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W2319314246 abstract "The ability of hematopoietic stem cells (HSC) to repopulate the hematopoietic and possibly other systems, combined with the opportunity to introduce exogenous genes into HSC, provides a unique opportunity for the treatment of hematological and non-hematological diseases. Recently, the first successful application of gene therapy in a clinical setting involved the transfer of the γ-chain of the interleukin (IL)-2 receptor gene (IL2RG) into HSC of patients with X-linked severe combined immunodeficiency-X1 (SCID-X1) [1Hacein-Bey-Abina S. Sustained correction of X-linked severe combined immunodeficiency by ex vivo gene therapy.N. Engl. J. Med. 2002; 346: 1185-1193Crossref PubMed Scopus (969) Google Scholar]. On March 21–23, over 150 researchers in the field of HSC gene therapy met for the Third Conference on Hematopoietic Stem Cell Gene Therapy: Biology and Technology, in Rockville, Maryland. Their goal was to summarize the status of the field, both basic science and clinical applications. A total of 42 scientific presentations, two workshops, and a poster session representing over 30 submitted abstracts were presented. This meeting was organized and chaired by George Stamatoyannopoulos (University of Washington, Seattle, WA), and sponsored by the National Institutes of Diabetes and Digestive and Kidney Diseases (NIDDK), Allergy and Infectious Diseases (NIAID), and the National Human Genome Research Institute (NHGRI). Several important themes emerged from the scientific presentations related to the biology of HSC, progress toward the successful application of gene therapy in several clinical settings, the further development of virus vector systems, and the regulation of virus vector expression. Many of the presentations focused on the biology and potential plasticity of hematopoietic stem cells (HSC). Several studies indicate that many different types of stem cells exhibit a specific flow cytometric pattern (known as the “side population”) when stained with the vital dye Hoechst 33342. Brian Sorrentino (St. Jude Children's Research Hospital, Memphis, TN) provided clear evidence that the ATP-binding cassette transporter ABCG2 is primarily responsible for the Hoechst 33342 dye-efflux properties of bone marrow HSC. The role of this transporter in HSC function is still under investigation. Connie Eaves (Terry Fox Laboratory, Vancouver, BC) presented evidence that cells expressing the pan-hematopoietic cell surface marker CD45 are present in mouse liver. These CD45+ cells have the ability to reconstitute the hematopoietic system of myeloablated recipients, and the CD45 donor cells that engraft in the liver of these primary recipients retain the ability to reconstitute the hematopoietic system of secondary recipients. She also presented evidence for the role for BCR-ABL in the control of lineage programming. Using transgenic donors or oncoretrovirus vector tagging, Thalia Papayannopoulou (University of Washington, Seattle, WA) described experiments that help define the cells from muscle that are able to reconstitute the hematopoietic system of myeloablated recipients. Consistent with recent reports from others, she found that these cells express the pan-hematopoietic cell surface marker CD45, and that they arise from the donor bone marrow. Upon transplantation, these cells can regenerate similar cells in both muscle and liver. Several researchers also presented evidence for the plasticity of HSC residing in the bone marrow. Donald Orlic (NHGRI, Bethesda, MD) used transgenic donors or oncoretrovirus vector tagging to demonstrate the ability of injected or mobilized mouse HSC to reconstitute the hematopoietic system and help regenerate damaged myocardium. The ability of individual HSC clones to give rise to both hematopoietic and non-hematopoietic tissues was further substantiated by studies presented by Saul Sharkis (Johns Hopkins Oncology Center, Baltimore, MD). In this case, male donor cells were found in several non-hematopoietic tissues of female recipients following secondary transplantation of marrow from female donors initially transplanted with a single male HSC. Work presented by Jan Nolta (Children's Hospital, Los Angeles, CA) indicated that human HSC transplanted into immunodeficient mice can also generate hepatocytes following chemical damage of the recipient liver. Finally, Stuart Orkin (Harvard Medical School, Boston, MA) demonstrated that plasticity is not necessarily restricted to primitive HSC and progenitors. By manipulating the levels of transcription factors involved in fate determination in hematopoiesis, he showed that early regulators can work in direct opposition to each other, enhancing one fate while actively retarding another fate pathway. Intervention in these pathways could allow for “retrograde” differentiation from one fate to another within the blood cell lineage. Claudio Bordignon (Istituto Scientifico H.S. Raffaele, Milan, Italy) described results from his most