Construction and Use of Retroviral Vectors Encoding the Toxic Gene Barnase

Agarwal, Sumit; Nikolai, Bryan C.; Yamaguchi, Tomoyuki; Lech, Patrycja; Somia, Nikunj V.

doi:10.1016/j.ymthe.2006.03.025

Cited by 30 publications

(34 citation statements)

References 37 publications

(32 reference statements)

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“…Lentiviral vector plasmids pCSIIEG, 21 pCSEPuro, 18 pOK/ EF1a-Artemis (including the EF1a intron 1 sequence), and pOK/PGK-Artemis 18 have been previously described. For construction of pOK/APro-Artemis, the EF1a promoter was excised from pOK/EF1a-Artemis with AgeI, yielding pOK/ Artemis.…”

Section: Plasmidsmentioning

confidence: 99%

Role of Transgene Regulation in Ex Vivo Lentiviral Correction of Artemis Deficiency

et al. 2015

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Artemis is a single-stranded endonuclease, deficiency of which results in a radiation-sensitive form of severe combined immunodeficiency (SCID-A) most effectively treated by allogeneic hematopoietic stem cell (HSC) transplantation and potentially treatable by administration of genetically corrected autologous HSCs. We previously reported cytotoxicity associated with Artemis overexpression and subsequently characterized the human Artemis promoter with the intention to provide Artemis expression that is nontoxic yet sufficient to support immunodevelopment. Here we compare the human Artemis promoter (APro) with the moderatestrength human phosphoglycerate kinase (PGK) promoter and the strong human elongation factor-1a (EF1a) promoter to regulate expression of Artemis after ex vivo lentiviral transduction of HSCs in a murine model of SCID-A. Recipient animals treated with the PGK-Artemis vector exhibited moderate repopulation of their immune compartment, yet demonstrated a defective proliferative T lymphocyte response to in vitro antigen stimulation. Animals treated with the EF1a-Artemis vector displayed high levels of T lymphocytes but an absence of B lymphocytes and deficient lymphocyte function. In contrast, ex vivo transduction with the AProArtemis vector supported effective immune reconstitution to wild-type levels, resulting in fully functional T and B lymphocyte responses. These results demonstrate the importance of regulated Artemis expression in immune reconstitution of Artemis-deficient SCID.

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Section: Plasmidsmentioning

confidence: 99%

Role of Transgene Regulation in Ex Vivo Lentiviral Correction of Artemis Deficiency

et al. 2015

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“…Lentiviral vector constructs were generated by standard molecular technique based on the pCSII and pCSIIEG lentiviral vectors, both of which have been previously described (Agarwal et al, 2006). pCSIIEG/EPuro.…”

Section: Lentiviral Vectorsmentioning

confidence: 99%

“…The Streptomyces alboniger puromycin-N-acetyltransferase gene was amplified by polymerase chain reaction (PCR) from the pPUR plasmid (Clontech, Mountain View, CA), using the following oligonucleotides: forward, 5 0 -TCTGCTAGCCATGGCCGAGTACAAGCCC-3 0 ; and reverse, 5 0 -GGCGACCGGTGGGGCACCGGGCTTGCGGG-3 0 . The amplified product was cleaved with NheI and AgeI (recognition sites underlined in the oligonucleotide sequences) and then ligated into pCSII-EF-MCS (Agarwal et al, 2006) to generate pCSIIEG/EPuro. pCSII/EAIG.…”

Section: Lentiviral Vectorsmentioning

confidence: 99%

“…pCSII/EAIG. The murine Artemis cDNA sequence was excised from pGEMT/mArt (Li et al, 2005) with NotI and cloned into pCSII/EF1a-MCS (Agarwal et al, 2006) at the NotI site to generate pCSII/EF1a-mArtemis. The internal ribosome entry site (IRES)-green fluorescent protein (GFP) sequence was excised from pCSII/CMV-I2G (Gori et al, 2007) with NheI and XbaI and cloned into the XbaI site of pCSII/EF1a-mArtemis to generate pCSII/EAIG.…”

