Design and in vivo characterization of self-inactivating human and non-human lentiviral expression vectors engineered for streptogramin-adjustable transgene expression

Abstract

Adjustable transgene expression is considered key for next-generation molecular interventions in gene therapy scenarios, therapeutic reprogramming of clinical cell phenotypes for tissue engineering and sophisticated gene-function analyses in the post-genomic era. We have designed a portfolio of latest generation self-inactivating human (HIV-derived) and non-human (EIAV-based) lentiviral expression vectors engineered for streptogramin-adjustable expression of reporter (AmyS(DeltaS), EYFP, SAMY, SEAP), differentiation-modulating (human C/EBP-alpha) and therapeutic (human VEGF) transgenes in a variety of rodent (CHO-K1, C2C12) and human cell lines (HT-1080, K-562), human and mouse primary cells (NHDF, PBMC, CD4+) as well as chicken embryos. Lentiviral design concepts include (i) binary systems harboring constitutive streptogramin-dependent transactivator (PIT) and PIT-responsive transgene expression units on separate lentivectors; (ii) streptogramin-responsive promoters (P(PIR8)) placed 5' of desired transgenes; (iii) within modified enhancer-free 3'-long terminal repeats; and (iv) bidirectional autoregulated configurations providing streptogramin-responsive transgene expression in a lentiviral one-vector format. Rigorous quantitative analysis revealed HIV-based direct P(PIR)-transgene configurations to provide optimal regulation performance for (i) adjustable expression of intracellular and secreted product proteins, (ii) regulated differential differentiation of muscle precursor cell lines into adipocytes or osteoblasts and (iii) conditional vascularization fine-tuning in chicken embryos. Similar performance could be achieved by engineering streptogramin-responsive transgene expression into an autoregulated one-vector format. Powerful transduction systems equipped with adjustable transcription modulation options are expected to greatly advance sophisticated molecular interventions in clinically and/or biotechnologically relevant primary cells and cell lines.

Poly(ADP-Ribose) Polymerase 1 Promotes Transcriptional Repression of Integrated Retroviruses

“…These cells efficiently support the early postentry steps of the gammaretrovirus and lentivirus life cycle (35)(36)(37)(38)(39). PARP-1 is the only member of the PARP family with catalytic activity in these cells, and DT40 cells are viable after PARP-1 knockout (34).…”

“…In addition, chicken cells can be used as a reliable model to study the molecular events that occur from entry to proviral transgene expression characteristic of the gammaretrovirus and lentivirus life cycle. Chicken cells are highly permissive to transduction with singleround infection by murine leukemia virus (MLV) and HIV reporter viruses (35)(36)(37)(38)(39), suggesting that the molecular mechanisms required for uncoating, reverse transcription, DNA integration, and transgene expression of these retroviruses are conserved in these cells. Further evidence supporting the conservation of the molecular mechanisms implicated in the early steps of HIV-1 infection in chicken cells indicates that, as in human cells, HIV integrates preferentially inside active transcription units in the chicken genome (35).…”

Gene Regulatable Lentiviral Vector System

“…Besides the Tet-MFP-, and ecdysone-regulated systems, different types of inducible lentiviral vectors have been generated based on the other chimeric regulatable systems, which include streptogramin-adjustable expression system derived from Streptomyces coelicolor (Mitta et al, 2004) and gaseous acetaldehyde-inducible expression system derived from Aspergillus nidulans (Hartenbach & Fussenegger, 2005). Although some endogenous cellular elements that respond to exogenous signals or stress have been adapted to HIV-1-and EIAV-derived lentiviral vectors (Beutelspacher et al, 2005;Hurttila et al, 2008;Parker et al, 2009), the pleiotropic effects exerted by the inducing agent would be a drawback in a human therapeutic context (Fussenegger, 2001).…”

Beyond retrovirus infection: HIV meets gene therapy

“…However, Yam et al (2002) reported that the CMV promoter showed low activity in CD34+, erythrocytic and myelomonocytic cells. In both cases, the promoter activity was constitutive and it seems that regulated lentiviral vectors better reflect physiological transgene expression levels (Mitta et al, 2004;Markusic et al, 2005). Despite these observations, the molecular context in which these promoters were evaluated is of major importance because cis elements can have synergistic effects on cell transduction efficiency.…”