recent clinical trial in which mobilized peripheral blood cells from two patients with adenenosine deaminase (ADA)-deficient SCID were transduced with an oncoretrovirus vector containing the ADA gene. Unlike previous trials for ADA-deficient SCID, the patients in this trial were not receiving exogenous ADA enzyme therapy at the time of transplantation, and the transduced CD34 cells were transplanted following a mild myelosuppressive treatment with the chemotherapeutic agent busulfan. The procedure was well tolerated, and gene marking rates as high as 11% in peripheral myeloid cells were detected. In this case, the overall level of transduced and engrafted HSC was sufficient to allow for the production of T, B, and NK cells at normal levels, the ability to respond to normal antigen stimulation, and the restoration of normal ADA metabolism. This study presents for the first time the use of a mild myelosuppressive regimen to enhance the engraftment of transduced autologous HSC in patients not already receiving high-dose myeloablative therapy as a mainline treatment for their disease. Donald Kohn (Children's Hospital, Los Angeles, CA) described the long-term follow up of a phase 1 trial of gene therapy for ADA-deficient SCID. Molecular analysis revealed very low rates of gene marking and oligo-or monoclonal reconstitution with provirus-marked cells. Although there appeared to be a selective expansion of transduced T cells (as expected with this disease), the overall level of transduced cells and vector expression was not sufficient to stop supplementing the patients with ADA enzyme therapy. After several improvements to the vector and transduction conditions, a new clinical trial has been initiated. As in the initial trial, recipients will be transplanted with transduced cells in the absence of myloablative conditioning and the presence of exogenous ADA enzyme therapy. Parameters to be tested include a comparison of bone marrow versus cord blood CD34 cells using genetically distinguishable vectors. A poster presented by Adrian Thrasher's group (Institute of Child Health, London, UK) described the second successful gene therapy of X-SCID, using a protocol similar to that used in the original successful trial by Cavazzana-Calvo et al. [1Hacein-Bey-Abina S. Sustained correction of X-linked severe combined immunodeficiency by ex vivo gene therapy.N. Engl. J. Med. 2002; 346: 1185-1193Crossref PubMed Scopus (969) Google Scholar]. Jennifer Puck (NHGRI, Bethesda, MD) described the development of a clinical trial to treat patients with SCID-X1 who fail allogeneic bone marrow transplantation. She also described an RD114-pseudotyped oncoretrovirus vector containing the gene IL2RG that she used to transduce CD34+ cells from SCID-X1 patients. Following transplantation into pre-immune fetal sheep, human T and B cells developed and, in addition, a high level of myeloid cells expressing the IL2RG was observed. Fabio Candotti (NHGRI, Bethesda, MD) described the development of an oncoretrovirus vector for the WAS gene that could correct the actin polymerization defect in T cells from patients with Wiskott–Aldrich syndrome. He also provided convincing evidence for in vivo selection for corrected T cells in patients with a somatic reversion of the Wiskott–Aldrich mutation. These data suggest that gene therapy may also be effective for this disease. Immune disorders caused by defects in myeloid cells have also been the subject of clinical trails. Dennis Hickstein (National Cancer Institute (NCI), Bethesda, MD) summarized results from a phase I trial with an oncoretrovirus vector for the CD18 gene to treat patients with leukocyte adhesion deficiency, and the development of a dog model for this disease in order to further the preclinical development of this approach. Two presentations focused on the development of retrovirus vectors for the gp91phox gene to treat chronic granulomatous disease. These included a lentivirus vector described by Didier Trono (University of Geneva, Switzerland) and an RD114-pseudo-typed oncoretrovirus vector described by Harry Malech (NIAID, Bethesda, MD), both of which were capable of expression at therapeutic levels. The powerful in vivo selection for corrected cells in the SCID disorders has overcome a relatively low frequency of stem cell transduction. If gene therapy is to be extended to diseases affecting myeloid cells, the frequency of gene transfer to HSC must be improved. Gene transfer into HSC with oncoretrovirus vectors as well as other vector systems is dependent in part on the source and cycling status of the targeted cell population. Donald Kohn described several combinatorial improvements to the culture conditions used to transduce human HSC as part of an ongoing clinical trial, including the use of several early acting cytokines and fibronectin fragment. Jan Nolta demonstrated that rates of gene transfer could be improved by inducing HSC to undergo cell division by inhibiting the cyclin-dependent kinases p15 and p27. In another approach, Cynthia