Genome replicating adenovirus

Abstract

The present invention provides a gutless vector with a minimal replication system. This vector is capable to amplify its own genome within the host cell, but is not able to generate new particles. Advantages of this vector include an increased longevity in dividing cell, an increased payload expression and no lysis of the host cell.

Description

[0001] Genome replicating adenovirus

[0002] Background of the invention

[0003] Adenoviral vectors are characterized by a comparably large cargo capacity, most notably for the helper-dependent version (see below), and a high efficiency of infection of different cell types. Moreover, technologies have been developed to steer them to infect certain cell types and avoid others. Adenoviral vectors, however, also have certain drawbacks, such as cytotoxicity (those of early generations) and a low expression of cargo genes (those that do not replicate).

[0004] In first-generation adenoviral vectors the Early 1 (El) and Early 3 (E3) regions are deleted. The El region encodes genetic information required for adenovirus survival and replication. Deletion of this region prevents uncontrolled adenoviral replication and formation of progeny particles and is therefore usually seen as an important safety factor. The E3 region encodes proteins that protects infected cells against the immune response and is often deleted in first generation vectors to increase the cargo capacity, because it is not essential for replication. By deleting El and E3 together, 5-6 kb of cargo capacity become available. First-generation adenoviral vectors are incapable of self-replication and require a packaging cell line expressing El protein for their production.

[0005] In second-generation adenoviral vectors, E2 and E4 deletions are introduced to attenuate the host cell immune response to the vector. The E2 region contains genes required for adenoviral DNA replication, and the E4 region encodes regulatory proteins for DNA transcription. Deleting these regions provides approximately 14 kb free space and significantly reduces native adenovirus protein synthesis. Nevertheless, second-generation adenoviruses still induce host immune responses through adenoviral proteins expressed by the remaining genes, ultimately resulting in reduced transgene expression in target cells.

[0006] Third-generation adenoviral vectors (also called "gutless" or high-capacity adenoviral vectors (HC- HAdVs)) have their entire native adenoviral genome removed, except for the inverted terminal repeats and i packaging signal, thereby increasing the cargo limitto 36 kb. This third-generation feature allows a relatively high-level expression of cargo proteins in host cells with no expression of viral proteins and little induction of an immune response. Nonetheless, since the genome is no longer replicated after infection of the target cell, the expression level of target genes can be lower than of other adenoviral vectors that still can replicate their genomes. Due to the lack of genes required for self-assembly, a "helper adenovirus" carrying the genes required for replication must be co-introduced into the packaging cells when producing third-generation adenoviral vectors.

[0007] Blood (2000) 95, 820-8 describes high-capacity (HC) adenoviral vectors. These vectors however do not contain any adenoviral genes, in particular not those of the minimal system described in the present invention. As the vectors of Blood (2000) 95(3), 820-8 do not carry all genes of a functional replication system they hence do not feature amplification of their delivered genome and increased payload expression upon transduction. To amplify their genome in a host cell, the vectors described in Blood (2000) 95(3), 820-8 require a helper virus or helper plasmid to trans-amplify their genome. The same applies to the vectors described in Exp Mol Med (2019) 51(10), 1-18. They represent conventional HC-HAdVs that do not feature amplification and increased payload expression upon transduction without formation of new progeny adenoviral particles. Lee et al. merely report in Exp Mol Med (2019) 51(10), 1-18 the production of a conventional HC-HAdV particle using a helper plasmid instead of a helper virus.

[0008] Cancer Gene Ther (2005) 13(2), 159-68 describes classical oncolytic viruses. Such viruses still carry essentially all viral genes required for (restricted) progeny particle formation and have a very limited cargo capacity of about 3 kb. These viruses do feature vector genome replication, however unlike the viral vectors of the present invention, the vectors described in Cancer Gene Ther (2005) 13(2), 159-68 are still capable of producing viral particles. The same applies to the adenoviral vectors described in J Virology (2000) 74(13) 6147-55.

[0009] The vectors of the present invention thereby differ from the vectors of the prior art for several reasons. They carry the full replication machinery, which enables them to amplify their own DNA and produce higher amounts of the encoded cargo proteins. At the same time they are not capable to produce any viral particles. Finally, they have a cargo capacity of 22 kb which is substantially more than that of any comparable vectors that feature genome replication. Advantages of this vector include an increased longevity in dividing cell and an increased payload expression.

[0010] Summary of the invention

[0011] The present disclosure relates to a minimal adenoviral vector capable of replicating its own DNA in a host cell. In certain embodiments, said minimal adenoviral vector is not capable to form viral particles. In certain embodiments, said minimal adenoviral vector has a cargo capacity of at least 17 kb, preferably 20 kb, more preferably 22 kb.

[0012] In certain embodiments, said minimal adenoviral vector contains 7 adenoviral genes encoding the following proteins: E1A-289R (SEQ. ID No. 2), E1B-19K (SEQ ID No. 4), E1B-55K (SEQ ID No. 6), DBP (SEQ ID No. 9), pTP (SEQ ID No. 12), Ad Pol (SEQ ID No. 15) and E4 ORF6 (SEQ ID No. 18), or functional variants or splice variants of any of aforementioned proteins. Splice variants of E1A-289R (SEQ ID No. 2) include E1A-243R (SEQ ID No. 29), E1A-217R (SEQ ID No. 30), E1A-171R (SEQ ID No. 31) and E1A-55R (SEQ ID No. 32).

[0013] Therefore, in certain embodiments, said minimal adenoviral vector contains 7 adenoviral genes encoding the following proteins: SEQ ID No. 2 (E1A-289R), SEQ ID No. 29 (E1A-243R), SEQ ID No. 30 (E1A-217R), SEQ ID No. 31 (E1A-171R), SEQ ID No. 32 (E1A-55R), SEQ ID No. 4 (E1B-19K), SEQ ID No. 6 (E1B-55K), SEQ ID No. 9 (DBP), SEQ ID No. 12 (pTP), SEQ ID No. 15 (Ad Pol) and SEQ I D No. 18 (E4 0RF6), or functional variants of any of aforementioned proteins

[0014] In certain embodiments, said minimal adenoviral vector contains the following adenoviral genes: E1A (SEQ ID No. 1), E1B-19K (SEQ ID No. 3), E1B-55K (SEQ ID No. 5), DBP (SEQ ID No. 7), pTP (SEQ ID No. 10 or 11), Ad Pol (SEQ ID No. 13 or 14) and E4 0RF6 (SEQ ID No. 16 or 17).

[0015] In certain embodiments, said minimal adenoviral vector additionally contains a L-ITR, a QJ encapsulation signal and a R-ITR. Preferably, said L-ITR comprises the nucleic acid sequence of SEQ ID No. 19, the QJ encapsulation signal comprises the nucleic acid sequence of SEQ ID No. 28 and the R-ITR comprises the nucleic acid sequence of SEQ ID No. 20.

[0016] In certain embodiments, said minimal adenoviral vector contains no adenoviral genes, other than the 7 adenoviral genes encoding the following proteins: E1A-289R (SEQ ID No. 2), E1B-19K (SEQ ID No. 4), E1B-55K (SEQ ID No. 6), DBP (SEQ ID No. 9), pTP (SEQ ID No. 12), Ad Pol (SEQ ID No. 15) and E4 0RF6 (SEQ ID No. 18), or functional variants or splice variants of any of aforementioned proteins, and optionally a L-ITR, a QJ encapsulation signal and a R-ITR. Splice variants of E1A-289R (SEQ ID No. 2) include E1A-243R (SEQ ID No. 29), E1A-217R (SEQ ID No. 30), E1A-171R (SEQ ID No. 31) and E1A-55R (SEQ ID No. 32).

[0017] In certain embodiments, said minimal adenoviral vector contains no adenoviral genes, other than the 7 adenoviral genes encoding the following proteins: E1A-289R (SEQ ID No. 2), SEQ ID No. 29 (E1A- 243R), SEQ ID No. 30 (E1A-217R), SEQ ID No. 31 (E1A-171R), SEQ ID No. 32 (E1A-55R), E1B-19K (SEQ ID No. 4), E1B-55K (SEQ ID No. 6), DBP (SEQ ID No. 9), pTP (SEQ ID No. 12), Ad Pol (SEQ ID No. 15) and E4 0RF6 (SEQ ID No. 18), or functional variants of any of aforementioned proteins, and optionally a L- ITR, a QJ encapsulation signal and a R-ITR.

[0018] In certain embodiments, said minimal adenoviral vector comprises a nucleic acid encoding a cargo gene to be expressed in said host cell. Preferably, said cargo gene that is to be expressed in said host cell encodes a therapeutically active polypeptide.

[0019] In certain embodiments, said minimal adenoviral vector is used in medicine. In preferred embodiments, said use in medicine is the treatment of a tumor or cancer.

[0020] In certain embodiments, said minimal adenoviral vector is used for the expression of a cargo gene in a host cell.

[0021] Figure legends

[0022] Figure 1 shows the number of viral genomes per HEK293 cells 4 hours and 72 hours after transduction with a gutless HAdV5 at an MOI of 1 and additionally with different plasmids carrying various adenoviral genes.

[0023] Figure 2 shows the magnitude of amplification of selected constructs after 72 hours. The underlying data are the same as in Figure 1.

[0024] Figure 3 shows the magnitude of amplification of viral genomes in A549 cells 72 hours after transduction with a gutless HAdV5 at an MOI of 1 and additionally with different plasmids carrying various adenoviral genes.

[0025] Figure 4 shows results based on experiments similar to those shown in Figure 3, but with different adenoviral genes.

[0026] Figure 5 shows that the minimal replication system leads to an efficient and quantifiable expression of the payload. It also shows that E4 ORF6 significantly contributes to an efficient payload expression.

[0027] Figure 6 shows results of the quantification of viral genomes per cell in HEK293T cells transfected with a minimal replication system with or without a co-transfection of E4 ORF6.

[0028] Figure 7 shows results of the quantification of GFP expression in HEK293T cells transfected with a minimal replication system with or without a co-transfection of E4 ORF6.

[0029] Figure 8 depicts the three different HC-HAdVs used in Example 7 to confirm cis-replication and increased reporter expression of the minimal adenoviral vectors of the present invention.

[0030] Figure 9 shows that while the conventionally (HV-based) produced HC-HAdV (GFP) expressed only the transgene GFP, but neither El, DBP or Fiber, transduction of BT-474 cells with replication-competent HAdV (GFP) results in expression of the GFP payload, along with the three E1A isoforms, DBP and the late protein Fiber. Transduction of BT-474 cells with HC-HAdV 3 (encoding the minimal replication system), confirms functional expression of the components of the minimal replication systems, such as E1A and DBP. Most importantly, no late gene expression (Fiber) is induced.

[0031] Figures 10 and 11 demonstrate the striking increase of payload expression due to AdV genome replication utilizing the minimal vector system of the present inventions as measured in Example 7.

[0032] Figures 12 and 13 demonstrate that the minimal replication system strongly boosts payload expression in El-complementing HEK293T, with no further increase by trans-complementation.

[0033] Definitions

[0034] The term "adenovirus" as used herein refers to any adenovirus, i.e. to human and non-human serotypes. The human isolates are classified into subgroups A-G. The term adenovirus of the present disclosure may refer to adenovirus subtype 5 and recombinant derivatives thereof, including the variant "HAdV-C5". HAdV-C5 includes modified version of the virus, such as one or more of the 4 mutations in the HVR7 (I421G, T423N, E424S and L426Y) of the hexon protein (Schmid et al. (2018). Nature Communications, 9(1), Article 1). The adenovirus according to the present disclosure also refers to a recombinant and infectious virus-like particle and / or a viral capsid and a packaged DNA genome. Said packaged DNA genome may comprise a sequence coding for a helper virus or a vector.

[0035] The terms "helper virus", "helper-virus" and "defective helper virus" refer to a virus defective in its replication and / or packaging as an infectious particle. The term also refers to a defective adenovirus and more specifically adenovirus serotype 5. The defective helper virus genome lacks at least one functional gene. One gene defect of a helper virus may be an E1 / E3 gene, which is required for viral replication. Another gene defect of a helper virus may comprise a packaging signal comprising recombination sites that can be site-specifica lly inactivated by excision using Cre recombinase.

[0036] The term "vector" as used herein in its most general meaning refers to any intermediate vehicles for a nucleic acid which, for example, enables said nucleic acid to be introduced into prokaryotic and / or eukaryotic host cells and, where appropriate, to be integrated into a genome. Such vectors are preferably replicated and / or expressed in the cell. Vectors comprise plasmids, phagemids, free nucleic acid molecules, virus-like particles (VLPs), enveloped delivery vehicles (EDVs), lipid nanoparticles (LNPs), adeno-associated viruses (AAVs) and adenoviruses. The term may also refer to helperdependent adenovirus vectors. A "minimal adenoviral vector" as used in the present disclosure refers to a vector which comprises only those adenoviral elements that are essential for the vector to replicate its own genome in a host cell, but not to produce new virions. A minimal adenoviral vector may however contain additional elements as cargo.

[0037] The terms "helper-dependent adenoviral vector(s)", "HdAd vector(s)", "high-capacity adenoviral vector(s)", "HC-HAdV(s)" and "gutless adenoviral vector" refer to a vector construct comprising adenoviral nucleotide sequences and optionally, one or more heterologous nucleotide sequences. A HdAd vector may comprise adenoviral nucleotide sequences that lack any homology to the helper adenovirus nucleic acid sequences. The HdAd vector may also comprise a functional packaging signal sequence without loxP recombination sites. The HdAd may encode a replication-defective adenovirus and may be engineered to comprise a mutated adenovirus genome by, e.g., introducing one or more mutations in an adenovirus genome (e.g., introducing deletions in one or more coding regions for adenoviral proteins).

[0038] The terms "virus-like particle" and "VLP" refers to a structure that in at least one attribute resembles a virus, but which has not been demonstrated to be able to propagate by replication in a host cell, as it contains no viral genetic material. A VLP may be a nonreplicating, non-infectious viral shell that contains a viral capsid but lacks all of the viral genome, in particular, the replicative components of the viral genome. VLPs are generally composed of one or more viral proteins, such as, but not limited to those proteins referred to as capsid, coat, shell, surface, and structural proteins. A VLP may also be infectious, representing infectious particles depending on receptors binding to a target cell for specific uptake.

[0039] The term "cell line" refers to an immortalized and transformed mammalian cell population that allows for continuous culture. A cell line may be a human cell line. One exemplary cell line is cell line HEK-293 (ATCC: CRL-1573), a derivative thereof, such as the cell line 293-F (Thermo Fischer), or a cell line encoding the adenoviral E1 region. A cell line may be able to grow in suspension culture in a costefficient chemically defined and animal component-free (CDACF) culture medium.

[0040] The term "transfection" refers to a process of deliberately introducing naked or purified nucleic acids into eukaryotic cells. Transfection may be carried out by physical, chemical, or viral methods. Physical methods may comprise electroporation, sonoparation, magnetofection or microinjection. Chemical methods of transfection may comprise calcium phosphate-mediated uptake, cationic polymer-mediated uptake or lipofection. Viral methods may comprise infection of cells with viral particles as a carrier for the genetic information to be transduced. More preferably, viral transfection (or adenofection) is achieved by transduction of cells with adenoviral vectors such as helperadenoviruses.

[0041] The term "expression cassette" as used herein refers to a nucleotide sequence capable of directing the transcription and translation of a heterologous coding sequence and the heterologous coding sequence to be expressed. An expression cassette comprises a regulatory element operably linked to a heterologous coding sequence to achieve expression of the protein product encoded by said heterologous coding sequence in the cell.

[0042] As used herein, the term "operably linked" refers to a linkage of polynucleotide elements in a functional relationship. A nucleic acid sequence is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. For instance, a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the coding sequence. Operably linked means that the nucleotide sequences being linked are typically contiguous. However, as enhancers generally function when separated from the promoter by several kilobases and intronic sequences may be of variable lengths, some polynucleotide elements may be operably linked but not directly flanked and may even function in trans from a different allele or chromosome.

[0043] The term "recombinant" as used in recombinant protein, recombinant protein domain and the like, means that said polypeptides are produced by the use of recombinant DNA technologies well known by the practitioner skilled in the relevant art. For example, a recombinant DNA molecule (e.g., produced by gene synthesis) encoding a polypeptide can be cloned into a bacterial expression plasmid (e.g. pQE30, Qiagen). When such a constructed recombinant expression plasmid is inserted into a host cell (e.g. E. coli), this host cell can produce the polypeptide encoded by this recombinant DNA. The correspondingly produced polypeptide is called a recombinant polypeptide.

[0044] The term "gene" as used herein refers to a particular nucleic acid sequence which is responsible for producing one or more cellular products such as polypeptides and / or RNA and / or for achieving one or more intercellular or intracellular functions. More specifically, said term relates to a nucleic acid section (typically DNA; but RNA in the case of RNA viruses) which comprises a nucleic acid coding for a specific protein or a functional or structural RNA molecule.

[0045] The term "cargo gene" as used herein refers to a nucleic acid sequence encoded on the minimal adenoviral vector. The cargo gene is not essential for replication of the minimal adenoviral vector in the host cell. The cargo gene is typically a gene encoding a therapeutically active polypeptide. The cargo gene may however be of other nature, such as a marker gene or a reporter gene.

[0046] The term "cargo capacity" as used herein refers to the maximum size of the cargo gene(s) contained on the minimal adenoviral vector. The term "infectious particles" or "infectious viral particles" as used herein refers to virus-like particles that are able to deliver a nucleic acid payload into a cell. In contrast to chemical transfection, infectious particles can enter the cell via a receptor-mediated uptake process.

[0047] As used herein, the term "E1A" (Early E1A protein) refers to the gene or its encoded protein(s) which plays a role in viral genome replication by driving entry of quiescent cells into the cell cycle. E1A protein has both transforming and trans-activating activities. The wild-type E1A proteins are encoded by the following nucleic acid sequence:

[0048] ATGAGACATATTATCTGCCACGGAGGTGTTATTACCGAAGAAATGGCCGCCAGTCTTTTGGAC CAGCTGATCGAAGAGGTACTGGCTGATAATCTTCCACCTCCTAGCCATTTTGAACCACCTACC CTTCACGAACTGTATGATTTAGACGTGACGGCCCCCGAAGATCCCAACGAGGAGGCGGTTTCG CAGATTTTTCCCGACTCTGTAATGTTGGCGGTGCAGGAAGGGATTGACTTACTCACTTTTCCG CCGGCGCCCGGTTCTCCGGAGCCGCCTCACCTTTCCCGGCAGCCCGAGCAGCCGGAGCAGAGA GCCTTGGGTCCGGTTTCTATGCCAAACCTTGTACCGGAGGTGATCGATCTTACCTGCCACGAG GCTGGCTTTCCACCCAGTGACGACGAGGATGAAGAGGGTGAGGAGTTTGTGTTAGATTATGTG GAGCACCCCGGGCACGGTTGCAGGTCTTGTCATTATCACCGGAGGAATACGGGGGACCCAGAT ATTATGTGTTCGCTTTGCTATATGAGGACCTGTGGCATGTTTGTCTACAGTAAGTGAAAATTA TGGGCAGTGGGTGATAGAGTGGTGGGTTTGGTGTGGTAATTTTTTTTTTAATTTTTACAGTTT TGTGGTTTAAAGAATTTTGTATTGTGATTTTTTTAAAAGGTCCTGTGTCTGAACCTGAGCCTG AGCCCGAGCCAGAACCGGAGCCTGCAAGACCTACCCGCCGTCCTAAAATGGCGCCTGCTATCC TGAGACGCCCGACATCACCTGTGTCTAGAGAATGCAATAGTAGTACGGATAGCTGTGACTCCG GTCCTTCTAACACACCTCCTGAGATACACCCGGTGGTCCCGCTGTGCCCCATTAAACCAGTTG CCGTGAGAGTTGGTGGGCGTCGCCAGGCTGTGGAATGTATCGAGGACTTGCTTAACGAGCCTG GGCAACCTTTGGACTTGAGCTGTAAACGCCCCAGGCCATAA ( SEQ ID No . 1 )

[0049] Alternative splicing of the ElA-encoding nucleic acid sequence results in 5 wild-type E1A protein isoforms (E1A-289R, E1A-243R, E1A-217R, E1A-171R, and E1A-55R) that have the following amino acid sequences:

[0050] E1A-289R:

[0051] MRHI ICHGGVITEEMAASLLDQLIEEVLADNLPPPSHFEPPTLHELYDLDVTAPEDPNEEAVS QI FPDSVMLAVQEGIDLLTFPPAPGSPEPPHLSRQPEQPEQRALGPVSMPNLVPEVIDLTCHE AGFPPSDDEDEEGEEFVLDYVEHPGHGCRSCHYHRRNTGDPDIMCSLCYMRTCGMFVYSPVSE PEPEPEPEPEPARPTRRPKMAPAILRRPTSPVSRECNSSTDSCDSGPSNTPPEIHPWPLCPI KPVAVRVGGRRQAVECIEDLLNEPGQPLDLSCKRPRP ( SEQ ID No . 2 )

[0052] E1A-243R:

[0053] MRHI ICHGGVITEEMAASLLDQLIEEVLADNLPPPSHFEPPTLHELYDLDVTAPEDPNEEAVS QI FPDSVMLAVQEGIDLLTFPPAPGSPEPPHLSRQPEQPEQRALGPVSMPNLVPEVIDLTCHE AGFPPSDDEDEEGPVSEPEPEPEPEPEPARPTRRPKMAPAILRRPTSPVSRECNSSTDSCDSG PSNTPPEIHPWPLCPIKPVAVRVGGRRQAVECIEDLLNEPGQPLDLSCKRPRP ( SEQ ID No . 29 )

[0054] E1A-217R:

[0055] MRHI ICHGGVITEEMAASLLDQLIEEPEQPEQRALGPVSMPNLVPEVIDLTCHEAGFPPSDDE DEEGEEFVLDYVEHPGHGCRSCHYHRRNTGDPDIMCSLCYMRTCGMFVYSPVSEPEPEPEPEP EPARPTRRPKMAPAILRRPTSPVSRECNSSTDSCDSGPSNTPPEIHPWPLCPIKPVAVRVGG RRQAVECIEDLLNEPGQPLDLSCKRPRP ( SEQ ID No . 30 )

[0056] E1A-171R:

[0057] MRHI ICHGGVITEEMAASLLDQLIEEPEQPEQRALGPVSMPNLVPEVIDLTCHEAGFPPSDDE DEEGPVSEPEPEPEPEPEPARPTRRPKMAPAILRRPTSPVSRECNSSTDSCDSGPSNTPPEIH PWPLCPIKPVAVRVGGRRQAVECIEDLLNEPGQPLDLSCKRPRP ( SEQ ID No . 31 )

[0058] E1A-55R:

[0059] MRHI ICHGGVITEEMAASLLDQLIEEVLCLNLSLSPSQNRSLQDLPAVLKWRLLS ( SEQ ID No . 32 )

[0060] As used herein, the term "E1B-19K" ( E1B-19 kDa protein) refers to the gene or its encoded protein which is a putative adenovirus Bcl-2 homolog that inhibits apoptosis induced by TNF or FAS pathways, as well as p53-mediated apoptosis. Without E1B 19K function, virus production is compromised because of premature death of host cell. Interacts with Bax protein in cell lysates. The wild-type E1B- 19K protein is encoded by the following nucleic acid sequence:

[0061] ATGGAGGCTTGGGAGTGTTTGGAAGATTTTTCTGCTGTGCGTAACTTGCTGGAACAGAGCTCT AACAGTACCTCTTGGTTTTGGAGGTTTCTGTGGGGCTCATCCCAGGCAAAGTTAGTCTGCAGA ATTAAGGAGGATTACAAGTGGGAATTTGAAGAGCTTTTGAAATCCTGTGGTGAGCTGTTTGAT TCTTTGAATCTGGGTCACCAGGCGCTTTTCCAAGAGAAGGTCATCAAGACTTTGGATTTTTCC ACACCGGGGCGCGCTGCGGCTGCTGTTGCTTTTTTGAGTTTTATAAAGGATAAATGGAGCGAA GAAACCCATCTGAGCGGGGGGTACCTGCTGGATTTTCTGGCCATGCATCTGTGGAGAGCGGTT GTGAGACACAAGAATCGCCTGCTACTGTTGTCTTCCGTCCGCCCGGCGATAATACCGACGGAG GAGCAGCAGCAGCAGCAGGAGGAAGCCAGGCGGCGGCGGCAGGAGCAGAGCCCATGGAACCCG AGAGCCGGCCTGGACCCTCGGGAATGA ( SEQ ID No . 3 )

[0062] The wild-type E1B-19K protein has the following amino acid sequence:

[0063] MEAWECLEDFSAVRNLLEQS SNSTSWFWRFLWGSSQAKLVCRIKEDYKWEFEELLKSCGELFD SLNLGHQALFQEKVIKTLDFSTPGRAAAAVAFLSFI KDKWSEETHLSGGYLLDFLAMHLWRAV VRHKNRLLLLSSVRPAI IPTEEQQQQQEEARRRRQEQSPWNPRAGLDPRE ( SEQ ID No .

