- Email: [email protected]
Gene Therapies for the Hemophilias
COMMENTARY
doi:10.1006/mthe.2000.0099, available online at http://www.idealibrary.com on IDEAL
Gene Therapies for the Hemophilias Arthur R. Thompson Puget Sound Blood Center and University of Washington, 921 Terry Avenue, Seattle, Washington 98104 Fax: 206-292-8030. E-mail: [email protected].
INTRODUCTION These are exciting times for those working toward a genetic cure for patients with hemophilia. Recent publicity surrounding the initial results of a phase I clinical trial of adeno-associated viral (AAV)-mediated delivery of the factor IX gene to skeletal muscle of patients with hemophilia B (1) is indeed gratifying. However, they only represent a slit-lamp view of the vast array of ongoing research progress in this field. To this end, a Third National Hemophilia Foundation Workshop on Gene Therapies for Hemophilia was held in Washington, DC, on March 24–25, 2000, and featured 40 invited speakers. This meeting was cosponsored by the National Institutes of Health, other governmental agencies, and representatives of industry (see //www.hemophilia.org under gene therapy). The talks focused on recombinant factors VIII and IX, hemophilic animal models, viral and nonviral vectors, immunologic consequences and immune responses to vectors and expressed factors, three current gene transfer clinical trials, and hepatitis as a prevalent complication from prior concentrate therapy and the potential for antihepatitis viral gene therapy. The following is a brief review that highlights the scope of issues covered; where references to specific data may be unclear from lack of a citation, a few of the presenters are identified by name. The entire program can be requested from the above website.
ANIMAL MODELS
OF
HEMOPHILIAS A
AND
B
Gene knockout mice and colonies of naturally occurring hemophilic dogs have provided the opportunity to examine the efficacy of factors VIII or IX gene therapy. They have also served as a valuable resource not only to examine the immune response to the expressed protein from the recombinant transgene, but also to provide insights into mechanisms involved in the frequent formation of alloantibodies in patients with hemophilia. Potential mutations to produce a “gain in function” included a factor VIII with covalent linkage of the A2 to A3 domains (referred to as an inactivation-resistant factor VIII or “IR8”) that is more stable in vitro (2). The altered protein was hemostatic with a shorter half-life in MOLECULAR THERAPY Vol. 2, No. 1, July 2000 Copyright The American Society of Gene Therapy
dogs with severe hemophilia A, as anticipated from reduced binding to von Willebrand factor. For hemophilia B, mutagenesis of sites of posttranslational modification of recombinant factor IX failed to improve its recovery in mice (R. G. Schaub, Boston, MA). As a model for alloimmune response, dogs with severe hemophilia A were multiply transfused with canine cryoprecipitate. Several assays were compared to determine their sensitivity D. Lillicrap, Kingston, ON, Canada). Contrary to a recent report, canine factor IX in the Chapel Hill colony (due to a missense mutation) accounts for severe hemophilia B without detectable factor IX antigen in affected animals; one commercial antiserum preparation gave false-positive Western blots (T. C. Nichols, Chapel Hill, NC). Gene therapy to primates has been impeded by a lack of deficient models and the difficulty in distinguishing the native primate factor from a highly homologous human clotting factor sequence. Nevertheless, primates can provide important information on dosing and are useful to screen for potential toxicities of therapeutic constructs. To address the issue of dose responses, Robert R. Montgomery (Medical College of Wisconsin, Milwaukee, WI) presented a novel assay capable of distinguishing even trace amounts of human factor VIII in macaque plasma. His laboratory utilized a species-specific monoclonal anti-human factor VIII antibody for capture and a coupled chromogenic factor X-activating assay to assess functional cofactor activity of the captured protein. Thus, the efficacy of human factor VIII gene transfer can be assessed in a primate background without requiring deficient animals.
VIRAL
AND
NONVIRAL VECTORS
A critical issue in gene therapy in general is to develop safe and robust vector systems. Several presentations focused on advances in vectors to improve efficiency of factor VIII or factor IX gene transfer into cells. Traditionally these have been of viral origin, derived from AAV, retroviral, including lentiviral, or adenoviral
5
COMMENTARY subtypes. Nonviral approaches may provide potentially safer alternatives and although highly inefficient in the past, several advances suggest that they may be approaching viral vectors in transfer efficiency. A final approach reviewed is direct gene repair of point mutations. AAV vectors, as used in the hemophilia B clinical trial (1), bind to cell-surface receptors with a heparan sulfate proteoglycan. R. Jude Samulski (University of North Carolina at Chapel Hill) added that receptor density correlated with transduction rates of different cells within tissues. Helper-free preparations were created with plasmids to provide helper gene functions and chromatographic concentration (3) removed expressed transgene protein. Similar measures are being incorporated into large-scale AAV vector production. Delayed expression from AAV vector transgenes can occur without secondstrand synthesis as complementary single strands can anneal. Using a small promoter, it has been possible to express the large B domain-deleted factor VIII cDNA in mice and expression was demonstrated in both hepatocytes and sinusoidal epithelial cells (4). Head-to-tail concatemers provide a strategy for AAV constructs with larger cDNAs (such as factor VIII) where an intron is inserted between the 3′ end of one and the 5′ end of a second vector and heterodimers with the proper orientation can express from the entire coding region. Traditional retroviral vectors have safer packaging cell lines and growth factors and tissue-specific promoters are being investigated (K. P. Ponder, St. Louis, MO). Engraftment of human marrow stromal cells in SCID factor VIII-deficient (knockout) mice provided expression of factor VIII but this was only transient due to promoter inactivation (5). Inder Verma (Salk Institute, La Jolla, CA) showed that with lentiviral variants, factor VIII knockout mice had prolonged expression with phenotypic correction. Regulatory sequences accounted in part for downregulation of expression following transduction. Intraperitoneal injections resulted in hepatic gene transfer in factor VIII gene knockout mice and caused less morbidity and mortality than the surgical preparation for portal vein infusions. Third-generation lentiviral vectors are being constructed to use plasmids and remove nonessential viral genes (6) and these are being transferred to preparative scale procedures to improve safety. Partial hepatectomy stimulates cell cycling and, at least in hepatocytes, enhances lentiviral vector transduction (7). In a separate presentation, a technique referred to as DNA “shuffling” (8) was proposed as potentially applicable to improve vector and/or transgene sequences. Adenoviral vectors expressed human or canine factor VIII in a canine hemophilic model and liver expression in mice was enhanced by an albumin promoter (9). However, Sheila Connelly (Genetic Therapy, Inc., Gaithersburg, MD) reported that these vectors produced dose-dependent, biphasic hepatotoxicity in mice, even after targeted disruption of E1, E2a, and E3 adenoviral genes. Similar toxicity was observed with adenoviral vectors in primates (10).