Dunbar (National Heart, Lung and Blood Institute (NHLBI), Bethesda, MD) described some recent competitive transduction assays in rhesus macaques in which she compared the transduction and engraftment of CD34 cells from bone marrow and from peripheral blood mobilized with G-CSF alone or in combination with SCF or Flt3-L. Her results suggest a distinct hierarchy with bone marrow and a G-CSF + SCF mobilized blood performing the best, and G-CSF and G-CSF + Flt3-L mobilized blood performing poorly. Taken together, these results emphasize the critical role that recombinant cytokines have in the optimization of HSC transduction rates. The sources and testing of recombinant cytokines for clinical gene therapy trials were the subject of a workshop held during the meeting (see below). The ability to amplify HSC would provide a powerful method to overcome the low frequency of gene transfer and engraftment of transduced HSC. Two presentations on this topic focused on manipulations of the early HSC regulatory homeobox gene HoxB4. In one study, Stefan Karlsson (Lund University, Sweden) presented evidence that mutating the HoxB4 gene in mice results in a decrease in HSC repopulating ability, and that this is due to a diminished rate of cell cycling. In another study, R. Keith Humphries (Terry Fox Laboratory, Vancouver, BC) presented new data demonstrating that overexpression of HoxB4 by oncoretrovirus vector transduction allows mouse reconstituting HSC to expand 80-fold in culture. He further showed that a similar approach could also be applied to human HSC using cord blood CD34 cells and immunodeficient mouse transplantation. Two other general approaches for the selection of transduced HSC were also discussed. The first involves the regulated use of cytokine receptor domains to provide a cell growth signal. In the cell growth switch model discussed by C. Anthony Blau (University of Washington, Seattle), the gene therapy vector encodes a fusion molecule composed of a signaling domain from the thrombopoietin receptor Mpl and a binding domain for a small drug molecule known as a chemical inducer of dimerization. Following bone marrow transduction and transplantation, exposure of transduced cells to this drug forces the fusion molecule to dimerize, which in turn results in an Mpl-like cell growth signal. In mice, this results in an selective expansion of transduced cells in the erythroid lineage, whereas in dogs there is a selective expansion in both red and selected white blood cells. In the selective amplifier system presented by Keiya Ozawa (Jichi Medical School, Japan), a similar approach is based on a fusion molecule composed of modified versions of the binding domain of the estrogen receptor and the signaling domain for the G-SCF receptor. In primate studies, this allowed for the selective expansion of transduced cells in the granulocyte lineage. More recent studies were also described using a newer fusion molecule composed of the binding domain for the erythropoietin receptor and the signaling domain for Mpl. It should be noted that neither of these approaches currently allow for selection at the level of primitive repopulating HSC. Alternatively, drug selection against untransduced HSC may allow a small number of transduced HSC containing a drug resistance gene to become dominant. Several researchers described the use of a modified version of the methylguanine methyltransferase (MGMT) drug resistance gene to allow for the selection of transduced primitive repopulating HSC. This modified gene confers a high degree of resistance to DNA-damaging agents such as 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) and temozolomide (TMZ), and is resistant to O6-benzylguanine (BG), which inactivates endogenous MGMT. In one set of experiments, Stanton Gerson (Case Western Reserve University, Cleveland, OH) showed that BG + BCNU treatment before and after transplantation of small numbers of transduced cells in mice can lead to a high degree of transduced HSC in the primary and secondary recipients. In a similar series of studies, Derek Persons (St. Jude Children's Research Hospital, Memphis, TN) described the use of this system to greatly expand the fraction of RBC in mice expressing a linked expression cassette for β-globin following low copy-number transduction of bone marrow with a lentivirus vector. Studies conducted by David Williams (Children's Hospital Medical Center, Cincinnati, OH) and others and presented by Patrick Kelly (Children's Hospital Medical Center, Cincinnati, OH) demonstrated that an MGMT selection approach is effective at selecting human HSC following transplantation into immunodeficient mice. The initial human HSC gene therapy trials revealed that conventional oncoretrovirus vectors and packaging systems were relatively inefficient at transduction of primitive, reconstituting HSC. This is due in part to the fact that the vectors used in these studies require cell division and active metabolism for efficient transduction, whereas human HSC are relatively