Doxycycline Regulatable Expression of Cytidine Deaminase Mediates Myeloprotection and Avoids Lymphotoxicity in a Murine Transplant Model

Abstract 2054

Hematopoietic stem cells (HSCs) genetically modified to overexpress drug resistance genes have been advocated to overcome chemotherapy induced myelosuppression. In this context, we demonstrated that overexpression of hCDD from a constitutive spleen focus forming virus (SFFV)-derived promoter protects hematopoietic cells from Ara-C toxicity in vitro and in vivo. However, these studies also indicated substantial lymphotoxicity by high level constitutive CDD expression (Rattmann et al. Blood, 2006). To circumvent this problem, we now have established a Doxycycline (Dox)-inducible (TET-ON) CDD-expression system and have evaluated this system in murine in vitro and in vivo protection assays.

In vitro CDD-mediated Ara-C resistance was evaluated in the hematopoietic cell line 32D as well as primary lineage negative (Lin-) clonogenic progenitor cells. In these studies cells were co-transduced with two lentiviral constructs expressing CDD (TET.CDD) or the reverse transactivator protein (rtTA3). In 32D cells, administration of 0.2 to 2.0 μg/ml Dox induced transgene expression to plateau levels within 24 hours. Rapid induction of the transgene also was observed at lower Dox concentrations, although exposure to 0.008 or 0.04 μg/ml led to notably reduced expression levels. Functionality of Dox-inducible hCDD expression was evaluated by exposing TET.CDD transgenic 32D cells to Ara-C. Transduced cells cultured in the presence of 2.0 μg/ml Dox proved completely resistant to Ara-C concentrations of up to 5000 nM, whereas eGFP- or not Dox treated control cells were susceptible to Ara-C exposure from 50 nM onwards. Again, hCDD-mediated drug resistance was dependent on the Dox dosage. When TET.CDD transduced 32D cells were exposed to Dox for 72h, marked protection from Ara-C was shown for Dox concentration of >0.2 μg/ml (LD₅₀ > 2.000 nM Ara-C). After Dox withdrawal transgene expression remained detectable for at least three days. Similar protection was observed in primary hematopoietic cells and progenitor cell derived colonies were protected from Ara-C doses of 300 to 600 nM while untransduced control cells did not yield colony growth at doses of 50 nM Ara-C or higher.

In vivo studies were performed by transplanting C57Bl/6 mice with Lin- cells from Rosa26-M2rtTA mice previously transduced with the TET.CDD or a control SIN lentiviral vector. Transgene expression was induced by Dox administration starting four weeks post transplantation. In this model Dox administration induced stable transgene expression in peripheral blood B, T, and myeloid cells peaking 15 days after start of administration and remaining detectable for 21 days after Dox withdrawal. No alterations in peripheral blood cell counts including the lymphocyte count were observed for up to eight weeks of Dox administration and also the relative contribution of gene modified cells to peripheral blood B, CD4⁺ or CD8⁺ T, and myeloid cells remained fairly constant during this time period. Even more important, a similar contribution of transduced cells was observed for the myeloid and the lymphoid cell compartment strongly arguing against a major lymphotoxicity. Also, no toxic effects of Dox-regulated hCDD expression was observed in other hematopoietic cell compartments including stem- or progenitor cells in various splenic, thymic and BM-derived hematopoietic cell compartments. Moreover, the TET.CDD vector conveyed significant protection against Ara-C (500 mg/kg, d1-4, i.v.) to the hematopoietic system as measured by granulocyte (0.26 +/−0.25 versus 0.8 +/−0, p=0.02) and platelet counts (584 +/−159 versus 883 +/−194, p=0.02) seven days after treatment. Furthermore, when the long term reconstitution potential of TET.CDD transduced Lin- cells was evaluated by secondary transplantation, robust, Dox-dependent transgenic eGFP expression was observed in peripheral blood B, CD4⁺ and CD8⁺ T, as well as myeloid cells of secondary recipients.

Taken together, our data demonstrate efficient Dox-inducible hCDD expression in 32D and primary murine bone marrow cells in vitro as well as in a murine in vivo bone marrow transplant gene transfer model. Most importantly, in the latter model Dox-inducible CDD expression not only allowed for significant protection from Ara-C induced myelotoxicity but also abrogated the lymphotoxicity observed previously with high and constitutive hCDD expression.