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Adenovirus serotype 34 vectors, nucleic acids and virus produced thereby

A technology of adenovirus and serotype, applied in the direction of virus/bacteriophage, virus, viral peptide, etc., can solve problems such as stimulating excessive immune response

Inactive Publication Date: 2006-04-12
MERCK & CO INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In addition, other serotypes have different tropisms, leading to eliciting exaggerated immune responses when used for vaccine or gene therapy purposes

Method used

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  • Adenovirus serotype 34 vectors, nucleic acids and virus produced thereby
  • Adenovirus serotype 34 vectors, nucleic acids and virus produced thereby
  • Adenovirus serotype 34 vectors, nucleic acids and virus produced thereby

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] Construction of pAd34ΔE1ΔE4Ad50rf6

[0051] To generate an E1-Ad34-based vector capable of propagating in an existing C / Ad5 E1 complementing cell line (293, PER.C6), Ad5 Orf6 was inserted into the native E4 region. To construct the Ad34 pre-adenoviral plasmid, the sequence homology between Ad34 and Ad35 was exploited. Cotransformation of BJ5183 bacteria with purified wild-type Ad34 viral DNA and an appropriately constructed Ad35 ITR expression cassette resulted in circularization of the viral genome due to homologous recombination. Construction of the Ad34-based preAd plasmid is outlined below:

[0052] We utilized the Ad35 ITR cassette to construct pAd34ΔE1ΔE4Ad50rf6 (Ad34 pre-Ad plasmid containing El and E4 deletions replaced with Ad5 Orf6). We anticipate that the sequence homology between Ad34 and Ad35 will allow homologous recombination to occur. The constructed Ad35 ITR cassette contained the right (bp 31599 to 31913 and bp 34419 to 34793) and left (bp4 to 456 a...

Embodiment 2

[0054] Rescue of pAd34AE1AE4Ad50rf6 into virus

[0055] To determine whether the pre-adenoviral plasmid pAd34ΔE1ΔE4Ad50rf6 could be rescued into virus and proliferate in the group CE1 complementation cell line, the plasmid was digested with PmeI and transfected into PER.C6 cells in T-25 flasks using the calcium phosphate co-precipitation technique ( Cell PhectTransfection Kit, Amersham Pharmacia Biotech Inc). After entry into the cell, Pme I digestion releases the viral genome from the plasmid sequence, allowing viral replication. Following transfection, a viral pathological effect (CPE) was observed indicating viral replication and amplification was occurring. Approximately 7-10 days after transfection, at the end of CPE, the infected cells and medium were harvested, freeze-thawed three times and the cell debris was pelleted by centrifugation. Approximately 1 ml of cell lysate was used to infect 80-90% confluent PER.C6 cells in T-225 flasks. Once CPE appeared, infected cel...

Embodiment 3

[0056] Example 3 Inserting the expression cassette into pAd34ΔE1ΔE4Ad50rf6

[0057] To introduce the gag or SEAP expression cassette (see Figures 8 and 9, respectively) into the El region of pAd34ΔE1ΔE4Ad50rf6, bacterial recombination was again used. A Gag expression cassette consisting of: 1) the human cytomegalovirus immediate early gene promoter, 2) the coding sequence of the human immunodeficiency virus type 1 (HIV-1) gag (strain CAM-1; 1526bp) gene, and 3) The bovine growth hormone polyadenylation signal sequence, cloned into the El deletion of the Ad35 shuttle plasmid, pNEBAd35-2 (precursor to the Ad35ITR cassette described above), yielded pNEBAd35CMVgagBGHpA. pNEBAd35-2 contains the Ad35 sequence (bp4-456 and bp3403-3886) from the left end of the genome, with a single SwaI site between the deletion site bp456-3403. The gag expression cassette was obtained from the previously constructed shuttle plasmid by EcoRI digestion. After digestion, the fragment of interest was ...

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Abstract

Adenoviral serotypes differ in their natural tropism. The various serotypes of adenovirus have been found to differ in at least their capsid proteins (e.g., penton-base and hexon proteins), proteins responsible for cell binding (e.g, fiber proteins), and proteins involved in adenovirus replication. This difference in tropism and capsid proteins among serotypes has led to the many research efforts aimed at redirecting the adenovirus tropism by modification of the capsid proteins. The present invention bypasses such requirement for capsid protein modification as it presents a recombinant, replication-defective adenovirus of serotype 34, a rare adenoviral serotype, and methods for generating the alternative, recombinant adenovirus. Additionally, means of employing the recombinant adenovirus for the delivery and expression of exogenous genes are provided.

Description

[0001] Cross References to Related Applications [0002] This application claims the benefit of US Provisional Patent Application 60 / 458,825, filed March 28,2003. Background of the invention [0003] The adenoviruses that have been identified in some birds and mammals are non-enveloped icosahedral viruses; Horne et al., 1959 J. Mol. Biol. 1:84-86; Horwitz, 1990 InVirology, eds. B.N. Fields and D.M. Knipe, pps. 1679-1721. The first human adenoviruses (Ads) were isolated more than 40 years ago. Since then, more than 100 different adenovirus serotypes have been isolated, which infect different mammalian species, 51 of which are of human origin; Straus, 1984, In The Adenoviruses, ed.H.Ginsberg, pps.451- 498, New York: Plenus Press; Hierholzer et al., 1988 J. Infect. Dis. 158: 804-813; Schnurr and Dondero, 1993, Intervirology; 36: 79-83; : 3940-5. Human serotypes have been classified into six subgenera (A-F) according to biological, chemical, immunological and structural criter...

Claims

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Application Information

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IPC IPC(8): C12N15/00C12N15/01C12N15/33A61K39/00A61K48/00C07K14/16C12N15/861
CPCC12N2740/16134C12N15/86A61K2039/5256C12N2740/16122C12N2710/10343A61K2039/53C07K14/005A61K48/00C12N2710/10371A61P31/18
Inventor E·A·埃米尼J·W·希弗A·J·贝特D·R·卡西米罗D·C·卡斯罗M·查斯泰恩
Owner MERCK & CO INC