quiescent. In addition, the envelope proteins used to package the vectors for these early studies targeted receptors that are expressed at very low levels on human HSC. To address these limitations, much work has focused on the development of vectors based on other viruses, such as the human immunodeficiency lentivirus, and packaging systems using alternative envelope pseudotypes, which target receptors which are expressed at higher levels on HSC such as GALV and VSVG. Several presentations focused on the abilities and limitations of vectors based on oncoviruses and lentiviruses. Using a novel method of inverse PCR, Christof von Kalle (University of Freiburg, Germany) provided clear evidence in several preclinical and clinical settings that oncoretrovirus vectors are capable of transducing polyclonal, multi-potential HSC of primates and humans. Luigi Naldini (University of Torino, Italy) presented several lines of evidence demonstrating the advantages of third-generation lentivirus vectors for the transduction of HSC, as well as the ability of lentivirus vectors to efficiently transduce primitive human stem/progenitor cells capable of multi-potential reconstitution in immunodeficient mice. Hans-Peter Kiem (Fred Hutchinson Cancer Research Center, Seattle, WA) reported very high rates of gene transfer following lentivirus vector transduction of baboon CD34 cells and transplantation into immunodeficient mice. However, when the same cells were transplanted back into myloablated autologous baboon recipients, the rates of long-term gene marking observed in peripheral blood cells were much lower. These data suggest that the high rates of gene transfer seen in immune-deficient mice and in surrogate assays for human HSC may not translate into high levels of gene transfer in clinical HSC gene therapy settings. Another cautionary note was provided by Christopher Baum (Hannover Medical School, Germany), who presented evidence documenting a case of leukemic transformation in mice as a result of proviral activation of an adjoining proto-oncogene. This finding serves to emphasize the potential hazards associated with randomly integrated provirus, and the need to minimize the number of integration events. This may be especially pertinent to the use of lentivirus vectors, as several investigators have found that lentivector transduction often leads to multiple integration events per cell. Hans-Peter Kiem also reported that oncoretrovirus vectors pseudotyped with the envelope from the feline leukemia virus RD114 allowed for high rates of gene transfer into reconstituting HSC of non-human primates, as they previously reported for dogs. High rates of gene transfer with RD114-pseudotyped oncoretrovirus vectors into human CD34 cells were also reported by Harry Malech and Arthur Bank (Columbia University, New York, NY) using transplantation into immunodeficient mice, and by Jennifer Puck using transplantation into pre-immune fetal sheep. Arthur Bank also described the development of a stable packaging line using the RD114 envelope, which should in turn help facilitate the use of this pseudotype in clinical trials. Wolfram Ostertag (Heinrich Pette Institut, Hamburg, Germany) reported several combinatorial improvements in the design of oncoretrovirus vectors, as well as the development of a new vector pseudotype based on LCMV. LCMV shares many of the same advantages for vector pseudotyping of VSVG, yet is less toxic. So far most researchers have focused on the use of retrovirus vectors based on oncoviruses and lentiviruses. As an alternative, David Russell (University of Washington, Seattle, WA) described the development of recombinant retrovirus vectors based on the spumavirus human foamy virus. This includes the development of a helper-free packaging system and the demonstration that these vectors can transduce at high levels mouse reconstituting HSC and human CD34 cells capable of reconstituting immunodeficient mice. Like lentivirus vectors, foamy virus vectors can transduce nondividing cells and can be concentrated by centrifugation. However, wild-type foamy virus has not been associated with human disease. In a very different approach, Dmitry Shayakhmetov (University of Washington, Seattle, WA) and Patricia Yotnda (Baylor College of Medicine, Houston, TX) separately described the development of adenovirus vectors for transient gene transfer into human HSC. This involves the generation of vectors using capsid elements from unconventional serotypes, including Ad35 and Ad11. Both researchers showed the ability of these vectors to transiently transduce human CD34 cells without diminishing viability. Capitalizing on this, Andre Lieber (University of Washington, Seattle, WA) described the development of a hybrid vector system based on deleted adenovirus and adenoassociated virus (ΔAd.AAV). These vectors retain the wide tropism, large payload, and genetic stability of adenoviruses, as well as the ability of AAV to integrate into the target cell genome. When packaged using the Ad35 serotype, these