Stem cell gene transfer—efficacy and safety in large animal studies

Abstract

The tremendous potential of stem cell-directed gene therapy has recently been demonstrated with the cure of children suffering from X-linked severe combined immunodeficiency (X-SCID) and adenosine deaminase deficiency [1Aiuti A. et al.Correction of ADA-SCID by stem cell gene therapy combined with nonmyeloablative conditioning.Science. 2002; 296: 2410-2413Crossref PubMed Scopus (786) Google Scholar, 2Cavazzana-Calvo M. et al.Gene therapy of human severe combined immunodeficiency (SCID)-X1 disease.Science. 2000; 288: 669-672Crossref PubMed Scopus (1815) Google Scholar, 3Hacein-Bey-Abina S. et al.Sustained correction of X-linked severe combined immunodeficiency by ex vivo gene therapy.N. Engl. J. Med. 2002; 346: 1185-1193Crossref PubMed Scopus (779) Google Scholar]. A new era of medicine appeared to be on the horizon, but enthusiasm was quickly dampened after it became evident that this clinical success had not come without severe adverse events (SAEs). Unfortunately, 2 of 10 children treated with gene therapy developed leukemia [4Hacein-Bey-Abina S. et al.LMO2-associated clonal T cell proliferation in two patients after gene therapy for SCID-X1.Science. 2003; 302: 415-419Crossref PubMed Scopus (2213) Google Scholar, 5Hacein-Bey-Abina S. et al.A serious adverse event after successful gene therapy for X-linked severe combined immunodeficiency.N. Engl. J. Med. 2003; 348: 255-256Crossref PubMed Scopus (1259) Google Scholar]. Thus, the very study that was initially regarded as the breakthrough for stem cell gene therapy is now considered by some as the breakdown for the field. To reach the point where both the great promise and the potential risk are equally apparent has taken over 20 years of developing and refining gene transfer techniques. A major obstacle has been the lack of tissue culture or rodent models predictive for stem cell gene transfer in humans. In this review we discuss the important contributions of large animal studies to the enormous progress in hematopoietic stem cell gene transfer and how large animal models can help to further improve efficiency and safety of stem cell gene therapy. Genetic modification of hematopoietic stem cells (HSCs) has the potential to cure a wide variety of genetic and malignant diseases affecting the hematopoietic system because HSCs provide a lifelong supply of circulating blood cells of all lineages. Genetic diseases such as Fanconi anemia, hemoglobinopathies, and immunodeficiencies, as well as acquired diseases such as AIDS and cancer, could potentially be treated by stem cell gene therapy. Although most of these diseases are curable by allogeneic stem cell transplantation, many patients lack a suitable donor, and the side effects of allogeneic stem cell transplantation such as graft-versus-host-disease make the transplantation of genetically modified autologous cells an attractive alternative. Furthermore,recent studies suggest that gene therapy may also enhance allogeneic stem cell transplantation [6Neff T. et al.Methylguanine methyltransferase-mediated in vivo selection and chemoprotection of allogeneic stem cells in a large-animal model.J. Clin. Invest. 2003; 112: 1581-1588Crossref PubMed Scopus (88) Google Scholar, 7Bank A. Hematopoietic stem cell gene therapy: selecting only the best.J. Clin. Invest. 2003; 112: 1478-1480Crossref PubMed Scopus (0) Google Scholar]. Hematopoietic stem cells have been targeted for genetic modification for over 2 decades. In the 1980s it was shown that mouse hematopoietic stem cells could be genetically modified with a degree of efficiency predicted to be therapeutic for many human diseases [8Joyner A. Keller G. Phillips R.A. Bernstein A. Retrovirus transfer of a bacterial gene into mouse haematopoietic progenitor cells.Nature. 1983; 305: 556-558Crossref PubMed Google Scholar, 9Williams D.A. Lemischka I.R. Nathan D.G. Mulligan R.C. Introduction of new genetic material into pluripotent haematopoietic stem cells of the mouse.Nature. 1984; 310: 476-480Crossref PubMed Scopus (0) Google Scholar]. These results prompted a high level of enthusiasm until it became apparent that the transducibility of hematopoietic stem cells is just another feature to distinguish mice from men. Because stem cell transduction protocols developed in the mouse did not translate into efficient gene transfer protocols in human studies, many investigators started to study stem cell transduction in dogs [10Kwok W.W. Storb R. 