Novel recombinant adenovirus vector having a reduced side effect
a technology of adenovirus and adenovirus gene, which is applied in the direction of viruses/bacteriophages, genetic material ingredients, dsdna viruses, etc., can solve the problems of inability to produce hd vectors in sufficient clinical applications, low vector productivity, and inability to maintain expression of transgenes, etc., to achieve the effect of enabling the persistence of transgene expression
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example 1
Construction of a Human Growth Hormone-Expressing Adenovirus Vector
[0128](1) Cloning of Human Growth Hormone cDNA
[0129]In order to clone human growth hormone cDNA by the PCR method, the following procedure was carried out.
[0130]Phage DNAs were prepared from a commercial human pituitary adenoma-derived cDNA library (CLONTECH), and used as a template DNA for PCR. Primers for PCR were designed so as to add a recognition site for a restriction enzyme on both ends, and the 5′-end primer contained the initiation codon and a NheI recognition site, and the 3′-end primer contained the termination codon and a SphI recognition site. The sequence of each primer is shown below:
5′-end primer5-TGGCTAGCTCACCTAGCGGCAATGGCT-3′(SEQ ID NO: 1) NheI Met3′-end primer5-CAGGCATGCCACCCGGGCAGCTAGAA-3′(SEQ ID NO: 2) SphI End
[0131]Using the above-mentioned cDNA library-derived DNA as a template, polymerase pfu (Takara Shuzo) was used in a standard PCR reaction to thereby obtain...
example 2
Construction of an E2A Gene-Deleted Adenovirus Vector Expressing hGH
[0135](1) Construction of a Recombinant Adenovirus Vector for constructing an E2A-deleted adenovirus vector
[0136]The cosmid vector pAx2LD3LCAwt (pAdex2LD3LCAwt in Japanese Unexamined Patent Publication (Kokai) No. 8-308585, on page 19, is identical with pAx2LD3LCAwt) is a derivative of the above-mentioned cosmid vector pAxCAwt. pAx2LD3LCAwt is a cosmid in which loxP sites have been inserted between the adenovirus L3 gene and the E2A gene (61.5 map units), and in the deletion site (78.0 map units) of the E3 gene, and the nucleotide sequence other than the loxP insertion sites is identical with pAxCAwt.
[0137]After the plasmid pUCHGH constructed in Example 1 was digested with NheI and BglI, it was blunt-ended to obtain an about 0.7 kb DNA fragment containing the coding region of hGH cDNA. The DNA fragment was inserted into the SwaI site between the promoter and the poly(A) sequence of the cosmid vector pAx2LD3LCAwt to ...
example 3
Investigation of Inflammation-Inducing Effect of an E2A-Deleted Adenovirus Vector
[0157]In order to determine whether the deletion of the adenovirus E2A gene leads to reduction of the inflammation-inducing effect, the hGH-expressing E2A-deleted adenovirus vector (ΔE2A-CAHGH) or a hGH-expressing first generation adenovirus vector (Ax1CAHGH) were injected to mice, and the effect of inducing inflammation in both adenovirus vectors was compared with the serum GPT levels (glutamic pyruvic transaminase) as an index. The method and the result are shown below.
(1) Method
[0158]Mice used were C57BL / 6 mice (7-week old, female). The adenovirus vector used was ΔE2A-CAHGH (Lot 3) or Ax1CAHGH (Lot C). The dosage of the adenovirus vector was 1×109 PFU, 3×108 PFU, or 1×108 PFU (five animals per group), and an adenovirus vector diluted in saline was administered via the tail vein at 0.2 ml per mouse. Three days before, and 3, 5, 7, 10, 14, 21, 28, 35, 42, 49, 56, and 63 days after the administration of...
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