Immune Responses to AAV in Clinical Trials
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Current Gene Therapy, 2011, 11, 321-330 321 Immune Responses to AAV in Clinical Trials Federico Mingozzi1 and Katherine A. High1,2,* 1Children’s Hospital of Philadelphia, Philadelphia, PA, USA; 2Howard Hughes Medical Institute, Philadelphia, PA, USA Abstract: Findings in the first clinical trial in which an adeno-associated virus (AAV) vector was introduced into the liver of human subjects highlighted an issue not previously identified in animal studies. Upon AAV gene transfer to liver, two subjects developed transient elevation of liver enzymes, likely as a consequence of immune rejection of transduced hepa- tocytes mediated by AAV capsid-specific CD8+ T cells. Studies in healthy donors showed that humans carry a population of antigen-specific memory CD8+ T cells probably arising from wild-type AAV infections. The hypothesis formulated at that time was that these cells expanded upon re-exposure to capsid, i.e. upon AAV-2 hepatic gene transfer, and cleared AAV epitope-bearing transduced hepatocytes. Other hypotheses have been formulated which include specific receptor- binding properties of AAV-2 capsid, presence of capsid-expressing DNA in AAV vector preparations, and expression of alternate open reading frames from the transgene; emerging data from clinical trials however fail to support these compet- ing hypotheses. Possible solutions to the problem are discussed, including the administration of a short-term immunosup- pression regimen concomitant with gene transfer, or the development of more efficient vectors that can be administered at lower doses. While more studies will be necessary to define mechanisms and risks associated with capsid-specific im- mune responses in humans, monitoring of these responses in clinical trials will be essential to achieving the goal of long- term therapeutic gene transfer in humans. Keywords: Adeno-associated virus, AAV, capsid, CD8 T cells, clinical trial, gene therapy, immune response, liver. INTRODUCTION nantly in a non-integrated form as episomes; a smaller pro- portion of genomes integrate within chromosomes, although Clinical gene therapy has made considerable progress in it is not clear at this point whether integration events are as- the past several years [1]. First-in-human studies have high- sociated with tumor formation [14, 15]. lighted some of the major challenges to be overcome in order to achieve safe, effective gene transfer and, as a result, al- A large number of studies in experimental animals have though to date there are still no licensed gene therapy prod- established the potential of AAV vectors as a therapeutic tool ucts in the U.S. or Europe, successful clinical trial results [4-8, 16-21]. However, translation of these results into clini- have recently been reported using both integrating [2] and cal studies revealed some of the limits of animal models in non-integrating viral vectors [3]. While for integrating vi- fully predicting outcomes in humans, a finding by no means ruses the biggest concern is insertional mutagenesis, for unique to gene transfer therapeutics [22]. adeno-associated virus (AAV) vectors, the most commonly Differences in mechanisms by which the immune system used vectors for in vivo gene transfer, host immune re- recognizes and reacts to the AAV capsid may account for sponses are still the main concern. some of these limitations. Early findings in a clinical trial in AAV vectors are vehicles of choice for in vivo gene which an AAV-2 vector was introduced into the liver of he- transfer, as they can transduce a wide variety of tissues, me- mophilia B subjects [23, 24] suggested that immune re- diating long-term expression of the donated gene after a sin- sponse directed to the AAV capsid represents one of the last gle in vivo administration [4-8]. Wild-type AAV is not asso- major roadblocks to the development of a successful thera- ciated with any pathology in humans, and is also naturally peutic platform based on AAV-mediated gene transfer. replication-defective, requiring a helper virus such as adeno- In recent years, in response to this hypothesis, a number virus to replicate [9]. The low efficiency in transducing pro- of investigators started to look prospectively at T cell re- fessional antigen presenting cells (e.g. macrophages or den- sponses to capsid after vector administration in humans. dritic cells) [10-12] perhaps contributes to the generally low While the emerging data lend credence to this original con- immunogenicity of AAV vectors. Recombinant AAV vectors cept, the debate has begun to shift from whether there is a are one of the simplest gene therapy vectors, containing only detectable T cell response to capsid, to what it means clini- the transgene expression cassette flanked by two non-coding cally. viral inverted terminal repeats (ITRs) enclosed in a capsid composed of three structural proteins, VP1, 2, and 3 [13]. In this article we shall review the data available on host Once within a cell, AAV vector genomes persist