Musculoskeletal

Microphysiological Systems to Model Genome Editing Outcomes

Genome editing technologies have significant potential to treat a variety of devastating human diseases and disorders. Howeverthere are many possible adverse consequences that are unique to genome editing tools, such as genome integrity, immune responses, and loss of therapeutic efficacy due to cell turnover, for which there are currently are no optimal systems for rigorous assessment. Moreover, these consequences are unique to human physiology, genome sequence, and immune systems, and therefore typical animal models are not completely informative. Using advanced genome editing strategies and methods for engineering human microphysiological tissue systems that recapitulate human physiology and function, we will systemically evaluate tissue physiology, genomic alterations, tissue regeneration, and immune response in response to various genome editing strategies and delivery methods. Specifically, we will determine the role of resident tissue stem cells, cell turnover, and tissue injury and regeneration in the stability of genome editing. We will incorporate immune cells into these microphysiological tissues to understand the consequences of immunity to bacteria-derived genome editing components.

Representative Publication: Saha et al. 2021. The NIH Somatic Cell Genome Editing program.  Nature. 2021 592(7853):195-204. doi: 10.1038/s41586-021-03191-1.

Disease Models

Rheumatoid Arthritis and Vascular Inflammation Cardiovascular disease (CVD) is accelerated by the high inflammatory burden induced by autoimmune diseases such as rheumatoid arthritis (RA). Systemic effects of RA include skeletal muscle, the largest tissue in the body.  In RA, inflammation-initiated cascades promote muscle atrophy, reduce hypertrophy, and lead to muscle protein turnover. Joint pain and loss of muscle function leads to physical inactivity that perpetuates poor muscle function. Even though systemic inflammation can be reduced by current RA medications, these agents target rapidly dividing immune cells and systemic cytokines rather than terminally differentiated skeletal myocytes, which release pro-inflammatory cytokines, limiting effectiveness on CVD. Importantly, exercise appears to reduce the severity of RA, in part, by improving skeletal muscle function. To evaluate skeletal muscle function under normal and disease conditions, we developed a human skeletal muscle microphysiological system (myobundles) using myoblasts from individuals with RA or age-matched controls. RA myobundles show greater sensitivity to IFNg than age-matched controls, and low doses of IFNg treatment selectively activate pro-inflammatory genes in RA myobundles and reduce expression of myosin heavy and light chain.  When RA myobundles are treated with IFN and then linked to tissue engineered blood vessels (TEBVs), the myobundles induce a pro-inflammatory response in the TEBVs. We will test the hypotheses that (1) cytokines and myokines released by inflamed RA skeletal muscle directly affect blood vessels, increasing inflammation and atherosclerosis predisposition, and (2) exercise reduces RA muscle inflammation by improving RA skeletal muscle function and altering the cytokine profile secreted by muscle.

Representative Publication: Oliver et al. 2022. Tissue engineered skeletal muscle model of rheumatoid arthritis using human primary skeletal muscle cells.  J Tissue Eng Regen Med., 16(2):128-139. doi: 10.1002/term.3266.

Juvenile Dermatomyositis Dermatomyositis (DM) is a rare inflammatory vasculopathic disease that causes muscle weakness and characteristic rashes in affected adults as well as children with the juvenile-onset form of the disease (JDM). Muscle and skin in DM/JDM patients exhibit autoantibodies, CD4+ T cells, plasmacytoid dendritic cells, and B cells and damage to muscle capillaries. Current treatments include broad immunosuppressive and immunomodulatory medications, particularly corticosteroids, that are associated with significant toxicity. Study of disease mechanisms and novel therapeutics is limited by DM/JDM’s rarity and the lack of animal models with testable relevant features. While the mechanisms underlying DM/JDM are not fully known, a critical step involves overexpression of type I interferon (IFN I) α-or-β in muscle by dendritic cells or myoblasts. This leads to expression of major histocompatibility complex (MHC) class I antigens on myocytes, increased expression of autoantigens, including MDA5, Mi-2, and TIF1gamma. NF-kB activation leads to the production of cytokines TNFα, IL6, and IL18, among others. MHC class I upregulation induces endoplasmic reticulum (ER) stress which results in the degradation of contractile proteins further reducing skeletal muscle function. Cytokines present in the muscle lead to endothelial activation and recruitment of monocytes, dendritic cells and T cells. To address the limitation of suitable models for DM/JDM, we have developed an in vitro human skeletal muscle engineered tissue model (myobundles) in a 6 well plate format that integrates common pathogenic features found in DM/JDM arising from the downstream effects of IFNβ. The myobundles resemble the architecture of native skeletal muscle and reproduce key physiological functions, specifically twitch and tetanus contraction in response to electrical stimulation simulating nerve conduction. Preliminary results show that myobundles treated with IFNβ exhibit reduced contractile force, upregulation of MHC I, endoplasmic reticulum stress, and the presence of antigens that elicit autoantibodies in JDM.

Representative Publication: Covert, L.T., Patel, H., Osman, A., Duncan, L., Dvergsten, J., and Truskey, G. 2023. Effect of type I interferon on engineered human skeletal muscle: A promising model for juvenile dermatomyositis. Rheumatology kead186. https://doi.org/10.1093/rheumatology/kead186.