Modified sequences (variants) of the adenovirus E2a gene for producing adeno-associated virus (AAV) based vectors.

Abstract

The present invention relates to the fields of genetic engineering, genetics, and biotechnology. More specifically, the present invention relates to a modified sequence variant of the adenovirus E2a gene, a helper plasmid containing the modified sequence variant of the adenovirus E2a gene for producing an adeno-associated virus-based vector, and the use of the modified sequence variant of the adenovirus E2a gene and the use of the helper plasmid containing this sequence for producing an adeno-associated virus-based vector.

Description

Technical Field

[0001] The present invention relates to the fields of genetic engineering, genetics, and biotechnology. More specifically, the present invention relates to variants of a modified sequence of the adenovirus E2a gene, helper plasmids comprising variants of the modified sequence of the adenovirus E2a gene for producing adeno-associated virus-based vectors, as well as to the use of variants of the modified sequence of the adenovirus E2a gene, and the use of helper plasmids comprising this sequence for producing adeno-associated virus-based vectors.

Background Art

[0002] To date, various drug delivery systems have been widely used, and in the field of gene therapy, there is the highest demand for delivery of gene therapy constructs using adeno-associated virus (AAV). AAV is a small non-enveloped virus of the family Parvoviridae, characterized by a linear single-stranded DNA genome. The AAV genome comprises, flanked by inverted terminal repeats (ITRs), a sequence of rep regulatory factors whose products are involved in the process of viral replication, and a sequence of the cap gene encoding the structural proteins VP1, VP2, and VP3 that form the AAV capsid. Adeno-associated virus cannot replicate on its own, and in order to effectively carry out its lytic life cycle, simultaneous co-infection of cells by a so-called helper virus (specifically adenovirus (Ad) or herpes simplex virus (HSV)) is required (Manuel AFV Goncalves, Adeno-associated virus:from defective virus to effective vector, Virol J. 2005 May 6;2:43).

[0003] Adenoviruses are members of the family Adenoviridae and are characterized by the presence of an icosahedral capsid lacking a lipoprotein envelope and a double-stranded DNA genome. Unlike AAVs, adenoviruses have a remarkably complex genome, with the viral gene sequence and numerous regulatory elements contained within nucleic acids whose size is limited due to sequence duplication. Generally, the genomes of members of the genus Mastadenovirus include sequences of early genes (e.g., E1-E4) encoding proteins involved in replication and transcription processes, flanked by inverted terminal repeats (ITRs), and sequences of later genes (e.g., L1-L5) that primarily encode structural viral proteins. The direct use of helper adenoviruses in the production of adeno-associated virus-based vectors has not found broad applications in the clinical development of gene therapies because various impurities were present, particularly in the form of adenovirus particles. Subsequently, to overcome the aforementioned shortcomings of this method, a three-plasmid adenovirus particle-free system for producing adeno-associated virus-based vectors has been developed, because it has already been found that the production of AAV requires and is sufficient to have functional sequences of adenovirus E1 genes (including E1a and E1b), E2a, E4, and VA (virus-associated) RNA (Juan Jose Aponte-Ubillus et al, Molecular design for recombinant adeno-associated virus (rAAV) vector production. Appl Microbiol Biotechnol, 2018;102(3):1045-1054). Therefore, E1a is considered a transactivator, specifically promoting the expression of AAV rep and cap genes, while E1b interacts with the adenovirus E4 gene, which promotes the intracellular transport of viral mRNA at the appropriate time. The latter is also involved in DNA replication.The E2a and VA RNA genes contribute to the stability of viral mRNA during the efficient translation process (Xiao Xiao et al, Production of High-Titer Recombinant Adeno-Associated Virus Vectors in the Absence of Helper Adenovirus. J Virol, 1998 Mar;72(3):2224-2232).

[0004] To date, the most common adenovirus particle-free system for producing adeno-associated virus-based vectors is a set of plasmids for transfecting animal cells containing an exogenous sequence of the adenovirus E1 gene. Most typically, this set of transfection plasmids consists of a plasmid containing a cassette with the target gene flanked by an ITR sequence, a plasmid containing the sequences of the AAV rep and cap genes, and a helper plasmid containing regions of the adenovirus genome containing the sequences of the adenovirus E2, E4, and VA RNA genes and regulatory elements necessary for AAV production. Many modern commercial companies possess such three-plasmid systems for producing AAV-based vectors.

[0005] Regarding the in vivo treatment of genetic disorders, a clear advantage is the use of drugs with high concentrations of delivery particles, which makes it possible to achieve therapeutic effects. To manufacture higher-concentration formulations of gene therapies, it is necessary to have a manufacturing method that yields the drug and / or its components (including delivery particles) in high quantities. The use of large plasmid vectors in experimental work can complicate the experimenter's work and may even directly affect the productivity of the process of manufacturing vectors based on adeno-associated virus particles. [Overview of the project] [Problems that the invention aims to solve]

[0006] Therefore, there is a constant need to develop novel systems for producing adeno-associated virus-based vectors. These systems would include a different set of plasmids from the components of existing systems, for example, by containing smaller nucleic acid sequences encoding adenovirus proteins. This would allow for increased productivity in AAV production compared to the use of larger plasmids in conventional commercially available systems, while maintaining the full functionality of the components of this system. [Means for solving the problem]

[0007] One of the most pressing research goals in the field of developing effective gene therapies is to create novel methods for manufacturing gene therapies and to improve existing methods in order to increase the yield of the products.

[0008] The inventors have developed a modified sequence variant of the adenovirus E2a gene and a helper plasmid for producing an adeno-associated virus-based vector containing the modified sequence of the adenovirus E2a gene. The inventors have discovered that the use of the modified sequence variant of the adenovirus E2a gene surprisingly increases the productivity level during AAV production. Definitions and general methods Unless otherwise defined herein, all technical and scientific terms used in connection with the present invention shall have the same meaning as those generally understood by those skilled in the art.

[0009] Furthermore, unless otherwise specified in context, singular terms shall include plural terms, and plural terms shall include singular terms. Typically, the classifications used herein, and the cell culture, molecular biology, microbiology, and genetics methods described herein, are well known and widely used by those skilled in the art. Enzymatic reactions and purification methods shall be carried out in accordance with the manufacturer's guidelines, either as is common in the art or as described herein.

[0010] The terms “naturally occurring,” “natural,” or “wild-type” are used to describe substances that can be found in nature, unlike those that are artificially manufactured. For example, proteins or nucleotide sequences present in living organisms (including viruses) that can be isolated from natural sources and have not been intentionally modified by humans in a laboratory are naturally occurring.

[0011] As used herein and in the claims, unless otherwise indicated by context, the words “include” and “comprise,” or variations thereof, such as “includes,” “including,” “comprises,” or “comprising,” are understood to mean the inclusion of the integer or group of integers being described, but not the exclusion of any other integer or group of integers. [Brief explanation of the drawing]

[0012] [Figure 1] Figure 1 is a graph showing the productivity of rAAV serotypes 2, 5, 6, and 9 generation in HEK293 cells after transfection with the complete sequence required for rAA generation (including sequences encoding the E2a gene and regulatory elements corresponding to the nucleic acids below): nucleic acid containing the native sequence of the adenovirus E2a gene (SEQ ID NO: 1), nucleic acid containing deletions of sequence regions corresponding to the 1696-2321 bp, 2400-2634 bp, and 2839-3492 bp regions in the SEQ ID NO: 1 (SEQ ID NO: 2), and nucleic acid containing deletions of sequence regions corresponding to the 1696-3492 bp region in the SEQ ID NO: 1 (SEQ ID NO: 3).

[0013] Productivity of rAAV generation after 3 plasmid transfection: 1. pHelper plasmid containing the naturally occurring sequences of E4, VA RNA, and the E2a gene (where the naturally occurring sequence of the adenovirus E2a gene includes SEQ ID NO: 1) along with regulatory elements; pRepCap plasmid encoding the Rep and Cap genes of serotype 2; plasmid encoding a transgene based on an antibody against VEGF / C5, 2. pHelper plasmid containing the naturally occurring sequence of the E4, VA RNA gene, and a modified sequence of the E2a gene (including deletions in the sequence region corresponding to the n. region at 1696-2321 bp, 2400-2634 bp, and 2839-3492 bp in the sequence of SEQ ID NO: 1, along with regulatory elements; pRepCap plasmid encoding the Rep and Cap genes of serotype 2; plasmid encoding a transgene based on an antibody against VEGF / C5, 3. pHelper plasmid containing the naturally occurring sequence of the E4, VA RNA gene, and a modified sequence of the E2a gene (including a deletion in the sequence region corresponding to the n. region of 1696-3492 bp in the sequence of SEQ ID NO: 1, and including the sequence of SEQ ID NO: 3) along with regulatory elements; pRepCap plasmid encoding the Rep and Cap genes of serotype 2; plasmid encoding a transgene based on an antibody against VEGF / C5, 4. pHelper plasmid containing naturally occurring sequences of E4, VA RNA, and E2a genes (where the naturally occurring sequence of the adenovirus E2a gene includes SEQ ID NO: 1) along with regulatory elements; pRepCap plasmid encoding the Rep gene for serotype 2 and the Cap gene for serotype 5; plasmid encoding a transgene based on coagulation factor IX protein, 5. pHelper plasmid containing the naturally occurring sequence of the E4, VA RNA gene, and a modified sequence of the E2a gene (including deletions in the sequence region corresponding to n. regions at 1696-2321 bp, 2400-2634 bp, and 2839-3492 bp in the sequence of SEQ ID NO: 1, along with regulatory elements; pRepCap plasmid encoding the Rep gene for serotype 2 and the Cap gene for serotype 5; plasmid encoding a transgene based on coagulation factor IX protein, 6. pHelper plasmid containing the naturally occurring sequence of the E4, VA RNA gene, and a modified sequence of the E2a gene (including a deletion in the sequence region corresponding to the n. region of 1696-3492 bp in the sequence of SEQ ID NO: 1, and including the sequence of SEQ ID NO: 3) along with regulatory elements; pRepCap plasmid encoding the Rep gene for serotype 2 and the Cap gene for serotype 5; when using plasmids encoding transgenes based on coagulation factor IX protein, 7. pHelper plasmid containing naturally occurring sequences of E4, VA RNA, and E2a genes (where the naturally occurring sequence of adenovirus E2a gene includes SEQ ID NO: 1) along with regulatory elements; pRepCap plasmid encoding the Rep gene for serotype 2 and the Cap gene for serotype 6; plasmid encoding a transgene based on coagulation factor VIII protein, 8. pHelper plasmid containing the naturally occurring sequence of the E4, VA RNA gene, and a modified sequence of the E2a gene (including deletions in the sequence region corresponding to the n. region of 1696-2321 bp, 2400-2634 bp, and 2839-3492 bp in the sequence of SEQ ID NO: 1, along with regulatory elements; pRepCap plasmid encoding the Rep gene for serotype 2 and the Cap gene for serotype 6; plasmid encoding a transgene based on coagulation factor VIII protein, 9. pHelper plasmid containing the naturally occurring sequence of the E4 VA RNA gene and a modified sequence of the E2a gene (including a deletion in the sequence region corresponding to the n. region of 1696-3492 bp in the sequence of SEQ ID NO: 1, and including the sequence of SEQ ID NO: 3) along with regulatory elements; pRepCap plasmid encoding the Rep gene for serotype 2 and the Cap gene for serotype 6; when using plasmids encoding transgenes based on coagulation factor VIII protein, 10. pHelper plasmid containing naturally occurring sequences of E4, VA RNA, and E2a genes (where the naturally occurring sequence of the adenovirus E2a gene includes SEQ ID NO: 1) along with regulatory elements; pRepCap plasmid encoding the Rep gene for serotype 2 and the Cap gene for serotype 9; when using plasmids encoding transgenes based on the survival motor neuron (SMN1) protein, 11. pHelper plasmid containing the naturally occurring sequence of the E4, VA RNA gene, and a modified sequence of the E2a gene (including deletions in the sequence region corresponding to n. regions at 1696-2321 bp, 2400-2634 bp, and 2839-3492 bp in the sequence of SEQ ID NO: 1, along with regulatory elements; pRepCap plasmid encoding the Rep gene for serotype 2 and the Cap gene for serotype 9; when using plasmids encoding transgenes based on the survival motor neuron SMN1 protein, 12. pHelper plasmid containing the naturally occurring sequence of the E4 VA RNA gene and a modified sequence of the E2a gene (including a deletion in the sequence region corresponding to the n. region of 1696-3492 bp in the sequence of SEQ ID NO: 1, and including the sequence of SEQ ID NO: 3) along with regulatory elements; pRepCap plasmid encoding the Rep gene for serotype 2 and the Cap gene for serotype 9; plasmid encoding a transgene based on the survival motor neuron SMN1 protein. [Figure 2]Figure 2 is a schematic diagram of the genome region of adenovirus serotype 2 containing the sequence of the E2a gene along with regulatory elements (A; corresponding to 22393bp~27214bp of NCBI reference number AC_000007.1) and the genome region of adenovirus serotype 54 containing the sequence of the E2a gene along with regulatory elements (B; corresponding to 21166bp~25369bp of NCBI reference number NC_012959.1), illustrating the sequence deletions described herein and their correspondences to each other. The sequence deletion regions are numbered relative to sequence number 1 (in the case of A) and sequence number 19 (in the case of B). [Modes for carrying out the invention]

