P. Herman

State-of-the-art lentiviral vectors for research use: risk assessment and biosafety recommendations.

Lentiviral vectors (LV) are competent gene transfer vehicles, as used for both research and gene therapy applications, because of their stable integration in non-dividing and dividing cells and long-term transgene expression. Along with our understanding that LV offer solutions for gene therapy, biosafety concerns have uncovered risks due to insertional mutagenesis, the generation of replication competent lentiviruses (RCL) and vector mobilization. Researchers therefore continue to devote significant efforts in designing LV with improved efficacy and biosafety features. The choice of a particular LV system for experimental studies is often driven by functional considerations, including increased productivity and/or transduction efficiency. The design of safer vectors has also directly benefited researchers allowing them to conduct experimental studies with lower risk. Currently, vectors combine improved safety features (that decrease the risk of recombination and vector mobilization) with increased transduction efficiency. Hence, risks associated with the inadvertent transduction of cells of the investigator gain greater importance in assessing the overall risk of these vectors and become an important biosafety concern. This review outlines the different strategies used to improve LV biosafety by comparing state-of-the-art and emerging LV production systems and highlighting biosafety issues that can arise during their contained use. The few existing national and international biosafety recommendations that specifically address the use of LV in research are discussed and recommendations for most common research activities using LV are proposed.

Engineering nucleases for gene targeting: safety and regulatory considerations

Nuclease-based gene targeting (NBGT) represents a significant breakthrough in targeted genome editing since it is applicable from single-celled protozoa to human, including several species of economic importance. Along with the fast progress in NBGT and the increasing availability of customized nucleases, more data are available about off-target effects associated with the use of this approach. We discuss how NBGT may offer a new perspective for genetic modification, we address some aspects crucial for a safety improvement of the corresponding techniques and we also briefly relate the use of NBGT applications and products to the regulatory oversight.

Genetic modification through oligonucleotide-mediated mutagenesis. A GMO regulatory challenge ?

In the European Union, the definition of a GMO is technology-based. This means that a novel organism will be regulated under the GMO regulatory framework only if it has been developed with the use of defined techniques. This approach is now challenged with the emergence of new techniques. In this paper, we describe regulatory and safety issues associated with the use of oligonucleotide-mediated mutagenesis to develop novel organisms. We present scientific arguments for not having organisms developed through this technique fall within the scope of the EU regulation on GMOs. We conclude that any political decision on this issue should be taken on the basis of a broad reflection at EU level, while avoiding discrepancies at international level.

Stromal factors support the expansion of the whole hemopoietic spectrum from bone marrow CD34+DR- cells and of some hemopoietic subsets from CD34+ and CD34+DR+ cells.

Ex vivo expanded bone marrow CD34+DR− cells could offer a graft devoid of malignant cells able to promptly reconstitute hemopoiesis after transplant. We investigated the specific expansion requirements of this subpopulation compared to the more mature CD34+ and CD34+DR+ populations. The role of stromal factors was assessed by comparing the expansion obtained when the cells were cultured in (1)u2002long-term bone marrow culture (LTBMC) medium conditioned by an irradiated human BM stroma (CM), (2)u2002medium supplemented with 15% FBS (FBSM) and (3)u2002non-conditioned LTBMC medium (LTM) for 21 days. The effect of the addition of G-CSF (G) and/or of MIP-1α (M) to a combination of IL-3, SCF, IL-6 and IL-11 (3, S, 6, 11) was analyzed. Compared to CD34+DR− cells, CD34+ and CD34+DR+ cells gave rise to a similar number of viable cells and to a lower progenitor expansion. The expansion potential of CD34+ and CD34+DR+ cells was equivalent in CM and in FBSM except for both the emergence of CD61+ megakaryocytic cells and LTC-IC maintenance which were improved by culture in CM. In contrast, expansion from CD34+DR− cells was enhanced by CM for all the parameters tested. Compared to FBSM, CM induced a higher level of CFU-GM and BFU-E expansion and allowed the emergence of CD61+ cells. HPP-CFC were maintained or expanded in CM but decreased in FBSM. Compared to input, the number of LTC-IC remaining after 21 days of CD34+DR− expansion culture was strongly decreased in FBSM and variably maintained or expanded in CM. Comparison with LTM indicated that stroma conditioning is responsible for this effect. G-CSF significantly improved CFU-GM and HPP-CFC expansion from CD34+DR− cells without being detrimental to the LTC-IC pool. The growth of CD61+ cells was significantly enhanced by G-CSF in CM. Addition of MIP-1α had no significant effect either on progenitor expansion or on LTC-IC, regardless of culture medium. We conclude that factors present in stroma- conditioned medium are necessary to support the expansion of the whole spectrum of hematopoietic cells from CD34+DR− cells and to support the expansion of cell subsets from CD34+ and CD34+DR+.