[0064] 4 )

[0065] As used herein, the term "E1B-55K" (E1B-55 kDa protein) refers to the gene or its encoded protein which plays a major role to prevent cellular inhibition of viral genome replication. It assembles an SCF- like E3 ubiquitin ligase complex based on the cellular proteins ELOB, ELOC, CUL5 and RBX1, in cooperation with viral E4 ORF6. This viral RING-type ligase ubiquitinates cellular substrates and targets them to proteasomal degradation: TP53 / p53, LIG4, MRE11-RAD50-NBS1 (MRN) complex, ITGA3, DAXX and BLM. Degradation of host TP53 / p53 activity is essential for preventing ElA-induced TP53 accumulation that would otherwise lead to cell apoptosis and growth arrest. The wild-type E1B-55K protein is encoded by the following nucleic acid sequence:

[0066] ATGGAGCGAAGAAACCCATCTGAGCGGGGGGTACCTGCTGGATTTTCTGGCCATGCATCTGTG GAGAGCGGTTGTGAGACACAAGAATCGCCTGCTACTGTTGTCTTCCGTCCGCCCGGCGATAAT ACCGACGGAGGAGCAGCAGCAGCAGCAGGAGGAAGCCAGGCGGCGGCGGCAGGAGCAGAGCCC ATGGAACCCGAGAGCCGGCCTGGACCCTCGGGAATGAATGTTGTACAGGTGGCTGAACTGTAT CCAGAACTGAGACGCATTTTGACAATTACAGAGGATGGGCAGGGGCTAAAGGGGGTAAAGAGG GAGCGGGGGGCTTGTGAGGCTACAGAGGAGGCTAGGAATCTAGCTTTTAGCTTAATGACCAGA CACCGTCCTGAGTGTATTACTTTTCAACAGATCAAGGATAATTGCGCTAATGAGCTTGATCTG CTGGCGCAGAAGTATTCCATAGAGCAGCTGACCACTTACTGGCTGCAGCCAGGGGATGATTTT GAGGAGGCTATTAGGGTATATGCAAAGGTGGCACTTAGGCCAGATTGCAAGTACAAGATCAGC AAACTTGTAAATATCAGGAATTGTTGCTACATTTCTGGGAACGGGGCCGAGGTGGAGATAGAT ACGGAGGATAGGGTGGCCTTTAGATGTAGCATGATAAATATGTGGCCGGGGGTGCTTGGCATG GACGGGGTGGTTATTATGAATGTAAGGTTTACTGGCCCCAATTTTAGCGGTACGGTTTTCCTG GCCAATACCAACCTTATCCTACACGGTGTAAGCTTCTATGGGTTTAACAATACCTGTGTGGAA GCCTGGACCGATGTAAGGGTTCGGGGCTGTGCCTTTTACTGCTGCTGGAAGGGGGTGGTGTGT CGCCCCAAAAGCAGGGCTTCAATTAAGAAATGCCTCTTTGAAAGGTGTACCTTGGGTATCCTG TCTGAGGGTAACTCCAGGGTGCGCCACAATGTGGCCTCCGACTGTGGTTGCTTCATGCTAGTG AAAAGCGTGGCTGTGATTAAGCATAACATGGTATGTGGCAACTGCGAGGACAGGGCCTCTCAG ATGCTGACCTGCTCGGACGGCAACTGTCACCTGCTGAAGACCATTCACGTAGCCAGCCACTCT CGCAAGGCCTGGCCAGTGTTTGAGCATAACATACTGACCCGCTGTTCCTTGCATTTGGGTAAC AGGAGGGGGGTGTTCCTACCTTACCAATGCAATTTGAGTCACACTAAGATATTGCTTGAGCCC GAGAGCATGTCCAAGGTGAACCTGAACGGGGTGTTTGACATGACCATGAAGATCTGGAAGGTG CTGAGGTACGATGAGACCCGCACCAGGTGCAGACCCTGCGAGTGTGGCGGTAAACATATTAGG AACCAGCCTGTGATGCTGGATGTGACCGAGGAGCTGAGGCCCGATCACTTGGTGCTGGCCTGC ACCCGCGCTGAGTTTGGCTCTAGCGATGAAGATACAGATTGA ( SEQ ID No . 5 )

[0067] The wild-type E1B-55K protein has the following amino acid sequence:

[0068] MERRNPSERGVPAGFSGHASVESGCETQESPATWFRPPGDNTDGGAAAAAGGSQAAAAGAEP MEPESRPGPSGMNWQVAELYPELRRILTITEDGQGLKGVKRERGACEATEEARNLAFSLMTR HRPECITFQQIKDNCANELDLLAQKYS IEQLTTYWLQPGDDFEEAIRVYAKVALRPDCKYKI S KLVNIRNCCYI SGNGAEVEI DTEDRVAFRCSMINMWPGVLGMDGWIMNVRFTGPNFSGTVFL ANTNLILHGVSFYGFNNTCVEAWTDVRVRGCAFYCCWKGWCRPKSRAS IKKCLFERCTLGIL SEGNSRVRHNVASDCGCFMLVKSVAVIKHNMVCGNCEDRASQMLTCSDGNCHLLKTIHVASHS RKAWPVFEHNILTRCSLHLGNRRGVFLPYQCNLSHTKILLEPESMSKVNLNGVFDMTMKIWKV LRYDETRTRCRPCECGGKHI RNQPVMLDVTEELRPDHLVLACTRAEFGSSDEDTD ( SEQ ID No . 6)

[0069] Of note, the E1B-19 kDa protein and the E1B-55 kDa protein are encoded by overlapping genes.

[0070] As used herein, the term "DBP" (DNA-binding protein) refers to the gene or its encoded protein which plays a role in the elongation phase of viral strand displacement replication by unwinding the template in an ATP-independent fashion, employing its capacity to form multimers. It also enhances the rate of initiation. It assembles in complex with viral pTP, Ad Pol, host NFIA and host POU2F1 / OCT1 on viral origin of replication and covers the whole ssDNA genome during synthesis. The DBP protein is encoded by the following nucleic acid sequence:

[0071] ATGGCTTCTAGAGAAGAGGAGCAGCGCGAAACCACCCCCGAGCGCGGACGCGGTGCGGCGCGA CGTCCCCCAACCATGGAGGACGTGTCGTCCCCGTCCCCGTCGCCGCCGCCTCCCCGGGCCCCC CCAAAAAAGCGGATGAGGCGGCGTATCGAGTCCGAGGACGAGGAAGACTCATCACAAGACGCG CTGGTGCCGCGCACACCCAGCCCGCGGCCATCGACCTCGGCGGCGGATTTGGCCATTGCGCCC AAGAAGAAAAAGAAGCGCCCTTCTCCCAAGCCCGAGCGCCCGCCATCACCAGAGGTAATCGTG GACAGCGAGGAAGAAAGAGAAGATGTGGCGCTACAAATGGTGGGTTTCAGCAACCCACCGGTG CTAATCAAGCATGGCAAAGGAGGTAAGCGCACAGTGCGGCGGCTGAATGAAGACGACCCAGTG GCGCGTGGTATGCGGACGCAAGAGGAAGAGGAAGAGCCCAGCGAAGCGGAAAGTGAAATTACG GTGATGAACCCGCTGAGTGTGCCGATCGTGTCTGCGTGGGAGAAGGGCATGGAGGCTGCGCGC GCGCTGATGGACAAGTACCACGTGGATAACGATCTAAAGGCGAACTTCAAACTACTGCCTGAC CAAGTGGAAGCTCTGGCGGCCGTATGCAAGACCTGGCTGAACGAGGAGCACCGCGGGTTGCAG CTGACCTTCACCAGCAACAAGACCTTTGTGACGATGATGGGGCGATTCCTGCAGGCGTACCTG CAGTCGTTTGCAGAGGTGACCTACAAGCATCACGAGCCCACGGGCTGCGCGTTGTGGCTGCAC CGCTGCGCTGAGATCGAAGGCGAGCTTAAGTGTCTACACGGAAGCATTATGATAAATAAGGAG CACGTGATTGAAATGGATGTGACGAGCGAAAACGGGCAGCGCGCGCTGAAGGAGCAGTCTAGC AAGGCCAAGATCGTGAAGAACCGGTGGGGCCGAAATGTGGTGCAGATTTCCAACACCGACGCA AGGTGCTGCGTGCACGACGCGGCCTGTCCGGCCAATCAGTTTTCCGGCAAGTCTTGCGGCATG TTCTTCTCTGAAGGCGCAAAGGCTCAGGTGGCTTTTAAGCAGATCAAGGCTTTTATGCAGGCG CTGTATCCTAACGCCCAGACCGGGCACGGTCACCTTTTGATGCCACTACGGTGCGAGTGCAAC TCAAAGCCTGGGCACGCGCCCTTTTTGGGAAGGCAGCTACCAAAGTTGACTCCGTTCGCCCTG AGCAACGCGGAGGACCTGGACGCGGATCTGATCTCCGACAAGAGCGTGCTGGCCAGCGTGCAC CACCCGGCGCTGATAGTGTTCCAGTGCTGCAACCCTGTGTATCGCAACTCGCGCGCGCAGGGC GGAGGCCCCAACTGCGACTTCAAGATTTCGGCCCCCGACCTGCTAAACGCATTGGTGATGGTG CGCAGCCTGTGGAGTGAAAACTTCACCGAGCTGCCGCGGATGGTTGTGCCTGAGTTTAAGTGG AGCACTAAACACCAGTATCGCAACGTGTCCCTGCCAGTGGCGCATAGCGATGCGCGGCAGAAC CCCTTTGATTTTTAA ( SEQ ID No . 7 )

[0072] The wild-type DBP protein has the following amino acid sequence:

[0073] MASREEEQRETTPERGRGAARRPPTMEDVSSPSPSPPPPRAPPKKRMRRRIESEDEEDSSQDA LVPRTPSPRPSTSAADLAIAPKKKKKRPSPKPERPPSPEVIVDSEEEREDVALQMVGFSNPPV LIKHGKGGKRTVRRLNEDDPVARGMRTQEEEEEPSEAESEITVMNPLSVPIVSAWEKGMEAAR ALMDKYHVDNDLKANFKLLPDQVEALAAVCKTWLNEEHRGLQLTFTSNKTFVTMMGRFLQAYL QSFAEVTYKHHEPTGCALWLHRCAEIEGELKCLHGS IMINKEHVIEMDVTSENGQRALKEQSS KAKIVKNRWGRNWQI SNTDARCCVHDAACPANQFSGKSCGMFFSEGAKAQVAFKQIKAFMQA LYPNAQTGHGHLLMPLRCECNSKPGHAPFLGRQLPKLTPFALSNAEDLDADL I SDKSVLASVH HPAL IVFQCCNPVYRNSRAQGGGPNCDFKI SAPDLLNALVMVRSLWSENFTELPRMWPEFKW STKHQYRNVSLPVAHSDARQNPFDF ( SEQ ID No . 9 )

[0074] As used herein, the term "pTP" (preterminal protein) refers to the gene or its encoded protein which is covalently bound to the viral DNA that acts as a primer for viral genomic replication by DNA strand displacement. It assembles on the viral origin of replication in an initiation complex with Ad Pol, DBP, host NFIA and host POU2F1 / OCT1. During initiation, the polymerase covalently couples the first dCTP with Ser-580 of pTP. The wild-type pTP protein is encoded by the following nucleic acid sequence:

[0075] ATGGCCTTGAGCGTCAACGATTGCGCGCGCCTGACCGGCCAGAGCGTCCCGACCATGGAGCAC

[0076] TTTTTGCCGCTGCGCAACATCTGGAACCGCGTCCGCGACTTTCCGCGCGCCTCCACCACCGCC GCCGGCATCACCTGGATGTCCAGGTACATCTACGGATATCATCGCCTTATGTTGGAAGATCTC GCCCCCGGAGCCCCGGCCACCCTACGCTGGCCCCTCTACCGCCAGCCGCCGCCGCACTTTTTG GTGGGATACCAGTACCTGGTGCGGACTTGCAACGACTACGTATTTGACTCGAGGGCTTACTCG CGTCTCAGGTACACCGAGCTCTCGCAGCCGGGTCACCAGACCGTTAACTGGTCCGTTATGGCC AACTGCACTTACACCATCAACACGGGCGCATACCACCGCTTTGTGGACATGGATGACTTCCAG TCTACCCTCACGCAGGTGCAGCAGGCCATATTAGCCGAGCGCGTTGTCGCCGACCTAGCCCTG CTTCAGCCGATGAGGGGCTTCGGGGTCACACGCATGGGAGGAAGAGGGCGCCACCTACGGCCA AACTCCGCCGCCGCCGCAGCGATAGATGCAAGAGATGCAGGACAAGAGGAAGGAGAAGAAGAA GTGCCGGTAGAAAGGCTCATGCAAGACTACTACAAAGACCTGCGCCGATGTCAAAACGAAGCC TGGGGCATGGCCGACCGCCTGCGCATTCAGCAGGCCGGACCCAAGGACATGGTGCTTCTGTCG ACCATCCGCCGTCTCAAGACCGCCTACTTTAATTACATCATCAGCAGCACCTCCGCCAGAAAC AACCCCGACCGCCGCCCGCTGCCGCCCGCCACGGTGCTCAGCCTACCTTGCGACTGTGACTGG TTAGACGCCTTTCTCGAGAGGTTTTCCGATCCGGTCGATGCGGACTCGCTCAGGTCCCTCGGC GGCGGAGTACCTACACAACAATTGTTGAGATGCATCGTTAGCGCCGTATCCCTGCCGCATGGC AGCCCCCCGCCAACCCATAACCGGGACATGACGGGCGGCGTCTTCCAACTGCGCCCCCGCGAG AACGGCCGCGCCGTCACCGAGACCATGCGCCGTCGCCGCGGGGAGATGATCGAGCGCTTTGTC GACCGCCTCCCGGTGCGCCGTCGTCGCCGCCGTGTCCCCCCTCCCCCACCGCCGCCAGAAGAA GAAGAAGGGGAGGCCCTTATGGAAGAGGAGATTGAAGAAGAAGAAGAGGCCCCTGTAGCCTTT GAGCGCGAGGTGCGCGACACTGTCGCCGAGCTCATCCGTCTTCTGGAGGAGGAGTTAACCGTG TCGGCGCGCAACTCCCAGTTTTTCAACTTCGCCGTGGACTTCTACGAGGCCATGGAGCGCCTT GAGGCCTTGGGGGATATCAACGAATCCACGTTGCGACGCTGGGTTATGTACTTCTTCGTGGCA GAACACACCGCCACCACCCTCAACTACCTCTTTCAGCGCCTGCGAAACTACGCCGTCTTCGCC CGGCACGTGGAGCTCAATCTCGCGCAGGTGGTCATGCGCGCCCGCGATGCCGAAGGGGGCGTG GTCTACAGCCGCGTCTGGAACGAGGGAGGCCTCAACGCCTTCTCGCAGCTCATGGCCCGCATT TCCAACGACCTCGCCGCCACCGTGGAGCGAGCCGGACGCGGAGATCTCCAGGAGGAAGAGATC GAGCAGTTCATGGCCGAGATCGCCTATCAAGACAACTCAGGAGACGTGCAGGAGATTTTGCGC CAGGCCGCCGTCAACGACACCGAAATTGATTCTGTCGAACTCTCTTTCAGGCTCAAGCTCACC GGGCCCGTCGTCTTCACGCAGAGGCGCCAGATTCAGGAGATCAACCGCCGCGTCGTCGCGTTC GCCAGCAACCTACGCGCGCAGCACCAGCTCCTGCCCGCGCGCGGCGCCGACGTGCCCCTGCCC CCTCTCCCGGCGGGTCCGGAGCCCCCCCTACCTCCGGGGGCTCGCCCGCGTCACCGCTTTTAG ( SEQ ID No . 10 )

[0077] A codon-optimized nucleic acid sequence encoding wild-type pTP protein is shown in the following:

[0078] ATGGCCCTGTCTGTTAACGACTGTGCTCGCCTCACTGGTCAATCCGTGCCTACGATGGAACAT TTCTTGCCTCTGAGAAATATATGGAATCGAGTGCGAGATTTCCCTCGGGCTTCTACTACTGCT GCAGGTATTACATGGATGAGTCGTTATATTTATGGATACCACAGGCTGATGCTTGAGGATTTA GCTCCTGGCGCTCCAGCGACATTGCGTTGGCCTCTTTATAGGCAGCCACCTCCCCACTTCCTG GTCGGCTATCAATATCTTGTAAGGACCTGTAATGATTATGTCTTCGATTCCCGTGCATATTCA CGCCTGCGCTATACTGAACTTTCCCAACCTGGGCATCAAACGGTCAACTGGAGCGTCATGGCT AATTGTACCTATACAATTAATACAGGTGCGTATCATAGGTTCGTAGATATGGACGATTTTCAA AGTACATTGACACAAGTTCAACAAGCGATTCTGGCAGAACGGGTAGTGGCTGATTTGGCACTC CTGCAACCCATGCGCGGTTTTGGCGTGACCAGAATGGGTGGGCGTGGTAGGCATTTAAGACCT AATAGTGCAGCTGCGGCGGCCATTGACGCCAGGGACGCTGGGCAGGAAGAGGGCGAGGAAGAG GTTCCCGTGGAGCGGCTGATGCAGGATTATTATAAGGATTTGAGGAGATGCCAGAATGAGGCT TGGGGTATGGCTGATCGTTTGCGGATACAACAAGCAGGGCCTAAAGATATGGTCCTGCTGAGC ACAATACGGAGACTGAAAACGGCGTATTTCAACTATATTATTTCTTCCACTTCAGCACGCAAT AATCCAGATAGGCGACCCTTGCCCCCTGCTACTGTACTGTCTCTGCCCTGTGATTGCGATTGG CTGGATGCATTCTTGGAACGATTCAGTGACCCAGTTGACGCAGATTCCCTTCGAAGCCTGGGG GGGGGCGTCCCCACCCAGCAGTTGCTGCGGTGTATAGTCAGTGCTGTTTCACTCCCCCACGGA AGTCCTCCTCCCACACACAATCGTGATATGACAGGAGGGGTATTTCAGCTCAGACCTAGAGAA AATGGACGGGCTGTGACAGAAACTATGCGACGGAGAAGGGGCGAAATGATAGAAAGATTCGTC GATAGGCTGCCCGTTCGTAGACGCAGAAGGAGAGTGCCGCCCCCTCCTCCCCCACCCGAAGAG GAAGAGGGAGAAGCTCTGATGGAGGAAGAAATCGAGGAAGAGGAAGAAGCGCCAGTTGCATTC GAACGGGAAGTAAGGGATACCGTGGCGGAACTGATACGGCTGTTGGAAGAAGAACTCACTGTA AGCGCAAGAAATAGCCAATTCTTTAATTTTGCGGTCGATTTCTATGAAGCTATGGAAAGGTTG GAAGCACTGGGCGACATAAATGAGAGCACTCTGAGGCGATGGGTGATGTATTTTTTTGTCGCT GAGCATACTGCGACTACTCTGAATTATCTTTTCCAACGATTGAGGAATTATGCAGTGTTTGCA AGACATGTCGAACTTAACCTGGCCCAAGTCGTGATGAGAGCTCGGGACGCTGAGGGCGGAGTC GTTTATTCCCGGGTGTGGAATGAAGGCGGGCTGAATGCATTTTCCCAACTTATGGCAAGAATA AGCAATGATCTGGCAGCGACTGTCGAAAGAGCGGGCAGAGGCGATTTACAAGAAGAGGAAATT GAACAATTTATGGCAGAAATTGCTTACCAGGATAATTCCGGCGATGTACAAGAAATCTTACGG CAAGCAGCGGTGAATGATACAGAGATAGACTCAGTAGAGTTGAGCTTTCGACTGAAATTGACG GGGCCAGTGGTTTTCACACAACGGAGACAAATACAAGAAATAAATAGGCGGGTGGTGGCCTTC GCTTCAAATTTAAGAGCCCAACATCAACTGCTTCCCGCTCGGGGAGCTGATGTTCCACTTCCG CCACTCCCTGCTGGCCCCGAACCCCCTTTACCCCCAGGTGCCAGACCCAGACACCGGTTC ( SEQ ID No . 11 )

[0079] The wild-type pTP protein has the following amino acid sequence:

[0080] MALSVNDCARLTGQSVPTMEHFLPLRNIWNRVRDFPRASTTAAGITWMSRYI YGYHRLMLEDL APGAPATLRWPLYRQPPPHFLVGYQYLVRTCNDYVFDSRAYSRLRYTELSQPGHQTVNWSVMA NCTYTINTGAYHRFVDMDDFQSTLTQVQQAILAERVVADLALLQPMRGFGVTRMGGRGRHLRP NSAAAAAIDARDAGQEEGEEEVPVERLMQDYYKDLRRCQNEAWGMADRLRIQQAGPKDMVLLS TIRRLKTAYFNYI I SSTSARNNPDRRPLPPATVLSLPCDCDWLDAFLERFSDPVDADSLRSLG GGVPTQQLLRCIVSAVSLPHGSPPPTHNRDMTGGVFQLRPRENGRAVTETMRRRRGEMIERFV DRLPVRRRRRRVPPPPPPPEEEEGEALMEEEIEEEEEAPVAFEREVRDTVAELIRLLEEELTV SARNSQFFNFAVDFYEAMERLEALGDINESTLRRWVMYFFVAEHTATTLNYLFQRLRNYAVFA RHVELNLAQWMRARDAEGGWYSRVWNEGGLNAFSQLMARISNDLAATVERAGRGDLQEEEI EQFMAEIAYQDNSGDVQEILRQAAVNDTEIDSVELS FRLKLTGPWFTQRRQIQEINRRVVAF ASNLRAQHQLLPARGADVPLPPLPAGPEPPLPPGARPRHRF ( SEQ ID No . 12 )

[0081] As used herein, the term "Ad Pol" (Adenovirus polymerase) refers to the gene or its encoded protein which is an eukaryotic-type DNA polymerase involved in viral genomic replication. DNA synthesis is protein primed, and acts in a strand displacement replication. It assembles in complex with viral pTP, DBP, host NFIA and host POU2F1 / OCT1 on viral origin of replication. The polymerase covalently transfers dCMP onto pTP, thereby initiating complementary strand synthesis. The wild-type Ad Pol protein is encoded by the following nucleic acid sequence: ATGGCCTTGGCTCAAGCTCACCGGGCCCGTCGTCTTCACGCAGAGGCGCCAGATTCAGGAGAT

