Extracellular vesicles produced by CRISPR-activated MSCs: A potential therapy for degenerative disc disease

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PROJECT SUMMARY Degenerative disc disease (DDD) is a major source of low back pain, which is not only the leading cause of activity limitation and work absence but also represents a tremendous financial burden to the society. DDD is defined as symptomatic intervertebral disc (IVD) degeneration with pronounced catabolic and inflammatory responses. Inflammatory mediators play an indispensable role in pain development during DDD. As current treatment strategies for DDD are limited, often associated with high risks, and are not always long-lasting, great hopes were once pinned on stem cells due to their regenerative and anti-inflammatory potential. However, various studies (including ours) have demonstrated that the success of mesenchymal stem cell (MSC)-based treatments for DDD is limited due to the harsh microenvironment of the degenerated IVD that hampers cell survival and functionality. More recently, extracellular vesicles (EVs) derived from MSCs have been suggested as a potential alternative treatment for DDD. Importantly, as an acellular approach, survival challenges associated with cell-based therapies are eliminated. EVs are lipid membrane particles released from cells that function as nanocarriers and, when derived from MSCs, are thought to have regenerative capacity. Targeted genetic modification of MSCs, with overexpression of TNFα-stimulated gene-6 (TSG6), may promote the regenerative capacity of the released EVs by altering their content. TSG6 is specifically promising as its expression has been identified as a crucial regulator of the regenerative and tissue-protective capacity of MSCs. The long-term goal of this research is to pave the way toward novel, non-opioid, pain management therapies for patients suffering from DDD. Specifically, this project will determine the potential of different EV subpopulations released from MSCs that underwent CRISPR/Cas9 activation of TSG6. We will address this goal by: (1) Establishing the TSG6-modified MSCs line and characterizing cells and released EVs; (2) Evaluating the effect of different EVs size subpopulations derived from TSG6-modified MSCs (which may differ in cargo/uptake/mobility) on degenerated disc cells. We will determine the therapeutic potential of these EVs by evaluating the expression of proinflammatory cytokines, catabolic enzymes, nerve factors, and matrix proteins. We will combine two recent biotechnological developments with outstanding therapeutic potential (MSC-derived EVs and CRISPR/Cas9 genome engineering) with a technical invention for the isolation of EV subpopulations (nanopocket membrane). The concept of using CRISPR/Cas9 to improve the cargo of MSC-derived EVs for improved DDD treatment is highly novel. If successful, it promises to provide a new therapy to combat DDD. The proposed project is impactful due to the urgent need for new, targeted, and non-opioid treatment options for DDD patients, and hence, fits the R21 mechanism well because of its high risk/high gain nature. Moreover, results and developments are likely to find applications in other degenerative-inflammatory diseases.