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

Abstract

Transduction with recombinant adenoassociated virus (AAV) vectors is limited by the need to convert its single-stranded (ss) genome to transcriptionally active double-stranded (ds) forms. For AAVmediated hemophilia B (HB) gene therapy, we have overcome this obstacle by constructing a liver-restricted mini-human factor IX (hFIX) expression cassette that can be packaged as complementary dimers within individual AAV particles. Molecular analysis of murine liver transduced with these self-complementary (sc) vectors demonstrated rapid formation of active ds-linear genomes that persisted stably as concatamers or monomeric circles. This unique property resulted in a 20-fold improvement in hFIX expression in mice over comparable ssAAV vectors. Administration of only 1 ؋ 10 10 scAAV particles led to expression of hFIX at supraphysiologic levels (8I U/mL) and correction of the bleeding diathesis in FIX knock-out mice. Of importance, therapeutic levels of hFIX (3%-30% of normal) were achieved in nonhuman primates using a significantly lower dose of scAAV than required with ssAAV. Furthermore, AAV5-pseudotyped scAAV vectors mediated successful transduction in macaques with pre-existing immunity to AAV8. Hence, this novel vector represents an important advance for hemophilia B gene therapy. IntroductionThe liver is an important target for gene therapy of a variety of genetic disorders, one of which is hemophilia B (HB), a lifethreatening bleeding disorder that arises from mutations in the blood coagulation factor IX (FIX) gene. By maintaining plasma FIX levels above 1% of normal (Ͼ 0.05 g/mL), the incidence of spontaneous hemorrhage is dramatically reduced and so the therapeutic end point for HB gene therapy is modest. 1 Currently, adeno-associated virus (AAV) vectors are the most promising for HB gene therapy and have been the focus of 2 recent clinical trials. 2 Efficient transduction with AAV is, however, limited by the need to convert its single-stranded (ss) genome into transcriptionally active double-stranded (ds) forms in target cells because of its dependence on host-cell-mediated DNA synthesis of the leading strand 3 or annealing of complementary genomes derived from separate virions. 4 Coinfection with adenovirus 5 or priming the target tissues with genotoxic agents 6,7 can enhance ds transgene formation, but the clinical use of these approaches is limited by potential toxicity. Rapid uncoating of the viral genome, as recently described with AAV8 vectors, allows efficient annealing of the ssAAV provirus to form double-stranded genomes. This unique biologic property is responsible for the 10-to 100-fold higher transduction of the liver with rAAV8 when compared with AAV2 vectors in murine models. [8][9][10] Even so, almost 10 13 AAV8 vector particles are required to achieve 100% hepatocyte transduction in mice, a level that is required for successful gene therapy of some metabolic disorders of the liver. 11 This high vector dose is problematic because it may (1) Supported by The Assisi Foundation of Memphis; the Ame...

Neutralizing antibodies against adeno-associated virus examined prospectively in pediatric patients with hemophilia

“…Specifically, human gene therapy trials using AAV2 and AAV8 serotypes for liver-directed correction of hemophilia are being performed, and preclinical approaches have used AAV2, AAV5 and AAV8 in animal models of hemophilia, directing therapy to the liver and to the joints. 3,23,24,26,[30][31][32] In addition, the onset of the most common complications of hemophilia (for example, inhibitor antibody formation and hemophilic joint disease) most commonly occur in childhood, so that gene therapy approaches to avoid or treat these complications ideally would be delivered to boys at this age. Boys entered the JOS trial at a very young age (o30 months of age) and regular prospective sample collection was performed for years during early childhood.…”

“…15 This problem is particularly relevant for patients with hemophilia because the most promising treatment relies on vector delivery to the liver through blood circulation. 3,23,24 The Joint Outcome Study (JOS) was a multi-institutional prospective clinical trial that randomized boys with hemophilia between 2 therapeutic approaches (preventive or on-demand clotting factor replacement) and followed patients prospectively over 3-5 years; JOS results demonstrating superior joint protection using a preventive approach have been reported previously. 1 Serial blood samples were collected throughout the JOS to examine exposure to viruses, including AAV.…”

“…Samples were assayed for NAbs directed against three different serotypes of AAV (AAV2, 5 and 8), each of which represents widely divergent clades of AAV phylogenetically, 6 and each of which has been proposed and/or tested as a potential clinical vector for gene therapy of hemophilia. 3,[23][24][25][26] …”

Single Amino Acid Modification of Adeno-Associated Virus Capsid Changes Transduction and Humoral Immune Profiles

“…However, studies in animals and humans have suggested that the AAV capsid mounts a very strong TD immune response. The evidence includes the following: (i) no NAb was detected after muscular injection, and AAV vector transgene expression was observed following readministration of AAV2 vector via muscular injection in MHC class II-deficient mice (42); (ii) transient immunosuppression with CD4 antibody treatment at the time of primary infection elicited no NAbs and allowed transgene expression in wild-type mice following readministration of AAV vectors (19,42,66); (iii) our prior work has demonstrated that a high NAb titer was achieved after immunization with AAV-pulsed dendritic cells in mice (34); (iv) IgG subclasses were produced in mice and primates with AAV administration (19,52,53,69); and (v) IgG subclasses were observed in humans and in patients following AAV vector treatment (6,51,54). In this study, isotype assays suggested that IgG2a is the predominant IgG subclass in all mice immunized with AAV1 to -5, which is consistent with other viruses that also elicit markedly increased IgG2a production in mice (20).…”

In utero administration of Ad5 and AAV pseudotypes to the fetal brain leads to efficient, widespread and long-term gene expression

“…45 The self-complimentary AAV2/5-GFP and AAV2/8-GFP were produced using the methods previously described by Nathwani et al 46 Vector genomes were calculated using the previously described slot-blot analysis with supercoiled plasmid DNA as standards. 47 The University of Pennsylvania Vector Core Facility supplied the self-complimentary AAV2/ 9-GFP virus and the details of vector production, titration, and quality control can be found on their website (http://www.med.upenn.edu/gtp/ vectorcore/).…”