Stress-Induced Mouse Model of the Cardiac Manifestations of Friedreich's Ataxia Corrected by AAV-mediated Gene Therapy.

Friedreich's ataxia (FA), an autosomal recessive disorder caused by a deficiency in the expression of frataxin (FXN), is characterized by progressive ataxia and hypertrophic cardiomyopathy. Although cardiac dysfunction is the most common cause of mortality in FA, the cardiac disease remains subclinical for most of the clinical course because the neurologic disease limits muscle oxygen demands. Previous FXN knockout mouse models exhibit fatal cardiomyopathy similar to human FA, but in contrast to the human condition, untreated mice become moribund by 2 months of age, unlike humans where the cardiac disease often does not manifest until the third decade. The study was designed to create a mouse model for early FA disease relevant to the time for which a gene therapy would likely be most effective. To generate a cardiac-specific mouse model of FA cardiomyopathy similar to the human disease, we used a cardiac promoter (αMyhc) driving CRE recombinase cardiac-specific excision of FXN exon 4 to generate a mild, cardiac-specific FA model that is normal at rest, but exhibits the cardiac phenotype with stress. The hearts of αMyhc mice had decreased levels of FXN and activity of the mitochondrial complex II/complex IV respiratory chain. At rest, αMyhc mice exhibited normal cardiac function as assessed by echocardiographic assessment of ejection fraction and fractional shortening, but when the heart was stressed chemically with dobutamine, αMyhc mice compared with littermate control mice had a 62% reduction in the stress ejection fraction (p < 2 × 10-4) and 71% reduction in stress-related fractional shortening (p < 10-5). When assessing functional cardiac performance using running on an inclined treadmill, αMyhc mice stayed above the midline threefold less than littermate controls (p < 0.02). A one-time intravenous administration of 1011 genome copies of AAVrh.10hFXN, an adeno-associated virus (AAV) serotype rh10 gene transfer vector expressing human FXN, corrected the stress-induced ejection fraction and fractional shortening phenotypes. Treated αMyhc mice exhibited exercise performance on a treadmill indistinguishable from littermate controls (p > 0.07). These αMyhc mice provide an ideal model to study long-term cardiac complications due to FA and AAV-mediated gene therapy correction of stress-induced cardiac phenotypes typical of human FA.

Biology of the Adrenal Gland Cortex Obviates Effective Use of Adeno-Associated Virus Vectors to Treat Hereditary Adrenal Disorders.

Congenital adrenal hyperplasia (CAH) is an autosomal recessive disorder occurring in 1:10,000 to 1:20,000 live births. In >95% of the cases, CAH results from mutations in the CYP21A2 gene, encoding the adrenal steroid enzyme 21-hydroxylase (21OH). Cardinal phenotypic features of CAH include genital ambiguity and sexual precocity, and in severe cases, neonatal salt loss and death. Current standard of care consists of lifelong oral steroid replacement to reverse the cortisol deficiency. Although significant advances in the treatment of CAH have been made, the burden of a lifelong therapeutic intervention is not ideal for quality of life. Gene therapy for CAH by adeno-associated virus (AAV) vectors has been shown to efficiently transduce the adrenal cortex, restoring normal steroidogenesis in the short term. However, adrenocortical cells are continuously renewed by stem cells located at the adrenal capsule, which differentiate as they centripetally migrate towards the adrenal medulla where they undergo apoptosis. In this context, we hypothesized that AAV-mediated genetic correction of the adrenal cortex will work short term but will eventually lead to a loss of correction. To test this hypothesis, we administered intravenously an AAV serotype rh.10 gene transfer vector (AAVrh.10-21OH-HA) to 21-hydroxylase deficient mice (21OH-/-). The data demonstrates that a single intravenous administration efficiently transduces adrenocortical cells leading to 21OH-HA expression and restoration of normal steroidogenesis. However, the duration of therapeutic efficacy lasted for only 8 weeks, accompanied by loss of 21OH-HA expression in the adrenal gland. Analysis in immunodeficient mice confirmed that the disappearance of transgene expression was not due to an antiviral/transgene immune response. Taken together, these results demonstrate that a single treatment with an adeno-associated viral vector expressing a functional copy of the mutated gene can only transiently treat adrenocortical hereditary disorders and that strategies to genetically modify the adrenocortical stem cells population will likely be required.

Anti-hIgE gene therapy of peanut-induced anaphylaxis in a humanized murine model of peanut allergy.

BACKGROUND: Peanuts are the most common food to provoke fatal or near-fatal anaphylactic reactions. Treatment with an anti-hIgE mAb is efficacious but requires frequent parenteral administration. OBJECTIVE: Based on the knowledge that peanut allergy is mediated by peanut-specific IgE, we hypothesized that a single administration of an adeno-associated virus (AAV) gene transfer vector encoding for anti-hIgE would protect against repeated peanut exposure in the host with peanut allergy. METHODS: We developed a novel humanized murine model of peanut allergy that recapitulates the human anaphylactic response to peanuts in NOD-scid IL2Rgammanull mice transferred with blood mononuclear cells from donors with peanut allergy and then sensitized with peanut extract. As therapy, we constructed an adeno-associated rh.10 serotype vector coding for a full-length, high-affinity, anti-hIgE antibody derived from the Fab fragment of the anti-hIgE mAb omalizumab (AAVrh.10anti-hIgE). In the reconstituted mice peanut-specific IgE was induced by peanut sensitization and hypersensitivity, and reactions were provoked by feeding peanuts to mice with symptoms similar to those of human subjects with peanut allergy. RESULTS: A single administration of AAVrh.10anti-hIgE vector expressed persistent levels of anti-hIgE. The anti-hIgE vector, administered either before sensitization or after peanut sensitization and manifestation of the peanut-induced phenotype, blocked IgE-mediated alterations in peanut-induced histamine release, anaphylaxis scores, locomotor activity, and free IgE levels and protected animals from death caused by anaphylaxis. CONCLUSION: If this degree of persistent efficacy translates to human subjects, AAVrh.10anti-hIgE could be an effective 1-time preventative therapy for peanut allergy and possibly other severe, IgE-mediated allergies.