Robert J. Davidson

Overcoming Preexisting Humoral Immunity to AAV Using Capsid Decoys

Capsid decoys enhance the efficacy of AAV vector transduction after systemic delivery in the presence of neutralizing antibodies. A Slight of Hand for Gene Therapy Gene therapy has been quite successful—in animal models. But when it comes to translating gene therapy to humans, there have only been a few shining successes. One limiting factor has been the vectors used. Adeno-associated virus (AAV) vectors are safe, noninvasive, and potentially effective; however, people who have been previously exposed to AAV have preexisting neutralizing antibodies that block gene delivery. Now, Mingozzi et al. trick these antibodies into binding empty viral capsid, overcoming their inhibitory effects. The authors hypothesized that introducing empty capsids along with the gene therapy vector would titrate out the neutralizing antibody response to AAV, allowing for successful gene therapy even in the presence of preexisting neutralizing antibodies. They found that varying the ratio of empty capsid to gene therapy vector could successfully inhibit the neutralizing antibody response in both human serum and a mouse model by serving as a decoy for antibody binding. The authors then mutated the receptor binding site of their capsid so that it could bind the neutralizing antibody but not target cells, further increasing the safety profile of this approach. These capsid decoys worked in a dose-dependent manner and were successful even with high antibody titers. What’s more, they were safe and effective in rhesus macaques. Although this approach remains to be tested in humans, tricking neutralizing antibodies with decoys may be the next step in advancing gene therapy in the clinic. Adeno-associated virus (AAV) vectors delivered through the systemic circulation successfully transduce various target tissues in animal models. However, similar attempts in humans have been hampered by the high prevalence of neutralizing antibodies to AAV, which completely block vector transduction. We show in both mouse and nonhuman primate models that addition of empty capsid to the final vector formulation can, in a dose-dependent manner, adsorb these antibodies, even at high titers, thus overcoming their inhibitory effect. To further enhance the safety of the approach, we mutated the receptor binding site of AAV2 to generate an empty capsid mutant that can adsorb antibodies but cannot enter a target cell. Our work suggests that optimizing the ratio of full/empty capsids in the final formulation of vector, based on a patient’s anti-AAV titers, will maximize the efficacy of gene transfer after systemic vector delivery.

In vivo genome editing of the albumin locus as a platform for protein replacement therapy.

Site-specific genome editing provides a promising approach for achieving long-term, stable therapeutic gene expression. Genome editing has been successfully applied in a variety of preclinical models, generally focused on targeting the diseased locus itself; however, limited targeting efficiency or insufficient expression from the endogenous promoter may impede the translation of these approaches, particularly if the desired editing event does not confer a selective growth advantage. Here we report a general strategy for liver-directed protein replacement therapies that addresses these issues: zinc finger nuclease (ZFN) -mediated site-specific integration of therapeutic transgenes within the albumin gene. By using adeno-associated viral (AAV) vector delivery in vivo, we achieved long-term expression of human factors VIII and IX (hFVIII and hFIX) in mouse models of hemophilia A and B at therapeutic levels. By using the same targeting reagents in wild-type mice, lysosomal enzymes were expressed that are deficient in Fabry and Gaucher diseases and in Hurler and Hunter syndromes. The establishment of a universal nuclease-based platform for secreted protein production would represent a critical advance in the development of safe, permanent, and functional cures for diverse genetic and nongenetic diseases.

Robust ZFN-mediated genome editing in adult hemophilic mice

Monogenic diseases, including hemophilia, represent ideal targets for genome-editing approaches aimed at correcting a defective gene. Here we report that systemic adeno-associated virus (AAV) vector delivery of zinc finger nucleases (ZFNs) and corrective donor template to the predominantly quiescent livers of adult mice enables production of high levels of human factor IX in a murine model of hemophilia B. Further, we show that off-target cleavage can be substantially reduced while maintaining robust editing by using obligate heterodimeric ZFNs engineered to minimize unwanted cleavage attributable to homodimerization of the ZFNs. These results broaden the therapeutic potential of AAV/ZFN-mediated genome editing in the liver and could expand this strategy to other nonreplicating cell types.

Modulation of CD8+ T cell responses to AAV vectors with IgG-derived MHC class II epitopes.

Immune responses directed against viral capsid proteins constitute a main safety concern in the use of adeno-associated virus (AAV) as gene transfer vectors in humans. Pharmacological immunosuppression has been proposed as a solution to the problem; however, the approach suffers from several potential limitations. Using MHC class II epitopes initially identified within human IgG, named Tregitopes, we showed that it is possible to modulate CD8+ T cell responses to several viral antigens in vitro. We showed that incubation of peripheral blood mononuclear cells with these epitopes triggers proliferation of CD4+CD25+FoxP3+ T cells that suppress killing of target cells loaded with MHC class I antigens in an antigen-specific fashion, through a mechanism that seems to require cell-to-cell contact. Expression of a construct encoding for the AAV capsid structural protein fused to Tregitopes resulted in reduction of CD8+ T cell reactivity against the AAV capsid following immunization with an adenoviral vector expressing capsid. This was accompanied by an increase in frequency of CD4+CD25+FoxP3+ T cells in spleens and lower levels of inflammatory infiltrates in injected tissues. This proof-of-concept study demonstrates modulation of CD8+ T cell reactivity to an antigen using regulatory T cell epitopes is possible.

