²late genes, ²and wherein one or more of the repressor elements are inserted downstream of ²the MLP ²TATA box.²2. An adenoviral vector comprising:²(a) a plurality of adenoviral early genes, and²(b) a plurality of adenoviral late genes under the control of a Major Late ²Promoter ²(MLP), and²(c) a transgene,²wherein the MLP comprises one or more repressor elements which are capable of ²regulating or controlling transcription of the adenoviral late genes, and ²wherein one or ²more of the repressor elements are inserted downstream of the MLP TATA box.²3. The adenoviral vector of claim 1 or 2, wherein the one or more repressor ²elements are ²inserted between the MLP TATA box and the +1 position of transcription.²4. The adenoviral vector of any one of claims 1 to 3, wherein the one or ²more repressor ²elements are capable of being bound by a repressor protein.²5. The adenoviral vector of any one of claims 1 to 4, wherein a gene ²encoding a repressor ²protein which is capable of binding to the one or more repressor elements is ²encoded ²within the adenoviral genome.²6. The adenoviral vector of claim 4 or 5, wherein the repressor protein is ²transcribed under ²the control of the MLP.²7. The adenoviral vector of any one of claims 4 to 6, wherein the repressor ²protein is the ²tetracycline repressor, the lactose repressor or the ecdysone repressor.²8. The adenoviral vector of claim 7, wherein the repressor protein is the ²tetracycline ²repressor (TetR).²<DP=2>²49²9. The adenoviral vector of any one of claims 1 to 8, wherein the one ²or more repressor²elements are a tetracycline repressor binding site comprising or consisting of ²the ²sequence set forth in SEQ ID NO: 2.²10. The adenoviral vector of any one of claims 1 to 9, wherein the nucleotide ²sequence of ²the MLP comprises or consists of the sequence set forth in SEQ ID NO: 6 or 7.²11. The adenoviral vector of any one of claims 1 to 10, wherein the presence ²of the one or ²more repressor elements does not affect production of the adenoviral E2B ²protein.²12. The adenoviral vector of any one of claims 1 to 11, wherein the adenoviral ²vector ²encodes the adenovirus L4 100K protein and wherein the L4 100K protein is not ²under ²control of the MLP.²13. The adenoviral vector of claim 2, wherein the transgene is inserted within ²one of the ²adenoviral early regions.²14. The adenoviral vector of claim 13, wherein the transgene is inserted ²within the ²adenoviral El region.²15. The adenoviral vector of claim 13 or 14, wherein the transgene comprises a ²Tripartite ²Leader (TPL) in its 5'-UTR.²16. The adenoviral vector of any one of claims 13 to 15, wherein the transgene ²encodes a ²therapeutic polypeptide.²17. The adenoviral vector of any one of claims 13 to 16, wherein the transgene ²encodes a ²virus protein.²18. The adenoviral vector of claim 17, wherein the virus protein is capable of ²assembly in or ²outside of a cell to produce a virus-like particle.²19. The adenoviral vector of claim 17 or 18, wherein the transgene encodes ²Norovirus VP1 ²or Hepatitis B HBsAG.²20. The adenoviral vector of any one of claims 13 to 15, wherein the transgene ²encodes an ²adeno-associated virus [AAV] Rep polypeptide, an AAV Cap polypeptide, an AAV ²Rep-Cap ²polypeptide and / or an AAV genome.²<DP=3>²50²21. A composition comprising an adenovirus particle comprising the adenoviral ²vector of any ²one of claims 1 to 20, together with one or more physiologically-acceptable ²carriers, ²excipients or diluents.²22. A kit comprising the adenoviral vector of any one of claims 1 to 20, ²wherein the kit ²additionally comprises one or more additional components selected from the ²group ²consisting of:²(i) a cell line that allows the adenoviral vector to infect the cells and ²replicate the ²adenovirus' genome, wherein the cell line represses the adenoviral vector's ²MLP ²and²(ii) one or more DNA plasmids for aiding in the construction of the adenoviral ²vector.²23. The adenoviral vector as defined in any one of claims 1 to 20 comprising ²the nucleotide ²sequence of SEQ ID NO: 6 or SEQ ID NO: 7.²24. A nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 6 ²or SEQ ID ²NO: 7.²25. An isolated mammalian cell comprising the adenoviral vector of any one of ²claims 1 to ²20.²26. The isolated mammalian cell of claim 25, wherein the cell is a HEK293, ²PerC6 or 911 cell ²which expresses the adenoviral E1A and E1B proteins.²27. A process for producing a modified mammalian cell, the process comprising ²the step: ²(a) infecting an isolated mammalian cell with the adenoviral vector of claim ²2, ²whereby the mammalian cell then comprises the adenoviral vector.²28. The process of claim 27, wherein the isolated mammalian cell to be ²infected is one ²which contains an adeno-associated virus [AAV] genome integrated into the ²genome of ²the cell.²29. The process of claim 28, wherein the AAV genome comprises left and right ²AAV inverted ²terminal repeats [ITRs].²30. The process of claim 28 or 29, wherein the transgene encodes an AAV Rep ²polypeptide, ²an AAV Cap polypeptide or an AAV Rep-Cap polypeptide.²<DP=4>²51²31. A process for producing a transgene product, the process comprising the ²steps:²(a) infecting mammalian cells with the adenoviral vector of claim 2;²(b) culturing the infected mammalian cells in a culture medium under ²conditions such ²that the transgene is expressed; and²(c) isolating or purifying transgene product from the cells or from the cell ²culture ²medium.²32. Use of the adenoviral vector of claim 2, in the production of a ²recombinant adeno-²associated virus (AAV).²33. The use of claim 32, wherein the transgene in the adenoviral vector ²encodes an AAV ²Rep-Cap polypeptide.²34. The use of claim 32 or 33, wherein the transgene encodes an AAV genome.²35. The use of claim 34, wherein the AAV genome comprises or consists of a 5' ²and 3' ²inverted terminal repeat [ITR] sequence from AAV with intervening sequences.²36. The use of any one of claims 32 to 35, wherein the transgene encodes a Rep-²Cap ²polypeptide from an AAV and an AAV genome with inverted terminal repeats ²[ITRs] ²flanking a sequence of interest.²37. A process for producing a modified mammalian cell, the process comprising ²the step: ²(a) infecting an isolated mammalian cell with the adenoviral vector of claim ²1, ²whereby the mammalian cell then comprises the adenoviral vector.²38. The process of claim 37, wherein the isolated mammalian cell to be ²infected is one ²which contains an adeno-associated virus [AAV] genome integrated into the ²genome of ²the cell.²39. The process of claim 38, wherein the AAV genome comprises left and right ²AAV inverted ²terminal repeats [ITRs]²40. The process of claim 38 or 39, wherein the adenoviral vector comprises a ²transgene ²which encodes an AAV Rep polypeptide, an AAV Cap polypeptide or an AAV Rep-Cap ²²polypeptide.²<DP=5>²52²41. Use of the adenoviral vector of any one of claims 1 to 20, in the ²production of a ²recombinant adeno-associated virus (AAV).²