An International Conversation on Stem Cell Gene Therapy
Authors: David A Williams
Journal: Molecular Therapy (2007)

The first studies targeting hematopoietic stem cells for genetic modification using retrovirus vectors were reported in the 1980s. It should be no surprise that significant progress has been made in this area during the ensuing 20 years. The progress, and many remaining challenges, were evident at a recent meeting organized by George Stamatoyannopoulos and Luigi Naldini. The 4th Conference on Stem Cell Gene Therapy, held in Thessaloniki, Greece, in September 2007, provided an ideal setting for both formal and informal discussions on the wide spectrum of state-of-the-art basic research, as well as current and future clinical trials. The conference highlighted new approaches to improving the safety and efficacy of multiple viral vector systems. Most impressive were the advances described in the exploitation of microRNA expression patterns for seemingly precise regulation of transgene expression by workers in Naldini's and Sadelain's groups. Specific microRNA target sequences are embedded into lentivirus vectors, leading to lineage-specific and even differentiation stage–specific inhibition of transgene expression. From a functional standpoint, an approach was described that used microRNA targeting for regulation of suicide gene expression in precise lineages and to reduce transgene expression in antigen-presenting cells with the goal of modulating potential immune responses. This work predicts a new generation of vectors by which expression can be fine-tuned for specific purposes. Progress was also clear in the development of safety-improved gamma retrovirus and lentivirus vectors that contain long terminal repeat enhancer deletions, weaker cellular promoters in internal configurations, stronger polyadenylation sequences to prevent read-through, and chromatin insulator sequences to “isolate” transcriptional activity of the integrated vector from neighboring genes. These modifications seem increasingly important to prevent insert-related genotoxicity, which was seen in both the French X-linked severe combined immunodeficiency (SCID-X1) and the German chronic granulomatous disease (CGD) trials. Interestingly, insertional mutagenesis is not only a potential side effect of retrovirus, lentivirus, and foamy virus vectors, but as reviewed at the conference by David Russell, it is also occurs with adeno-associated virus (AAV)-derived vectors. As they relate to insertion-induced cell transformation, interesting data were presented by Fulvio Mavilio that suggested that overrepresentation of transcriptionally “active” genes by insertions of Moloney murine leukemia retroviruses (MLVs) compared with lentivirus vectors may be significant but not very impressive. However, using “functional relationship” analysis, MLV insertions are enriched in genes involved in hematopoietic cell-specific functions, proto-oncogenes, and intracellular signaling cascades, and the integration patterns also appear to be “cell-specific,” as if the retrovirus exploits the gene machinery that is critical for a particular cell type. This suggests that some other order of integrase-specific interaction between virus and cell target may be present. As in other areas, new research will almost certainly define the mechanisms of these preliminary observations. An additional consideration, touched on by several speakers, is that not all target cells may be equally susceptible to transformation by insertional activation of genes. The most often cited example of this is the previous experience in T-cell transductions in immune deficiency diseases and addressed at this conference experimentally by Dorothy von Laer. This concept as it relates to red cell progenitors may be an important consideration in the design of vectors for globin expression in the current and future trials for thalassemia and sickle cell anemia, as suggested by Michel Sadelain. However, it would seem that the occurrence of erythroleukemia in both mice and humans may predict otherwise, and it is yet to be determined how important insulator sequences, which tend to have adverse effects on recombinant titer, will be in treating these diseases. Important advances appear to have been made using newer vector systems. Dennis Hickstein reported successful correction of canine leukocyte adhesion deficiency due to deficiency of the integrin CD18, a fatal disease in dogs. Andre Lieber used new Ad 5/35 hybrid vectors to direct site-specific integration in human CD34+ cells via the CD46 receptor. Preliminary results using globin vectors also showed that 50% of Mo7e cells expressed gamma globin and were protected against position effects. To me, the most revealing discussions involved the issue of engraftment of transduced hematopoietic stem and progenitor cells for diseases in which myeloid cells must be phenotypically corrected. In short, a review of the published and unpublished data suggests that none of the currently used cytoreduction protocols (lower doses of busulfan and melphalan used in several European trials and now being adopted in trials in the United States at Children's Hospital of Los Angeles, the National Institutes of Health (NIH), and Cincinnati Children's Hospital) leads to engraftment of significant numbers of transduced cells with long-term myeloid cell potential. In this sense it is interesting to speculate that the apparent higher level of engraftment of marked myeloid cells in the adenosine deaminase (ADA) deficiency–SCID trial may suggest a previously unsuspected and subtle stem cell defect in this disease. Indeed, published and unpublished data from multiple trials (including cancer trials using drug resistance genes) suggest that none of these trials, with the possible exception of the ADA trial and two specific patients in the German CGD trial (of several patients in CGD trials in London, Germany, and the NIH unreported to date), have consistent, long-term engraftment of myeloid cells over 1%. The two exceptional patients in the German CGD trial both had insertions in EVI-1 and have subsequently developed monosomy 7. An additional confounding issue is the reliance on quantitative PCR methods to measure gene marking in human clinical trials, which leads to difficulty comparing marking data from one trial to another. These low marking results do not reflect the experience in large animals—canines and nonhuman primates—but the latter studies generally use more aggressive preparative regimens. Efficient transduction and engraftment of human long-lived and pluripotent hematopoietic stem cells (HSC) are critical issues as they relate to success of future human gene therapy trials outside applications in immunodeficiency diseases, in which gene therapy has clearly succeeded in infants and young children. In particular, as presented by several investigators at this conference, although human clinical trials for hemoglobinopathies may be a reality owing to success in the development of lentivirus vectors that allow high-level and lineage-specific expression of globin, the 500-pound nonhuman primate (pardon the pun) in the room is the stem cell–transduction issue. Based on a panel discussion organized to address the use of cytoreduction to improve engraftment of gene-modified HSC, significant progress needs to be made in this area before we can anticipate success in gene therapy protocols outside a limited number of diseases for which very strong pressure is available to select and expand a limited number of engrafted cells. This conference was one of several ongoing series of small international gatherings that effectively facilitate an international discussion of gene therapy topics. Another example is the Stem Cell Clonality and Genotoxicity Retreat held every 18 months and organized by an international group of investigators (a review of the most recent meeting was published in Molecular Therapy 1Frederickson RM Integrating ideas on insertional mutagenesis by gene transfer vectors.Mol Ther. 2007; 15: 1228-1232Abstract Full Text Full Text PDF PubMed Scopus (4) Google Scholar). These smaller meetings are often exemplary in that they encourage informal and yet serious discussions about the issues facing the field. Finally, it was remarkable to note the very large number of Molecular Therapy citations in the presentations—truly many more than for any other journal. This supports our view that Molecular Therapy is the leading journal of the field and publishes the best science from all over the world.