[0082] CAACCGCCGCGTCGTCGCGTTCGCCAGCAACCTACGCGCGCAGCACCAGCTCCTGCCCGCGCG

[0083] CGGCGCCGACGTGCCCCTGCCCCCTCTCCCGGCGGGTCCGGAGCCCCCCCTACCTCCGGGGGC

[0084] TCGCCCGCGTCACCGCTTTTAGATGCATCATCCAAGGACACCCCCGCGGCCCACCGCCCGCCG

[0085] CGCGGTACCGTAGTCGCGCCGCGGGGATGCGGCCTCTTGCAAGCCATCGACGCCGCCACCAAC

[0086] CAGCCCCTGGAAATTAGGTATCACCTGGATCTAGCCCGCGCCCTGACCCGTCTATGCGAGGTA

[0087] AACCTGCAGGAGCTCCCGCCTGACCTGACGCCGCGGGAGCTCCAGACCATGGACAGCTCCCAT

[0088] CTGCGCGATGTTGTCATCAAGCTCCGACCGCCGCGCGCGGACATCTGGACTTTGGGCTCGCGC

[0089] GGCGTGGTGGTCCGATCCACCGTAACTCCCCTCGAGCAGCCAGACGGTCAAGGACAAGCAGCC

[0090] GAAGTAGAAGACCACCAGCCAAACCCGCCAGGCGAGGGGCTCAAATTCCCACTCTGCTTCCTT

[0091] GTGCGCGGTCGTCAGGTCAACCTCGTGCAGGATGTACAGCCCGTGCACCGCTGCCAGTACTGC

[0092] GCACGTTTTTACAAAAGCCAGCACGAGTGTTCGGCCCGTCGCAGGGACTTCTACTTTCACCAC

[0093] ATCAATAGCCACTCCTCCAATTGGTGGCGGGAGATCCAGTTCTTCCCGATCGGCTCGCATCCT

[0094] CGCACCGAGCGTCTCTTTGTCACCTACGATGTAGAGACCTATACTTGGATGGGGGCCTTTGGG

[0095] AAGCAGCTCGTGCCCTTCATGCTGGTCATGAAGTTCGGCGGAGATGAGCCTCTAGTGACTGCC

[0096] GCGCGAGACCTAGCCGCGAACCTTGGATGGGACCGCTGGGAACAAGACCCGCTTACCTTCTAC

[0097] TGCATCACCCCAGAAAAAATGGCCATAGGTCGCCAGTTTAGGACCTTTCGCGACCACCTGCAA

[0098] ATGCTAATGGCCCGTGACCTGTGGAGCTCATTCGTCGCTTCCAACCCTCATCTTGCAGACTGG

[0099] GCCCTTTCAGAGCACGGGCTCAGCTCCCCTGAAGAGCTCACCTACGAGGAACTTAAAAAATTG

[0100] CCTTCCATCAAGGGCATCCCGCGCTTCTTGGAACTTTACATTGTGGGCCACAACATCAACGGC

[0101] TTTGACGAGATCGTGCTCGCCGCCCAGGTAATTAACAACCGTTCCGAGGTGCCGGGACCCTTC

[0102] CGCATCACACGCAACTTTATGCCTCGCGCGGGAAAGATACTCTTCAACGATGTCACCTTCGCC

[0103] CTGCCAAATCCGCGTTCCAAAAAGCGCACGGACTTTTTGCTCTGGGAGCAGGGCGGATGCGAC

[0104] GACACTGACTTCAAATACCAGTACCTCAAAGTCATGGTCAGGGACACCTTTGCGCTCACCCAC

[0105] ACCTCGCTCCGGAAGGCCGCGCAGGCATACGCGCTACCCGTAGAAAAGGGATGCTGCGCCTAC

[0106] CAGGCCGTCAACCAGTTCTACATGCTAGGCTCTTACCGTTCGGAGGCCGACGGGTTTCCGATC

[0107] CAAGAGTACTGGAAAGACCGCGAAGAGTTTGTCCTCAACCGCGAGCTGTGGAAAAAAAAGGGA

[0108] CAGGATAAGTATGACATCATCAAGGAAACCCTGGACTACTGCGCCCTAGACGTGCAGGTCACC

[0109] GCCGAGCTGGTCAACAAGCTGCGCGACTCCTACGCCTCCTTCGTGCGTGACGCGGTAGGTCTC

[0110] ACAGACGCCAGCTTCAACGTCTTCCAGCGTCCAACCATATCATCCAACTCACATGCCATCTTC

[0111] AGGCAGATAGTCTTCCGAGCAGAGCAGCCCGCCCGTAGCAACCTCGGTCCCGACCTCCTCGCT

[0112] CCCTCGCACGAACTATACGATTACGTGCGCGCCAGCATCCGCGGTGGAAGATGCTACCCTACA

[0113] TATCTTGGAATACTCAGAGAGCCCCTCTACGTTTACGACATTTGCGGCATGTACGCCTCCGCG

[0114] CTCACCCACCCCATGCCATGGGGTCCCCCACTCAACCCATACGAGCGCGCGCTTGCCGCCCGC

[0115] GCATGGCAGCAGGCGCTAGACTTGCAAGGATGCAAGATAGACTACTTCGACGCGCGCCTGCTG

[0116] CCCGGGGTCTTTACCGTGGACGCAGACCCCCCGGACGAGACGCAGCTAGACCCCCTACCGCCA

[0117] TTCTGCTCGCGCAAGGGCGGCCGCCTCTGCTGGACCAACGAGCGCCTACGCGGAGAGGTAGCC

[0118] ACCAGCGTTGACCTTGTCACCCTGCACAACCGCGGTTGGCGCGTGCACCTGGTGCCCGACGAG

[0119] CGCACCACCGTCTTTCCCGAATGGCGGTGCGTTGCGCGCGAATACGTGCAGCTAAACATCGCG

[0120] GCCAAGGAGCGCGCCGATCGCGACAAAAACCAAACCCTGCGCTCCATCGCCAAGTTGCTGTCC

[0121] AACGCCCTCTACGGGTCGTTTGCCACCAAGCTTGACAACAAAAAGATTGTCTTTTCTGACCAG

[0122] ATGGATGCGGCCACCCTCAAAGGCATCACCGCGGGCCAGGTGAATATCAAATCCTCCTCGTTT

[0123] TTGGAAACTGACAATCTTAGCGCAGAAGTCATGCCCGCTTTTCAGAGGGAGTACTCACCCCAA

[0124] CAGCTGGCCCTCGCAGACAGCGATGCGGAAGAGAGTGAGGACGAACGCGCCCCCACCCCCTTT

[0125] TATAGCCCCCCTTCAGGAACACCCGGTCACGTGGCCTACACCTACAAACCAATCACCTTCCTT

[0126] GATGCCGAAGAGGGCGACATGTGTCTTCACACCCTGGAGCGAGTGGACCCCCTAGTGGACAAC

[0127] GACCGCTACCCCTCCCACTTAGCCTCCTTCGTGCTGGCCTGGACGCGAGCCTTTGTCTCAGAG

[0128] TGGTCCGAGTTTCTATACGAGGAGGACCGCGGAACACCGCTCGAGGACAGGCCTCTCAAGTCT GTATACGGGGACACGGACAGCCTTTTCGTCACCGAGCGTGGACACCGGCTCATGGAAACCAGA GGTAAGAAACGCATCAAAAAGCATGGGGGAAACCTGGTTTTTGACCCCGAACGGCCAGAGCTC ACCTGGCTCGTGGAATGCGAGACCGTCTGCGGGGCCTGCGGCGCGGATGCCTACTCCCCGGAA TCGGTATTTCTCGCGCCCAAGCTCTACGCCCTCAAAAGTCTGCACTGCCCCTCGTGCGGCGCC TCCTCCAAGGGCAAGCTGCGCGCCAAGGGCCACGCCGCGGAGGGGCTGGACTATGACACCATG GTCAAATGCTACCTGGCCGACGCGCAGGGCGAAGACCGGCAGCGCTTCAGCACCAGCAGGACC AGCCTCAAGCGCACCCTGGCCAGCGCGCAGCCCGGAGCGCACCCCTTCACCGTGACCCAGACT ACGCTGACGAGGACCCTGCGCCCGTGGAAAGACATGACCCTGGCCCGTCTGGACGAGCACCGA CTACTGCCGTACAGCGAAAGCCGCCCCAACCCGCGAAACGAGGAGATATGCTGGATCGAGATG CCGTAG ( SEQ I D No . 13 )

[0129] A codon-optimized nucleic acid sequence encoding wild-type Ad Pol protein is shown in the following:

[0130] ATGGCCTTAGCCCAAGCCCATCGAGCACGCCGGTTACATGCGGAAGCGCCTGACTCTGGTGAC CAGCCACCCAGACGCAGGGTACGGCAACAACCCACTCGTGCTGCTCCTGCCCCCGCAAGGGCA CGAAGAAGACGAGCACCCGCCCCATCACCGGGTGGGTCAGGGGCTCCACCAACCAGTGGCGGA AGTCCTGCAAGCCCCCTGTTGGACGCCAGCAGCAAAGATACTCCTGCAGCCCATAGACCACCC AGGGGCACAGTGGTGGCTCCCCGAGGCTGTGGTCTGCTGCAGGCTATTGATGCGGCTACAAAT CAACCTCTGGAGATCCGTTACCATTTAGACCTGGCACGAGCATTGACTAGGCTGTGTGAAGTG AATTTACAAGAACTGCCCCCGGATTTGACTCCACGTGAACTGCAAACAATGGATTCCAGTCAC TTACGTGACGTAGTTATAAAACTGCGGCCACCCAGAGCAGATATTTGGACACTGGGTTCAAGG GGTGTCGTCGTTCGTTCTACAGTGACACCTCTGGAACAACCGGATGGACAGGGCCAGGCTGCT GAGGTCGAGGATCATCAACCTAATCCCCCTGGTGAAGGTTTGAAGTTTCCGCTGTGTTTTCTC GTCCGGGGCAGACAAGTTAATTTAGTCCAAGACGTTCAACCAGTTCATAGGTGTCAATATTGT GCGCGATTCTATAAGTCCCAACATGAATGCTCTGCGCGGCGGAGAGACTTTTATTTCCATCAT ATTAACTCTCATAGTAGCAACTGGTGGAGAGAAATTCAATTCTTTCCCATAGGTTCTCACCCA CGAACAGAAAGATTGTTCGTTACGTATGACGTCGAAACATACACCTGGATGGGCGCATTCGGA AAACAACTTGTACCTTTTATGCTTGTTATGAAATTTGGTGGGGACGAACCACTTGTAACGGCA GCCCGGGATCTGGCTGCTAATCTCGGGTGGGATCGGTGGGAGCAGGACCCACTGACATTTTAT TGTATTACACCTGAGAAGATGGCAATCGGAAGGCAATTCCGCACATTCAGGGATCATCTCCAG ATGCTGATGGCTAGGGATCTCTGGTCCAGTTTTGTGGCCAGCAATCCACACCTGGCCGATTGG GCACTGAGCGAACATGGACTTTCTAGCCCCGAGGAACTGACCTATGAAGAGTTAAAGAAGCTC CCAAGCATAAAAGGAATACCCCGATTTCTGGAGCTGTATATAGTAGGACATAATATTAATGGT TTCGATGAAATTGTCCTTGCAGCACAAGTTATCAATAATCGCTCTGAAGTCCCCGGCCCTTTT CGTATTACCAGGAATTTCATGCCGCGGGCCGGTAAAATTCTGTTTAATGACGTGACATTTGCA TTGCCTAACCCCCGGAGTAAGAAACGTACCGATTTCCTGCTGTGGGAACAAGGCGGGTGTGAT GATACAGATTTTAAGTATCAATATCTGAAGGTAATGGTGAGAGATACTTTCGCTCTTACTCAT ACAAGCCTTAGAAAAGCTGCCCAAGCTTATGCCTTGCCAGTTGAGAAAGGGTGTTGTGCATAT CAAGCTGTGAATCAATTTTATATGCTGGGAAGTTATCGAAGCGAAGCAGATGGCTTCCCCATT CAGGAAT AT T GGAAGGATCGAGAGGAAT T T GT GT TGAAT CGT GAAT T AT GGAAGAAGAAAGGC

[0131] CAAG AC AAAT AC GAT AT AAT T AAAGAGAC T T T GGAT TATTGTGCTCTGGATGTC CAAGT G AC A GCAGAACTTGTGAATAAATTGCGGGATAGTTATGCATCATTTGTCCGCGATGCCGTCGGGCTG ACTGATGCATCTTTTAATGTGTTTCAACGGCCTACAATCAGCAGCAATTCTCACGCTATATTT AGACAAATTGTGTTTCGTGCCGAACAACCTGCACGCAGTAATTTAGGGCCAGATTTGCTGGCC CCTAGCCATGAGCTTTATGACTATGTTAGAGCATCTATAAGGGGCGGGCGGTGTTATCCCACT TACCTGGGGATTCTGCGAGAACCACTTTATGTCTATGACATCTGTGGGATGTATGCGAGCGCT CTGACGCATCCTATGCCTTGGGGACCACCCCTGAATCCCTATGAAAGAGCATTAGCGGCGAGA GCTTGGCAACAAGCCCTGGATCTGCAGGGCTGTAAAATTGATTATTTTGATGCTCGGCTCCTG CCTGGAGTGTTCACTGTAGATGCTGATCCACCAGATGAAACCCAATTAGATCCATTGCCCCCA

[0132] TTTTGTAGTCGGAAAGGCGGCAGGTTGTGTTGGACAAATGAAAGATTGCGGGGCGAAGTCGCT

[0133] ACTTCAGTAGATTTAGTTACGCTCCATAATCGGGGATGGAGAGTACATTTAGTTCCTGATGAA

[0134] CGAACAACAGTGTTCCCAGAGTGGAGATGTGTGGCACGGGAGTATGTACAACTTAATATTGCC

[0135] GCTAAAGAAAGGGCAGACAGGGATAAGAATCAGACGTTGAGGAGTATTGCAAAACTCCTCTCT

[0136] AATGCGCTTTATGGAAGTTTCGCAACTAAGCTGGATAATAAGAAAATAGTGTTCAGTGACCAA

[0137] ATGGACGCCGCGACTTTGAAGGGGATTACTGCAGGGCAAGTTAATATTAAGTCTAGTAGCTTC

[0138] CTGGAGACAGATAACCTCTCTGCCGAGGTGATGCCAGCCTTCCAACGGGAATATTCCCCACAG

[0139] CAACTTGCGCTGGCCGATTCCGACGCAGAGGAATCTGAAGATGAGAGAGCTCCTACTCCTTTC

[0140] TACTCACCACCCTCTGGCACCCCTGGGCATGTCGCTTATACTTATAAGCCCATTACGTTTCTC

[0141] GACGCTGAGGAAGGGGATATGTGCCTGCATACACTTGAAAGGGTAGATCCTCTTGTCGATAAT

[0142] GATCGGTATCCAAGCCATCTTGCTTCATTTGTCTTGGCATGGACCAGGGCATTCGTGTCCGAA

[0143] TGGTCAGAGTTCCTGTATGAAGAAGATCGAGGGACCCCCTTAGAAGATCGTCCCTTGAAAAGC

[0144] GTGTATGGAGATACCGATTCATTGTTTGTTACAGAACGAGGTCATCGCCTGATGGAGACAAGG

[0145] GGCAAGAAGCGGATTAAGAAACACGGCGGCAATTTAGTATTCGATCCAGAGAGGCCGGAATTG

[0146] ACATGGTTAGTCGAGTGTGAAACTGTGTGTGGTGCTTGTGGAGCCGACGCATATAGCCCTGAG

[0147] TCCGTCTTCCTGGCCCCAAAACTTTATGCACTGAAGAGCCTTCATTGTCCATCTTGTGGCGCA

[0148] AGCAGTAAAGGTAAATTGCGTGCGAAAGGACATGCAGCCGAAGGCCTCGATTACGATACTATG

[0149] GTAAAGTGTTATCTTGCGGATGCCCAAGGTGAGGATAGGCAAAGATTTAGTACATCACGGACT

[0150] AGCTTGAAAAGAACATTGGCTTCTGCACAACCAGGGGCTCATCCTTTTACTGTTACACAAACG

[0151] ACTTTGACCCGGACGCTTCGACCCTGGAAGGATATGACATTGGCACGCCTCGATGAACATAGG

[0152] CTGCTTCCCTATTCTGAGTCACGCCCGAATCCAAGGAATGAAGAAATCTGTTGGATAGAAATG CCTTAA ( SEQ I D No . 14 )

[0153] The wild-type Ad Pol protein has the following amino acid sequence:

[0154] MALAQAHRARRLHAEAPDSGDQPPRRRVRQQPTRAAPAPARARRRRAPAPSPGGSGAPPTSGG

[0155] SPAS PLLDASSKDTPAAHRPPRGTWAPRGCGLLQAIDAATNQPLEIRYHLDLARALTRLCEV

[0156] NLQELPPDLTPRELQTMDSSHLRDWIKLRPPRADIWTLGSRGVWRSTVTPLEQPDGQGQAA

[0157] EVEDHQPNPPGEGLKFPLCFLVRGRQVNLVQDVQPVHRCQYCARFYKSQHECSARRRDFYFHH

[0158] INSHSSNWWREIQFFPIGSHPRTERLFVTYDVETYTWMGAFGKQLVPFMLVMKFGGDEPLVTA

[0159] ARDLAANLGWDRWEQDPLTFYCITPEKMAIGRQFRTFRDHLQMLMARDLWSS FVASNPHLADW

[0160] ALSEHGLSSPEELTYEELKKLPS IKGI PRFLELYIVGHNINGFDEIVLAAQVINNRSEVPGPF

[0161] RITRNFMPRAGKILFNDVTFALPNPRSKKRTDFLLWEQGGCDDTDFKYQYLKVMVRDTFALTH

[0162] TSLRKAAQAYALPVEKGCCAYQAVNQFYMLGSYRSEADGFPIQEYWKDREEFVLNRELWKKKG

[0163] QDKYDI IKETLDYCALDVQVTAELVNKLRDSYASFVRDAVGLTDASFNVFQRPTISSNSHAI F

[0164] RQIVFRAEQPARSNLGPDLLAPSHELYDYVRAS IRGGRCYPTYLGILREPLYVYDICGMYASA

[0165] LTHPMPWGPPLNPYERALAARAWQQALDLQGCKIDYFDARLLPGVFTVDADPPDETQLDPLPP

[0166] FCSRKGGRLCWTNERLRGEVATSVDLVTLHNRGWRVHLVPDERTTVFPEWRCVAREYVQLNIA

[0167] AKERADRDKNQTLRSIAKLLSNALYGSFATKLDNKKIVFSDQMDAATLKGITAGQVNIKS SSF

[0168] LETDNLSAEVMPAFQREYSPQQLALADSDAEESEDERAPTPFYSPPSGTPGHVAYTYKPI TFL

[0169] DAEEGDMCLHTLERVDPLVDNDRYPSHLASFVLAWTRAFVSEWSEFLYEEDRGTPLEDRPLKS

[0170] VYGDTDSLFVTERGHRLMETRGKKRIKKHGGNLVFDPERPELTWLVECETVCGACGADAYSPE

[0171] SVFLAPKLYALKSLHCPSCGASSKGKLRAKGHAAEGLDYDTMVKCYLADAQGEDRQRFSTSRT

[0172] SLKRTLASAQPGAHPFTVTQTTLTRTLRPWKDMTLARLDEHRLLPYSESRPNPRNEEICWIEM P ( SEQ ID No . 15 )

[0173] As used herein, the term "E4 ORF6" (Early 4 ORF6 protein) refers to the gene or its encoded protein which plays a major role to prevent cellular inhibition of viral genome replication by nuclear bodies. It assembles an SCF-like E3 ubiquitin ligase complex based on the cellular proteins ELOB, ELOC, CUL5 and RBX1, in cooperation with viral E1B-55K. The wild-type E4 ORF6 protein is encoded by the following nucleic acid sequence:

[0174] ATGACTACGTCCGGCGTTCCATTTGGCATGACACTACGACCAACACGATCTCGGTTGTCTCGG CGCACTCCGTACAGTAGGGATCGTCTACCTCCTTTTGAGACAGAAACCCGCGCTACCATACTG GAGGATCATCCGCTGCTGCCCGAATGTAACACTTTGACAATGCACAACGTGAGTTACGTGCGA GGTCTTCCCTGCAGTGTGGGATTTACGCTGATTCAGGAATGGGTTGTTCCCTGGGATATGGTT CTAACGCGGGAGGAGCTTGTAATCCTGAGGAAGTGTATGCACGTGTGCCTGTGTTGTGCCAAC ATTGATATCATGACGAGCATGATGATCCATGGTTACGAGTCCTGGGCTCTCCACTGTCATTGT TCCAGTCCCGGTTCCCTGCAGTGTATAGCCGGCGGGCAGGTTTTGGCCAGCTGGTTTAGGATG GTGGTGGATGGCGCCATGTTTAATCAGAGGTTTATATGGTACCGGGAGGTGGTGAATTACAAC ATGCCAAAAGAGGTAATGTTTATGTCCAGCGTGTTTATGAGGGGTCGCCACTTAATCTACCTG CGCTTGTGGTATGATGGCCACGTGGGTTCTGTGGTCCCCGCCATGAGCTTTGGATACAGCGCC TTGCACTGTGGGATTTTGAACAATATTGTGGTGCTGTGCTGCAGTTACTGTGCTGATTTAAGT GAGATCAGGGTGCGCTGCTGTGCCCGGAGGACAAGGCGCCTTATGCTGCGGGCGGTGCGAATC ATCGCTGAGGAGACCACTGCCATGTTGTATTCCTGCAGGACGGAGCGGCGGCGGCAGCAGTTT ATTCGCGCGCTGCTGCAGCACCACCGCCCTATCCTGATGCACGATTATGACTCTACCCCCATG TAG ( SEQ ID No . 16)

[0175] A codon-optimized nucleic acid sequence encoding wild-type E4 ORF6 protein is shown in the following:

[0176] ATGACAACCTCAGGTGTGCCTTTCGGAATGACCCTGCGTCCCACGCGCAGCCGACTGAGCCGC

[0177] AGGACACCCTATTCCCGCGACCGGCTTCCACCGTTCGAAACTGAGACTAGGGCCACTATTCTC GAAGACCACCCTCTTCTTCCTGAGTGCAATACCCTGACCATGCATAATGTATCCTATGTTAGG GGACTGCCTTGTTCAGTTGGCTTCACCCTTATCCAAGAGTGGGTCGTGCCTTGGGATATGGTC TTGACAAGAGAAGAATTGGTCATATTAAGAAAATGCATGCATGTCTGTCTCTGCTGCGCTAAT ATCGACATTATGACATCTATGATGATTCACGGCTATGAATCATGGGCCTTGCATTGCCACTGC AGCTCACCTGGGAGCTTGCAATGCATTGCTGGCGGTCAAGTGCTGGCTTCTTGGTTCCGGATG GTCGTCGATGGAGCGATGTTCAACCAAAGATTCATCTGGTATAGAGAAGTTGTCAACTATAAT ATGCCGAAGGAAGTGATGTTCATGAGCTCAGTCTTCATGAGAGGGAGACATCTCATTTATTTG CGGCTGTGGTACGACGGACATGTTGGCTCCGTTGTGCCAGCAATGTCATTCGGCTATTCTGCT CTGCATTGCGGAATCCTGAATAACATCGTCGTCCTTTGTTGTTCTTATTGCGCAGACCTCAGC GAAATTCGCGTTAGGTGTTGCGCTAGGCGCACTCGGCGTCTGATGCTTAGGGCTGTCCGCATA ATTGCAGAAGAAACGACCGCGATGCTGTACAGTTGTCGTACAGAACGAAGAAGACAACAATTC ATCAGGGCACTTCTCCAACATCATAGGCCAATTCTCATGCATGACTACGATAGCACACCGATG TAA ( SEQ ID No . 17 )

[0178] The wild-type E4 ORF6 protein has the following amino acid sequence:

[0179] MTTSGVPFGMTLRPTRSRLSRRTPYSRDRLPPFETETRATILEDHPLLPECNTLTMHNVSYVR GLPCSVGFTLIQEWWPWDMVLTREELVILRKCMHVCLCCANIDIMTSMMIHGYESWALHCHC SSPGSLQCIAGGQVLASWFRMWDGAMFNQRFIWYREWNYNMPKEVMFMSSVFMRGRHL IYL RLWYDGHVGSWPAMSFGYSALHCGILNNIWLCCSYCADLSEIRVRCCARRTRRLMLRAVRI IAEETTAMLYSCRTERRRQQFIRALLQHHRPILMHDYDSTPM ( SEQ ID No . 18 ) The proteins encoded on the vector system of the present disclosure may additionally comprise a protein tag which may be used to detect expression of the respective proteins. An exemplary protein tag is the FLAG tag (DYKDDDDK - SEQ. ID No. 8).

[0180] The term "packaged" refers to the result of virus genome or vector packaging into viral particles. The packaging may occur randomly in some cases, but may also be guided by the presence of a specific packaging signal in the nucleic acid molecule to be packaged.

