KR20070073299A - Recombinant adenovirus vectors inhibit the production of replicable adenoviruses - Google Patents

Abstract

The present invention relates to an optimal novel adenovirus vector that inhibits the production of replication-competent adenovirus (RCA) capable of replicating in 293 cells.

Description

Recombinant adenoviral vector for reducing replication-competent adenovirus formation

1 relates to the nucleic acid sequence of degenerate synIX and the corresponding IX nucleic acid sequence of the original adenovirus used in the present invention.

Figure 2 relates to the nucleic acid sequence of degenerate synIVa2 and the corresponding IVa2 nucleic acid sequence of the original adenovirus used in the present invention.

3 is a schematic diagram of various modified vectors used in the present invention.

4 is a schematic diagram of an experiment for quantifying replicable adenovirus (RCA) used in the present invention.

The present invention relates to recombinant adenovirus vectors in which production of replicable adenovirus (RCA) is inhibited.

Gene therapy is the delivery of functional genes to tissues that have lost or lost gene activity. In gene therapy, genetically engineered recombinant viral vectors have been used.

Among the recombinant viral vectors, adenoviruses are i) not pathogenic, ii) are not integrated into the genome of the infected host, and iii) replicate equally well in dividing or inactive cells. And iv) it has a large host range and can be infected with a wide variety of cells, making it the vector of choice for gene therapy or vaccine applications.

The production of replication deficient adenoviruses useful for gene therapy or vaccine applications requires the use of cell lines capable of supplying E1 proteins lacking in these recombinant adenoviruses. Currently used cell lines are 293 cell lines and have about 11-12% of the left hand end of the adenovirus type 5 genome (4.3 kb, including the portion of the lef ITR, encapsidation signal, E1, pIX, and IVa2). .

The DNA sequence of the adenovirus vector which is mainly used currently has a homologous region of about 1 kb with the adenovirus DNA sequence of the 293 cell line, which is a cell line supplying the E1 gene deficient in the vector. Due to sequence similarity between the packaging cell and the vector, when the virus is produced in the 293 cell line, recombination between the virus and the adenovirus sequence of the cell line produces a wild type virus containing the E1 gene derived from the 293 cell line instead of the gene to be transported. Wild-type viruses are replicable adenoviruses (RCAs) and can cause safety issues.

Cell- or vector-based solutions have been introduced to solve the above problems. A representative example of cell-based solutions is the Per.C6 cell line engineered to have only minimal E1 sequences in human embryonic retinoblasts. No formation of replicable adenovirus (RCA) was detected in adenovirus production in this cell line.

Vector-based solutions include the following attempts. First, there was a method of removing or relocating the pIX gene to the right end of the viral chromosome (J. Virol 1996; 70: 8459-8467). According to this report, the production of three replicable adenoviruses (RCA) was observed for adenoviruses with Ad2-based pIX removed or rearranged at a large-scale preparation of 30 or more. Thus, elimination or rearrangement of pIX reduces the production of replicable adenovirus (RCA) but does not completely inhibit the production of replicable adenovirus (RCA) because a portion of the IVa2 gene (~ 250bp) remains.

US6410298 also provides a recombinant adenovirus expression vector in which part or all of the pIX DNA present in the homologous similarity is deleted to inhibit the production of wild-type adenovirus. The pIX gene encodes the minor component of the external adenovirus capsid that stabilizes the group-of-nine hexson, which accounts for the majority of the virus's capsid. The deleted adenovirus of some or all of the pIX used here proliferated well in the 293 cell line and the productivity and infectious function were not significantly different from the undeleted virus and could possibly reduce the production of replicable adenovirus (RCA). Was not experimentally proven. In addition, since the virus still has a homologous similarity (~ 250bp) of a portion corresponding to the IVa2 gene, replicable adenovirus (RCA) can be produced by homologous recombination.

Another approach based on vector-based solutions for reducing replicable adenovirus (RCA) production is to reduce homology between the adenovirus vector and the 293 cell line by substituting the nucleic acid sequence without changing the amino acid. (Gene Therapy (2001) 8, 1713-1720). In this report, pIX and IVa2 genes in homologous similarity regions were introduced to reduce the homologous similarity region by introducing a codon sequence modification (silent modification) without amino acid change, thereby reducing the production of replicable adenovirus (RCA). The adenovirus introduced with the degenerate gene sequence used here was tested for the production of replicable adenovirus (RCA) after seven passages, whereas conventional adenoviruses produced replicable adenovirus (RCA). It has a limitation that it cannot be suppressed before deformation.