6
There remains concern that persons whose livers harbor hepatitis C would be at high risk of sustained hepatotoxicity. Adenoviral vectors with no detectable viral gene expression should be safer. Reactions to the helperproduced adenoviral capsid proteins may still occur. Minimal, transient liver toxicity occurred in one mouse; this was not observed after 3 1012 particles per kilogram of “gutless” adenoviral particles were infused into dogs (11). Relevant to the development of such “mini” adenoviral vectors, a construct with the large dystrophin gene with muscle-specific promoters (12) expressed sufficient dystrophin for 4 months in affected mice to partially restore muscle function. Although previously plagued by low transduction efficiencies and in vitro transfection, nonviral approaches to gene therapy in vivo have been successful in achieving prolonged expression of therapeutic levels of a variety of proteins. One approach is to use electroporation applied to muscles of SCID mice to enhance injected plasmid transfer of human factor IX cDNA. Muscle cells were able to express factor IX for several months (13). In dogs with severe hemophilia B, expression of human factor IX was transient due to an immune response. For species-specific transgenes, this approach should allow repeat procedures without an immunogenic viral vector. Furthermore, the size of the transgene is not limited as it is in AAV or retroviral vectors. A second approach is to use transposon sequences that integrate into a limited number of insertion sites. Transposons have successfully transferred human factor IX into immune-competent mice and therapeutic levels of human factor IX were observed in these animals, persisting for 1 year (14). Furthermore, repeat administrations were successful. Gene repair by hybrid RNA/DNA oligonucleotides has provided successful conversion in the majority of genes tested in cell culture, although the percentage of cells converted is often low. Phenotypic success with this approach was reported as in vivo repair of a frameshift mutation in the UDP-glucuronosyltransferase gene in the Gunn rat (15). According to R. Michael Blaese (Kimeragen, Newtown, PA), increased size of the oligonucleotides, having the correct sequence only on the side with the full DNA sequence, and use of RNA hairpin turns improved efficiency. More compact DNA hairpin structures were also found to facilitate conversion (16). Although there are multiple point mutations in hemophilic families and this approach could not correct a common, recurrent factor VIII gene inversion, repair would avoid potential complications from random integration.
IMMUNOLOGIC CONSEQUENCES
AND IMMUNE
RESPONSES
Alloimmune inhibitors occur in 10–30% of patients with severe hemophilia A. A major concern for gene therapy of hemophilia is whether the expressed recombinant protein would be even more immunogenic. Gene transfer to naive patients could evoke the same or even highMOLECULAR THERAPY Vol. 2, No. 1, July 2000 Copyright The American Society of Gene Therapy
COMMENTARY er frequencies of immunity to an expressed, B domaindeleted protein and the cells or tissues used for expression may influence the response. Alternatively, cellular expression might be the equivalent of a continuous infusion that could promote tolerance. Although usually seen in severe patients, there are several more recent reports of high-titer inhibitors occurring in patients with mild hemophilia A. The latter frequently occur after major trauma or surgery with a few to several weeks of intense factor replacement therapy. These occurrences are consistent with a response to trauma serving as an adjuvant and leading to loss of tolerance to a mildly deficient factor VIII. Types of hemophilic mutations vary in their incidence of alloimmune responses and high-titer inhibitors are seen at least 10-fold more frequently in severe hemophilia A than B. Within hemophilic brother pairs, it is not unusual for only one to develop an inhibitor implicating other acquired and hereditary influences in inhibitor development. Factor VIII gene knockout mice with targeted disruption of internal exons circulate trace levels of amino-terminal heavy chains (17). Murine factor VIII cDNA in an adenoviral vector corrected the bleeding tendency transiently for 1 month. Cytotoxic and humoral immune responses occurred and anti-murine CD4 antibodies suppressed splenic cytotoxic T-cell response. Some surviving animals showed reappearance of circulating low factor VIII levels. Delivery of human factor IX to murine muscles by adenoviral vectors created an inflammatory response with fiber necrosis and the occurrence of cytotoxic Tlymphocytes specific to cells making factor IX (18). With AAV vector, only a humoral response to human factor IX was seen. However, the humoral responses were different, with IgG subclass 1 being predominant after AAV versus 2c following adenoviral vectors. This response did not occur in CD4-deficient mice. In a mouse model of erythropoietin (EPO) transfer, animals receiving replication-deficient adenoviral vectors expressing EPO gave a brisk antihuman EPO response versus low antimurine EPO (19). An AAV vector had lower levels of inflammatory cytokines/chemokines in this system. Parenthetically, it was noted that some delivery catheters or stainless-steel needles inactivated viral vectors unless coated with albumin, giving the impression of less efficient transduction. Antigens represent the initial signal for an immune response and can be either foreign or self proteins. Expressed costimulatory molecules (e.g., B7.1 or B7.2) provide a second signal on dendritic or antigen-presenting cells. A third, “danger” signal arises from inflammatory responses and stimulates immature antigen-presenting cells to increase their surface-exposed MHC class II molecules (20). Presentation is to naive T-cells that process the antigen into peptides; for MHC I, they are usually about 9 amino acids in length. Tissue necrosis as opposed to apoptosis provides potent costimulatory danger signals as an adjuvant (21). Cyclosporin blocks the MOLECULAR THERAPY Vol. 2, No. 1, July 2000 Copyright The American Society of Gene Therapy
first signal. However, tolerance results when the first but not the second signal occurs. Continued boosting and high doses block the second signal. Currently, blockade of the costimulatory pathways is the best approach to alter immune responsiveness because of redundancy in danger signals. The proliferative responses of CD4 lymphocytes from patients with allo- or autoantibodies to recombinant factor VIII fluctuated over time and were occasionally detectable in healthy subjects (22). Overlapping factor VIII peptides of 20 amino acids showed a high frequency of stimulation by sequences from A2, C2, and A3 domains in patients’ cells.
