Hans-Georg Eckert

Influence of multiplicity of infection and protein stability on retroviral vector-mediated gene expression in hematopoietic cells.

Using retroviral vectors encoding enhanced green fluorescent protein (egfp), we addressed to what extent expression of retroviral transgenes in hematopoietic cells depends on the multiplicity of infection (moi) and on the half-life of the encoded protein. we show that an elevation of the moi not only elevates the frequency of transduced cells, but also increases transgene expression levels and reduces interanimal variability in vivo (hematopoietic cells of c57bl/6j mice analyzed 13 weeks after transplantation). this suggests that the moi has to be carefully controlled and should be adapted as desired for clinical studies when evaluating vector performance in preclinical models. the impact of protein stability is demonstrated by comparing vectors expressing egfp or a destabilized variant with a c-terminal pest-sequence, d2egfp. the loss of expression with d2egfp was more pronounced in terminally differentiated cells of the peripheral blood (>30 fold) than in progenitor cells (five- to 10-fold), indicating a stronger transcription of the retroviral promoter in progenitor cells and a predominant role of protein inheritance over de novo synthesis of transgenic protein in mature blood cells. This analysis reveals an important and differentiation-dependent contribution of protein half-life to the expression of retroviral vectors in hematopoietic cells, establishes d2EGFP as a more accurate reporter for determination of vector transcription, and also suggests that preclinical data obtained under conditions of high transduction rates or with vectors expressing stable reporter proteins require careful interpretation.

Multidrug Resistance 1 Gene Transfer Can Confer Chemoprotection to Human Peripheral Blood Progenitor Cells Engrafted in Immunodeficient Mice

Myelosuppression is the main side effect of cancer chemotherapy. An improved rate of retroviral vector-mediated gene transfer to hematopoietic stem cells, shown in more recent clinical trials, has created the basis to test the concept of myeloprotective gene therapy. We transplanted clinical-scale human peripheral blood progenitor cell grafts (n = 2) transduced with retroviral vector SF91m3, which contains the human multidrug resistance 1 gene (MDR1), into nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice. Engrafted mice of one cohort were protected from paclitaxel toxicity (p < 0.05) and we noted a similar trend in the second cohort. In paclitaxel-treated mice that had received gene-transduced cells we found a significant increase in gene marking (p < 0.05 - p < 0.01) or P-glycoprotein expression (p < 0.01) compared with their chemotherapy-naive counterparts. This is the first report showing that cytostatic drug resistance gene therapy can mediate chemoprotection of human clinically relevant stem cell populations with marrow engraftment potential.

Improved post-transcriptional processing of an MDR1 retrovirus elevates expression of multidrug resistance in primary human hematopoietic cells

we describe the functional analysis of a novel retroviral vector, sf91m3, which was designed for improved expression of the in vivo selectable marker, multidrug resistance 1 gene (mdr1), in hematopoietic cells. sf91m3 combines several promising features. the vector backbone lacks viral coding sequences and aug-start codons 5′ of the mdr1 cdna. a point mutation of a cryptic splice acceptor of the mdr1 cdna increases the probability of transferring an intact provirus. the titer of a pg13 packaging cell clone containing a single proviral integration is high (>2 × 106 particles/ml from frozen stocks of serum-free vector harvests). human hematopoietic cells transduced with sf91m3 reliably express mdr1 before and after passage through nod/scid mice, as shown by quantitative pcr and efflux assays with rhodamine 123 or hoechst 33342. finally, sf91m3 mediates resistance to escalated doses of cytotoxic agents, as shown by survival and differentiation of transduced colony-forming cells in the presence of colchicine at 48 ng/ml (>10 × IC50). Thus, SF91m3 may represent an interesting candidate for future trials addressing the safety and utility of MDR1 gene transfer; moreover, this study demonstrates that sequence alterations improving post-transcriptional processing of retroviral vectors have a substantial impact for gene expression in hematopoietic cells.

Establishment of an optimised gene transfer protocol for human primary T lymphocytes according to clinical requirements

Current gene therapeutic protocols directed towards the treatment of inherited disorders (eg ADA-SCID) and viral infections (eg AIDS), as well as adoptive immunotherapy approaches are based on the use of genetically modified lymphocytes. Since only insufficient transduction of T cells is obtained using existing techniques, the development of more efficient gene transfer protocols into these cells is of great importance. We present here a protocol for the highly efficient transduction of human primary T cells at high densities (1 × 106/ml) by retroviral infection. Using retroviral vectors encoding a truncated human low-affinity nerve growth factor receptor (ΔLNGFR) as a gene transfer marker, we obtained transduction frequencies of more than 70% of CD3+ cells after two cycles of infection. Our protocol is based on the use of FBS-free media for both the production of retrovirus-containing supernatant and the cultivation of the primary T cells. Since the protocol presented here works just as efficiently under large-scale conditions, it may be easily adapted to clinical needs and ‘good manufacturing practice’ (GMP) standards.

