The development of the conditionally replication-competent adenovirus: replacement of E4 orf1-4 region by exogenous gene.
BACKGROUND
Tumor or tissue specific replicative adenovirus armed with a therapeutic gene has shown a promising anti-cancer therapeutic modality. However, because the genomic packaging capacity is constrained, only a few places inside it are available for transgene insertion. In the present study, we introduce a novel strategy utilizing the early E4 region for the insertion of therapeutic gene(s).


METHODS
We constructed the conditionally replication-competent adenovirus (CRAd), Ad5E4(mRFP) by: (i) replacing the E4/E1a promoter by the prostate-specific enhancer element; (ii) inserting mRFP inside the E4orf1-4 deletion region; and (iii) sub-cloning enhanced green fluorescent protein controlled by cytomegalovirus promoter in the left end of the viral genome. Subsequently, we evaluated its replication abilities and killing activities in vitro, as well as its in vivo anti-tumor efficacy in CWR22rv xenografts.


RESULTS
When infected with Ad5E4(mRFP), the number and intensity of the mRFP gene products increased in a prostate cancer cell-specific manner as designed, suggesting that the mRFP gene and E4orfs other than E4orf1-4 were well synthesized from one transcript via alternative splicing as the recombinant adenovirus replicated. As expected from the confirmed virus replication capability, Ad5E4(mRFP) induced cell lysis as potent as the wild-type adenovirus and effectively suppressed tumor growth when tested in the CWR22rv xenografts in nude mice. Furthermore, Ad5E4(endo/angio) harboring an endostatin-angiostatin gene in E4orf1-4 was able to enhance CRAd by replacing mRFP with a therapeutic gene.


CONCLUSIONS
The approach employed in the present study for the insertion of a therapeutic transgene in CRAd should facilitate the construction of CRAd containing multiple therapeutic genes in the viral genome that may have the potential to serve as highly potent cancer therapeutic reagents.
Nam
Jae-Kook
JK
Genitourinary Cancer Branch, National Cancer Center, 111 Jungbalsan-ro, Ilsandong-gu, Gyeonggi-do, Republic of Korea.
Lee
Mi-Hyang
MH
Seo
Hae-Hyun
HH
Kim
Seok-Ki
SK
Lee
Kang-Huyn
KH
Kim
In-Hoo
IH
Lee
Sang-Jin
SJ
D000256:Adenoviridae / Q000235:genetics* / Q000502:physiology*; D017504:Adenovirus E4 Proteins / Q000235:genetics*; D000818:Animals; D016923:Cell Death; D045744:Cell Line, Tumor; D004274:DNA, Recombinant / Q000235:genetics; D005818:Genetic Engineering; D006801:Humans; D008164:Luminescent Proteins / Q000235:genetics*; D008297:Male; D051379:Mice; D008819:Mice, Nude; D016366:Open Reading Frames / Q000235:genetics*; D011471:Prostatic Neoplasms / Q000473:pathology; D011995:Recombination, Genetic; D014779:Virus Replication / Q000502:physiology*; D023041:Xenograft Model Antitumor Assays; D000097573:Red Fluorescent Protein
D017504:Adenovirus E4 Proteins; D004274:DNA, Recombinant; D008164:Luminescent Proteins; D000097573:Red Fluorescent Protein