CLINICAL TRIALS PATIENTS
OF
FACTORS VIII
OR
IX
IN
HEMOPHILIC
Three current phase I clinical trials were reviewed after hearing from hemophilic parents who remain excited by the prospects of gene therapy but desire caution in research, recognizing that safety is the major concern. Muscle gene therapy with an adeno-associated viral vector factor IX cDNA has been given to five patients and no humoral immune response nor complications were observed; the transgene was demonstrated in biopsied muscle tissues that were also positive for immunohistochemical staining of factor IX protein (1). A retroviral construct with factor VIII cDNA (23) has been administered to 10 subjects. For the third study presented, Richard Selden (Transkaryotic Therapies, Inc., Cambridge, MA) described that autologous fibroblasts were harvested from skin biopsies, transfected by electroporation, and a single high-expressing clone grown in culture. Six severe hemophilia A patients received autologous implants into their omental fat pads. Again, no adverse events related to the procedure were observed. In each of these three studies some subjects report fewer bleeding episodes and occasionally have very low levels of clotting factor activity detected. Reproducibility remains to be demonstrated and dose-escalation studies are in progress. Absence of detectable germline sequences was as anticipated, given the compactness of spermatocyte DNA (24).
HEPATITIS,
A
FREQUENT COMPLICATION
OF
HEMOPHILIA
Hepatitis B (HBV) and especially chronic C (HCV) infections affect nearly all adult hemophilic patients, having been transmitted by plasma-derived factor concentrates prior to effective viral inactivation methods introduced 10–15 years ago. Concerning attempts at gene therapy, this necessitates the consideration of potential interactions of the vector strategy to prevent hepatic toxicity. This requires a greater understanding of the natural history of HCV. A second area where gene therapy may impact patients with hepatitis and hemophilia is its potential for use to introduce virucidal therapy. Although HCV can lead to progressive liver disease and has an increased incidence of hepatoma, there are few data relevant to its natural history. In a recent half-
7
COMMENTARY century look-back study of samples from military subjects, HCV often persists subclinically for decades (25). Current estimates are that within a lifetime, about onethird of infected patients will show progression. HIV coinfection and alcohol consumption are important additive risks; lack of clearing an initial infection is associated with increased age and male sex. In an animal model of bile duct ligation, Margaret V. Ragni (University of Pittsburgh School of Medicine) reported that liver disease is associated with secretion of proinflammatory cytokines, raising the possibility of identifying HCVinfected patients at risk of progressive liver disease. Transgenic mice that express HBV provide a model for partial viral replication. INFγ primarily mediates cytotoxic T-lymphocytes that abolish viral replication by noncytopathic mechanisms. When transgenic HBV mice were crossed with a murine INFγ knockout, HBV replication was enhanced (26). Primary human hepatocytes were transplanted beneath murine renal capsules after anti-c-Met preparation. HBV infection with a complete life cycle and superinfection with HDV occurred within the human hepatocytes in this xenotransplantation model (27). Ribozymes have been targeted to digest the 5′ ends of HCV RNAs. Once an animal model of HCV infection is established, gene transfer of these ribozymes should allow a more complete evaluation of this novel therapeutic strategy.
SUMMARY A wide variety of viral and nonviral vector strategies are being explored for gene transfer of factors VIII and IX, and expression systems are being optimized. Although gene repair is often problematic, efforts to develop this area may yield a very safe approach for patients with point mutations. Indeed, a variety of approaches may be useful for different patients. Although factor replacement therapy is currently effective at managing most bleeding episodes after they occur, not all complications are prevented. Gene therapy for the hemophilias could provide continuous protection if not a cure. This is especially critical for the two-thirds of hemophilic patients in countries where no concentrate therapy is available. If cost-effective efforts to identify and properly diagnose these patients could be followed by a simple procedure capable of creating prolonged in vivo expression, gene therapy for the hemophilias will have a significant worldwide impact.
