Efficient retrovirus-mediated transfer and expression of a human adenosine deaminase gene in diploid skin fibroblasts from an adenosine deaminase-deficient human.

Palmer, Theo D.; Hock, Rick S.; Osborne, William; Miller, A. Dusty

doi:10.1073/pnas.84.4.1055

Cited by 134 publications

(31 citation statements)

References 32 publications

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“…Knockout and wild-type control keratinocytes were infected 2 days after plating with a high titer helper-free recombinant retrovirus based on the LXCN vector (Palmer et al 1987) and transducing the v-Ha-ras oncogene (Dotto et al 1985). At 6 days after infection, cells were collected by trypsinization, counted, and injected subcutaneoulsy into nude mice at the indicated concentrations.…”

Section: Tumorigenicity Assaysmentioning

confidence: 99%

The absence of p21Cip1/WAF1 alters keratinocyte growth and differentiation and promotes ras-tumor progression.

Missero¹,

Cunto²,

Kiyokawa³

et al. 1996

Genes Dev.

305

271

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p21Cip1/WAF1 was the first cyclin-dependent kinase (CDK) inhibitor to be identified, as a mediator of p53 in DNA damage-induced growth arrest, cell senescence, and direct CDK regulation. p21 may also play an important role in differentiation-associated growth arrest, as its expression is augmented in many terminally differentiating cells. A general involvement of p21 in growth/differentiation control and tumor suppression has been questioned, as mice lacking p21 undergo a normal development, harbor no gross alterations in any of their organs, and exhibit no increase in spontaneous tumor development. However, a significant imbalance between growth and differentiation could be unmasked under conditions where normal homeostatic mechanisms are impaired. We report here that primary keratinocytes derived from p21 knockout mice, transformed with a ras oncogene, and injected subcutaneously into nude mice exhibit a very aggressive tumorigenic behavior, which is not observed with wild-type control keratinocytes nor with keratinocytes with a disruption of the closely related p27 gene. p21 knockout keratinocytes tested under well-defined in vitro conditions show a significantly increased proliferative potential, which is also observed but to a lesser extent with p27 knockout cells. More profound differences were found in the differentiation behavior of p21 versus p27 knockout keratinocytes, with p21 (but not p27) deficiency causing a drastic down-modulation of differentiation markers linked with the late stages of the keratinocyte terminal differentiation program. Thus, our results reveal a so far undetected role of p21 in tumor suppression, demonstrate that this function is specific as it cannot be attributed to the closely related p27 molecule, and point to an essential involvement of p21 in terminal differentiation control, which may account for its role in tumor suppression.

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Section: Tumorigenicity Assaysmentioning

confidence: 99%

The absence of p21Cip1/WAF1 alters keratinocyte growth and differentiation and promotes ras-tumor progression.

Missero¹,

Cunto²,

Kiyokawa³

et al. 1996

Genes Dev.

305

271

View full text Add to dashboard Cite

show abstract

“…49 We have previously estimated the number of LNSA-transduced patient fibroblasts necessary to provide treatment at 4 × 10 8 , based on data from red cell therapy. 27 As LNFnA and LNSA vectors express similar amounts of ADA in transduced fibroblasts, then 4 × 10 8 cells expressing ADA from the fibronectin promoter would provide enzyme to metabolize the cytotoxic ADA substrates deoxyadenosine and adenosine that accumulate in ADA deficiency.…”

Section: Discussionmentioning

confidence: 99%

“…27 As LNFnA and LNSA vectors express similar amounts of ADA in transduced fibroblasts, then 4 × 10 8 cells expressing ADA from the fibronectin promoter would provide enzyme to metabolize the cytotoxic ADA substrates deoxyadenosine and adenosine that accumulate in ADA deficiency. 49,50 Transfer of this number of cells is feasible using skin equivalent grafts. It is likely that the calculated number is an overestimate as genetically modified cells continually produce ADA, whereas transfused red cells have a 20-to 30-day half-life with diminishing therapeutic ADA activity.…”

