Consistent, persistent expression from modified retroviral vectors in murine hematopoietic stem cells

Abstract

Retroviral vectors based on the Moloney murine leukemia virus (MoMuLV) have shown inconsistent levels and duration of expression as well as a propensity for the acquisition of de novo methylation in vivo. MoMuLV-based vectors are known to contain sequences that are capable of suppressing or preventing expression from the long terminal repeat. Previously, we constructed a series of modified retroviral vectors and showed that they function significantly better than MoMuLV-based vectors in vitro. To test the efficacy of the modified vectors in hematopoietic stem cells in vivo, we examined gene expression and proviral methylation in differentiated hematopoietic colonies formed in the spleens of mice after serial transplantation with transduced bone marrow (2°CFU-S). We found a significant increase in the frequency of expression with our modified vectors (>90% expression in vector DNA containing 2°CFU-S) over the frequency observed with the standard MoMuLV-based vector (28% expression in vector containing 2°CFU-S). Expression from the modified vectors was highly consistent, with expression in >50% of the vector-containing 2°CFU-S from all 20 transplant recipients analyzed, whereas expression from the standard MoMuLV-based vector was inconsistent, with expression in 0-10% of the vector containing 2°CFU-S from 8 recipients and expression in >50% of the vector-containing 2°CFU-S from 4 other recipients. In addition, we established that the modified vectors had a lower level of DNA methylation than the control vector. These findings represent significant advances in the development and evaluation of effective retroviral vectors for application in vivo.

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 MND vector is also based on Mo-MuLV, is permissive for expression in most cells, and also contains multiple alterations. 24,32 The PBS is the permissive sequence found in the dl58rev vector, the YY1 region was deleted, the enhancer was deleted, and Thy-1, a positive regulatory element reported to decrease methylation, was added to the LTR. In comparison, in L⌬4pQ-H␤H the dl58rev permissive PBS is also used, but the entire segment of the YY1, G/C repeats, enhancer and promoter of the LTR were deleted.…”

“…In addition, a permissive primer binding site (PBS) was used, originally described in the dl58rev vector 29 and used in the MSCV and MND vectors. 24,[30][31][32] In comparison to the L YY1 BGSN vector, where the YY1 site was altered to a non-binding sequence, in the L⌬4pQ-H␤H vector, the YY1 binding site was removed as were the G/C repeat unit and the promoter and enhancer elements. Although the LTR deletion was made up to the U3/R junction, the end filling and blunt end ligation restored the complete R1 region, thus infectious vector virus was produced by the packaging cells (titer у10 5 ).…”

Dangerous liaisons: the role of “danger” signals in the immune response to gene therapy

“…81,82 These cells have been shown to offer significant advantages over the use of differentiated cell types and may be capable of inducing specific tolerance to transgenic proteins. [83][84][85] In experiments carried out by the Dunbar laboratory, a retroviral neoexpression vector was delivered, ex vivo, to both lymphocytes and hematopoietic stem cells (HSCs) and subsequently reinjected into Rhesus monkeys. 86 The modified lymphocytes were quickly rejected by the host, whereas transfer of the genetically modified HSCs resulted in long-term engraftment and tolerance to the neopeptide.…”

A role of cyclin G in the process of apoptosis

“…pMND-X-SN, the parental retrovirus vector, contains modi®ed LTRs that are active in embryonic carcinoma cells (Robbins et al, 1997). To construct the cyclin G-expressing retrovirus vector, pMND-cycG-SN, the EcoRI ± BamHI fragment containing the complete open reading frame of cyclin G was inserted into the same sites in the pMND-X-SN.…”