[0014] Detailed description of the invention nucleic acid In one aspect, the present invention relates to a nucleic acid comprising a modified sequence of the adenovirus E2a gene, wherein the sequence is among the sequences homologous to sequence number 1, with a degree of identity of at least 63%: i. Deletion of sequence regions corresponding to 1696-2321 bp, 2400-2634 bp, and 2839-3492 bp; or ii. Deletion of the sequence region corresponding to 1696~3492bp; or iii. Deletion of the sequence region corresponding to 1696~2321bp; or iv. Deletion of the sequence region corresponding to 2400~2634bp; or Deletion of the sequence region corresponding to v.2839~3492bp; or vi. Deletion of sequence regions corresponding to 1696~2321bp and 2400~2634bp; or vii. Deletion of sequence regions corresponding to 1696~2321bp and 2839~3492bp; or viii. Deletion of sequence regions corresponding to 2400-2634 bp and 2839-3492 bp This relates to nucleic acids, including modifications selected from the following.

[0015] The terms "nucleic acid", "polynucleotide", "oligonucleotide" and "polynucleotide sequence", which are used synonymously herein, refer to the exact sequence (whether modified or unmodified) of nucleotides that identifies a fragment or region of a nucleic acid (with or without unnatural nucleotides) and is either double-stranded DNA or RNA, single-stranded DNA or RNA, or a transcription product of said DNA.

[0016] As used herein, a polynucleotide includes all nucleic acid sequences obtained by any means available in the art, by way of non-limiting example, including, by way of non-limiting example, recombinant means, i.e., cloning of nucleic acid sequences from recombinant libraries or cell genomes using conventional cloning techniques, PCR and the like, and by synthetic means.

[0017] It should be noted that the present invention does not relate to a nucleotide sequence in its natural chromosomal environment (i.e., the natural state). Thus, an isolated nucleic acid, produced for example by recombinant genetics using a host cell or by chemical synthesis, is intended to be included within the present invention.

[0018] Unless otherwise indicated, the term "nucleotide sequence" encompasses the complement thereof. Therefore, it should be understood that a nucleic acid having a specific sequence encompasses the complementary strand having the complementary sequence thereto.

[0019] The term "homologous sequence" refers to two or more nucleotide sequences that are at least 61 percent identical, specifically 61 percent, 62 percent, 63 percent, 64 percent, 65 percent, 66 percent, 67 percent, 68 percent, 69 percent, 70 percent, 71 percent, 72 percent, 73 percent, 74 percent, 75 percent, 76 percent, 77 percent, 78 percent, 79 percent, 80 percent, 81 percent, 82 percent, 83 percent, 84 percent, 85 percent, 86 percent, 87 percent, 88 percent, 89 percent, 90 percent, 91 percent, 92 percent, 93 percent, 94 percent, 95 percent, 96 percent, 97 percent, 98 percent, 99 percent, and 100 percent. Therefore, according to the present invention, homologous nucleotide sequences may have a common evolutionary origin or may be generated by codon optimization in the initial sequence.

[0020] The term "identity" refers to a comparison of the nucleotide sequences of two nucleic acids. Identity is determined based on a reference sequence. The sequence of the adenovirus E2a gene is used as the reference sequence in accordance with this invention. Algorithms for sequence analysis are well known in the art.

[0021] One of the characteristics of the genetic code is degeneracy, that is, the ability of different codons (trinucleotides) to code for the same amino acid. Such codons that translate to the same amino acid are called synonymous codons. In natural sequences, one of the synonymous codons is randomly selected during the course of evolution, but the usage frequencies of synonymous codons differ, with some being more preferred and others less preferred for each amino acid. Codon optimization is a widely used technique to increase the productivity of protein molecules, and it provides a rational mapping of one of the appropriate synonymous codons to each amino acid in a protein sequence. One of the general principles of codon optimization is to use the most frequent codon, and other approaches such as harmonization (reproducing the distribution of codon usage frequencies) have been introduced later, but they do not necessarily increase productivity. In addition to codon frequency, the GC content of a sequence (the ratio of guanine and cytosine to the total length of the sequence) can also affect production efficiency. Specifically, in mammalian cells, high GC content has been shown to be associated with increased mRNA expression levels (Grzegorz Kudla ET AL., High Guanine and Cytosine Content Increases mRNA Levels in Mammalian Cells, June 2006, Volume 4, Issue 6, e180, pp.933-942). Furthermore, it is noteworthy that stable secondary structural elements of mRNA (i.e., those with low free folding energy) can reduce efficiency.

[0022] The term "adenovirus" refers to viruses of the family Adenoviridae. Viruses of this family are viral particles containing a double-stranded DNA genome and an icosahedral capsid lacking a lipoprotein envelope. The Adenoviridae family can be classified into six genera, specifically including the genus Mastadenoviruses, which infect mammals. The criteria for the boundaries of this family are phylogenetic analysis, characteristics of genomic composition, and viral infection in specific animals (Maria Benko et al, ICTV Virus Taxonomy Profile: Adenoviridae 2022, J Gen Virol. 2022 Mar;103(3):001721). Within the genus Mastadenoviruses, a group of primate adenoviruses is distinguished, and seven subgroups or species of adenoviruses (HAdV-A to G) are combined. Considering the presence of various biological characteristics, serotypes of the viruses are also characterized within these subgroups of adenoviruses (e.g., serotypes 2 and 5 in Mastadenovirus C, and serotype 54 in Mastadenovirus D). Adenoviruses infect the respiratory tract, gastrointestinal tract, and kidneys, and primarily enter cells using CAR receptors (coxsackievirus and AdV receptors) (Urs F. Greber, Adenoviruses-Infection, pathogenesis and therapy, FEBS Lett. 2020 Jun;594(12):1818-1827). More detailed information on various members of the Adenoviridae family is available in the literature (Balazs Harrach et al, Adenoviruses across the animal kingdom: a walk in the zoo, FEBS Lett. 2019 Dec;593(24):3660-3673).

[0023] The organization of adenovirus genomes is characterized by remarkable intergeneric diversity in both the content of functional and regulatory elements and genome size. Generally, adenovirus genomes contain sequences of early genes (E1-E4), intermediate genes (transcripts IX and IVa2), and late genes (L1-L5) flanked by inverted terminal repeats (ITRs). ITRs contain conserved sequences and function as origins for viral replication. Depending on the adenovirus species, virus-associated RNA (VA RNA) genes may also be represented in the genome (Thomas Lion, Adenovirus Infections in Immunocompetent and Immunocompromised Patients, Clin Microbiol Rev. 2014 Jul;27(3):441-462).

[0024] Adenovirus replication involves preterminal proteins (pTPs), adenovirus polymerase (Adv Pol), and DAN-binding proteins (encoded by the E2 gene), as well as several cellular transcription factors. In the early stages of replication, these proteins bind to various ITRs of the virus, thereby determining the formation of the pre-initiation complex, and subsequently the formation of a new double-stranded genome (Rob C. Hoeben et al., Adenovirus DNA Replication, Cold Spring Harb Perspect Biol. 2013 Mar;5(3):a013003). The adenovirus protein encoded by the E1 gene is required for the activation of the initial gene promoter during the viral lysis cycle; furthermore, transcription is also activated by the physical interaction of the E1 and E4 gene products. On the other hand, E4 encodes a complex of protein products that promote transcription and are involved in RNA splicing and transport (M Bondesson, Adenovirus E4 open reading frame 4 protein autoregulates E4 transcription by inhibiting E1A transactivation of the E4 promoter, J Virol. 1996 Jun;70(6):3844-51).

[0025] The terms "adenovirus" or "Ad" are used synonymously in this specification. The most efficient and widely used method for producing rAAV vectors is by transfection of mammalian cells, such as HEK293 cells, using a three-plasmid system. The first plasmid in such a system contains an expression cassette with the gene of interest (GOI) operably linked to an expression regulatory sequence, flanked by viral inverted terminal repeats (ITRs). ITRs are essential components for viral replication and packaging of the expression cassette into a viral capsid. The second plasmid contains the nucleotide sequences of the adeno-associated virus rep and cap genes, which encode the viral replication enzyme and the structural protein of its capsid for the desired serotype. The third plasmid (also known as the helper plasmid) contains adenovirus genes that facilitate replication and packaging of the recombinant AAV genome (Parminder Singh Chahal et al., Production of adeno-associated virus (AAV) serotypes by transient transfection of HEK293 cell suspension cultures for gene delivery, J Virol Methods. 2014 Feb;196:163-173).

[0026] A group of variants obtained as a result of a modified sequence of the adenovirus E2a gene, which are in the sequence of SEQ ID NO: 1 or sequences homologous to the sequence of SEQ ID NO: 1 with a degree of identity of at least 63%: i. Deletion of sequence regions corresponding to 1696-2321 bp, 2400-2634 bp, and 2839-3492 bp; or ii. Deletion of the sequence region corresponding to 1696~3492bp; or iii. Deletion of the sequence region corresponding to 1696~2321bp; or iv. Deletion of the sequence region corresponding to 2400~2634bp; or Deletion of the sequence region corresponding to v.2839~3492bp; or vi. Deletion of sequence regions corresponding to 1696~2321bp and 2400~2634bp; or vii. Deletion of sequence regions corresponding to 1696~2321bp and 2839~3492bp; or viii. Deletion of sequence regions corresponding to 2400-2634 bp and 2839-3492 bp Variants having regulatory elements including modifications selected from resulted in increased production levels during AAV-based vector generation compared to the sequence of SEQ ID NO: 1 or sequences homologous to SEQ ID NO: 1 with a degree of identity of at least 63%. Deletion of the said region does not adversely affect the expression of the E2a gene or the function of each protein.

[0027] Sequence ID 1 is the sequence of a wild-type gene containing regulatory elements, which encodes the adenovirus E2a protein and corresponds to the 22393bp–27214bp sequence of the adenovirus serotype 2 (NCBI reference number AC_000007.1) genome.

[0028] In one aspect of the present invention, a sequence homologous to the sequence of Sequence ID No. 1 with a degree of identity of at least 63% is the sequence of the E2a gene of an adenovirus serotype other than the serotype having Sequence ID No. 1, or a codon-optimized variant of the sequence of Sequence ID No. 1.

