Self-complementary adeno-associated virus (scAAV) vectors can significantly minimize the vector load required to achieve sustained transgene expression. In this study, transcriptional regulatory elements were systematically screened to produce constitutive and liver-specific scAAV factor IX (FIX) expression cassettes. In addition, optimization of GC content, cis- regulatory elements, and codon usage in the human FIX (hFIX) transgene increased expression 4-20-fold. A vector was developed that was capable of expressing high FIX levels in comparison with the single-stranded (ss) AAV vector used in a recent clinical trial. The ssAAV and scAAV vectors display different transgene expression and genome stability patterns in the liver, as determined by immunohistochemical staining, in situ messenger RNA (mRNA) hybridization and vector genome quantitation. The ssAAV2 vector promoted strong FIX expression in only a subset of hepatocytes. The scAAV2-hFIX vector showed widespread ( approximately 80% of hepatocytes), moderate FIX expression levels similar to normal livers with correction of coagulation function in FIX-deficient mice. The ability of low dose scAAV-FIX vectors to achieve near-physiological expression may circumvent inflammatory responses in the liver. In addition to providing an improved scAAV vector for potential application in future hemophilia B clinical trials and liver-directed gene delivery, these studies underscore the need for rigorous analysis and optimization of vector genome cassettes.

Delivery of genes that are larger than the wild-type adeno-associated virus (AAV) 4,681 nucleotide genome is inefficient using AAV vectors. We previously demonstrated in vitro that concurrent proteasome inhibitor (PI) treatment improves transduction by AAV vectors encoding oversized transgenes. In this study, an AAV vector with a 5.6 kilobase (kb) factor VIII expression cassette was used to test the effect of an US Food and Drug Administration-approved PI (bortezomib) treatment concurrent with vector delivery in vivo. Intrahepatic vector delivery resulted in factor VIII expression that persisted for >1 year in hemophilia mice. Single-dose bortezomib given with AAV2 or AAV8 factor VIII vector enhanced expression on average ~600 and ~300%, respectively. Moreover, coadministration of AAV8.canineFVIII (1 × 10(13) vg/kg) and bortezomib in hemophilia A dogs (n = 4) resulted in normalization of the whole blood clotting time (WBCT) and 90% reduction in hemorrhages for >32 months compared to untreated hemophilia A dogs (n = 3) or dogs administered vector alone (n = 3). Demonstration of long-term phenotypic correction of hemophilia A dogs with combination adjuvant bortezomib and AAV vector expressing the oversized transgene establishes preclinical studies that support testing in humans and provides a working paradigm to facilitate a significant expansion of therapeutic targets for human gene therapy.

Hemophilic bleeding into joints causes synovial and microvascular proliferation and inflammation (hemophilic synovitis) that contribute to end-stage joint degeneration (hemophilic arthropathy), the major morbidity of hemophilia. New therapies are needed for joint deterioration that progresses despite standard intravenous (IV) clotting factor replacement. To test whether factor IX within the joint space can protect joints from hemophilic synovitis, we established a hemophilia B mouse model of synovitis. Factor IX knockout (FIX ؊/؊ ) mice received a puncture of the knee joint capsule with a needle to induce hemarthrosis; human factor IX (hFIX) was either injected through the needle into the joint space (intraarticu- larly IntroductionThe most significant morbidity resulting from congenitally deficient factor VIII or IX activity (hemophilia A or hemophilia B) is the progressive destruction of joints resulting from recurrent intraarticular (IA) hemorrhage. Although bleeding at other sites does occur in persons with hemophilia, the musculoskeletal system is by far the most common site; 85% of all bleeding events occur in joints, and 80% of these affect 6 problem joints: the elbows, knees, and ankles. 1 Joint hemorrhage is treated by intravenous (IV) infusion of clotting factor to raise the circulating plasma activity. There is a need for adjunctive therapies directed specifically to the pathology within the hemophilic joint.An understanding of the pathophysiology of hemophilic joint disease is only now emerging. [2][3][4] Joint bleeding results in a chronic inflammatory disorder known as hemophilic synovitis, which in time evolves into a complex arthritis termed hemophilic arthropathy, in which the synovial disease is accompanied by degenerative changes in cartilage and underlying bone. 3,4 As the inflammatory environment that develops in response to blood in a joint stimulates neoangiogenesis of fragile blood vessels, one or more "target" joints for recurrent bleeding develop. Joint-surface erosions secondary to chronic synovitis often occur in early childhood. 5 If aggressive early prophylactic factor replacement is not instituted, 90% of persons with severe hemophilia (Ͻ 1% factor VIII or factor IX activity) will have chronic degenerative changes in 1 to 6 joints by 25 years of age.A limited number of treatment options exist for recurrent joint bleeding and hemophilic synovitis. 6 The mainstay of therapy is replacement clotting factor dosed to achieve a circulating plasma activity level adequate to provide hemostasis throughout the body. Factor replacement in response to ongoing bleeding does not halt the progression of existing arthropathy. 5 Instead, institution of uninterrupted preventive (prophylactic) factor infusions at an early age, before the onset of recurrent joint bleeding, should be the standard of care. 7 The major costs of hemophilia to the healthcare system in dollars, to society in lost productivity and to the person with hemophilia in terms of quality of life, result from bleeding into joints. 8 F...

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Vector capsid dose-dependent inflammation of transduced liver has limited the ability of adeno-associated virus (AAV) factor IX (FIX) gene therapy vectors to reliably convert severe to mild hemophilia B in human clinical trials. These trials also identified the need to understand AAV neutralizing antibodies and empty AAV capsids regarding their impact on clinical success. To address these safety concerns, we have used a scalable manufacturing process to produce GMP-grade AAV8 expressing the FIXR338L gain-of-function variant with minimal (<10%) empty capsid and have performed comprehensive dose–response, biodistribution, and safety evaluations in clinically relevant hemophilia models. The scAAV8.FIXR338L vector produced greater than 6-fold increased FIX specific activity compared with wild-type FIX and demonstrated linear dose responses from doses that produced 2–500% FIX activity, associated with dose-dependent hemostasis in a tail transection bleeding challenge. More importantly, using a bleeding model that closely mimics the clinical morbidity of hemophilic arthropathy, mice that received the scAAV8.FIXR338L vector developed minimal histopathological findings of synovitis after hemarthrosis, when compared with mice that received identical doses of wild-type FIX vector. Hemostatically normal mice (n=20) and hemophilic mice (n=88) developed no FIX antibodies after peripheral intravenous vector delivery. No CD8+ T cell liver infiltrates were observed, despite the marked tropism of scAAV8.FIXR338L for the liver in a comprehensive biodistribution evaluation (n=60 animals). With respect to the role of empty capsids, we demonstrated that in vivo FIXR338L expression was not influenced by the presence of empty AAV particles, either in the presence or absence of various titers of AAV8-neutralizing antibodies. Necropsy of FIX–/– mice 8–10 months after vector delivery revealed no microvascular or macrovascular thrombosis in mice expressing FIXR338L (plasma FIX activity, 100–500%). These preclinical studies demonstrate a safety:efficacy profile supporting an ongoing phase 1/2 human clinical trial of the scAAV8.FIXR338L vector (designated BAX335).