Genetic protection of repopulating hematopoietic cells with an improved MDR1‐retrovirus allows administration of intensified chemotherapy following stem cell transplantation in mice

This study was undertaken to analyze the hematotoxicity of paclitaxel (Taxol®) and to test whether transduction of repopulating hematopoietic cells with a retroviral vector (SF1m) expressing the human multidrug resistance 1 gene (MDR1) would permit dose intensification following bone marrow transplantation (BMT). While the regimen chosen (8×20 mg/kg i.p. within 12 days) produced a non‐lethal, reversible hematotoxicity in mice with steady‐state hematopoiesis, only 35.3% (6/17) of control mice survived when treated starting 14 days post BMT. In contrast, 83.3% (15/18) of mice transplanted with SF1m‐transduced cells survived, owing to a significant protection against severe acute myelotoxicity (as determined by neutrophil counts, white and red blood cell counts and values for hemoglobin and hematocrit). After recovery from chemotherapy, an increase of myeloid cells that were resistant to colchicine and effluxed the fluorochrome Rhodamine 123 was observed in SF1m‐mice, but not in controls. These results reveal that the lethal, dose‐limiting hematotoxicity of an intensified post‐transplantation chemotherapy with paclitaxel can be prevented by retroviral transfer of the MDR1 gene to a minor proportion of repopulating cells. Our mouse model, mimicking clinically achievable gene transfer rates, thus suggests that bone marrow chemoprotection may widen the therapeutic window and permit an earlier onset of post‐transplantation chemotherapy.

Down-regulation of retroviral transgene expression during differentiation of progenitor-derived dendritic cells

OBJECTIVE Hematopoietic progenitor cells are a promising source for generation of genetically modified dendritic cells. A prerequisite for using these cells in therapeutic approaches is stable vector-mediated transgene expression during and after cell maturation. We investigated the expression of enhanced green fluorescence protein (EGFP) mediated by retroviral vectors in dendritic cells and other hematopoietic cells differentiated in vitro. MATERIAL AND METHODS CD34(+) cells were efficiently transduced with retroviral vector constructs known to mediate different expression levels due to distinct cis-acting elements. EGFP(+) cells were purified by cell sorting and differentiated to monocytes, granulocytes, dendritic cells, and erythrocytes. Coexpression of EGFP and cell type-specific markers was analyzed by flow cytometry. RESULTS Transgene expression from various retroviral vectors was silenced exclusively in dendritic cells, but not in other mature myeloid cells. Loss of EGFP was most pronounced in cells initially displaying low expression levels. This was confirmed by using a retroviral vector coding for a variant of EGFP with significantly reduced half-life. In contrast, a majority of dendritic cells showed stable expression when a self-inactivating retroviral construct using an internal cytomegalovirus promotor was used. CONCLUSIONS We suggest that expression from the retroviral long terminal repeat is silenced during dendritic cell differentiation in vitro. High levels of stable transgene product in progenitor cells may mask a loss of expression. An improvement of retroviral vectors mediating stable transgenic expression is necessary for therapeutic approaches using gene-modified dendritic cells.

Cis-Active Elements of Friend Spleen Focus-Forming Virus: From Disease Induction to Disease Prevention

The polycythemic strain of the Friend spleen focus-forming virus (SFFVp) is a replication-defective, acutely transforming retrovirus inducing a bistage erythroleukemia in susceptible mice. The first stage of the disease is an acute polyclonal erythroblastosis induced by the proliferation-promoting effect of gp55. gp55 is expressed from a spliced subgenomic message of SFFVp and stimulates the cellular receptor for erythropoietin. Using a selectable SFFVp that otherwise mimics the specificity of the disease, we demonstrate that the kinetics of the polyclonal expansion depends on the transcriptional strength of the retroviral cis-active elements. By exchanging gp55 for apathogenic genes, we show that SFFVp enhancer and splice signals can be successfully utilized for the development of retroviral vectors mediating very efficient transgene expression in hematopoietic cells. Apathogenic selectable SFFVp-based vectors carrying distinct enhancer alterations are a valuable tool to analyze transcriptional control of leukemia viruses in the absence of oncogenic proteins. Moreover, they might have therapeutic potential.

Contribution of the Retroviral Leader to Gene Transfer and Expression in Packaging Cells and Myeloid Stem and Progenitor Cells

Retroviral vectors designed to transduce hemopoietic stem cells have to be optimised with respect to basic requirements for gene transfer and expression, (i) The cis-regulatory elements of the genomic vector RNA interacting with retroviral proteins supplied from packaging cells must allow for optimal packaging, reverse transcription and integration into the genome of the target cell, (ii) The cis-regulatory elements of the integrated ’proviral’ vector DNA interacting with the cellular transcriptional machinery must guarantee efficient and long-lasting gene expression in the target cell and its progeny.