Haemopoietic defect and decreased expansion potential of bone marrow autografts from patients with acute myeloid leukaemia in first remission.

Autologous bone marrow (BM) transplantation for acute myeloid leukaemia (AML) in complete remission (CR) is frequently followed by a slow haemopoietic recovery. We assessed the haemopoietic capacity of purified BM stem cell (CD34+DR−) and progenitor cell (CD34+DR+) populations from patients with AML in CR, and compared these data with those of normal BM. The feasibility of ex vivo expansion in stroma‐conditioned medium supplemented with cytokines was also investigated.

Animal Cell Cultures: Risk Assessment and Biosafety Recommendations

During the last three decades, animal cell culturing has been essential for biomedical research and biotechnological activities in general. Along with this increasing importance, biosafety concerns have pointed to the risks of manipulating animal cell cultures for human health and the environment. A maximal reduction of these risks necessitates a thorough risk assessment of the cell cultures used. It involves an evaluation of both the intrinsic properties of the cell culture, including subsequent properties acquired as a result of genetic modification, and the possibility that the cell culture may inadvertently or deliberately become contaminated with pathogens. The latter is a major hazard associated with the manipulation of animal cell cultures, as adventitious agents may be pathogenic and have a better capacity to survive in unfavorable conditions. Consequently, most of the containment measures primarily aim at protecting cells from adventitious contamination. Therefore, a comprehensive evaluation of the risks encountered during the handling of cell cultures should include considerations regarding the type of manipulation as well. As a rule, cell cultures known to harbor an infectious etiologic agent should be manipulated in compliance with containment measures recommended for the etiologic agent. With the exception of very well-characterized cell cultures for which the use of a type II biosafety cabinet depends on the origin of the cells, work with cell cultures from human or primate origin should generally and minimally be performed under containment level 2 using a type II biosafety cabinet. In every case, containment measures should minimize adventitious contamination of the cell cultures and offer a maximal protection of human health and the environment.

In vitro evaluation of the haemopoietic defect of CD34+ cells from patients with acute myeloid leukaemia in first remission

Haemopoietic cells from patients with acute myeloid leukaemia (AML) in first complete remission (CR1) show in vitro a haemopoietic defect and a decreased expansion potential. To better characterize this haemopoietic defect, CR1 AML and normal CD34+ cells were analysed for immunophenotype, viability, cell cycle and progenitor content before and during expansion culture in stroma‐conditioned medium supplemented with cytokines. The production of haemopoiesis inhibitor by patient cells and the influence of high concentrations of stem cell factor (SCF) and Flt3‐ligand (FL) on cell survival and ex vivo expansion potential were also studied. Before expansion, patient CD34+ cells showed viability and cell‐cycle phase distribution similar to normal but lower percentages of CD34+DR− or CD34+CD38− cells and lower progenitor content. After 48u2003h of culture ±30% of patient cells had died regardless of the cytokine combination used, whereas only 15% of normal cells died. After 7u2003d of culture, viability and cell cycle analyses showed comparable data for normal and patient samples. Co‐culture of patient and normal cells did not show any evidence for haemopoiesis inhibitor production by patient cells. Even at high cytokine concentrations, a low progenitor expansion and a decrease in CD34+ cell numbers was observed for patient samples in contrast to normal samples. In conclusion, CR1 AML CD34+ cells showed excessive early cell mortality. No evidence for cell‐cycle arrest or haemopoiesis inhibitor production was shown. SCF and FL used at high concentrations did not correct the patient cell expansion defect.