CRISPR-dCAS9 genomic engineering for boosting the therapeutic potential of Extracellular Vesicles

Degenerative disc disease is a major contributor to low back pain, characterized by inflammation of the intervertebral disc (IVD), degradation of the extracellular matrix, loss of hydration, and cell death. Current therapies fail to address these underlying mechanisms, underscoring the need for regenerative strategies. Mesenchymal stem cells (MSCs) exhibit immunomodulatory and regenerative potential, but their efficacy is hampered by the harsh microenvironment of the degenerated IVD. Acellular MSC-derived extracellular vesicles (EVs) have shown therapeutic potential and offer a promising alternative for IVD regeneration. Here, we explore CRISPR-dCas9 mediated activation of TSG6 and STEAP3 to boost the therapeutic potency and biogenesis of MSC-derived EVs, respectively. This project seeks to determine the feasibility of using CRISPR-dCas9 activation to enhance the regenerative capacity of MSC-EVs in an IVD (Aim 1) and a macrophage treatment model (Aim 2), and to determine the feasibility of using CRISPR multiplexing for advancing EV therapy (Aim 3). CRISPRa-mediated activation of TSG-6 in MSCs produced EVs that exerted anti-inflammatory effects on both human IVD cells and macrophages, partially exceeding those of control MSCs. Small RNA-seq and proteomic analyses of these EVs confirmed enrichment of anti-inflammatory microRNAs and proteins. Proof-of-concept multiplexed activation of TSG-6 and STEAP3 in pluripotent stem cells was successfully achieved, yielding a modest increase in EV production upon differentiation into MSCs. These findings validate CRISPR-dCas9 activation as a robust strategy to improve the therapeutic efficacy and production of MSC-derived EVs, and they establish the feasibility of gene-multiplexing approaches for future regenerative-medicine applications.