Development of a Novel Gene Therapy & Investigation of Synthetic Gene Therapy Delivery Systems

Abstract

Dystroglycanopathies are a family of neuromuscular disorders, in which enzymes that glycosylate the protein dystroglycan and therefore play a key role in muscle structure, have reduced or nonexistent activity. For example, Limb-girdle muscular dystrophy type R9 is caused by a mutation in the FKRP gene, that encodes one of various enzymes that glycosylates the muscle membrane protein dystroglycan. The result is muscle degeneration and weakness, and palliative care is presently the only available treatment for dystroglycanopathy patients. We approached the need for treatment from a gene therapy perspective, focusing on two main ideas: 1) the development of a novel AAV gene therapy with which to treat limb-girdle muscular dystrophy type R9, and 2) the evolution of a synthetic nanoparticle with a long-range goal of improving tissue targeting and therapeutic gene delivery. Our research into AAV gene therapy led us to determine that removal of the untranslated regions of the FKRP gene increases protein expression. Following these in vitro results, we further verified the restoration of muscle strength and health in a 10-month-old LGMDR9 mouse model. Additionally, potential deleterious effects of AAV-FKRP gene therapy has created controversy in the field, and our data suggest that this is not an issue at the doses and vectors tested, as treated WT mice show no physiological evidence of harmful effects. However, AAVs are unavailable as a treatment for patients with preexisting immunity to the vector, such that alternative gene therapy delivery systems must be considered. Using customizable synthetic nanoparticles bearing a library of surface miniproteins that encapsulate their own mRNA, we selected for desired characteristics (i.e. tissue tropism) over multiple rounds of selection in vivo. This genetically coded library consisted of millions of nanoparticles, which we injected into mice for two rounds of in vivo selection for binding to specific cell types, such as skeletal muscle. Following each round, we sequenced nanoparticle mRNA in desired tissues, from which we then created a new library to be evaluated in vivo again. The results of this suggest common binding moieties in mini-protein binders on the surface of the nanoparticles. The goal is to identify synthetic particles bearing surface proteins that have high affinity for selected tissues that could eventually be used as gene therapy delivery mechanisms for neuromuscular disorders.