Safe Passage: A Plea for Safety in Hemophilia Gene Therapy
Authors: Margaret V. Ragni
Journal: Molecular Therapy (2002)

Hemophilia has attracted widespread interest as a model for gene therapy. An X-linked bleeding disorder caused by deficient or defective factor VIII (F.VIII) or factor IX (F.IX) and characterized by bleeding into joints (hemarthroses) and muscles (hematomas), hemophilia is considered to be an ideal target for gene transfer for several reasons. First, the disease is monogenic: the severity of the disease is directly related to the level of clotting F.VIII or F.IX, encoded on the long arm of the X chromosome [1Hoyer L.W. Hemophilia A.N. Engl. J. Med. 1994; 330: 38-47Crossref PubMed Scopus (369) Google Scholar]. Successful gene therapy treatment in hemophilia is defined as prevention of hemorrhage and is thus straightforward. Second, a precise level of F.VIII or F.IX is not crucial. As long as at least 1–5% F.VIII or F.IX clotting activity is achieved [1Hoyer L.W. Hemophilia A.N. Engl. J. Med. 1994; 330: 38-47Crossref PubMed Scopus (369) Google Scholar, 2Mannucci P.M. Tuddenheim E.G.D. Medical progress: the hemophilias—from royal genes to gene therapy.N. Engl. J. Med. 2001; 344: 1773-1779Crossref PubMed Scopus (810) Google Scholar, 3Lofqvist T. Nilsson I.M. Berntorp E. Pettersson H. Hemophilia prophylaxis in young patients—a long term follow up.J. Int. Med. 1997; 241: 395-400Crossref PubMed Scopus (295) Google Scholar, 4Leisner R.J. Khair K. Hann I.M. The impact of prophylactic treatment on children with severe hemophilia.Br. J. Hematol. 1996; 92: 973-978Crossref PubMed Scopus (167) Google Scholar], spontaneous bleeding, which leads to most of the complications of the disease, will be prevented. Third, the precise site of F.VIII and F.IX production is not crucial: as long as the F.VIII or F.IX protein gains access to the circulation, hemostasis will be achieved. Fourth, clotting F.VIII and F.IX protein levels are easily quantitated by standard clotting assays [1Hoyer L.W. Hemophilia A.N. Engl. J. Med. 1994; 330: 38-47Crossref PubMed Scopus (369) Google Scholar]. Fifth, achieving adequate circulating levels of F.VIII or F.IX has been shown to be effective in the treatment and prevention of symptoms of the disease. Thus, hemophilia has become a powerful disease model for gene therapists. With the initiation of phase I clinical trials in hemophilia gene transfer, it is timely to consider the risks and benefits of gene transfer, as well as the risks and benefits associated with past milestones in hemophilia treatment. From plasma to recombinant factors, with each new technological advance, there have been major setbacks. Consider the toll that AIDS [5Ragni M.V. AIDS retrovirus antibodies in hemophiliacs treated with factor VIII or factor IX concentrates, cryoprecipitate, or fresh frozen plasma: prevalence, seroconversion rate, and clinical correlations.Blood. 1986; 67: 592-595PubMed Google Scholar], hepatitis C [6Ragni M.V. Belle S.H. Impact of human immunodeficiency virus (HIV) on progression to end-stage liver disease in individuals with hemophilia and hepatitis C.J. Infect. Dis. 2001; 183: 1112-1115Crossref PubMed Scopus (188) Google Scholar], inhibitor formation [7Ragni M.V. Hord J.D. Blatt J. Central venous catheter infection in hemophiliacs undergoing prophylaxis or immune tolerance.Haemophilia. 1997; 3: 90-95Crossref PubMed Scopus (29) Google Scholar], systemic infection [8Journeycake J.M. Quinn C.T. Miller K.L. Zajac J.L. Buchanan G.R. Catheter-related deep venous thrombosis in children with hemophilia.Blood. 2001; 98: 1727-1731Crossref PubMed Scopus (106) Google Scholar], and thrombosis [9Ehrenforth S. Incidence of development of FVIII and factor IX inhibitors in hemophiliacs.Lancet. 1992; 339: 594-598Abstract PubMed Scopus (519) Google Scholar] have taken on this population, the potential threat of new transmissible agents [10Evatt B. Creutzfeldt-Jakob disease and haemophilia: assessment of risk.Haemophilia. 2000; 6: 94-99Crossref PubMed Scopus (10) Google Scholar], the ever-increasing high cost of product [11Lipton R.A. The economics of factor VIII inhibitor treatment.Semin. Hematol. 1994; 31: 37-38PubMed Google Scholar, 12Kessler C.M. Aledort L.M. The economics of immune tolerance and treatment of hemophiliacs with inhibitors.Vox Sang. 1996; 70: 74-76Crossref PubMed Google Scholar], and the unfortunate and unpredictable product shortages [13Hemophilia (Minimal) Data Set. Federally Funded Hemophilia Treatment Centers, National Summary, March 29, 1999Google Scholar, 14Soucie J.M. Jackson D. Evatt B. Jackson D. Occurrence of hemophilia in the United States.Am. J. Hematol. 