[0029] Those skilled in the art will readily understand that the genome sequences of various serotypes of adenoviruses (in particular, the genome sequences of various adenovirus serotypes of the same genus) may exhibit remarkable similarity and be characterized by a high degree of identity. Therefore, while adenovirus serotypes 1, 5, 6, 7, 11, 54, and 89 belong to the genus Mastadenovirus, the sequence of Sequence ID No. 1 is homologous to the sequence of the E2a gene containing the regulatory element of adenovirus serotype 1, and this sequence is 98% identical to Sequence ID No. 32; the sequence of the E2a gene containing the regulatory element of adenovirus serotype 5 is homologous to the sequence of the E2a gene containing the regulatory element of adenovirus serotype 5, and this sequence is 97% identical to Sequence ID No. 33; and the sequence of the E2a gene containing the regulatory element of adenovirus serotype 6 is homologous to the sequence of the E2a gene containing the regulatory element of adenovirus serotype 6, and this sequence is 99% identical to... Sequence ID 34 is homologous to the sequence of the E2a gene with regulatory elements for adenovirus serotype 7, and this sequence is sequence ID 35 with 64% identity; sequence ID 36 is homologous to the sequence of the E2a gene with regulatory elements for adenovirus serotype 11, and this sequence is sequence ID 19 with 63% identity; and sequence ID 37 is homologous to the sequence of the E2a gene with regulatory elements for adenovirus serotype 89, and this sequence is sequence ID 37 with 98% identity. In this case, the sequence of human adenovirus serotype 2 serves as a reference sequence for the nucleic acid positions of all other adenovirus genome sequences mentioned herein. The position of each adenovirus nucleic acid can be determined by conventional sequence alignment using publicly available BlastP software from the National Center for Biotechnology Information (NCBI).

[0030] Therefore, the group of sequences homologous to sequence number 1, with a degree of identity of at least 63%: i. Deletion of sequence regions corresponding to 1696-2321 bp, 2400-2634 bp, and 2839-3492 bp; or ii. Deletion of the sequence region corresponding to 1696~3492bp; or iii. Deletion of the sequence region corresponding to 1696~2321bp; or iv. Deletion of the sequence region corresponding to 2400~2634bp; or Deletion of the sequence region corresponding to v.2839~3492bp; or vi. Deletion of sequence regions corresponding to 1696~2321bp and 2400~2634bp; or vii. Deletion of sequence regions corresponding to 1696~2321bp and 2839~3492bp; or viii. Deletion of sequence regions corresponding to 2400-2634 bp and 2839-3492 bp Adenovirus E2a genes selected from and including modifications can be encoded by a wide range of diverse nucleotide sequences representing the sequence of the E2a gene having regulatory elements for adenovirus serotypes other than the serotype having Sequence ID No. 1. Such nucleotide sequence variants are within the scope of the present invention.

[0031] In some aspects of the present invention, the variant of the sequence of SEQ ID NO: 1 (a naturally occurring sequence of the E2a gene having a regulatory element for adenovirus serotypes other than the serotype having SEQ ID NO: 1) is the sequence of SEQ ID NO: 19 and corresponds to the sequence from 21166 bp to 25369 bp of the genome of adenovirus serotype 54 (NCBI reference sequence: NC_012959.1). The naturally occurring variant of the sequence of the E2a gene for adenovirus serotypes other than the serotype having SEQ ID NO: 1, having the sequence of SEQ ID NO: 19, is provided for illustrative purposes only and should not be interpreted as limiting the naturally occurring variants of the sequence of the E2a gene for adenovirus serotypes other than the serotype having SEQ ID NO: 1. Furthermore, the sequence of SEQ ID NO: 1 i. Deletion of sequence regions corresponding to 1696-2321 bp, 2400-2634 bp, and 2839-3492 bp; or ii. Deletion of the sequence region corresponding to 1696~3492bp; or iii. Deletion of the sequence region corresponding to 1696~2321bp; or iv. Deletion of the sequence region corresponding to 2400~2634bp; or Deletion of the sequence region corresponding to v.2839~3492bp; or vi. Deletion of sequence regions corresponding to 1696~2321bp and 2400~2634bp; or vii. Deletion of sequence regions corresponding to 1696~2321bp and 2839~3492bp; or viii. Deletion of sequence regions corresponding to 2400-2634 bp and 2839-3492 bp This is in the sequence of sequence number 19. i. Deletion of sequence regions corresponding to 1534-2036 bp, 2115-2340 bp, and 2545-3198 bp; or ii. Deletion of the sequence region corresponding to 1534~3198bp; or iii. Deletion of the sequence region corresponding to 1534~2036bp; or iv. Deletion of the sequence region corresponding to 2115~2340 bp; or Deletion of the sequence region corresponding to v.2545~3198bp; or vi. Deletion of sequence regions corresponding to 1534-2036 bp and 2115-2340 bp; or vii. Deletion of sequence regions corresponding to 1534-2036 bp and 2545-3198 bp; or viii. Deletion of sequence regions corresponding to 2115~2340bp and 2545~3198bp This corresponds to the specified value (see Figure 2).

[0032] Group in the codon-optimized variant of sequence 1: i. Deletion of sequence regions corresponding to 1696-2321 bp, 2400-2634 bp, and 2839-3492 bp; or ii. Deletion of the sequence region corresponding to 1696~3492bp; or iii. Deletion of the sequence region corresponding to 1696~2321bp; or iv. Deletion of the sequence region corresponding to 2400~2634bp; or Deletion of the sequence region corresponding to v.2839~3492bp; or vi. Deletion of sequence regions corresponding to 1696~2321bp and 2400~2634bp; or vii. Deletion of sequence regions corresponding to 1696~2321bp and 2839~3492bp; or viii. Deletion of sequence regions corresponding to 2400-2634 bp and 2839-3492 bp The adenovirus E2a gene, including modifications selected from the above, can also be encoded by a wide range of distinct nucleotide sequences due to genetic coding redundancy. Constructing these alternative sequences that encode the same one-and-the-same amino acid sequence is well within the scope of the art. Such nucleotide sequence variants are within the scope of the present invention.

[0033] In some aspects of the present invention, the codon-optimized variant of the sequence of sequence number 1 is the sequence of sequence number 10. The sequence having sequence number 1 and its codon-optimized variant having the sequence of sequence number 10 have 92% identity. The codon-optimized variant of the sequence of sequence number 1 having the sequence of sequence number 10 is provided for illustrative purposes only and should not be construed as limiting the codon-optimized variant of the sequence of sequence number 1.

[0034] Therefore, nucleic acids containing a modified sequence of the adenovirus E2a gene, wherein this sequence is homologous to the sequence of Sequence ID No. 1 with a degree of identity of at least 63%: i. Deletion of sequence regions corresponding to 1696-2321 bp, 2400-2634 bp, and 2839-3492 bp; or ii. Deletion of the sequence region corresponding to 1696~3492bp; or iii. Deletion of the sequence region corresponding to 1696~2321bp; or iv. Deletion of the sequence region corresponding to 2400~2634bp; or Deletion of the sequence region corresponding to v.2839~3492bp; or vi. Deletion of sequence regions corresponding to 1696~2321bp and 2400~2634bp; or vii. Deletion of sequence regions corresponding to 1696~2321bp and 2839~3492bp; or viii. Deletion of sequence regions corresponding to 2400-2634 bp and 2839-3492 bp Nucleic acids, including modifications selected from the present invention, are also part of the present invention.

[0035] In some aspects of the present invention, a nucleic acid comprising a modified sequence of the adenovirus E2a gene, wherein the sequence comprises deletions of sequence regions homologous to the 1696-2321 bp, 2400-2634 bp, and 2839-3492 bp regions in the sequence of SEQ ID NO: 1 or in a sequence homologous to the sequence of SEQ ID NO: 1 with a degree of identity of at least 63%, wherein the nucleic acid comprises the sequence of SEQ ID NO: 2 or comprises a sequence homologous to the sequence of SEQ ID NO: 2 with a degree of identity of at least 62%.

[0036] According to the present invention, a sequence homologous to the sequence of Sequence ID No. 2, having a degree of identity of at least 62%, is either a sequence of the E2a gene of an adenovirus serotype other than the serotype having Sequence ID No. 2, or a codon-optimized variant of the sequence of Sequence ID No. 2.

[0037] In some aspects of the present invention, a nucleic acid comprising a modified sequence of the adenovirus E2a gene other than the serotype having SEQ ID NO: 2, wherein this sequence includes deletions of sequence regions corresponding to the regions 1696-2321 bp, 2400-2634 bp, and 2839-3492 bp in the sequence of SEQ ID NO: 1, is the sequence of SEQ ID NO: 20. The sequence of SEQ ID NO: 20 is a nucleic acid comprising a modified sequence of the E2a gene of adenovirus serotype 54, wherein this sequence includes deletions of sequence regions corresponding to the regions 1534-2036 bp, 2115-2340 bp, and 2545-3198 bp in the sequence of SEQ ID NO: 19. The sequence of the E2a gene of the adenovirus serotype having SEQ ID NO: 2 and the sequence of the E2a gene of the serotype having SEQ ID NO: 20 have 62% identity. The modified sequence variants of the E2a gene of adenovirus serotypes other than the serotype having sequence number 2, which have sequence number 20, are provided for illustrative purposes only and should not be interpreted as restricting the sequence variants of the E2a gene of adenovirus serotypes other than the serotype having sequence number 2.

[0038] In some aspects of the present invention, the codon-optimized variant of the sequence of sequence number 2 is the sequence of sequence number 11. The sequence having sequence number 2 and its codon-optimized variant having the sequence of sequence number 11 have 88% identity. The codon-optimized variant of the sequence of sequence number 2 having the sequence of sequence number 11 is provided for illustrative purposes only and should not be construed as limiting the codon-optimized variant of the sequence of sequence number 2.

[0039] In some aspects of the present invention, a nucleic acid comprising a modified sequence of the adenovirus E2a gene, wherein the sequence comprises a deletion of a sequence region corresponding to a 1696-3492 bp region in the sequence of SEQ ID NO: 1 or in a sequence homologous to the sequence of SEQ ID NO: 1 with a degree of identity of at least 63%, wherein the nucleic acid comprises the sequence of SEQ ID NO: 3 or comprises a sequence homologous to the sequence of SEQ ID NO: 3 with a degree of identity of at least 61%.

[0040] According to the present invention, a sequence homologous to the sequence of Sequence ID No. 3, having a degree of identity of at least 61%, is either the sequence of the E2 gene of an adenovirus serotype other than the serotype having Sequence ID No. 3, or a codon-optimized variant of the sequence of Sequence ID No. 3.

[0041] In some aspects of the present invention, a nucleic acid comprising a modified sequence of the adenovirus E2a gene other than the serotype having SEQ ID NO: 3, wherein the sequence includes a deletion of a sequence region corresponding to the 1696-3492 bp region in the sequence of SEQ ID NO: 1, is the sequence of SEQ ID NO: 21. The sequence of SEQ ID NO: 21 is a nucleic acid comprising a modified sequence of the E2a gene of adenovirus serotype 54, wherein the sequence includes a deletion of a sequence region corresponding to the 1534-3198 bp region in the sequence of SEQ ID NO: 19. The sequence of the E2a gene of the adenovirus serotype having SEQ ID NO: 3 and the sequence of the E2a gene of the serotype having SEQ ID NO: 21 have 61% identity. The variant of the modified sequence of the E2a gene of the adenovirus serotype other than the serotype having SEQ ID NO: 3, which comprises the sequence of SEQ ID NO: 21, is provided for illustrative purposes only and should not be interpreted as limiting the variants of the E2a gene of the adenovirus serotype other than the serotype having SEQ ID NO: 3.

[0042] In some aspects of the present invention, the codon-optimized variant of the sequence of sequence number 3 is the sequence of sequence number 12. The sequence having sequence number 3 and its codon-optimized variant having the sequence of sequence number 12 have 87% identity. The codon-optimized variant of the sequence of sequence number 3 having the sequence of sequence number 12 is provided for illustrative purposes only and should not be construed as limiting the codon-optimized variant of the sequence of sequence number 3.

[0043] In some aspects of the present invention, a nucleic acid comprising a modified sequence of the adenovirus E2a gene, wherein the sequence comprises a deletion of a sequence region corresponding to a 1696-2321 bp region in the sequence of SEQ ID NO: 1 or in a sequence homologous to the sequence of SEQ ID NO: 1 with a degree of identity of at least 63%, wherein the nucleic acid comprises the sequence of SEQ ID NO: 4 or comprises a sequence homologous to the sequence of SEQ ID NO: 4 with a degree of identity of at least 64%.

[0044] According to the present invention, a sequence homologous to the sequence of Sequence ID No. 4, having at least 64% identity, is either the sequence of the E2a gene of an adenovirus serotype other than the serotype having Sequence ID No. 4, or a codon-optimized variant of Sequence ID No. 4.