2Pipe, S. W., and Kaufman, R. J. (1997). Characterization of a genetically engineered inactivation-resistant coagulation factor VIIIa. Proc. Natl. Acad. Sci. USA 94: 11851–11856. 3Zolotukhin, S., et al. (1999). Recombinant adeno-associated virus purification using novel methods improves infectious titer and yield. Gene Ther. 6: 973–985. 4Chao, H. J., Nao, L., Bruce, A. T., and Walsh, C. E. (2000). Sustained expression of human factor VIII in mice using a parvovirus-based vector. Blood 95: 1594–1599. 5Chuah, M. K. L., et al. (2000). Long-term persistence of human bone marrow stromal cells transduced with factor VIII-retroviral vectors and transient production of therapeutic levels of human factor VIII in nonmyeloablated immunodeficient mice. Hum. Gene. Ther. 11: 729–738. 6Kafri, T., van Praag, H., Ouyang, L., Gage, F. H., and Verma, I. M. (1999). A packaging cell line for lentivirus vectors. J. Virol. 73: 576–584. 7Park, F., Ohashi, K., Chiu, W., Naldini, L., and Kay, M. A. (2000). Efficient lentiviral transduction of liver requires cell cycling in vivo. Nat. Genet. 24: 49–52. 8Minshull, J., and Stemmer, W. P. (1999). Protein evolution by molecular breeding. Curr. Opin. Chem. Biol. 3: 284–290. 9Gallo-Penn, A. M., et al. (1999). In vivo evaluation of an adenoviral vector encoding canine factor VIII: High-level, sustained expression in hemophiliac mice. Hum. Gene Ther. 10: 1791–1802. 10Lozier, J. N., Metzger, M. E., Donahue, R. E., and Morgan, R. A. (1999). Adenovirusmediated expression of human coagulation factor IX in the rhesus macaque is associated with dose-limiting toxicity. Blood 94: 3968–3975. 11Balague, C., et al. (2000). Sustained high-level expression of full-length human factor VIII and restoration of clotting activity in hemophilic mice using a minimal adenovirus vector. Blood 95: 820–828. 12Hartigan–O’Connor, D., and Chamberlain, J. S. (2000). Developments in gene therapy for muscular dystrophy. Microsc. Res. Tech. 48: 223–238. 13Mahato, R. I., Smith, L. C., and Rolland, A. (1999). Pharmaceutical perspectives of nonviral gene therapy. Adv. Genet. 41: 95–156. 14Yant, S. R., Meuse, L., Chiu, W., Ivics, Z., Izsvak, Z., and Kay, M. A. (2000). Somatic integration and long-term transgene expression in normal and haemophilic mice using a DNA transposon system. Nat. Genet. 25: 35–41. 15Kren, B. T., et al. (1999). Correction of the UDP-glucuronosyltransferase gene defect in the Gunn rat model of Crigler–Najjar syndrome type I with a chimeric oligonucleotide. Proc. Natl. Acad. Sci. USA 96: 10349–10354. 16Miao, C. H., Chou, S. H., Nichols, T. C., and Thompson, A. R. (1999). Chimeric RNA/DNA hybrid dumbbells capped with compact trinucleotide loops more consistently correct the factor in point mutation IX canine hemophilic fibroblasts. Blood 94: 373a. 17Sarkar, R., Gao, G. P., Chirmule, N., Tazelaar, J., and Kazazian, H. H. (2000). Partial correction of murine hemophilia A with neo-antigenic murine factor VIII. Hum. Gene Ther. 11: 881–894. 18Fields, P. A., et al. (2000). Role of vector in activation of T cell subsets in immune responses against the secreted transgene product factor IX. Mol. Ther. 1: 225–235. 19Tripathy, S. K., Black, H. B., Goldwasser, E., and Leiden, J. M. (1996). Immune responses to transgene-encoded proteins limit the stability of gene expression after injection of replication-defective adenovirus vectors. Nat. Med. 2: 545–550. 20Pasquini, S., et al. (1997). Cytokines and costimulatory molecules as genetic adjuvants. Immunol. Cell Biol. 75: 397–401. 21Gallucci, S., Lolkema, M., and Matzinger, P. (1999). Natural adjuvants: Endogenous activators of dendritic cells. Nat. Med. 5: 1249–1255. 22Reding, M. T., et al. (1999). CD4+ cell response to factor VIII in hemophilia A, acquired hemophilia, and healthy subjects. Thromb. Haemost. 82: 509–515. 23Greengard, J. S., and Jolly, D. J. (1999). Animal testing of retroviral-mediated gene therapy for factor VIII deficiency. Thromb. Haemost. 82: 555–561. 24Gordon, J. W. (1998). Germline alteration by gene therapy: Assessing and reducing the risks. Mol. Med. Today 4: 468–470. 25Seeff, L., et al. (2000). 45-year follow-up of hepatitis C virus infection in healthy young adults. Ann. Int. Med. 132: 105–111. 26McClary, H., Koch, R., Chisari, F. V., and Guidotti, L. G. (2000). Relative sensitivity of hepatitis B virus and other hepatotropic viruses to the antiviral effects of cytokines. J. Virol. 74: 2255–2264. 27Ohashi, K., et al. (2000). Sustained survival of human hepatocytes in mice: A model for in vivo infection with human hepatitis B and hepatitis delta viruses. Nat. Med. 6: 327–331.
REFERENCES 1Kay, M. A., et al. (2000). Evidence for gene transfer and expression of factor IX in haemophilia B patients treated with an AAV vector. Nat. Genet. 24: 257–261.