[0181] The terms "packaging signal IP" and "encapsulation signal <P" are used interchangeably and refer to a nucleic acid sequence that is required for packaging a given nucleic acid molecule into a virion or VLP. Many viruses comprise a specific packaging signal to prefer packaging of their own genome instead of host nucleic acid molecules. The viral packaging signal sequence is often further designated by the Greek letter Psi (QJ). Adenoviruses comprise packaging signal sequences in their DNA genome for specific packaging into virus-like particles. Recombinant vectors comprising a packaging signal QJ are packaged into viral-like particles. The packaging domain of HAdV-C5 is located between 220 and 400 bp of the viral genome and consists of seven AT-rich repeats, referred to as A-repeats. An exemplary encapsulation signal has the following nucleic acid sequence:

[0182] GGTTTTAGGCGGATGTTGTAGTAAATTTGGGCGTAACCGAGTAAGATTTGGCCATTTT CGCGGGAAAACTGAATAAGAGGAAGTGAAATCTGAATAATTTTGTGTTACTCATAGCG CGTAATATTTGTCTAGGGCCGCGGGGACTTTGACCGTTTACGTGGAGACTCGCCCAGG TGTTTTT ( SEQ ID No . 28 )

[0183] The term "inverted terminal repeat" and "ITR" as used herein refer to the art-recognized regions found at the 5' ("L-ITR") and 3' ("R-ITR") termini of the adenoviral genome which function as origins of DNA replication.

[0184] All rAdVs used for genetic engineering of cells share the serotype-specific inverted terminal repeats (ITRs) flanking both sides of the adenovirus genome as one common feature in their genomic composition. It has been established that the ITRs play an important role in adenovirus DNA replication. Dependent on the serotype, the ITRs are approximately 160 bp in length, and sequence comparisons revealed that the first 50 bp from the ends in particular contain sequences that have been well conserved during adenovirus evolution (Ishino et al., 1987; Tolun et al., 1979).

[0185] ITR sequences that find use herein may be full length, wild-type adenoviral ITRs or fragments thereof that retain functional capability, or may be sequence variants of full-length, wild-type adenoviral ITRs that are capable of functioning as origins of replication, adenoviral ITRs useful in the adenoviral vectors of the embodiments provided herein may be derived from any known adenoviral serotype. An exemplary ITR has the following nucleic acid sequence: CATCATCAATAATATACCTTATTTTGGATTGAAGCCAATATGATAATGAGGGGGTGGAGTTTG

[0186] TGACGTGGCGCGGGGCGTGGGAACGGGGCGGGTGACGTAG ( SEQ ID No . 19 )

[0187] Since the R-ITR is the reverse complement of the L-ITR, the respective nucleic acid has the following sequence:

[0188] CTACGTCACCCGCCCCGTTCCCACGCCCCGCGCCACGTCACAAACTCCACCCCCTCATTATCA

[0189] TATTGGCTTCAATCCAAAATAAGGTATATTATTGATGATG ( SEQ ID No . 20 )

[0190] The complete nucleic acid sequence of a genome self-replicating HC-HAdV , minimal adenoviral vector, of the present disclosure is as follows:

[0191] CATCATCAATAATATACCTTATTTTGGATTGAAGCCAATATGATAATGAGGGGGTGGAGTTTG

[0192] TGACGTGGCGCGGGGCGTGGGAACGGGGCGGGTGACGTAGTAGTGTGGCGGAAGTGTGATGTT

[0193] GCAAGTGTGGCGGAACACATGTAAGCGACGGATGTGGCAAAAGTGACGTTTTTGGTGTGCGCC

[0194] GGTGTACACAGGAAGTGACAATTTTCGCGCGGTTTTAGGCGGATGTTGTAGTAAATTTGGGCG

[0195] TAACCGAGTAAGATTTGGCCATTTTCGCGGGAAAACTGAATAAGAGGAAGTGAAATCTGAATA

[0196] ATTTTGTGTTACTCATAGCGCGTAATATTTGTCTAGGGCCGCGGGGACTTTGACCGTTTACGT

[0197] GGAGACTCGCCCAGGTGTTTTTCTCAGGTGTTTTCCGCGTTCCGGGTCAAAGTTGGCGTTTTA

[0198] TTATTATAGTCAGCTGACGTGTAGTGTATTTATACCCGGTGAGTTCCTCAAGAGGCCACTCTT

[0199] GAGTGCCAGCGAGTAGAGTTTTCTCCTCCGAGCCGCTCCGACACCGGGACTGAAAATGAGACA

[0200] TATTATCTGCCACGGAGGTGTTATTACCGAAGAAATGGCCGCCAGTCTTTTGGACCAGCTGAT

[0201] CGAAGAGGTACTGGCTGATAATCTTCCACCTCCTAGCCATTTTGAACCACCTACCCTTCACGA

[0202] ACTGTATGATTTAGACGTGACGGCCCCCGAAGATCCCAACGAGGAGGCGGTTTCGCAGATTTT

[0203] TCCCGACTCTGTAATGTTGGCGGTGCAGGAAGGGATTGACTTACTCACTTTTCCGCCGGCGCC

[0204] CGGTTCTCCGGAGCCGCCTCACCTTTCCCGGCAGCCCGAGCAGCCGGAGCAGAGAGCCTTGGG

[0205] TCCGGTTTCTATGCCAAACCTTGTACCGGAGGTGATCGATCTTACCTGCCACGAGGCTGGCTT

[0206] TCCACCCAGTGACGACGAGGATGAAGAGGGTGAGGAGTTTGTGTTAGATTATGTGGAGCACCC

[0207] CGGGCACGGTTGCAGGTCTTGTCATTATCACCGGAGGAATACGGGGGACCCAGATATTATGTG

[0208] TTCGCTTTGCTATATGAGGACCTGTGGCATGTTTGTCTACAGTAAGTGAAAATTATGGGCAGT

[0209] GGGTGATAGAGTGGTGGGTTTGGTGTGGTAATTTTTTTTTTAATTTTTACAGTTTTGTGGTTT

[0210] AAAGAATTTTGTATTGTGATTTTTTTAAAAGGTCCTGTGTCTGAACCTGAGCCTGAGCCCGAG

[0211] CCAGAACCGGAGCCTGCAAGACCTACCCGCCGTCCTAAAATGGCGCCTGCTATCCTGAGACGC

[0212] CCGACATCACCTGTGTCTAGAGAATGCAATAGTAGTACGGATAGCTGTGACTCCGGTCCTTCT

[0213] AACACACCTCCTGAGATACACCCGGTGGTCCCGCTGTGCCCCATTAAACCAGTTGCCGTGAGA

[0214] GTTGGTGGGCGTCGCCAGGCTGTGGAATGTATCGAGGACTTGCTTAACGAGCCTGGGCAACCT

[0215] TTGGACTTGAGCTGTAAACGCCCCAGGCCATAAGGTGTAAACCTGTGATTGCGTGTGTGGTTA

[0216] ACGCCTTTGTTTGCTGAATGAGTTGATGTAAGTTTAATAAAGGGTGAGATAATGTTTAACTTG

[0217] CATGGCGTGTTAAATGGGGCGGGGCTTAAAGGGTATATAATGCGCCGTGGGCTAATCTTGGTT

[0218] ACATCTGACCTCATGGAGGCTTGGGAGTGTTTGGAAGATTTTTCTGCTGTGCGTAACTTGCTG

[0219] GAACAGAGCTCTAACAGTACCTCTTGGTTTTGGAGGTTTCTGTGGGGCTCATCCCAGGCAAAG

[0220] TTAGTCTGCAGAATTAAGGAGGATTACAAGTGGGAATTTGAAGAGCTTTTGAAATCCTGTGGT

[0221] GAGCTGTTTGATTCTTTGAATCTGGGTCACCAGGCGCTTTTCCAAGAGAAGGTCATCAAGACT

[0222] TTGGATTTTTCCACACCGGGGCGCGCTGCGGCTGCTGTTGCTTTTTTGAGTTTTATAAAGGAT

[0223] AAATGGAGCGAAGAAACCCATCTGAGCGGGGGGTACCTGCTGGATTTTCTGGCCATGCATCTG

[0224] TGGAGAGCGGTTGTGAGACACAAGAATCGCCTGCTACTGTTGTCTTCCGTCCGCCCGGCGATA

[0225] ATACCGACGGAGGAGCAGCAGCAGCAGCAGGAGGAAGCCAGGCGGCGGCGGCAGGAGCAGAGC CCATGGAACCCGAGAGCCGGCCTGGACCCTCGGGAATGAATGTTGTACAGGTGGCTGAACTGT

[0226] ATCCAGAACTGAGACGCATTTTGACAATTACAGAGGATGGGCAGGGGCTAAAGGGGGTAAAGA

[0227] GGGAGCGGGGGGCTTGTGAGGCTACAGAGGAGGCTAGGAATCTAGCTTTTAGCTTAATGACCA

[0228] GACACCGTCCTGAGTGTATTACTTTTCAACAGATCAAGGATAATTGCGCTAATGAGCTTGATC

[0229] TGCTGGCGCAGAAGTATTCCATAGAGCAGCTGACCACTTACTGGCTGCAGCCAGGGGATGATT

[0230] TTGAGGAGGCTATTAGGGTATATGCAAAGGTGGCACTTAGGCCAGATTGCAAGTACAAGATCA

[0231] GCAAACTTGTAAATATCAGGAATTGTTGCTACATTTCTGGGAACGGGGCCGAGGTGGAGATAG

[0232] ATACGGAGGATAGGGTGGCCTTTAGATGTAGCATGATAAATATGTGGCCGGGGGTGCTTGGCA

[0233] TGGACGGGGTGGTTATTATGAATGTAAGGTTTACTGGCCCCAATTTTAGCGGTACGGTTTTCC

[0234] TGGCCAATACCAACCTTATCCTACACGGTGTAAGCTTCTATGGGTTTAACAATACCTGTGTGG

[0235] AAGCCTGGACCGATGTAAGGGTTCGGGGCTGTGCCTTTTACTGCTGCTGGAAGGGGGTGGTGT

[0236] GTCGCCCCAAAAGCAGGGCTTCAATTAAGAAATGCCTCTTTGAAAGGTGTACCTTGGGTATCC

[0237] TGTCTGAGGGTAACTCCAGGGTGCGCCACAATGTGGCCTCCGACTGTGGTTGCTTCATGCTAG

[0238] TGAAAAGCGTGGCTGTGATTAAGCATAACATGGTATGTGGCAACTGCGAGGACAGGGCCTCTC

[0239] AGATGCTGACCTGCTCGGACGGCAACTGTCACCTGCTGAAGACCATTCACGTAGCCAGCCACT

[0240] CTCGCAAGGCCTGGCCAGTGTTTGAGCATAACATACTGACCCGCTGTTCCTTGCATTTGGGTA

[0241] ACAGGAGGGGGGTGTTCCTACCTTACCAATGCAATTTGAGTCACACTAAGATATTGCTTGAGC

[0242] CCGAGAGCATGTCCAAGGTGAACCTGAACGGGGTGTTTGACATGACCATGAAGATCTGGAAGG

[0243] TGCTGAGGTACGATGAGACCCGCACCAGGTGCAGACCCTGCGAGTGTGGCGGTAAACATATTA

[0244] GGAACCAGCCTGTGATGCTGGATGTGACCGAGGAGCTGAGGCCCGATCACTTGGTGCTGGCCT

[0245] GCACCCGCGCTGAGTTTGGCTCTAGCGATGAAGATACAGATTGAGGTACTGAAATGTGTGGGC

[0246] GTGGCTTAAGGGTGGGAAAGAATATATAAGGTGGGGGTCTTATGTAGTTTTGTATCTGTTTTG

[0247] CAGCAGCCGCCGCCGCCATGAGCACCAACTCGTTTGATGGAAGCATTGTGAGCTCATATTTGA

[0248] CAACGCGCATGCCCCCATGGGCCGGGGTGCGTCAGAATGTGATGGGCTCCAGCATTGATGGTC

[0249] GCCCCGTCCTGCCCGCAAACTCTACTACCTTGACCTACGAGACCGTGTCTGGAACGCCGTTGG

[0250] AGACTGCAGCCTCCGCCGCCGCTTCAGCCGCTGCAGCCACCGCCCGCGGGATTGTGACTGACT

[0251] TTGCTTTCCTGAGCCCGCTTGCAAGCAGTGCAGCTTCCCGTTCATCCGCCCGCGATGACAAGT

[0252] TGACGGCTCTTTTGGCACAATTGGATTCTTTGACCCGGGAACTTAATGTCGTTTCTCAGCAGC

[0253] TGTTGGATCTGCGCCAGCAGGTTTCTGCCCTGAAGGCTTCCTCCCCTCCCAATGCGGTTTAAA

[0254] ACATAAATAAAAAACCAGACTCTGTTTGGATTTGGATCAAGCAAGTGTCTTGCTGTCTTTATT

[0255] TAGGGGTTTTGCGCGCGCGGTAGGCCCGGGACCAGCGGTCTCGGTCGTTGAGGGTCCTGTGTA

[0256] TTTTTTCCAGGACGTGGTAAAGGTGACTCTGGATGTTCAGATACATGGGCATAAGCCCGTCTC

[0257] TGGGGTGGAGGTAGCACCACTGCAGAGCTTCATGCTGCGGGGTGGTGTTGTAGATGATCCAGT

[0258] CGTAGCAGGAGCGCTGGGCGTGGTGCCTAAAAATGTCTTTCAGTAGCAAGCTGATTGCCATGA

[0259] GACCATGTAACAGAAATGAGCAGAGAAAACCCAAGAAAGCGAACTGGAGACTTGATGAGTGTG

[0260] TCAAAGATGCTCAGAGTCCAAGGTCCTCTGTGGCTCACTGACTTCAGAAGCCAACCTCCGTTG

[0261] TTCAAGTCACTTGTGAGGTTACTATGCTAGAGCACTAAATATTATCCAGATAGCCCAAAGAGG

[0262] TGAAGGCAGACATGTGGAAGAACCTGGATTTTTGGGCAAGTTGATGAATGCCTCCTGCCCCAT

[0263] ATCAAAGAGAGGTGATAGGAGACAACTTTGTGAATTTGAAATAATGCACCGGGGAAACAGGAA

[0264] AACGTAATGTAAGTCACGCTTCTTGGCTTTTTTCTTGCCATTACCCTACTTGGCCAAGTGCAA

[0265] ATGGGATTTCAATATATCATAAGTATGCATCTATTAATAACAATGCAAGAAAGCTGTACAACT

[0266] GAAGTCTGAGATTTTGTAAGAAACAGAATCTCTGTAAGCATCACCATCCAACAGAACTTCCTG

[0267] AGTTGATGCTGAATCATCCCAGAAAGAAGGCGCGCCGCTGACACCCAGCCTGGGTGCCGGTGG

[0268] CCAGCTCTGCAGGATCTTCAAGTCTGGGGTGCCACCAGCAAGCGACGGTCCTCCATGGGCTCT

[0269] TCACCTTACGGCAGTGTCCAGAGGCACCGCCAGTCCTCTGCTCCTATGCTGGTCCTGCTGTCC

[0270] CTGGCAAAAGGAGCCAGAGCATTCTCTCCAGGCCTCCCGAGGAGGCTGCTTCCTTTGTTTTGC

[0271] AGATGGAGGCTCCCATCCTCTGTTCTGAATCAATGTGCTCCAAAGATAAGCCCCAAGAAAACA

[0272] GTTGTTGCCTTTTGACACTGACAATTAGAATCGTTGGAAAATGGAGAAAACAGGAAATGGCAA

[0273] ATGGTTTCAGTGACCAGGAGGAAACCGTGCCTGAAAGTTGCTGCTTAGTGACTGGGACACTCG

[0274] CTTTCTGCTCTCTTATGAAGGACAGCCTAGGCCGTGTGGCCTTTTATAAACAAAGCTATGAAG GGGTCGTCAAATTTTCTAGGGCTGCAACTGTGGCACTACGTCCTGTTGTGCCAGGTGACACTG

[0275] ACAAGCAGCACTGAGTTCTATGCAAGCCCAGGTGTGCTTCTCTCATGGTGACCCCCAGAGAAC

[0276] TAAGGCCCAGCTCTTCCTCTGTCACACCCCTCCCAGCCCCCACTGTCAGACAAGGGACCACAT

[0277] TCACAGACAGTCTCAGCCAAGATGGCAACCTTGGAAGTCCTGGGGATGCCTTTCTAGAAGCTT

[0278] TAGGCTGACGCCAGGGAGCTGTAAAGCCCCCACCTGTCCCTGGGGGTTGTGTGCTGGGCAGGA

[0279] GAGAGGGGAAAGAGCCCAAACTCCACCACTGCCTGCTGGCACAACTGAGCCCACGCCAGGCAC

[0280] TGCACCAGCTTCACTCACCAGCTACCACATGCTAATGTCTCCTGTTTACCCAGGTGCAATCAC

[0281] CTGAGTGCTGCTTTCTCACCACTGCATGGGAAGAACGATCTCATTACCAATTCAACCACTTAA

[0282] GCAGGCAAAAGGACTCCAACGGGGGAAAGGCAGAGGACAAACGATTCGGGGACTGAGACAGAT

[0283] GCCCACAAAATGTTCCTGGATTTTAGCCGGATTCGGGCTGAAGTTCCTTGCCCTATGAGCCCT

[0284] TGGAGGAGAGCATCCTGACTCAGACAAGAATTCAAATGACAAGACTTCTGGGGGATGGCAGTG

[0285] AATGGATCCAAAGCCTTGTTTTATAAAGAATGTGGAGGAAGGGAGGAAGGAAGGGAGGGAAGG

[0286] GGAGACTGAGAGGGAGAGAGAATAAAGAAGAGGAAGAGAAGGAAGAGGAGGAGGAGGAAGGGG

[0287] AGGGGAGAGGAAAAAGGGGAGGGTAGAGGAGGAGGGAGAAGGGGAAGGGAGGACAGAAGAGGG

[0288] GAGGGAGCGGGGAGATTATCTACCCCTCCATGCTAGGGAACTCCTGTCTCCTTTTCCAGCAGA

[0289] CACTGCTCCATGAGTGCCCCCACAGAGAGCCAGCCTGCCACACAGCAGAAGGTGCTCAGCTAT

[0290] CTGTCCCAAAGGTTGAGCACTAGTGTATACCACAAGGAAAAAAACCTTCTTTTGTACTGTTTG

[0291] GAAGCCAGAATTAGAAACAACTCGTTCACCCTTCTGACTTTCCCTTTGCAATGACAGGAAGAT

[0292] AGTATACTGCATGGGACCACCACACATTCAAGATCTGAGTTGTGGGGACCCCCACCCCCACAT

[0293] GGGTGGAGCCCCCTGAGACCCGTGGGCTCTGACTGGCCATGGCCCATAGATAGTGACAACATA

[0294] CACACTGCTATCTGAAGCCTAGTTGGAGGACGGATGGATGCTTTTAGGAAAAAATTCCCTTTA

[0295] GTCGTCCCTCAGAAACTACAACGGCTTCCTAAAATGAATGGCAGGCAAGTTTTGATTGTTTCC

[0296] TAAGTTCCCCCTAACTCTCAGAGCCTGTGACTCAGCTTCTCCAGAATGCCTGCAGGGAGGCAG

[0297] GACCACTGGGAGTAATGAGCACGAATTGGGCCACTTCTCCCAGTGGACTGTTAACGCCGAGAT

[0298] CTGGGGTGGAACCCACTGAACACCTGCCCTTTTGCGTTGCCCAGAATGCAGCCAGAGGGATAC

[0299] GGGCCACAGTAACTCTGGGGCCCCTGAACCTACCTTCCCAAGACCCTGACGGGAAGGTATTGC

[0300] TTACATGACAGTCCAGTCAGTCCCTCTGTCACTCAGTTAACAAACATGGTCATGGGTCTACTA

[0301] TGGGCCAGGCTCTGTTCCGGGCATGGGGATAACACCGTGTGTCTCTACTCTTATGGACACAAG

[0302] TAAATCAATCAAAGCAGATAATGTTCTAATGACCACCCAGTAGGGTGAAGGGATGGAGGGTGG

[0303] GCAGGTGCTGGCAGAAACCTAAGGTCAAGTCATGGGACCCCAATGGGGAACTGGAAACTGCCA

[0304] CACCATTTTCTATGTTAGGGGTCTTTCCTTGCCCTTCTAGAGACCTCACCCATGGTAATGATG

[0305] CTGCCCTCAGGTTTGAACTGGTCTGAAGCTGTCACTGGGGGCTTCACAAAATGAATGCCACTG

[0306] AAAGCAAACGGCAGAGATCTGTATATGAAATTCTTTTTACCCTTTGTTTTCTGTCTTCCCACA

[0307] TGATCTATGACTGCTGCAAACTCTATTCTGACTCCTCCTCAGTCTTGGCCGGTCTGGGAAGAA

[0308] CTTGGTTTAGATGCTGGGAATGATCCCGGGCAAAGGAAACAAATATATTTCCAGCTTGCTTTT

[0309] CTTCTTCCCCTGTGGTGCCTGCATCCTCTGTCTCGCACTGACTCACAGGCTGGACTGACAAAG

[0310] TGGCTGTGTTGACAGGGAGGATTTAGTGGATGCCAGGCCTGACCCTCAAAGGCCCTCAGGTGG

[0311] GGCCAATTTCCAAGGTTCTTAGAGAAGATTTGGGGCCACAAATCTGCTCTGCCACTTCACTGC

[0312] TGGGTGAAAGCTGTTGGCTTGAGAAGAGAGAGAATAGGTGAGGGACAATAATTCTCAAGGCAA

[0313] GCTAGCTGATATCCTTTCATTTGTGGCTTCCAAAGACAGGGAAAGGCAAGGCAGACCAGGGTG

[0314] GAAACAGCGACATCCTGAGGGGACGACGTCCGTTTTGCTAATTCTGTTTCCCTGGCAGCTTTG

[0315] TGCTGGACAAGATTTCTCTGGTTTCAGCTGAAGAGTGAACAGCTGAGGAGCTCTGTGACTCTG

[0316] AGAGAGAGAGAAATCAATAGCCCTTGAAAGGAAAGCACTGCTTGTTCTTCCAGGCCAGGGCAG

[0317] GCAGGATAAAGAGAACAAGGCTGTACATACCAAAGCCGAATTTCCATTAAAGGCCAGAATCCT

[0318] GGGAGGCCCTTTCAGCTTAATGACCATCTTCTGATCACTATCACTTAGAAAATGTTCATGTCT

[0319] CCAGTTTTCTCTGCAGATAGTGCAGACATCCTGTGCACATTGATATTTTATGCACCAGATGCA

[0320] ATTCTCTGCAAAGGTCCCCTCTGGCTGGTGTCCATCCGCTTGCTCTCATGACCACTGATGCGA

[0321] CTCTGGGGGCAAGTCAAGCCATTCCTTCTAGACTCTAGAATCTAGAGGATGATCCCAACTCAC

[0322] AGGCCTCTTGGACAAGCTTGCTTTGGATTCCTTCTGCCTGTACTACCCAGACTGCCAGTCCTC

[0323] CAGCAGGGCAATCCAACTTCCTGCCTCTAGGGTGTGGGAGTCCTGGGCGGGGTTTGGAATCCA CAGCAGATGGCTGGCAGGAGAGGCAGTTCCCGAGCACCCACTAGGTGCCAAGCAGGCAGGTAT