[0045] In some aspects of the present invention, a nucleic acid comprising a modified sequence of the adenovirus E2a gene other than the serotype having SEQ ID NO: 4, wherein the sequence includes a deletion of a sequence region corresponding to the 1696-2321 bp region in the sequence of SEQ ID NO: 1, is the sequence of SEQ ID NO: 22. The sequence of SEQ ID NO: 22 is a nucleic acid comprising a modified sequence of the E2a gene of adenovirus serotype 54, wherein the sequence includes a deletion of a sequence region corresponding to the 1534-2036 bp region in the sequence of SEQ ID NO: 19. The sequence of the E2a gene of the adenovirus serotype having SEQ ID NO: 4 and the sequence of the E2a gene of the serotype having SEQ ID NO: 22 have 64% identity. The variant of the modified sequence of the E2a gene of the adenovirus serotype other than the serotype having SEQ ID NO: 4, which comprises the sequence of SEQ ID NO: 22, is provided for illustrative purposes only and should not be interpreted as limiting the variants of the E2a gene of the adenovirus serotype other than the serotype having SEQ ID NO: 4.

[0046] In some aspects of the present invention, the codon-optimized variant of the sequence of sequence number 4 is the sequence of sequence number 13. The sequence having sequence number 4 and its codon-optimized variant having the sequence of sequence number 13 have 91% identity. The codon-optimized variant of the sequence of sequence number 4 having the sequence of sequence number 13 is provided for illustrative purposes only and should not be construed as limiting the codon-optimized variant of the sequence of sequence number 4.

[0047] In some aspects of the present invention, a nucleic acid comprising a modified sequence of the adenovirus E2a gene, wherein the sequence comprises a deletion of a sequence region corresponding to a 2400-2634 bp region in the sequence of SEQ ID NO: 1 or in a sequence homologous to the sequence of SEQ ID NO: 1 with a degree of identity of at least 63%, wherein the nucleic acid comprises the sequence of SEQ ID NO: 5 or comprises a sequence homologous to the sequence of SEQ ID NO: 5 with a degree of identity of at least 64%.

[0048] According to the present invention, a sequence homologous to the sequence of Sequence ID No. 5, having at least 64% identity, is either a sequence of the E2a gene of an adenovirus serotype other than the serotype having Sequence ID No. 5, or a codon-optimized variant of the sequence of Sequence ID No. 5.

[0049] In some aspects of the present invention, a nucleic acid comprising a modified sequence of the adenovirus E2a gene of a serotype other than the serotype having SEQ ID NO: 5, wherein the sequence includes a deletion of a sequence region corresponding to the 2400-2634 bp region in the sequence of SEQ ID NO: 1, is the sequence of SEQ ID NO: 23. The sequence of SEQ ID NO: 23 is a nucleic acid comprising a modified sequence of the E2a gene of adenovirus serotype 54, wherein the sequence includes a deletion of a sequence region corresponding to the 2115-2340 bp region in the sequence of SEQ ID NO: 19. The sequence of the E2a gene of the adenovirus serotype having SEQ ID NO: 5 and the sequence of the E2a gene of the serotype having SEQ ID NO: 23 have 64% identity. The variant of the modified sequence of the E2a gene of an adenovirus serotype other than the serotype having SEQ ID NO: 5, which comprises the sequence of SEQ ID NO: 23, is provided for illustrative purposes only and should not be interpreted as limiting the variants of the E2a gene of an adenovirus serotype other than the serotype having SEQ ID NO: 5.

[0050] In some aspects of the present invention, the codon-optimized variant of the sequence of sequence number 5 is the sequence of sequence number 14. The sequence having sequence number 5 and its codon-optimized variant having the sequence of sequence number 14 have 91.5% identity. The codon-optimized variant of the sequence of sequence number 5 having sequence number 14 is provided for illustrative purposes only and should not be construed as limiting the codon-optimized variant of the sequence of sequence number 5.

[0051] In some aspects of the present invention, a nucleic acid comprising a modified sequence of the adenovirus E2a gene, wherein the sequence comprises a deletion of a sequence region corresponding to a 2839-3492 bp region in the sequence of SEQ ID NO: 1 or in a sequence homologous to the sequence of SEQ ID NO: 1 with a degree of identity of at least 63%, wherein the nucleic acid comprises the sequence of SEQ ID NO: 6 or comprises a sequence homologous to the sequence of SEQ ID NO: 6 with a degree of identity of at least 61%.

[0052] According to the present invention, a sequence homologous to the sequence of SEQ ID NO: 6, having at least 61% identity, is either the sequence of the E2a gene of an adenovirus serotype other than the serotype having SEQ ID NO: 6, or a codon-optimized variant of the sequence of SEQ ID NO: 6.

[0053] In some aspects of the present invention, a nucleic acid comprising a modified sequence of the adenovirus E2a gene other than the serotype having SEQ ID NO: 6, wherein the sequence includes a deletion of a sequence region corresponding to the 2839-3492 bp region in the sequence of SEQ ID NO: 1, is the sequence of SEQ ID NO: 24. The sequence of SEQ ID NO: 24 is a nucleic acid comprising a modified sequence of the E2a gene of adenovirus serotype 54, wherein the sequence includes a deletion of a sequence region corresponding to the 2545-3198 bp region in the sequence of SEQ ID NO: 19. The sequence of the E2a gene of the adenovirus serotype having SEQ ID NO: 6 and the sequence of the E2a gene of the serotype having SEQ ID NO: 24 have 61% identity. The variant of the modified sequence of the E2a gene of the adenovirus serotype other than the serotype having SEQ ID NO: 6, which comprises the sequence of SEQ ID NO: 24, is provided for illustrative purposes only and should not be interpreted as limiting the variants of the E2a gene of the adenovirus serotype other than the serotype having SEQ ID NO: 6.

[0054] In some aspects of the present invention, the codon-optimized variant of the sequence of sequence number 6 is the sequence of sequence number 15. The sequence having sequence number 6 and its codon-optimized variant having the sequence of sequence number 15 have 91% identity. The codon-optimized variant of the sequence of sequence number 6 having the sequence of sequence number 15 is provided for illustrative purposes only and should not be construed as limiting the codon-optimized variant of the sequence of sequence number 6.

[0055] In some aspects of the present invention, a nucleic acid comprising a modified sequence of the adenovirus E2a gene, wherein the sequence comprises a deletion of a sequence region corresponding to the 1696-2321 bp and 2400-2634 bp regions in the sequence of SEQ ID NO: 1 or in a sequence homologous to the sequence of SEQ ID NO: 1 with a degree of identity of at least 63%, wherein the nucleic acid comprises the sequence of SEQ ID NO: 7 or comprises a sequence homologous to the sequence of SEQ ID NO: 7 with a degree of identity of at least 63%.

[0056] According to the present invention, a sequence homologous to the sequence of SEQ ID NO: 7, having at least 63% identity, is either the sequence of the E2 gene of an adenovirus serotype other than the serotype having SEQ ID NO: 7, or a codon-optimized variant of the sequence of SEQ ID NO: 7.

[0057] In some aspects of the present invention, a nucleic acid comprising a modified sequence of the adenovirus E2a gene other than the serotype having SEQ ID NO: 7, wherein this sequence includes deletions of sequence regions corresponding to the 1696-2321 bp and 2400-2634 bp regions in the sequence of SEQ ID NO: 1, is the sequence of SEQ ID NO: 25. The sequence of SEQ ID NO: 25 is a nucleic acid comprising a modified sequence of the E2a gene of adenovirus serotype 54, wherein this sequence includes deletions of sequence regions corresponding to the 1534-2036 bp and 2115-2340 bp regions in the sequence of SEQ ID NO: 19. The sequence of the E2a gene of the adenovirus serotype having SEQ ID NO: 7 and the sequence of the E2a gene of the serotype having SEQ ID NO: 25 have 63% identity. The modified sequence variants of the E2a gene of adenovirus serotypes other than the serotype having sequence number 7, which have sequence number 25, are provided for illustrative purposes only and should not be interpreted as restricting the sequence variants of the E2a gene of adenovirus serotypes other than the serotype having sequence number 7.

[0058] In some aspects of the present invention, the codon-optimized variant of the sequence of sequence number 7 is the sequence of sequence number 16. The sequence having sequence number 7 and its codon-optimized variant having the sequence of sequence number 16 have 90% identity. The codon-optimized variant of the sequence of sequence number 7 having the sequence of sequence number 16 is provided for illustrative purposes only and should not be construed as limiting the codon-optimized variant of the sequence of sequence number 7.

[0059] In some aspects of the present invention, a nucleic acid comprising a modified sequence of the adenovirus E2a gene, wherein the sequence comprises a deletion of a sequence region corresponding to the regions 1696-2321 bp and 2839-3492 in the sequence of SEQ ID NO: 1 or a sequence homologous to the sequence of SEQ ID NO: 1 with a degree of identity of at least 63%, wherein the nucleic acid comprises the sequence of SEQ ID NO: 8 or a sequence homologous to the sequence of SEQ ID NO: 8 with a degree of identity of at least 62%.

[0060] According to the present invention, a sequence homologous to the sequence of SEQ ID NO: 8, having at least 62% identity, is either the sequence of the E2a gene of an adenovirus serotype other than the serotype having SEQ ID NO: 8, or a codon-optimized variant of SEQ ID NO: 8.

[0061] In some aspects of the present invention, a nucleic acid comprising a modified sequence of the adenovirus E2a gene other than the serotype having SEQ ID NO: 8, wherein this sequence includes deletions of sequence regions corresponding to the 1696-2321 bp and 2839-3492 bp regions in the sequence of SEQ ID NO: 1, is the sequence of SEQ ID NO: 26. The sequence of SEQ ID NO: 26 is a nucleic acid comprising a modified sequence of the E2a gene of adenovirus serotype 54, wherein this sequence includes deletions of sequence regions corresponding to the 1534-2036 bp and 2545-3198 bp regions in the sequence of SEQ ID NO: 19. The sequence of the E2a gene of the adenovirus serotype having SEQ ID NO: 8 and the sequence of the E2a gene of the serotype having SEQ ID NO: 26 have 62% identity. The modified sequence variants of the E2a gene of adenovirus serotypes other than the serotype having sequence number 8, which have sequence number 26, are provided for illustrative purposes only and should not be interpreted as restricting the sequence variants of the E2a gene of adenovirus serotypes other than the serotype having sequence number 8.

[0062] In some aspects of the present invention, the codon-optimized variant of the sequence of sequence number 8 is the sequence of sequence number 17. The sequence having sequence number 8 and its codon-optimized variant having the sequence of sequence number 17 have 89% identity. The codon-optimized variant of the sequence of sequence number 8 having the sequence of sequence number 17 is provided for illustrative purposes only and should not be construed as limiting the codon-optimized variant of the sequence of sequence number 8.

[0063] In some aspects of the present invention, a nucleic acid comprising a modified sequence of the adenovirus E2a gene, wherein the sequence comprises deletions of sequence regions corresponding to the 2400-2634 bp and 2839-3492 bp regions in the sequence of SEQ ID NO: 1 or in a sequence homologous to the sequence of SEQ ID NO: 1 with a degree of identity of at least 63%, wherein the nucleic acid comprises the sequence of SEQ ID NO: 9 or comprises a sequence homologous to the sequence of SEQ ID NO: 9 with a degree of identity of at least 61%.

[0064] According to the present invention, sequences homologous to the sequence of Sequence ID No. 9, having at least 61% identity, are either sequences of the E2a gene of adenovirus serotypes other than the serotype having Sequence ID No. 9, or codon-optimized variants of the sequence of Sequence ID No. 9.

[0065] In some aspects of the present invention, a nucleic acid comprising a modified sequence of the adenovirus E2a gene other than the serotype having SEQ ID NO: 9, wherein this sequence includes deletions of sequence regions corresponding to the 2400-2634 bp and 2839-3492 bp regions in the sequence of SEQ ID NO: 1, is the sequence of SEQ ID NO: 27. The sequence of SEQ ID NO: 27 is a nucleic acid comprising a modified sequence of the E2a gene of adenovirus serotype 54, wherein this sequence includes deletions of sequence regions corresponding to the 2115-2340 bp and 2545-3198 bp regions in the sequence of SEQ ID NO: 19. The sequence of the E2a gene of the adenovirus serotype having SEQ ID NO: 9 and the sequence of the E2a gene of the serotype having SEQ ID NO: 27 have 61% identity. The modified sequence variants of the E2a gene of adenovirus serotypes other than the serotype having sequence number 9, which have sequence number 27, are provided for illustrative purposes only and should not be interpreted as restricting the sequence variants of the E2a gene of adenovirus serotypes other than the serotype having sequence number 9.

