Special Poster Session 50th International Society for the Study of the Lumbar Spine Annual Meeting 2024

INTRODUCTION

Degenerative Disc Disease (DDD), a major contributor to low back pain, is characterized by the expression of pro-inflammatory and pro-catalytic genes in the Intervertebral Disc (IVD). Mesenchymal stem cells (MSCs) have displayed promise treating IVD degeneration due to their ability to promote tissue regeneration and reduce inflammation. However, the degenerated microenvironment of the IVD limits the effectiveness of cellular therapy. MSC-derived Extracellular Vesicles (EV) are a promising approach for an acellular regenerative treatment of the IVD. EVs are lipid membrane nanoparticles with crucial roles in cellular communication. MSC-derived EVs have been shown to regulate inflammation and senescence in IVD cells, potentially overcoming the limitations of cellular regenerative therapy. CRISPR-based genomic engineering could be used to increase the therapeutic effect of EVs. Specifically,  a dCas9-CRISPR activation system could induce the overexpression of genes involved in the therapeutic effect of MSC-derived EVs. Gene TSG6 has been identified as a critical regulator enhancing the regenerative and protective abilities of MSCs. Hence, the primary objective of this study is to augment the therapeutic impact of  EVs by amplifying TSG6 expression within MSCs through a CRISPR activation system.

METHODS

Immortalized human MSCs were transduced with a CRISPR activation lentivirus and gRNA vectors targeting gene TSG6 . Transduction activation efficiency was evaluated through RT-qPCR (n=3) and Western Blot (WB) analyses. Extracellular vesicles (EVs) derived from the TSG6-activated MSCs were isolated by ultracentrifugation. EV particle number and size distribution were determined using nanoparticle tracking analysis (NTA). The morphology and composition of the EVs were assessed through TEM and WB, respectively. Human IVD cells obtained from patients undergoing spinal surgery were isolated, expanded, and subjected to IL-1β pre-stimulation (2.5 ng/ml). Subsequently, these cells were treated with MSC-derived EVs (1000 particles per cell) (n=5). The gene expression levels of pro-inflammatory markers were measured using RT-qPCR.

RESULTS

The expression levels of TSG6 showed a significant increase of 130.6 ± 12.67-fold (n=3; p<0.05) in transduced MSCs compared to controls. IL-1ß pre-stimulation further increased TSG6 expression to 184-fold ± 39.11 (n=3; p<0.05) [FIG.1A]. Protein expression visibly increased in TSG6 transduced cells compared to control [FIG.1B].  Preliminary data suggests that these EVs have the capacity to decrease the expression of pro-inflammatory markers such as IL-6, IL-8, IL-1β, and COX2 in human IVD cells. The strongest effects were observed on IL-8 IL-1ß, and COX-2 [FIG.2].

DISCUSSION

To summarize, we've successfully created a lineage of MSCs that exhibit enhanced TSG6 expression, allowing for the extraction of EVs displaying distinctive traits in morphology, marker expression, and effectiveness in IVD cell activity. Despite observing variability in therapeutic effectiveness among donors, CRISPR-modified MSC-derived EVs exhibit potential for addressing disc degeneration. Our ongoing and forthcoming experiments aim to delve deeper into the potential of these EVs, including examining prolonged changes in IVD gene expression, alterations in extracellular matrix (ECM) composition, as well as assessing cell survival and proliferation.

The development of an MSC lineage that overexpresses TSG6 to generate EVs marks a potential paradigm shift in treating not only IVD degeneration but also various other degenerative ailments.