BAX 335 hemophilia B gene therapy clinical trial results: potential impact of CpG sequences on gene expression.

Gene therapy has the potential to maintain therapeutic blood clotting factor IX (FIX) levels in patients with hemophilia B by delivering a functional human F9 gene into liver cells. This phase 1/2, open-label dose-escalation study investigated BAX 335 (AskBio009, AAV8.sc-TTR-FIXR338Lopt), an adeno-associated virus serotype 8 (AAV8)-based FIX Padua gene therapy, in patients with hemophilia B. This report focuses on 12-month interim analyses of safety, pharmacokinetic variables, effects on FIX activity, and immune responses for dosed participants. Eight adult male participants (aged 20-69 years; range FIX activity, 0.5% to 2.0%) received 1 of 3 BAX 335 IV doses: 2.0 × 1011; 1.0 × 1012; or 3.0 × 1012 vector genomes/kg. Three (37.5%) participants had 4 serious adverse events, all considered unrelated to BAX 335. No serious adverse event led to death. No clinical thrombosis, inhibitors, or other FIX Padua-directed immunity was reported. FIX expression was measurable in 7 of 8 participants; peak FIX activity displayed dose dependence (32.0% to 58.5% in cohort 3). One participant achieved sustained therapeutic FIX activity of ∼20%, without bleeding or replacement therapy, for 4 years; in others, FIX activity was not sustained beyond 5 to 11 weeks. In contrast to some previous studies, corticosteroid treatment did not stabilize FIX activity loss. We hypothesize that the loss of transgene expression could have been caused by stimulation of innate immune responses, including CpG oligodeoxynucleotides introduced into the BAX 335 coding sequence by codon optimization. This trial was registered at www.clinicaltrials.gov as #NCT01687608.

Employing a gain-of-function factor IX variant R338L to advance the efficacy and safety of hemophilia B human gene therapy: preclinical evaluation supporting an ongoing adeno-associated virus clinical trial.

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).

Long-term follow-up after gene therapy for canavan disease.

Canavan disease is a hereditary leukodystrophy caused by mutations in the aspartoacylase gene (ASPA), leading to loss of enzyme activity and increased concentrations of the substrate N-acetyl-aspartate (NAA) in the brain. Accumulation of NAA results in spongiform degeneration of white matter and severe impairment of psychomotor development. The goal of this prospective cohort study was to assess long-term safety and preliminary efficacy measures after gene therapy with an adeno-associated viral vector carrying the ASPA gene (AAV2-ASPA). Using noninvasive magnetic resonance imaging and standardized clinical rating scales, we observed Canavan disease in 28 patients, with a subset of 13 patients being treated with AAV2-ASPA. Each patient received 9 × 10(11) vector genomes via intraparenchymal delivery at six brain infusion sites. Safety data collected over a minimum 5-year follow-up period showed a lack of long-term adverse events related to the AAV2 vector. Posttreatment effects were analyzed using a generalized linear mixed model, which showed changes in predefined surrogate markers of disease progression and clinical assessment subscores. AAV2-ASPA gene therapy resulted in a decrease in elevated NAA in the brain and slowed progression of brain atrophy, with some improvement in seizure frequency and with stabilization of overall clinical status.

Phase 1 gene therapy for Duchenne muscular dystrophy using a translational optimized AAV vector.

Efficient and widespread gene transfer is required for successful treatment of Duchenne muscular dystrophy (DMD). Here, we performed the first clinical trial using a chimeric adeno-associated virus (AAV) capsid variant (designated AAV2.5) derived from a rational design strategy. AAV2.5 was generated from the AAV2 capsid with five mutations from AAV1. The novel chimeric vector combines the improved muscle transduction capacity of AAV1 with reduced antigenic crossreactivity against both parental serotypes, while keeping the AAV2 receptor binding. In a randomized double-blind placebo-controlled phase I clinical study in DMD boys, AAV2.5 vector was injected into the bicep muscle in one arm, with saline control in the contralateral arm. A subset of patients received AAV empty capsid instead of saline in an effort to distinguish an immune response to vector versus minidystrophin transgene. Recombinant AAV genomes were detected in all patients with up to 2.56 vector copies per diploid genome. There was no cellular immune response to AAV2.5 capsid. This trial established that rationally designed AAV2.5 vector was safe and well tolerated, lays the foundation of customizing AAV vectors that best suit the clinical objective (e.g., limb infusion gene delivery) and should usher in the next generation of viral delivery systems for human gene transfer.