[0066] In some aspects of the present invention, the codon-optimized variant of the sequence of sequence number 9 is the sequence of sequence number 18. The sequence having sequence number 9 and its codon-optimized variant having the sequence of sequence number 18 have 90% identity. The codon-optimized variant of the sequence of sequence number 9 having sequence number 18 is provided for illustrative purposes only and should not be construed as limiting the codon-optimized variant of the sequence of sequence number 9.

[0067] In one aspect, the present invention relates to a nucleic acid comprising a modified sequence of the adenovirus E2a gene, wherein the sequence comprises the sequence of Sequence ID No. 2, or a sequence homologous to the sequence of Sequence ID No. 2 with at least 62% identity. According to the present invention, the sequence homologous to the sequence of Sequence ID No. 2 with at least 62% identity is either each sequence of the E2a gene of an adenovirus serotype other than the serotype having Sequence ID No. 2, or a codon-optimized variant of the sequence of Sequence ID No. 2.

[0068] In one aspect, the present invention relates to a nucleic acid comprising a modified sequence of the adenovirus E2a gene, wherein the sequence comprises the sequence of SEQ ID NO: 3, or a sequence homologous to the sequence of SEQ ID NO: 3 with at least 61% identity. According to the present invention, the sequence homologous to the sequence of SEQ ID NO: 3 with at least 61% identity is either each sequence of the E2a gene of an adenovirus serotype other than the serotype having SEQ ID NO: 3, or a codon-optimized variant of the sequence of SEQ ID NO: 3.

[0069] In one aspect, the present invention relates to a nucleic acid comprising a modified sequence of the adenovirus E2a gene, wherein the sequence comprises the sequence of SEQ ID NO: 4, or a sequence homologous to the sequence of SEQ ID NO: 4 with at least 64% identity. According to the present invention, the sequence homologous to the sequence of SEQ ID NO: 4 with at least 64% identity is either each sequence of the E2a gene of an adenovirus serotype other than the serotype having SEQ ID NO: 4, or a codon-optimized variant of the sequence of SEQ ID NO: 4.

[0070] In one aspect, the present invention relates to a nucleic acid comprising a modified sequence of the adenovirus E2a gene, wherein the sequence comprises the sequence of SEQ ID NO: 5, or a sequence homologous to the sequence of SEQ ID NO: 5 with at least 64% identity. According to the present invention, the sequence homologous to the sequence of SEQ ID NO: 5 with at least 64% identity is either each sequence of the E2a gene of an adenovirus serotype other than the serotype having SEQ ID NO: 5, or a codon-optimized variant of the sequence of SEQ ID NO: 5.

[0071] In one aspect, the present invention relates to a nucleic acid comprising a modified sequence of the adenovirus E2a gene, wherein the sequence comprises the sequence of SEQ ID NO: 6, or a sequence homologous to the sequence of SEQ ID NO: 6 with at least 61% identity. According to the present invention, the sequence homologous to the sequence of SEQ ID NO: 6 with at least 61% identity is either each sequence of the E2a gene of an adenovirus serotype other than the serotype having SEQ ID NO: 6, or a codon-optimized variant of the sequence of SEQ ID NO: 6.

[0072] In one aspect, the present invention relates to a nucleic acid comprising a modified sequence of the adenovirus E2a gene, wherein the sequence comprises the sequence of SEQ ID NO: 7, or a sequence homologous to the sequence of SEQ ID NO: 7 with at least 63% identity. According to the present invention, the sequence homologous to the sequence of SEQ ID NO: 7 with at least 63% identity is either each sequence of the E2a gene of an adenovirus serotype other than the serotype having SEQ ID NO: 7, or a codon-optimized variant of the sequence of SEQ ID NO: 7.

[0073] In one aspect, the present invention relates to a nucleic acid comprising a modified sequence of the adenovirus E2a gene, wherein the sequence comprises the sequence of SEQ ID NO: 8, or a sequence homologous to the sequence of SEQ ID NO: 8 with at least 62% identity. According to the present invention, the sequence homologous to the sequence of SEQ ID NO: 8 with at least 62% identity is either each sequence of the E2a gene of an adenovirus serotype other than the serotype having SEQ ID NO: 8, or a codon-optimized variant of the sequence of SEQ ID NO: 8.

[0074] In one aspect, the present invention relates to a nucleic acid comprising a modified sequence of the adenovirus E2a gene, wherein the sequence comprises the sequence of SEQ ID NO: 9, or a sequence homologous to the sequence of SEQ ID NO: 9 with at least 61% identity. According to the present invention, the sequence homologous to the sequence of SEQ ID NO: 9 with at least 61% identity is either each sequence of the E2a gene of an adenovirus serotype other than the serotype having SEQ ID NO: 9, or a codon-optimized variant of the sequence of SEQ ID NO: 9.

[0075] In some embodiments, the present invention is an isolated nucleic acid. An "isolated" nucleic acid molecule is one that has been identified and separated from at least one nucleic acid molecular impurity. Isolated nucleic acid molecules differ from the forms or aggregates found under natural conditions. Therefore, isolated nucleic acid molecules differ from nucleic acid molecules that exist in cells under natural conditions.

[0076] In the array of sequence number 1 i. 1696~2321bp, 2400~2634bp, 2839~3492bp (Sequence ID 2), ii. 1696~3492bp (SEQ ID NO: 3), iii. 1696~2321bp (Sequence ID 4), iv.2400~2634bp (SEQ ID NO: 5) v.2839~3492bp (Sequence ID 6), vi.1696~2321bp, 2400~2634bp (Sequence ID 7), vii. 1696~2321bp, 2839~3492bp (Sequence ID 8), viii. 2400~2634bp, 2839~3492bp (Sequence ID 9) The sequences of SEQ ID NOs: 2, 3, 4, 5, 6, 7, 8, and 9, which represent modified sequences of the adenovirus E2a gene having deletions in the sequence region corresponding to the region, are provided herein and below for illustrative purposes only. As previously stated, those skilled in the art will recognize the group in the sequence of SEQ ID NO: 1: i. Deletion of sequence regions corresponding to 1696-2321 bp, 2400-2634 bp, and 2839-3492 bp; or ii. Deletion of the sequence region corresponding to 1696~3492bp; or iii. Deletion of the sequence region corresponding to 1696~2321bp; or iv. Deletion of the sequence region corresponding to 2400~2634bp; or Deletion of the sequence region corresponding to v.2839~3492bp; or vi. Deletion of sequence regions corresponding to 1696~2321bp and 2400~2634bp; or vii. Deletion of sequence regions corresponding to 1696~2321bp and 2839~3492bp; or viii. Deletion of sequence regions corresponding to 2400-2634 bp and 2839-3492 bp You will understand that the sequence with the selected modifications can mean any sequence that is close to sequence numbers 2, 3, 4, 5, 6, 7, 8, and 9.

[0077] In the codon-optimized sequence of sequence number 10 i. 1696~2321bp, 2400~2634bp, 2839~3492bp (Sequence ID 11), ii. 1696~3492bp (Sequence ID 12), iii. 1696~2321bp (Sequence ID 13), iv.2400~2634bp (SEQ ID NO: 14) v.2839~3492bp (Sequence ID 15), vi. 1696~2321bp, 2400~2634bp (Sequence ID 16), vii. 1696~2321bp, 2839~3492bp (Sequence ID 17), viii. 2400~2634bp, 2839~3492bp (Sequence ID 18) The codon-optimized sequences of SEQ ID NOs: 11, 12, 13, 14, 15, 16, 17, and 18, which represent modified sequences of the adenovirus E2a gene having deletions of sequence regions corresponding to the region, are provided herein and below for illustrative purposes only. As previously stated, those skilled in the art will recognize the group in the sequence of SEQ ID NO: 10: i. Deletion of sequence regions corresponding to 1696-2321 bp, 2400-2634 bp, and 2839-3492 bp; or ii. Deletion of the sequence region corresponding to 1696~3492bp; or iii. Deletion of the sequence region corresponding to 1696~2321bp; or iv. Deletion of the sequence region corresponding to 2400~2634bp; or Deletion of the sequence region corresponding to v.2839~3492bp; or vi. Deletion of sequence regions corresponding to 1696~2321bp and 2400~2634bp; or vii. Deletion of sequence regions corresponding to 1696~2321bp and 2839~3492bp; or viii. Deletion of sequence regions corresponding to 2400-2634 bp and 2839-3492 bp You will understand that the sequence with the selected modifications can mean any sequence close to sequence numbers 11, 12, 13, 14, 15, 16, 17, and 18.

[0078] In the sequence of sequence number 19 i. 1534~2036bp, 2115~2340bp, 2545~3198bp (Sequence ID 20), ii. 1534~3198bp (Sequence ID 21), iii. 1534~2036bp (Sequence ID 22), iv.2115~2340bp (Sequence ID 23), v.2545~3198bp (Sequence ID 24), vi.1534~2036bp, 2115~2340bp (Sequence ID 25), vii. 1534~2036bp, 2545~3198bp (Sequence ID 26), viii. 2115~2340bp, 2545~3198bp (Sequence ID 27) The sequences of SEQ ID NOs. 20, 21, 22, 23, 24, 25, 26, and 27, which represent modified sequences of the adenovirus E2a serotype 54 gene having deletions of sequence regions corresponding to the region, are provided herein and below for illustrative purposes only. As previously stated, those skilled in the art will recognize the group in the sequence of SEQ ID NO. 19: i. Deletion of sequence regions corresponding to 1534-2036 bp, 2115-2340 bp, and 2545-3198 bp; or ii. Deletion of the sequence region corresponding to 1534~3198bp; or iii. Deletion of the sequence region corresponding to 1534~2036bp; or iv. Deletion of the sequence region corresponding to 2115~2340 bp; or Deletion of the sequence region corresponding to v.2545~3198bp; or vi. Deletion of sequence regions corresponding to 1534-2036 bp and 2115-2340 bp; or vii. Deletion of sequence regions corresponding to 1534-2036 bp and 2545-3198 bp; or viii. Deletion of sequence regions corresponding to 2115~2340bp and 2545~3198bp You will understand that the sequence with the selected modification can mean any sequence close to sequence numbers 20, 21, 22, 23, 24, 25, 26, and 27.

[0079] All nucleic acids containing a modified sequence of the adenovirus E2a gene according to the present invention encode the adenovirus E2a protein. In some embodiments, an adeno-associated virus-based vector is produced using a nucleic acid comprising a modified sequence of the adenovirus E2a gene according to the present invention. Helper plasmid In one aspect, the present invention relates to a helper plasmid for producing an adeno-associated virus-based vector comprising a nucleic acid containing a modified sequence of the adenovirus E2a gene, wherein the sequence is from the sequence of SEQ ID NO: 1 or from a group of sequences homologous to the sequence of SEQ ID NO: 1 with a degree of identity of at least 63%: i. Deletion of sequence regions corresponding to 1696-2321 bp, 2400-2634 bp, and 2839-3492 bp; or ii. Deletion of the sequence region corresponding to 1696~3492bp; or iii. Deletion of the sequence region corresponding to 1696~2321bp; or iv. Deletion of the sequence region corresponding to 2400~2634bp; or Deletion of the sequence region corresponding to v.2839~3492bp; or vi. Deletion of sequence regions corresponding to 1696~2321bp and 2400~2634bp; or vii. Deletion of sequence regions corresponding to 1696~2321bp and 2839~3492bp; or viii. Deletion of sequence regions corresponding to 2400-2634 bp and 2839-3492 bp This relates to helper plasmids, including modifications selected from the following.

[0080] The terms "helper plasmid" and "pHelper" are used synonymously herein. In this invention, "helper plasmid" and "pHelper" refer to plasmids having the functionality necessary for producing AAV-based vectors.

