A Barrel of Monkeys: scAAV8 Gene Therapy for Hemophilia in Nonhuman Primates
Authors: Randy J Chandler, Charles P Venditti
Journal: Molecular Therapy (2011)

In this issue of Molecular Therapy, Nathwani et al. present outstanding nonhuman primate (NHP) experiments1Nathwani AC Rosales C McIntosh J Rastegarlari G Nathwani D Raj D et al.Long-term safety and efficacy following systemic administration of a self-complementary AAV vector encoding human FIX pseudotyped with serotype 5 and 8 capsid proteins.Mol Ther. 2011; 19: 876-885Abstract Full Text Full Text PDF PubMed Scopus (239) Google Scholar performed as a prelude to a human trial that has already been hailed as achieving the “holy grail” of hemophilia B gene therapy.2Ponder KP Hemophilia gene therapy: a holy grail found.Mol Ther. 2011; 19: 427-428Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar The aims of the preclinical studies were to determine the safety, efficacy, dose requirements, and immune responses after peripheral intravenous delivery of a self-complementary adeno-associated viral vector (AAV), sc-AAV-LP1-hFIXco, pseudotyped with either AAV8 or AAV5 capsid. These studies describe the treatment and long-term outcome of sc-AAV2/8-LP1-hFIXco gene therapy in one of the largest NHP cohorts studied to date. Because these experiments underlie the first test of an AAV serotype 8 vector in human clinical trials, they have particular relevance not only to hemophilia, but to any disorder that may be treated by liver-directed gene therapy. Hemophilia A and B are bleeding disorders caused by mutations in the clotting factors VIII and IX (FIX), respectively. In severe cases of hemophilia, prolonged and life-threatening spontaneous bleeds can occur in the absence of prophylactic treatment. These disorders have long been considered ideal candidates for gene therapy. Small and large animal models exist for both types of hemophilia, and this has afforded extensive preclinical experimentation with diverse vector delivery systems.3Murphy SL High KA Gene therapy for haemophilia.Br J Haematol. 2008; 140: 479-487Crossref PubMed Scopus (81) Google Scholar Because the defective clotting factors are expressed in the liver, the tissue mass that must be targeted is reduced and the amount of enzymatic activity required to achieve a therapeutic affect is low; on the order of 1% of wild-type FIX expression can substantially ameliorate disease complications. The effectiveness of gene therapy for hemophilia can be readily evaluated by robust and validated clotting assays that require the collection of a small amount of blood. Because hemophilia is relatively common, severe, and treatable only by the expensive and invasive procedures to replace absent clotting factors, there are strong, practical justifications for the use of alternative therapies to treat this disorder. Hemophilia B therapy using integrating and adenoviral vectors, nonviral gene delivery, and cellular transplantation have proven to be modestly successful in animal studies. However, AAV vectors have emerged as the most promising delivery agents for the treatment of hemophilia by gene therapy, culminating in several human trials that used AAV vectors.4Kay MA Manno CS Ragni MV Larson PJ Couto LB McClelland A et al.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 (882) Google Scholar,5Manno CS Chew AJ Hutchison S Larson PJ Herzog RW Arruda VR et al.AAV-mediated factor IX gene transfer to skeletal muscle in patients with severe hemophilia B.Blood. 2003; 101: 2963-2972Crossref PubMed Scopus (603) Google Scholar,6Manno CS Pierce GF Arruda VR Glader B Ragni M Rasko JJ et al.Successful transduction of liver in hemophilia by AAV-Factor IX and limitations imposed by the host immune response.Nat Med. 2006; 12: 342-347Crossref PubMed Scopus (1603) Google Scholar Unfortunately, none of the patients with hemophilia who received AAV gene therapy achieved persistent, therapeutic expression of the viral transgene. Direct injection into the skeletal muscle yielded low transduction and subtherapeutic FIX expression,4Kay MA Manno CS Ragni MV Larson PJ Couto LB McClelland A et al.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 (882) Google Scholar,5Manno CS Chew AJ Hutchison S Larson PJ Herzog RW Arruda VR et al.AAV-mediated factor IX gene transfer to skeletal muscle in patients with severe hemophilia B.Blood. 