Replication of enhancer-deficient amphotropic murine leukemia virus in human cells

Reuss, Frank U.; Berdel, Bianca; Ploss, Martin; Heber, Ricarda

doi:10.1073/pnas.191182098

Cited by 8 publications

(32 citation statements)

References 34 publications

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“…The findings presented here for FV-based vectors are different from those described for MLV vectors, 9,10,35 since RCRs appeared more rapidly with FFV SIN vectors. This is surprising since the FFV SIN deletion removed promoter sequences from À18 to À725, whereas those for MLV were much smaller (À150 to À357) removing only enhancer elements and hardly affecting the core promoter.…”

Section: Features Of Foamy Virus Sin Vectors P Bastone and M Löcheltcontrasting

confidence: 99%

“…[4][5][6] For retroviruses, partial or complete deletion of the U3 promoter of the 3 0 long terminal repeat (LTR) is one way to directly abrogate infectivity resulting in self-inactivating (SIN) retroviral vectors; 7 however, recombination events may lead to replication-competent revertants (RCRs). [8][9][10][11] Such RCRs can be expected to pose, besides the intrinsic insertional mutagenesis potential of retroviral vectors, added risk of altering the expression of cellular genes. Vectors based on apathogenic viruses already possess a substantial degree of biological safety, but under rare conditions genetic manipulation and insertion of therapeutically relevant genes into apparently innocuous viruses can still result in disease potential.…”

Section: Introductionmentioning

confidence: 99%

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Kinetics and characteristics of replication-competent revertants derived from self-inactivating foamy virus vectors

Bastone

Löchelt

2004

Gene Ther

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In this study, self-inactivating (SIN) retroviral vectors based on feline foamy virus (FFV) were constructed and analysed. The FFV SIN vectors were devoid of the core FFV long terminal repeat promoter plus upstream sequences but contained all structural and regulatory genes. This design allowed sensitive detection of replication-competent revertants (RCRs). The FFV SIN vectors efficiently transduced the green fluorescence protein into recipient cells. However, RCRs appeared after serial passages of transduced cells. In all RCR clones analysed, parts of the heterologous cytomegalovirus immediate early promoter, originally driving expression of the FFV vector genome, were taken up to restore the deleted SIN promoter function required for replication competence. The RCRs were strongly reduced in replication capacity compared with the parental replication-competent vectors containing the FFV promoter. In all RCR genomes analysed, the uptake of the heterologous promoter was accompanied by deletion of almost the complete marker gene. Although the RCRs described in this study may not have the capacity to spread in humans and animals, they may pose a theoretical risk, for instance during transduction of haematopoietic stem cells. Thus, FV-based SIN vectors require additional genetic modifications in order to avoid RCRs.

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Section: Features Of Foamy Virus Sin Vectors P Bastone and M Löcheltcontrasting

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Kinetics and characteristics of replication-competent revertants derived from self-inactivating foamy virus vectors

Bastone

Löchelt

2004

Gene Ther

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“…13 In brief, the provirus MLV-(MOA) is based on the Moloney-MLV clone 63-2, 14 but contains the (Figure 1) is characterized by a deletion that extends from position Ϫ357 to Ϫ150 in the U3 region and removes both enhancer copies at positions Ϫ341 to Ϫ257 and Ϫ266 to Ϫ182. 15 We have demonstrated that amphotropic MLV replication in most transformed human cells tested requires the presence of these 75 bp enhancer elements.…”

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confidence: 99%

“…16 However, in specific human fibrosarcoma and lymphoma lines virus replication is possible in the absence of these enhancers. 13 One of these lines, the fibrosarcoma HT-1080 is the basis of the FLY family of packaging cells. 17 In contrast to these human cells, murine NIH/3T3 cells are not permissive for Mo-MLV mutants that lack both 75 bp enhancer elements.…”

