No abstract

Adeno-associated viral (AAV) vectors are an extensively studied and highly used vector platform for gene therapy applications. We hypothesize that in the first clinical trial using AAV to treat hemophilia B, AAV capsid proteins were presented on the surface of transduced hepatocytes, resulting in clearance by antigen-specific CD8+ T cells and consequent loss of therapeutic transgene expression. It has been previously shown that proteasome inhibitors can have a dramatic effect on AAV transduction in vitro and in vivo. Here, we describe using the US Food and Drug Administration-approved proteasome inhibitor, bortezomib, to decrease capsid antigen presentation on hepatocytes in vitro, whereas at the same time, enhancing gene expression in vivo. Using an AAV capsid-specific T-cell reporter (TCR) line to analyze the effect of proteasome inhibitors on antigen presentation, we demonstrate capsid antigen presentation at low multiplicities of infection (MOIs), and inhibition of antigen presentation at pharmacologic levels of bortezomib. We also demonstrate that bortezomib can enhance Factor IX (FIX) expression from an AAV2 vector in mice, although the same effect was not observed for AAV8 vectors. A pharmacological agent that can enhance AAV transduction, decrease T-cell activation/proliferation, and decrease capsid antigen presentation would be a promising solution to obstacles to successful AAV-mediated, liver-directed gene transfer in humans.

The assessment of the risk of germline transmission of vector-coded sequences is critical for clinical translation of gene transfer strategies. We used rabbit models to analyze the risk of germline transmission of adeno-associated viral (AAV) vectors. Intravenous injection of AAV-2 or AAV-8 resulted in liver-mediated, long-term expression of therapeutic levels of human factor IX (hFIX) in a dose-dependent manner. In high-dose cohorts, AAV-8 resulted in twofold higher levels of circulating hFIX and of vector DNA in liver compared to AAV-2. Vector sequences were found in the semen of all rabbits. The kinetics of vector clearance from semen was dose- and time-dependent but serotype-independent. No late recurrence of AAV-8 sequences was found in the semen over several consecutive cycles of spermatogenesis. In a novel rabbit model, AAV-2 or AAV-8 sequences were detected in the semen of vasectomized animals that lack germ cells. Therefore, structures of the genitourinary (GU) tract, as well as the testis, contribute significantly to vector shedding in the semen. Collectively, data from these two models suggest that the risk of inadvertent germline transmission in males by AAV-8 vectors is low, similar to that of AAV-2, and that AAV dissemination to the semen is in part modulated by host-dependent factors.

In vivo gene transfer to the ischemic heart via electroporation holds promise as a potential therapeutic approach for the treatment of heart disease. In the current study, we investigated the use of in vivo electroporation for gene transfer using 3 different penetrating electrodes and one non-penetrating electrode. The hearts of adult male swine were exposed through a sternotomy. Eight electric pulses synchronized to the rising phase of the R wave of the ECG were administered at varying pulse widths and field strengths following an injection of either a plasmid encoding luciferase or one encoding green fluorescent protein. Four sites on the anterior wall of the left ventricle were treated. Animals were euthanized 48 hours after injection and electroporation and gene expression was determined. Results were compared to sites in the heart that received plasmid injection but no electric pulses or were not treated. Gene expression was higher in all electroporated sites when compared to injection only sites demonstrating the robustness of this approach. Our results provide evidence that in vivo electroporation can be a safe and effective non-viral method for delivering genes to the heart, in vivo.

Catalytic domain variants of activated factor VII (FVIIa) with enhanced hemostatic properties are highly attractive for the treatment of bleeding disorders via genebased therapy. To explore this in a hemophilic mouse model, we characterized 2 variants of murine activated FVII (mFVIIa-VEAY and mFVIIa-DVQ) with modified catalytic domains, based on recombinant human FVIIa (rhFVIIa) variants. Using purified recombinant proteins, we showed that murine FVIIa (mFVIIa) and variants had comparable binding to human and murine tissue factor (TF) and exhibited similar extrinsic coagulant activity. In vitro in the absence of TF, the variants showed a 6-to 17-fold enhanced proteolytic and coagulant activity relative to mFVIIa, but increased inactivation by antithrombin. Gene delivery of mFVIIa-VEAY resulted in long-term, effective hemostasis at 5-fold lower expression levels relative to mFVIIa in hemophilia A mice or in hemophilia B mice with inhibitors to factor IX. However, expression of mFVIIa-VEAY at 14-fold higher than therapeutic levels resulted in a progressive mortality to 70% within 6 weeks after gene delivery. IntroductionOver the past 3 decades, discoveries in multiple disciplines have been instrumental in the molecular dissection of hemophilia. These resulted in the development of novel therapies for the disease, including more effective treatment for the most serious complication in factor replacement, the development of inhibitory antibodies to factor VIII (FVIII) or IX (FIX). In particular, rhFVIIa (commercially known as NovoSeven [Novo Nordisk]) has a proven record of successful treatment for hemophilia patients with inhibitors, by providing localized hemostasis when administered at supraphysiologic doses of 90-110 g/kg. 1 However, its short plasma half-life (2.7 hours) necessitates frequent infusions, resulting in high treatment costs 2 and preventing its use as a universal hemostatic agent.For pharmacologic intervention, efforts to enhance the procoagulant activity of rhFVIIa have focused on chemical modification/ formulation 3,4 or the rational design of novel, high-specific activity variants of rhFVIIa. This has been achieved by enhancing the phospholipid binding or increasing the catalytic activity of rhFVIIa. [5][6][7][8] Although data from human subjects suggest that a single-dose of a rhFVIIa variant (rhFVIIa-DVQ, also known as NN1731) with enhanced, TF-independent activity is safe, 9 efficacy and long-term safety data in hemophilic patients are limited. Moreover, the majority of its in vivo efficacy data have been derived using nonhomologous animal models in short-term studies, where potential species incompatibilities between human FVIIa and endogenous (animal) TF 10 may affect the hemostatic outcomes. 11,12 As an alternative to rhFVIIa infusion, we have demonstrated in animal models the efficacy of gene-based FVIIa therapy for inherited bleeding disorders. In a pilot study, we generated a murine factor VII (mFVII) transgene (mFVII-2RKR) that can be secreted in its active form (mFVIIa) and, after co...