[0324] CAGGTGCAGGGCTTCGGAGGTGCAGAAGCACAGCCCCTGTCCCCCTCAGGTTCCCAGTCTGGC

[0325] CTCAGGGACAGACAGGCAGGTAGGTATGGAAACGGGGTACTGGAAGGCATACAGCTGAGGGAG

[0326] AGAGCAGCTGCACCCTGGTCAATCCCCCACTCATTTCCCTGGAGGTGGGGCTGAGGCACCTGG

[0327] AGCACTCTGCTTGCTTGGGTTGTCCCTTTGCCAAAGAATGCCGGGCAGAAAGTAGAGAACAAG

[0328] CAGGCCCAGGAAGCACTAGAAGTCCCTCATGGGTGGACAGAGCTTTACCTTCGGAGCTGTCAC

[0329] CTCCTGTGTTCCTCCCAACAACCCTGTGAGGCAGGCGGGCAGGCTGAGCAGGGGACTGTGCCC

[0330] CGTTGACAGATGAGGAAAGCGAGGCTCAGGGGGAAGTGACTTACCTGTGGCGTCGCAGCTAGG

[0331] CAGTAGTGGAGCGGGCACCTGCCTCCAGGCCTTCGGATCCCAGCTGCCCCAGGGCGCTGCCTC

[0332] TGAGGTGCTGTACGAGGAGGTGACTCGGGCCAGCTCATGAGCAAAGGAGCTGCCGGGCAGGAA

[0333] GGACTCACTCCCCAGGGCCTGGCAGGACGGGGGCTGTCGCCCGAGGCTGACCTGGGGGTGCCT

[0334] GGCTTCCCCAAGCTTGCAGGCTTTGTCTACATGAGTCTACAACAACCGATTGAACAATCCATC

[0335] AGCTGCTTGGCCACAAAATGGTTCTGACTGATCTTGTCACTGCCCTGAGTGTCTAGCTGGGTC

[0336] ATCTGGGTGTTGTGGTTATACCCACCAACTGCTTTGGTGGAACGAAACCAAACAGCTTTGTGA

[0337] TCTTAGCTTGTGCGGCCGCGGGATCATTCCTGGACTCAGATTGTTCTGAAGAAGCCCAGTTCT

[0338] GGGTGGCATCAAGTGCTTGCTAGATGGGGGGCTTGCCTTGATCCGGCTACACTTGGAGGTGAC

[0339] TTGTTCTTGGACGGCTACATACAGAAAGAGAGAAGTGGGGATGAGTTCCAAAGGCATCCTCGA

[0340] CTTGGGCTGTGGCCACCGGAGGGTAGCTCCTGGCCCAACACGGACTTCTCACCTCCCGCCCTT

[0341] GGCTCTCTACTGAGCTCCCCCCTGCTCCCCAGTTCCTCGCCATTCCCCTCATTTCTCTGCCCT

[0342] CAGCCTGGACTGCAGTTCTTCTGGGAAGCTGCCCCAACTCCCTAGGTCTGTGCTCACCAAGAG

[0343] CAGATCACACTGGACTGAAATGCCAGCTGATTTGTCTCTTCAAGAAAATTGGAAGCTCCTGGA

[0344] GGTCAGGGTCCATGTCTGCTTTTACACTCAGTGCTCTGTATGCAGGCCTGGCACTGCCCACCC

[0345] TTTGACAGGTGGTGCATATTTTGTAGAAGGAAGGAAGGGGCCAGGTGGGGTGGGCTGGGCTGG

[0346] TGGCGGGAGCTAGCTCAGCCTCTTAGATTCTCTACCCGATGGATGTGACCTGGGACAGCAAGT

[0347] GAGTGTGGTGAGTGAGTGCAGACGGTGCTTTGTTCCCCTCTTGTCTCATAGCCTAGATGGCCT

[0348] CTGAGCCCAGATCTGGGGCTCAGACAACATTTGTTCAACTGAACGGTAATGGGTTTCCTTTCT

[0349] GAAGGCTGAAATCTGGGAGCTGACATTCTGGACTCCCTGAGTTCTGAAGAGCCTGGGGATGGA

[0350] GAGACACGGAGCAGAAGATGGAAGGTAGAGTCCCAGGTGCCTAAGATGGGGAATACATCTCCC

[0351] CTCATTGTCATGAGAGTCCACTCTAGCTGATATCTACTGTGGCCAATATCTACCGGTACTTTT

[0352] TTGGGGTGGACACTGAGTCATGCAGCAGTCTTATGGTTTACCCAAGGTCAGGTAGGGGAGACA

[0353] GTGCAGTCAGAGCACAAGCCCAGTGTGTCTGACCCACCCAAGAATCCATGCTCGTATCTACAA

[0354] AAATGATTTTTTCTCTTGTAATGGTGCCTAGGTTCTTTTATTATCATGGCATGTGTATGTTTT

[0355] TCAACTAGGTTACAATCTGGCCTTATAAGGTTAACCTCCTGGAGGCCACCAGCCTTCCTGAAA

[0356] CTTGTCTGTGCTGTCCCTGCAACTGGAGTGTGCCTGATGTGGCACTCCAGCCTGGACAAGTGG

[0357] GACACAGACTCCGCTGTTATCAGGCCCAAAGATGTCTTCCATAAGACCAGAAGAGCAATGGTG

[0358] TAGAGGTGTCATGGGCTACAATAAAGATGCTGACCTCCTGTCTGAGGGCAAGCAGCCTCTTCT

[0359] GGCCCTCAGACAAATGCTGAGTGTTCCCAAGACTACCCTCGGCCTGGTCCAATCTCATCCCAC

[0360] TGGTGCGTAAGGGTTGCTGAACTCATGACTTCTTGGCTAGCCTGCAACCTCCACGGAGTGGGA

[0361] ACTACATCAGGCATTTTGCTAACTGCTGTATCCTAGGCCAATAAATGTTGATCACATTTATAG

[0362] CTGCCATGGTAGGGTGGGGACCCCTGCTATCTATCTGTGGAGGCTCTGGGAGCCCCTGACACA

[0363] AACTTTCTGAAGCAGAGCCTCCCCAACCCCTTTTCCATTCCCTATACCTGACAGATGGCCCAG

[0364] GAACCCATTAGAAATGGAAGGTCACTGCAGCAGTATGTGAATGTGCGTGTGGGAGAAGGGCAG

[0365] GATCAGAGCCCTGGGGGTGTGGCAGCCCCCAAGTGATTCTAATCCAGATCCTAGGGTTGTTTC

[0366] CCTGTCCCATTGAAATAGCTGCTTTAAGGGGCCTGACTCAGGGAAATCAGTCTCTTGAATTAA

[0367] GTGGTGATTTTGGAGTCATTTAGACCAGGCCTTCAATTGGGATCCTGCTCTTAGAGTTGGATG

[0368] AATTATTTAACTGATTTTCAGATCTCCTCTTTCTCAATGCTTTCAGAAGCACAGTAACTGCTT

[0369] ACTCTGAAATGAATTCTCACCCCACTTCCACATATGCACCCCTTGCCCACCCCTTTGGGAACA

[0370] CTGGCCTTAACTGCTTACCTTCAAATGGACTCATCTGTTGGGAGATATATGCATTCTGCCGTT

[0371] CAGGGGTCATTGCCATAAGACCTGATCTCTGTTCCTCTTGCTAAACAGAAGATGAAAAAGACA

[0372] AATTAGATTACAGCTACCAATTAATAATTAGCCTTAGGATCGCTGCGTGGGGACCTAGGACTT GGCTTTGGTGCAGCAGAAAGCATGAATAAACACACCAGCATACACTCGCATGCATGCCCCACC CTCTCGAGCAAAATTCCACAGGTATAAATAAAGTAAGATTCTGCACCTGGGTTAAAAACACAA CT GC AACAGCAT AGAAT GGGGCAGGAGAGACAGAAC T TAATAGCAAGAGCAC ACAGAAAAAAG TTTTAGGCATTTTGGATGTCCATCTGCTCAGGATGGGTCAGCAGTGAGATGCGGTCACCAAAA GAACAAATGTAACATTAGGCTGCATTAATAGAAGCAGAGTATGTAGAAGGAGGGAGGTGACAG TCCTATGCTAACTCTGCCTTGGCCAGACTATACCCACAGGAGTCTGGGCATGCCAGTCTCAGG GAGACCCAGACAGACTGGCTGCATTCAGAGGATGGTAAGTAATGAGAGTGGGGATTGGACTTC AAACTACCCAGACAAAGAATGGCTGAGCAAGCCAAGGATGCTGTGGCTGGGGCAGAGCAGACT GTGGGCTATGTAGTGGTGGATACCTAGCCTCTGCAGGGCTGTCATAGGGAAAGGACATTGAGA AGAGGACTGAGGCTTGTTCCTGGTGGTCCTGGCATGAACGGCCAGATGATCACATGGTCAGGT GGACACAGTCTCCAACACTGGGAGTAGCCAAACACTTACTGCCAACCTCCCGCCCTTCTCCTG ACTAGTTGCAGCATAGGCAATTGGGAGGAGCTTCCTGTCTCCATCTGAAAGCTGGCTGGGTGG GCAGGGGGAGGAGCGAGCCAAGTTTCAAGGCCGCAGTTTCAGCACTCAGTCTGGGATCGGCTC AAGGAGCAAAGGGGAAGAACATAGCCAGGAGGGAATAACATGAAGGCCCCCAGACCCAGAAAA GGCATGACTTGCTCTGAGACCCTCAGCCAGTTGGTGTCAGGTTGTGACTCGGATCCAGGTCTG ACTCCCAGTCCAGTGCTTGAAGCCTCACCCCACACAGTGAGGGGAGCCCGGCCATCTCTGCTC AACTGCTGCCATCTCTCTCCCCTTCTCAACCACCAAGGCAGCTCTGTCTGGGAGCACAAGCTC CAAGTCCACTTTCTGGTCTGTGTCCCCCCCAAGATGCCAGAGGACTTGCCTCTACAACACGGG CTGCCCGTGCAGTGCCTGCTTTTCCAGCAAAGGGCTTCTGGGAACCCTTCTCTGCACTCAGTG GGGCTGGTGGGAGTGGGGCGGGGTAGCGACCCAGTGCTTGGGACTGTGCCCGGCTCTCAGGCC TGGCAGCAGTTCCTGGCCTTGGTTCCTGCCAAGGCAGAGAGGACAAACACATGGCACCGGGAA GACTACACCAGAAGCGATTCCACCAGACTGGGGTTTGCTTTTCTATCCCGCCCTTAGCCTGCT TCCTGTCCTGGTCCCTGCCTCCCCCTCCACTGGAGCTGCCATGTGGGCAGTGAGGGGCTGTTT CTCAGCTGCCCTATGGAGCTGCCCTCTCCCTGCCAAAGCATTGGCAAGGCGGCAAGGGGTGGG GGTGGGGATGGGGGGTGGGATCTGCCTTCTCAAGCTCTCATTATACTGAGCACGTCTCACCCA TTATTTTATGTCATCTAGCAACACCCCATGTGGACACTGAGGAGCACGGGGGTCACATGACCA CTGCCCAAGGCCACACCATCCGGATCTGCCTGAGATGGTCAGGGTTGGCAGCCATTTCTGAAG GCAGTCCTTTCGCTTTGGCTCTTCTTGTACCAGTCTCAGGACATCAGGGCAGAAGATCTACAG TCCCCAGCTTACTGATGTGACAGCAGAGGCTCAGAGAGGTTAAATGACTTGCCCAAGGTGACA CGGCTAAGAAGTACAGTATCTCCTAACTGCAGACCAGGTGCTTCTGCTGCTTCTGGGGACAGA TTCCTGCGTGGCTGGCTAGGTCTAAACGGTCCTTAACTCCATCCCCACCGGTTGCTGCATTAG TT T CAT CAAAT AACACAGT T GT ACAGAGGT AGGGGT T CAGGGGCAGGGGCAGAT GGAGGC TGG AGAGTGTGACTAAGGAAACAGCAGGGGAAGTGCGGTAAAGTCCGAAGGGAGGGACGGAAAGAG AAAGCCAAGCCCAGGGGCGTGCCAGACAAAAGGAAAGGCCACGCCGGGGCAGGGCAGGCTTCA GCGGGTGCTGGGGCGTCTTCATCCCGGGAAGCACACATTCCAGAGGACCCCGGAGTCTAATGG AAAAGCTGGCCAGCCTATCACTATGGAAACTGCCAAGGCCACACAGCGCTACGCGTATTTAAA TCGCGAGTGCTGGAGTGTTGAGATACTGTAGTGGTGGGATGCTTAAGTGCTGGGGTGCTGGGT GTTGGGATGCCTAGGTGCTGGGGTGCCGAGATGCTGGAGTACTAGTGTGCTGGGATGCTGAAG TGCTGGGGTCCTGAGATGCTGCAGTGCTAGGGTGCTGAGATTCTGGGCTGCTGGAGTGTTGGG GTGCTGGGATGCTGGAGTGCTGAGATGCTTGGACAATGGGGTGCTGGAATACTATGGTGCTGG GGTGCTGGGGTATTGAGATGCTAGGGTACTGGGATGCTGAAGTGCTGAGATCCTGGAGTGCTG GGCTGCTGGGCCACAGGCTCTTGAATCCATTCGTCTGCCCAGGGGAAGAAACCAGAAGATAAA GAGCTAATGAAGGAGCTTTGGTTGAGAGGGAGGAAGTAATGGAAGGAGCAACATCTTGTGGAG GAGCAGGAGAGAATGGACCTCAGGTTGGGAGAGAGGGCCAGGCTACAGGCCAGAGAGGCAGAA GGATTCCAGCAGAGTGTGGGCTCCAGGAGCCAAGGGGAAACAGGTTTCTGGGAGGAGAGAGTC CAGTACTGCTGAAGTGGCAAGTCCGCTGAGGACCAGGAAGCTTCATTTGGCTTTATGACCAGG AGGAATTTGGAACTGTGACTAGAGTACTTAGGGGGAAGGAGGCAAGACTGGAGCCAGATTGCT CTGGGTTGAGGGGTGAGTGGGAGGTGAAGCAGGGCACTGTCACTCCTTTGAAGGGTGGCAGAG AGCTGGAATTGGTGCTGGATGGGCTGTGGGGTGACAGGGTCATGTGGAAAGCCCCTGGGGGGC ACCTGGAAAAGGAGAAGCTGACAGTACAGTGAGAGGACAGCTAAGGGAAAGCGGAATGGCAGA ACACGCACTGCCAGGAGGAATGAGGATAGGGTCAGGAGTGCCAGGGGCAGTGAGGCCAGCCTG GGGTCAGGTGGCAGGACGTGTCCAGGAAGCTGGTCTGCACTGCAGCCCACACTGGCTCAGCCT TGAGGTTCCCTGTGTGGTTGGGGTAGGAAGTTGAACCCTCTGGGAATGGAAGATGGAACCAGC TCTGCGAGCCAAGCTCAGCTTTTATCTATGGGTCTCTGAGGGCTGGCAGAGCTGAGTGGGGAC AACTGTGATCCGTGAGGCTCTCAGGTTGAGGTGGCCCCTCCGGGAGGGCTTCATTTTCCCAGC GGGTAGGTTCTAAGCAGCAGTGGCTGGGCAGGTGGGTCCAACACAGAGCCAGAGAAGGGTGAA TGGGCCTCCTGGCACCCCACCCCTGCTGCCCCTGAGCTCAGTGATGGAGGGGGACAGCACAGC TGAGCCCAAGTGCTTTGGTGTGGCCCTGAGGGAAAGCTGCAGCCTGCCTGGGGCCTGGCATGG ATGGGACACTTGAGGCAGAGGGACAATAGTGGGCGCTGCAGTGAGGTTGGCTCTTGGAGAGGT TTCCTGAGGAGTGCTGCCTGAGACGGGCAGGGAGAACAGAGACAAAGTTGGTGACAGGGAATG AAAGCTGACTGAAGGACTTTACCCAGACCTATGAGGATATCTCTCTCAGCAGGAAGCAGGAGG GGACTGTGTGAGGACTGGCCAAGAGCTGGAGTGTTGGGAAAATGACTCTTTCTCCGACCCCTC TGTCCTAGCTCTGGCCCCTGGACTGCGGAGGTCTGCTTCCACCCCCATTGGTCGATCGTTGTC CCTTGTCACAGCCATTGAGAATTTTGGCAGGGAGCATGTTCTTAGAGCATTTTTAGGCTCTGC GGGACATAACAGCTCTGCCTCAGAGCACATGCCTTTCTCAGCTCCTGAAAGCCACTGATCAAA TTGGAACATTTTGTACCTTAGGGATGAGGATATCAACTCTCCCAGCCACTTAGAGGGATAAAT GTGATGATGCATTCAATTGTGACTACATCTGATCCCAACTGTTGCTTCAGCTGCTCTCCTATA GCACATGGCGGGAGGCGTGCATCCCAGTAGCTACCTCCCCACTTTTGGGGAGATGTGGTTCCA TCCATGAAACCTGGGTACCCGCCTACCAGGTCCTGGCCTATCAGGTGGCAGGGTCTGGTCAAA GAAGGGCATGTGTGGTCTTCAGCAAGGGAGACAGGACGGTGGTGCAGAGCGTCTAGACCCTCA GGGCAAGTCTCCCCCACACCTGCTCCCGGGGCAGTTGTCTTTGTGACCTCCCATCCCCCTCTG TTTCATCCTCTATAAAATGAGGGGCTGAGCCCCAAAATAACAGGCTTCTTTGCCATGATGCAA AACTGCTGAATCTTTCTTTCTGACACACAAGGCATCGAGCAGCCTCTGAAAGAACCAAAGCCA CTAGCAGGCTTCCTGACTTGGGTTTGTAGGTACTGAATACTCCCTTGAAAAATAAAAACATAG AGGCACTTTTCTCCTGGCTGTTTATTACAGAACGAAGAAAAAACACACTGGCTTGAAACAGAC GCCAGATTTCAAATGTAGAGGTGAAATACGAGGTGGCAATTAAAATGTGATTACAGAAAGTCT GGACACTGAGAAAAGTTTACAGGACAGTGGGTGTGGGTTTTCTATAACAGACACTTAAATATA CAT GAG GAT AAT T GCAGAT AGAAACC AT C AAAGACAAAC C CC AAAT C AAC T AAT AAT GT T TAG AGAT GTTCCCCCC C AAACC ACAGAGC C T TAG AT C AAAAC AAAT AC T GAAAGGC T T T AAAC CAG GAACAGCTCGCCTTAACCCCACGAGGGTGCACACAAGCTGGGCTTTTTCTCTCGGTCTGAATG GTAAAGGGAGGAGGATACTCTAGCTCCTCCAGGTGGATTGCTGAGACAGGGCTCGGCTCACAC ACTGTCTCTGCGCCTCTCCCAAATCTGGAGAACTCTCCCAGCCTCCTGGTAAAGTGTCTCTGT GGGGCACTTAACGATAAAACAGCTTCTGCTGTAAAGCTCATTAGGAAAGAGCTAGCGGAGACT GAAAGGT T C GC AAAAGAGAT T AAGAAT C AC AC AAGG C AAT AGGAT T T T T AGT GAAC AT AG AAA TAAATGGCCAAGTGGTTTTCTATTTGGCATTTGTCAACTTGCACAACAACTCTTGGTCATATC CACATTGCTCATTGCATTAAAACCATAAGCGACTCAGCCACCTAGCTTAACAAGGTATCACTG GAGCAAACAACACGGTCTGCATATTTGTAACATTGTATAATAAACACAAAACAATGCATAGTA AACACAACT CT ACT GAAACAAAAGCCGT CGCT T T AT T TACAAAGT CACAAAAT GAAGTAT AAA TACTTCTGTCATTAATGTTTAGGAAAACCATTTACAAAATTTTCAAATATGTACACGTAGCTT GAAAAATCACCAGCTTTCCATTTTGTCACAGGTAGAGAGAGGGATAAGCATGGGCTGACAACA CCACTCAAATTGTAACGGGAGACAACTGCGGGTATGGATCGACACCACTTCCTAGAGTGATGT CACCATGGGGGTTTCTATGGGCATCCTGCTCAGATTTAAAGTGCCCCAGCATCCTGGGTGACT TGCCCAGAATTCTGGGCTGTGGCATACGCGTGATTCTGCTGGTTCACAAACTGGATGGCCACA ACGCCTTCTCCTGCATCCCGATCTTTGTCCCCCTTTGGCTCTCGTTGTTCACGCTGATGGCAA CCACATTTGGACAGAAGGGAGGAAACACCGCGGGATCCCGGTAAAATACAGCATAGCAAAACT TTAACCTCCAAATCAAGCCTCTACTTGAATCCTTTTCTGAGGGATGAATAAGGCATAGGCATC AGGGGCTGTTGCCAATGTGCATTAGCTGTTTGCAGCCTCACCTTCTTTCATGGAGTTTAAGAT ATAGTGTATTTTCCCAAGGTTTGAACTAGCTCTTCATTTCTTTATGTTTTAAATGCACTGACC T C C C AC AT TCCCTTTT T AGT AAAAT AT T C AGAAAT AAT T T AAAT AC AT C AT T G C AAT GAAAAT AAATGTTTTTTATTAGGCAGAATCCAGATGCTCAAGGCCCTTCATAATATCCCCCAGTTTAGT AGTTGGACTTAGGGAACAAAGGAACCTTTAATAGAAATTGGACAGCAAGAAAGCGAGCTTCTA GCCAATTGCTGCACAGGCGGGGAGGCGGCCCAAAGGGAGATCCGACTCGTCTGAGGGCGAAGG CGAAGACGCGGAAGAGGCCGCAGAGCCGGCAGCAGGCCGCGGGAAGGAAGGTCCGCTGGATTG AGGGCCGAAGGGACGTAGCAGAAGGACGTCCCGCGCAGAATCCAGGTGGCAACACAGGCGAGC AGCCATGGAAAGGACGTCAGCTTCCCCGACAACACCACGGAATTGTCAGTGCCCAACAGCCGA GCCCCTGTCCAGCAGCGGGCAAGGCAGGCGGCGATGAGTTCCGCCGTGGCAATAGGGAGGGGG AAAGCGAAAGTCCCGGAAAGGAGCTGACAGGTGGTGGCAATGCCCCAACCAGTGGGGGTTGCG TCAGCAAACACAGTGCACACCACGCCACGTTGCCTGACAACGGGCCACAACTCCTCATAAAGA GACAGCAACCAGGATTTATACAAGGAGGAGAAAATGAAAGCCATACGGGAAGCAATAGCATGA TACAAAGGCAT T AAAGCAGC GT AT CCACAT AGCGTAAAAGGAGCAACAT AGT T AAGAAT ACCA GTCAATCTTTCACAAATTTTGTAATCCAGAGGTTGATTAGCTTAAGGCATTTCTATCCAACAG ATTTCTTCATTCCTTGGATTCGGGCGTGACTCAGAATAGGGAAGCAGCCTATGTTCATCGAGG CGTGCCAATGTCATATCCTTCCAGGGTCGAAGCGTCCGGGTCAAAGTCGTTTGTGTAACAGTA AAAGGATGAGCCCCTGGTTGTGCAGAAGCCAATGTTCTTTTCAAGCTAGTCCGTGATGTACTA AATCTTTGCCTATCCTCACCTTGGGCATCCGCAAGATAACACTTTACCATAGTATCGTAATCG AGGCCTTCGGCTGCATGTCCTTTCGCACGCAATTTACCTTTACTGCTTGCGCCACAAGATGGA CAATGAAGGCTCTTCAGTGCATAAAGTTTTGGGGCCAGGAAGACGGACTCAGGGCTATATGCG TCGGCTCCACAAGCACCACACACAGTTTCACACTCGACTAACCATGTCAATTCCGGCCTCTCT GGATCGAATACTAAATTGCCGCCGTGTTTCTTAATCCGCTTCTTGCCCCTTGTCTCCATCAGG CGATGACCTCGTTCTGTAACAAACAATGAATCGGTATCTCCATACACGCTTTTCAAGGGACGA TCTTCTAAGGGGGTCCCTCGATCTTCTTCATACAGGAACTCTGACCATTCGGACACGAATGCC CTGGTCCATGCCAAGACAAATGAAGCAAGATGGCTTGGATACCGATCATTATCGACAAGAGGA TCTACCCTTTCAAGTGTATGCAGGCACATATCCCCTTCCTCAGCGTCGAGAAACGTAATGGGC TTATAAGTATAAGCGACATGCCCAGGGGTGCCAGAGGGTGGTGAGTAGAAAGGAGTAGGAGCT CTCTCATCTTCAGATTCCTCTGCGTCGGAATCGGCCAGCGCAAGTTGCTGTGGGGAATATTCC CGTTGGAAGGCTGGCATCACCTCGGCAGAGAGGTTATCTGTCTCCAGGAAGCTACTAGACTTA ATATTAACTTGCCCTGCAGTAATCCCCTTCAAAGTCGCGGCGTCCATTTGGTCACTGAACACT ATTTTCTTATTATCCAGCTTAGTTGCGAAACTTCCATAAAGCGCATTAGAGAGGAGTTTTGCA ATACTCCTCAACGTCTGATTCTTATCCCTGTCTGCCCTTTCTTTAGCGGCAATATTAAGTTGT ACATACTCCCGTGCCACACATCTCCACTCTGGGAACACTGTTGTTCGTTCATCAGGAACTAAA TGTACTCTCCATCCCCGATTATGGAGCGTAACTAAATCTACTGAAGTAGCGACTTCGCCCCGC AATCTTTCATTTGTCCAACACAACCTGCCGCCTTTCCGACTACAAAATGGGGGCAATGGATCT AATTGGGTTTCATCTGGTGGATCAGCATCTACAGTGAACACTCCAGGCAGGAGCCGAGCATCA AAATAATCAATTTTACAGCCCTGCAGATCCAGGGCTTGTTGCCAAGCTCTCGCCGCTAATGCT CTTTCATAGGGATTCAGGGGTGGTCCCCAAGGCATAGGATGCGTCAGAGCGCTCGCATACATC CCACAGATGTCATAGACATAAAGTGGTTCTCGCAGAATCCCCAGGTAAGTGGGATAACACCGC CCGCCCCTTATAGATGCTCTAACATAGTCATAAAGCTCATGGCTAGGGGCCAGCAAATCTGGC CCTAAATTACTGCGTGCAGGTTGTTCGGCACGAAACACAATTTGTCTAAATATAGCGTGAGAA TTGCTGCTGATTGTAGGCCGTTGAAACACATTAAAAGATGCATCAGTCAGCCCGACGGCATCG CGGACAAATGATGCATAACTATCCCGCAATTTATTCACAAGTTCTGCTGTCACTTGGACATCC AGAGCACAATAATCCAAAGTCTCTTTAATTATATCGTATTTGTCTTGGCCTTTCTTCTTCCAT AATTCACGATTCAACACAAATTCCTCTCGATCCTTCCAATATTCCTGAATGGGGAAGCCATCT GCTTCGCTTCGATAACTTCCCAGCATATAAAATTGATTCACAGCTTGATATGCACAACACCCT TTCTCAACTGGCAAGGCATAAGCTTGGGCAGCTTTTCTAAGGCTTGTATGAGTAAGAGCGAAA GTATCTCTCACCATTACCTTCAGATATTGATACTTAAAATCTGTATCATCACACCCGCCTTGT TCCCACAGCAGGAAATCGGTACGTTTCTTACTCCGGGGGTTAGGCAATGCAAATGTCACGTCA TTAAACAGAATTTTACCGGCCCGCGGCATGAAATTCCTGGTAATACGAAAAGGGCCGGGGACT TCAGAGCGATTATTGATAACTTGTGCTGCAAGGACAATTTCATCGAAACCATTAATATTATGT CCTACTATATACAGCTCCAGAAATCGGGGTATTCCTTTTATGCTTGGGAGCTTCTTTAACTCT TCATAGGTCAGTTCCTCGGGGCTAGAAAGTCCATGTTCGCTCAGTGCCCAATCGGCCAGGTGT GGATTGCTGGCCACAAAACTGGACCAGAGATCCCTAGCCATCAGCATCTGGAGATGATCCCTG AATGTGCGGAATTGCCTTCCGATTGCCATCTTCTCAGGTGTAATACAATAAAATGTCAGTGGG TCCTGCTCCCACCGATCCCACCCGAGATTAGCAGCCAGATCCCGGGCTGCCGTTACAAGTGGT TCGTCCCCACCAAATTTCATAACAAGCATAAAAGGTACAAGTTGTTTTCCGAATGCGCCCATC CAGGTGTATGTTTCGACGTCATACGTAACGAACAATCTTTCTGTTCGTGGGTGAGAACCTATG GGAAAGAATTGAATTTCTCTCCACCAGTTGCTACTATGAGAGTTAATATGATGGAAATAAAAG TCTCTCCGCCGCGCAGAGCATTCATGTTGGGACTTATAGAATCGCGCACAATATTGACACCTA TGAACTGGTTGAACGTCTTGGACTAAATTAACTTGTCTGCCCCGGACGAGAAAACACAGCGGA AACTTCAAACCTTCACCAGGGGGATTAGGTTGATGATCCTCGACCTCAGCAGCCTGGCCCTGT CCATCCGGTTGTTCCAGAGGTGTCACTGTAGAACGAACGACGACACCCCTTGAACCCAGTGTC CAAATATCTGCTCTGGGTGGCCGCAGTTTTATAACTACGTCACGTAAGTGACTGGAATCCATT GTTTGCAGTTCACGTGGAGTCAAATCCGGGGGCAGTTCTTGTAAATTCACTTCACACAGCCTA GTCAATGCTCGTGCCAGGTCTAAATGGTAACGGATCTCCAGAGGTTGATTTGTAGCCGCATCA ATAGCCTGCAGCAGACCACAGCCTCGGGGAGCCACCACTGTGCCCCTGGGTGGTCTATGGGCT GCAGGAGTATCTTTGCTGCTGGCGTCCAACAGGGGGCTTGCAGGACTTCCGCCACTGGTTGGT GGAGCCCCTGACCCACCCGGTGATGGGGCGGGTGCTCGTCTTCTTCGTGCCCTTGCGGGGGCA GGAGCAGCACGAGTGGGTTGTTGCCGTACCCTGCGTCTGGGTGGCTGGTCACCAGAGTCAGGC GCTTCCGCATGTAACCGGCGTGCTCGATGGGCTTGGGCTAAGGCCATTGGGCCGGGATTTTCC TCCACGTCCCCGCATGTTAGAAGACTTCCCCTGCCCTCGCCGGAGCCGAACCGGTGTCTGGGT CTGGCACCTGGGGGTAAAGGGGGTTCGGGGCCAGCAGGGAGTGGCGGAAGTGGAACATCAGCT CCCCGAGCGGGAAGCAGTTGATGTTGGGCTCTTAAATTTGAAGCGAAGGCCACCACCCGCCTA TTTATTTCTTGTATTTGTCTCCGTTGTGTGAAAACCACTGGCCCCGTCAATTTCAGTCGAAAG CTCAACTCTACTGAGTCTATCTCTGTATCATTCACCGCTGCTTGCCGTAAGATTTCTTGTACA TCGCCGGAATTATCCTGGTAAGCAATTTCTGCCATAAATTGTTCAATTTCCTCTTCTTGTAAA TCGCCTCTGCCCGCTCTTTCGACAGTCGCTGCCAGATCATTGCTTATTCTTGCCATAAGTTGG GAAAATGCATTCAGCCCGCCTTCATTCCACACCCGGGAATAAACGACTCCGCCCTCAGCGTCC CGAGCTCTCATCACGACTTGGGCCAGGTTAAGTTCGACATGTCTTGCAAACACTGCATAATTC CT CAAT CGT T GGAAAAGAT AAT T CAGAGT AGT CGCAGTAT GCT CAGCGACAAAAAAATAC AT C ACCCATCGCCTCAGAGTGCTCTCATTTATGTCGCCCAGTGCTTCCAACCTTTCCATAGCTTCA TAGAAATCGACCGCAAAATTAAAGAATTGGCTATTTCTTGCGCTTACAGTGAGTTCTTCTTCC AACAGCCGTATCAGTTCCGCCACGGTATCCCTTACTTCCCGTTCGAATGCAACTGGCGCTTCT TCCTCTTCCTCGATTTCTTCCTCCATCAGAGCTTCTCCCTCTTCCTCTTCGGGTGGGGGAGGA GGGGGCGGCACTCTCCTTCTGCGTCTACGAACGGGCAGCCTATCGACGAATCTTTCTATCATT TCGCCCCTTCTCCGTCGCATAGTTTCTGTCACAGCCCGTCCATTTTCTCTAGGTCTGAGCTGA AATACCCCTCCTGTCATATCACGATTGTGTGTGGGAGGAGGACTTCCGTGGGGGAGTGAAACA GCACTGACTATACACCGCAGCAACTGCTGGGTGGGGACGCCCCCCCCCAGGCTTCGAAGGGAA TCTGCGTCAACTGGGTCACTGAATCGTTCCAAGAATGCATCCAGCCAATCGCAATCACAGGGC AGAGACAGTACAGTAGCAGGGGGCAAGGGTCGCCTATCTGGATTATTGCGTGCTGAAGTGGAA GAAATAATATAGTTGAAATACGCCGTTTTCAGTCTCCGTATTGTGCTCAGCAGGACCATATCT TTAGGCCCTGCTTGTTGTATCCGCAAACGATCAGCCATACCCCAAGCCTCATTCTGGCATCTC CTCAAATCCTTATAATAATCCTGCATCAGCCGCTCCACGGGAACCTCTTCCTCGCCCTCTTCC TGCCCAGCGTCCCTGGCGTCAATGGCCGCCGCAGCTGCACTATTAGGTCTTAAATGCCTACCA CGCCCACCCATTCTGGTCACGCCAAAACCGCGCATGGGTTGCAGGAGTGCCAAATCAGCCACT ACCCGTTCTGCCAGAATCGCTTGTTGAACTTGTGTCAATGTACTTTGAAAATCGTCCATATCT ACGAACCTATGATACGCACCTGTATTAATTGTATAGGTACAATTAGCCATGACGCTCCAGTTG ACCGTTTGATGCCCAGGTTGGGAAAGTTCAGTATAGCGCAGGCGTGAATATGCACGGGAATCG AAGACAT AAT CAT T ACAGGT CCT T ACAAGATAT T GAT AGCCGACCAGGAAGT GGGGAGGT GGC TGCCTATAAAGAGGCCAACGCAATGTCGCTGGAGCGCCAGGAGCTAAATCCTCAAGCATCAGC CTGTGGTATCCATAAATATAACGACTCATCCATGTAATACCTGCAGCAGTAGTAGAAGCCCGA GGGAAATCTCGCACTCGATTCCATATATTTCTCAGAGGCAAGAAATGTTCCATCGTAGGCACG GATTGACCAGTGAGGCGAGCACAGTCGTTAACAGACAGGGCCATGGTGGGTAGGTCGAAAGGC CCGGAGATGAGGAAGAGGAGAACAGCGCGGCAGACGTGCGCTTTTGAAGCGTGCAGAATGCCG GGCCTCCGGAGGACCTTCGGGCGCCCGCCCCGCCCCTGAGCCCGCCCCTGAGCCCGCCCCCGG ACCCACCCCTTCCCAGCCTCTGAGCCCAGAAAGCGAAGGAGCAAAGCTGCTATTGGCCGCTGC CCCAAAGGCCTACCCGCTTCCATTGCTCAGCGGTGCTGTCCATCTGCACGAGACTAGTGAGAC GTGCTACTTCCATTTGTCACGTCCTGCACGACGCGAGCTGCGGGGCGGGGGGGAACTTCCTGA CTAGGGGAGGAGTAGAAGGTGGCGCGAAGGGGCCACCAAAGAACGGAGCCGGTTGGCGCCTAC CGGTGGATGTGGAATGTGTGCGAGGCCAGAGGCCACTTGTGTAGCGCCAAGTGCCCAGCGGGG CTGCTAAAGCGCATGCTCCAGACTGCCTTGGGAAAAGCGCCTCCCCTACCCGGTAGAAATAGG GCCCCTTGCCATAGAGCCCACCGCATCCCCAGCATGCCTGCTATTGTCTTCCCAATCCTCCCC CTTGCTGTCCTGCCCCACCCCACCCCCCAGAATAGAATGACACCTACTCAGACAATGCGATGC AATTTCCTCATTTTATTAGGAAAGGACAGTGGGAGTGGCACCTTCCAGGGTCAAGGAAGGCAC GGGGGAGGGGCAAACAACAGATGGCT GGCAACT AGAAGGCACAAT CGAT T T AAAAAT CAAAGG GGTTCTGCCGCGCATCGCTATGCGCCACTGGCAGGGACACGTTGCGATACTGGTGTTTAGTGC TCCACTTAAACTCAGGCACAACCATCCGCGGCAGCTCGGTGAAGTTTTCACTCCACAGGCTGC GCACCATCACCAATGCGTTTAGCAGGTCGGGGGCCGAAATCTTGAAGTCGCAGTTGGGGCCTC CGCCCTGCGCGCGCGAGTTGCGATACACAGGGTTGCAGCACTGGAACACTATCAGCGCCGGGT GGTGCACGCTGGCCAGCACGCTCTTGTCGGAGATCAGATCCGCGTCCAGGTCCTCCGCGTTGC TCAGGGCGAACGGAGTCAACTTTGGTAGCTGCCTTCCCAAAAAGGGCGCGTGCCCAGGCTTTG AGTTGCACTCGCACCGTAGTGGCATCAAAAGGTGACCGTGCCCGGTCTGGGCGTTAGGATACA GCGCCTGCATAAAAGCCTTGATCTGCTTAAAAGCCACCTGAGCCTTTGCGCCTTCAGAGAAGA ACATGCCGCAAGACTTGCCGGAAAACTGATTGGCCGGACAGGCCGCGTCGTGCACGCAGCACC TTGCGTCGGTGTTGGAAATCTGCACCACATTTCGGCCCCACCGGTTCTTCACGATCTTGGCCT TGCTAGACTGCTCCTTCAGCGCGCGCTGCCCGTTTTCGCTCGTCACATCCATTTCAATCACGT GCTCCTTATTTATCATAATGCTTCCGTGTAGACACTTAAGCTCGCCTTCGATCTCAGCGCAGC GGTGCAGCCACAACGCGCAGCCCGTGGGCTCGTGATGCTTGTAGGTCACCTCTGCAAACGACT GCAGGTACGCCTGCAGGAATCGCCCCATCATCGTCACAAAGGTCTTGTTGCTGGTGAAGGTCA GCTGCAACCCGCGGTGCTCCTCGTTCAGCCAGGTCTTGCATACGGCCGCCAGAGCTTCCACTT GGTCAGGCAGTAGTTTGAAGTTCGCCTTTAGATCGTTATCCACGTGGTACTTGTCCATCAGCG CGCGCGCAGCCTCCATGCCCTTCTCCCACGCAGACACGATCGGCACACTCAGCGGGTTCATCA CCGTAATTTCACTTTCCGCTTCGCTGGGCTCTTCCTCTTCCTCTTGCGTCCGCATACCACGCG CCACTGGGTCGTCTTCATTCAGCCGCCGCACTGTGCGCTTACCTCCTTTGCCATGCTTGATTA GCACCGGTGGGTTGCTGAAACCCACCATTTGTAGCGCCACATCTTCTCTTTCTTCCTCGCTGT CCACGATTACCTCTGGTGATGGCGGGCGCTCGGGCTTGGGAGAAGGGCGCTTCTTTTTCTTCT TGGGCGCAATGGCCAAATCCGCCGCCGAGGTCGATGGCCGCGGGCTGGGTGTGCGCGGCACCA GCGCGTCTTGTGATGAGTCTTCCTCGTCCTCGGACTCGATACGCCGCCTCATCCGCTTTTTTG GGGGGGCCCGGGGAGGCGGCGGCGACGGGGACGGGGACGACACGTCCTCCATGGTTGGGGGAC GTCGCGCCGCACCGCGTCCGCGCTCGGGGGTGGTTTCGCGCTGCTCCTCTTCTCTAGAAGCCA TGGTGGCGGGTTCTCTCTGAGTCTGTGGGGACCAGAAGAATACTCGAGGCCAGCTTGGGTCTC CCTATAGTGAGTCGTATTAATTTCGATAAGCCAGTAAGCAGTGGGTTCTCTAGTTAGCCAGAG AGCTCTGCTTATATAGACCTCCCACCGTACACGCCTACCGCCCATTTGCGTCAATGGGGCGGA GTTGTTACGACATTTTGGAAAGTCCCGTTGATTTTGGTGCCAAAACAAACTCCCATTGACGTC AATGGGGTGGAGACTTGGAAATCCCCGTGAGTCAAACCGCTATCCACGCCCATTGATGTACTG CCAAAACCGCATCACCATGGTAATAGCGATGACTAATACGTAGATGTACTGCCAAGTAGGAAA GTCCCATAAGGTCATGTACTGGGCATAATGCCAGGCGGGCCATTTACCGTCATTGACGTCAAT