1998; 59: 288-294Crossref PubMed Scopus (286) Google Scholar]. In fact, what is unique to hemophilia is that the complications of hemophilia treatment have been far worse than the disease itself, greatly increasing the morbidity and mortality of the disease [15Soucie J.M. Mortality among males with hemophilia: relations with source of medical care.Blood. 2000; 96: 437-442PubMed Google Scholar]. A major concern, therefore, in achieving successful gene therapy for hemophilia is safety. It is the purpose of this brief commentary to review the complications of past hemophilia treatments and to consider the potential risks of gene therapies for hemophilia, with emphasis on the need for safety. Safe passage on the long-awaited journey to achieving a “cure” by gene therapy for hemophilia has been a goal of the National Hemophilia Foundation (NHF), its membership, physicians, consumers, and scientists [16Thompson A.R. Gene therapies for the hemophilias.Mol. Ther. 2000; 2: 5-8Abstract Full Text Full Text PDF PubMed Scopus (4) Google Scholar, 17Kelley K.C. An introduction to gene therapy.Haemophilia. 2000; 6: 110-114Crossref PubMed Scopus (3) Google Scholar, 18National Hemophilia Foundation Website, Gene transfer clinical trials in hemophilia, http://www.hemophilia.org/bdi/bdi_gene.htm.Google Scholar], and the basis for recommendations by the NHF Medical and Scientific Advisory Council and the Gene Therapy Working Group [19Recommendations for Conducting Gene Therapy Clinical Trials in Persons with Bleeding Disorders, MASAC Recommendation #120. The Medical and Scientific Advisory Council (MASAC) of the National Hemophilia Foundation (NHF), August 8, 2001, http://www.hemophilia.org/programs/masc/masac120.htm.Google Scholar]. Historically, hemophilia is a story of progress associated with severe setbacks. In the 1950s, the first breakthrough in treatment was the use of whole blood, which effected a several percent increase of factor levels. Its use was, however, complicated by allergic reactions, volume overload, and hepatitis. The introduction in 1964 of cryoprecipitate, a cold precipitate of plasma with a more concentrated level of F.VIII, followed by the availability of clotting factor concentrates in the 1970s, resulted in the achievement of effective clotting factor levels and effective treatment and prevention of bleeding symptoms, but at the expense of inhibitor formation, HIV, hepatitis A, B, and C, and chronic hepatitis in the majority of patients. With the introduction of recombinant technology in the 1990s, an unprecedented treatment safety was realized, with freedom from HIV and hepatitis. Yet even with these advances, the use of animal tissue cultures in the manufacture of recombinant factor has raised concerns, albeit theoretic, about the potential for slow virus infection (for example, transmissible spongiform encephalopathies (TSEs)) or other unknown pathogens. Thus, the hemophilia community, which now has the tools to accomplish early, safe, and effective prevention and treatment of hemorrhages, is, unfortunately, left with a myriad of complications of treatment. Thus, the evolution of clotting factor treatment for the hemophilias has been associated with medical complications more disabling than the disease itself, two of which, AIDS and hepatitis, are the leading cause of death in this population [15Soucie J.M. Mortality among males with hemophilia: relations with source of medical care.Blood. 2000; 96: 437-442PubMed Google Scholar]. The development of neutralizing antibodies to infused F.VIII occurs in up to 15–25% of individuals with hemophilia [7Ragni M.V. Hord J.D. Blatt J. Central venous catheter infection in hemophiliacs undergoing prophylaxis or immune tolerance.Haemophilia. 