The production and stability of recombinant retroviral vecwas reached between retroviral vector production and tors was examined at various temperatures. The two studdecay. Maximal accumulated recombinant retroviral titers ied recombinant retroviral vectors, based on different packwere five-to ten-fold increased after a medium incubation aging cell lines, exhibited a four-fold increased half-life at at 32°C as compared to 37°C. Furthermore, multiple cycles 32°C as compared to 37°C. Surprisingly, this increased of freezing and thawing of retroviral vector supernatants stability at 32°C was only observed within a very narrow hardly affected the recombinant retroviral vector titer, indetemperature window. At 30°C and 34°C, retroviral vector pendent of the presence of serum. This knowledge on half-lives were quite similar to that at 37°C. Regardless of characteristics of recombinant retroviral vectors has practithe vector half-life, retroviral vectors accumulated in the cal implications for the manufacturing of these viruses for culture medium for a period of 48 h before an equilibrium clinical gene therapy protocols.Keywords: retroviral vector titer; retroviral vector stability; gene therapyRecombinant retroviral vectors are widely used to introretroviral vectors at different temperatures, culture volumes, and producer cell densities. duce genes into cells. At present recombinant retroviral vectors are being employed in the majority of clinicalIn the design of our experiments we included intermediate storage steps in liquid nitrogen. Therefore, we gene therapy protocols. The success of such therapies is critically dependent on the transduction efficiency.first investigated the effect of repeated freezing (in liquid nitrogen) and thawing (at 37°C) of vector supernatant Among other factors, this transduction efficiency will be influenced by the titer of vector preparations employed.batches on their retroviral vector titer. The vector supernatant batches were obtained by performing a 2 h harvest Several methods have been described to obtain high recombinant retroviral vector titers. These methods from the recombinant amphotropic neo-vector producing cell line POPA/neo-4 at 37°C. One batch contained 10% include redesign of the retroviral vector, 1-3 the generation of producer cells with increased recombinant retrovirus newborn calf serum (NBCS) and a second was kept serum-free. The vector supernatant batches were divided copy numbers, 4,5 changing the retroviral vector harvest procedure by adding sodium butyrate or dexamethasone over several ampoules that were frozen and thawed for different numbers of cycles, ranging from 0 to 16 times. to the culture medium 6,7 or by using a culture temperature of 32°C instead of 37°C, 6,8,9 and down-stream conNext, the end-point retroviral vector titer was determined on NIH/3T3 cells. As can be seen in Figure 1, the first centration of retroviral vectors in the vector supernatant batch. 9,10 In general, each of these methods results in an two to four cycles had the largest impact ...

Viruses are the most commonly used vectors for clinical gene therapy. The risk of dissemination of a viral vector into the environment via excreta from the treated patient, a phenomenon called shedding, is a major safety concern for the environment. Despite the significant number of clinical gene therapy trials that have been conducted worldwide, there is currently no overview of actual shedding data available. In this article, an inventory of shedding data obtained from a total of 100 publications on clinical gene therapy trials using retroviral, adenoviral, adeno-associated viral and pox viral vectors is presented. In addition, the experimental set-up for shedding analysis including the assays used and biological materials tested is summarized. The collected data based on the analysis of 1619 patients in total demonstrate that shedding of these vectors occurs in practice, mainly determined by the type of vector and the route of vector administration. Due to the use of non-quantitative assays, the lack of information on assay sensitivity in most publications, and the fact that assay sensitivity is expressed in various ways, general conclusions cannot be made as to the level of vector shedding. The evaluation of the potential impact and consequences of the observations is complicated by the high degree of variety in the experimental design of shedding analysis between trials. This inventory can be supportive to clinical gene therapy investigators for the establishment of an evidence-based risk assessment to be included in a clinical protocol application, as well as to national regulatory authorities for the ongoing development of regulatory guidelines regarding gene therapy.

We have generated a cell line, designated POAM-P1, shedding amphotropic recombinant retroviruses carrying the human adenosine deaminase (hADA) gene. It exhibits a 1 log increased retrovirus titer on NIH-3T3 cells and a five-fold more efficient transduction of human ADA-deficient T lymphocytes, as compared to the previously generated cell line POC-1 which produces the same recombinant hADA retrovirus. To study whether the titer of retrovirus-producing cell lines influences the transduction efficiency of hematopoietic stem cells in a co-culture setting, we compared the POAM-P1 and POC-1 cell lines with respect to their gene transfer efficiency on rhesus monkey bone marrow. Following co-cultivation of rhesus monkey bone marrow with POAM-P1 cells, successful transduction could be demonstrated in approximately 10% of myeloid progenitor colonies (CFU-C) and 0.1% of peripheral blood mononuclear cells (PBMC) and granulocytes in vivo until > 1 year after autologous transplantation. In addition, the presence of functional hADA enzyme was detected in red blood cells, PBMC, and granulocytes. Monkeys receiving POC-1 co-cultured bone marrow carried transduced blood cells for > 2 years after transplantation. Despite the higher retrovirus titer of POAM-P1 cells as compared to POC-1 cells, no difference was observed in gene transfer efficiency into CFU-C and long-term repopulating stem cells. This shows that in our co-cultivation procedure the retrovirus titer was not limiting the transduction efficiency of primate hematopoietic stem cells.

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