[0373] AGGGGGCGTACTTGGCATATGATACACTTGATGTACTGCCAAGTGGGCAGTTTACCGTAAATA CTCCACCCATTGACGTCAATGGAAAGTCCCTATTGGCGTTACTATGGGAACATACGTCATTAT TGACGTCAATGGGCGGGGGTCGTTGGGCGGTCAGCCAGGCGGGCCATTTACCGTAAGTTATGT AACGCGGAACTCCATATATGGGCTATGAACTAATGACCCCGTAATTGATTACTATTAATAACT AGT T GGAAACAGACAT GAT AAGAT ACAT T GAT GAGT T TGGACAAACCACAAC T AGAATGC AGT GAAAAAAATGCTTTATTTGTGAAATTTGTGATGCTATTGCTTTATTTGTAACCATTATAAGCT

[0374] GCAATAAACAAGTCCTGCAGGGGTGTGGCTGGCACGAAATTGACCAACCCTGGGGTTAGTATA

[0375] GCTTAGTTAAACTTTCGTTTATTGCTAAAGGTTAATCACTGCTGTTTCCCGTGGGGGTGTGGC

[0376] TAGGCTAAGCGTTTTGAGCTGCATTGCTGCGTGCTTGGGAGGTGTCTGGAACTAGCAGAGGTG

[0377] GTGAGTGGGGCAGGTGGAGGTGGGAGCATACCTGGGACCCGAGGTCGGGGGAGACTCGGGGTA

[0378] CCCAGGACGGGAAAGGGGCAGCTAGCATTGCGTGCATGCAGTACCAGCTCGACGGACCGTCAT

[0379] TACATCGGTGTGCTATCGTAGTCATGCATGAGAATTGGCCTATGATGTTGGAGAAGTGCCCTG

[0380] ATGAATTGTTGTCTTCTTCGTTCTGTACGACAACTGTACAGCATCGCGGTCGTTTCTTCTGCA

[0381] ATTATGCGGACAGCCCTAAGCATCAGACGCCGAGTGCGCCTAGCGCAACACCTAACGCGAATT

[0382] TCGCTGAGGTCTGCGCAATAAGAACAACAAAGGACGACGATGTTATTCAGGATTCCGCAATGC

[0383] AGAGCAGAATAGCCGAATGACATTGCTGGCACAACGGAGCCAACATGTCCGTCGTACCACAGC

[0384] CGCAAATAAATGAGATGTCTCCCTCTCATGAAGACTGAGCTCATGAACATCACTTCCTTCGGC

[0385] ATATTATAGTTGACAACTTCTCTATACCAGATGAATCTTTGGTTGAACATCGCTCCATCGACG

[0386] ACCATCCGGAACCAAGAAGCCAGCACTTGACCGCCAGCAATGCATTGCAAGCTCCCAGGTGAG

[0387] CTGCAGTGGCAATGCAAGGCCCATGATTCATAGCCGTGAATCATCATAGATGTCATAATGTCG

[0388] ATATTAGCGCAGCAGAGACAGACATGCATGCATTTTCTTAATATGACCAATTCTTCTCTTGTC

[0389] AAGACCATATCCCAAGGCACGACCCACTCTTGGATAAGGGTGAAGCCAACTGAACAAGGCAGT

[0390] CCCCTAACATAGGATACATTATGCATGGTCAGGGTATTGCACTCAGGAAGAAGAGGGTGGTCT

[0391] TCGAGAATAGTGGCCCTAGTCTCAGTTTCGAACGGTGGAAGCCGGTCGCGGGAATAGGGTGTC

[0392] CTGCGGCTCAGTCGGCTGCGCGTGGGACGCAGGGTCATTCCGAAAGGCACACCTGAGGTTGTC

[0393] ATGGTGGCGGCGCCGGGGTCTTTATAAAAAAGGGCCAAGTGCAGAGCGAGTATATATAGGACT

[0394] AAAAAATGACGTAACGGTTAAAGTCCACAAAAAACACCCAGAAAACCGCACGCGAACCTACGC

[0395] CCAGAAACGAAAGCCAAAAAACCCACAACTTCCTCAAATCGTCACTTCCGTTTTCCCACGTTA

[0396] CGTAACTTCCCATTTTAAGAAAACTACAATTCCCAACACATACAAGTTACTCCGCCCTAAAAC

[0397] GTCGACTACGTCACCCGCCCCGTTCCCACGCCCCGCGCCACGTCACAAACTCCACCCCCTCAT

[0398] TATCATATTGGCTTCAATCCAAAATAAGGTATATTATTGATGATG ( SEQ ID No . 27 )

[0399] Embodiments of the invention

[0400] HAdV5 offers various advantages as a gene delivery vehicle for the in-situ production of synergistic protein-based therapeutics. Vectorized and engineered derivatives of the HAdV5 are characterized by an intrinsic transgene packaging capacity of up to 37 kb and the ability to transduce both dividing and quiescent cells. They are also extensively characterized and exhibit a high clinical safety margin due to their non-integrating genome and negligible risk of oncogenesis. HAdV5 can furthermore be engineered to mask the HAdV5's natural liver tropism and allow retargeting to defined tumor biomarkers. The combination of high-capacity HAdV5 (HC-HAdV5), capsid engineering, retargeting and shielding strategies is already a major improvement in HAdV5-based gene therapy approaches.

[0401] In vivo experiments, however, have demonstrated that transgene expression levels can be limited. Loss and / or degradation of the transduced payload is often observed after around 2 weeks, thereby often preventing long-term expression of therapeutic payloads. Additionally, potent tumor regression requires multiple consecutive intratumoral injections of high vector titers. The present invention addresses these problems by providing a novel and universally applicable adenoviral vector that contains an intrinsic DNA replication system. Of importance, the vector only replicates the genetic material, but no virus particles are produced. Self-induced and -sustained payload replication increases the copy number of the encoded transgenes, thereby leading to an elevated expression and secretion of the transgenes in the tumor microenvironment. The continuous amplification of the transgene provides a sufficiently high concentration of the mRNA and translated protein payload within the cell (and secretion from the cell, if applicable), ensuring long-lasting expression. At the same time, the increased secretion level of the payload, e.g., into the tumor microenvironment from suitable infected cells, allows a dose reduction of the injected vector, thereby increasing the safety of the vector.

[0402] Wild-type HAdV5 is capable to generate up to 1,000,000 genome copies per host cell, starting from a single template molecule. In contrast, the current generation of high-capacity HAdV5 (HC-HAdV5) vectors lead to only a few transgene copies per cell, namely the ones that have been taken up, since the vector is deficient for DNA replication. The present invention therefore dissected the endogenous WT HAdV5 replication machinery, in order to identify all proteins that are strictly required for efficient replication. This minimal DNA replication system can then form the basis for a new vector that is able to deliver gene payloads into cells that are transcribed and translated, while at the same time the replication machinery is expressed, self-assembled and amplifying the vector genome, leading to an increased expression of the payload.

[0403] The viral proteins which are directly associated with the replication machinery are located in the transcriptional unit E2 of the WT HAdV5, and their genetic structure and functional properties are well- described. However, further genes are reported (in particular in the E4 transcriptional unit) to be of great importance for this process. The identification of all required components necessary for efficient replication and genetic assembly is therefore crucial. The vector can further be equipped with elements that control the activity of the replication system, such as tumor-specific promoters (e.g. the telomerase reverse transcriptase promoter, hTERT).

[0404] The adenoviral genome encodes about 40 genes and is classified according to their temporal gene expression into early and late genes. Early genes are categorized into four transcriptional units (TUs), namely E1-E4. Importantly, all early genes are expressed prior to replication of the viral genome, whereas the late genes are characterized by expression post-replication. While the early TUs are controlled by individual promoter sequences, transcription of all late TUs (LI- L5) is driven by a single promoter, called Major Late Promoter (MLP). Transcription from the MLP generates a single pre- mRNA, and by several splicing events, the mature mRNA is generated, leading to the synthesis of the late genes by the cellular ribosomal machinery. Besides early and late genes, the HAdV genome carries an encapsulation signal QJ and non-coding inverted terminal repeats (ITRs). QJ directs efficient packaging of the amplified viral genome into newly assembled virions, whereas the ITRs are required for viral DNA amplification and protection of the viral genome from degradation by the host cell.

[0405] Upon infection, protein E1A is immediately expressed, driven by ubiquitous, cellular transcription factors. E1A interacts with a plethora of important cellular proteins. Binding to the tumor suppressor Rb results in sequestration of the transcription factors E2F. This induces a cell cycle progression from Go / Gito the S phase. In this highly active "synthesis state", the cell provides the molecular machinery to supply numerous building blocks, such as nucleotides and amino acids. Furthermore, E1A is required to transactivate all further TUs (E2-E4 and all late TUs via activation of the MLP), highlighting ElA's fundamental function during the viral life cycle.

