Globin Gene Therapy: One (Seemingly) Small Vector Change, One Giant Leap in Optimism
Authors: David Bodine
Journal: Molecular Therapy (2000)

In the July 6, 2000 issue of Nature, May et al. report the successful generation of a lentiviral vector containing a human β-globin gene that can be used to rescue a mouse model of β-thalassemia (1May C. et al.Therapeutic haemoglobin synthesis in beta-thalassaemic mice expressing lentivirus-encoded human beta-globin.Nature. 2000; 406: 82-86Crossref PubMed Scopus (496) Google Scholar). This is a significant achievement for the research team lead by Michel Sadelain and brings to an end more than a decade of frustration regarding a critical step in the development of gene therapy for hemoglobin disorders. Hemoglobinopathies are relatively common disorders, allowing gene therapy to now be proposed for a large number of patients. In the late 1980s, hemoglobinopathies were considered prime candidates for gene addition therapy. The genes were in hand, the molecular defects were understood, and several important features of globin gene regulation were known. Importantly, analyses of patients indicated that synthesis of 15–20% of the normal amount of β-globin could result in a substantial clinical improvement. Initial studies demonstrated that human β-globin genes could be inserted into oncoretroviral vectors and transduced into the hematopoietic stem cells (HSC) where they were expressed in differentiated red blood cells at low levels (2Dzierzak E.A. Papayannopoulou Th. Mulligan R.C. Lineage-specific expression of a human beta-globin gene in murine bone marrow transplant recipients reconstituted with retrovirus-transduced stem cells.Nature. 2000; 331: 35-41Crossref Scopus (230) Google Scholar, 3Bodine D.M. Karlsson S. Nienhuis A.W. Combination of interleukins 3 and 6 preserves stem cell function in culture and enhances retrovirus-mediated gene transfer into hematopoietic stem cells.Proc. Natl. Acad. Sci. USA. 2000; 86: 8897-8901Crossref Scopus (244) Google Scholar, 4Bender M.A. Gelinas R.E. Miller A.D. A majority of mice show long-term expression of a human beta-globin gene after retrovirus transfer into hematopoietic stem cells.Mol. Cell. Biol. 2000; 9: 1426-1434Crossref Scopus (119) Google Scholar). At the same time, the β-globin locus control region (LCR), a 25-kb sequence containing DNase I hypersensitive sites located ~60 kb upstream of the adult β-globin gene, was shown to be required for high-level expression of β-globin genes in transgenic mice (5van Grosveld F. Assendelft G.B. Greaves D.R. Kollias G. Position-independent, high-level expression of the human beta-globin gene in transgenic mice.Cell. 1987; 51: 975-985Abstract Full Text PDF PubMed Scopus (1442) Google Scholar). The critical regions of the LCR were rapidly identified, and in this optimistic environment it seemed that the simple addition of small LCR sequences to the globin retroviral vector would allow high-level expression of a transduced globin gene. Unfortunately, the next decade was spent demonstrating that retroviral vectors containing globin genes and LCR sequences were not able to introduce unrearranged copies of the transgene to target cells at high frequency (6Novak U. Harris E.A. Forrester W. Groudine M. Gelinas R. High-level beta-globin expression after retroviral transfer of locus activation region-containing human beta-globin gene derivatives into murine erythroleukemia cells.Proc. Natl. Acad. Sci. USA. 1990; 87: 3386-3390Crossref PubMed Scopus (105) Google Scholar). A variety of approaches were taken to solve this problem by a group of dedicated investigators. Removal of part of the second intervening sequence (IVS2) of the β-globin gene improved the stability of LCR/globin retrovirus vectors (7Sadelain M. Wang C.H. Antoniou M. Grosveld F. Mulligan R.C. Generation of a high-titer retroviral vector capable of expressing high levels of the human beta-globin gene.Proc. Natl. Acad. Sci. USA. 1995; 92: 6728-6732Crossref PubMed Scopus (182) Google Scholar, 8Li Q. Emery D.W. Fernandez M. Han H. Stamatoyannopoulos G. Development of viral vectors for gene therapy of beta-chain hemoglobinopathies: Optimization of a gamma-globin gene expression cassette.Blood. 1999>; 93: 2208-2216PubMed Google Scholar, 9Ren S. Wong B.Y. Li J. Luo X.N. Wong P.M. Atweh G.F. Production of genetically stable high-titer retroviral vectors that carry a human gamma-globin gene under the control of the alpha-globin locus control region.Blood. 1996; 87: 2518-2524PubMed Google Scholar). Specific contexts of globin genes and LCR fragments also contributed to more stable retrovirus vectors (7Sadelain M. Wang C.H. Antoniou M. Grosveld F. Mulligan R.C. Generation of a high-titer retroviral vector capable of expressing high levels of the human beta-globin gene.Proc. Natl. Acad. Sci. USA. 1995; 92: 6728-6732Crossref PubMed Scopus (182) Google Scholar). Leboulch et al. hypothesized that processing of the genomic LCR/globin retroviral RNA at cryptic sites was responsible for internal deletions of the globin gene. Mutagenesis of many of these sites improved the stability of LCR/globin retrovirus vectors (10Leboulch P. et al.Mutagenesis of retroviral vectors transducing human beta-globin gene and beta-globin locus control region derivatives results in stable transmission of an active transcriptional structure.EMBO J. 1994; 13: 3065-3076Crossref PubMed Scopus (156) Google Scholar). These improvements were sufficient to introduce unrearranged LCR/globin cassettes into mouse HSC, only to reveal a second problem. As had been observed in transgenic mice, the expression of the transferred globin genes with small LCR sequences varied depending on the position of integration in the target cell genome (position effects) (7Sadelain M. Wang C.H. Antoniou M. Grosveld F. Mulligan R.C. Generation of a high-titer retroviral vector capable of expressing high levels of the human beta-globin gene.Proc. Natl. Acad. Sci. USA. 1995; 92: 6728-6732Crossref PubMed Scopus (182) Google Scholar, 11Plavec I. Papayannopoulou Th. Maury C. Meyer F. A human beta-globin gene fused to the human beta-globin locus control region is expressed at high levels in erythroid cells of mice engrafted with retrovirus-transduced hematopoietic stem cells.Blood. 