Gene Therapy for Hemophilia: A Step Closer to Reality
Authors: Gilbert C. White, Harold R. Roberts
Journal: Molecular Therapy (2000)

Hemophilia B is an X-linked disorder caused by a defect or deficiency of coagulation factor IX. The disease is characterized by recurrent, spontaneous hemorrhage in joints, soft tissue, and, less commonly, internal organs. Current treatment of hemophilia B is with intravenous plasma-derived or recombinant forms of replacement factor. Although these treatments can be effective in the treatment of acute hemorrhagic episodes and in prophylaxis, especially for young patients, exogenous factor IX administration is not a cure, as it does not provide long-term, continuous levels of the coagulation factor. For this reason, gene therapy is considered an attractive approach to the treatment of hemophilia. Two recent publications, one in Nature Genetics by Kay et al. (1Kay M.A. Manno C.S. Ragni M.V. Larson P.J. Couto L.B. McClelland A. Glader B. Chew A.J. Tai S.J. Herzog R.W. Arruda V. Johnson F. Scallan C. Skarsgard E. Flake A.W. High K.A. Evidence for gene transfer and expression of factor IX in haemophilia B patients treated with an AAV vector.Nat. Genet. 2000; 24: 257-261Crossref PubMed Scopus (878) Google Scholar) and one in Molecular Therapy by Wang et al. (2Wang L. Nichols T.C. Read M.S. Bellinger D.A. Verma I.M. Sustained expression of therapeutic level of factor IX in hemophilia B dogs by AAV-mediated gene therapy in liver.Mol. Ther. 2000; 1: 154-158Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar), bring curative gene therapy for hemophilia a step closer to reality. Kay et al. describe early results from treatment of three patients with severe hemophilia B (due to missense mutations) with intramuscular injections of adeno-associated virus (AAV) vector carrying the gene encoding human factor IX under control of the CMV promoter (1Kay M.A. Manno C.S. Ragni M.V. Larson P.J. Couto L.B. McClelland A. Glader B. Chew A.J. Tai S.J. Herzog R.W. Arruda V. Johnson F. Scallan C. Skarsgard E. Flake A.W. High K.A. Evidence for gene transfer and expression of factor IX in haemophilia B patients treated with an AAV vector.Nat. Genet. 2000; 24: 257-261Crossref PubMed Scopus (878) Google Scholar). These are the first three patients enrolled in a phase I, dose-escalation trial and all three patients received the lowest dose of vector, 2 × 1011 vector genomes per kilogram of body weight. All three patients demonstrate gene transfer and expression by Southern blot on DNA extracted from muscle biopsies and by RT-PCR. One of the three had a small but reproducible increase in baseline factor IX levels and a reduction in exogenous factor IX requirement. It is of more than passing interest that this patient has a qualitative abnormality of factor IX, with baseline factor IX antigen levels of 24%. Although the authors suggest that saturation of extravascular binding sites in this patient by circulating endogenous factor IX antigen may result in higher levels of donated gene product, it is also possible that gene therapy somehow caused derepression of the endogenous factor IX gene, resulting in higher levels of the severely dysfunctional molecule. Measurement of factor IX antigen levels after gene therapy would have been interesting. A second patient demonstrated a reduction in factor requirement despite levels of factor IX that were persistently <1%. It is important to note that, as a phase I trial, the study reports no toxicities, although more data to document this are needed. A significant concern going into gene therapy of hemophilia is the development of immunological responses to the transduced factor IX. Exogenous factor IX administered intravenously is processed primarily through MHC class II mechanisms. There has been a concern that transduced factor IX will be processed through MHC class I mechanisms and that this may lead to the formation of inhibitory antibodies in patients who were previously tolerant to exogenous factor IX. It is encouraging that no such antibodies have been encountered in the three patients reported. Another concern is germline transmission, but despite low-level vector signals in serum, saliva, and urine 24 h after injection serial semen samples have been persistently negative for vector sequences. The work by Wang et al. (2Wang L. Nichols T.C. Read M.S. Bellinger D.A. Verma I.M. Sustained expression of therapeutic level of factor IX in hemophilia B dogs by AAV-mediated gene therapy in liver.Mol. Ther. 2000; 1: 154-158Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar) presages the next significant advance in the use of AAV as a vector: the achievement of higher expression levels. Using an AAV vector encoding canine factor IX cDNA under the control of a synthetic liver-specific promoter, the authors describe long-term expression of factor IX after a single intraportal injection in two dogs with hemophilia B. In dogs receiving 5 × 1012 particles, low-level expression was observed for up to a year. In dogs receiving an AAV vector encoding canine factor IX cDNA under the control of the same liver-specific promoter and a woodchuck hepatitis posttranscriptional element in a dose of 8.7 × 1013 particles, expression has been achieved for over 6 months up to an average of 218 ng/ml (5% of normal levels). This level is over three times the levels of factor IX observed in previous studies with AAV, which translated into humans might provide normal factor IX levels—an exciting prospect, indeed. Other gene therapy studies in hemophilia are currently under way. In the first clinical trial of gene therapy in human hemophilia, Transkaryotic Therapies, Inc. (TKT) has used a nonviral ex vivo approach in severe hemophilia A (factor VIII deficiency). Dermal skin fibroblasts obtained by biopsy from patients and electroporated ex vivo with a B-domain deleted factor VIII expression plasmid