[0406] ElA-induced cell cycle progression into the S phase (transformation) promotes accumulation and activation of the tumor suppressor p53, which usually causes induction of apoptosis. To prevent p53- mediated cell death, HAdV5 has evolved the TU E1B which encodes the two proteins E1B-55K and E1B- 19K. E1B-19K efficiently inhibits host cell apoptosis by suppressing both intrinsic and extrinsic apoptotic signaling. E1B-55K complements E1B-19K by inhibiting different apoptotic pathways and more importantly, it inhibits p53-mediated apoptosis through two distinct mechanisms. First, E1B-55K binds to the N-terminal transactivation domain of p53 and thereby ablates p53's ability to activate proapoptotic genes. Second, E1B-55K forms a heterodimer with the protein E4 ORF6 and highjacks the cellular E3 ligase Cu IlinS to ubiquitinate cellular proteins, such as Mrell, Daxx and p53. Ubiquitination targets proteins to the proteasome where they get degraded through the ubiquitin-proteasome pathway (UPP), resulting in decreased levels of activated p53.

[0407] The transcriptional unit E2 is organized in E2A and E2B. The E2 TU provides proteins that are directly involved in the adenoviral DNA replication machinery, comprising DNA-binding protein (DBP), precursor terminal protein (pTP) and the adenoviral DNA polymerase (Ad Pol). The ratiometric abundance of all E2 gene products seems to be important for efficient replication, consequently, their expression is tightly regulated.

[0408] The E3 TU encodes seven gene products. All of them carry out immunomodulatory functions to preserve cellular viability during persistent infections. E3 proteins comprise both soluble and membrane proteins that facilitate host cell immune defense evasion by inhibition of antigen presentation, downregulation of apoptosis receptors and suppression of TNFa signaling.

[0409] E4 represents the last TU that gets expressed prior to amplification of the viral DNA. E4 expresses 6 proteins (E4 ORF1, 2, 3, 4, 6, 6 / 7) with discrete function. The protein E4 ORF1 enhances cellular glucose uptake, flux and metabolism and glucose intermediates are used to augment nucleotide synthesis, supporting the ElA-induced S phase transition.

[0410] The gene product of E4 ORF6 co-localizes with nuclear multiprotein complexes, so-called PML nuclear bodies (PML-NB). PML-NBs are implicated in DNA damage repair, transcriptional regulation, regulation of apoptosis and antiviral response. E4 ORF3 drives the reorganization of PML-NBs into discrete viral replication foci. E4 ORF3 causes inhibition of the tumor suppressor p53 and the DNA repair machinery Mrell-Rad50-Nbsl (MRN), thereby allowing efficient viral replication by shielding the viral replicon from host cell defense mechanisms, both functionally and spatially. Inhibition of the MRN-complex has been shown to be crucial since this complex mediates the end-to-end ligation of the viral genomes and effectively inhibits replication of viral DNA.

[0411] E4 ORF6 represents yet another mechanism to mediate host cell defense. In conjunction with E1B- 55K it promotes degradation of p53, Daxx, DNA Ligase IV and components of MRN.

[0412] The last protein in the E4 TU is the gene product of the E4 ORF6 / 7. This fusion protein of E4 ORF6 linked to the unique E4 ORF7 sequence is described to transactivate the expression of E2 products and thereby enhance viral DNA replication. Even though E4 ORF6 / 7 is described in several studies to promote ElA-independent expression of E2 proteins, it is still debatable if E4 ORF6 / 7 can fully compensate for the loss of the El TU.

[0413] Whilst there is an emerging knowledge with respect to the adenoviral genes and their function, there are also still many open questions. The present invention aimed to identify those components that are crucial for replication. This was achieved by a two-tiered approach. In a top-down approach genes were systematically eliminated from the wild-type in order to identify those genes that are required for viral replication. In a bottom-up approach the genes identified were then reconstituted into a synthetic minimal replication system and their functionality was experimentally tested.

[0414] In certain embodiments, the present disclosure relates to a minimal adenoviral vector capable of replicating its own DNA in a host cell. Said minimal adenoviral vector comprises the molecular machinery that enables to replicate its own DNA in a host cell transduced with the vector. Said minimal adenoviral vector is however not capable to form viral particles.

[0415] Therefore, in certain embodiments the present disclosure relates to a minimal adenoviral vector capable of replicating its own DNA in a host cell, wherein said vector is not capable to form viral particles.

[0416] In certain embodiments, the present disclosure relates to a minimal adenoviral expression system capable of replicating its own DNA in a host cell, wherein said vector is not capable to form viral particles.

[0417] Since the adenoviral vector of the present invention is devoid of all adenoviral genes that are not absolutely required for replication it has a large cargo capacity. Cargo capacity can be as large as 17 kb, preferably 18 kb, more preferably 19 kb, even more preferably 20 kb, and most preferably 22 kb. Therefore, in certain embodiments the present disclosure relates to a minimal adenoviral vector capable of replicating its own DNA in a host cell, and wherein said vector has a cargo capacity of at least 17 kb, at least 18 kb, at least 19 kb or at least 20 kb.

[0418] In other embodiments the present disclosure relates to a minimal adenoviral vector capable of replicating its own DNA in a host cell, wherein said vector is not capable to form viral particles, and wherein said vector has a cargo capacity of at least 17 kb, at least 18 kb, at least 19 kb or at least 20 kb.

[0419] Experimentally, the following proteins were identified that are required for replication: E1A-289R (SEQ ID No. 2), E1B-19K (SEQ ID No. 4), E1B-55K (SEQ ID No. 6), DBP (SEQ ID No. 9), pTP (SEQ ID No. 12), Ad Pol (SEQ ID No. 15) and E4 ORF6 (SEQ ID No. 18). It will be understood that also functional variants or splice variants of aforementioned proteins may be used when practicing the present invention. Splice variants of E1A-289R (SEQ ID No. 2) include E1A-243R (SEQ ID No. 29), E1A-217R (SEQ ID No. 30), E1A-171R (SEQ ID No. 31) and E1A-55R (SEQ ID No. 32).

[0420] Therefore, in certain embodiments the present disclosure relates to a minimal adenoviral vector capable of replicating its own DNA in a host cell, wherein said minimal adenoviral vector comprises genes encoding the following proteins: E1A-289R (SEQ ID No. 2), SEQ ID No. 29 (E1A-243R), SEQ ID No. 30 (E1A-217R), SEQ ID No. 31 (E1A-171R), SEQ ID No. 32 (E1A-55R), E1B-19K (SEQ ID No. 4), E1B-55K (SEQ ID No. 6), DBP (SEQ ID No. 9), pTP (SEQ ID No. 12), Ad Pol (SEQ ID No. 15) and E4 ORF6 (SEQ ID No. 18), or functional variants thereof.

[0421] In certain embodiments the present disclosure relates to a minimal adenoviral vector capable of replicating its own DNA in a host cell, wherein said minimal adenoviral vector contains genes encoding the following proteins: E1A-289R (SEQ ID No. 2), SEQ ID No. 29 (E1A-243R), SEQ ID No. 30 (E1A-217R), SEQ ID No. 31 (E1A-171R), SEQ ID No. 32 (E1A-55R), E1B-19K (SEQ ID No. 4), E1B-55K (SEQ ID No. 6), DBP (SEQ I D No. 9), pTP (SEQ ID No. 12), Ad Pol (SEQ ID No. 15) and E4 ORF6 (SEQ ID No. 18), or functional variants thereof.

[0422] In certain embodiments the present disclosure relates to a minimal adenoviral vector capable of replicating its own DNA in a host cell, wherein said minimal adenoviral vector contains genes encoding the following proteins: E1A-289R (SEQ ID No. 2), SEQ ID No. 29 (E1A-243R), SEQ ID No. 30 (E1A-217R), SEQ ID No. 31 (E1A-171R), SEQ ID No. 32 (E1A-55R), E1B-19K (SEQ ID No. 4), E1B-55K (SEQ ID No. 6), DBP (SEQ I D No. 9), pTP (SEQ ID No. 12), Ad Pol (SEQ ID No. 15) and E4 ORF6 (SEQ ID No. 18), or functional variants of any of aforementioned proteins.

[0423] In certain embodiments the present disclosure relates to a minimal adenoviral vector capable of replicating its own DNA in a host cell, wherein said minimal adenoviral vector consists of 7 adenoviral genes encoding the proteins of SEQ ID No. 2 (E1A-289R), SEQ ID No. 29 (E1A-243R), SEQ ID No. 30 (E1A-217R), SEQ ID No. 31 (E1A-171R), SEQ ID No. 32 (E1A-55R), SEQ ID No. 4 (E1B-19K), SEQ ID No. 6 (E1B-55K), SEQ ID No. 9 (DBP), SEQ ID No. 12 (pTP), SEQ ID No. 15 (Ad Pol) and SEQ ID No. 18 (E4 ORF6), or functional variant or splice variants s of any of aforementioned proteins.

[0424] Splice variants of the E1A gene (SEQ ID No. 1) include the proteins E1A-289R (SEQ ID No. 2), E1A- 243R (SEQ ID No. 29), E1A-217R (SEQ ID No. 30), E1A-171R (SEQ ID No. 31) and E1A-55R (SEQ ID No. 32).

[0425] In certain embodiments the present disclosure relates to a minimal adenoviral vector capable of replicating its own DNA in a host cell, wherein said minimal adenoviral vector comprises the following adenoviral genes: E1A (SEQ ID No. 1), E1B-19K (SEQ ID No. 3), E1B-55K (SEQ ID No. 5), DBP (SEQ ID No. 7), pTP (SEQ ID No. 10 or 11), Ad Pol (SEQ ID No. 13 or 14) and E4 0RF6 (SEQ ID No. 16 or 17).

[0426] In certain embodiments the present disclosure relates to a minimal adenoviral vector capable of replicating its own DNA in a host cell, wherein said minimal adenoviral vector comprises the following adenoviral genes: E1A (SEQ ID No. 1), E1B-19K (SEQ ID No. 3), E1B-55K (SEQ ID No. 5), DBP (SEQ ID No. 7), pTP (SEQ ID No. 10 or 11), Ad Pol (SEQ ID No. 13 or 14) and E4 0RF6 (SEQ ID No. 16 or 17), or variants of said genes encoding functional variants of the proteins encoded by aforementioned genes.

[0427] In certain embodiments the present disclosure relates to a minimal adenoviral vector capable of replicating its own DNA in a host cell, wherein said minimal adenoviral vector consists of the following adenoviral genes: SEQ ID No. 1 (E1A), SEQ I D No. 3 (E1B-19K), SEQ ID No. 5 (E1B-55K), SEQ ID No. 7 (DBP), SEQ ID No. 10 or 11 (pTP), SEQ ID No. 13 or 14 (Ad Pol) and SEQ ID No. 16 or 17 (E4 ORF6), or adenoviral genes encoding functional variants of the encoded proteins.

[0428] In certain embodiments the present disclosure relates to a minimal adenoviral vector capable of replicating its own DNA in a host cell, wherein said vector is not capable to form viral particles, and wherein said minimal adenoviral vector contains genes encoding the following proteins: E1A-289R (SEQ ID No. 2), SEQ ID No. 29 (E1A-243R), SEQ ID No. 30 (E1A-217R), SEQ ID No. 31 (E1A-171R), SEQ ID No. 32 (E1A-55R), E1B-19K (SEQ ID No. 4), E1B-55K (SEQ ID No. 6), DBP (SEQ ID No. 9), pTP (SEQ ID No. 12), Ad Pol (SEQ ID No. 15) and E4 ORF6 (SEQ I D No. 18), or functional variants of any of aforementioned proteins.

[0429] In certain embodiments, the present disclosure relates to a minimal adenoviral vector capable of replicating its own DNA in a host cell, wherein said vector is not capable to form viral particles, wherein said minimal adenoviral vector contains genes encoding the following proteins: E1A-289R (SEQ ID No. 2), SEQ ID No. 29 (E1A-243R), SEQ ID No. 30 (E1A-217R), SEQ ID No. 31 (E1A-171R), SEQ ID No. 32 (E1A-55R), E1B-19K (SEQ ID No. 4), E1B-55K (SEQ ID No. 6), DBP (SEQ ID No. 9), pTP (SEQ ID No. 12), Ad Pol (SEQ ID No. 15) and E4 ORF6 (SEQ ID No. 18), or functional variants of any of aforementioned proteins, and wherein said vector has a cargo capacity of at least 17 kb, at least 18 kb, at least 19 kb or at least 20 kb.

[0430] In certain embodiments, the present disclosure relates to a minimal adenoviral vector containing genes encoding the following proteins: E1A-289R (SEQ ID No. 2), SEQ ID No. 29 (E1A-243R), SEQ ID No. 30 (E1A-217R), SEQ. ID No. 31 (E1A-171R), SEQ ID No. 32 (E1A-55R), E1B-19K (SEQ ID No. 4), E1B-55K (SEQ ID No. 6), DBP (SEQ ID No. 9), pTP (SEQ ID No. 12), Ad Pol (SEQ ID No. 15) and E4 0RF6 (SEQ ID No. 18), or functional variants of any of aforementioned proteins, and no additional adenoviral genes.

[0431] Splice variants of the E1A gene (SEQ ID No. 1) include the proteins E1A-289R (SEQ ID No. 2), E1A- 243R (SEQ ID No. 29), E1A-217R (SEQ ID No. 30), E1A-171R (SEQ ID No. 31) and E1A-55R (SEQ ID No. 32).

[0432] The adenoviral vector also may contain a L-ITR, a QJ encapsulation signal and a R-ITR. Any suitable ITR may be used in connection with the present disclosure. An exemplary L-ITR comprises the nucleic acid sequence of SEQ ID No. 19. An exemplary R-ITR comprises the nucleic acid sequence of SEQ ID No. 20. Likewise, any suitable QJ encapsulation signal may be used in connection with the present disclosure. An exemplary QJ encapsulation signal comprises the nucleic acid sequence of SEQ ID No. 28.

[0433] Therefore, in certain embodiments the present disclosure relates to a minimal adenoviral vector capable of replicating its own DNA in a host cell, wherein said minimal adenoviral vector comprises a L-ITR, a QJ encapsulation signal and a R-ITR. In certain embodiments the present disclosure relates to a minimal adenoviral vector capable of replicating its own DNA in a host cell, wherein said minimal adenoviral vector comprises a L-ITR, a QJ encapsulation signal and a R-ITR, wherein said L-ITR comprises the nucleic acid sequence of SEQ ID No. 19, the QJ encapsulation signal comprises the nucleic acid sequence of SEQ ID No. 28 and the R-ITR comprises the nucleic acid sequence of SEQ I D No. 20.

[0434] In certain embodiments the present disclosure relates to a minimal adenoviral vector capable of replicating its own DNA in a host cell, wherein said vector is not capable to form viral particles, wherein said minimal adenoviral vector contains genes encoding the following proteins: E1A-289R (SEQ ID No. 2), SEQ ID No. 29 (E1A-243R), SEQ ID No. 30 (E1A-217R), SEQ ID No. 31 (E1A-171R), SEQ ID No. 32 (E1A-55R), E1B-19K (SEQ ID No. 4), E1B-55K (SEQ ID No. 6), DBP (SEQ ID No. 9), pTP (SEQ ID No. 12), Ad Pol (SEQ ID No. 15) and E4 ORF6 (SEQ ID No. 18), or functional variants of any of aforementioned proteins, wherein said vector has a cargo capacity of at least 17 kb, at least 18 kb, at least 19 kb or at least 20 kb, and wherein said minimal adenoviral vector comprises a L-ITR, a QJ encapsulation signal and a R-ITR.

[0435] The arrangement of the seven genes on the minimal adenoviral vector may principally be in any order. The genes may also be encoded on the sense strand or the antisense strand. In certain embodiments, the present disclosure relates to a minimal adenoviral vector capable of replicating its own DNA in a host cell, wherein said vector is not capable to form viral particles, wherein said minimal adenoviral vector contains genes encoding the following proteins: E1A-289R (SEQ ID No. 2), SEQ ID No. 29 (E1A-243R), SEQ. ID No. 30 (E1A-217R), SEQ ID No. 31 (E1A-171R), SEQ ID No. 32 (E1A-55R), E1B- 19K (SEQ ID No. 4), E1B-55K (SEQ ID No. 6), DBP (SEQ ID No. 9), pTP (SEQ ID No. 12), Ad Pol (SEQ ID No. 15) and E4 ORF6 (SEQ ID No. 18), or functional variants of any of aforementioned proteins, and wherein aforementioned genes are located on the sense strand.

[0436] In certain embodiments, the present disclosure relates to a minimal adenoviral vector capable of replicating its own DNA in a host cell, wherein said vector is not capable to form viral particles, wherein said minimal adenoviral vector contains genes encoding the following proteins: E1A-289R (SEQ ID No. 2), SEQ ID No. 29 (E1A-243R), SEQ ID No. 30 (E1A-217R), SEQ ID No. 31 (E1A-171R), SEQ ID No. 32 (E1A-55R), E1B-19K (SEQ ID No. 4), E1B-55K (SEQ ID No. 6), DBP (SEQ ID No. 9), pTP (SEQ ID No. 12), Ad Pol (SEQ ID No. 15) and E4 0RF6 (SEQ ID No. 18), or functional variants of any of aforementioned proteins, and wherein aforementioned genes are located on the antisense strand.

[0437] In certain embodiments, the present disclosure relates to a minimal adenoviral vector capable of replicating its own DNA in a host cell, wherein said vector is not capable to form viral particles, wherein said minimal adenoviral vector contains genes encoding the following proteins: E1A-289R (SEQ ID No. 2), SEQ ID No. 29 (E1A-243R), SEQ ID No. 30 (E1A-217R), SEQ ID No. 31 (E1A-171R), SEQ ID No. 32 (E1A-55R), E1B-19K (SEQ ID No. 4), E1B-55K (SEQ ID No. 6), DBP (SEQ ID No. 9), pTP (SEQ ID No. 12), Ad Pol (SEQ ID No. 15) and E4 ORF6 (SEQ ID No. 18), or functional variants of any of aforementioned proteins, and wherein some genes are located on the sense strand and some genes are located on the antisense strand.

[0438] In certain embodiments, the present disclosure relates to a minimal adenoviral vector capable of replicating its own DNA in a host cell, wherein said vector is not capable to form viral particles, wherein said minimal adenoviral vector contains genes encoding the following proteins: E1A-289R (SEQ ID No. 2), SEQ ID No. 29 (E1A-243R), SEQ ID No. 30 (E1A-217R), SEQ ID No. 31 (E1A-171R), SEQ ID No. 32 (E1A-55R), E1B-19K (SEQ ID No. 4), E1B-55K (SEQ ID No. 6), DBP (SEQ ID No. 9), pTP (SEQ ID No. 12), Ad Pol (SEQ ID No. 15) and E4 ORF6 (SEQ ID No. 18), or functional variants of any of aforementioned proteins, and wherein the E1A, the E1B-19K and the E1B-55K genes located on the sense strand and the DBP, the pTP, the Ad Pol and the E4 ORF6 genes are located on the antisense strand.

[0439] Splice variants of the E1A gene (SEQ ID No. 1) include the proteins E1A-289R (SEQ ID No. 2), E1A- 243R (SEQ ID No. 29), E1A-217R (SEQ ID No. 30), E1A-171R (SEQ ID No. 31) and E1A-55R (SEQ ID No. 32).

[0440] The genes of the minimal adenoviral vector may contain their own individual promoters or may be transcribed as dicistronis, triscistronic or multicistronic units.

[0441] The genes of the minimal adenoviral vector may also overlap, provided the proteins encoded by such overlapping cistrons are functional. In fact, the E1B-19K and the E1B-55K genes as naturally occurring and as used herein do overlap.

[0442] The minimal adenoviral vector of the present disclosure may be used for the high level expression of any protein of interest in a host cell. Preferred proteins of interest are therapeutically active proteins or polypeptides. The protein or polypeptide to be expressed in the host cell is also encoded on the minimal adenoviral vector.

[0443] Therefore in certain embodiments, the present disclosure relates to a minimal adenoviral vector capable of replicating its own DNA in a host cell, wherein said vector comprises a nucleic acid encoding a cargo gene to be expressed in said host cell.

[0444] In certain embodiments, the present disclosure relates to a minimal adenoviral vector capable of replicating its own DNA in a host cell, wherein said vector is not capable to form viral particles, and wherein said vector comprises a nucleic acid encoding a cargo gene to be expressed in said host cell.

[0445] In certain embodiments, the present disclosure relates to a minimal adenoviral vector comprising a nucleic acid encoding a cargo gene, wherein said cargo gene is a non-adenoviral gene.

[0446] In certain embodiments, the present disclosure relates to a minimal adenoviral vector capable of replicating its own DNA in a host cell, wherein said vector is not capable to form viral particles, wherein said minimal adenoviral vector contains genes encoding the following proteins: E1A-289R (SEQ ID No. 2), SEQ. ID No. 29 (E1A-243R), SEQ ID No. 30 (E1A-217R), SEQ ID No. 31 (E1A-171R), SEQ ID No. 32 (E1A-55R), E1B-19K (SEQ ID No. 4), E1B-55K (SEQ I D No. 6), DBP (SEQ ID No. 9), pTP (SEQ ID No. 12), Ad Pol (SEQ ID No. 15) and E4 ORF6 (SEQ ID No. 18), or functional variants of any of aforementioned proteins, and wherein said vector comprises a nucleic acid encoding a cargo gene to be expressed in said host cell.

[0447] In certain embodiments, the present disclosure relates to a minimal adenoviral vector capable of replicating its own DNA in a host cell, wherein said vector is not capable to form viral particles, wherein said minimal adenoviral vector contains genes encoding the following proteins: E1A-289R (SEQ ID No. 2), SEQ ID No. 29 (E1A-243R), SEQ ID No. 30 (E1A-217R), SEQ ID No. 31 (E1A-171R), SEQ ID No. 32 (E1A-55R), E1B-19K (SEQ ID No. 4), E1B-55K (SEQ ID No. 6), DBP (SEQ ID No. 9), pTP (SEQ ID No. 12), Ad Pol (SEQ ID No. 15) and E4 ORF6 (SEQ ID No. 18), or functional variants of any of aforementioned proteins, wherein said minimal adenoviral vector comprises a L-ITR, a QJ encapsulation signal and a R- ITR, and wherein said vector comprises a nucleic acid encoding a cargo gene to be expressed in said host cell.

[0448] In certain embodiments, the present disclosure relates to a minimal adenoviral vector capable of replicating its own DNA in a host cell, wherein said vector is not capable to form viral particles, wherein said minimal adenoviral vector contains genes encoding the following proteins: E1A-289R (SEQ ID No. 2), SEQ ID No. 29 (E1A-243R), SEQ ID No. 30 (E1A-217R), SEQ ID No. 31 (E1A-171R), SEQ ID No. 32 (E1A-55R), E1B-19K (SEQ. ID No. 4), E1B-55K (SEQ ID No. 6), DBP (SEQ ID No. 9), pTP (SEQ ID No. 12), Ad Pol (SEQ ID No. 15) and E4 0RF6 (SEQ ID No. 18), or functional variants of any of aforementioned proteins, wherein said minimal adenoviral vector comprises a L-ITR, a QJ encapsulation signal and a R- ITR, wherein said vector comprises a nucleic acid encoding a cargo gene to be expressed in said host cell, and wherein said cargo gene encodes a therapeutically active polypeptide.

[0449] Splice variants of the E1A gene (SEQ ID No. 1) include the proteins E1A-289R (SEQ ID No. 2), E1A- 243R (SEQ ID No. 29), E1A-217R (SEQ ID No. 30), E1A-171R (SEQ ID No. 31) and E1A-55R (SEQ ID No. 32).

[0450] The minimal adenoviral vectors of the present disclosure may be used in medicine. Such use in medicine includes the use for the treatment of a tumor or cancer. The minimal adenoviral vectors of the present disclosure may also be used for the expression of a cargo gene in a host cell.

[0451] Examples

[0452] Example 1: General methods, cell lines and constructs utilized

[0453] Cell lines

[0454] For experiments in which replication of the constructs of the present invention was tested, HEK293 cells (ATCC: CRL-1573) and A549 cells (ATCC: CCL-185) were used. HEK293 cells carry the adenoviral El gene, whereas A549 cells do not. For experiments in which an increase in payload expression was tested, HEK293T / 17 (ATCC: CRL-11268) were transfected instead of HEK293.