[0081] The term "adeno-associated virus" refers to viruses of the Parvoviridae family. The genomic makeup of all known AAV serotypes is very similar. The AAV genome is a linear, single-stranded DNA molecule with a length of less than 5000 nucleotides (nt). Inverted terminal repeats (ITRs) flank the intrinsic coding nucleotide sequences for the replication of non-structural proteins (Rep) and structural proteins (Cap). The Cap gene encodes the VP proteins (VP1, VP2, and VP3) that form the capsid. The last 145 nucleotides are self-complementary and constructed to allow for the formation of an energetically stable intramolecular double helix that forms a T-shaped hairpin. Such a hairpin structure functions as an origin for viral DNA replication and acts as a primer for cellular DNA polymerase complexes. After wild-type AAV (wtAAV) infects mammalian cells, Rep genes (e.g., Rep78 and Rep52) are expressed using the P5 and P19 promoters, respectively, and both Rep proteins have specific functions in viral genome replication. As a result of splicing events in the Rep open reading frame (Rep ORF), four Rep proteins (e.g., Rep78, Rep68, Rep52, and Rep40) are actually expressed. However, it has been shown that unspliced ​​mRNA encoding the Rep78 and Rep52 proteins is sufficient for AAV vector production in mammalian cells (see U.S. Patent Application Publication No. 20160032254).

[0082] In this specification, "adeno-associated virus" and "AAV" are used synonymously. The terms "adeno-associated virus-based vector," "AAV-based vector," and "rAAV" are used synonymously in this specification.

[0083] In some aspects of the present invention, a helper plasmid for producing an adeno-associated virus-based vector comprises a nucleic acid containing a modified sequence of the adenovirus E2a gene, wherein the sequence comprises deletions of sequence regions corresponding to the 1696-2321 bp, 2400-2634 bp, and 2839-3492 bp regions in the sequence of SEQ ID NO: 1 or in a sequence homologous to the sequence of SEQ ID NO: 1 with a degree of identity of at least 63%, and the sequence of SEQ ID NO: 2, or a sequence homologous to the sequence of SEQ ID NO: 2 with a degree of identity of at least 62%. As described above, the sequence homologous to the sequence of SEQ ID NO: 2 with a degree of identity of at least 62% is either the sequence of the E2a gene of an adenovirus serotype other than the serotype having SEQ ID NO: 2, or a codon-optimized variant of the sequence of SEQ ID NO: 2.

[0084] In some aspects of the present invention, a helper plasmid for producing an adeno-associated virus-based vector is a nucleic acid comprising a modified sequence of the adenovirus E2a gene, wherein the sequence comprises a deletion of a sequence region corresponding to a 1696-3492 bp region in the sequence of SEQ ID NO: 1 or in a sequence homologous to the sequence of SEQ ID NO: 1 with a degree of identity of at least 63%, and the sequence of SEQ ID NO: 3, or a sequence homologous to the sequence of SEQ ID NO: 3 with a degree of identity of at least 61%. As described above, the sequence homologous to the sequence of SEQ ID NO: 3 with a degree of identity of at least 61% is either the sequence of the E2a gene of an adenovirus serotype other than the serotype having SEQ ID NO: 3, or a codon-optimized variant of the sequence of SEQ ID NO: 3.

[0085] In some aspects of the present invention, a helper plasmid for producing an adeno-associated virus-based vector is a nucleic acid comprising a modified sequence of the adenovirus E2a gene, wherein the sequence comprises a deletion of a sequence region corresponding to a 1696-2321 bp region in the sequence of SEQ ID NO: 1 or in a sequence homologous to the sequence of SEQ ID NO: 1 with a degree of identity of at least 63%, and the sequence of SEQ ID NO: 4, or a sequence homologous to the sequence of SEQ ID NO: 4 with a degree of identity of at least 64%. As described above, the sequence homologous to the sequence of SEQ ID NO: 4 with a degree of identity of at least 64% is either the sequence of the E2a gene of an adenovirus serotype other than the serotype having SEQ ID NO: 4, or a codon-optimized variant of the sequence of SEQ ID NO: 4.

[0086] In some aspects of the present invention, a helper plasmid for producing an adeno-associated virus-based vector is a nucleic acid comprising a modified sequence of the adenovirus E2a gene, wherein the sequence comprises a deletion of a sequence region corresponding to a 2400-2634 bp region in the sequence of SEQ ID NO: 1 or in a sequence homologous to the sequence of SEQ ID NO: 1 with a degree of identity of at least 63%, and the sequence of SEQ ID NO: 5, or a sequence homologous to the sequence of SEQ ID NO: 5 with a degree of identity of at least 64%. As described above, the sequence homologous to the sequence of SEQ ID NO: 5 with a degree of identity of at least 64% is either the sequence of the E2a gene of an adenovirus serotype other than the serotype having SEQ ID NO: 5, or a codon-optimized variant of the sequence of SEQ ID NO: 5.

[0087] In some aspects of the present invention, a helper plasmid for producing an adeno-associated virus-based vector is a nucleic acid comprising a modified sequence of the adenovirus E2a gene, wherein the sequence comprises a deletion of a sequence region corresponding to a 2839-3492 bp region in the sequence of SEQ ID NO: 1 or in a sequence homologous to the sequence of SEQ ID NO: 1 with a degree of identity of at least 63%, and the sequence of SEQ ID NO: 6, or a sequence homologous to the sequence of SEQ ID NO: 6 with a degree of identity of at least 61%. As described above, the sequence homologous to the sequence of SEQ ID NO: 6 with a degree of identity of at least 61% is either the sequence of the E2a gene of an adenovirus serotype other than the serotype having SEQ ID NO: 6, or a codon-optimized variant of the sequence of SEQ ID NO: 6.

[0088] In some aspects of the present invention, a helper plasmid for producing an adeno-associated virus-based vector is a nucleic acid comprising a modified sequence of the adenovirus E2a gene, wherein the sequence comprises a deletion of a sequence region corresponding to the 1696-2321 bp and 2400-2634 bp regions in the sequence of SEQ ID NO: 1 or in a sequence homologous to the sequence of SEQ ID NO: 1 with a degree of identity of at least 63%, and the sequence of SEQ ID NO: 7, or a sequence homologous to the sequence of SEQ ID NO: 7 with a degree of identity of at least 63%. As described above, the sequence homologous to the sequence of SEQ ID NO: 7 with a degree of identity of at least 63% is either the sequence of the E2a gene of an adenovirus serotype other than the serotype having SEQ ID NO: 7, or a codon-optimized variant of the sequence of SEQ ID NO: 7.

[0089] In some aspects of the present invention, a helper plasmid for producing an adeno-associated virus-based vector is a nucleic acid comprising a modified sequence of the adenovirus E2a gene, wherein the sequence comprises a deletion of a sequence region corresponding to the 1696-2321 bp and 2839-3492 bp regions in the sequence of SEQ ID NO: 1 or in a sequence homologous to the sequence of SEQ ID NO: 1 with a degree of identity of at least 63%, and the sequence of SEQ ID NO: 8, or a sequence homologous to the sequence of SEQ ID NO: 8 with a degree of identity of at least 62%. As described above, the sequence homologous to the sequence of SEQ ID NO: 8 with a degree of identity of at least 62% is either the sequence of the E2a gene of an adenovirus serotype other than the serotype having SEQ ID NO: 8, or a codon-optimized variant of the sequence of SEQ ID NO: 8.

[0090] In some aspects of the present invention, a helper plasmid for producing an adeno-associated virus-based vector is a nucleic acid comprising a modified sequence of the adenovirus E2a gene, wherein the sequence comprises a deletion of a sequence region corresponding to the 2400-2634 bp and 2839-3492 bp regions in the sequence of SEQ ID NO: 1 or in a sequence homologous to the sequence of SEQ ID NO: 1 with a degree of identity of at least 63%, and the sequence of SEQ ID NO: 9, or a sequence homologous to the sequence of SEQ ID NO: 9 with a degree of identity of at least 61%. As described above, the sequence homologous to the sequence of SEQ ID NO: 9 with a degree of identity of at least 61% is either each sequence of the E2a gene of an adenovirus serotype other than the serotype having SEQ ID NO: 9, or a codon-optimized variant of the sequence of SEQ ID NO: 9.

[0091] In one aspect, the present invention relates to a helper plasmid for producing an adeno-associated virus-based vector comprising a nucleic acid containing a modified sequence of the adenovirus E2a gene, wherein the sequence comprises the sequence of SEQ ID NO: 2 or a sequence homologous to the sequence of SEQ ID NO: 2 with at least 62% identity.

[0092] In one aspect, the present invention relates to a helper plasmid for producing an adeno-associated virus-based vector comprising a nucleic acid containing a modified sequence of the adenovirus E2a gene, wherein the sequence comprises the sequence of SEQ ID NO: 3 or a sequence homologous to the sequence of SEQ ID NO: 3 with at least 61% identity.

[0093] In one aspect, the present invention relates to a helper plasmid for producing an adeno-associated virus-based vector comprising a nucleic acid containing a modified sequence of the adenovirus E2a gene, wherein the sequence comprises the sequence of SEQ ID NO: 4 or a sequence homologous to the sequence of SEQ ID NO: 4 with at least 64% identity.

[0094] In one aspect, the present invention relates to a helper plasmid for producing an adeno-associated virus-based vector comprising a nucleic acid containing a modified sequence of the adenovirus E2a gene, wherein the sequence comprises the sequence of SEQ ID NO: 5 or a sequence homologous to the sequence of SEQ ID NO: 5 with at least 64% identity.

[0095] In one aspect, the present invention relates to a helper plasmid for producing an adeno-associated virus-based vector comprising a nucleic acid containing a modified sequence of the adenovirus E2a gene, wherein the sequence comprises the sequence of SEQ ID NO: 6 or a sequence homologous to the sequence of SEQ ID NO: 6 with at least 61% identity.

[0096] In one aspect, the present invention relates to a helper plasmid for producing an adeno-associated virus-based vector comprising a nucleic acid containing a modified sequence of the adenovirus E2a gene, wherein the sequence comprises the sequence of SEQ ID NO: 7 or a sequence homologous to the sequence of SEQ ID NO: 7 with at least 63% identity.

[0097] In one aspect, the present invention relates to a helper plasmid for producing an adeno-associated virus-based vector comprising a nucleic acid containing a modified sequence of the adenovirus E2a gene, wherein the sequence comprises the sequence of SEQ ID NO: 8 or a sequence homologous to the sequence of SEQ ID NO: 8 with at least 62% identity.

[0098] In one aspect, the present invention relates to a helper plasmid for producing an adeno-associated virus-based vector comprising a nucleic acid containing a modified sequence of the adenovirus E2a gene, wherein the sequence comprises the sequence of SEQ ID NO: 9 or a sequence homologous to the sequence of SEQ ID NO: 9 with at least 61% identity.

[0099] In some aspects of the present invention, a helper plasmid for producing an adeno-associated virus-based vector comprises a nucleic acid (any of the above variants) containing a modified sequence of the adenovirus E2a gene, a nucleic acid containing a sequence of the adenovirus VA RNA gene, and a nucleic acid containing a sequence of the adenovirus E4 gene.

[0100] In some aspects of the present invention, the sequence of the adenovirus VA RNA gene includes sequence number 28. In some aspects of the present invention, the sequence of the adenovirus E4 gene is the naturally occurring sequence of the full length of the E4 gene, the sequence of E4 or f6 / 7, or the sequence of E4 or f6.

[0101] In some aspects of the present invention, the naturally occurring full-length sequence of the adenovirus E4 gene includes SEQ ID NO: 29. In some aspects of the present invention, the sequence of adenovirus E4 orf6 / 7 is the sequence encoding sequence number 30.

[0102] In some aspects of the present invention, the sequence of adenovirus E4 orf6 is the sequence encoding sequence number 31. use In one aspect, the present invention relates to the use of a nucleic acid comprising any one of the modified sequence variants of the adenovirus E2a gene described herein, or to the use of a helper plasmid comprising a nucleic acid comprising any one of the modified sequence variants of the adenovirus E2a gene described herein, for the production of an adeno-associated virus-based vector.

[0103] In one aspect, the present invention relates to the use of nucleic acids comprising any one of the modified sequence variants of the adenovirus E2a gene described herein for the production of vectors based on adeno-associated virus.

[0104] In one aspect, the present invention relates to the use of a helper plasmid comprising a nucleic acid containing one of the modified sequence variants of the adenovirus E2a gene described herein for the production of an adeno-associated virus-based vector.