2003; 101: 2963-2972Crossref PubMed Scopus (603) Google Scholar whereas portal-vein delivery engendered something previously unappreciated in human translation—specifically, that a T-cell response directed against the AAV capsid could result in the targeting of transduced hepatocytes and the loss of FIX expression.6Manno CS Pierce GF Arruda VR Glader B Ragni M Rasko JJ et al.Successful transduction of liver in hemophilia by AAV-Factor IX and limitations imposed by the host immune response.Nat Med. 2006; 12: 342-347Crossref PubMed Scopus (1603) Google Scholar The previous hemophilia AAV trial has convincingly illustrated that murine and canine models have not accurately mimicked the immune response observed in humans following AAV gene therapy and that preexisting immunity to the AAV capsid must be circumvented or avoided for AAV gene therapy to be successful in humans. To overcome practical and theoretical barriers for AAV gene delivery as a treatment for hemophilia, several technical advances in vector design were utilized by Nathwani et al. This novel design included the use of a self-complementary vector that yields more rapid conversion to double-stranded DNA and greater FIX expression than single-strand vectors.7Nathwani AC Gray JT Ng CY Zhou J Spence Y Waddiington SN et al.Self-complementary adeno-associated virus vectors containing a novel liver-specific human factor IX expression cassette enable highly efficient transduction of murine and nonhuman primate liver.Blood. 2006; 107: 2653-2661Crossref PubMed Scopus (330) Google Scholar In addition, the FIX coding sequence was “codon optimized” to increase translational efficiency of the AAV transgene. An AAV8 capsid protein was selected because it targets the liver with great efficiency8Gao GP Alvira MR Wang L Calcedo R Johnston J Wilson JM Novel adeno-associated viruses from rhesus monkeys as vectors for human gene therapy.Proc Natl Acad Sci USA. 2002; 99: 11854-11859Crossref PubMed Scopus (1243) Google Scholar and is rapidly removed from the target cell surface as compared with the AAV2 serotype used in prior studies.6Manno CS Pierce GF Arruda VR Glader B Ragni M Rasko JJ et al.Successful transduction of liver in hemophilia by AAV-Factor IX and limitations imposed by the host immune response.Nat Med. 2006; 12: 342-347Crossref PubMed Scopus (1603) Google Scholar,9Thomas CE Storm TA Huang Z Kay MA Rapid uncoating of vector genomes is the key to efficient liver transduction with pseudotyped adeno-associated virus vectors.J Virol. 2004; 78: 3110-3122Crossref PubMed Scopus (303) Google Scholar The practical implications of these vector improvements include the ability to deliver the virus into the peripheral circulation, as opposed to the hepatic artery, and still achieve efficient hepatic transduction. The improved vector also produced transduction at relatively low doses, minimizing the chances of an inactivating T-cell immune response to the capsid. Nathwani and colleagues1Nathwani AC Rosales C McIntosh J Rastegarlari G Nathwani D Raj D et al.Long-term safety and efficacy following systemic administration of a self-complementary AAV vector encoding human FIX pseudotyped with serotype 5 and 8 capsid proteins.Mol Ther. 2011; 19: 876-885Abstract Full Text Full Text PDF PubMed Scopus (239) Google Scholar report the results from the treatment of 24 NHPs. Sixteen animals were infused by the peripheral intravenous route using variable doses ranging from 2 × 109 to 2 × 1012 pcr-vg/kg of sc-AAV2/8-LP1-hFIXco. At the three highest doses, the animals (n = 11) achieved peak FIX expression that ranged from 3–10% to 274–580%. One animal developed nephrotic syndrome associated with the development of an anti-human FIX inhibitor and was euthanized. Two others were euthanized for toxicology and vector biodistribution studies. The remaining NHPs in this group (n = 8) have continued to express therapeutic levels of FIX for longer than 22 weeks without complications. Follow-up studies from an additional eight animals previously treated10Nathwani AC Gray JT McIntosh J Ng CY Zhou J Spence Y et al.Safe and efficient transduction of the liver after peripheral vein infusion of self-complementary AAV vector results in stable therapeutic expression of human FIX in nonhuman primates.Blood. 