The adenovirus described above did not show replicable adenovirus (RCA) production, showing a decrease in replicable adenovirus (RCA). However, this strategy is still used for gene therapy or vaccine applications that have already been used, such as the creation of replicable adenovirus (RCA) by homologous recombination, since it still has about 70% homology similar to codon sequence modifications. Since adenovirus vectors have safety problems due to the production of replicable adenovirus (RCA) due to the potential recombination potential during virus propagation in 293 cell lines, efforts to further reduce the possibility of producing replicable adenovirus are required. .

Against this background, the inventors found that among the pVIII and IVa2 genes of the adenovirus vectors corresponding to the sequences homologous to the E1 gene of the packaging cells providing the E1 gene, the pⅨ gene is deleted and the IVa2 gene has a reduced homology with the E1 gene. In case of using the recombinant adenovirus vector Ad5.CMV / DC92 substituted with the above-described adenovirus vector, the nucleic acid sequence degenerates not only the existing adenovirus vector Ad5.CMV but also the entire Ad5.CMV / D9, pIX gene and a part of the IVa2 gene from which the pIX gene was deleted. The present invention was completed by confirming that the production of replicable adenovirus (RCA) is suppressed more than the adenovirus vector Ad5.CMV / C92.

One object of the present invention is (i) in the pVIII gene, in which the pV gene is substituted without modification of the amino acid sequence with a nucleic acid sequence that is deleted in its entirety or has reduced homology with the adenovirus E1 gene, and (ii) in the IVa2 gene, 4091 to To provide an adenovirus vector that inhibits the production of replication-competent adenovirus, wherein the nucleic acid sequence corresponding to 4344 comprises the IVa2 gene substituted with the nucleic acid sequence having reduced homology with the adenovirus E1 gene. .

Another object of the present invention is to provide a method for generating a replication defective adenovirus comprising the step of introducing the recombinant adenovirus vector into the packaging cells.

In one embodiment, the present invention provides a kit comprising: (i) in the pVIII gene, in which the pV gene is substituted without modification of the amino acid sequence with a nucleic acid sequence that is deleted in its entirety or has reduced homology with the adenovirus E1 gene, and (ii) in the IVa2 gene, 4091 To provide an adenovirus vector that inhibits the production of replication-competent adenovirus, wherein the nucleic acid sequence corresponding to 4344 comprises the IVa2 gene substituted with the nucleic acid sequence having reduced homology with the adenovirus E1 gene. to be.

The present invention relates to adenovirus vectors, wherein adenovirus includes all adenoviruses, including mastadenovirus and aviadenovirus. Preferably, the adenovirus is serogroup C adenovirus, more preferably serotype 2: Ad2 or celltype 5 (serotype 5: Ad5).

As used herein, the term "self-replicating" refers to a new synthesis of infectious adenovirus in cells infected with a virus. In the present invention, a system using adenovirus vectors and packaging cells recombined so as to delete the system required for the virus to be replicated in order to prevent self-replicating of the virus was used. Preferably, the adenovirus vector is a vector from which one or more genes selected from the E1 and E3 genes have been removed, more preferably the vector from which both the E1 and E3 genes have been deleted. Preferred packaging cells are 293 cells comprising the E1 gene.

In the system using the adenovirus vector / packaging cell, in order to solve the problem that a replicable adenovirus is generated by homologous recombination between the genome of the packaging cell and the adenovirus vector, the sequence corresponds to a sequence homologous to the E1 gene of the packaging cell. PⅨ and IVa2 genes on the vector were modified

Specifically, the p Ⅸ gene of the vector was deleted without substitution or modification of the amino acid sequence with a nucleic acid sequence having reduced homology with the whole. In the case of the IVa2 gene of the vector, the nucleic acid sequence corresponding to 4091 to 4344 was replaced with a nucleic acid sequence having reduced homology with the adenovirus E1 gene without modification of the amino acid sequence.