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confidence: 99%

Enhancer-deficient amphotropic murine leukemia virus and recombinants with heterologous transcription elements can be efficiently amplified and detected in Mus dunni fibroblasts

et al. 2002

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Amphotropic murine leukemia virus (MLV) replicates in cells from various mammalian species, including humans, and is a potential contaminant in MLV vector preparations for human gene transfer studies. Mus dunni fibroblasts are routinely used for amplification and detection of contaminating virus. We have recently characterized an amphotropic MLV mutant lacking the 75-bp viral enhancer elements and spontaneous MLV-(RCMV) recombinants that have acquired cytomegalovirus (CMV) transcription elements. Both of these viruses replicate in specific human cell types. To test Keywords: MLV; murine leukemia virus; amphotropic; Mus dunni; replication; detectionAmphotropic murine leukemia virus was originally isolated from feral mice and found to replicate in cells from various mammalian species, including humans.1,2 The env gene of isolate 4070A 3 was subsequently used for transduction of primate cells with the newly developed murine leukemia virus (MLV) vectors. 4,5 Human gene transfer studies frequently use MLV-based vectors with amphotropic host range to introduce and express markeror therapeutic genes. 6 Recombinant, replication-competent amphotropic murine leukemia virus is a potential contaminant and safety risk in these vector preparations.7,8 Replication-competent virus can potentially arise at all stages of vector production by recombination between vector and packaging constructs or host DNA, such as endogenous retroviruses. The probability of such a recombination and the exact genotype of the recombinant virus depends on the design of the production process and the nature of packaging-and vector constructs. Considerable progress has been made during the past years to exclude the generation of replication competent virus (for a recent review see Ref. 10). The US Food and Drug Administration (FDA) has recently released 'Guidance on testing for replication competent retrovirus in retroviral vector-based gene therapy products and during follow-up of patients in clinical trials using retroviral vec- 11 As a routine procedure the FDA recommends testing samples of the cell banks, vector supernatant and ex vivo transduced cells by infection on a Mus dunni fibroblast line 12 and subsequent detection in a PG-4 S+LϪ indicator assay.We used an amphotropic host range mutant of Moloney MLV, MLV-(MOA) (Figure 1), to study replication and gene expression of amphotropic MLV in human cells. 13 In brief, the provirus MLV-(MOA) is based on the Moloney-MLV clone 63-2, 14 but contains the

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“…Recent reports of replication-competent retroviruses with enhancer deletion underscore these concerns. 43 …”

Section: Discussionmentioning

confidence: 99%

Retroviral hybrid LTR vector strategy: functional analysis of LTR elements and generation of endothelial cell specificity

2004

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Transcriptional targeting is an important aspect of developing gene therapy vectors in order to restrict transgene expression to selected target cells. One approach, when using retroviral vectors, is to replace viral transcriptional control elements within the long terminal repeat (LTR) with sequences imparting the desired specificity. We have developed such hybrid LTR retroviruses, incorporating sequences from each of the human promoters for flt-1, ICAM-2 and KDR, as part of our antivascular cancer gene therapy strategy targeting tumour endothelial cells. The chosen fragments were used to replace the enhancer or combined enhancer and proximal promoter regions of the viral LTR. All showed activity in primary human breast microvascular endothelial cells, with viruses incorporating ICAM-2 sequences exhibiting the greatest specificity versus nonendothelial cells in vitro and a marked alteration of specificity towards endothelial cells in a subcutaneous xenograft model in vivo. Moreover, our study documents the effect of enhancer and/or proximal promoter deletion on LTR activity and reports that differential dependence in different cell lines can give the false impression of specificity if experiments are not adequately controlled. This finding also has implications for other retroviral vector designs seeking to provide transcriptional specificity and for their safety with respect to prevention of gene activation at sites of proviral integration.

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Replication of enhancer-deficient amphotropic murine leukemia virus in human cells

Cited by 8 publications

References 34 publications

Kinetics and characteristics of replication-competent revertants derived from self-inactivating foamy virus vectors

Kinetics and characteristics of replication-competent revertants derived from self-inactivating foamy virus vectors

Enhancer-deficient amphotropic murine leukemia virus and recombinants with heterologous transcription elements can be efficiently amplified and detected in Mus dunni fibroblasts

Retroviral hybrid LTR vector strategy: functional analysis of LTR elements and generation of endothelial cell specificity

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