predomi- immune responses to the AAV capsid in humans, the current hypothesis formulated to explain differences between hu- *Address correspondence to this author at the Children’s Hospital of Phila- mans and experimental animals, and strategies proposed to delphia, Philadelphia, PA, USA; Tel: +1 2155904521; Fax: +1 2155903660; avoid capsid recognition by the human immune system. E-mail: [email protected] 1566-5232/11 $58.00+.00 © 2011 Bentham Science Publishers Ltd. 322 Current Gene Therapy, 2011, Vol. 11, No. 4 Mingozzi and High IMMUNE RESPONSES TO AAV CAPSID IN LIVER tolerant to the transgene product [5, 38-41]. Finally, beyond GENE TRANSFER: THE HEMOPHILIA B TRIALS hemophilia, the establishment of a gene transfer technology platform for hepatic gene transfer would allow the treatment The lack of circulating functional coagulation factor VIII of a wide range of genetic disorders and infectious diseases. (F.VIII) or factor IX (F.IX) results in the X-linked inherited disease hemophilia A or B, respectively [25, 26]. Hemophilia The pre-clinical work supporting AAV-mediated gene is characterized by defective coagulation, resulting in in- transfer to liver for hemophilia B is compelling [4, 5, 40, 42, creased risk of bleeding into joint spaces, with consequent 43]. Long-term expression (>9 years) at therapeutic levels arthropathy, or in bleeding in other internal sites, exposing (6-8% of normal levels) in hemophilic dogs was achieved patients to life-threatening hemorrhagic episodes. Although a after infusing a dose of 1x1012 vector genomes (vg)/kg of conventional protein replacement therapy is available to AAV-canine F.IX into the portal vein without production of manage hemophilia, several limitations of this treatment neutralizing antibodies to the transgene product [5]. prompted the study of a gene replacement approach to treat With the expectation that immune responses to the trans- the disease [27]. Furthermore, hemophilia represents an ideal gene product would be avoided in a liver-directed approach, disease model for gene therapy as: a) clinical endpoints are the first dose escalation study of hepatic artery delivery of an well defined and b) these are easily measured; c) circulating AAV-2 vector expressing human F.IX under the control of a levels between 5 and 100% of normal result in significant liver-specific promoter was initiated [23]. Subjects affected amelioration of the disease phenotype and are not associated by severe hemophilia B (pre-treatment F.IX levels <1% of with side effects; d) functional clotting factors can be pro- normal) were enrolled into three dose cohorts, receiving duced by a variety of tissues, including liver, muscle, and 8x1010 to 2x1012 vg/kg. Vector infusion proceeded unevent- fibroblasts [27-30]. However, one severe complication of fully for all subjects. both conventional, protein replacement therapy, and (poten- tially) gene therapy is the development of inhibitory antibod- None of the subjects enrolled in the first two dose cohorts ies (termed inhibitors) to the coagulation factor, which se- showed evidence of vector-related toxicity or efficacy de- verely complicate clinical management of the disease [31, fined as levels of human F.IX expression >1% of normal. th 32]. The 5 subject, subject E, was the first enrolled at what was expected to be a therapeutic dose (2x1012 vg/kg) based on Initial efforts to develop an AAV gene transfer approach the canine studies. This individual did indeed show therapeu- to treat hemophilia B focused on skeletal muscle as the target tic levels of F.IX expression initially, in the range of 10- cell [33, 34]. Clinical translation of results to affected hu- 12%, sufficient to convert his disease phenotype from severe mans resulted in long-term detection of F.IX transgene prod- to mild. However, beginning 4 weeks after vector infusion, uct in muscle biopsies [35-37]; however, at the doses tested, F.IX levels began to fall, and gradually returned to baseline circulating F.IX levels did not reliably rise above 1%. No (<1%) at 10 weeks after vector infusion. Concurrently, liver acute or long-term toxicity was observed following vector enzymes, which had been normal for the first few weeks administration, documented by normal clinical laboratory after vector infusion, began to rise, then slowly returned to testing, including absence of inhibitory and non-inhibitory normal without medical intervention. No inhibitory or non- antibodies to the secreted transgene product, absence of inhibitory antibodies to the F.IX transgene product were muscle enzyme elevation, and absence of lymphocytic infil- measured in this subject. Possible toxic or infectious causes trates in repeated muscle biopsies analyzed over several of the increase in liver enzymes were sought and excluded. years. Exposure to HIV, HBV, and/or HCV as a conse- At the request of regulatory agencies, the next subject stud- quence of prior infusion with infected plasma-derived prod- ied, subject G, was infused at a 5-fold lower dose than E and ucts did not alter the outcome of gene transfer.