Preclinical assessment of an optimized AAV-FVIII vector in mice and non-human primates for the treatment of hemophilia A.

Extensive clinical data from liver-mediated gene therapy trials have shown that dose-dependent immune responses against the vector capsid may impair or even preclude transgene expression if not managed successfully with prompt immune suppression. The goal of this preclinical study was to generate an adeno-associated viral (AAV) vector capable of expressing therapeutic levels of B-domain deleted factor VIII (FVIII) at the lowest possible vector dose to minimize the potential Risk of a capsid-mediated immune response in the clinical setting. Here, we describe the studies that identified the investigational agent SPK-8011, currently being evaluated in a phase 1/2 study (NCT03003533) in individuals with hemophilia A. In particular, the potency of our second-generation expression cassettes was evaluated in mice and in non-human primates using two different bioengineered capsids (AAV-Spark100 and AAV-Spark200). At 2 weeks after gene transfer, primates transduced with 2 × 1012 vg/kg AAV-Spark100-FVIII or AAV-Spark200-FVIII expressed FVIII antigen levels of 13% ± 2% and 22% ± 6% of normal, respectively. Collectively, these preclinical results validate the feasibility of lowering the AAV capsid dose for a gene-based therapeutic approach for hemophilia A to a dose level orders of magnitude lower than the first-generation vectors in the clinic.

Activated protein C has a regulatory role in factor VIII function.

Mechanisms thought to regulate activated factor VIII (FVIIIa) cofactor function include A2-domain dissociation and activated protein C (APC) cleavage. Unlike A2-domain dissociation, there is no known phenotype associated with altered APC cleavage of FVIII, and biochemical studies have suggested APC plays a marginal role in FVIIIa regulation. However, the in vivo contribution of FVIIIa inactivation by APC is unexplored. Here we compared wild-type B-domainless FVIII (FVIII-WT) recombinant protein with an APC-resistant FVIII variant (FVIII-R336Q/R562Q; FVIII-QQ). FVIII-QQ demonstrated expected APC resistance without other changes in procoagulant function or A2-domain dissociation. In plasma-based studies, FVIII-WT/FVIIIa-WT demonstrated dose-dependent sensitivity to APC with or without protein S, whereas FVIII-QQ/FVIIIa-QQ did not. Importantly, FVIII-QQ demonstrated approximately fivefold increased procoagulant function relative to FVIII-WT in the tail clip and ferric chloride injury models in hemophilia A (HA) mice. To minimize the contribution of FV inactivation by APC in vivo, a tail clip assay was performed in homozygous HA/FV Leiden (FVL) mice infused with FVIII-QQ or FVIII-WT in the presence or absence of monoclonal antibody 1609, an antibody that blocks murine PC/APC hemostatic function. FVIII-QQ again demonstrated enhanced hemostatic function in HA/FVL mice; however, FVIII-QQ and FVIII-WT performed analogously in the presence of the PC/APC inhibitory antibody, indicating the increased hemostatic effect of FVIII-QQ was APC specific. Our data demonstrate APC contributes to the in vivo regulation of FVIIIa, which has the potential to be exploited to develop novel HA therapeutics.

An engineered human albumin enhances half-life and transmucosal delivery when fused to protein-based biologics.

Needle-free uptake across mucosal barriers is a preferred route for delivery of biologics, but the efficiency of unassisted transmucosal transport is poor. To make administration and therapy efficient and convenient, strategies for the delivery of biologics must enhance both transcellular delivery and plasma half-life. We found that human albumin was transcytosed efficiently across polarized human epithelial cells by a mechanism that depends on the neonatal Fc receptor (FcRn). FcRn also transported immunoglobulin G, but twofold less than albumin. We therefore designed a human albumin variant, E505Q/T527M/K573P (QMP), with improved FcRn binding, resulting in enhanced transcellular transport upon intranasal delivery and extended plasma half-life of albumin in transgenic mice expressing human FcRn. When QMP was fused to recombinant activated coagulation factor VII, the half-life of the fusion molecule increased 3.6-fold compared with the wild-type human albumin fusion, without compromising the therapeutic properties of activated factor VII. Our findings highlight QMP as a suitable carrier of protein-based biologics that may enhance plasma half-life and delivery across mucosal barriers.