8
MOLECULAR THERAPY Vol. 2, No. 1, July 2000 Copyright The American Society of Gene Therapy
8
doi:10.1006/mthe.2000.0099, available online at http://www.idealibrary.com on IDEAL
Gene Therapies for the Hemophilias Arthur R. Thompson Puget Sound Blood Center and University of Washington, 921 Terry Avenue, Seattle, Washington 98104 Fax: 206-292-8030. E-mail: [email protected].
INTRODUCTION These are exciting times for those working toward a genetic cure for patients with hemophilia. Recent publicity surrounding the initial results of a phase I clinical trial of adeno-associated viral (AAV)-mediated delivery of the factor IX gene to skeletal muscle of patients with hemophilia B (1) is indeed gratifying. However, they only represent a slit-lamp view of the vast array of ongoing research progress in this field. To this end, a Third National Hemophilia Foundation Workshop on Gene Therapies for Hemophilia was held in Washington, DC, on March 24–25, 2000, and featured 40 invited speakers. This meeting was cosponsored by the National Institutes of Health, other governmental agencies, and representatives of industry (see //www.hemophilia.org under gene therapy). The talks focused on recombinant factors VIII and IX, hemophilic animal models, viral and nonviral vectors, immunologic consequences and immune responses to vectors and expressed factors, three current gene transfer clinical trials, and hepatitis as a prevalent complication from prior concentrate therapy and the potential for antihepatitis viral gene therapy. The following is a brief review that highlights the scope of issues covered; where references to specific data may be unclear from lack of a citation, a few of the presenters are identified by name. The entire program can be requested from the above website.
ANIMAL MODELS
OF
HEMOPHILIAS A
AND
B
Gene knockout mice and colonies of naturally occurring hemophilic dogs have provided the opportunity to examine the efficacy of factors VIII or IX gene therapy. They have also served as a valuable resource not only to examine the immune response to the expressed protein from the recombinant transgene, but also to provide insights into mechanisms involved in the frequent formation of alloantibodies in patients with hemophilia. Potential mutations to produce a “gain in function” included a factor VIII with covalent linkage of the A2 to A3 domains (referred to as an inactivation-resistant factor VIII or “IR8”) that is more stable in vitro (2). The altered protein was hemostatic with a shorter half-life in MOLECULAR THERAPY Vol. 2, No. 1, July 2000 Copyright The American Society of Gene Therapy
dogs with severe hemophilia A, as anticipated from reduced binding to von Willebrand factor. For hemophilia B, mutagenesis of sites of posttranslational modification of recombinant factor IX failed to improve its recovery in mice (R. G. Schaub, Boston, MA). As a model for alloimmune response, dogs with severe hemophilia A were multiply transfused with canine cryoprecipitate. Several assays were compared to determine their sensitivity D. Lillicrap, Kingston, ON, Canada). Contrary to a recent report, canine factor IX in the Chapel Hill colony (due to a missense mutation) accounts for severe hemophilia B without detectable factor IX antigen in affected animals; one commercial antiserum preparation gave false-positive Western blots (T. C. Nichols, Chapel Hill, NC). Gene therapy to primates has been impeded by a lack of deficient models and the difficulty in distinguishing the native primate factor from a highly homologous human clotting factor sequence. Nevertheless, primates can provide important information on dosing and are useful to screen for potential toxicities of therapeutic constructs. To address the issue of dose responses, Robert R. Montgomery (Medical College of Wisconsin, Milwaukee, WI) presented a novel assay capable of distinguishing even trace amounts of human factor VIII in macaque plasma. His laboratory utilized a species-specific monoclonal anti-human factor VIII antibody for capture and a coupled chromogenic factor X-activating assay to assess functional cofactor activity of the captured protein. Thus, the efficacy of human factor VIII gene transfer can be assessed in a primate background without requiring deficient animals.
VIRAL
AND
NONVIRAL VECTORS
A critical issue in gene therapy in general is to develop safe and robust vector systems. Several presentations focused on advances in vectors to improve efficiency of factor VIII or factor IX gene transfer into cells. Traditionally these have been of viral origin, derived from AAV, retroviral, including lentiviral, or adenoviral
5
COMMENTARY subtypes. Nonviral approaches may provide potentially safer alternatives and although highly inefficient in the past, several advances suggest that they may be approaching viral vectors in transfer efficiency. A final approach reviewed is direct gene repair of point mutations. AAV vectors, as used in the hemophilia B clinical trial (1), bind to cell-surface receptors with a heparan sulfate proteoglycan. R. Jude Samulski (University of North Carolina at Chapel Hill) added that receptor density correlated with transduction rates of different cells within tissues. Helper-free preparations were created with plasmids to provide helper gene functions and chromatographic concentration (3) removed expressed transgene protein. Similar measures are being incorporated into large-scale AAV vector production. Delayed expression from AAV vector transgenes can occur without secondstrand synthesis as complementary single strands can anneal. Using a small promoter, it has been possible to express the large B domain-deleted factor VIII cDNA in mice and expression was demonstrated in both hepatocytes and sinusoidal epithelial cells (4). Head-to-tail concatemers provide a strategy for AAV constructs with larger cDNAs (such as factor VIII) where an intron is inserted between the 3′ end of one and the 5′ end of a second vector and heterodimers with the proper orientation can express from the entire coding region. Traditional retroviral vectors have safer packaging cell lines and growth factors and tissue-specific promoters are being investigated (K. P. Ponder, St. Louis, MO). Engraftment of human marrow stromal cells in SCID factor VIII-deficient (knockout) mice provided expression of factor VIII but this was only transient due to promoter inactivation (5). Inder Verma (Salk Institute, La Jolla, CA) showed that with lentiviral variants, factor VIII knockout mice had prolonged expression with phenotypic correction. Regulatory sequences accounted in part for downregulation of expression following transduction. Intraperitoneal injections resulted in hepatic gene transfer in factor VIII gene knockout mice and caused less morbidity and mortality than the surgical preparation for portal vein infusions. Third-generation lentiviral vectors are being constructed to use plasmids and remove nonessential viral genes (6) and these are being transferred to preparative scale procedures to improve safety. Partial hepatectomy stimulates cell cycling and, at least in hepatocytes, enhances lentiviral vector transduction (7). In a separate presentation, a technique referred to as DNA “shuffling” (8) was proposed as potentially applicable to improve vector and/or transgene sequences. Adenoviral vectors expressed human or canine factor VIII in a canine hemophilic model and liver expression in mice was enhanced by an albumin promoter (9). However, Sheila Connelly (Genetic Therapy, Inc., Gaithersburg, MD) reported that these vectors produced dose-dependent, biphasic hepatotoxicity in mice, even after targeted disruption of E1, E2a, and E3 adenoviral genes. Similar toxicity was observed with adenoviral vectors in primates (10).