1993; 81: 1384-1392PubMed Google Scholar, 12Raftopoulos H. Ward M. Leboulch P. Bank A. Long-term transfer and expression of the human beta-globin gene in a mouse transplant model.Blood. 1997; 90: 3414-3422PubMed Google Scholar). In addition, silencing of LCR/globin transgenes expressed from retrovirus vectors was frequently observed (7Sadelain M. Wang C.H. Antoniou M. Grosveld F. Mulligan R.C. Generation of a high-titer retroviral vector capable of expressing high levels of the human beta-globin gene.Proc. Natl. Acad. Sci. USA. 1995; 92: 6728-6732Crossref PubMed Scopus (182) Google Scholar). Although frustrating to those observing the field, much of what was learned from these studies led to the recent success. The development and characterization of lentivirus vectors for gene transfer (13Naldini L. et al.In vivo gene delivery and stable transduction of nondividing cells by a lentiviral vector.Science. 1996; 272: 263-267Crossref PubMed Scopus (4034) Google Scholar) offered the break that the field needed, and Sadelain and co-workers took advantage of the opportunity. Sadelain recognized that one of the functions of the HIV rev protein, which is expressed during the packaging of lentivirus vectors, was to suppress splicing of the viral RNA genome. Sadelain hypothesized that the presence of rev would prevent splicing of the LCR/globin cassette. Furthermore, lentivirus vectors allow the packaging of larger constructs than oncoretroviral vectors. Sadelain hypothesized that larger LCR fragments would be more resistant to position effects as they appeared to be in transgenic mice. Finally, Sadelain noted that silencing of expression from lentivirus vectors over time was much less frequent than with comparable oncoretrovirus vectors. The research team led by Chad May linked a β-globin gene with the IVS2 sequence deletion to larger fragments of the LCR and inserted this construct into a lentivirus vector. VSV-G pseudotyped lentivirus vectors were generated and used to transduce HSC from both normal mice and mice heterozygous for the thalassemia-inducing knockout of the β-globin genes. After transduction, the HSC were transplanted into recipient mice whose endogenous marrow had been destroyed by radiation. The results described in the Nature paper are nothing short of outstanding. In mice repopulated with normal cells exposed to the virus, human β-globin mRNA was present at levels that were nearly 15% of the level of the endogenous mouse β-globin mRNA after adjustment for the ratio of marked to unmarked cells. This value was consistent from mouse to mouse, suggesting that no significant position effects were encountered and that few of the vectors were silenced. Hemoglobin tetramers consisting of two mouse α-globin chains and two human β-globin chains were easily detectable in red blood cells. In β-thalassemic mice repopulated with autologous cells exposed to the vector, the results were even more striking. Due to the reduced number of mouse β-globin chains per cell, mouse α2/human β2 tetramers represented about 25% of the hemoglobin molecules. This additional hemoglobin caused significant increases in the number of red blood cells and the amount of hemoglobin per red blood cell in the treated animals. While not entirely cured, the mouse model of β-thalassemia was clearly and significantly improved by gene therapy. The findings by May et al. (1May C. et al.Therapeutic haemoglobin synthesis in beta-thalassaemic mice expressing lentivirus-encoded human beta-globin.Nature. 2000; 406: 82-86Crossref PubMed Scopus (496) Google Scholar) have given a badly needed shot in the arm to globin gene therapy research and should energize the field for years to come. These findings have made gene therapy for thalassemia a realistic goal that will be aggressively pursued. As discussed in the manuscript, several problems remain to be addressed. In vivo selection for corrected red blood cells is not likely to be as powerful as the selection for corrected lymphocytes demonstrated in the recent successful cure of X-linked SCID (14Cavazzana-Calvo M. et al.Gene therapy of human severe combined immunodeficiency (SCID)-X1 disease.Science. 2000; 288: 669-672Crossref PubMed Scopus (2253) Google Scholar). Therefore, for gene therapy to have a realistic chance at improving a human hemoglobinopathy, gene transfer into human HSC must become more efficient than in previous human stem cell marking trials. One idea is to use a dominant selectable marker gene such as dihydrofolate reductase in the vector and in vivo selection after repopulation with methotrexate (15Allay J.A. Sorrentino B.P. In vivo selection of retrovirally transduced hematopoietic stem cells.Nat. Med. 1998; 4: 1136-1143Crossref PubMed Scopus (180) Google Scholar). This concept has been successfully demonstrated in mouse models and could be combined into the Sadelain vector design. Second, the degree and method of bone marrow ablation needed to give globin gene marked stem cells a competitive advantage over their endogenous counterparts must be determined. The mouse models may be excellent vehicles to determine how much marrow ablation and how many corrected cells are required for an effective cure. Finally, it would be even better if even more β-like globin could be expressed in red blood cells. For example, if a γ-globin gene could be expressed to approximately 25% of the level of the sickle β-globin mRNA, gene therapy could be extended to sickle cell disease. Through the dedication of Michel Sadelain and his research team, optimism about globin gene therapy has returned. It is sincerely hoped that frustration of the past 12 years will be replaced with success during the next decade.