Immunity and AAV-Mediated Gene Therapy for Muscular Dystrophies in Large Animal Models and Human Trials

Adeno-associated viral (AAV) vector-mediated gene replacement for the treatment of muscular dystrophy represents a promising therapeutic strategy in modern medicine. One major obstacle in using AAV vectors for in vivo gene delivery is the development of host immune responses to the viral capsid protein and transgene products as evidenced in animal models and human trials for a range of genetic diseases. Here, we review immunity against AAV vector and transgene in the context of gene delivery specific to muscles for treating muscular dystrophies and non-muscle diseases in large animal models and human trials, factors that influence the intensity of the immune responses, and immune modulatory strategies to prevent unwanted immune responses and induce tolerance to the vector and therapeutic gene for a successful gene therapy

Duchenne muscular dystrophy

Progress in understanding the role of dystrophin raises promising hopes for a treatment for Duchenne muscular dystrophy. In addition, great improvements have been made in the ability to diagnose this disease using simple molecular methods.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/29288/1/0000349.pd

Immune Responses to rAAV6: The Influence of Canine Parvovirus Vaccination and Neonatal Administration of Viral Vector

Recombinant adeno-associated viral (rAAV) vectors promote long-term gene transfer in many animal species. Significant effort has focused on the evaluation of rAAV delivery and the immune response in both murine and canine models of neuromuscular disease. However, canines provided for research purposes are routinely vaccinated against canine parvovirus (CPV). rAAV and CPV possess significant homology and are both parvoviruses. Thus, any immune response generated to CPV vaccination has the potential to cross-react with rAAV vectors. In this study, we investigated the immune response to rAAV6 delivery in a cohort of CPV-vaccinated canines and evaluated multiple vaccination regimens in a mouse model of CPV-vaccination. We show that CPV-vaccination stimulates production of neutralizing antibodies with minimal cross-reactivity to rAAV6. In addition, no significant differences were observed in the magnitude of the rAAV6-directed immune response between CPV-vaccinated animals and controls. Moreover, CPV-vaccination did not inhibit rAAV6-mediated transduction. We also evaluated the immune response to early rAAV6-vaccination in neonatal mice. The influence of maternal hormones and cytokines leads to a relatively permissive state in the neonate. We hypothesized that immaturity of the immune system would permit induction of tolerance to rAAV6 when delivered during the neonatal period. Mice were vaccinated with rAAV6 at 1 or 5 days of age, and subsequently challenged with rAAV6 exposure during adulthood via two sequential IM injections, 1 month apart. All vaccinated animals generated a significant neutralizing antibody response to rAAV6-vaccination that was enhanced following IM injection in adulthood. Taken together, these data demonstrate that the immune response raised against rAAV6 is distinct from that which is elicited by the standard parvoviral vaccines and is sufficient to prevent stable tolerization in neonatal mice

Cross-species conservation of a polymorphic dinucleotide repeat in the dystrophin gene

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/46994/1/335_2004_Article_BF00292158.pd

Dystrophin‐deficient mdx mice display a reduced life span and are susceptible to spontaneous rhabdomyosarcoma

Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/154288/1/fsb2fj067353com.pd

Onset of experimental severe cardiac fibrosis is mediated by overexpression of angiotensin-converting enzyme 2

Angiotensin-converting enzyme (ACE) 2 is a recently identified homologue of ACE. There is great interest in the therapeutic benefit for ACE2 overexpression in the heart. However, the role of ACE2 in the regulation of cardiac structure and function, as well as maintenance of systemic blood pressure, remains poorly understood. In cell culture, ACE2 overexpression led to markedly increased myocyte volume, assessed in primary rabbit myocytes. To assess ACE2 function in vivo, we used a recombinant adeno-associated virus 6 delivery system to provide 11-week overexpression of ACE2 in the myocardium of stroke-prone spontaneously hypertensive rats. ACE2, as well as the ACE inhibitor enalapril, significantly reduced systolic blood pressure. However, in the heart, ACE2 overexpression resulted in cardiac fibrosis, as assessed by histological analysis with concomitant deficits in ejection fraction and fractional shortening measured by echocardiography. Furthermore, global gene expression profiling demonstrated the activation of profibrotic pathways in the heart mediated by ACE2 gene delivery. This study demonstrates that sustained overexpression of ACE2 in the heart in vivo leads to the onset of severe fibrosis

Inhibitory Control Over Ca2+ Sparks via Mechanosensitive Channels Is Disrupted in Dystrophin Deficient Muscle but Restored by Mini-Dystrophin Expression

Background: In dystrophic skeletal muscle, osmotic stimuli somehow relieve inhibitory control of dihydropyridine receptors (DHPR) on spontaneous sarcoplasmic reticulum elementary Ca release events (ECRE) in high Ca external environments. Such 'uncontrolled' Ca sparks were suggested to act as dystrophic signals. They may be related to mechanosensitive pathways but the mechanisms are elusive. Also, it is not known whether truncated dystrophins can correct the dystrophic disinhibition. Methodology/Principal Findings: We recorded ECRE activity in single intact fibers from adult wt, mdx and mini-dystrophin expressing mice (MinD) under resting isotonic conditions and following hyper-/ hypo-osmolar external shock using confocal microscopy and imaging techniques. Isotonic ECRE frequencies were small in wt and MinD fibers, but were markedly increased in mdx fibers. Osmotic challenge dramatically increased ECRE activity in mdx fibers. Sustained osmotic challenge induced marked exponential ECRE activity adaptation that was three times faster in mdx compared to wt and MinD fibers. Rising external Ca concentrations amplified osmotic ECRE responses. The eliminated ECRE suppression in intact osmotically stressed mdx fibers was completely and reversibly resuscitated by streptomycine (200 μM), spider peptide GsMTx-4 (5 μM) and Gd (20 μM) that block unspecific, specific cationic and Ca selective mechanosensitive channels (MsC), respectively. ECRE morphology was not substantially altered by membrane stress. During hyperosmotic challenge, membrane potentials were polarised and a putative depolarisation through aberrant MsC negligible excluding direct activation of ECRE through tubular depolarisation. Conclusions/Significance: Dystrophin suppresses spontaneous ECRE activity by control of mechanosensitive pathways which are suggested to interact with the inhibitory DHPR loop to the ryanodine receptor. MsC-related disinhibition prevails in dystrophic muscle and can be resuscitated by transgenic mini-dystrophin expression. Our results have important implications for the pathophysiology of DMD where abnormal MsC in dystrophic muscle confer disruption of microdomain Ca homeostasis. MsC blockers should have considerable therapeutic potential if more muscle specific compounds can be found