6
There remains concern that persons whose livers harbor hepatitis C would be at high risk of sustained hepatotoxicity. Adenoviral vectors with no detectable viral gene expression should be safer. Reactions to the helperproduced adenoviral capsid proteins may still occur. Minimal, transient liver toxicity occurred in one mouse; this was not observed after 3 1012 particles per kilogram of “gutless” adenoviral particles were infused into dogs (11). Relevant to the development of such “mini” adenoviral vectors, a construct with the large dystrophin gene with muscle-specific promoters (12) expressed sufficient dystrophin for 4 months in affected mice to partially restore muscle function. Although previously plagued by low transduction efficiencies and in vitro transfection, nonviral approaches to gene therapy in vivo have been successful in achieving prolonged expression of therapeutic levels of a variety of proteins. One approach is to use electroporation applied to muscles of SCID mice to enhance injected plasmid transfer of human factor IX cDNA. Muscle cells were able to express factor IX for several months (13). In dogs with severe hemophilia B, expression of human factor IX was transient due to an immune response. For species-specific transgenes, this approach should allow repeat procedures without an immunogenic viral vector. Furthermore, the size of the transgene is not limited as it is in AAV or retroviral vectors. A second approach is to use transposon sequences that integrate into a limited number of insertion sites. Transposons have successfully transferred human factor IX into immune-competent mice and therapeutic levels of human factor IX were observed in these animals, persisting for 1 year (14). Furthermore, repeat administrations were successful. Gene repair by hybrid RNA/DNA oligonucleotides has provided successful conversion in the majority of genes tested in cell culture, although the percentage of cells converted is often low. Phenotypic success with this approach was reported as in vivo repair of a frameshift mutation in the UDP-glucuronosyltransferase gene in the Gunn rat (15). According to R. Michael Blaese (Kimeragen, Newtown, PA), increased size of the oligonucleotides, having the correct sequence only on the side with the full DNA sequence, and use of RNA hairpin turns improved efficiency. More compact DNA hairpin structures were also found to facilitate conversion (16). Although there are multiple point mutations in hemophilic families and this approach could not correct a common, recurrent factor VIII gene inversion, repair would avoid potential complications from random integration.
IMMUNOLOGIC CONSEQUENCES
AND IMMUNE
RESPONSES
Alloimmune inhibitors occur in 10–30% of patients with severe hemophilia A. A major concern for gene therapy of hemophilia is whether the expressed recombinant protein would be even more immunogenic. Gene transfer to naive patients could evoke the same or even highMOLECULAR THERAPY Vol. 2, No. 1, July 2000 Copyright The American Society of Gene Therapy
COMMENTARY er frequencies of immunity to an expressed, B domaindeleted protein and the cells or tissues used for expression may influence the response. Alternatively, cellular expression might be the equivalent of a continuous infusion that could promote tolerance. Although usually seen in severe patients, there are several more recent reports of high-titer inhibitors occurring in patients with mild hemophilia A. The latter frequently occur after major trauma or surgery with a few to several weeks of intense factor replacement therapy. These occurrences are consistent with a response to trauma serving as an adjuvant and leading to loss of tolerance to a mildly deficient factor VIII. Types of hemophilic mutations vary in their incidence of alloimmune responses and high-titer inhibitors are seen at least 10-fold more frequently in severe hemophilia A than B. Within hemophilic brother pairs, it is not unusual for only one to develop an inhibitor implicating other acquired and hereditary influences in inhibitor development. Factor VIII gene knockout mice with targeted disruption of internal exons circulate trace levels of amino-terminal heavy chains (17). Murine factor VIII cDNA in an adenoviral vector corrected the bleeding tendency transiently for 1 month. Cytotoxic and humoral immune responses occurred and anti-murine CD4 antibodies suppressed splenic cytotoxic T-cell response. Some surviving animals showed reappearance of circulating low factor VIII levels. Delivery of human factor IX to murine muscles by adenoviral vectors created an inflammatory response with fiber necrosis and the occurrence of cytotoxic Tlymphocytes specific to cells making factor IX (18). With AAV vector, only a humoral response to human factor IX was seen. However, the humoral responses were different, with IgG subclass 1 being predominant after AAV versus 2c following adenoviral vectors. This response did not occur in CD4-deficient mice. In a mouse model of erythropoietin (EPO) transfer, animals receiving replication-deficient adenoviral vectors expressing EPO gave a brisk antihuman EPO response versus low antimurine EPO (19). An AAV vector had lower levels of inflammatory cytokines/chemokines in this system. Parenthetically, it was noted that some delivery catheters or stainless-steel needles inactivated viral vectors unless coated with albumin, giving the impression of less efficient transduction. Antigens represent the initial signal for an immune response and can be either foreign or self proteins. Expressed costimulatory molecules (e.g., B7.1 or B7.2) provide a second signal on dendritic or antigen-presenting cells. A third, “danger” signal arises from inflammatory responses and stimulates immature antigen-presenting cells to increase their surface-exposed MHC class II molecules (20). Presentation is to naive T-cells that process the antigen into peptides; for MHC I, they are usually about 9 amino acids in length. Tissue necrosis as opposed to apoptosis provides potent costimulatory danger signals as an adjuvant (21). Cyclosporin blocks the MOLECULAR THERAPY Vol. 2, No. 1, July 2000 Copyright The American Society of Gene Therapy
first signal. However, tolerance results when the first but not the second signal occurs. Continued boosting and high doses block the second signal. Currently, blockade of the costimulatory pathways is the best approach to alter immune responsiveness because of redundancy in danger signals. The proliferative responses of CD4 lymphocytes from patients with allo- or autoantibodies to recombinant factor VIII fluctuated over time and were occasionally detectable in healthy subjects (22). Overlapping factor VIII peptides of 20 amino acids showed a high frequency of stimulation by sequences from A2, C2, and A3 domains in patients’ cells.