hybrid vectors can stably transduce human cell lines and primary hematopoietic progenitors. In addition to high rates of gene transfer, many potential HSC gene therapy applications require high-level, persistent expression, often in a regulated or tissue-specific fashion. Presentations related to this topic focused on vector biology and various approaches to reducing silencing position effects. It has been known for years that virus elements remaining in recombinant virus vectors can have a significant impact on the expression of transduced genes. In the case of recombinant vectors based on oncoretrovirus vectors, Wolfram Ostertag discussed the role and interaction of elements in the LTR, primer binding site, and sequences surrounding the primer binding site and elsewhere on expression of the transferred gene. Donald Kohn discussed the results of his latest studies into the effects of various sequences in the oncoretrovirus vectors, which serve as targets for silencing. Several presentations also discussed the apparent advantage of lentivirus vectors for achieving high-level, long-term expression. Didier Trono and Luigi Naldini both presented evidence that lentivirus vectors exhibit a very low incidence of silencing following transduction of human CD34 cells and transplantation into immunodeficient mice. However, in all of these cases there were several copies of provirus per transduced cell, making it difficult to directly assess the incidence of provirus silencing. To address this issue more directly, Robert Hawley (American Red Cross, Rockville, MD) derived a series of cell lines and human CD34 cell cultures transduced with single copies of lentivirus vectors. In contrast to the results described above, he found a low but distinct incidence of vector silencing, and a more pronounced variation in the level of expression between various clones. These results were also consistent with studies reported by Punam Malik (Children's Hospital, Los Angeles, CA), in which expression of lentivector provirus was found to be highly variable in mice following secondary bone marrow transplantation. These results suggest that lentivirus vectors may exhibit a lower rate of overt gene silencing, but may still be sensitive to the effects of surrounding chromatin on vector expression. One of the highlights of the meeting was the progress in developing vectors to express globin genes at high levels. These vectors allow the possibility to treat the most common inherited blood diseases, the hemoglobinopathies, by gene therapy. Michel Sadelain (Memorial Sloan-Kettering Cancer Center, New York, NY) described lentivirus vectors containing the human β-globin gene that could cure mice with moderate β-thalassemia and dramatically improve the condition of mice with severe β-thalassemia. Using this vector system it is now possible to measure the hemoglobin in animals in terms of grams of hemoglobin per deciliter relative to the number of virus copies. Robert Pawliuk (Massachusetts Institute of Technology, Cambridge, MA) and Derek Persons presented similar studies using lentivirus vectors containing genes for either an anti-sickling form of β-globin or γ-globin. Expression levels were sufficient to inhibit some of the hemoglobin precipitation and sickling in mouse models of sickle cell disease. One global approach to the prevention of silencing position effects involves the use of chromatin insulators. As expertly summarized by Gary Felsenfeld (NIDDK, Bethesda, MD), these are naturally occurring elements that form boundaries between differentially regulated chromatin loci. There is a wide variety of chromatin insulators using a variety of mechanisms. Recent studies indicate that the ability of such elements to block the interaction between promoters and enhancers is probably distinct from the ability to form a boundary against the encroachment of silencing heterochromatin. One recent model suggests that the HS4 chromatin insulator from the chicken β-globin loci may function to block encroaching heterochromatin by recruiting proteins that acetylate adjoining histones. David Bodine (NHGRI, Bethesda, MD) presented evidence that position effects could be suppressed in transgenic mice by linking the γ-globin gene to a promoter from the erythroid-specific ankyrin gene. Naturally occurring point mutations in the ankyrin promoter associated with ankyrin deficiency caused position effect variegation in transgenic mice, which could be reversed by the addition of the chicken HS4 insulator to the mutant ankyrin/γ-globin construct. The highest levels of γ-globin expression were observed when the promoter from another erythroid-specific gene, Band-3, was linked to the γ-globin gene. Position-independent expression assayed in transgenic mice could again be achieved by flanking these later cassettes with the chicken HS4 insulator. Building on previous studies, David Emery (University of Washington, Seattle, WA) presented evidence that the chicken HS4 element can significantly reduce the incidence of