1997; 3: 90-95Crossref PubMed Scopus (29) Google Scholar]. The presence of these alloantibodies is associated with poor response to standard treatment and often severe bleeding. Bleeding complications are higher than in those without inhibitors, and treatment remains difficult, complex, and costly. Standard treatment with clotting factor is not effective because the inhibitor blocks its effect, so alternative approaches to treatment must be used, potentially thrombogenic active F.IX products or costly factor recombinant factor F.VIIa products [1Hoyer L.W. Hemophilia A.N. Engl. J. Med. 1994; 330: 38-47Crossref PubMed Scopus (369) Google Scholar, 2Mannucci P.M. Tuddenheim E.G.D. Medical progress: the hemophilias—from royal genes to gene therapy.N. Engl. J. Med. 2001; 344: 1773-1779Crossref PubMed Scopus (810) Google Scholar]. Although early studies of drugs to effect an immune blockade (that is, CTLA4 Ig and anti-CD40 ligand) were promising in animals, trials in humans have been hampered by drug toxicities. Chronic hepatitis C virus infection affects 80% of adults with hemophilia, and chronic liver disease is the second leading cause of death in this population [6Ragni M.V. Belle S.H. Impact of human immunodeficiency virus (HIV) on progression to end-stage liver disease in individuals with hemophilia and hepatitis C.J. Infect. Dis. 2001; 183: 1112-1115Crossref PubMed Scopus (188) Google Scholar, 15Soucie J.M. Mortality among males with hemophilia: relations with source of medical care.Blood. 2000; 96: 437-442PubMed Google Scholar]. Now after 20–30 years of chronic hepatitis C, as many as 20% or more may have cirrhosis [6Ragni M.V. Belle S.H. Impact of human immunodeficiency virus (HIV) on progression to end-stage liver disease in individuals with hemophilia and hepatitis C.J. Infect. Dis. 2001; 183: 1112-1115Crossref PubMed Scopus (188) Google Scholar]. Yet treatment is suboptimal, with few active drugs identified that lead to durable HCV suppression, and few treatment options once cirrhosis and fibrosis occur [20Poynard T. Bedossa P. Opoion P. Natural history of liver fibrosis progression in patients with chronic hepatitis C.Lancet. 1997; 349: 825-832Abstract Full Text Full Text PDF PubMed Scopus (2800) Google Scholar]. The presence of blood rich in proteases in the joint with each recurrent bleed destroys the lining synovial tissue and leads to chronic arthropathy. Worsening disability and pain lead to joint replacement, often in the third or fourth decade of life [1Hoyer L.W. Hemophilia A.N. Engl. J. Med. 1994; 330: 38-47Crossref PubMed Scopus (369) Google Scholar, 2Mannucci P.M. Tuddenheim E.G.D. Medical progress: the hemophilias—from royal genes to gene therapy.N. Engl. J. Med. 2001; 344: 1773-1779Crossref PubMed Scopus (810) Google Scholar]. This plight has led to the use of prophylaxis, that is, the prevention of hemorrhages through prophylactic factor infusion at least 3–5 times weekly. By maintaining a level of Γ 1% [3Lofqvist T. Nilsson I.M. Berntorp E. Pettersson H. Hemophilia prophylaxis in young patients—a long term follow up.J. Int. Med. 1997; 241: 395-400Crossref PubMed Scopus (295) Google Scholar, 4Leisner R.J. Khair K. Hann I.M. The impact of prophylactic treatment on children with severe hemophilia.Br. J. Hematol. 1996; 92: 973-978Crossref PubMed Scopus (167) Google Scholar], spontaneous bleeding may be avoided and joint damage averted. The use of every other day treatment has been very effective in prevention of joint bleeding, but at a cost, both financial and medical. With prophylaxis, there is as much as a fivefold increase in factor use over standard “reactive” treatment approach, that is, every other day treatment comes to 15 times per month rather than the standard four or five per month, leading to higher cost [11Lipton R.A. The economics of factor VIII inhibitor treatment.Semin. Hematol. 1994; 31: 37-38PubMed Google Scholar, 12Kessler C.M. Aledort L.M. The economics of immune tolerance and treatment of hemophiliacs with inhibitors.Vox Sang. 1996; 70: 74-76Crossref PubMed Google Scholar]. Further, the use of central lines is often required for venous access, especially among children, and these devices are associated with significant morbidity, primarily infection. Studies have found that over half of the children using long-term central venous catheters or ports develop central line infection [7Ragni M.V. Hord J.D. Blatt J. Central venous catheter infection in hemophiliacs undergoing prophylaxis or immune tolerance.Haemophilia. 1997; 3: 90-95Crossref PubMed Scopus (29) Google Scholar] and thrombosis [8Journeycake J.M. Quinn C.T. Miller K.L. Zajac J.L. Buchanan G.R. Catheter-related deep venous thrombosis in children with hemophilia.Blood. 