[0105] In some variants of use, a helper plasmid containing a nucleic acid with one of the modified sequence variants of the adenovirus E2a gene described herein is used to produce an adeno-associated virus-based vector by transfecting a host cell with this helper plasmid, as well as by transfecting it with a plasmid containing the sequence of the gene of interest and the gene encoding the AAV protein.

[0106] The terms "manufacture" and "obtain" are used synonymously in this specification. In some aspects of the present invention, a modified sequence variant of the adenovirus E2a gene according to the present invention can be incorporated into the genome of a host cell to produce a packaging cell line or a producing cell line for viral vectors. Such approaches for producing viral vectors are well known to those skilled in the art and have been described in the literature (Imre Kovesdi et al., Adenoviral producer cells, Viruses. 2010 Aug;2(8):1681-1703).

[0107] In one aspect, the present invention relates to the use of modified sequence variants of the adenovirus E2a gene according to the present invention for producing adenovirus-based recombinant viral vectors by incorporating a portion of an adenovirus genome containing any one of the modified sequence variants of the adenovirus E2a gene described herein into the genome of a host cell, and similarly by incorporating a replication-deficient recombinant adenovirus of the gene of interest into the genome.

[0108] In some respects, the present invention relates to the use of modified adenovirus E2a gene variants according to the present invention for producing adenovirus-based recombinant viral vectors by incorporating a portion of the adenovirus genome lacking the E2a gene sequence into the genome of a host cell, and similarly, by incorporating into the genome of a replication-deficient recombinant adenovirus containing either the gene of interest or a modified sequence variant of the adenovirus E2a gene according to the present invention. Such approaches are well known to those skilled in the art and have been described in the literature (MI Gorziglia et al., Generation of an adenovirus vector lacking E1, e2a, E3, and all of E4 except open reading frame 3, J Virol. 1999 Jul;73(7):6048-55; H Zhou et al., A new vector system with inducible E2a cell line for production of higher titer and safer adenoviral vectors, Virology. 2000 Sep 30;275(2):348-57).

[0109] In one aspect, the present invention relates to the use of modified sequence variants of the adenovirus E2a gene according to the present invention for producing a recombinant viral vector based on adeno-associated virus by incorporating a portion of an adenovirus genome containing a nucleic acid comprising any one of the modified sequence variants of the adenovirus E2a gene according to the present invention into the genome of a host cell, and / or by incorporating a portion of an adeno-associated virus genome and / or an expression cassette having the target gene and ITR into the genome of a host cell. Such approaches are well known to those skilled in the art and have been described in the literature (see Satoki Nakamura et al., Development of packaging cell lines for generation of adeno-associated virus vectors by lentiviral gene transfer of trans-complementary components, 2004 Oct;73(4):285-94; see abstract; Nagarathinam Selvaraj et al., Detailed Protocol for the Novel and Scalable Viral Vector Upstream Process for AAV Gene Therapy Manufacturing, Hum Gene Ther. August 2021;32(15-16):850-861). [Examples]

[0110] Examples (Implementation of the present invention) The following examples are provided to better illustrate the present invention. These examples are for illustrative purposes only and should not be construed as limiting the scope of the present invention in any way.

[0111] Although the aforementioned invention has been described in some detail by description and examples for the purpose of clarifying understanding, it will be readily apparent to those skilled in the art, in consideration of the teachings of the present invention, that certain changes and modifications to the present invention can be made without departing from the spirit and scope of the appended embodiments. Materials and general methods Recombinant DNA technology DNA was manipulated using standard methods described in Sambrook, J. et al., Molecular cloning: A laboratory manual; Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 2012. Molecular biology reagents were used according to the manufacturer's protocols. Briefly, plasmid DNA was prepared for further manipulation in *E. coli* cells grown under selective antibiotic pressure to prevent plasmid loss within the cell population. The inventors isolated plasmid DNA from cells using commercially available kits, measured its concentration, and used it for cloning by restriction endonuclease treatment or PCR amplification. DNA fragments were ligated together and transformed into bacterial cells for clone selection and further construction. All resulting gene constructs were confirmed by restriction pattern and complete Sanger sequencing. DNA sequencing DNA sequences were determined using Sanger sequencing. DNA and protein sequences were analyzed, and the sequence data was processed using SnapGene Viewer 4.2 or later to create, map, analyze, annotate, and visualize the sequences. Cell culture This experiment used the HEK293 cell line (human fetal kidney clone 293). Suspension HEK293 cells used to produce AAV were cultured under standard conditions of 37°C and 5% CO2 in complete culture medium without FBS and antibiotics. Cell viability was assessed using Trypan Blue staining and a disposable cell counting chamber with an automated Countess II counter. Generation of viral particles from AAV recombinant vectors To produce recombinant AAV virus particles containing transgenes based on various proteins, the inventors used HEK293-producing cells transfected with the following three plasmids: • Plasmid containing an AAV expression cassette for transgene expression; • A plasmid containing the Cap gene for serotype 2, 5, 6, or 9, and the Rep gene for the AAV2 serotype. Each gene encodes several protein products using an alternative reading frame; • Plasmid containing adenovirus Ad2 genes: E2a, E4, and VA RNA, necessary for constructing and packaging AAV capsids.

[0112] The titer of the viral particles was determined by quantitative PCR using primers and samples specific to the region of the recombinant viral genome, and expressed as the copy number of the viral genome per 1 ml. Statistical data analysis The results are shown as mean ± standard deviation (SD). The experimental results were compared using one-way analysis of variance (ANOVA) and subsequent Dunnett's multiple pairwise comparisons, and were determined to be statistically significant.

[0113] Example 1. Evaluation of the productivity of AAV-based recombinant viral vector generation using a modified adenovirus E2a gene sequence and a helper plasmid based thereon. The following deletions are found in naturally occurring sequences of the regulatory adenovirus E2a gene, which contain sequence number 1 or sequences homologous to sequence number 1 with a degree of identity of at least 63%: i. 1696~2321bp, 2400~2634bp, 2839~3492bp; or ii. 1696~3492bp We implemented it.

[0114] Modified sequence variants of the adenovirus E2a gene containing regulatory elements were constructed and further tested for productivity levels, while simultaneously generating vectors based on adeno-associated virus serotypes rAAV2, rAAV5, rAAV6, and rAAV9.

[0115] To demonstrate improved productivity levels during rAAV generation, we constructed plasmids encoding target gene constructs, specifically including the following: a. An E2a gene sequence having a regulatory element, which includes SEQ ID NO: 1 or a sequence homologous to the sequence of SEQ ID NO: 1 with a degree of identity of at least 63%. b. Modified sequences of the E2a gene having regulatory elements, which include deletions of sequence regions corresponding to the regions of 1696-2321 bp, 2400-2634 bp, and 2839-3492 bp in the sequence of sequence 1 or in a sequence homologous to the sequence of sequence number 1 with a degree of identity of at least 63%, and which include the sequence of sequence number 2, or which include a sequence homologous to the sequence of sequence number 2 with a degree of identity of at least 62%, and c. A sequence of the E2a gene having a regulatory element, which includes a deletion of a sequence region corresponding to a 1696-3492 bp region in the sequence of sequence 1 or in a sequence homologous to the sequence of sequence number 1 with a degree of identity of at least 63%, and which includes the sequence of sequence number 3, or a sequence homologous to the sequence of sequence number 3 with a degree of identity of at least 61%.

[0116] The following were produced in a similar manner: i. A viral vector based on adeno-associated virus serotype 2, carrying a nucleotide sequence encoding an antibody against VEGF / C5. ii. A viral vector based on adeno-associated virus serotype 5, carrying a nucleotide sequence encoding coagulation factor IX (FIX) protein. iii. A viral vector based on adeno-associated virus serotype 6 that carries a nucleotide sequence encoding coagulation factor VIII (FVIII) protein, and iv. Viral vectors based on adeno-associated virus serotype 9 that carry the nucleotide sequence encoding the survival motor neuron (SMN1) protein. These were the remaining plasmids needed for the generation of [the substance].

[0117] The modified sequences encoding the developed E2a gene and its regulatory elements were compared to similar initial sequences encoding the E2a gene and regulatory elements having Sequence ID No. 1. - During the generation of rAAV serotype 2 using a transgene based on an antibody against VEGF / C5, a 2.41-fold increase is obtained when using an adenovirus E2a gene sequence containing SEQ ID NO: 2, which has deletions in the sequence regions corresponding to 1696-2321 bp, 2400-2634 bp, and 2839-3492 bp in the sequence of SEQ ID NO: 1, and a 3.02-fold increase is obtained when using an adenovirus E2a gene sequence containing SEQ ID NO: 3, which has deletions in the sequence regions corresponding to 1696-3492 bp in the sequence of SEQ ID NO: 1; - When generating rAAV serotype 5 using a transgene based on coagulation factor IX protein, use an adenovirus E2a gene sequence containing SEQ ID NO: 2, which has deletions in the sequence regions corresponding to 1696-2321 bp, 2400-2634 bp, and 2839-3492 bp in the sequence of SEQ ID NO: 1.45 times; and when using an adenovirus E2a gene sequence containing SEQ ID NO: 3, which has deletions in the sequence region corresponding to 1696-3492 bp in the sequence of SEQ ID NO: 1, which has deletions in the sequence regions corresponding to 1696-3492 bp in the sequence of SEQ ID NO: 1, which has deletions in the sequence regions corresponding to 1696-3492 bp in the sequence of SEQ ID NO: 1, which has deletions in the sequence of SEQ ID NO: 3690 times; - When generating rAAV serotype 6 using a transgene based on coagulation factor VIII protein, a 1.81-fold increase is obtained when using an adenovirus E2a gene sequence containing SEQ ID NO: 2, which has deletions in the sequence regions corresponding to 1696-2321 bp, 2400-2634 bp, and 2839-3492 bp in the sequence of SEQ ID NO: 1, and a 1.83-fold increase is obtained when using an adenovirus E2a gene sequence containing SEQ ID NO: 3, which has deletions in the sequence region corresponding to 1696-3492 bp in the sequence of SEQ ID NO: 1; - When generating rAAV serotype 9 using a transgene based on the survival motor neuron (SMN1) protein, the ratio is 1.8 times when using an adenovirus E2a gene sequence containing SEQ ID NO: 2, which has deletions in the sequence regions corresponding to 1696-2321 bp, 2400-2634 bp, and 2839-3492 bp in the sequence of SEQ ID NO: 1, and 1.97 times when using an adenovirus E2a gene sequence containing SEQ ID NO: 3, which has deletions in the sequence region corresponding to 1696-3492 bp in the sequence of SEQ ID NO: 1. This has been shown to lead to increased productivity in rAAV generation (Figure 1).

[0118] Similar data was obtained after comparing the codon-optimized variant of the adenovirus E2a gene having SEQ ID NO: 10 with its modified variant. This modified variant includes i. deletions at 1696-2321 bp, 2400-2634 bp, and 2839-3492 bp (SEQ ID NO: 11), or ii. deletions at 1696-3492 bp (SEQ ID NO: 12). Similar data was also obtained after comparing the sequence of the adenovirus E2a gene of a serotype other than SEQ ID NO: 1 having SEQ ID NO: 19 with its modified variant. This modified variant includes i. deletions at 1534-2036 bp, 2115-2340 bp, and 2545-3198 bp (SEQ ID NO: 20), or ii. deletions at 1534-3198 bp (SEQ ID NO: 21).

[0119] Therefore, the E2a gene and its regulatory elements are encoded in the sequence of SEQ ID NO: 1, or in sequences homologous to the sequence of SEQ ID NO: 1 with a degree of identity of at least 63%. i. 1696~2321bp, 2400~2634bp, 2839~3492bp; or ii. 1696~3492bp Nucleic acids containing deletions of the sequence region corresponding to the specified region increase the productivity of recombinant viral vectors based on various serotypes of AAV compared to the natural sequence of the adenovirus E2a gene containing SEQ ID NO: 1, or sequences homologous to the SEQ ID NO: 1 sequence with a degree of identity of at least 63%.