CLINICAL TRIALS PATIENTS
OF
FACTORS VIII
OR
IX
IN
HEMOPHILIC
Three current phase I clinical trials were reviewed after hearing from hemophilic parents who remain excited by the prospects of gene therapy but desire caution in research, recognizing that safety is the major concern. Muscle gene therapy with an adeno-associated viral vector factor IX cDNA has been given to five patients and no humoral immune response nor complications were observed; the transgene was demonstrated in biopsied muscle tissues that were also positive for immunohistochemical staining of factor IX protein (1). A retroviral construct with factor VIII cDNA (23) has been administered to 10 subjects. For the third study presented, Richard Selden (Transkaryotic Therapies, Inc., Cambridge, MA) described that autologous fibroblasts were harvested from skin biopsies, transfected by electroporation, and a single high-expressing clone grown in culture. Six severe hemophilia A patients received autologous implants into their omental fat pads. Again, no adverse events related to the procedure were observed. In each of these three studies some subjects report fewer bleeding episodes and occasionally have very low levels of clotting factor activity detected. Reproducibility remains to be demonstrated and dose-escalation studies are in progress. Absence of detectable germline sequences was as anticipated, given the compactness of spermatocyte DNA (24).
HEPATITIS,
A
FREQUENT COMPLICATION
OF
HEMOPHILIA
Hepatitis B (HBV) and especially chronic C (HCV) infections affect nearly all adult hemophilic patients, having been transmitted by plasma-derived factor concentrates prior to effective viral inactivation methods introduced 10–15 years ago. Concerning attempts at gene therapy, this necessitates the consideration of potential interactions of the vector strategy to prevent hepatic toxicity. This requires a greater understanding of the natural history of HCV. A second area where gene therapy may impact patients with hepatitis and hemophilia is its potential for use to introduce virucidal therapy. Although HCV can lead to progressive liver disease and has an increased incidence of hepatoma, there are few data relevant to its natural history. In a recent half-
7
COMMENTARY century look-back study of samples from military subjects, HCV often persists subclinically for decades (25). Current estimates are that within a lifetime, about onethird of infected patients will show progression. HIV coinfection and alcohol consumption are important additive risks; lack of clearing an initial infection is associated with increased age and male sex. In an animal model of bile duct ligation, Margaret V. Ragni (University of Pittsburgh School of Medicine) reported that liver disease is associated with secretion of proinflammatory cytokines, raising the possibility of identifying HCVinfected patients at risk of progressive liver disease. Transgenic mice that express HBV provide a model for partial viral replication. INFγ primarily mediates cytotoxic T-lymphocytes that abolish viral replication by noncytopathic mechanisms. When transgenic HBV mice were crossed with a murine INFγ knockout, HBV replication was enhanced (26). Primary human hepatocytes were transplanted beneath murine renal capsules after anti-c-Met preparation. HBV infection with a complete life cycle and superinfection with HDV occurred within the human hepatocytes in this xenotransplantation model (27). Ribozymes have been targeted to digest the 5′ ends of HCV RNAs. Once an animal model of HCV infection is established, gene transfer of these ribozymes should allow a more complete evaluation of this novel therapeutic strategy.
SUMMARY A wide variety of viral and nonviral vector strategies are being explored for gene transfer of factors VIII and IX, and expression systems are being optimized. Although gene repair is often problematic, efforts to develop this area may yield a very safe approach for patients with point mutations. Indeed, a variety of approaches may be useful for different patients. Although factor replacement therapy is currently effective at managing most bleeding episodes after they occur, not all complications are prevented. Gene therapy for the hemophilias could provide continuous protection if not a cure. This is especially critical for the two-thirds of hemophilic patients in countries where no concentrate therapy is available. If cost-effective efforts to identify and properly diagnose these patients could be followed by a simple procedure capable of creating prolonged in vivo expression, gene therapy for the hemophilias will have a significant worldwide impact.