2001; 98: 1727-1731Crossref PubMed Scopus (106) Google Scholar]. The long-term consequences of these vascular problems are not yet known. Although not known to be transmitted through blood or blood products, there is a potential risk to hemophiliacs receiving blood products of exposure to slow virus agents. Such agents include Creutzfeldt-Jakob disease (CJD), new variant CJD and other forms of TSEs (that is, mad cow disease), and other prion diseases, which are caused by cell proteins called prions [10Evatt B. Creutzfeldt-Jakob disease and haemophilia: assessment of risk.Haemophilia. 2000; 6: 94-99Crossref PubMed Scopus (10) Google Scholar]. So far, there have been no cases of blood-borne transmission of slow viruses identified in the United States or Europe. However, for those reliant on blood product treatment, this remains a potential concern [10Evatt B. Creutzfeldt-Jakob disease and haemophilia: assessment of risk.Haemophilia. 2000; 6: 94-99Crossref PubMed Scopus (10) Google Scholar]. TABLE 1Gene transfer in hemophiliaThe Challenge of Gene Transfer in Hemophilia Avoid disabling complications of innovative therapiesComplications of Treatment of Hemophilia Inhibitor formation Chronic arthropathy Central venous catheter infection, thrombosis Hepatitis and ESLD AIDS, HIV infection Cost ShortagesPotential Complications of Hemophilia Gene Therapy Inflammation Hepatotoxicity Inhibitor formation Germline transmission Insertional mutagenesis ThrombosisThe Ideal Gene Transfer for Hemophilia Nonimmunogenic Nontoxic Noninfectious Affordable Safe at all ages Effective Single, noninvasive dose Open table in a new tab Over 40% of adults with hemophilia have evidence of HIV infection, most now with advanced disease 20 years after initial infection [5Ragni M.V. AIDS retrovirus antibodies in hemophiliacs treated with factor VIII or factor IX concentrates, cryoprecipitate, or fresh frozen plasma: prevalence, seroconversion rate, and clinical correlations.Blood. 1986; 67: 592-595PubMed Google Scholar]. Despite reduction of AIDS-related morbidity and mortality with potent highly active antiretroviral therapy (HAART), drug toxicity is not uncommon. Despite these advances, AIDS remains the leading cause of death among individuals with hemophilia [6Ragni M.V. Belle S.H. Impact of human immunodeficiency virus (HIV) on progression to end-stage liver disease in individuals with hemophilia and hepatitis C.J. Infect. Dis. 2001; 183: 1112-1115Crossref PubMed Scopus (188) Google Scholar, 15Soucie J.M. Mortality among males with hemophilia: relations with source of medical care.Blood. 2000; 96: 437-442PubMed Google Scholar]. With the advances afforded by HAART treatment, many hemophiliacs are now surviving AIDS, only to die of end-stage liver disease (ESLD) [6Ragni M.V. Belle S.H. Impact of human immunodeficiency virus (HIV) on progression to end-stage liver disease in individuals with hemophilia and hepatitis C.J. Infect. Dis. 2001; 183: 1112-1115Crossref PubMed Scopus (188) Google Scholar]. In those with chronic hepatitis C infection, HIV infection has been associated with a three- to fourfold increase in the risk of progression to ESLD in those with hemophilia [6Ragni M.V. Belle S.H. Impact of human immunodeficiency virus (HIV) on progression to end-stage liver disease in individuals with hemophilia and hepatitis C.J. Infect. Dis. 2001; 183: 1112-1115Crossref PubMed Scopus (188) Google Scholar]. Unfortunately, antiviral treatment of hepatitis C is limited by lack of tissue histology in most, due to avoidance of liver biopsy in this population and exclusion of this population from participation in clinical trials. Although HAART may improve AIDS survival, treatment of HCV co-infection may be poorly tolerated because of complicating drug interactions and worsening liver function and fibrosis [20Poynard T. Bedossa P. Opoion P. Natural history of liver fibrosis progression in patients with chronic hepatitis C.Lancet. 1997; 349: 825-832Abstract Full Text Full Text PDF PubMed Scopus (2800) Google Scholar]. Among those who progress to ESLD, transplantation may not be possible, however, as AIDS is considered an absolute contraindication [21Gordon, R. D., van Thiel, D. H., and Starzl, T. E.1993. Liver transplantation. In Diseases of the Liver, 7th edition L. Schiff and E. R. Schiff, Eds., pp. 1218. J.B. Lippincott Co. Philadelphia.Google Scholar] to transplantation. This is because survival following liver transplantation was dismal before the availability of antiretroviral therapy [22Ragni M.V. Bontempo F.A. Lewis J.H. Organ transplantation in HIVpositive patients with hemophilia.N. Engl. J. Med. 1990; 322: 1886-1887PubMed Google Scholar]. Preliminary data from those undergoing the procedure with postoperative antiretroviral therapy [23Ragni M.V. Dodson S.F. Hunt S. Bontempo F.A. Fung J.J. Liver transplantation in a hemophilia patient with AIDS and hepatitis C.Blood. 