Nonintegrating lentiviral (NIL) vectors were produced from HIV-1-based lentiviral vectors by introducing combinations of mutations made to disable the integrase protein itself and to alter the integrase recognition sequences (att) in the viral LTR. NIL vectors with these novel combinations of mutations were used to transduce the human T lymphoid cell line Jurkat and primary human CD34(+) hematopoietic progenitor cells to assess their efficacy measured through transient expression of the enhanced green fluorescent protein (eGFP) reporter gene. The most disabled NIL vectors resulted in initial high levels of eGFP expression (approximately 90% of cells), but expression was transient, diminishing toward background (<0.5%) within less than 1 month. Southern blot analyses of transduced Jurkat cells confirmed the loss of detectable NIL vector sequence (linear form and one- and two-LTR circles) by 1 month. There were low residual levels of integration by NIL vectors (reduced approximately 10(4)-fold compared to wild-type vectors), despite any combination of the engineered changes. Based upon analysis of the sequences of the DNA from the junctions of the vector LTR and cellular chromosomes, these rare integrated NIL vector sequences were not mediated by an integrase-driven mechanism due to reversion of the engineered mutations, but more likely were produced by background recombination events. The development of NIL vectors provides a novel tool for efficient transient gene expression in primary stem cells and hematopoietic and lymphoid cells.

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.

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.