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Promoterless cassettes for expression of alphavirus structural proteins

A technology of structural proteins and non-structural proteins, which is applied in the field of preparation of recombinant alpha virus particles, can solve the problems of lack, reduction of the theoretical number of functional recombination events, and decline in the theoretical prediction of the formation rate of replicable alpha viruses, etc.

Active Publication Date: 2010-08-11
ALPHAVAX INC
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0013] The present invention provides alphavirus RNA helper molecules encoding alphavirus structural proteins that lack promoter sequences, thereby significantly reducing the theoretical number of functional recombination events that can occur between the helper molecule and the replicon vector, resulting in recombinant alphavirus particles A theoretically predicted decline in the rate of replication-competent alphavirus formation during production

Method used

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  • Promoterless cassettes for expression of alphavirus structural proteins
  • Promoterless cassettes for expression of alphavirus structural proteins
  • Promoterless cassettes for expression of alphavirus structural proteins

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0111] Embodiment 1: Construction of dHcap and dHgp helper

[0112] Primers (capsid F (SEQ ID NO: 98), GPF (SEQ ID NO: 60) and 13-101.pr4 (SEQ ID NO: 6) (Table 1) were designed for use from the VEE helper plasmid (for The capsid helper, referred to as "13.2.2", for the glycoprotein helper, referred to as "13.4.6") amplifies the capsid and glycoprotein (GP) genes, as described in U.S. Patent No. 5,792,462, Pushko et al. , 1997 (Virology 239:389-401) and PCT Publication WO02 / 03917 (Olmsted et al.). These primers each provide a RsrII restriction site and also bind to the beginning of the capsid or glycoprotein coding sequence. References cited above The DNA plasmids described in are a convenient source for obtaining structural protein coding fragments, e.g., by PCR amplification. Alternatively, these coding fragments can be obtained from full-length clones of VEE or attenuated variants thereof (seeing, U.S. Patent No. 5,185,440 ; U.S. Patent 5,505,947).

[0113]Amplification us...

Embodiment 2

[0115] Example 2. The expression analysis method of promoter-free auxiliary expression cassette

[0116] To determine how well the Δ26S helper constructs described here express structural proteins, each helper was electroporated into Vero cells along with the VEE replicon vector described above. To demonstrate the capability of the novel promoterless structural protein expression cassette of the present invention, the GFP or botulinum neurotoxin coding sequence was inserted into the cloning site of the VEE replicon vector. Expression of these coding sequences from particles using various combinations of the promoterless structural protein expression cassettes described herein demonstrates the utility and novelty of these cassettes.

[0117] Use RiboMAX T7 according to the manufacturer's procedure The transcription kit (Promega Corporation, Madison, WI) transcribes RNA from each helper and replicon vector by runaway transcription. Prior to electroporation, helper and replico...

Embodiment 3

[0124] Example 3: Expression analysis of full-length and truncated Δ26S helpers

[0125] The dHcap(FL) and dHgp(FL) helpers expressed protein as determined by IFA and Western blotting, and replicated efficiently as demonstrated by Northern blotting.

[0126] The complete set of truncated Δ26S replicons (deletions 1-8) for capsid and GP were analyzed, protein expression was analyzed by IFA, and how well each was expressed and replicated by Northern blotting. Each dHcap helper RNA was combined with VEE replicon RNA and 13.4.6 glycoprotein helper RNA, and the three RNAs were electroporated into Vero cells. Northern analysis and IFA were performed as described above. The results of IFA using capsid-specific antibodies are shown in Table 2. All dHcap helpers were positive for capsid expression by IFA, although the dHcap8 helper was only weakly positive.

[0127] Northern analysis of RNA extracted from electroporated cells indicated that all truncated capsid Δ26S helpers replicat...

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Abstract

The present invention provides an isolated RNA molecule comprising: a) an alphavirus 5' replication recognition sequence, wherein at least one initiation codon has been removed from the 5' replication recognition sequence; b) a nucleotide sequence encoding an alphavirus structural protein; and c) an alphavirus 3' replication recognition sequence, with the proviso that the RNA molecule does not contain a promoter that directs transcription of the nucleotide sequence of (b), and wherein the alphavirus 5' and 3' replication recognition sequences of (a) and (c) direct replication of the RNA molecule in the presence of alphavirus nonstructural proteins.

Description

[0001] priority statement [0002] This application claims the benefit of U.S. Provisional Application No. 60 / 936,637, filed June 21, 2007, under 35 U.S.C. §119(e), which is hereby incorporated by reference in its entirety. [0003] Statement of Government Funding [0004] Aspects of this invention were supported with funding under Grant No. 5 UO1A1057286-03 from the National Institutes of Health. field of invention [0005] The present invention relates to improved constructs for making recombinant alphavirus particles and methods of making recombinant alphavirus particles. Background of the invention [0006] α病毒目前用作研发传染病和癌症疫苗的载体平台(例如,参见U.S.专利No.5,792,462;6,156,558;5,811,407;6,531,135;6,541,010;6,783,939;6,844,188;6,982,087;7,045,335;5,789,245;6,015,694;5,739,026;Pushko等,Virology 239(2): 389-401 (1997), Frolov et al., J. Virol. 71(1): 248-258 (1997); Smerdou and Liljestrom, J. Virol. 73(2): 1092-1098 (1999) ). Alphaviruses comprise the genus in the Togaviridae family, ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/63C12N15/40C07K14/705A61K38/00
CPCA61K39/285C12N15/86C12N2710/24134A61K2039/5256C12N2770/36143C12N2770/36134A61K39/12A61P31/00A61P31/12A61P31/14A61P35/00A61P37/04
Inventor K·I·卡姆鲁德J·F·史密斯M·莫汉
Owner ALPHAVAX INC
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