Multiple modifications in cis elements of the long terminal repeat of retroviral vectors lead to increased expression and decreased DNA methylation in embryonic carcinoma cells

Abstract

Infection by murine retroviruses in embryonic carcinoma (EC) and embryonic stem cells is highly restricted. The transcriptional unit of the Moloney murine leukemic virus (MoMuLV) long terminal repeat (LTR) is inactive in EC and embryonic stem cells in association with increased proviral methylation. In this study, expression in F9 EC cells was achieved from novel retroviral vectors containing three modifications in the MoMuLV-based retroviral vector: presence of the myeloproliferative sarcoma virus LTR, substitution of the primer binding site, and either deletion of a negative control region at the 5 end of the LTR or insertion of a demethylating sequence. We conclude that inhibition of expression from the MoMuLV LTR in EC cells is mediated through the additive effects of multiple cis-acting elements affecting the state of methylation of the provirus.), and the G1Na plasmid was provided by P. Tolstechev (Genetic Therapy, Inc., Gaithersburg, Md.). The LN vector was constructed and packaged in the laboratory of A. Dusty Miller (Fred Hutchinson Cancer Center, Seattle, Wash.).The MPSV LTR was used to replace the 3Ј MoMuLV LTR of G1Na to make MPneo. The NCR was removed from the MPSV LTR as an NheI (at nucleotide 33 in the LTR)-to-Sau3a (at nucleotide 97 in the LTR) fragment. The cut ends of the LTR were ligated together after fill-in by Klenow DNA polymerase to make the MPncr 3Ј LTR; this was then used to replace the 3Ј LTR of G1Na, yielding MPncrneo. The Thy-1 fragment in the plasmid Bluescript was opened at the SmaI site immediately 3Ј of the insert, and a synthetic oligonucleotide encoding an XbaI site (New England Biolabs, Beverly, Mass.) was ligated in place. The Thy-1 piece was isolated as an XbaI-XbaI fragment and cloned into the NheI site of the MPSV LTR in Bluescript. The Thy-1-substituted MPSV LTR was inserted in place of the 3Ј LTR in G1Na to make MPthyneo.The 5Ј LTR and psi region from plasmid LN was subcloned as an EcoRI-EcoRI fragment. The KpnI-SpeI fragment encompassing the PBS was removed and replaced by the KpnI-SpeI fragment from dl587rev. The 5Ј LTR/leader region fragment containing the dl587rev PBS was then returned to the MPneo, MPthyneo, and MPncrneo plasmids to produce MPdlneo, MPthydlneo, and MPncrdlneo.To make LNncrneo, the NCR (NheI at position 33 to Sau3a at position 97; Fig.

Efficient Lentiviral Transduction and Improved Engraftment of Human Bone Marrow Mesenchymal Cells

“…The MND-GFP-SN vector, which expresses the GFP from the modified MND long-terminal repeat (LTR) and contains a simian virus-40 neo R cassette, was a kind gift of Dr. D. Kohn (Children's Hospital of Los Angeles, Los Angeles, CA) ( Fig. 1A) [13]. The oncoretroviral vector expressing the B-domain deleted FVIII cDNA from the murine leukemia virus (MLV) LTR was described previously [14].…”

“…In a separate experiment, an oncoretroviral vector was used (MND-GFP-SN), which expressed GFP from a modified LTR that confers long-term expression in various stem cells (Fig. 1A) [13]. Confocal microscopical analysis and flow cytometry indicated that transduction of the human BM mesenchymal cells was significantly more efficient with the lentiviral HIV SIN -CMV-GFP vector (Fig.…”

Aberrant Clonal Hematopoiesis Following Lentiviral Transduction of HSPC in a Rhesus Macaque

“…Because RV promoter/enhancers included in LV vectors promote high constitutive transgene expression, two clinical trials for adrenoleukodystrophy have included a RV promoter/enhancer similar to MSCV, termed MND, in the LV to drive high expression of the transgene 4,5,45,46 . In contrast to the vector utilized in ZL34, the adrenoleukodystrophy trial LV placed the RV promoter/enhancer internally, instead of within the LV LTR 4,5,45,46 . Tumor-prone mouse models have suggested that an internal strong promoter/enhancer is less genotoxic than the same promoter/enhancer placed within the LV LTR 39 .…”

Gene Therapy for Pediatric AIDS

“…These vectors were based on a modified MLV vector backbone that we developed, named MND. 12 The MND vector, like the MSCV vector, has modifications in the cis-acting transcriptional elements that lead to expression in a higher percentage of hematopoietic and lymphoid cells, at higher levels and with greater persistence over time, compared to the standard MLV vectors. [13][14][15] Additionally, we have included the Woodchuck Hepatitis Virus posttranscriptional regulatory element (WPRE) in some vectors, because of its ability to increase the levels of vector-derived transcripts, which leads to improved titers and gene expression.…”