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Hexon isolated from simian adenovirus serotype 19, hypervariable region thereof and chimeric adenovirus using the same

a technology of chimeric adenovirus and simian adenovirus, which is applied in the field of hexon, can solve the problems of adenoviruses infecting the liver, antibodies reducing the efficiency of viral replication, and the generation of antigenic epitopes of adenoviral capsids

Inactive Publication Date: 2013-03-07
MOGAM BIOTECH RES INST
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

The present invention provides a new hexon protein and its corresponding DNA for use in creating a chimeric adenovirus. This new protein helps to better prepare and control the virus for use in gene therapy and other medical applications. Additionally, the invention also provides a composition containing the chimeric adenovirus. Overall, this new technology helps to improve the efficiency and effectiveness of adenovirus-based treatments.

Problems solved by technology

However, one problem encountered with the use of adenoviral vectors for gene transfer in vivo is the generation of antibodies to antigenic epitopes on adenoviral capsid proteins.
It is also known that the antibodies reduce the efficiency of viral replication by way of inhibiting the infection of host cells (Wohlfart, C.
In addition to the above mentioned problems associated with the neutralizing antibodies, it has been reported that adenoviruses infect the liver when exposing systemically.
Such liver selectivity is a major problem in achieving efficient cure by systemic administration of an adenoviral therapeutic agent (Worgall, S., et al.
However, a method to achieve complete hexon exchange for modifying hexon protein has the problem of deteriorated productivity due to the fact that subtle structural differences in binding hexon to penton and fiber induce instability of the adenoviral capsid structure (Roberts, D. M., et al.

Method used

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  • Hexon isolated from simian adenovirus serotype 19, hypervariable region thereof and chimeric adenovirus using the same
  • Hexon isolated from simian adenovirus serotype 19, hypervariable region thereof and chimeric adenovirus using the same
  • Hexon isolated from simian adenovirus serotype 19, hypervariable region thereof and chimeric adenovirus using the same

Examples

Experimental program
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example 1

Identification of Hexon Gene of SAd19

[0081]A hexon gene was amplified from SAd19 using PCR, cloned into a pGEM-T easy vector, and sequenced with an ABI automatic DNA sequencer.

Identification of Hexon Gene

[0082]The genome of SAd19 was isolated using a DNeasy Tissue Kit (QIAGEN, Germany) and used as a template for the amplification of a hexon gene via PCR with a primer set of SEQ ID NOs: 1 and 2. The amplified hexon gene was purified by Wizard® SV Gel and PCR Clean-Up system (Promega, WI, USA) and inserted into a pGEM-T easy vector (Promega, WI, USA) with the aid of T4 DNA ligase (Roche, Switzerland). The resulting recombinant vector was named pGEM-SAd19 Hexon vector. After transformation of E. coli cells with the vector, the vector DNAs were extracted from 10 clones of the transformants and sequenced by employing an ABI automatic DNA sequencer. Among the 10 base sequences was selected the one which had the highest sequence homology with others, thereby determining the nucleotide seq...

example 2

Preparation of Chimeric Adenovirus with Hexon of SAd19 Substituted Therein

[0084]A shuttle vector for exchanging a hexon gene was constructed and named pHex vector, which is carrying left- and right-extended regions of hexon gene for homologous recombination. The hexon gene of SAd19 was cloned into unique restriction site of pHex vector, locating between left- and right-extended arms of hexon to afford a recombinant vector, named pHex-SAd19 Hexon. SphI-linearized pHex-SAd19 Hexon was subject to homologous recombination with AsiSI-linearized pAd H5—8DS in BJ5183 (Stratagene, CA, USA) to give a SAd19 Hexon-carrying recombinant vector, named pAd H5 / S19—8DS. After being linearized with Pad, the pAd H5 / S19—8DS was transfected into A549 cells to generate novel chimeric adenovirus with the hexon of SAd19 anchored therein (Ad H5 / S19—8DS).

Construction of Shuttle Vector

[0085]A shuttle vector suitable for the substitution of a hexon gene through homologous recombination was constructed. In thi...

example 3

In Vitro Study of Chimeric Adenovirus Having the Hexon of SAd19

[0096]Ad H5 / S19—8DS virus was in vitro assayed for selectivity for lung cancer by comparing expression levels of E1A and LK8 in the lung cancer cell line A549 and the normal cell line MRCS both of which were infected at various MOIs with the virus. In addition, Ad H5 / S19—8DS virus was measured for affinity for the coagulation factors which mediate blood-circulating virus to liver.

MOI-dependent E1A Expression

[0097]A549 lung cancer cells and MRCS normal cells, which were both grown at about 80% confluency in 6-well plates, were infected with Ad H5 / S19—8DS virus at an MOI of 100, 25, 10 and 1. After incubation at 37° C. for 24 hrs, the cells were harvested by centrifugation at 3,000 rpm for 5 min, suspended in 1×SDS-PAGE buffer (50 mM Tris(pH6.8), 2% SDS, 100 mM dithiothreitol, 0.1% bromophenol blue, 10% Glycerol), heated at 100° C. for 5 min in a water bath, and centrifuged at 10,000 rpm for 2 min. The resulting supernata...

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Abstract

Novel hexon isolated from simian adenovirus serotype 19 encoded in the polynucleotide defined as SEQ ID NO: 3, hepervariable region thereof, chimeric adenovirus comprising the same, and therapeutic use thereof provides a solution to the problem of safety and effective systemic treatment for developing gene therapeutic agents using adenovirus.

Description

FIELD OF THE INVENTION[0001]The present invention relates to novel hexon isolated from simian adenovirus serotype 19 (“SAd19”), hypervariable region (“HVR”) thereof, chimeric adenovirus using the same, and a therapeutic use thereof.BACKGROUND OF THE INVENTION[0002]Adenovirus belongs to the family Adenoviridae first isolated in 1953. Human adenoviruses are categorized into six (6) subgenera (A through F) based on the genome similarity, oncogenecity, and blood coagulation characteristics. Adenoviruses infect most non-divided cells such as muscle, lung, brain, and cardiac cells, and its molecular biological characteristics are well known in the art. Its genome is composed of a linear, double-stranded DNA of 35 kb and its replication in the host cell depends on viral protein, E1A.[0003]Above characteristics of adenovirus can be exploited by using a nonreplicative vector having E1A deleted therefrom. Since the development of HEK293 cell line in which the adenoviral E1 gene is inserted, t...

Claims

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Application Information

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IPC IPC(8): A61K35/76C12N15/34C12N5/10C12N15/861A61P35/00C07K14/075C12N7/01
CPCC07K14/005C12N2710/10322C12N2810/6018C12N2710/10343C12N2710/10332A61P35/00A61K39/001128A61K39/001182A61K39/0011A61K39/001151A61K39/001129C07K14/075C12N15/11C12N15/861C12N7/00C12N15/86C12N2710/10345A61K2039/5256C12N2710/10321C12N2710/10342
Inventor LEE, KYUHYUNYUN, SEONGTAEKOH, DAEKYUNGLEE, HONG-KYUJO, EUI-CHEOL
Owner MOGAM BIOTECH RES INST
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