Sixty percent of mutations causing Duchenne muscular dystrophy (DMD) occur within introns 44-55 of the DMD gene. The Spencer and Pyle Labs have designed a CRISPR/Cas9 platform that deletes introns 44 and 55 of the DMD gene to mimic a mild Becker muscular dystrophy (BMD) phenotype. This study compares the efficacy of two different Cas9 nucleases (either eSpCas9 or HypaCas9) in vitro to determine their respective efficiencies in comparison to wild type SpCas9. The data showed that editing efficiency of HypaCas9 was less efficient than either eSpCas9 or wild type SpCas9. Upon sequencing, it was revealed that the eSpCas9 plasmid sent from a company was actually identical to wild type SpCas9. Therefore, though we were not able to assess the efficiency of eSpCas9, the data revealed that hypaCas9 will not be a suitable substitute for wild type Cas9 due to its low efficiency.

Duchenne muscular dystrophy (DMD) is a progressive muscle wasting disorder with no cure. Patients with DMD typically have out-of-frame mutations in the DMD gene, which leads to lack of dystrophin protein. However, there is an allelic, milder disease, Becker muscular dystrophy (BMD), where in-frame mutations allow for some production of an internally deleted dystrophin, which is at least somewhat functional. Thus, a therapeutic strategy is to restore the DMD reading frame, thereby making a DMD mutation into a BMD mutation and likely leading to a milder clinical phenotype. Gene editing tools such as the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated protein (Cas) 9 system would allow for permanent gene reframing. Here we describe development of a CRISPR/Cas9 gene editing platform which deletes DMD exons 45-55, creating an in-frame mutation associated with a very mild disease course in BMD patients. This region also encompasses a hotspot of approximately half of all DMD patient mutations, meaning this single platform would be therapeutically relevant for a large percentage of patients.

We first demonstrate in vitro applicability of our CRISPR/Cas9 platform in DMD human induced pluripotent stem cells where we show restored dystrophin protein and function in cardiac and skeletal muscle cells in vitro and after engraftment in vivo. An alternative route of delivery besides cell therapy is direct delivery of CRISPR/Cas9 to muscle in vivo. To that end, we created a novel humanized dystrophic mouse model containing an out-of-frame DMD gene that we can target with our CRISPR/Cas9 platform, which is human sequence specific. We demonstrate restored dystrophin protein after application of our CRISPR/Cas9 platform through electroporation, viral delivery, and nanoparticle delivery in vivo. Although we have achieved highest efficiency of dystrophin generation using adeno-associated virus (AAV) as a delivery vehicle, this work also provides proof-of-principle development of novel nanoparticles as an alternative means to AAV to deliver gene editing platforms to muscle in vivo. Non-viral delivery strategies may be advantageous due to their potential to evade an immune response and achieve stem cell targeting. Future work will continue to improve both approaches for effective direct delivery of our CRISPR platform, which reframes the DMD gene for a large percentage of Duchenne patients.

Osteopontin (OPN) is one of the most highly up-regulated genes in Duchenne Muscular Dystrophy (DMD) patients and also in the mdx mouse. Single nucleotide polymorphisms in the SPP1 gene (OPN gene) are associated with changes in muscle strength and age of loss of ambulation in DMD patients. Previous work in the lab has shown that ablation of OPN in mdx mice significantly ameliorates their dystrophic phenotype by decreasing muscle fibrosis and increasing muscle regeneration and strength in young mice. These changes in the pathology of mdx mice were associated with a decrease in NKT and Gr-1+ cell populations, and decreased intramuscular TGF-β.

In this project, we expanded on these previous observations and further explored the ways in which OPN exerts its regulatory role in the dystrophic pathology. Our current study shows that OPN ablation in mdx mice induces long term benefits on dystrophic muscles. OPN-/-mdx (up to more than one year old) display increased muscle mass and myofiber size, which translates to an improved performance in wire, grip and pulmonary function tests in dystrophic mice.

We show here that OPN expression in dystrophic muscle impairs muscle regeneration indirectly, by skewing the macrophage population toward a pro-inflammatory/pro-fibrotic phenotype. OPN ablation in mdx mice leads to reductions in M1 and M2a macrophages and increases in M2c. These changes were also associated with increased macrophage expression of pro-regenerative factors, without significantly changing pro-fibrotic factor expression. Moreover, OPN may impact regeneration directly by impairing terminal differentiation of myoblasts. In addition, we showed that OPN may promote muscle fibrosis in dystrophic muscle by directly interacting with fibroblasts and increasing collagen expression. This effect of OPN in fibroblast cultures is likely dependent in cell-type specific post-translational modifications.

