Equal potency of gammaretroviral and lentiviral SIN vectors for expression of O6-methylguanine–DNA methyltransferase in hematopoietic cells

Schambach, Axel; Bohne, Jens; Chandra, Satya V.; Will, Elke; Margison, Geoffrey P.; Williams, David A.; Baum, Christopher

doi:10.1016/j.ymthe.2005.08.012

Cited by 176 publications

(160 citation statements)

References 42 publications

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“…To this end, we used a gammaretroviral self-inactivating (SIN) vector (SRS.SF), which contains the spleen focus-forming virus enhancer/promoter as internal promoter. 42 Figure 3 CTF/NF1 proteins mediate the enhancer-blocking activity of their cognate DNA binding sites. (a) The enhancer-blocking properties of the CTF/ NF1 binding sites were quantitated as described in the legend of Figure 1f in comparison with that of a 250-bp DNA control spacer.…”

Section: Ctf/nf1 As Insulators For Gene Transfer a Gaussin Et Almentioning

confidence: 99%

“…Gamma-retroviral supernatant production was performed using 293T cells as previously described, with coexpression of ecotropic envelope proteins. 42,52 Cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum, 100 U ml À1 penicillin/streptomycin and 2 mM glutamine. Viral titers, determined on SC-1 cells by flow cytometry, were in the range of 5Â10 6 to 2Â10 7 IU ml À1 in un-concentrated supernatants.…”

Section: Retroviral Vectorsmentioning

confidence: 99%

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CTF/NF1 transcription factors act as potent genetic insulators for integrating gene transfer vectors

Gaussin

Modlich

Bauche³

et al. 2011

Gene Ther

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Gene transfer-based therapeutic approaches have greatly benefited from the ability of some viral vectors to efficiently integrate within the cell genome and ensure persistent transmission of newly acquired transgenes to the target cell progeny. However, integration of provirus has been associated with epigenetic repercussions that may influence the expression of both the transgene and cellular genes close to vector integration loci. The exploitation of genetic insulator elements may overcome both issues through their ability to act as barriers that limit transgene silencing and/or as enhancer-blockers preventing the activation of endogenous genes by the vector enhancer. We established quantitative plasmid-based assay systems to screen enhancerblocker and barrier genetic elements. Short synthetic insulators that bind to nuclear factor-I protein family transcription factors were identified to exert both enhancer-blocker and barrier functions, and were compared to binding sites for the insulator protein CTCF (CCCTC-binding factor). Gamma-retroviral vectors enclosing these insulator elements were produced at titers similar to their non-insulated counterparts and proved to be less genotoxic in an in vitro immortalization assay, yielding lower activation of Evi1 oncogene expression and reduced clonal expansion of bone marrow cells.

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Section: Ctf/nf1 As Insulators For Gene Transfer a Gaussin Et Almentioning

confidence: 99%

Section: Retroviral Vectorsmentioning

confidence: 99%

CTF/NF1 transcription factors act as potent genetic insulators for integrating gene transfer vectors

Gaussin

Modlich

Bauche³

et al. 2011

Gene Ther

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“…Having established conditions for high-titer production and dose-controlled use of SIN alpharetroviral vectors, we explored their potency in a direct comparison with lentiviral (advanced HIV-1-based SIN design [5,45]) and gammaretroviral (advanced MLV-based SIN design [31,33]) vectors. All three SIN vectors contained the same internal expression cassette (SFFV-EGFP-wPRE) (Fig.…”

mentioning

confidence: 99%

Self-Inactivating Alpharetroviral Vectors with a Split-Packaging Design

Suerth

Maetzig

Galla

et al. 2010

J Virol

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Accidental insertional activation of proto-oncogenes and potential vector mobilization pose serious challenges for human gene therapy using retroviral vectors. Comparative analyses of integration sites of different retroviral vectors have elucidated distinct target site preferences, highlighting vectors based on the alpharetrovirus Rous sarcoma virus (RSV) as those with the most neutral integration spectrum. To date, alpharetroviral vector systems are based mainly on single constructs containing viral coding sequences and intact long terminal repeats (LTR). Even though they are considered to be replication incompetent in mammalian cells, the transfer of intact viral genomes is unacceptable for clinical applications, due to the risk of vector mobilization and the potentially immunogenic expression of viral proteins, which we minimized by setting up a split-packaging system expressing the necessary viral proteins in trans. Moreover, intact LTRs containing transcriptional elements are capable of activating cellular genes. By removing most of these transcriptional elements, we were able to generate a self-inactivating (SIN) alpharetroviral vector, whose LTR transcriptional activity is strongly reduced and whose transgene expression can be driven by an internal promoter of choice. Codon optimization of the alpharetroviral Gag/Pol expression construct and further optimization steps allowed the production of high-titer self-inactivating vector particles in human cells. We demonstrate proof of principle for the versatility of alpharetroviral SIN vectors for the genetic modification of murine and human hematopoietic cells at a low multiplicity of infection.