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These initial difficulties in achieving high-level stem cell transduction demonstrated that results from large animal models were much more predictive of human stem cell gene therapy studies than mouse studies and suggested that these models might be more appropriate assays for the optimization of clinical gene therapy protocols. Two technical advances have substantially increased the information gained from large animal studies, namely the development of fluorescent marker proteins and the use of competitive repopulation assays: The competitive repopulation assay allows for efficient evaluation of different gene transfer conditions within one animal and reduces the otherwise substantial interanimal variability in outbred large animals. The cells to be used for transplantation are split into two or more equal aliquots, which are transduced under different conditions using distinguishable vectors. After transduction, all cells are pooled and reinfused into the autologous host, where gene marking derived from the different experimental arms is monitored in the marrow and/or peripheral blood after engraftment (Fig. 1A). A drawback of this assay is that it cannot distinguish between the efficiency of gene transfer to stem cells during ex vivo transduction and the subsequent engraftment of transduced stem cells. Early [10Kwok W.W. Storb R. Miller A.D. 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Fluorescent proteins also allow for easy purification of live transduced populations by FACS. Furthermore, they allow for the assessment of gene expression in nonnucleated cells such as circulating red blood cells and platelets. Since large animal models are expensive and very labor intensive, investigators have developed surrogate assays such as xenotransplantation assays to study human candidate stem cells [38McCune J.M. Namikawa R. Kaneshima H. Shultz L.D. Lieberman M. Weissman I.L. The SCID-hu mouse: murine model for the analysis of human hematolymphoid differentiation and function.Science. 1988; 241: 1632-1639Crossref PubMed Google Scholar, 39Nolta J.A. Hanley M.B. Kohn D.B. Sustained human hematopoiesis in immunodeficient mice by cotransplantation of marrow stroma expressing human interleukin-3: analysis of gene transduction of long-lived progenitors.Blood. 1994; 83: 3041-3051Crossref PubMed Google Scholar, 40Zanjani E.D. Pallavicini M.G. Harrison M.R. Tavassoli M. 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In addition, integration site analysis revealed common proviral integrants in SRCs and in the baboon at 6 weeks but not at 6 months after transplantation [[44]Horn P.A. Thomasson B.M. Wood B.L. Andrews R.G. Morris J.C. Kiem H.-P. Distinct hematopoietic stem/progenitor cell populations are responsible for repopulating NOD/SCID mice compared with nonhuman primates.Blood. 2003; 102: 4329-4335Crossref PubMed Scopus (0) Google Scholar]. Thus, while the NOD/SCID model has provided useful data for the transduction of early human progenitors available data suggest that the NOD/SCID model assays short-term rather than long-term repopulating cells. Studies in the NOD/SCID model may therefore not translate directly into clinical stem cell gene therapy trials and studies in large animals and in NOD/SCID mice should probably be viewed as complementary approaches to optimize gene transfer to true multilineage long-term repopulating stem cells. The majority of clinical gene therapy trials and preclinical gene transfer studies have utilized oncoretroviral vectors based on the Moloney murine leukemia virus (MoMLV) to transfer genes stably to hematopoietic stem cells. One of the major disadvantages of oncoretroviral vectors is their need for target cell division for transduction [45Miller D.G. Adam M.A. Miller A.D. Gene transfer by retrovirus vectors occurs only in cells that are actively replicating at the time of infection.Mol. Cell. Biol. 1990; 10: 4239-4242Crossref PubMed Google Scholar, 46Lewis P.F. Emerman M. Passage through mitosis is required for oncoretroviruses but not for the human immunodeficiency virus.J. Virol. 1994; 68: 510-516Crossref PubMed Google Scholar, 47Andreadis S. Fuller A.O. Palsson B.O. Cell cycle dependence of retroviral transduction: an issue of overlapping time scales.Biotechnol. Bioeng. 1998; 58: 272-281Crossref PubMed Scopus (0) Google Scholar, 48Roe T. Reynolds T.C. Yu G. Brown P.O. 