Claims

1. A nucleic acid comprising a modified sequence of the adenovirus E2a gene, wherein the sequence is from the group of sequences homologous to sequence number 1, with a degree of identity of at least 63%: i. Deletion of sequence regions corresponding to 1696–2321 bp, 2400–2634 bp, and 2839–3492 bp; or ii. Deletion of the sequence region corresponding to 1696–3492 bp; or iii. Deletion of the sequence region corresponding to 1696–2321 bp; or iv. Deletion of the sequence region corresponding to 2400–2634 bp; or v. Deletion of the sequence region corresponding to 2839–3492 bp; or vi. Deletion of sequence regions corresponding to 1696–2321 bp and 2400–2634 bp; or vii. Deletion of sequence regions corresponding to 1696–2321 bp and 2839–3492 bp; or viiii. Deletion of sequence regions corresponding to 2400–2634 bp and 2839–3492 bp Nucleic acids containing modifications selected from.

2. The nucleic acid according to claim 1, wherein the sequence homologous to the sequence of SEQ ID NO: 1, having a degree of identity of at least 63%, is the sequence of the E2a gene of an adenovirus serotype other than the serotype having SEQ ID NO: 1, or is a codon-optimized variant of the sequence of SEQ ID NO:

1.

3. The nucleic acid according to claim 1 or 2, comprising a modified sequence of the adenovirus E2a gene, wherein the modified sequence includes deletions of sequence regions corresponding to the regions of 1696-2321 bp, 2400-2634 bp, and 2839-3492 bp in the sequence of SEQ ID NO: 1 or a sequence homologous to the sequence of SEQ ID NO: 1 with a degree of identity of at least 63%, and also includes the sequence of SEQ ID NO: 2, or includes a sequence homologous to the sequence of SEQ ID NO: 2 with a degree of identity of at least 62%.

4. The nucleic acid according to claim 3, wherein the sequence homologous to the sequence of Sequence ID No. 2, having a degree of identity of at least 62%, is the sequence of the E2a gene of an adenovirus serotype other than the serotype having Sequence ID No. 2, or is a codon-optimized variant of the sequence of Sequence ID No.

2.

5. The nucleic acid according to claim 1 or 2, comprising a modified sequence of the adenovirus E2a gene, wherein the modified sequence comprises a deletion of a sequence region corresponding to the 1696-3492 bp region in the sequence of SEQ ID NO: 1 or in a sequence homologous to the sequence of SEQ ID NO: 1 with a degree of identity of at least 63%, and the sequence of SEQ ID NO: 3 or a sequence homologous to the sequence of SEQ ID NO: 3 with a degree of identity of at least 61%.

6. The nucleic acid according to claim 5, wherein the sequence homologous to the sequence of SEQ ID NO: 3, having a degree of identity of at least 61%, is either the sequence of the E2a gene of an adenovirus serotype other than the serotype having SEQ ID NO: 3, or a codon-optimized variant of the sequence of SEQ ID NO:

3.

7. The nucleic acid according to claim 1 or 2, comprising a modified sequence of the adenovirus E2a gene, wherein the modified sequence comprises a deletion of a sequence region corresponding to the 1696-2321 bp region in the sequence of SEQ ID NO: 1 or a sequence homologous to the sequence of SEQ ID NO: 1 with a degree of identity of at least 63%, and the sequence of SEQ ID NO: 4 or a sequence homologous to the sequence of SEQ ID NO: 4 with a degree of identity of at least 64%.

8. The nucleic acid according to claim 7, wherein the sequence homologous to the sequence of SEQ ID NO: 4, which has at least 64% identity, is the sequence of the E2a gene of an adenovirus serotype other than the serotype having SEQ ID NO: 4, or is a codon-optimized variant of the sequence of SEQ ID NO:

4.

9. The nucleic acid according to claim 1 or 2, comprising a modified sequence of the adenovirus E2a gene, wherein the modified sequence comprises a deletion of a sequence region corresponding to a 2400-2634 bp region in the sequence of sequence number 1 or in a sequence homologous to sequence number 1 with a degree of identity of at least 63%, and the sequence of sequence number 5 or a sequence homologous to sequence number 5 with a degree of identity of at least 64%.

10. The nucleic acid according to claim 9, wherein the sequence homologous to the sequence of Sequence ID No. 5, which has at least 64% identity, is the sequence of the E2a gene of an adenovirus serotype other than the serotype having Sequence ID No. 5, or is a codon-optimized variant of the sequence of Sequence ID No.

5.

11. The nucleic acid according to claim 1 or 2, comprising a modified sequence of the adenovirus E2a gene, wherein the modified sequence comprises a deletion of a sequence region corresponding to the 2839-3492 bp region in the sequence of SEQ ID NO: 1 or a sequence homologous to the sequence of SEQ ID NO: 1 with a degree of identity of at least 63%, and the sequence of SEQ ID NO: 6 or a sequence homologous to the sequence of SEQ ID NO: 6 with a degree of identity of at least 61%.

12. The nucleic acid according to claim 11, wherein the sequence homologous to the sequence of SEQ ID NO: 6, which has at least 61% identity, is either the sequence of the E2a gene of an adenovirus serotype other than the serotype having SEQ ID NO: 6, or a codon-optimized variant of the sequence of SEQ ID NO:

6.

13. The nucleic acid according to claim 1 or 2, comprising a modified sequence of the adenovirus E2a gene, wherein the modified sequence comprises a deletion of a sequence region corresponding to the regions of 1696-2321 bp and 2400-2634 bp in the sequence of SEQ ID NO: 1 or a sequence homologous to the sequence of SEQ ID NO: 1 with a degree of identity of at least 63%, and the sequence of SEQ ID NO: 7 or a sequence homologous to the sequence of SEQ ID NO: 7 with a degree of identity of at least 63%.

14. The nucleic acid according to claim 13, wherein the sequence homologous to the sequence of SEQ ID NO: 7, which has at least 63% identity, is either the sequence of the E2a gene of an adenovirus serotype other than the serotype having SEQ ID NO: 7, or a codon-optimized variant of the sequence of SEQ ID NO:

7.

15. The nucleic acid according to claim 1 or 2, comprising a modified sequence of the adenovirus E2a gene, wherein the modified sequence comprises a deletion of a sequence region corresponding to the regions 1696-2321 bp and 2839-3492 bp in the sequence of SEQ ID NO: 1 or a sequence homologous to the sequence of SEQ ID NO: 1 with a degree of identity of at least 63%, and the sequence of SEQ ID NO: 8 or a sequence homologous to the sequence of SEQ ID NO: 8 with a degree of identity of at least 62%.

16. The nucleic acid according to claim 15, wherein the sequence homologous to the sequence of SEQ ID NO: 8, which has at least 62% identity, is either the sequence of the E2a gene of an adenovirus serotype other than the serotype having SEQ ID NO: 8, or a codon-optimized variant of the sequence of SEQ ID NO:

8.

17. The nucleic acid according to claim 1 or 2, comprising a modified sequence of the adenovirus E2a gene, wherein the modified sequence comprises a deletion of a sequence region corresponding to the regions of 2400-2634 bp and 2839-3492 bp in the sequence of SEQ ID NO: 1 or a sequence homologous to the sequence of SEQ ID NO: 1 with a degree of identity of at least 63%, and the sequence of SEQ ID NO: 9 or a sequence homologous to the sequence of SEQ ID NO: 9 with a degree of identity of at least 61%.

18. The nucleic acid according to claim 17, wherein the sequence homologous to the sequence of SEQ ID NO: 9, which has at least 61% identity, is one of the E2a gene sequences of an adenovirus serotype other than the serotype having SEQ ID NO: 9, or a codon-optimized variant of the sequence of SEQ ID NO:

9.

19. Nucleic acids containing modified sequences of the adenovirus E2a gene, group: i. The sequence of sequence number 2, or a sequence homologous to the sequence of sequence number 2 with at least 62% identity; or ii. The sequence of sequence number 3, or a sequence homologous to the sequence of sequence number 3 with at least 61% identity; or iii. The sequence of sequence number 4, or a sequence homologous to the sequence of sequence number 4 with at least 64% identity; or iv. The sequence of sequence number 5, or a sequence homologous to the sequence of sequence number 5 with at least 64% identity; or v. The sequence of sequence number 6, or a sequence homologous to the sequence of sequence number 6 with at least 61% identity; or vi. The sequence of sequence number 7, or a sequence homologous to the sequence of sequence number 7 with at least 63% identity; or vii. The sequence of sequence number 8, or a sequence homologous to the sequence of sequence number 8 with at least 62% identity; or viiii. The sequence of sequence number 9, or a sequence homologous to the sequence of sequence number 9 with at least 61% identity. A nucleic acid containing a sequence selected from the following.

20. i. The sequences homologous to the sequence of Sequence ID No. 2, which have at least 62% identity, are the respective sequences of the E2a gene of adenovirus serotypes other than the serotype having Sequence ID No. 2, or codon-optimized variants of the sequence of Sequence ID No. 2; or ii. The sequences homologous to the sequence of Sequence ID No. 3, which have at least 61% identity, are the sequences of the E2a gene of adenovirus serotypes other than the serotype having Sequence ID No. 3, or codon-optimized variants of the sequence of Sequence ID No. 3; or iii. The sequences homologous to the sequence of Sequence ID No. 4, which have at least 64% identity, are the sequences of the E2a gene of adenovirus serotypes other than the serotype having Sequence ID No. 4, or codon-optimized variants of the sequence of Sequence ID No. 4; or iv. The sequences homologous to the sequence of Sequence ID No. 5, which have at least 64% identity, are the sequences of the E2a gene of adenovirus serotypes other than the serotype having Sequence ID No. 5, or codon-optimized variants of the sequence of Sequence ID No. 5; or v. The sequences homologous to the sequence of SEQ ID NO: 6, which have at least 61% identity, are the sequences of the E2a gene of adenovirus serotypes other than the serotype having SEQ ID NO: 6, or codon-optimized variants of the sequence of SEQ ID NO: 6; or vi. The sequences homologous to the sequence of SEQ ID NO: 7, which have at least 63% identity, are the sequences of the E2a gene of adenovirus serotypes other than the serotype having SEQ ID NO: 7, or codon-optimized variants of the sequence of SEQ ID NO: 7; or vii. The sequences homologous to the sequence of SEQ ID NO: 8, which have at least 62% identity, are the sequences of the E2a gene of adenovirus serotypes other than the serotype having SEQ ID NO: 8, or codon-optimized variants of the sequence of SEQ ID NO: 8; or viiii. The sequences homologous to the sequence of Sequence ID No. 9, which have at least 61% identity, are the respective sequences of the E2a gene of adenovirus serotypes other than the serotype having Sequence ID No. 9, or codon-optimized variants of the sequence of Sequence ID No.

9. The nucleic acid according to claim 19.

21. A helper plasmid for producing an adeno-associated virus-based vector, comprising a nucleic acid containing a modified sequence of the adenovirus E2a gene according to any one of claims 1 to 20.

22. A helper plasmid for producing an adeno-associated virus-based vector according to claim 21, comprising a nucleic acid containing the sequence of the adenovirus VA RNA gene and a nucleic acid containing the sequence of the adenovirus E4 gene.

23. A helper plasmid for producing an adeno-associated virus-based vector according to claim 22, wherein the sequence of the adenovirus VA RNA gene includes sequence number 28.

24. A helper plasmid for producing an adeno-associated virus-based vector according to claim 22, wherein the sequence of the adenovirus E4 gene is the naturally occurring full-length sequence of the E4 gene, the sequence of E4 or f6 / 7, or the sequence of E4 or f6.

25. The full-length, naturally occurring sequence of the adenovirus E4 gene comprises SEQ ID NO: 29, a helper plasmid for producing an adeno-associated virus-based vector according to claim 24.

26. A helper plasmid for producing an adeno-associated virus-based vector according to claim 24, wherein the sequence of adenovirus E4 orf6 / 7 is the sequence encoding sequence number 30.

27. The helper plasmid for producing an adeno-associated virus-based vector according to claim 24, wherein the sequence of adenovirus E4 orf6 is the sequence encoding sequence number 31.

28. Use of a nucleic acid comprising a modified sequence of the adenovirus E2a gene according to any one of claims 1 to 20, or a helper plasmid according to any one of claims 21 to 27, for producing a vector based on adeno-associated virus.

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