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“…(Table 1). Thus, the combination IC 50 for HR'SIN-MDR1 transduced cells is 1.6-fold (P ¼ 0.05), for HR'SIN-MGMT P140K 2.1-fold (P ¼ 0.05) and for HR'SIN-MDR1-IRES-MGMT P140K 6.2-fold (P ¼ 0.05) higher than in control cells (Figure 1d). Cells transduced with the combination vector showed also in comparison with the single vectors a significant survival advantage (3.9-fold in comparison to HR'SIN-MDR1, P ¼ 0.05 and threefold to HR'SIN-MGMT P140K , P ¼ 0.05).…”

Section: Transduction Of Hl60 Cells With Lentiviral Vectorsmentioning

confidence: 90%

“…49 Besides these aspects concerning the safety of vectors, a direct comparison of gammaretroviral and lentiviral SIN-vectors showed no differences regarding the expression of MGMT P140K and the protection levels after exposure to O 6 -BG/BCNU in hematopoietic cells. 50 However, for the clinical use of our bicistronic lentiviral vector more intense safety analyses will be required.…”

Section: Discussionmentioning

confidence: 99%

Chemoprotection of human hematopoietic stem cells by simultaneous lentiviral overexpression of multidrug resistance 1 and O6-methylguanine-DNA methyltransferaseP140K

et al. 2009

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Myelotoxicity is a dose-limiting effect of many chemotherapeutic regimens. Thus, there is great interest in protecting human hematopoietic stem cells by the transfer of drug resistance genes. The main focus of this study was the simultaneous overexpression of multidrug resistance 1 (MDR1) and the O 6 -benzylguanine (O 6 -BG)-resistant mutant MGMT P140K (O 6 -methylguanine-DNA methyltransferase) with a bicistronic lentiviral vector (HR 0 SIN-MDR1-IRES-MGMT P140K ), with regard to the capability to convey chemoprotection in the leukemia cell line, HL60, and human hematopoietic stem cells (CD34 + ). Combination therapy with O 6 -BG/1-(2-chloroethyl)-3-(4-amino-2-methyl pyrimidine-5-yl)methyl-1-nitrosourea) (ACNU) plus paclitaxel showed a significant survival advantage of HL60 cells transduced with this combination vector. In CD34 + cells, monotherapy with O 6 -BG/temozolomide (TMZ) resulted in an increased percentage of MGMT-positive cells (vs untreated cells) after transduction with HR 0 SIN-MDR1-IRES-MGMT P140K (28.3%). For combination therapy with O 6 -BG/temozolomide plus paclitaxel the increase was higher with the combination vector (52.8%) than with a vector expressing MGMT P140K solely (29.1%). With regard to MDR1-positive cells the protective effect of the combination vector (88.5%) was comparable to the single vector HR 0 SIN-MDR1 (90.0%) for monotherapy with paclitaxel and superior for combination therapy with O 6 -BG/temozolomide plus paclitaxel (84.6 vs 69.7%). In conclusion, the combination vector presents simultaneous protective effects of two drugresistance genes, offering an opportunity to increase the cancer therapeutic index.

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Equal potency of gammaretroviral and lentiviral SIN vectors for expression of O6-methylguanine–DNA methyltransferase in hematopoietic cells

Cited by 176 publications

References 42 publications

CTF/NF1 transcription factors act as potent genetic insulators for integrating gene transfer vectors

CTF/NF1 transcription factors act as potent genetic insulators for integrating gene transfer vectors

Self-Inactivating Alpharetroviral Vectors with a Split-Packaging Design

Chemoprotection of human hematopoietic stem cells by simultaneous lentiviral overexpression of multidrug resistance 1 and O6-methylguanine-DNA methyltransferaseP140K

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