Improved gene expression upon transfer of the adenosine deaminase minigene outside the transcriptional unit of a retroviral vector.

Abstract

This study describes a type of retroviral vector called double-copy (DC) vector that was designed to improve the expression of transduced genes. The unique feature of DC vectors is that the transduced gene is inserted within the

Modification of multiple transcriptional regulatory elements in a Moloney murine leukemia virus gene transfer vector circumvents silencing in fibroblast grafts and increases levels of expression of the transferred enzyme

“…The deletion in the LTR could also accommodate additional positive regulatory elements since large inserts in this location have been shown to be replicated. 33,34 …”

Comparison of methotrexate resistance conferred by a mutated dihydrofolate reductase (DHFR) cDNA in two different retroviral vectors

“…The double copy vector was designed to eliminate or at least reduce the negative effects of the retroviral transcriptional unit by placing the gene of interest outside the retroviral transcriptional unit. 6 We observed an 8-fold difference (the ED 50 of DC/ SV6S31 and SFGS31 was increased 5-fold and 40-fold compared with mock) in protection from cytotoxicity of MTX between DC/SV6S31 and SFGS31 constructs in NIH3T3 cells. Northern blot analysis as well as quantitative RT-PCR showed a 22-to 38-fold difference in mRNA levels between DC/SV6S31 and SFGS31 (SFGS31 Ͼ DC/SV6S31).…”

“…Thus, upon infection of target cells, this expression unit is duplicated in the 5ЈLTR to form a double copy structure. 6 Figure 1a shows this construct. The SFG-based dicistronic retroviral vector containing mutant DHFR cDNA (S31) also contained the IRES of encephalomyocarditis virus and the neo gene.…”

“…Consequently, this inserted transcription unit (S31 DHFR in our case) in the 3ЈU3 region is duplicated in the target cell and creates a double copy in the target cell. 6 The SFG-based dicistronic vector used has an internal ribosomal entry site (IRES) that allows transgenes to be expressed under LTR transcriptional control only. 5,7 In this study, we compared the resistance to MTX conferred by DC/SV6S31 and SFGS31 retroviral constructs in NIH3T3 cells and in mouse and human hematopoietic progenitor cells.…”

An in vitro system for efficiently evaluating gene therapy approaches to hemoglobinopathies

“…Important components of the Luv vector included: (1) utilization of an extended packaging signal which includes a portion of the gag coding sequence to enhance packaging of the viral genomic RNA and increase titer; 24 (2) utilization of the wild-type MoMLV splice signals and the viral envelope start codon (ATG) for initiation of translation of vector encoded transgenes to facilitate viral RNA processing and transgene expression; 15 (3) disruption of the Pr65 gag open reading frame with a stop codon to minimize the possibility of translating gag peptides which could contribute to vector immunogenicity and toxicity; (4) elimination of any MoMLV envelope sequences which could contribute to vector immunogenicity and toxicity; and (5) inclusion of a multiple cloning site within the 3Ј LTR U3 region for development of double copy vectors. 25 In order to identify cells transduced with Luv-derived vectors, a truncated version of the nerve growth factor receptor (NGFR) gene as well as the optimized green fluorescence protein (GFP) were cloned into the Luv vector to yield LuvNM and LuvGM, respectively. [26][27][28] These marker genes permit rapid determination of retroviral titer and evaluation of transduced cell populations using multiparameter FACS analysis.…”