Advocacy Beyond Identity: A Dutch Gay/Lesbian Organization's Embrace of a Public Policy Strategy.

The gay/lesbian social movement has primarily been understood as an identity movement. This article contributes to expanding understandings of the gay/lesbian movement by following the advocacy of the Dutch Association for the Integration of Homosexuality COC (COC) as a case of a gay/lesbian movement organization's expansion of its action repertoire to include public policy goals. On the basis of archival and interview data, this article identifies several factors that enabled the COC to see the Dutch government as a potential public policy partner. Previous legal successes and facilitation by the institutionalized wing of the women's movement, coupled with a constitutional change, resulted in the COC's development of a policy strategy. By tracing the history of the COC's strategic interactions, this article demonstrates that, while an identity strategy was constant throughout the COC's advocacy, the organization could combine an identity strategy with strategies of legal change, cultural change, and public policy.

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.

Overcoming preexisting humoral immunity to AAV using capsid decoys.

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.

Development and characterization of xenograft model systems for adenoid cystic carcinoma.

Adenoid cystic carcinoma (ACC) is one of the most common malignancies to arise in human salivary glands, and it also arises in the glandular tissue of other organ systems. To address the paucity of experimental model systems for this tumor type, we have undertaken a program of transplanting tissue samples of human ACC into immunodeficient nu/nu mice to create xenograft model systems. In 17 of 23 attempts (74%), xenograft tumors were successfully grown. In all cases, the histologic appearance of the donating tumor was recapitulated in the subsequent xenograft. Characterization of a subset of xenograft models by immunohistochemical biomarkers and by RNA transcript microarray analysis showed good fidelity in the recapitulation of gene expression patterns in the xenograft tumors compared with the human donor tumors. As ACC is known to frequently contain a t(6;9) translocation that fuses the MYB and NFIB genes, fluorescence in situ hybridization (FISH) of 12 ACC xenograft models was performed that assayed MYB locus break-apart and MYB-NFIB locus fusion. Of 12 xenograft models, 11 (92%) revealed MYB locus rearrangement and 10 (83%) showed evidence of fusion of the MYB and NFIB loci. The two related xenograft models (derived from primary and metastatic tumors, respectively, of the same human subject) were karyotyped, showing a t(1;6) translocation, suggesting MYB translocation to a novel fusion partner gene. Overall, our results indicate that ACC is amenable to xenografting and that ACC xenograft models recapitulate the molecular and morphologic characteristics of human tumors, suggesting utility as valid experimental and preclinical model systems for this disease.

Androgen-independent growth and tumorigenesis of prostate cancer cells are enhanced by the presence of PKA-differentiated neuroendocrine cells.

The neuroendocrine status of prostatic adenocarcinomas is considered a prognostic indicator for development of aggressive, androgen-independent disease. Neuroendocrine-like cells are thought to function by providing growth and survival signals to surrounding tumor cells, particularly following androgen ablation therapy. To test this hypothesis directly, LNCaP cells were engineered to inducibly express a constitutively activated form of the cyclic AMP-dependent protein kinase A catalytic subunit (caPKA), which was previously found upon transient transfection to be sufficient for acquisition of neuroendocrine-like characteristics and loss of mitotic activity. Clonal cells that inducibly expressed caPKA enhanced the growth of prostate tumor cells in anchorage-dependent and anchorage-independent in vitro assays as well as the growth of prostate tumor xenografts in vivo, with the greatest effects seen under conditions of androgen deprivation. These results suggest that neuroendocrine-like cells of prostatic tumors have the potential to enhance androgen-independent tumor growth in a paracrine manner, thereby contributing to progression of the disease.