Muscle injury treatment by gene therapy associated with alginate hydrogel and autophagy induction

Abstract

Fibrosis is characterized by an excessive accumulation of extracellular matrix components and is one of the main sequelae acquired after muscle injury. It is most prevalent in severe injuries in which healing occurs slowly and prevents proper muscle contraction - which can lead to muscle contractures and chronic pain. Besides being an important aggravating factor of muscular dystrophies, it is also one of the main causes that lead professional athletes to abandon their activities early.The study of factors leading to the reduction of fibrosis and muscle regeneration is of great relevance. The use of angiotensin AT1 receptor blockers and the induction of the autophagy pathway have shown benefits in tissue regeneration. However, there is a need to better understand this mechanism in skeletal muscle. Several cells such as myoblasts, fibroblasts and stem cells participate in the process of muscle regeneration and it is important to understand the effect of silencing the AT1 receptor and the autophagy pathway in these cells so that it is possible to seek therapy. Therefore, in the in vitro part of the project, primary cultures of fibroblast, myoblasts, satellite cells, muscle-derived stem cell-like cells (MuSCs) and murine injury-derived stem cell-like cells (iMuSCs) will be established. These will be transduced with lentivirus expressing microRNA for the AT1a receptor and the effect of the microRNA on the autophagic flow will be observed. We will evaluate cell proliferation and migration as well as the expression of genes and proteins related to autophagy, fibrogenesis and myogenesis. In the in vivo stage of the project, we will propose a combined therapy for the treatment of anterior tibial muscle laceration (TA) in a murine model. Combinated therapy will be performed by injecting the lentivetor expressing the microRNA to AT1a receptor, via alginate hydrogel, associated with the autophagy pathway through the protein-poor diet (DPP). Histomorphometric, molecular and functional parameters will be evaluated at this stage. Thus, the project aims to clarify the role of AT1a receptor silencing and the induction of autophagy pathway in vitro and in vivo in cells derived from skeletal muscle, in addition to proposing a combined therapy for the treatment of muscle injury.