Tightly regulated 'all-in-one' lentiviral vectors for protection of human hematopoietic cells from anticancer chemotherapy.

Successful application of gene therapy strategies may require stringently regulated transgene expression. Along this line, we describe a doxycycline (Dox)-inducible 'all-in-one' lentiviral vector design using the pTET-T11 (TII) minimal-promoter and a reverse transactivator protein (rtTA2S-M2) driven by the phosphoglycerate kinase promoter allowing for tight regulation of transgene expression (Lv.TII vectors). Vector design was evaluated in human hematopoietic cells in the context of cytidine deaminase (hCDD)-based myeloprotective gene therapy. Upon Dox administration, a rapid (16-24 h) and dose-dependent (>0.04 µg ml(-1) Dox) onset of transgene expression was detected in Lv.TII.CDD gene-modified K562 cells as well as in primary human CD34(+) hematopoietic cells. Importantly, in both cell models low background transgene expression was observed in the absence of Dox. Functionality of Dox-inducible hCDD expression was demonstrated by >10-fold increase in cytosine arabinoside (1-ß-d-arabinofuranosylcytosine, Ara-C) resistance of Lv.TII.CDD-transduced K562 cells. In addition, Lv.TII.CDD-transduced CD34(+)-derived myeloid cells were protected from up to 300 nm Ara-C (control affected from 50 nm onwards). These data clearly demonstrate the suitability of our self-inactivating lentiviral vector to induce robust, tightly regulated transgene expression in human hematopoietic cells with minimal background activity and highlight the potential of our construct in myeloprotective gene therapy strategies.

Efficient in vivo regulation of cytidine deaminase expression in the haematopoietic system using a doxycycline-inducible lentiviral vector system.

Regulated transgene expression may reduce transgene-specific and genotoxic risks associated with gene therapy. To prove this concept, we have investigated the suitability of doxycycline (Dox)-inducible human cytidine deaminase (hCDD) overexpression from lentiviral vectors to mediate effective myeloprotection while circumventing the lymphotoxicity observed with constitutive CDD activity. Rapid Dox-mediated transgene induction associated with a 6-17-fold increase in drug resistance was observed in 32D and primary murine bone marrow (BM) cells. Moreover, robust Dox-regulated transgene expression in the entire haematopoietic system was demonstrated for primary and secondary recipients of hCDD-transduced R26-M2rtTA transgenic BM cells. Furthermore, mice were significantly protected from myelosuppressive chemotherapy as evidenced by accelerated recovery of granulocytes (1.9±0.6 vs 1.3±0.3, P=0.034) and platelets (883±194 vs 584±160 10(3) per µl, P=0.011). Minimal transgene expression in the non-induced state and no overt cellular toxicities including lymphotoxicity were detected. Thus, using a relevant murine transplant model our data provide conclusive evidence that drug-resistance transgenes can be expressed in a regulated fashion in the lymphohaematopoietic system, and that Dox-inducible systems may be used to reduce myelotoxic side effect of anticancer chemotherapy or to avoid side effects of high constitutive transgene expression.

Shuttle of lentiviral vectors via transplanted cells in vivo.

Lentiviral vectors have turned out to be an efficient method for stable gene transfer in vitro and in vivo. Not only do fields of application include cell marking and tracing following transplantation in vivo, but also the stable delivery of biological active proteins for gene therapy. A variety of cells, however, need immediate transplantation after preparation, for example, to prevent cell death, differentiation or de-differentiation. Although these cells are usually washed several times following lentiviral transduction, there may be the risk of viral vector shuttle via transplanted cells resulting in undesired in vivo transduction of recipient cells. We investigated whether infectious lentiviral particles are transmitted via ex vivo lentivirally transduced cells. To this end, we explored potential viral shuttle via ex vivo lentivirally transduced cardiomyocytes in vitro and following transplantation into the brain and peripheral muscle. We demonstrate that, even after extensive washing, infectious viral vector particles can be detected in cell suspensions. Those lentiviral vector particles were able to transduce target cells in transwell experiments. Moreover, transmitted vector particles stably transduced resident cells of the recipient central nervous system and muscle in vivo. Our results of lentiviral vector shuttle via transduced cardiomyocytes are significant for both ex vivo gene therapy and for lentiviral cell tracing, in particular for investigation of stem cell differentiation in transplantation models and co-cultivation systems.