Determining the efficacy of a novel muscle specific gene therapy for Kennedy’s Disease

Kennedy’s Disease (KD) is a progressive neuromuscular disorder primarily affecting men and is characterised by progressive weakness of the arms, legs and head region, leading to a loss of mobility and alteration of speech, swallowing and breathing, causing significant disability. KD is caused by a repeat expansion mutation within the Androgen Receptor (AR) gene, found on the X chromosome. All KD patients suffer from the same abnormal CAG repeat expansion of the AR gene, with expansions of >38 repeats resulting in KD. A successful gene therapy approach would therefore be applicable to all KD patients. Though reported to have a 1:50,000 male prevalence, our recent genetic investigations have revealed the predicted prevalence based on genetic data to be 1:6,887 males, suggesting many undiagnosed cases and extending the impact of a successful therapy. Currently, there are no effective treatments available for KD. The weakness in KD is caused by the degeneration of muscles and motor neurons innervating them. Substantial evidence from KD mouse models has surprisingly demonstrated that without expression of the mutant AR in skeletal muscle, there is no development of disease phenotype including no loss of motoneurons. With further evidence from patients, it is now well established that skeletal muscle is a primary and early site of pathology. Manifestation of KD is triggered by binding of expanded AR to androgens. Whilst androgen-reducing therapies are effective in disease models, they are limited in the clinic by unacceptable side effects associated with lowering testosterone levels long-term in males. Attempts to reduce mutant AR in KD mouse models, using antisense oligonucleotides (ASOs) have shown promise for reducing disease phenotypes. However, unlike our novel strategy, AR reduction was not specific to skeletal muscle, making occurrence of secondary side-effects in patients likely, as the AR has crucial roles in multiple tissues. Due to these secondary side effects, AR silencing in peripheral tissues is particularly undesirable when carried out long-term, as is necessary for treating KD given that patients usually experience a normal life expectancy. We have already developed innovative tools to reduce AR expression specifically in skeletal muscle approach and to date we have: 1) developed a system for reducing expression of AR - using a novel approach based on CasRx, a small ribonuclease that can be programmed to bind and cleave AR RNA. 2) established a strategy for delivering and restricting therapeutics specifically to skeletal muscle - by combining a novel AAV vector known as MyoAAV with a muscle-specific promoter. Our current challenge, addressed by this project, is that we need to demonstrate efficacy of our proposed therapeutic approach. We will test this a) in vitro in patient derived myogenic cells to first demonstrate functionality of our strategy within the context of disease and also to examine off-target effects of RNA silencing within an appropriate genetic background; and b) in vivo in a mouse model of KD which expresses the causative mutation resulting in the development of progressive neuromuscular deficits. We aim to develop a novel genetic-based therapeutic approach for treating the neurodegenerative phenotype of KD.