Assessment of hippocampal adeno-associated viral vector gene delivery via frameless stereotaxis in a nonhuman primate.

BACKGROUND/AIMS: Expression of the neuropeptide galanin in hippocampal neurons reduces seizures in the kainic acid rodent model of epilepsy. In order to translate these findings into a human clinical trial, the safety and feasibility of hippocampal adeno-associated viral (AAV) vector expression must be demonstrated in a nonhuman primate model. METHODS: The Stealth Frameless Stereotactic System and Navigus Biopsy Appliance (Medtronic) were used to inject self-complementary AAV2 carrying the gene for green fluorescent protein (GFP) into monkey hippocampi. Using a single occipital trajectory per side (n = 8 trajectories), multiple injections spaced by 5 mm were delivered to each hippocampus. RESULTS: GFP was expressed in both neuronal and glial cells. Injections led to nonhomogeneous gene expression, suggesting closer spacing of injections may lead to more gene expression. Increasing injection volumes entailed a general increase in volume of expression, but there was no overlap of expression within the 5-mm injection interval. Efforts to avoid the occipital horn failed to prevent leaking of vector into the ventricle, and resulted in deviation of the trajectory at proximal points from the hippocampus. CONCLUSION: Using the occipital approach, adequate cannulation of the monkey hippocampus will require transventricular trajectories.

Adeno-associated virus for the treatment of muscle diseases: toward clinical trials.

Muscle diseases include muscular dystrophies, cardiomyopathies, neuromuscular and metabolic disorders. The loss of normal muscle structure and function is associated with significant morbidity and mortality. Patients with Duchenne muscular dystrophy usually lose ambulation in their teenage years, and frequently experience severe respiratory problems and heart failure in later stages of life. These unmet medical needs have encouraged the development of genetic strategies targeting the underlying muscle disease processes. Adeno-associated virus (AAV) vectors have been identified as promising gene delivery candidates because of their ability to transduce muscle tissue efficiently while transporting a genetic payload. There is currently significant momentum in the research of AAV-mediated delivery of muscle genes. Various AAV-based therapeutic strategies are undergoing preclinical and clinical testing, including the use of miniaturized and codon-optimized transgenes, exon skipping expression cassettes, novel tissue-specific promoters, AAV capsid mutants and chimeras, and localized intravascular administration procedures. These advancements in gene delivery have led to the generation of AAV vectors with targeted transgene expression, tissue-selective tropism and minimal off-target effects. This review describes advances in AAV gene therapy that are specific to the treatment of muscle diseases, and discusses the implications of their clinical application.

Dystrophin immunity in Duchenne's muscular dystrophy.

We report on delivery of a functional dystrophin transgene to skeletal muscle in six patients with Duchenne's muscular dystrophy. Dystrophin-specific T cells were detected after treatment, providing evidence of transgene expression even when the functional protein was not visualized in skeletal muscle. Circulating dystrophin-specific T cells were unexpectedly detected in two patients before vector treatment. Revertant dystrophin fibers, which expressed functional, truncated dystrophin from the deleted endogenous gene after spontaneous in-frame splicing, contained epitopes targeted by the autoreactive T cells. The potential for T-cell immunity to self and nonself dystrophin epitopes should be considered in designing and monitoring experimental therapies for this disease. (Funded by the Muscular Dystrophy Association and others; ClinicalTrials.gov number, NCT00428935.).

Novel role for aspartoacylase in regulation of BDNF and timing of postnatal oligodendrogenesis.

Neuronal growth factors are thought to exert a significant degree of control over postnatal oligodendrogenesis, but mechanisms by which these factors coordinateoligodendrocyte development with the maturation of neural networks are poorly characterized. We present here a developmental analysis of aspartoacylase (Aspa)-null tremor rats and show a potential role for this hydrolytic enzyme in the regulation of a postnatal neurotrophic stimulus that impacts on early stages of oligodendrocyte differentiation. Abnormally high levels of brain-derived neurotrophic factor (BDNF) expression in the Aspa-null Tremor brain are associated with dysregulated oligodendrogenesis at a stage in development normally characterized by high levels of Aspa expression. BDNF promotes the survival of proliferating cells during the early stages of oligodendrocyte maturation in vitro, but seems to compromise the ability of these cells to populate the cortex in vivo. Aspartoacylase activity in oligodendrocytes is shown to provide for the negative regulation of BDNF in neurons, thereby determining the availability of a developmental stimulus via a mechanism that links oligodendroglial differentiation with neuronal maturation.