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Biotechnol. 1999; 10: 448-453Crossref PubMed Scopus (26) Google Scholar]) was therefore thought to overcome the most significant obstacle to stem cell gene therapy, the need to induce cycling of stem cells to allow transduction. Stable transduction of nondividing human hematopoietic cells by HIV-1-based lentiviral vectors has been reported [51Uchida N. et al.HIV, but not murine leukemia virus, vectors mediate high efficiency gene transfer into freshly isolated G0/G1 human hematopoietic stem cells.Proc. Natl. Acad. Sci. USA. 1998; 95: 11939-11944Crossref PubMed Scopus (240) Google Scholar, 52Case S.S. et al.Stable transduction of quiescent CD34(+)CD38(−) human hematopoietic cells by HIV-1-based lentiviral vectors.Proc. Natl. Acad. Sci. USA. 1999; 96: 2988-2993Crossref PubMed Scopus (0) Google Scholar]; however, whether lentiviral vectors are capable of transducing truly quiescent, metabolically inactive cells, is still a matter of debate [[53]Emerman M. Learning from lentiviruses.Nat. Genet. 2000; 24: 8-9Crossref PubMed Scopus (0) Google Scholar]. Enthusiasm for lentiviral vectors rose when highly efficient gene transfer into human NOD/SCID repopulating cells using lentiviral vectors was reported [54Miyoshi H. Smith K.A. Mosier D.E. Verma I.M. Torbett B.E. Transduction of human CD34+ cells that mediate long-term engraftment of NOD/SCID mice by HIV vectors.Science. 1999; 283: 682-686Crossref PubMed Scopus (522) Google Scholar, 55Woods N.B. et al.Lentiviral gene transfer into primary and secondary NOD/SCID repopulating cells.Blood. 2000; 96: 3725-3733PubMed Google Scholar]. Surprisingly, results from two different laboratories using lentiviral vectors in primates suggest that they are in fact inferior to oncoretroviral vectors with respect to gene transfer to long-term repopulating cells [56An D.S. et al.Marking and gene expression by a lentivirus vector in transplanted human and nonhuman primate CD34(+) cells.J. 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In addition, studies in the dog model using HIV-1-based vectors have shown relatively efficient gene transfer after an overnight transduction culture with in vivo gene marking levels of up to 12% [[64]Horn P.A. et al.Efficient lentiviral gene transfer to canine repopulating cells using an overnight transduction protocol.Blood. 2004; 103: 3710-3716Crossref PubMed Scopus (56) Google Scholar]. In summary, species-specific effects impact on the effectiveness of lentiviral vectors in large animal studies and it appears that clinical trials will be necessary to determine definitively the success of these vectors in stem cell gene therapy. Of note, inborn antiviral defense mechanisms against HIV have been described in human cells [[65]Turelli P. et al.Cytoplasmic recruitment of INI1 and PML on incoming HIV preintegration complexes: interference with early steps of viral replication.Mol. Cell. 2001; 7: 1245-1254Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar]. 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Lentiviral vectors were first described in 1996 [[49]Naldini L. et al.In vivo gene delivery and stable transduction of nondividing cells by a lentiviral vector.Science. 1996; 272: 263-267Crossref PubMed Google Scholar], and only now are we beginning to see reliable and efficient marking in canine and nonhuman primate transplantation studies [36Hanawa H. et al.Efficient gene transfer into rhesus repopulating hematopoietic stem cells using a simian immunodeficiency virus-based lentiviral vector system.Blood. 2004; 103: 4062-4069Crossref PubMed Scopus (124) Google Scholar, 64Horn P.A. et al.Efficient lentiviral gene transfer to canine repopulating cells using an overnight transduction protocol.Blood. 2004; 103: 3710-3716Crossref PubMed Scopus (56) Google Scholar]. Available preclinical data therefore suggest that lentiviral vectors will be an efficient means for clinical gene transfer to hematopoietic stem cells. 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Cell cycle requirements for transduction by foamy virus vectors compared to those of oncovirus and lentivirus vectors.J. Virol. 2004; 78: 2327-2335Crossref PubMed Scopus (80) Google Scholar]. Foamy virus vectors have been shown to transduce human NOD/SCID repopulating cells efficiently [43Josephson N.C. et al.Transduction of human NOD/SCID-repopulating cells with both lymphoid and myeloid potential by foamy virus vectors.Proc. Natl. Acad. Sci. USA. 2002; 99: 8295-830