When inducing a silent mutation in which only the nucleic acid sequence is replaced with a sequence homologous to the E1 gene without modification of the amino acid sequence, the present invention is characterized by replacing with an optimized codon showing high expression in packaging cells. The pIX gene was replaced with a nucleic acid sequence having an optimized codon of SEQ ID NO: 5, and the IVa2 gene was replaced with a nucleic acid sequence having an optimized codon of SEQ ID NO: 6.

In the adenovirus vector, since the IVa2 protein involved in the packaging signal is expressed, the adenovirus is normally produced, but the generated virus is not a replicable adenovirus (RCA), and thus can be safely used for various medicinal purposes.

In a specific embodiment of the present invention, the inventors have identified an adenovirus Ad5.CMV / D9, pIX, which has deleted pIX in an adenovirus vector, and IVa2, wherein the sequence corresponding to 4091-4344 is replaced with the nucleic acid sequence of SEQ ID NO: 6. Adenovirus Ad5.CMV / C92 was prepared by replacing the virus Ad5.CMV / DC92, pIX with the nucleic acid sequence of SEQ ID NO: 5 and IVa2 with the nucleic acid sequence of SEQ ID NO: 6. As a result, it was confirmed that virus production using the modified vector is normal and production of replicable adenovirus (RCA) is suppressed. In particular, the adenosine in which the pIX gene is deleted and a part of the IVa2 gene is substituted with the degenerate nucleic acid sequence is confirmed. Virus Ad5.CMV / DC92 has a high inhibitory effect on the production of replicable adenovirus (RCA).

Vectors that inhibit the production of replicable adenovirus (RCA) of the present invention can be safely applied to the field of gene therapy or vaccine.

To this end, nucleic acids encoding one or more therapeutic proteins and / or antigenic peptides to be delivered and expressed in cells, organs or organisms can be inserted into the vector. For example, hormones, cytokines, enzymes, antibodies, growth factors, transcriptional regulators, blood factors, vaccines, structural proteins, ligand proteins and receptors, cell surface antigens, receptor antagonists used to treat or prevent human diseases And various bioactive peptides such as derivatives thereof. It may also include antisense nucleic acids or siRNAs expressing genes that regulate gene expression in a target cell.

Vectors provided herein can include expression control elements such as promoters, operators, start codons, stop codons, polyadenylation signals and enhancers that are not derived from adenoviruses.

In another aspect, the present invention relates to a method for producing a replication defective adenovirus comprising introducing the recombinant adenovirus vector into a packaging cell.

The harvesting of the virus from the culture supernatant can be performed under conditions obtainable with high yields in terms of purity, stability, morphology and infectivity.

Adenoviruses expressing the above-mentioned therapeutic proteins can be used for the treatment or prevention of various diseases such as genetic diseases, cancer, viral infections, and the like.

Hereinafter, the present invention will be described in more detail in the following examples. However, these examples are only illustrative of the present invention and do not limit the scope of the present invention.

Example  1: General Molecular Biology

Treatment of restriction enzymes (all restriction enzymes from New England Biolab), agarose gel electrophoresis, gel extraction kit (Solgent gel extraction kit), protein extraction using phenol / chloroform, ethanol precipitation, conjugation of DNA fragments And methods commonly used in molecular biology such as E. coli transformation, pure plasmid DNA purification, and polymerase chain reactions (all primers synthesized from Genome) are described in Molecular Cloning (Second Edition) by Sambrook et al. The field was carried out with the least modification.

Example  2 : Degenerate Sequence  design

In the open-reading frames of the pIX and IVa2 regions, we tried to introduce a homologous similarity between the DNA of the adenovirus vector and the adenovirus DNA of the 293 cell line through codon substitution. Priority of codon substitution was introduced according to the following rules.

i) Substitution with the highest priority codon according to the frequency of codon usage in humans (JOURNAL OF VIROLOGY, May 2000, Vol. 74, No. 10, p. 4839-4852)

ii) if the highest priority codon is already used in the adenovirus sequence, replace it with the second priority codon

iii) in some cases, substituting for a third priority codon when its frequency is similar to that of the second priority

