Transient Gene Expression by Nonintegrating Lentiviral Vectors

Abstract

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.

Cellular Restriction of Retrovirus Particle-Mediated mRNA Transfer

“…The continued expression in more than 1% of the target cell population was suggestive of residual integration events, as previously described (Fig. 1C) (20). The residual integration of the D64V mutant may be circumvented by using a double or triple mutant at the DDE catalytic site.…”

“…We also produced a third-generation self-inactivating lentiviral vector (RRL.PPT.SF.GFP.pre) expressing EGFP under the control of the strong enhancer-promoter derived from the long terminal repeat of the MLV spleen focus-forming virus (SFFV). Additionally, integration-defective lentivirus particles, competent to form episomal DNA by using an integrase-deficient variant of the lentiviral gag-pol plasmid (integrase D64V), were produced (20,26).…”

Protein transduction from retroviral Gag precursors

“…As envelope proteins can be modified to deliver cytokine signals (47), it may be possible to engineer cells using multifunctional retroviral particle preparations, which elicit a cascade of immediate, transient, and permanent effects (48). Underlining the versatility of this concept, intermediate steps of the retroviral life cycle can be exploited to deliver episomal DNA (14,15,49) or mRNA (13,50).…”

“…Interestingly, the transgene-dependent expression of GFP (ILV and NILV) started to accumulate beyond 10 h posttransduction, exactly when the polyprotein-mediated transduction began to decrease. Compared with the polyprotein-mediated delivery, NILV-transduced cells showed a much longer duration of expression (declining 50 h posttransduction) with persistence in a subset of cells, as described (13)(14)(15). Thus, rapid onset and full reversion was only obtained with protein transduction.…”

SIVMAC Vpx improves the transduction of dendritic cells with nonintegrative HIV-1-derived vectors

“…To circumvent this problem, several groups have evaluated the use of integration-incompetent LVs. [3][4][5][6][7][8] Gene expression from these vectors is driven from viral DNA molecules that remain in an episomal form after their entry into the nucleus as 1 or 2 long terminal repeat circles (1-or 2-LTRs, respectively). These episomes are considered as a deadend product in the normal retroviral infection process, but they are a functional substrate for the transcriptional machinery.…”