A rapid pro-hemostatic approach to overcome direct oral anticoagulants

Direct inhibitors of coagulation factor Xa (FXa) or thrombin are promising oral anticoagulants that are becoming widely adopted. The ability to reverse their anticoagulant effects is important when serious bleeding occurs or urgent medical procedures are needed. Here, using experimental mouse models of hemostasis, we show that a variant coagulation factor, FXaI16L, rapidly restores hemostasis in the presence of the anticoagulant effects of these inhibitors. The ability of FXaI16L to reverse the anticoagulant effects of FXa inhibitor depends, at least in part, on the ability of the active site inhibitor to hinder antithrombin-dependent FXa inactivation, paradoxically allowing uninhibited FXa to persist in plasma. Because of its inherent catalytic activity, FXaI16L is more potent (by >50-fold) in the hemostasis models tested than a noncatalytic antidote that is currently in clinical development. FXaI16L also reduces the anticoagulant-associated bleeding in vivo that is induced by the thrombin inhibitor dabigatran. FXaI16L may be able to fill an important unmet clinical need for a rapid, pro-hemostatic agent to reverse the effects of several new anticoagulants.

Novel factor VIII variants with a modified furin cleavage site improve the efficacy of gene therapy for hemophilia A

Essentials Factor (F) VIII is an inefficiently expressed protein. Furin deletion FVIII variants were purified and characterized using in vitro and in vivo assays. These minimally modified novel FVIII variants have enhanced function. These variants provide a strategy for increasing FVIII expression in hemophilia A gene therapy.

52. Therapeutic Factor VIII Expression After AAV Delivery in Non-Human Primates

Adeno-associated viral (AAV) vector delivery of factor VIII (FVIII) has been challenging due to its intrinsic properties that result in inefficient expression compared to similarly sized proteins. Early studies of AAV delivery in hemophilia A mice and dogs suggested that the therapeutic vector dose for FVIII will be higher than for factor IX. However, higher vector loads may induce stronger immune responses against capsid antigens, as evidenced in the clinical studies of AAV delivery for hemophilia B. The use of codon-optimization and novel FVIII variants with enhanced biological properties may provide strategies to increase FVIII expression or secretion to support clinical studies for hemophilia A. One published study has reported clinically relevant levels of hFVIII following AAV-hFVIII delivery in non-human primates (NHPs). This study utilized a hFVIII variant that included a 17 amino acid synthetic sequence within the 14 amino acid B-domain region that increased hFVIII expression compared to the parental B-domain deleted FVIII-SQ transgene (McIntosh, 2013). While this and other variants may increase expression after AAV delivery, the use of non-native FVIII sequences may also increase the risk of development of neutralizing antibodies to potential neoantigens. In order to generate an AAV-hFVIII vector capable of expressing therapeutic levels of FVIII at a clinically relevant vector dose without introduction of any neoantigens, 28 hFVIII-SQ sequences were generated and introduced into our optimized expression cassette containing a modified transthyretin (TTRm) promoter. The constructs were initially screened by hydrodynamic delivery of plasmid DNA which identified 11 candidates that expressed FVIII 2-7 fold higher than our first generation codon optimized construct, CO3. AAV vectors (n=9) were generated using a novel AAV capsid, Spark100, with the best performing FVIII constructs. Hem A/CD4 KO mice were administered the vectors alongside CO3 (4×10e12vg/kg). At 8 weeks post vector administration, 2/9 expressed hFVIII similar to CO3, 5/9 were 4-8 fold higher than CO3 while 2/9 (SPK-8003 and SPK-8005) were >10 fold more potent than CO3. SPK-8005 was then evaluated in a dose escalation study in cynomologus macaques (n=3/group) treated with 3 doses: 2×10e12, 5×10e12 and 1×10e13 vg/kg and compared to vehicle controls (n=2). At 2 weeks post AAV administration, average hFVIII levels in the low, mid and high dose cohorts were 12.7 ± 2.1, 22.6 ± 0.8 and 54.1 ± 15.6 percent of normal, respectively. By 3-4 weeks, hFVIII expression started to decline in most of the animals concomitant with generation of antibodies against human FVIII. Of note, this is an expected and well-described observation that occurs in immune competent animal models due to differences between human and endogenous FVIII protein sequences. The 2 macaques that did not develop anti-hFVIII antibodies had sustained FVIII expression through the last time point evaluated. Finally, no vector-related toxicity events were observed. In summary, extensive codon-optimization identified novel AAV-hFVIII constructs capable of achieving therapeutic FVIII levels in macaques at clinically relevant doses. To our knowledge, the hFVIII levels observed in this study are the highest reported in a large animal model after treatment with an AAV vector expressing an unmodified FVIII-SQ protein. These safety and efficacy results in NHPs support the use of SPK-8005 hepatic gene transfer for the potential treatment of hemophilia A.