2Pipe, S. W., and Kaufman, R. J. (1997). Characterization of a genetically engineered inactivation-resistant coagulation factor VIIIa. Proc. Natl. Acad. Sci. USA 94: 11851–11856. 3Zolotukhin, S., et al. (1999). Recombinant adeno-associated virus purification using novel methods improves infectious titer and yield. Gene Ther. 6: 973–985. 4Chao, H. J., Nao, L., Bruce, A. T., and Walsh, C. E. (2000). Sustained expression of human factor VIII in mice using a parvovirus-based vector. Blood 95: 1594–1599. 5Chuah, M. K. L., et al. (2000). Long-term persistence of human bone marrow stromal cells transduced with factor VIII-retroviral vectors and transient production of therapeutic levels of human factor VIII in nonmyeloablated immunodeficient mice. Hum. Gene. Ther. 11: 729–738. 6Kafri, T., van Praag, H., Ouyang, L., Gage, F. H., and Verma, I. M. (1999). A packaging cell line for lentivirus vectors. J. Virol. 73: 576–584. 7Park, F., Ohashi, K., Chiu, W., Naldini, L., and Kay, M. A. (2000). Efficient lentiviral transduction of liver requires cell cycling in vivo. Nat. Genet. 24: 49–52. 8Minshull, J., and Stemmer, W. P. (1999). Protein evolution by molecular breeding. Curr. Opin. Chem. Biol. 3: 284–290. 9Gallo-Penn, A. M., et al. (1999). In vivo evaluation of an adenoviral vector encoding canine factor VIII: High-level, sustained expression in hemophiliac mice. Hum. Gene Ther. 10: 1791–1802. 10Lozier, J. N., Metzger, M. E., Donahue, R. E., and Morgan, R. A. (1999). Adenovirusmediated expression of human coagulation factor IX in the rhesus macaque is associated with dose-limiting toxicity. Blood 94: 3968–3975. 11Balague, C., et al. (2000). Sustained high-level expression of full-length human factor VIII and restoration of clotting activity in hemophilic mice using a minimal adenovirus vector. Blood 95: 820–828. 12Hartigan–O’Connor, D., and Chamberlain, J. S. (2000). Developments in gene therapy for muscular dystrophy. Microsc. Res. Tech. 48: 223–238. 13Mahato, R. I., Smith, L. C., and Rolland, A. (1999). Pharmaceutical perspectives of nonviral gene therapy. Adv. Genet. 41: 95–156. 14Yant, S. R., Meuse, L., Chiu, W., Ivics, Z., Izsvak, Z., and Kay, M. A. (2000). Somatic integration and long-term transgene expression in normal and haemophilic mice using a DNA transposon system. Nat. Genet. 25: 35–41. 15Kren, B. T., et al. (1999). Correction of the UDP-glucuronosyltransferase gene defect in the Gunn rat model of Crigler–Najjar syndrome type I with a chimeric oligonucleotide. Proc. Natl. Acad. Sci. USA 96: 10349–10354. 16Miao, C. H., Chou, S. H., Nichols, T. C., and Thompson, A. R. (1999). Chimeric RNA/DNA hybrid dumbbells capped with compact trinucleotide loops more consistently correct the factor in point mutation IX canine hemophilic fibroblasts. Blood 94: 373a. 17Sarkar, R., Gao, G. P., Chirmule, N., Tazelaar, J., and Kazazian, H. H. (2000). Partial correction of murine hemophilia A with neo-antigenic murine factor VIII. Hum. Gene Ther. 11: 881–894. 18Fields, P. A., et al. (2000). Role of vector in activation of T cell subsets in immune responses against the secreted transgene product factor IX. Mol. Ther. 1: 225–235. 19Tripathy, S. K., Black, H. B., Goldwasser, E., and Leiden, J. M. (1996). Immune responses to transgene-encoded proteins limit the stability of gene expression after injection of replication-defective adenovirus vectors. Nat. Med. 2: 545–550. 20Pasquini, S., et al. (1997). Cytokines and costimulatory molecules as genetic adjuvants. Immunol. Cell Biol. 75: 397–401. 21Gallucci, S., Lolkema, M., and Matzinger, P. (1999). Natural adjuvants: Endogenous activators of dendritic cells. Nat. Med. 5: 1249–1255. 22Reding, M. T., et al. (1999). CD4+ cell response to factor VIII in hemophilia A, acquired hemophilia, and healthy subjects. Thromb. Haemost. 82: 509–515. 23Greengard, J. S., and Jolly, D. J. (1999). Animal testing of retroviral-mediated gene therapy for factor VIII deficiency. Thromb. Haemost. 82: 555–561. 24Gordon, J. W. (1998). Germline alteration by gene therapy: Assessing and reducing the risks. Mol. Med. Today 4: 468–470. 25Seeff, L., et al. (2000). 45-year follow-up of hepatitis C virus infection in healthy young adults. Ann. Int. Med. 132: 105–111. 26McClary, H., Koch, R., Chisari, F. V., and Guidotti, L. G. (2000). Relative sensitivity of hepatitis B virus and other hepatotropic viruses to the antiviral effects of cytokines. J. Virol. 74: 2255–2264. 27Ohashi, K., et al. (2000). Sustained survival of human hepatocytes in mice: A model for in vivo infection with human hepatitis B and hepatitis delta viruses. Nat. Med. 6: 327–331.
REFERENCES 1Kay, M. A., et al. (2000). Evidence for gene transfer and expression of factor IX in haemophilia B patients treated with an AAV vector. Nat. Genet. 24: 257–261.
8
MOLECULAR THERAPY Vol. 2, No. 1, July 2000 Copyright The American Society of Gene Therapy
8