Effect of the tripartite leader on synthesis of a non-viral protein in an adenovirus 5 recombinant

Berkner, Kathleen L.; Sharp, Phillip A.

doi:10.1093/nar/13.3.841

Cited by 90 publications

(64 citation statements)

References 18 publications

(20 reference statements)

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“…19 The higher expression of the HSVtk gene is likely due to vector replication and its preceding by the Ad5 TPL. 20,21 This suggests that the level of HSVtk expression as previously reported 22 and the presence of E1a, independent of its role in adenoviral replication, are not responsible for the significantly enhanced antitumor effect, compared with either treatment used alone, when GCV was given to Ad.TK RC -treated mice after allowing time for viral replication. This observation demonstrates the importance of the amplification and spread of the viral inoculum beyond the initially infected cells by in situ conversion of the originally transduced cells into adenoviral producer cells.…”

Section: Discussionsupporting

confidence: 55%

Adenoviral vectors capable of replication improve the efficacy of HSVtk/GCV suicide gene therapy of cancer

et al. 1999

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A major obstacle to the success of gene therapy strategies with ME180 tumors. Treatment of tumors with Ad.TK RC that directly target cancer cells is the poor vector distriwithout GCV resulted in a similar antitumor effect, conbution within solid tumors. To address this problem, we firming that the replicating vector has an oncolytic effect. developed an E1b 55 kDa attenuated, replicationWhen GCV was initiated 3 days after Ad.TK RC injection, competent adenovirus (Ad.TK RC ) which expresses the hersurvival of mice with each tumor type was greatly propes simplex-1 thymidine kinase (HSVtk) gene to sensitize longed, with 60% of animals with ME180 tumors surviving tumors to ganciclovir (GCV). Efficacy of this combined for over 160 days. These results confirm that both the strategy was tested in nude mice with subcutaneous oncolysis caused by a replicating virus and suicide/prodrug human A375 melanoma and ME180 cervical carcinomas.gene therapy with HSVtk/GCV have potent antitumor Intratumoral injection of a replication-defective adenoviral effects. When combined, these two approaches are compvector expressing HSVtk (Ad.TK) followed by GCV treatlementary resulting in a significantly improved treatment ment resulted in doubling of the survival time of mice bearoutcome. ing A375 tumors and 20% long-term survival of mice

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Section: Discussionsupporting

confidence: 55%

Adenoviral vectors capable of replication improve the efficacy of HSVtk/GCV suicide gene therapy of cancer

et al. 1999

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“…Consequently, we attempted to produce replication-defective Ad vectors in which the expression of RG was driven by the strong MCMV promoter (Dorsch-Hasler et al, 1985 ;Addison et al, 1997) in combination with an intron sequence previously used in Ad vectors (Berkner & Sharp, 1985). The construction of plasmid pMH5(I)Fx is described in Methods and shown in Fig.…”

Section: Resultsmentioning

confidence: 99%

“…1). The Ad-immunoglobulin hybrid intron from plasmid pMLPsp1a (Kaufman & Sharp, 1982 ;Berkner & Sharp, 1985) was isolated by PCR amplification and inserted into the EcoRI site of pMH5 to generate pMH5(I). The RG gene was cloned into pMH5(I) downstream of the MCMV promoter and the intron to produce plasmid pMH5(I)Fx.…”

Section: Methodsmentioning

confidence: 99%

Development and use of a 293 cell line expressing lac repressor for the rescue of recombinant adenoviruses expressing high levels of rabies virus glycoprotein.

Matthews¹,

Cummings²,

Evelegh³

et al. 1999

Journal of General Virology

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An expression cassette designed for high-level production of rabies virus glycoprotein (RG) could not be rescued into a replication-defective, adenovirus-based vector using standard procedures. To overcome this difficulty, a 293-based cell line, designated 293LAP13, was constructed that contained and expressed a derivative of the lac repressor protein. The lac operator sequence, to which the repressor binds, was incorporated into an expression cassette, containing a promoter and intron, designed for high-level production of RG. Insertion of a single operator sequence immediately downstream of the transcription start site and the use of the 293LAP13 cell line allowed recombinant viruses that could not be isolated with 293 cells to be rescued efficiently. The operator-containing virus reached higher titres in 293LAP13 than in parental 293 cells and also produced plaques more efficiently in 293LAP13 cells. Moreover, in non-complementing human and canine cell lines, adenovirus vectors with a promoter-intron expression cassette expressed RG at much higher levels than vectors lacking the intron. These observations, together with the demonstration that expression of RG by operator-containing vectors was repressed markedly in 293LAP13 cells and that this inhibition was relieved at least partly by IPTG, suggest that the 293LAP13 cell line may be useful for the rescue and propagation of many vectors in which high expression of the desired protein prevents vector rescue in 293 cells.

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“…The activity of the parental plasmid, pBLCAT6, was then normalized to 1 in all cell lines. As a positive control, pD5CAT, which contains the SV40 promoter/enhancer region upstream of the CAT gene was used (Berkner and Sharp, 1985, data not shown). The most active Ron/CAT promoter construct in CMT-93 cells had approximately 10% of the amount of CAT protein produced compared to the positive control.…”

Section: Deletion Analyses Of the 5'¯anking Dna Of The Mouse Ron Genementioning

confidence: 99%

Characterization of the mouse Ron/Stk receptor tyrosine kinase gene

et al. 1998

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In an e ort to understand the mechanisms governing the regulation of the mouse Ron receptor gene, a mouse genomic library was screened and overlapping clones coding for the Ron gene and¯anking DNA were identi®ed. Continuous DNA sequence was obtained for approximately 16.4 kilobases. The gene, from the initiator methionine to the polyadenylation site, is contained within 13 244 basepairs and contains 19 exons. Primer extension analyses were performed to determine the transcription start site of the mouse Ron transcript. Multiple transcription start sites were found which also appear to be used in transfected reporter constructs containing Ron 5'¯anking DNA. To determine the location of sites which may be critical for the function of the Ron gene promoter, a series of chimeric genes containing serial deletions of the Ron gene promoter fused to the coding sequences for the chloramphenicol acetyltransferase gene were constructed. Transient transfection analyses of these hybrid genes into various cell lines demonstrated that two regions of the Ron gene promoter, encompassing nucleotides 7585 to 7465 and from 7465 to 7285, are important for expression of this transcript in CMT-93 cells. Further analysis of the Ron promoter utilizing gel mobility shift analyses suggests that regions encompassing nucleotides 7585 to 7508 and nucleotides 7375 to 7285 appear to bind speci®c proteins which may be involved in the negative and positive regulation, respectively, of the mouse Ron gene.

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Effect of the tripartite leader on synthesis of a non-viral protein in an adenovirus 5 recombinant

Cited by 90 publications

References 18 publications

Adenoviral vectors capable of replication improve the efficacy of HSVtk/GCV suicide gene therapy of cancer

Adenoviral vectors capable of replication improve the efficacy of HSVtk/GCV suicide gene therapy of cancer

Development and use of a 293 cell line expressing lac repressor for the rescue of recombinant adenoviruses expressing high levels of rabies virus glycoprotein.

Characterization of the mouse Ron/Stk receptor tyrosine kinase gene

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