1999; 93: 1113-1114PubMed Google Scholar] suggest that survival in those with AIDS is similar to that in HIV-negative individuals with ESLD [24Ragni M.V. Early mortality among HIV+ liver transplant recipients. 2002Google Scholar]. Moreover, it should be pointed out that liver transplantation does cure hemophilia [25Bontempo F.A. Liver transplantation in hemophilia A.Blood. 1987; 69: 1721-1724Crossref PubMed Google Scholar], further validating hemophilia as a model for gene therapy. Crucial to effective intervention of these potential complications is early recognition. Yet this is often not possible, given the many medical problems individuals with hemophilia may experience, the common exclusion from clinical trials of new drugs, and the paucity of specialists in some geographic areas. Further, treatment of the many complications of hemophilia, for example AIDS and hepatitis, has been associated with increasing morbidity associated with adverse drug reactions [26Sulkowski M.S. Thomas D.L. Chaisson R.E. Moore R.D. Hepatotoxicity associated with antiretoviral therapy in adults infected with human immunodeficiency virus and the role of hepatitis C or B virus infection.JAMA. 2000; 283: 74-80Crossref PubMed Scopus (899) Google Scholar], including lactic acidosis due to antiretroviral therapy, hemolytic anemia with ribavirin, and myalgias and depression with interferon. In addition to transmissible infection, invasiveness, and inconvenience of a treatment given intravenously 3–4 times monthly as treatment or 3–4 times weekly as prevention [3Lofqvist T. Nilsson I.M. Berntorp E. Pettersson H. Hemophilia prophylaxis in young patients—a long term follow up.J. Int. Med. 1997; 241: 395-400Crossref PubMed Scopus (295) Google Scholar, 4Leisner R.J. Khair K. Hann I.M. The impact of prophylactic treatment on children with severe hemophilia.Br. J. Hematol. 1996; 92: 973-978Crossref PubMed Scopus (167) Google Scholar], two other disadvantages of current clotting factor treatment affect those at greatest risk. First, clotting factor treatment is expensive. A 70-kilogram male who uses 4000 units 3–4 times monthly for treatment of spontaneous bleeds requires between 50,000 and 100,000 units per year, which, at $1 per unit, is $50,000 to $100,000 per year [11Lipton R.A. The economics of factor VIII inhibitor treatment.Semin. Hematol. 1994; 31: 37-38PubMed Google Scholar, 12Kessler C.M. Aledort L.M. The economics of immune tolerance and treatment of hemophiliacs with inhibitors.Vox Sang. 1996; 70: 74-76Crossref PubMed Google Scholar]; this is unaffordable by the uninsured, by those on assistance, and by 80% of the world's hemophilia population [2Mannucci P.M. Tuddenheim E.G.D. Medical progress: the hemophilias—from royal genes to gene therapy.N. Engl. J. Med. 2001; 344: 1773-1779Crossref PubMed Scopus (810) Google Scholar]. A second disadvantage of current protein treatment is the unpredictable nature of factor shortages. For example, during the last two years there was close to a 50% decline in available clotting factor as a result of various regulatory setbacks, including pharmaceutical deficiencies, delaying approval of new factor products, and production delays and production slowdowns of older products in anticipation of new product availability [13Hemophilia (Minimal) Data Set. Federally Funded Hemophilia Treatment Centers, National Summary, March 29, 1999Google Scholar, 14Soucie J.M. Jackson D. Evatt B. Jackson D. Occurrence of hemophilia in the United States.Am. J. Hematol. 1998; 59: 288-294Crossref PubMed Scopus (286) Google Scholar]. The latter led to nationwide rationing of product, fewer treatments, cessation of prophylaxis for some, use of plasma-derived products by some, and an increase in potentially preventable “breakthrough” bleeds [13Hemophilia (Minimal) Data Set. Federally Funded Hemophilia Treatment Centers, National Summary, March 29, 1999Google Scholar, 14Soucie J.M. Jackson D. Evatt B. Jackson D. Occurrence of hemophilia in the United States.Am. J. Hematol. 