Section: Lentiviral Vectorsmentioning

confidence: 99%

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Cytotoxicity Associated with Artemis Overexpression After Lentiviral Vector-Mediated Gene Transfer

et al. 2010

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Artemis is a hairpin-opening endonuclease involved in nonhomologous end-joining and V(D)J recombination. Deficiency of Artemis results in radiation-sensitive severe combined immunodeficiency (SCID) characterized by complete absence of T and B cells due to an arrest at the receptor recombination stage. We have generated several lentiviral vectors for transduction of the Artemis sequence, intending to complement the deficient phenotype. We found that transduction by a lentiviral vector in which Artemis is regulated by a strong EF-1a promoter resulted in a dose-dependent loss of cell viability due to perturbed cell cycle distribution, increased DNA damage, and increased apoptotic cell frequency. This toxic response was not observed in cultures exposed to identical amounts of control vector. Loss of cell viability was also observed in cells transfected with an Artemis expression construct, indicating that toxicity is independent of lentiviral transduction. Reduced toxicity was observed when cells were transduced with a moderate-strength phosphoglycerate kinase promoter to regulate Artemis expression. These results present a novel challenge in the establishment of conditions that support Artemis expression at levels that are nontoxic yet sufficient to correct the T À B À phenotype, crucial for preclinical studies and clinical application of Artemis gene transfer in the treatment of human SCID-A.

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“…Recombinant plasmids were generated using standard molecular cloning techniques. Lentivirus vector plasmids pCSIIEG and pCSII have been described previously (Agarwal et al, 2006). To construct pEFDIG, a Tyr22DHFR coding sequence was obtained as a XhoI-ClaI (blunt) fragment from pLasBD (Zhao et al, 1997) and cloned between XhoI and BamHI (blunt) of pCSII-CMV-12G (N. Somia, unpublished data) to form pCCDG.…”

Section: Methodsmentioning

confidence: 99%

Protection of Mice from Methotrexate Toxicity by ex Vivo Transduction Using Lentivirus Vectors Expressing Drug-Resistant Dihydrofolate Reductase

Gori

Podetz-Pedersen

Swanson

et al. 2007

J Pharmacol Exp Ther

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Methotrexate (MTX) dose-escalation studies were conducted in C57BL/6 mice to determine the chemoprotective effect of transplantation using bone marrow transduced with lentivirus vectors expressing a drug-resistant variant of murine dihydrofolate reductase (DHFR). Methotrexate-resistant dihydrofolate reductase [tyrosine-22 (Tyr22)DHFR] and enhanced green fluorescent protein (GFP) coding sequences were inserted into self-inactivating lentiviral vectors as part of a genetic fusion or within the context of a bicistronic expression cassette. MTXtreated animals that received Tyr22DHFR-transduced marrow recovered to normal hematocrit levels by 3 weeks post-transplant and exhibited significant GFP marking in myeloid and lymphoid lineage-derived peripheral blood mononuclear cells (PBMCs). In contrast, MTX-treated animals transplanted with control GFP-transduced marrow exhibited extremely reduced hematocrits with severe marrow hypoplasia and did not survive MTX dose escalation. To minimize cell manipulation, we treated unfractionated marrow in an overnight exposure. Transduction at a multiplicity of infection of 10 resulted in up to 11% vectormodified PBMCs in primary recipients and successful repopulation of secondary recipients with vector-marked cells. Experimental cohorts exhibited sustained proviral expression with stable GFP fluorescence intensity. These results demonstrate the effectiveness of lentivirus vectors for chemoprotection in a well developed animal model, with the potential for further preclinical development toward human application.

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Construction and Use of Retroviral Vectors Encoding the Toxic Gene Barnase

Cited by 30 publications

References 37 publications

Role of Transgene Regulation in Ex Vivo Lentiviral Correction of Artemis Deficiency

Role of Transgene Regulation in Ex Vivo Lentiviral Correction of Artemis Deficiency

Cytotoxicity Associated with Artemis Overexpression After Lentiviral Vector-Mediated Gene Transfer

Protection of Mice from Methotrexate Toxicity by ex Vivo Transduction Using Lentivirus Vectors Expressing Drug-Resistant Dihydrofolate Reductase

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