have been laparoscopically injected intraperitoneally. A total of 6 patients have been treated in this study, which is now over a year in progress. In another phase I, single-dose, dose-escalation, peripheral vein injection study sponsored by Chiron Corp. using a replication-deficient, human complement-resistant Moloney-based retrovirus vector containing the B-domain deleted form of factor VIII, a total of 10 patients with severe hemophilia A have been enrolled at four vector doses. Both trials are farther along than the Kay et al. trial, but results are not yet published on either study. However, there are no preliminary reports of toxicity, in the form of either antibodies or other complications. One might ask, amid all of the controversy surrounding the gene therapy trials at the University of Pennsylvania and the reports of complications in other trials (3Weiss R. Nelson D. Teen dies undergoing experimental gene therapy.The Washington Post. 1999; Google Scholar, 4Lasalandra M. Deaths of patients cloud experiments in gene therapy.The Boston Herald. 1999; Google Scholar, 5Adams C. NIH not told of bad events in gene trials.The Wall Street Journal. 2000; Google Scholar), if this is the right time for a report as preliminary as that of Kay et al. and would it not be better to complete the study prior to publication? The factor IX levels that are being reported are low, the observation period is short, and the toxicity studies are incomplete. Without doubt, it is certainly the right time for the initiation of these trials. The trial by Kay et al. and the other clinical trials of gene therapy in hemophilia were preceded by extensive preclinical trials in transgenic mice and hemophilic dogs, two different and highly suitable animal models (6Snyder R.O. Miao C.H. Patijn G.A. Spratt S.K. Danos O. Nagy D. Gown A.M. Winther B. Meuse L. Cohen L.K. Thompson A.R. Kay M.A. Persistent and therapeutic concentrations of human factor IX in mice after hepatic gene transfer of recombinant AAV vectors.Nat. Genet. 1997; 16: 270-276Crossref PubMed Scopus (497) Google Scholar, 7Monahan P.E. Samulski R.J. Tazelaar J. Xiao X. Nichols T.C. Bellinger D.A. Read M.S. Walsh C.E. Direct intramuscular injection with recombinant AAV vectors results in sustained expression in a dog model of hemophilia.Gene Ther. 1998; 5: 40-49Crossref PubMed Scopus (177) Google Scholar, 8Nakai H. Herzog R.W. Hagstrom J.N. Walter J. Kung S.H. Yang E.Y. Tai S.J. Iwaki Y. Kurtzman G.J. Fisher K.J. Colosi P. Couto L.B. High K.A. Adeno-associated viral vector-mediated gene transfer of human blood coagulation factor IX into mouse liver.Blood. 1998; 91: 4600-4607Crossref PubMed Google Scholar, 9Wang L. Takabe K. Bidlingmaier S.M. Ill C.R. Verma I.M. Sustained correction of bleeding disorder in hemophilia B mice by gene therapy.Proc. Natl. Acad. Sci. USA. 1999; 96: 3906-3910Crossref PubMed Scopus (197) Google Scholar, 10Herzog R.W. Yang E.Y. Couto L.B. Hagstrom J.N. Elwell D. Fields P.A. Burton M. Bellinger D.A. Read M.S. Brinkhous K.M. Podsakoff G.M. Nichols T.C. Kurtzman G.J. High K.A. Long-term correction of canine hemophilia B by gene transfer of blood coagulation factor IX mediated by adeno-associated viral vector.Nat. Med. 1999; 5: 56-63Crossref PubMed Scopus (468) Google Scholar, 11Chao H. Samulski R. Bellinger D. Monahan P. Nichols T. Walsh C. Persistent expression of canine factor IX in hemophilia B canines.Gene Ther. 1999; 6: 1695-1704Crossref PubMed Scopus (90) Google Scholar, 12Snyder R.O. Miao C. Meuse L. Tubb J. Donahue B.A. Lin H.F. Stafford D.W. Patel S. Thompson A.R. Nichols T. Read M.S. Bellinger D.A. Brinkhous K.M. Kay M.A. Correction of hemophilia B in canine and murine models using recombinant adeno-associated viral vectors.Nat. Med. 1999; 5: 64-70Crossref PubMed Scopus (316) Google Scholar). The trials were properly scrutinized by the Food and Drug Administration and by the Recombinant DNA Advisory Committee and were strongly supported by the hemophilia community. Perhaps it is precisely because of the current controversy that the trial by Kay et al. needs reporting even in its preliminary state. Reaction to the unfortunate death of Jesse Gelsinger has resulted in an environment where clinical trials such as the TKT trial are suspended despite the absence of complications (13Saltus R. Beth Israel halts trial of gene therapy, cites national debate over safety.The Boston Globe. 2000; Google Scholar). Clearly, oversight of gene therapy clinical trials must be improved, but it is important that clinical trials with gene therapy proceed. The potential of this form of treatment for genetic disorders like hemophilia remains very great. Because of the current environment, the gene therapy field needs positive findings, and the observations of Kay et al. are positive and thus provide a strong argument for continued clinical trials in hemophilia. In closing, these reports should be taken in the historical perspective of those who have worked in the field for many decades. Kenneth M. Brinkhous, a vital collaborator of both the Kay and Verma labs in preclinical studies, entered the hemophilia field in the 1930s when there was no treatment for the disease. He first characterized factor VIII, so-called antihemophilic factor (AHF), as a component of plasma necessary for the conversion of prothrombin to thrombin, developed the first purified plasma-derived concentrates of factor VIII, identified canine models of hemophilia that have been essential to the development of gene therapy, and contributed to the development of recombinant forms of factor VIII. His response to the papers by Kay et al. and Wang et al.: “It is not often that an individual lives to see the whole spectrum of a disease, from biochemical characterization, to molecular definition, to correction of the disease. I am gratified to see the day when a permanent cure of hemophilia is at hand.” So are we!