[0455] Viral vector generation using helper virus:

[0456] High-capacity adenoviral vectors (HC-HAdV) do not encode any viral DNA and generally are produced using a helper virus (HV) that supplies all adenoviral proteins during the production process. HC-HAdVs were produced as described by Brtlcher et al. (Mol Ther Methods Clin Dev (2021) 20:572- 86). Briefly, the reporter expression cassettes (e.g. CMV - eGFP - BGH Poly(A)) were subcloned into the pC4HSU plasmid, which will constitute the HC-HAdV genome in the process of vector production. Upon linearization of the pC4HSU plasmid and ethanol precipitation, the pC4HSU DNA was transfected into the producer cell line "116" (termed 116 cells hereafter), and simultaneously, 116 cells were cotransduced with Ad5 helper virus. HC-HAdV titer was sequentially increased over multiple passages by releasing the HC-HAdV from the producer cells and re-transduction of 116 cells together with HV. Purification of HC-HAdV was performed via ultracentrifugation with two CsCI gradients at 250,000 x g as described by Briicher et al. (Mol Ther Methods Clin Dev (2021) 20:572-86).

[0457] Viral vector generation using helper plasmids:

[0458] High-capacity adenoviral vectors (HC-HAdV) can alternatively be produced using helper plasmids instead of helper virus. The transfected helper plasmids supply all adenoviral proteins during the production process. Indicated HC-HAdVs were produced as described by Lee et al. Exp Mol Med (2019) 51(10), 1-18.

[0459] Helper plasmids are only required to trans-complement all adenoviral genes to produce functional HC-HAdV, however, it is crucial to prevent formation of helper virus upon transfection of helper plasmids. Therefore, all helper plasmids are generally devoid of the L-ITR, R-ITR, encapsulation signal QJ, E1A- and ElB-coding regions. Due to the lack of ITRs and encapsulation signal QJ, it is virtually impossible to package the helper plasmid into a capsid and to thus generate helper virus.

[0460] To reduce the risk of homologous recombination and formation of replication-competent particles during the HC-HAdV production process, two different helper plasmids were cloned. The first helper plasmid encodes all adenoviral genes, except the El and E3 region, and the E4 promoter is replaced with a PGK promoter. The second helper plasmid is additionally devoid of the DBP-coding sequence and the entire E4 region. Both helper plasmids encode a plasmid backbone that contains an SV40 origin of replication to boost expression of the encoded adenoviral proteins.

[0461] For production of HC-HAdV, 12xl06HEK293T cells were seeded in a 150 mm dish 24 h prior to transfection. The day of transfection, 22.5 pg of the Pacl-linearized pC4HSU genome (encoding either HC-HAdV 1, 2, or 3) is mixed together with 52.5 pg of helper plasmid 1, 1, and 2, respectively in serum- free Opti-MEM. Next, 112.5 pl of TranslT-293 (Mirus) transfection reagent (1:1.5 ratio) is added and transfection mix is vigorously mixed and incubated for 30 min at room temperature. Then, HEK293T producer cells are transfected by addition of the entire mix to the cell culture media. 60 h post transfection, cells are harvested, pelleted (centrifuged at 300 x g for 3 min), supernatant is aspirated and cell pellet is resuspended in 500 pl of fresh cell culture media. Produced HC-HAdV particles are released by three consecutive freeze-thawing cycles using liquid nitrogen and a 37°C water bath. Free DNA molecules are digested by addition of 250 U Benzonase (Merck) and incubation at 37°C for 1 h, followed by removal of cell debris by centrifugation at 800 x g for 5 min. Supernatant contains produced HC-HAdV and can be used for quantification and transduction assays.

[0462] Replication Assay in HEK293 or A549: To identify all adenoviral protein components that are required for adenoviral DNA replication within the host cell, HEK293 or A549 cells were transfected. 24 h prior to transfection 360,000 HEK293 cells were seeded per well in 12 well dishes. The next day, HEK293 cells of each well were transfected with a total of 1800 ng DNA using the TranslT-293 (Mirus) transfection reagent at a ratio of 1:1.5 (pg DNA : pl TransIT, e.g. per 1.8 pg DNA, 2.7 pl TransIT were used to form the transfection complex). Different mammalian expression plasmids were added to the transfection mix, encoding either a single adenoviral protein (e.g. DBP only) or an entire adenoviral genome (e.g. AE1, A3-deleted adenoviral genome). Equimolar amounts of each plasmid (2xlO10copies per plasmid) were transfected. The transfection mix was prepared in serum-free Opti-MEM. After adding all expression plasmids to the transfection mix, pcDNA3.1 empty plasmid (only circular expression plasmid without any relevant inserts, such as promoters or genes) was added to result in a total amount of DNA of 1800 ng. As plasmid amounts are now normalized, identical amounts of TransIT were added to each transfection reaction, vigorously mixed, and the transfection mix was incubated for 30 mins and then added to the cells. 2 h post-transfection, HC-HAdVs that are only encoding a reporter gene and no adenoviral genes, were added to the cells at an MOI 1. 4 h later (6 h post-transfection) the entire medium was removed and fresh growth medium was added to the cells. Replicates of each condition were harvested for DNA extraction, as this short time period represents the amount of adenoviral genome copy numbers in the nucleus without any DNA replication. Cells were trypsinized, centrifuged at 800 x g for 5 min and washed twice with PBS. 48 h post-transduction the second set of replicates of each condition were harvested and washed twice with PBS. DNA then was extracted so that the increase in viral copy numbers can be quantified via qPCR.

[0463] Transfection of different combinations of expression plasmids encoding different adenoviral proteins allows for screening and identification of the minimal set of adenoviral proteins that are required for potent replication of transduced adenoviral genomes.

[0464] Transfection for replication assays in the El-non-complementing cell line A549 was performed similarly as in HEK293 cells (described above). 110,000 A549 cells were seeded per well in a 12 well dish, 24 h prior to transfection. 24 h after seeding, cells of each well were transfected with a total of 2500 ng DNA and 3.6xlO10copies of each plasmid that was probed for its involvement in adenoviral genome replication. Similarly to the transfection of HEK293 cells, A549 cells were transduced 2 h posttransfection with a HC-HAdV encoding a reporter gene (MOI 1) and cells were either harvested by trypsinization or washed and replenished with fresh growth media 4 h post-transduction. Replicates of each transfection were harvested 72 h post-transduction and subjected to qPCR analysis.

[0465] Quantification of transduced cells: Cells were harvested as described above. Total DNA was extracted using the DNeasy Blood & Tissue Kit (Qiagen). Extracted DNA was used for qPCR analysis on a Mx300 (Agilent) to determine the increase in vector copy number due to adenoviral DNA replication. qPCR analysis was performed in a 96-well qPCR plate (Thermo Fisher) in a total volume of 20 pl per well. Each qPCR reaction was performed with 5 pl of extracted total DNA, 10 pl 2x qPCR Mastermix (Powerllp SYBR Green Mastermix, Thermo Fisher), forward and reverse primer (500 nM final concentration for each primer) and water. The qPCR cycling parameters were as follows: Polymerase was first activated for 2 min at 50°C, followed by 2 min at 95°C. Amplification was performed for 40 cycles, starting with 15 s denaturation at 95°C, followed by annealing and extension for 1 min at 60°C. Lastly, a dissociation step was performed by slowly increasing (1.6°C per s) the temperature to 95°C for 15 s, slowly decreasing (1.6°C per s) to 60°C for 1 min and a final increase to 95°C for 15 s (increase of 0.15°C per s).

[0466] Absolute quantification of copy numbers was obtained by comparing the Ct values of each qPCR reaction to the Ct values of a standard with defined copy numbers (0, 10°, lO O7copies / pl). For copy number quantification of HC-HAdV encoding a GFP reporter, primer GFP_Fwd. (CCGACAAGCAGAAGAACGGC (SEQ ID No. 21)) and GFP_Rev. (GTGATCGCGCTTCTCGTTGG (SEQ ID No. 22)) were used, for HC-HAdV encoding a firefly luciferase, primer ffLuc_Fwd. (CACCGTGGTGTTCGTGTCTA (SEQ I D No. 23)) and ffLuc_Rev. (GCTGGTCACGAAGGTGTACA (SEQ ID No.

[0467] 24)) were used, respectively. HC-HAdV copy numbers were normalized to copies / cell by quantifying cellular DNA copy numbers using the primers GAPDH_Fwd. (AATTCCATGGCACCGTCAAG (SEQ ID No.

[0468] 25)) and GAPDH_Rev. (ATCGCCCCACTTGATTTTGG (SEQ ID No. 26)).

[0469] Replication Assay combined with reporter quantification:

[0470] To test if adenoviral genome replication results in enhanced payload expression due to the increased vector copy number, a modified replication assay was developed. As high-level and potent transfection of the replication machinery into the host cell is key, HEK293T / 17 instead of HEK293 cells were transfected. Additionally, each of the mammalian expression plasmids that were transfected encodes in the backbone an SV40 origin of replication to further increase expression of the encoded viral protein(s).

[0471] In brief, 300,000 HEK293T / 17 cells per well were seeded into a 12 well dish. 24 h later, the transfection was performed similarly as described above. 3.75xlO10copies per plasmid were added to the transfection mix and a total of 1800 ng of DNA was transfected per well after the transfection reagent TranslT-293 was added at a ratio of 1:1.5 (see above). 2 h post-transfection, a reporter HC- HAdV was added (encoding either GFP or firefly luciferase) and 4 h post-transduction samples were harvested and washed twice with PBS , and medium was replaced for the respective replicates that undergo vector DNA replication. 48 h post-transduction the remaining replicates were harvested and washed twice with PBS. Harvested cells were split in half and 50% of the sample were subjected to qPCR analysis (to determine vector genome amplification), while the other 50% were used to quantify the reporter expression. GFP expression was monitored via flow cytometry.

[0472] Flow cytometry:

[0473] For GFP quantification, the cells were resuspended in PBS containing 4% PFA and fixed for 10 min at room temperature. Cells were centrifuged for 5 min at 800 x g and cells were washed in PBS. After centrifuging, PBS was aspirated, and cells were resuspended in PBS containing 1% BSA and 0.05% azide. GFP fluorescence was measured on a BD FACSymphony (BD Bioscience) using the high-throughput sampler and flow cytometric analysis was performed using FlowJo software (BD Bioscience).

[0474] Constructs

[0475] Among others, the following construct were utilized:

[0476] Table 1:

[0477] Example 2: Systematic deletion of adenoviral genes - Step 1

[0478] In a first experiment, the HEK293 cell line was used which carries the adenoviral El gene. This gene therefore does not need to be supplemented. HEK293 cells were transduced with a gutless HAdV5 at an MOI of 1 and additionally transfected with different plasmids carrying various adenoviral genes. The following constructs were tested.

[0479] Table 2: 4 hours and 48 hours post-transduction, the number of viral genomes per cell was quantified. Figure 1 shows the number of viral genomes per cell, Figure 2 shows the magnitude of amplification of selected constructs after 72 hours. It can be concluded that all E2 genes (DBP, pTP and Ad Pol), as well as E4 ORF6 are required for potent replication of the adenoviral genome.

[0480] Example 3: Systematic deletion of adenoviral genes - Step 2

[0481] In the next experiment, A549 cells were used. A549 cells do not carry the adenoviral El gene which hence needs to be provided separately. Like in Example 1, cells were transduced with a gutless HAdV5 at an MOI of 1 and additionally transfected with different plasmids carrying various adenoviral genes. The following constructs were tested.

[0482] Table 3:

[0483] Again, 4 hours and 72 hours post-transduction, the number of viral genomes per cell was quantified. Figure 3 shows the magnitude of amplification of the constructs after 72 hours. It could be concluded that the El genes are essential for efficient viral replication. Furthermore, the importance of E2 genes (DBP, pTP and Ad Pol) could be confirmed.

[0484] Example 4: Systematic deletion of adenoviral genes - Step 3 In the next experiment, the relevance of the different open reading frames of the E4 genes was tested. The experimental set-up was identical to Example 3 A549 cells were used. The following constructs were tested.

[0485] Table 4:

[0486] Again, 4 hours and 72 hours post-transduction, the number of viral genomes per cell was quantified.

[0487] Figure 4 shows the magnitude of amplification of the constructs after 72 hours. It could be concluded that ORF6 of the E4 contributes most to viral replication.

[0488] Example 5: Assembly and testing of the viral replication system using eGFR expression as payload

[0489] Based on the experiments performed so far, it was evident that seven genes are required for an efficient viral replication system: E1A, E1B-19K, E1B-55K, DBP, pTP, Ad Pol and E4 ORF6. In a next step, eGFP as payload was integrated into the gutless HAdV5 in order to investigate if the replication system indeed leads to an increased payload expression. In this experiment, HEK293T cells were used. In a first experiment, again the contribution of various open reading frames of E4 was investigated. The minimal replication system was also compared to plasmids encoding all adenoviral genes except deletions in El, E3 and E4. The following constructs were tested.

[0490] Table 5:

[0491] The percentage of GFP-expressing cells was measured 48 hours after transduction. Results are shown in Figure 5. It could be confirmed that the minimal replication system leads to an efficient and quantifiable increase in payload expression. It could furthermore be confirmed that E4 ORF6 significantly contributes to an efficient payload expression.

[0492] Example 6: Characterization of the viral replication system

[0493] In this experiment the minimal replication system including E4 of ORF6 was characterized and compared to a gutless adenoviral system. Measured was the amplification of viral genomes in the target cells (HEK293T), as well as the percentage of cells expressing GFP. The following constructs were tested:

[0494] Table 6: Results of the quantification of viral genomes per cell are shown in Figure 6. Results of the quantification of GFP expressing cells are shown in Figure 7. Transfection of the minimal replication system in combination with E4 ORF6 drives more than 3000-fold genome amplification and increases the GFP+population size from 15 % to 30%.

[0495] Example 7: C / 's-replication and increased reporter expression

[0496] The minimal adenoviral vector contains the seven adenoviral genes E1A, E1B-19K, E1B-55K, DBP, pTP, Ad Pol and E4 ORF6 or equivalents thereof, encoding the respective functional proteins. The arrangement of these seven genes may principally be in any order. The genes may also be encoded on the sense strand or the antisense strand. In fact, in certain examples herein the E1A, the E1B-19K and the E1B-55K gene located on the sense strand directly adjacent to the L-ITR, and the DBP, the pTP, the Ad Pol and the E4 ORF6 gene are located on the antisense strand directly adjacent to the R-ITR (see Figure 8 bottom). The genes may also contain their individual promoters or may be transcribed as dicistronis, triscistronic or multicistronic units. The genes may also overlap, provided the proteins encoded by such overlapping cistrons are functional. In fact, the E1B-19K and the E1B-55K genes as naturally occurring and as used herein do overlap.

[0497] To test our hypothesis if the developed minimal replication system indeed promotes cis- amplification and an enhanced payload expression, three different HC-HAdVs were cloned and produced (Figure 8). Two control vectors either only encode a GFP reporter cassette (HC-HAdV 1, Figure 8, top) or a GFP reporter cassette and additionally the native El-coding region of the HAdV5 (HC-HAdV 2, Figure 8, middle). As E1A and E1B are described to potently transactivate numerous host transcription factors, HC-HAdV 2 contributes to elucidate if elevated payload expression is indeed a direct result from HC-HAdV genome replication or rather the consequence of El-mediated transcription factor transactivation.

[0498] HC-HAdV 3 (Figure 8, bottom) depicts the vector of the present invention that encodes a GFP reporter and the final minimal replication system, including the native El-coding region, an E2 expression system and the E4 ORF6 expression cassette. The E2 expression system (comprising DNA- binding protein (DBP), precursor terminal protein ( pTP) and the adenoviral DNA polymerase (Ad Pol)) express DBP from a CMV promoter, while pTP and AdV Pol are transcribed as dicistronic expression cassette from a PGK promoter and a T2A ribosomal skipping site.

[0499] In order to exclude trans-activation of helper virus (HV) by El-encoding HC-HAdV, a helper plasmid system was used instead of a helper virus as previously described (Exp Mol Med (2019) 51, 1-18). This ensures that absolutely no HV contamination can occur during production of HC-HAdV.

[0500] HC-HAdV titer was quantified and El-non-complementing cells BT-474 (DSMZ: ACC-64) were transduced at an MOI 5, alongside with a conventionally produced HC-HAdV (G FP, produced using HV) and a replication-competent HAdV (GFP). Forty-eight hours post-transduction protein expression was analyzed using western blotting and immunostaining to confirm genome identity of produced vectors. While the conventionally (HV-based) produced HC-HAdV (GFP) expressed only the transgene GFP, but neither El, DBP or Fiber, transduction of BT-474 cells with replication-competent HAdV (GFP) resulted in potent expression of the GFP payload, along with the three E1A isoforms, DBP and Fiber (Figure 9).

[0501] This observation underscores that HC-HAdVs do not express any viral genes, whereas replication- competent HAdVs undergo the complete viral life cycle. Similarly to transduction with the conventionally produced HC-HAdV (GFP), transduction with HC-HAdV 1 resulted solely in GFP expression, with no early and late genes detectable.

[0502] Next, BT-474 were transduced with HC-HAdV 2 and HC-HAdV 3 and Western Blot analysis demonstrated that GFP, E1A, and GFP, E1A, and DBP, respectively were expressed upon transduction. Except after transduction with replication-competent HAdV, Fiber expression was never apparent, suggesting that no formation of replication-competent AdV (RCA) or HV occurred.

[0503] Next, BT-474 cells were again transduced at an MOI of 5 and cells were harvested 72 h posttransduction. Payload expression was analyzed via flow cytometry and genome replication was quantified by qPCR. Transduction with conventionally produced HC-HAdV (GFP) resulted in ~11.5% of GFP+cells with an MFI of ~350 whereas transduction with replication-competent HAdV (GFP) led to almost all cells being GFP+and displaying a more than 11-fold increased MFI (~4000) (Figures 10 and 11, left panel), highlighting again the striking increase in payload expression due to AdV genome replication.

[0504] Transduction with HC-HAdV 1, 2, and 3 yielded to an MFI of 100, 96, and 354, respectively, pointing to increased GFP levels due to the minimal replication system. Increased GFP expression upon transduction with HC-HAdV 3 was also exhibited in the flow cytometry scatter plot, as a high GFP- expressing population emerges (Figure 10, bottom right panel). Although the conventionally produced HC-HAdV (GFP) and helper plasmid-produced HC-HAdV 1 encode the same viral genome, HV-produced HC-HAdV (GFP) displays a higher MFI. Considering that HV-produced HC-HAdV (GFP) was purified from cell lysate, while helper plasmid-produced HC-HAdV (GFP) was directly quantified from lysate of the producer cells, this difference in MFI is most likely a consequence from inaccurate vector titer quantification. This hypothesis was further corroborated by the observation, that HV-produced HC- HAdV (GFP) exhibits a 100% larger GFP+population size (11.6% GFP+) while helper-plasmid produced HC-HAdV 1 (GFP) only displays a GFP+population of ~5.7%.

[0505] Orthogonal HAdV genome copy number quantification by qPCR analysis confirmed more than ~100, 000-fold genome replication upon BT-474-transduction with replication-competent HAdV (GFP) (Figure 11, right panel). Transduction with HC-HAdV 1 and 2 resulted in ~0.1 vector copies / cell 72 h post-transduction, while transduction with the self-replicating HC-HAdC 3 displayed a vector copy number of ~30 genomes / cell. This suggests that the minimal replication system is indeed functional and promotes more than 300-fold replication of delivered genomes per cell. HV-produced HC-HAdV (GFP) exhibited ~0.7 vector copies / cell, validating the hypothesis that HC-HAdV (GFP) only features an increased MFI because cells were initially transduced at a higher MOI.

[0506] Example 8: The minimal replication system strongly boosts payload expression in El- complementing HEK293T, with no further increase by trans-complementation

[0507] In this experiment a sAEl,AE3 vDNA) establishes ~3, 500-fold genome amplification which in turn promotes enhanced GFP expression as reflected by an ~18-fold increase in MFI. Pre-transfection of HEK293T cells with other AdV replication system confirms previous data and GFP expression is correlating with genome copy number. Transduction with HC-HAdV 3 upon mock transfection results in ~2, 300-fold replication, showcasing that genome amplification is purely driven by the c / s-encoded minimal replication system, as only non-coding vDNA was pre-transfected. Most importantly, neither genome replication nor payload expression could be further increased by pre-transfection with replication systems. This observation confirms that the c / s-encoded minimal replication system on the HC-HAdV indeed expresses sufficient protein levels of the replication machinery to function at its maximal capacity, and thus can't be further enhanced through trans-complementation.

[0508] Altogether, these data show that self-sustained HC-HAdV genome replication is an elegant opportunity to effortlessly increase payload expression. By encoding only seven adenoviral genes on the vector genome, it is shown that these few gene products are sufficient to assemble a functional replication machinery within the host cell which induces potent in situ vector genome amplification, driving enhanced payload expression. Additional trans-complementation does not further increase genome replication and transgene expression suggesting that the encoded minimal replication system is indeed capable to induce maximal vector genome replication.

[0509] The described minimal replication system can be simply encoded on a cloning plasmid, allowing insertions of any kind of payloads and production of those designed vectors (Mol Ther Methods Clin Dev (2021) 20, 572-586, and Exp Mol Med (2019) 51(10), 1-18) Importantly, the developed minimal replication system features a very compact genetic architecture, still retaining more than 22 kb transgene capacity on the HC-HAdV which is plenty of coding space to deliver the genetic blueprint for a variety of therapeutic proteins, allowing the expression of multiple biologicals for combination therapy.

Claims

Claims1. A minimal adenoviral vector capable of replicating its own DNA in a host cell, wherein said vector is not capable to form viral particles and has a cargo capacity of at least 17 kb, preferably 20 kb, more preferably 22 kb.

2. The adenoviral vector according to claim 1, wherein said vector consists of the following adenoviral genes: SEQ ID No. 1 (E1A), SEQ ID No. 3 (E1B-19K), SEQ ID No. 5 (E1B-55K), SEQ ID No. 7 (DBP), SEQ. ID No. 10 or 11 (pTP), SEQ ID No. 13 or 14 (Ad Pol) and SEQ ID No. 16 or 17 (E4 ORF6), or adenoviral genes encoding functional variants of the encoded proteins.

3. The adenoviral vector according to any one of the preceding claims, wherein said vector consists of 7 adenoviral genes encoding the proteins of SEQ ID No. 2 (E1A-289R), SEQ ID No. 29 (E1A- 243R), SEQ ID No. 30 (E1A-217R), SEQ ID No. 31 (E1A-171R), SEQ ID No. 32 (E1A-55R), SEQ ID No. 4 (E1B-19K), SEQ ID No. 6 (E1B-55K), SEQ ID No. 9 (DBP), SEQ ID No. 12 (pTP), SEQ ID No. 15 (Ad Pol) and SEQ ID No. 18 (E4 ORF6), or functional variants or splice variants of any of aforementioned proteins.

4. The adenoviral vector according to any one of the preceding claims, wherein said vector contains a L-ITR, a QJ encapsulation signal and a R-ITR.

5. The adenoviral vector according to claim 4, wherein said L-ITR comprises the nucleic acid sequence of SEQ ID No. 19, the QJ encapsulation signal comprises the nucleic acid sequence of SEQ ID No. 28 and the R-ITR comprises the nucleic acid sequence of SEQ ID No. 20.

6. The adenoviral vector according to claim 5, wherein said vector contains no additional adenoviral genes.

7. The adenoviral vector according to any one of the preceding claims, wherein the genes encoding proteins E1A, E1B-19K and E1B-55K are located on the sense strand and the genes encoding proteins DBP, pTP, Ad Pol and E4 ORF6 are located on the antisense strand.

8. The adenoviral vector according to any one of the preceding claims, wherein said vector comprises a nucleic acid encoding a cargo gene to be expressed in said host cell.

9. The adenoviral vector according to claim 9, wherein said cargo gene is a non-adenoviral gene.

10. The adenoviral vector according to claim 8 or 9, wherein said cargo gene that is to be expressed in said host cell encodes a therapeutically active polypeptide.

11. The adenoviral vector according to any one of the preceding claims for use in medicine.

12. The adenoviral vector for use according to claim 11, wherein said use in medicine is the treatment of a tumor or cancer.

13. The adenoviral vector according to any one of claims 1-10 for use in the expression of a cargo gene in a host cell.

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