1998; 59: 288-294Crossref PubMed Scopus (286) Google Scholar]. In times of shortage, with less product available, a patient previously on prophylaxis (that is, 3–4 times weekly treatment to prevent bleeds) may experience bleeds otherwise prevented. These bleeds thereby “break through” treatment, when the factor level falls below 1% and spontaneous bleeds, previously prevented by prophylaxis, occur. Progress in hemophilia has come at a high price; thus, in considering gene transfer for hemophilia, there is concern regarding yet more potential complications of new therapeutics in this population. Whether the F.VIII or F.IX gene is delivered by a retroviral vector [27Powell J.S. Results from one year followup of a phase 1 trial of FVIII gene transfer for severe hemophilia A using a retroviral construct administered by peripheral intravenous infusion.Blood. 2001; 98: 535aGoogle Scholar], adeno-associated viral (AAV) vector [28Kay M.A. Evidence for gene transfer and expression of factor IX in haemophilia B patients treated with an AAV vector.Nature. 2000; 24: 257-261Google Scholar], lentiviral vector [29Kafri T. Blomer U. Peterson D.A. Gage F.H. Verma I.M. Sustained expression of genes delivered directly into liver and muscle by lentiviral vectors.Nat. Genet. 1997; 17: 314-317Crossref PubMed Scopus (548) Google Scholar], gutted adenoviral vector [30Zhang W. Development and application of a minimal-adenoviral vector system for gene therapy of hemophilia A.Thromb. Hemostas. 1999; 82: 562-571PubMed Google Scholar], naked DNA plasmid [31Roth D.A. Tawa N.E. O'Brien J.M. Treco D.A. Selden R.F. Nonviral transfer of the gene encoding coagulation factor VIII in patients with severe hemophilia A.N. Engl. J. Med. 2001; 344: 1735-1742Crossref PubMed Scopus (316) Google Scholar], or other technology, the goal of hemophilia gene therapy is not only to achieve a 5% F.VIII or F.IX level in the bloodstream [3Lofqvist T. Nilsson I.M. Berntorp E. Pettersson H. Hemophilia prophylaxis in young patients—a long term follow up.J. Int. Med. 1997; 241: 395-400Crossref PubMed Scopus (295) Google Scholar, 4Leisner R.J. Khair K. Hann I.M. The impact of prophylactic treatment on children with severe hemophilia.Br. J. Hematol. 1996; 92: 973-978Crossref PubMed Scopus (167) Google Scholar] to eliminate spontaneous bleeding (for example, central nervous system bleeding and disabling recurrent joint bleeding), but also to achieve it safely. Among the potential risks associated with gene transfer are inflammation, hepatotoxicity, germline transmission, insertional mutagenesis, and either inadequate or excessive transgene expression. An inflammatory response to the vector or gene infused is a potential risk with any new transgene. If a severe inflammatory response occurs, symptoms may include fever, abdominal pain, hepatitis, and liver or kidney dysfunction. Potential concern would be if this occurred in a patient with underlying hepatitis or past alcoholism [6Ragni M.V. Belle S.H. Impact of human immunodeficiency virus (HIV) on progression to end-stage liver disease in individuals with hemophilia and hepatitis C.J. Infect. Dis. 2001; 183: 1112-1115Crossref PubMed Scopus (188) Google Scholar]. The prior evaluation of the gene vector in animal models, initiation of phase I trials at doses lower than a comparable safe dose in animal models, and careful, methodic monitoring during and after vector administration should help to minimize this risk [32High K.A. AAV-mediated gene transfer for hemophilia.Ann. NY Acad. Sci. 2001; 953: 64-74Crossref PubMed Scopus (39) Google Scholar]. One potential adverse event that might occur with gene transfer, especially gene transfer directed to the liver, is liver damage. Among hemophiliacs with chronic hepatitis C from past blood product exposures [6Ragni M.V. Belle S.H. Impact of human immunodeficiency virus (HIV) on progression to end-stage liver disease in individuals with hemophilia and hepatitis C.J. Infect. Dis. 2001; 183: 1112-1115Crossref PubMed Scopus (188) Google Scholar], there is risk for potential hepatotoxicity or for preexisting hepatitis to adversely affect the efficiency of gene transfer to the hepatocyte [32High K.A. AAV-mediated gene transfer for hemophilia.Ann. NY Acad. Sci. 2001; 953: 64-74Crossref PubMed Scopus (39) Google Scholar]. Studies in animal models of liver injury [33Ezure T. The development and compensation of biliary cirrhosis in interleukin-6 deficient mice.Am. J. Pathol. 