Laying the Foundation for Neuromuscular Disease Gene Therapy

Neuromuscular diseases are inherited disorders that primarily affect the nervous system and muscle. Most neuromuscular diseases have no effective therapy. Advances in adeno-associated virus (AAV)-mediated gene therapy are now bringing hopes to alleviating these diseases at the genetic root. ^1,2 An outstanding example is the approval of Zolgensma, an AAV drug that changes the disease course of type I spinal muscular atrophy (SMA) patients by one-time intravenous administration, on May 24, 2019 by the U.S. Food and Drug Administration. ³ Despite the promise, there are still enormous challenges ahead due to the inherent complexity of individual neuromuscular disease, the scarcity of animal models, the difficulty in crossing the blood–brain barrier, the necessity for bodywide therapy, the immune toxicity associated with high-dose systemic delivery, and the small packaging capacity of the AAV vector. Preclinical animal studies are essential to establish the proof-of-principle and refine the gene therapy protocol before conducting human trials. In this issue of Human Gene Therapy, a collection of articles is presented to highlight some of the effort in addressing various barriers in neuromuscular disease gene therapy. ^{4 –6}

Friedreich's ataxia (FRDA) affects 1 in 20,000 to 50,000 people. It is caused by frataxin deficiency due to pathological expansion of guanine-adenine-adenine (GAA) trinucleotides in the intron 1 of the frataxin gene. Late-onset heart failure is a prominent cause of mortality. Yet, there is no good model that can reproduce the subclinical nature of the heart disease in human patients. The Ronald Crystal laboratory developed a cardiac-specific FRDA model that exhibited stress-induced cardiac manifestations reminiscent of the patient phenotype. ⁴ The authors then tested an AAVrh10 frataxin vector in the new model. Intravenous injection of 1 × 10¹⁰ viral genome particles of the vector effectively attenuated the heart disease. ⁴ The new FRDA model opens the door for testing and polishing gene therapy strategies for FRDA-associated cardiomyopathy.

The use of the endogenous promoter can limit untoward off-target expression and prevent toxic supraphysiological expression. However, endogenous promoters often exceed the packaging capacity of the AAV vector. Development of the highly abbreviated gene-specific promoter, although not a trivial task, would bring in obvious therapeutic advantages. To develop a minimized endogenous promoter for FRDA gene therapy, Marek Napierala and colleagues designed 19 constructs based on the frataxin gene sequence. ⁶ After testing in human cells (including cell lines and induced pluripotent stem cells) and mice, the authors successfully identified the desired promoter suitable for future FRDA gene therapy.

Route of delivery may profoundly impact gene therapy outcome. Superoxide dismutase 1 (SOD1) gene mutation causes amyotrophic lateral sclerosis (ALS). A promising therapy is to reduce expression of the mutant SOD1 protein with microRNA. Previous studies suggest that systemic delivery of a silencing microRNA with AAV prolonged the lifespan but did not improve respiratory function. Since ALS patients often die from respiratory failure, creative approaches are needed to overcome this hurdle. Nicole Nichols and colleagues hypothesized that intralingual injection can lead to mutant SOD1 silencing in the tongue and, through retrograde transport, in the hypoglossal motor neurons in the medulla. ⁵ This is expected to improve ventilation and swallowing. The authors detected abundant AAV in the tongue but not the medulla. Despite the lack of improvement in swallowing, respiration was significantly enhanced. ⁵ These results suggest that intralingual injection may be used to supplement systemic injection. Concomitant local and systemic therapy will likely maximize therapeutic benefits.

For ages, many neuromuscular diseases are considered incurable. This may no longer be that case in the era of AAV gene therapy. However, we must remember there are still a lot of unknowns in the process of benchside to bedside translation. Unpleasant surprises are an inherent feature of high-risk high-reward neuromuscular disease gene therapy. This is clearly evidenced by recently surfaced severe adverse events (renal failure in the Duchenne muscular dystrophy trial and death in the X-linked myotubular myopathy trial) following high-dose systemic AAV injection. ^{7 –10} There is no doubt that in-depth probing of available clinical data will shed light on the underlying cause(s). However, it is equally important to bring these clinical observations back to the laboratory to try to reproduce (or partially reproduce) patient findings in research animals. These animal models can then be used to test innovative strategies that are designed to reduce toxicity of systemic AAV gene therapy. Basic research is needed more than ever to eventually realize the immense therapeutic potential of gene therapy in neuromuscular disease patients.