Clinical Trials Targeting Muscle as a Metabolic Factory

Adeno-associated virus (AAV)-based vectors have been shown to transduce skeletal muscle with high efficiency and result in sustained transgene expression. Preclinical studies using muscle-targeted therapeutic rAAV vectors for the treatment of other forms of metabolic disease including hemophilia B and alpha-1-

antitrypsin (AAT)-deficiency have been very successful and have lead to testing in Phase I clinical trials.

Hemophilia B is an X-linked recessive disorder resulting from a deficiency of functional coagulation factor IX and results in prolonged bleeding and spontaneous bleeding episodes into joints and soft tissue and increased risk of intracranial and intraperitoneal bleeding. Treatment of hemophilia B consists of repeated therapeutic and prophylactic infusions of recombinant factor IX (F.IX) protein. As little as 1% of wild-type levels of circulating factor IX, can mediate significant therapeutic benefits in affected individuals. Factor IX is normally synthesized in the liver; however, preclinical studies in both murine and canine models of the disease have shown that skeletal muscles can also act as a depot organ for factor IX expression and secretion and provide long-term therapeutic levels of correction.

A phase I dose-escalation clinical study was performed in 8 subjects with hemophilia A, testing a therapeutic rAAV serotype 2-based vector (rAAV2-CMV-F.IX) delivered via multiple intramuscular injections. 1.2 x 1012 vector genomes were injected per site with the numbers of injection sites ranging from 10 to 12 (low dose), 30-50 (middle dose), or 80-90 (high dose). No significant toxicities were noted and the highest dose of 1.8 x 1012 vg/kg was well-tolerated. Circulating F.IX levels increased above baseline levels in 4 of the 8 subjects, however levels remained below the therapeutic threshold of 1% normal, except in one patient who demonstrated a transient level of 1.4% normal at 12 weeks post-injection. F.IX expression was noted in the regions of injection sites for all subjects at 2 months post-injection. Although all but 2 subjects opted out of subsequent muscle biopsy procedures after the first one, both samples showed continued F.IX expression at 6 and 10 months post-injection (Kay et al. 2000; Manno et al. 2003). In one subject, local F.IX expression in the injection site could still be detected 3.7 years post-treatment (Jiang et al. 2006). Since the initiation of the clinical studies, improvements in muscle-targeted gene therapy strategies have emerged. In one study, Arruda et al. demonstrated that an intravascular method of vector delivery could increase circulating F.IX levels by almost tenfold as compared to multiple intramuscular injections of the same vector (Arruda et al. 2005). In another study, Arruda et al. demonstrated that approximately 50-fold higher levels of F.IX could be expressed in the canine model of the disease using a pseudotyped rAAV2/1 vector (Arruda et al. 2004). Of note, with the higher levels of transgene expression, new complications such as immune response to the expressed therapeutic protein, not previously seen at the lower transduction levels, emerged, thus highlighting the complexities in developing effective therapies for metabolic diseases. However, the overall improvements in transduction levels achieved with the newer vectors or delivery mechanisms may eventually translate into better clinical outcomes.

Alpha-one antitrypsin is a protease inhibitor produced normally in the liver and plays a role in the maintenance of structure and function of the lung. AAT-deficiency results in the disruption of the normal balance of proteases and antipro-teases and affected individuals become prone to lung disease. Protein replacement therapy is provided to prevent progression of lung disease in AAT-deficient individuals. Initial Phase I dose escalation studies utilized a therapeutic rAAV2 vector which was administered intramuscularly to 12 subjects. Similar to the findings from the hemophilia B trial, the rAAV2 vector was found to be safe with minimal toxicities (Brantly et al. 2006). More recently, a rAAV2/1-based dose escalation Phase I clinical trial was initiated and is currently active ( Identifier: NCT00430768). Nine subjects were enrolled in which rAAV2/1 vector encoding normal (M) AAT was injected intramuscularly at doses ranging from 6.9 x 1012 to 6 x 1013 vector genome particles per subject. Initial results indicate that the vector is well-tolerated and safe and furthermore, that 6 of 6 subjects within the two highest dose cohorts had M-specific AAT expression levels above the background, and sustained at least 90 days, indicating that more efficient vector-mediated transduction can result in improved outcomes (Brantly et al. 2009; Mueller and Flotte 2008).

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