2000; 156: 1627-1639Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar] suggest that a single “hit” to the liver, such as with hepatitis C, results in transient inflammation followed by repair, a regulated process. However, if a second “hit,” such as HIV [6Ragni M.V. Belle S.H. Impact of human immunodeficiency virus (HIV) on progression to end-stage liver disease in individuals with hemophilia and hepatitis C.J. Infect. Dis. 2001; 183: 1112-1115Crossref PubMed Scopus (188) Google Scholar] or a gene vector, is sustained, the inflammatory and repair process might become unregulated and potentially accelerate progression of liver disease [33Ezure T. The development and compensation of biliary cirrhosis in interleukin-6 deficient mice.Am. J. Pathol. 2000; 156: 1627-1639Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar]. The potential risk of hepatotoxicity with liver-based gene therapy has prompted the implementation of liver biopsy at baseline, before gene transfer, with close monitoring of liver function tests [32High K.A. AAV-mediated gene transfer for hemophilia.Ann. NY Acad. Sci. 2001; 953: 64-74Crossref PubMed Scopus (39) Google Scholar]. It is possible that neutralizing antibodies may develop after exposure to the F.VIII or F.IX produced with gene transfer. Hemophilia gene therapy in humans has not yet led to inhibitor formation, but it is anticipated, given that neutralizing antibodies have developed to every new protein factor treatment modality thus far. One potential approach to minimize this risk is to limit gene transfer to only those with over 10–20 previous exposures to factor protein, the median time to development of inhibitors in previously untreated patients [1Hoyer L.W. Hemophilia A.N. Engl. J. Med. 1994; 330: 38-47Crossref PubMed Scopus (369) Google Scholar, 34Addiego J. Frequency of inhibitor development in haemophiliacs treated with low-purity factor VIII.Lancet. 1993; 342: 462-464Abstract PubMed Scopus (161) Google Scholar]. Whether gene therapy approaches that require repeat infusions of the vector will sensitize subjects and induce neutralizing antibodies remains unknown. Unfortunately, treatment of inhibitors remains complex, difficult, and costly [11Lipton R.A. The economics of factor VIII inhibitor treatment.Semin. Hematol. 1994; 31: 37-38PubMed Google Scholar, 12Kessler C.M. Aledort L.M. The economics of immune tolerance and treatment of hemophiliacs with inhibitors.Vox Sang. 1996; 70: 74-76Crossref PubMed Google Scholar]. One potential adverse consequence of gene therapy is germline transmission, that is, insertion of the gene into the germline cells (sperm cells). Gene transfer may potentially allow the transmission of genetic material to future offspring. The concern is that the transferred DNA could disrupt normal genes and result in serious birth defects or even fetal death, or cause long-term effects such as cancer. It has been estimated, however, that the frequency of spontaneous mutation in the human genome, 1 in 109 per nucleotide per year, or one genomic insertion in every 50–100 individuals, is far greater than the estimated rate of exogenous gene insertion, one in 6000 sperm [35Kazazian H.H. An estimated frequency of endogenous insertional mutations in humans.Nat. Genet. 1999; 22: 130Crossref PubMed Scopus (128) Google Scholar]. The true frequency of exogenous germline transmission associated with hemophilia gene transfer remains unknown; thus, young men with hemophilia who choose to undergo gene transfer must seriously consider sperm banking to avoid these potential risks. The use of double barrier contraception has also been recommended for protection against germline transmission, and as a protection against the theoretical risk that vector in semen could transform somatic cells in sexual partners [32High K.A. AAV-mediated gene transfer for hemophilia.Ann. NY Acad. Sci. 2001; 953: 64-74Crossref PubMed Scopus (39) Google Scholar]. Another potential risk of gene therapy relates to the theoretical risk that the gene vector could either activate an oncogene due to the placement of a strong promoter, or inactivate a tumor-suppressor gene and result in malignancy [32High K.A. AAV-mediated gene transfer for hemophilia.Ann. NY Acad. Sci. 2001; 953: 64-74Crossref PubMed Scopus (39) Google Scholar]. It is clear that long-term follow-up of a large number of gene transfer recipients will be required to determine the risk of insertional mutagenesis in hemophiliacs as well as other groups at risk. Previous studies of mortality [36Rosendaal F.R. Mortality and course of death in Dutch hemophiliacs, 1973-86.Br. J. Haematol. 