Article

Plasmapheresis eliminates the negative impact of AAV antibodies on micro-dystrophin gene expression following vascular delivery.

1] Departments of Pediatrics and Neurology, The Ohio State University and Nationwide Children's Hospital, Columbus, OH, USA, [2] Centers for Gene Therapy and Vaccines and Immunity at The Research Institute at Nationwide Children's Hospital, Columbus, OH. [3] Integrated Biomedical Science Graduate Program, College of Medicine, The Ohio State University, Columbus, OH.
Molecular Therapy (Impact Factor: 6.43). 10/2013; DOI: 10.1038/mt.2013.244
Source: PubMed

ABSTRACT Duchenne muscular dystrophy (DMD) is a monogenic disease potentially treatable by gene replacement. Use of recombinant adeno-associated virus will ultimately require a vascular approach to broadly transduce muscle. We tested the impact of pre-existing AAV antibodies on micro-dystrophin expression following vascular delivery to non-human primates. Rhesus macaques were treated by isolated limb perfusion via a fluoroscopically guided catheter. In addition to serostatus stratification, the animals were placed into one of three immune suppression groups: no immune suppression, prednisone, and triple immune suppression (prednisone, tacrolimus and mycophenolate mofetil). The animals were analyzed for transgene expression at 3 or 6 months. Micro-dystrophin expression was visualized in AAVrh.74 sero-negative animals (mean 48.0% ± 20.8) that was attenuated in sero-positive animals (19.6 ± 18.7%). Immunosuppression did not affect transgene expression. Importantly, removal of AAV binding antibodies by plasmapheresis in AAV sero-positive animals resulted in high level transduction (60.8 ± 18.0%), which is comparable to that of AAV sero-negative animals (53.7 ± 7.6%). While non-pheresed sero-positive animals demonstrated significantly lower transduction levels (10.1 ± 6.0%). These data support the hypothesis that removal of AAV binding antibodies by plasmapheresis permits successful and sustained gene transfer in the presence of pre-existing immunity (natural infection) to AAV.Molecular Therapy (2013); doi:10.1038/mt.2013.244.

0 Followers
 · 
84 Views
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Objective Dysferlinopathies are a family of untreatable muscle disorders caused by mutations in the dysferlin gene. Lack of dysferlin protein results in progressive dystrophy with chronic muscle fiber loss, inflammation, fat replacement, and fibrosis; leading to deteriorating muscle weakness. The objective of this work is to demonstrate efficient and safe restoration of dysferlin expression following gene therapy treatment.Methods Traditional gene therapy is restricted by the packaging capacity limit of adeno-associated virus (AAV), however, use of a dual vector strategy allows for delivery of over-sized genes, including dysferlin. The two vector system (AAV.DYSF.DV) packages the dysferlin cDNA utilizing AAV serotype rh.74 through the use of two discrete vectors defined by a 1 kb region of homology. Delivery of AAV.DYSF.DV via intramuscular and vascular delivery routes in dysferlin deficient mice and nonhuman primates was compared for efficiency and safety.ResultsTreated muscles were tested for dysferlin expression, overall muscle histology, and ability to repair following injury. High levels of dysferlin overexpression was shown for all muscle groups treated as well as restoration of functional outcome measures (membrane repair ability and diaphragm specific force) to wild-type levels. In primates, strong dysferlin expression was demonstrated with no safety concerns.InterpretationTreated muscles showed high levels of dysferlin expression with functional restoration with no evidence of toxicity or immune response providing proof of principle for translation to dysferlinopathy patients.
    03/2015; 2(3). DOI:10.1002/acn3.172
  • [Show abstract] [Hide abstract]
    ABSTRACT: Recently adeno-associated virus (AAV) became the first clinically approved gene therapy product in the western world. To develop AAV for future clinical application in a widespread patient base, particularly in therapies which require intravenous (i.v.) administration of vector, the virus must be able to evade pre-existing antibodies to the wild type virus. Here we demonstrate that in mice, AAV vectors associated with extracellular vesicles (EVs) can evade human anti-AAV neutralizing antibodies. We observed different antibody evasion and gene transfer abilities with populations of EVs isolated by different centrifugal forces. EV-associated AAV vector (ev-AAV) was up to 136-fold more resistant over a range of neutralizing antibody concentrations relative to standard AAV vector in vitro. Importantly in mice, at a concentration of passively transferred human antibodies which decreased i.v. administered standard AAV transduction of brain by 80%, transduction of ev-AAV transduction was not reduced and was 4000-fold higher. Finally, we show that expressing a brain targeting peptide on the EV surface allowed significant enhancement of transduction compared to untargeted ev-AAV. Using ev-AAV represents an effective, clinically relevant approach to evade human neutralizing anti-AAV antibodies after systemic administration of vector.
    Biomaterials 06/2014; 35(26). DOI:10.1016/j.biomaterials.2014.05.032 · 8.31 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Gene therapy strategies for congenital myopathies may require repeat administration of adeno-associated viral (AAV) vectors due to aspects of the clinical application, such as: (i) administration of doses below therapeutic efficacy in patients enrolled in early phase clinical trials; (ii) progressive reduction of the therapeutic gene expression over time as a result of increasing muscle mass in patients treated at a young age; and (iii) a possibly faster depletion of pathogenic myofibers in this patient population. Immune response triggered by the first vector administration, and to subsequent doses, represents a major obstacle for successful gene transfer in young patients. Anti-capsid and anti-transgene product related humoral and cell-mediated responses have been previously observed in all preclinical models and human subjects who received gene therapy or enzyme replacement therapy (ERT) for congenital myopathies. Immune responses may result in reduced efficacy of the gene transfer over time and/or may preclude for the possibility of re-administration of the same vector. In this study, we evaluated the immune response of a Pompe patient dosed with an AAV1-GAA vector after receiving Rituximab and Sirolimus to modulate reactions against ERT. A key finding of this single subject case report is the observation that B-cell ablation with rituximab prior to AAV vector exposure results in non-responsiveness to both capsid and transgene, therefore allowing the possibility of repeat administration in the future. This observation is significant for future gene therapy studies and establishes a clinically relevant approach to blocking immune responses to AAV vectors.
    08/2014; 1:14033. DOI:10.1038/mtm.2014.33

Full-text

Download
5 Downloads
Available from
Apr 17, 2015