Section: Discussionmentioning

confidence: 99%

Sustained gene expression in transplanted skin fibroblasts in rats

et al. 1999

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Retrovirus-mediated gene transfer into adult skin fibroviral LTR promoter (LASN) were compared for their ability blasts has provided measurable amounts of therapeutic to express ADA from transduced primary rat skin fibroproteins in animal models. However, the major problem blasts in vivo. Skin grafts formed from fibroblasts transemerging from these experiments was a limited time of duced with LNFnA showed strong human ADA enzyme vector encoded gene expression once transduced cells activity from 1 week to 3 months. In contrast, skin grafts were engrafted. We hypothesized that sustained trans-containing LASN-transduced fibroblasts tested positive for duced gene expression in quiescent fibroblasts in vivo human ADA for weeks 1 and 2, were faintly positive at might be obtained by using a fibronectin (Fn) promoter.week 3 and showed no human ADA expression at 1, 2 and Fibronectin plays a key role in cell adhesion, migration and 3 months. Thus, a fibronectin promoter provided sustained wound healing and is up-regulated in quiescent fibroblasts.transduced gene expression at high levels for at least 3 Retroviral vectors containing human adenosine deaminase months in transplanted rat skin fibroblasts, perhaps permit-(ADA) cDNA linked to rat fibronectin promoter (LNFnA) or ting the targeting of this tissue for human gene therapy.

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“…The LHL vector carries the hygromycin B resistance marker driven by the Moloney murine leukaemia virus LTR and contains normal splice donor and acceptors. 35 The ecotropic receptor gene on the vector pJET (kindly supplied by JM Cunningham) was used to construct a vector for high-level expression of the ecotropic receptor with a selectable marker gene. This vector, pMETbsr, was generated using the plasmids pMSG and pSV2bsr, which contain the MMTVLTR promoter and a blasticidin selectable marker respectively.…”

Section: Infection Of V79 Cells By Lhl Ecotropic Virusesmentioning

confidence: 99%

Mutational effects of retrovirus insertion on the genome of V79 cells by an attenuated retrovirus vector: implications for gene therapy

et al. 2003

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Attenuated retroviruses are currently the most widely used vectors in clinical gene therapy because of their potential to effect stable and permanent gene transfer. Since gene delivery is accompanied by random insertion of foreign genetic material into the recipient chromosomal DNA, the potential for insertional mutagenesis exists. In this study, we used a defective retrovirus vector containing a selectable marker, the hygromycin phosphotransferase gene, to investigate the mutagenic effects of vector integration on the mammalian genome. V79 Chinese hamster cells were infected with virus supernatants or by coculture with virus producer cells, and provirus insertion events occurred at low and high frequencies, respectively. The frequency of hprt mutagenesis was increased by a factor of 2.3 over the spontaneous hprt mutation frequency only following multiple provirus insertions/cell genome. Multiple provirus insertions (43/genome) resulted in instability at the hprt locus in 63% of the virally induced hprt mutants, as indicated by rearrangements at the molecular level, whereas no rearrangements were found when the provirus copy number was 1-2/genome. To demonstrate direct proviral involvement in mutagenesis, the defective MLV vector was retrieved along with flanking genomic hprt sequences from one mutant, and localized within intron 5 of the hprt gene. These data suggest that provirus copy number is a key factor when considering the potential hazards of using retrovirus vectors for gene therapy.

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Efficient retrovirus-mediated transfer and expression of a human adenosine deaminase gene in diploid skin fibroblasts from an adenosine deaminase-deficient human.

Cited by 134 publications

References 32 publications

The absence of p21Cip1/WAF1 alters keratinocyte growth and differentiation and promotes ras-tumor progression.

The absence of p21Cip1/WAF1 alters keratinocyte growth and differentiation and promotes ras-tumor progression.

Sustained gene expression in transplanted skin fibroblasts in rats

Mutational effects of retrovirus insertion on the genome of V79 cells by an attenuated retrovirus vector: implications for gene therapy

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