1989; 71: 71-76Crossref PubMed Scopus (183) Google Scholar, 37Triemstra M. Mortality in patients with hemophilia.Ann. Int. Med. 1995; 123: 823-827Crossref PubMed Scopus (157) Google Scholar] have documented the low frequency of deaths from myocardial infarction and coronary disease in those with hemophilia. Atherosclerotic plaques do form in the arteries of hemophiliacs, as demonstrated at autopsy (M.V.R., unpublished data). Yet, given that elevated F.VIII levels are a risk factor for cardiovascular disease [38Rumley A. Factor VIII, von Willebrand factor and the risk of major ischaemic heart disease in the Caerphilly Heart Study.Br. J. Haematol. 1999; 105: 110-116Crossref PubMed Scopus (163) Google Scholar], it is unknown whether increasing the F.VIII or F.IX levels above 5% or higher could promote formation of a fibrin clot after plaque-induced vascular endothelial damage and platelet activation, and result in formation of a coronary thrombus and symptomatic disease. The latter is hypothetical and there is no evidence that gene transfer may lead to greater cardiovascular morbidity among hemophiliacs by increasing factor levels. However, these potential risks emphasize the critical importance of careful monitoring of individuals with hemophilia for signs of cardiovascular disease during long-term post-gene transfer follow-up. With the possibility that several potential gene therapy approaches for hemophilia may be developed, it may be possible to choose the least potentially problematic therapy. For example, those with advanced HIV disease might avoid retroviral vector-based gene transfer, as antiretroviral therapy could potentially destroy the vector. Those with advanced ESLD might consider avoiding liver-based vector therapies. Those at high risk for inhibitor formation, that is, those with familial history of inhibitors and African Americans [1Hoyer L.W. Hemophilia A.N. Engl. J. Med. 1994; 330: 38-47Crossref PubMed Scopus (369) Google Scholar, 34Addiego J. Frequency of inhibitor development in haemophiliacs treated with low-purity factor VIII.Lancet. 1993; 342: 462-464Abstract PubMed Scopus (161) Google Scholar, 39Ragni M.V. Inhibitor formation in African-Americans with hemophilia A.Blood. 1999; 94: 642aPubMed Google Scholar], might select gene transfer techniques that do not involve re-dosing. Children who undergo gene transfer might avoid treatment until after 20 or more exposures [1Hoyer L.W. Hemophilia A.N. Engl. J. Med. 1994; 330: 38-47Crossref PubMed Scopus (369) Google Scholar, 32High K.A. AAV-mediated gene transfer for hemophilia.Ann. NY Acad. Sci. 2001; 953: 64-74Crossref PubMed Scopus (39) Google Scholar] to avoid inhibitor formation. Affordability will be another issue for consideration for all potential subjects, including those who are uninsured or under-insured: whatever the cost, gene transfer will likely be less costly in a lifetime than years of high-cost, protein-based therapies. The availability of insurance coverage and risk of potential disclosure of genetic information also loom ahead. Finally, it will be essential to determine how to balance safety and efficacy considerations in clinical trials, such that a minimum of subjects receive low dose vector in which efficacy is considered unlikely, while at the same time sufficient clinical information is generated to define the minimum effective dose to durably and reliably prevent spontaneous bleeding in this population. The interested reader is directed to the RAC web site for a safety discussion of vectors in human clinical trials [40ORDA Website. (Fourth National Gene Transfer Safety Symposium: Safety considerations in the use of AAV vectors in gene transfer clinical trials, March 27, 2001, http://www.4.od.nih.gov/oba/rac/transcript3-7-01.pdf., First National Gene Transfer Safety Symposium: Internally deleted, helper-dependent adenoviral vectors, http://www.4.od.nih.gov/oba/rac/3-8-00EXCERPT.pdf.Google Scholar]. Notwithstanding the above issues, first and foremost safety is and must continue to be the major consideration for the introduction of new gene transfer techniques in the hemophilia population. The ideal gene transfer therapy should be nonimmunogenic, nontoxic, noninfectious, affordable, and effective in a single, noninvasive dose. It is hoped that effective gene transfer technology will lead to phenotypic cure of the disorder within the next 5 to 10 years. Yet, whatever gene technologies are developed, the major challenge for those with hemophilia will be to avoid disabling complications, such as those that occurred with past innovative therapies.