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 TNFa-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.
