Oncolytic adenoviruses for the treatment of cancer

An oncolytic adenovirus, adenovirus technology, applied in the field of tumor biology, can solve problems such as lack of potency, genome instability, and interference with promoter virus sequences

Inactive Publication Date: 2009-07-15
DNATRIX
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the application of cellular promoters to a viral background has revealed an important problem: the presence of viral sequences that interfere with the correct regulation of the promoter and reduce selectivity 16，17
Furthermore, it has also been reported that even small modifications in the E4 region can lead to genome instability, thus strategies based on E4 region modification have been abandoned. 22
Besides selectivity, another problem with the E2F1 promoter is the lack of potency

Method used

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  • Oncolytic adenoviruses for the treatment of cancer
  • Oncolytic adenoviruses for the treatment of cancer
  • Oncolytic adenoviruses for the treatment of cancer

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0065] Oncolytic adenoviruses with E1a regulated by the E2F1 promoter separated by MD sequences express E1a and replicate selectively in tumor cells.

[0066] Adenoviruses with E1a regulated by the E2F1 promoter separated by MD sequences were constructed as follows: For the production of ICOVIR-1 (Ad-E2F-Δ24RGD), the human E2F1 promoter was obtained by PCR of human peripheral blood mononuclear cells using Oligonucleotides extending from -218 bp to +51 bp of the E2F1 promoter (position +1 indicates start of transcription). The oligonucleotides contained KpnI and HindIII restriction targets for cloning into plasmid pGL3 (Promega, Southampton, UK). The resulting plasmid was named pGL3-E2F. Thus, by removing (nt) 122 and 129 nucleotides of E1a (derived from pXC1-Δ24, which has a HindIII site between nt 348 and nt 522 of the Ad5 genome) 9 ) except for the 5766-base plasmid was recombined to obtain pE2F-Δ24. pE2F-Δ24 and pShuttle 41 Recombination was performed to obtain pShuttle...

Embodiment 2

[0070] The kozak sequence enhances E1a expression in oncolytic adenoviruses in which E1a is regulated by the E2F1 promoter separated by MD.

[0071]Oncolytic adenoviruses were constructed with E1a regulated by the E2F1 promoter separated by the MD sequence and a kozak sequence that enhances its translation. To this end, a DNA fragment containing the MD sequence, the E2F1 promoter and E1a was isolated from pShuttle-MD-E2F-D24 described in Example 1 by KpnI restriction digestion and subcloned into pGEM3Z (Promega) to obtain the plasmid pGEM- E2F-d24. This plasmid was used to replace the E1a translation origin with an oligonucleotide containing the kozak sequence to obtain pGEM-E2F-KD24. The thus modified KpnI fragment was recloned into KpnI of pShuttle-MD-E2F-D24 to obtain pShuttle-DM-E2F-KD24. Finally, pShuttle-MD-E2F-KD24 was recombined with pVK503 to obtain pICOVIR5. ICOVIR5 virus was generated by digesting this plasmid with PacI and transfected into HEK293 cells. ICOVIR5...

Embodiment 3

[0074] The kozak sequence confers increased oncolytic potency of adenovirus whose E1a expression is regulated by the E2F1 promoter separated by MD.

[0075] We cultured cells from the tumor cell lines SKMel-28 and FaDu in 96-well plates, where visible reduction of ICOVIR2 replication ability (as in Example 1 and Figure 4 described in ). These cells were infected with increasing amounts of ICOVIR5, ICOVIR2 and AdwtRGD (the latter serving as a control for maximal lytic capacity). Five days after infection, the amount of protein was measured spectroscopically as a reflection of cell survival. The results in the present invention Figure 6 displayed in . The cleavage ability of ICOVIR5 in SKMel-28 was the same as that of AdwtRGD and higher than that of ICOVIR2. In FaDu, it is also higher than ICOVIR2, although not to the level of AdwtRGD.

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Abstract

The invention relates to an oncolytic adenovirus for the treatment of cancer, containing a human DNA sequence isolating a promoter conferring selective expression on an adenoviral gene. Said adenovirus can also contain a sequence that optimises the protein translation of an adenoviral gene regulated by a promoter conferring tumour selectivity. The invention is suitable for use in the treatment of cancer.

Description

technical field [0001] The field of the invention relates primarily to the field of tumor biology. In particular, the present invention relates to adenoviruses that replicate selectively in tumors (called oncolytic adenoviruses) and their use for inhibiting cancer. Background technique [0002] Existing treatments for cancer are mainly based on chemotherapy, radiation therapy and surgery. Although early stage cancers have a high cure rate, most cases of advanced stage cancers are incurable because they cannot be removed surgically or because their doses are limited due to the toxicity of radiation therapy or chemotherapy to normal cells . To alleviate this situation, biotechnologies have been developed that seek to improve the efficacy and selectivity of cancer treatments. Among them, gene therapy and virotherapy use viruses to treat cancer. In gene therapy, viruses are modified to prevent their replication and to serve as vehicles or carriers of therapeutic genetic mate...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/861A61K35/761
CPCC12N2710/10332A61K35/761C12N2820/007C12N2710/10343A61K35/76C12N7/00C12N15/86A61P35/00C12N15/8613C12N2710/10021C12N2710/10032C12N2710/10043C12N2710/10321
Inventor 拉蒙·阿莱马尼博纳斯特雷马内尔·马里亚·卡斯卡洛皮克拉斯胡安·乔斯·罗哈斯埃克斯波西托
Owner DNATRIX
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