silencing position effects on expression of oncoretrovirus vectors for both reporter genes and γ-globin. However, there are topological constraints on the use of this element, including the distance between the element and target promoter and interaction between internal promoters and the LTR enhancers. Studies presented by Robert Hawley demonstrated that the chicken HS4 element could also reduce the sensitivity of lentivirus vectors to position effects, especially when combined with a scaffold-attachment region (SAR) element from the human β-interferon gene. The incidence of silencing also differed depending on the promoter used. Studies presented by Carolyn Lutzko (Children's Hospital, Los Angeles, CA) also indicated that this SAR element could reduce silencing of both oncoretrovirus and lentivirus vectors following transduction of human CD34 cells and transplantation in immunodeficient mice. Her studies also indicate that this SAR function was dependent on orientation. Christopher Lowrey (Dartmouth Medical School, Hanover, NH) presented evidence that a composite construct containing critical elements from the β-globin locus control region, termed a hypersensitive site forming element, can also reduce the incidence of silencing observed with an oncoretrovirus vector in an erythroid cell line. The meeting also included two workshops designed to discuss issues related to clinical applications of HSC gene therapy. Two main issues emerged from these discussions. The first is the use of non-ablative myelosuppression to enhance the engraftment of transduced HSC in patients who would otherwise not receive such treatment. Ample evidence indicates that the level of engraftment that can be achieved with transduced HSC in the absence of a strong selective pressure or some form of myelosuppression is exceedingly low. The successful clinical trials described by Claudio Bordignon (above) provide the first evidence that mild myelosuppression can be used to enhance the engraftment of transduced HSC in patients without serious adverse effects. During the discussion at least two other clinical HSC gene therapy trials involving the use of mild myelosuppression were revealed. After a long discussion of exactly what constituted “mild” myelosuppression, it was the consensus of the participants that this approach could now be considered in other HSC gene therapy applications on a case-by-case basis. The other main issue discussed at these workshops focused on the availability of recombinant cytokines for HSC gene therapy applications. This includes the use of cytokines as culture supplements to enhance the survival and transduction of HSC, as well as the use of cytokines as agents to mobilize HSC. As discussed by Carolyn Wilson from the Food and Drug Administration, these agents must be produced following current Good Manufacturing Practice (cGPM) standards, because they either are administered directly to patients or are used in the processing of cells that are administered to patients. (Guidance documents for these standards can be found at http://www.fda.gov/cber/reading.htm.) However, in many cases these agents are not commercially available in a pharmaceutical form, so special arrangements must be made to manufacture cGMP-grade lots for initial clinical trials. This can be very expensive, and is often complicated by patent and intellectual property issues, which can restrict the availability of proprietary manufacturing processes and reagents. As introduced by Donald Kohn, there are three general approaches to overcoming these limits: 1) negotiations between the HSC gene therapy research community and the pharmaceutical industry; 2) contract manufacturing; and 3) centralized sources manufactured and/or distributed through the NIH. Two potential candidate resources at the NCI, including the RAID and CTEP programs, already exist. The RAID program (Rapid Access to Intervention Development) is designed to mediate preclinical contract research resources to the academic and small business community, whereas CTEP (Cancer Therapy Evaluation Program) is designed to distribute rare pharmaceutical reagents provided by the pharmaceutical industry for clinical trials. However, both of these programs must still negotiate complicated intellectual property issues with the pharmaceutical companies that hold certain critical patents, potentially limiting their services. (More information on these programs can be found at http://dtp.nci.nih.gov.) Another workshop on this critical topic is being planned for the upcoming meeting of the American Society of Gene Therapy, to be held in Boston on June 5–9, 2002, and interested parties are invited to attend." @default.
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W2319314246 title "The Third Conference on Hematopoietic Stem Cell Gene Therapy: Biology and Technology" @default.
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W2319314246 doi "https://doi.org/10.1006/mthe.2002.0617" @default.
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