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Production of recombinant AAV using adenovirus comprising AAV rep/cap genes

a technology of aav and adenovirus, which is applied in the field of adenoviruses, can solve the problems of not being able to easily scale up for industrial production, not being able to produce high levels of raav in packaging cell lines, and not being able to meet the requirements of large-scale industrial applications, and achieves high levels of raav. high yield, high yield of raav vector

Inactive Publication Date: 2007-03-22
GENOVO
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

The invention provides a novel method for producing high yields of rAAV vector using a novel adenovirus vector. The vector contains the AAV rep and cap genes and can be used for large-scale industrial applications. The vector is infected into cells containing a nucleic acid sequence flanked by AAV ITRs, resulting in high levels of rAAV. The vector can be easily scaled for industrial production of rAAV. The method involves infecting the host cell with an adenovirus vector containing a minimal promoter or no promoter for rep and cap genes, followed by infection with an rAAV genome. The vector can also contain a second helper virus or a stably expressed rAAV genome to supply the deleted adenoviral sequences. The invention also provides a recombinant virus carrying the rep gene in which rep interferes with its replication.

Problems solved by technology

However, the disadvantage of the methods taught by U.S. Pat. No. 5,753,500 and Li et al. is that co-transfection of two plasmids along with infection by a helper virus is inefficient, may exhibit poor reproducibility, may result in generation of pseudo-wildtype replication-competent AAV (rcAAV), and cannot be easily scaled up for industrial production of rAAV. rcAAV, comprising rep and cap flanked by ITRs, is produced when the rep and cap genes recombine with the ITRs flanking the transgene which results in deletion of the transgene.
The disadvantages of the first method are shared with this method.
The disadvantage of this method is that it requires making a cell line that expresses sufficient levels of rep and cap, and requires multiple components—including the cell line, the rAAV genome, and an adenovirus—to produce rAAV, which do not lend themselves to easy and convenient downstream manufacturing processes.
In addition, some of these packaging cell lines do not produce high levels of rAAV.
U.S. Pat. No. 5,354,678 does not disclose the levels of rAAV, if any, produced by this method.
As described above, current rAAV production methods are not amenable for production of sufficient rAAV for pharmaceutical applications in a convenient manner.

Method used

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  • Production of recombinant AAV using adenovirus comprising AAV rep/cap genes
  • Production of recombinant AAV using adenovirus comprising AAV rep/cap genes
  • Production of recombinant AAV using adenovirus comprising AAV rep/cap genes

Examples

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example 1

Cell Lines and Viruses and Maintenance and Propagation Thereof

[0119] All cell lines are maintained in Dulbecco's Modified Eagle's Medium (DMEM; Gibco BRL) supplemented with 10% FBS (Hyclone) and 50 μg / ml of penicillin, 50 μg / ml of streptomycin, and 10 μg / ml of neomycin (Gibco BRL). Human embryonic kidney cell line 293 is obtained from ATCC (CRL 1573). 293-derived 84-31 cells (1) which express adenovirus E1 / E4orf6 proteins, and HeLa-derived B50 cells (7) which express AAV-2 Rep and Cap proteins from the native p5 promoter, are obtained from Dr. Guangping Gao, Institute for Human Gene Therapy, University of Pennsylvania. 293-CG3 is a 293-derived cell line carrying stably integrated copies of AAV ITRs flanking GFP as marker gene (Chen et al., unpublished data). Human adenovirus type 5 (ATCC VR-5) and derived recombinant adenoviruses are propagated on 293 cells and purified through CsCl gradient centrifugation according to the method of Jones and Shenk with modification (2).

example 2

Construction of Plasmids and Generation of Recombinant Adenoviruses

[0120] Standard recombinant DNA techniques are employed to create recombinant plasmids (3). DNA containing the rep and cap sequences of pAV2 (ATCC 37216) between DraIII site (nucleotide 241, upstream of the AAV-2 p5 promoter) and NcoI site (nucleotide 4489, downstream of the polyA signal) is removed and replaced through multiple cloning steps with a DNA cassette containing GFP under the transcriptional control of elongation factor 1 alpha (EF1α) promoter and upstream of the SV40 polyA signal to create pAV2cisEFGFP (FIG. 1). The AAV-2 rep and cap genes located between a Dra III site (nucleotide 241, upstream of the p5 promoter) and a BsaI site (nucleotide 4464, downstream of the polyA signal) are further subcloned to obtain pAd-p5-RC (FIG. 1). A small DNA fragment between nucleotides 241 and 287 of pAd-p5-RC containing the p5 promoter is removed and replaced with a Drosophila melanogaster minimal heat shock protein (...

example 3

Transfection of 293 Cells and Selection of the 293-CG3 Stable Cell Line

[0121] 293 cells are grown to ˜70% confluency in 6-cm tissue culture dishes and co-transfected overnight with 1 μg pIRES1neo and 10 μg pAV2cisEFGFP by the calcium phosphate transfection method. The monolayer is replenished with fresh medium containing 10% FBS and cultured for 24 hours. Following trypsinization, cells are seeded at a 1:20 dilution in fresh medium containing 10% FBS. After incubation for another 24 hours, fresh medium containing 1,250 μg / ml of G418 (Gibco BRL) is added to the cell monolayer for genetic selection of G418-resistant cells. The medium containing G418 is replaced every 3-4 days to allow formation of G418-resistant cell colonies. A total of fifty colonies are picked, six of which demonstrate constitutive GFP expression. These six clones are expanded and tested for their ability to rescue functional rAAV by transfection with pBV-EiOV-RC, a plasmid that carries adenovirus E2A, E4ORF6, and...

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Abstract

This invention relates to novel adenoviruses useful in the production of high titers of recombinant adeno-associated virus (rAAV) comprising a foreign DNA inert and methods of making these adenoviruses. The adenovirus comprises the AAV rep gene in which the p5 promoter of rep is replaced by a minimal promoter or by no promoter. The invention also provides methods of producing high levels of rAAV as a substantially homogeneous preparation and compositions of rAAV.

Description

TECHNICAL FIELD OF THE INVENTION [0001] This invention relates to novel adenoviruses useful in the production of high titers of recombinant adeno-associated virus (rAAV) comprising a foreign DNA insert and methods of making these adenoviruses. The adenovirus comprises the AAV rep gene in which the p5 promoter of rep is replaced by a minimal promoter or by no promoter. The invention also provides methods of producing high levels of rAAV as a substantially homogeneous preparation and compositions of rAAV. BACKGROUND OF THE INVENTION [0002] A recombinant virus carrying a foreign DNA insert may be used to deliver genes to cells, where the gene may be expressed, if desired, to permit production of recombinant proteins in vitro or in vivo, vaccination of human and non-human mammals, or treatment or amelioration of diseases or genetic defects in humans or in non-human mammals. One may treat or ameliorate diseases or genetic defects by providing some effective level of normal gene products,...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): A61K48/00C12N15/861
CPCA61K2039/5256C12N15/86C12N2710/10344C12N2750/14143C12N2840/203C12N2830/42C12N2830/75C12N2840/20C12N2830/002
Inventor CHEN, HAIFENGKURTZMAN, GARY
Owner GENOVO
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