According to the above rules, the gene was synthesized from genscript and inserted into the SmaI site of the vector pUC57 to replace the coding sequence of the pIX gene without modification of amino acids, to generate EcoRV and BamHI at the 5 'end and BglII at the 3' end for cloning. This was called vector pUC57-synIX. The nucleic acid sequence of degenerate synIX and the corresponding nucleic acid sequence (wt) of the original adenovirus are shown in FIG. 1. In addition, according to the above rules, a gene was synthesized from genscript to replace the coding sequence of the IVa2 gene without modification of amino acids, and to generate EcoRV and BamHI at the 5 'end and BstXI at the 3' end for cloning, and inserted into the SmaI site of the vector pUC57. This was called vector pUC57-synIVa2. The nucleic acid sequence of degenerate synIVa2 and the corresponding nucleic acid sequence (wt) of the original adenovirus are shown in FIG. 2.

Example  3: Adenovirus  Homologous similarity sites in the vector Transformed  Manufacture of vectors

i) Preparation of pShuttleCMV / D9-EGFP

Vector pShuttleCMV-EGFP containing the EGFP gene in the plasmid pShuttleCMV (FIG. 3) was used as an initial product for vector modification of the present invention. Using this plasmid as a template, 5'-SV40pA (5'-cga cgc ggc cgc aat gca att gtt gtt gtt aac ttg-3 'SEQ ID NO: 1) and 3'-SV40pA (5'-tcg gat atc ccc aga tcc gcg tta PCR aga tac att gat SEQ ID NO: 2), the product (0.15kb) was cleaved with NotI, EcoRV, and then the larger fragment (6.1kb) of the two fragments of the plasmid pShuttleCMV-EGFP treated with the same restriction enzyme was isolated and joined. After obtaining pShuttleCMV / M-EGFP with restriction enzyme site at the back of vector SV40 polyA, the plasmid pShuttleCMV was used as a template. Vector 3) and 3'-ΔIX (5'-cca ttg cca tca tta tgg acg-3 'SEQ ID NO: 4) primers and the product (0.51kb) was digested with EcoRV, BstXI and treated with the same restriction enzyme pShuttleCMV / D9-EGFP (FIG. 3) was obtained by inserting into pShuttleCMV / 1-EGFP and removing the pIX gene present in the homologous similarity region.

ii) Preparation of pShuttleCMV / DC92-EGFP

The smaller fragment (0.28kb) of the two fragments cut out with EcoRV and BstXI for the vector pUC57-synIVa2 with the degenerate IVa2 gene was transferred to the larger one (6.52kb) of the two fragments of the vector pShuttleCMV / D9-EGFP treated with the same restriction enzyme. Insertion yielded pShuttleCMV / DC92-EGFP with the pIX gene removed and a portion of IVa2 replaced with the degenerate nucleic acid sequence (FIG. 3).

iv) Preparation of pShuttleCMV / C92-EGFP

The pUC57-synIX of the vector pUC57-synIX with the left pIX gene was cut into EcoRV and BglII, and the small fragment (0.54 kb) was inserted into the vector pShuttleCMV / DC92-EGFP cut with EcoRV and BamHI to degenerate the pIX and IVa2 genes. A vector pShuttlemCMV / C92-EGFP was obtained that was substituted with nucleic acid sequence (about 60% homology) (FIG. 3).

Each modified vector is a plasmid pAd5.CMV-EGFP, pAd5.CMV / D9-EGFP, pAd5.CMV / DC92-EGFP, pAd5.CMV with adenovirus genome modified by homologous recombination with the pAdeasy vector. Each modified adenovirus was produced in 293 cell lines after / C92-EGFP was obtained.