This study validates OPN as a therapeutic target in DMD and lays a foundation for therapies involving pharmacological targeting of OPN, by providing a mechanistic understanding of OPN’s relationship to the processes of immune response, regeneration and fibrosis in dystrophic muscle.

Duchenne muscular dystrophy (DMD) is an x-linked recessive lethal muscle wasting disease with no cure. DMD is often caused by out-of-frame mutations that result in a loss of dystrophin protein. However, there is an allelic and milder form of the disease, Becker muscular dystrophy (BMD), which is typically caused by in-frame mutations that result in somewhat functional dystrophin protein. Therefore, a promising strategy to treat DMD is to utilize gene editing tools such as the clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (CRISPR/Cas9) system to convert a DMD mutation into a BMD mutation resulting in a clinically milder phenotype. However, achieving safe and efficient systemic delivery of the CRISPR/Cas9 system remains a significant challenge for DMD as skeletal muscle comprises ~40% of the total body mass. Here, we develop and optimize non-viral nanoparticles and adeno-associated viral (AAV) carriers as approaches to achieve safe and efficient systemic CRISPR/Cas9 delivery. We successfully demonstrate CRISPR/Cas9 delivery in vitro using iteratively optimized polymer-based nanoparticles. While these nanocarriers perform well in vitro, they inefficiently traffic to skeletal muscle after systemic delivery in vivo. In order to improve nanoparticle-mediated delivery to muscle and muscle stem cells, we developed and screened an AAV peptide display library for potential peptide motif ligands that may mediate skeletal muscle and muscle stem cell entry. We identify novel AAV variants highly enriched in skeletal muscle and muscle stem cells. Future work will focus on validating AAV variants and examining whether the muscle-specific peptide motif ligands may improve nanoparticle entry in muscle. While nanoparticles are promising carriers of CRISPR/Cas9 due to their low immunogenicity, AAV carriers are typically administered at high viral doses and often lead to AAV-induced immunotoxicities that pose life threatening risks. Therefore, we characterize AAV-mediated immune responses that arise in response to double dosing AAV in a dystrophic mouse model, in which the first dose effectively immunizes the mice. We reveal the production of AAV-specific antibodies that leads to subsequent activation of the classical complement pathway and induction of pro-inflammatory cytokines only after the second administration of AAV. Single cell RNA-sequencing (scRNA-seq) similarly confirms pronounced activation primarily after the second dose of AAV. Future work will continue to understand and identify targetable genes, pathways, or immune cell subtypes that may facilitate development of mitigation strategies to improve the safety and efficacy of AAV-based therapies.

Duchenne muscular dystrophy (DMD) is one of the most common inherited, lethal childhood diseases. This X-linked recessive disorder is often caused by mutations in the DMD gene that leads to loss of functional dystrophin protein and results in myofibers that are susceptible to membrane rupture. Sarcolemmal fragility leads to chronic cycles of muscle degeneration and regeneration and a subsequent reprogramming of the muscle niche that includes unresolved inflammation, defective muscle stem cell activity, aberrant pathological fibrosis, intramuscular adipose tissue (IMAT) accumulation and ultimately failed regeneration. Our lab has shown that osteopontin (SPP1) is a critical regulator of DMD disease progression that links many of these processes through its complex biology. SPP1 is multifunctional matricelluar protein, encoded by the Spp1 gene, that is post-translationally modified in many ways, expressed by a host of different cells in the muscle niche and can bind to a plethora of different receptors. This complexity can greatly impact how SPP1 acts on target cells and the signaling cascades that result in DMD disease progression. In this dissertation, we utilized single cell RNA sequencing to unbiasedly explore how two cell-specific sources of SPP1, macrophage-derived and muscle stem cell-derived, affect cell-cell interactions in dystrophic muscle.Here we show that macrophage-derived SPP1 is an autocrine regulator of macrophage TGFβ1. We identified two novel adipogenically primed stromal cell populations that are regulated by macrophage-derived TGFβ and contribute to IMAT. Our work established a link between macrophage-derived SPP1, reduced macrophage-derived TGFβ and ectopic fatty infiltration that is a hallmark of progressive DMD. Additionally, we showed that muscle stem cell (MuSC)-specific SPP1 has an autocrine inhibitory effect on MuSC stemness and positive effect on the fibrotic and inflammatory phenotype of MuSCs. Endothelial cells greatly expand in the MuSC Spp1 cKO showing a pro-angiogenic, anti-inflammatory state. We hypothesize that MuSC-derived SPP1 crosstalks with endothelial cells (ECs), leading to reduced EC expansion and an anti-angiogenic, pro-inflammatory, activated state. Furthermore, we provide evidence that this MuSC-EC regulatory axis affects macrophage phenotype. Altogether, this work advances knowledge of cell-specific SPP1 biology that drives DMD disease progression.