2007; 109: 1414-1421Crossref PubMed Scopus (221) Google Scholar with sc-AAV2/8-LP1-hFIXco (n = 5) or AAV2/5-LP1-hFIXco (n = 3), or both (n = 1), were likewise encouraging and showed therapeutic production of FIX as long as 5.5 years after gene transfer. The vector resulted in rapid expression of the FIX gene, achieving ∼20% of normal FIX levels within 24 hours of delivery in the highest dose cohort. This observation provides strong support for the extension of a similar gene delivery strategy to treat conditions with acute presentations, such as inborn errors of intermediary metabolism. Necropsies performed on animals at various time points were entirely unrevealing. Nonetheless, sc-AAV2/8-LP1-hFIXco treatment led to minor but noteworthy complications. Three animals in the lowest-dose cohorts did not achieve detectable serum FIX. Two animals developed anti-human factor IX antibodies; one responded to rituximab and cyclophosphamide treatment. Consistent with other studies of AAV8-mediated gene transfer,8Gao GP Alvira MR Wang L Calcedo R Johnston J Wilson JM Novel adeno-associated viruses from rhesus monkeys as vectors for human gene therapy.Proc Natl Acad Sci USA. 2002; 99: 11854-11859Crossref PubMed Scopus (1243) Google Scholar,11Wang L Calcedo R Wang H Bell P Grant R Vandenberghe LH et al.The pleiotropic effects of natural AAV infections on liver-directed gene transfer in macaques.Mol Ther. 2010; 18: 126-134Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar ∼100-fold increased transduction of the liver compared to other tissues was documented. Ongoing surveillance of the treated NHP group for as long as 66 months using abdominal ultrasonography and random liver biopsies has not revealed any liver tumors or mitotic transformation of hepatocytes. A series of supportive murine studies are also presented1Nathwani AC Rosales C McIntosh J Rastegarlari G Nathwani D Raj D et al.Long-term safety and efficacy following systemic administration of a self-complementary AAV vector encoding human FIX pseudotyped with serotype 5 and 8 capsid proteins.Mol Ther. 2011; 19: 876-885Abstract Full Text Full Text PDF PubMed Scopus (239) Google Scholar to address concerns about the development of hepatic carcinomas in mice following neonatal treatment with AAV.12Donsante A Miller DG Li Y Vogler C Brunt EM Russell DW et al.AAV vector integration sites in mouse hepatocellular carcinoma.Science. 2007; 317: 477Crossref PubMed Scopus (483) Google Scholar Nathwani et al. did not observe a statistically significant increase in the occurrence of hepatic carcinoma in mice treated with sc-AAV2/8-LP1-hFIXco. The encouraging results reported here provide practical guidance for other groups that hope to bring AAV8 to the clinic. Whereas other studies have suggested that AAV8 transduction may have limitations in the delivery and expression of viral transgenes in the NHP liver, Nathwani et al. have convincingly demonstrated the importance of careful pretreatment screening for humoral immunity to AAV8 before gene delivery. Their results prove how important this is for effectiveness: in the 11 NHPs treated with sc-AAV2/8-LP1-hFIXco at doses above the treatment threshold (i.e., >2 × 1010 pcr-vg/kg), all achieved therapeutic FIX expression. It is perhaps equally significant that none of the animals that received the highest dose of vector developed a detectable T-cell response to the AAV8 capsid. Although the clinical ramifications of cellular mediated immunity to AAV in human gene therapy have been variable,13Mingozzi F Meulenberg JJ Hui DJ Basner-Tschakarjan E Hasbrouck NC Edmonson SA et al.AAV-1-mediated gene transfer to skeletal muscle in humans results in dose-dependent activation of capsid-specific T cells.Blood. 2009; 114: 2077-2086Crossref PubMed Scopus (223) Google Scholar,14Brantly ML Chulay JD Wang L Mueller C Humphries M Spencer LT et al.Sustained transgene expression despite T lymphocyte responses in a clinical trial of rAAV1-AAT gene therapy.Proc Natl Acad Sci USA. 2009; 106: 16363-16368Crossref PubMed Scopus (267) Google Scholar T cells reactive against the AAV2 capsid were associated with transaminitis and loss of transgene expression in the prior trial of liver-directed gene therapy for hemophilia B.6Manno CS Pierce GF Arruda VR Glader B Ragni M Rasko JJ et al.Successful transduction of liver in hemophilia by AAV-Factor IX and limitations imposed by the host immune response.Nat Med. 2006; 12: 342-347Crossref PubMed Scopus (1603) Google Scholar The emerging results from these NHP studies and the ongoing clinical experiences highlight the need for extensive humoral and cellular immune prescreening of participants in future AAV clinical trials.