Example  4 : Transformed Adenovirus  Replica Adenovirus  ( RCA ) Creation  compare

The degree of production of replicable adenovirus (RCA) was propagated in 293 cell lines of each adenovirus Ad5.CMV-EGFP, Ad5.CMV / D9-EGFP, Ad5.CMV / DC92-EGFP, Ad5.CMV / C92-EGFP. It was analyzed later. To this end, five plaques for each adenovirus were selected and propagated in 293 cell lines through 12 or more passages as shown in FIG. 4. Generation of replicable adenovirus (RCA) in each adenovirus from samples of each passaged passage was determined by quantitative analysis in non-transcomplementing cell lines (eg, A549). Plaque formation was observed after 14 days of 3 x 10 ¹¹ vp infection with each assay and agar layer over it. Because of infection with non-transcomplementing cell lines, plaque formation means the production of replicable adenovirus (RCA) by homologous recombination between viral vectors and 293 cells. Experiments with detection of replicable adenoviruses (RCAs) showed that replication of adenoviruses derived from modified adenoviruses than the conventional adenovirus Ad5.CMV-EGFP suppressed the production of replicable adenoviruses (RCA). Adenoviruses with the removed and degenerate IVa2 genes were most effective in inhibiting replication of adenovirus (RCA) production.

Viruses that inhibit the production of replication-competent adenovirus (RCA) of the present invention can be safely used for gene therapy and cell therapy of various diseases.

<110> POSTECH ACADEMY-INDUSTRY FOUNDATION          POSTECH FOUNDATION          Genexine Inc. <120> Recombinant adenoviral vector for reducing replication-competent          adenovirus formation <160> 6 <170> KopatentIn 1.71 <210> 1 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> 5'-SV40pA primer <400> 1 cgacgcggcc gcaatgcaat tgttgttgtt aacttg 36 <210> 2 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> 3'-SV40pA primer <400> 2 tcggatatcc ccagatccgc gttaagatac attgat 36 <210> 3 <211> 38 <212> DNA <213> Artificial Sequence <220> <223> 5'-del-IX primer <400> 3 cccgatatcg gatccaccat aaataaaaaa ccagactc 38 <210> 4 <211> 21 <212> DNA <213> Artificial Sequence <220> 3223-del-IX primer <400> 4 ccattgccat cattatggac g 21 <210> 5 <211> 423 <212> DNA <213> Artificial Sequence <220> <223> pIX <400> 5 atgagtacga atagcttcga cggcagtatc gtctcaagct acctgaccac caggatgcca 60 ccctgggctg gcgtccgcca aaacgtcatg gggagctcaa tcgacggcag gccagtgctc 120 ccagccaata gcaccacgct gacgtatgaa acggtcagcg gcacccccct ggaaaccgcc 180 gctagcgctg ctgccagcgc tgccgccgct acggctaggg gcatcgtcac cgatttcgcc 240 tttctctcac ccctggcctc aagcgccgcc agccgcagca gcgctaggga cgataaactg 300 accgccctgc tggcccagct ggacagcctg acgcgcgagc tgaacgtggt gagccaacaa 360 ctcctggacc tcaggcaaca agtgagcgct ctcaaagcca gcagcccccc aaacgccgtg 420 taa 423 <210> 6 <211> 276 <212> DNA <213> Artificial Sequence <220> <223> IVa2 <400> 6 ggcctcccac tcgccatatc actgctgctc aaggacatct tccgccatca tgctcaaaga 60 agctgttatg attggataat atataatacg acgccccaac acgaggccct ccaatggtgt 120 tatctgcatc cacgcgatgg cctgatgcca atgtacctca atatacaaag ccatctgtat 180 catgtgctcg agaagatcca tcgcacgctg aatgatcgcg ataggtggag ccgcgcttat 240 cgtgccagga agacgcccaa gtaaggatcc gatatc 276

Claims (5)

a nucleic acid sequence corresponding to 4091-4344 in (i) the pVIII gene, without substitution of the amino acid sequence in its entirety deleted or with reduced nucleic acid homology with the adenovirus E1 gene, and (ii) in the IVa2 gene An adenovirus vector that inhibits the production of a replication-competent adenovirus comprising an IVa2 gene substituted with a nucleic acid sequence having reduced homology with the adenovirus E1 gene. The adenovirus vector according to claim 1, wherein the pIX gene is substituted with the nucleic acid sequence of SEQ ID NO: 5. The adenovirus vector according to claim 1, wherein the IVa2 gene is substituted with the nucleic acid sequence of SEQ ID NO.6. A method of producing a replication defective adenovirus comprising the step of introducing the recombinant adenovirus vector of claim 1 into a packaging cell. The method of claim 4, wherein the packaging cell is a cell comprising the E1 gene.

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