(J-389) CRISPRa of Novel Gene, BLST, Amplifies Cartilage Deposition in Engineered Disc

Introduction:: CRISPRa screens have led to our lab's discovery of a previously unknown function for a zinc finger protein, which we are calling BLST. BLST appears to regulate senescence, cell activity, and protein processing with the potential to be used as a novel cell engineering tool. Disc-like angle ply structures (DAPS) are artificial total IVD replacements that have shown promise as therapeutic options to treat severe cases of degenerative disc disease. Here we are testing BLST’s ability to amplify cell phenotype with targeted gene expression using CRISPR-activation (CRISPRa). Utilizing synergistic CRISPRa upregulation of aggrecan (ACAN), collagen-II (Col2), and BLST in ASCs, we hypothesize that we can amplify cartilaginous ECM deposition in DAPS without the use of growth factors.

Materials and Methods:: ACAN/Col2-NTC and BLST upregulated human adipose-derived stem cells (ASCs) were pellet cultured for 21-days after which pellets were harvested, fixed, and submitted for histology (n=5), or designated for biochemical analysis (n=10). For DAPS seeding, ACAN/Col2-NTC and -BLST edited cells were seeded onto electrospun polycaprolactone (PCL) annulus fibrosus and agarose nucleus pulposus scaffolds and cultured for 2.5 weeks prior to combining scaffolds and culturing for an additional 2.5 weeks (Figure 1I, n=6-9). After 5 total weeks of culture DAPS were harvested, fixed, and submitted for histology.


Results, Conclusions, and Discussions:: BLST ­­upregulated ASCs show increases in cartilage ECM deposition in pellet culture (Figure 1A/B) showing significant increases in size (Figure 1C) with no significant increase in number of cells per pellet (Figure 1D), indicating BLST-edited cells are significantly more efficient at producing cartilage compared to the -NTC. BLST-edited pellets show significant increases in collagen and sGAG deposition per pellet, as well as the percent of collagen and sGAG retained per pellet (Figure 1E-H). BLST-edited cells were able to produce 9 successful DAPS after 5-weeks of culture compared to the -NTC which initially seeded 6 DAPS and 3 failing, due to annulus layers not adhering to one another, prior to the 5-week time point (Figure 1J). The resulting data indicates BLST upregulation increases proliferation rates of ACAN/Col2/BLST-ASCS, improving the production rate of DAPS. Furthermore, BLST-edited DAPS show significant increases in collagen and sGAG deposition after 5-weeks of culture compared to the -NTC (Figure 1K-M). BLST upregulation shows promise as a tool to amplify cartilage cell phenotype and improve cartilage deposition in cell therapy and tissue engineering strategies, such as DAPS. This research examines the effects of CRISPRa upregulation of ACAN/Col2/BLST in ASCs for use in a total IVD cartilage tissue engineering scaffold. Our results indicate that synergistic upregulation of ACAN/Col2/BLST drastically increases cartilage deposition in DAPS without growth factors. The identification of a novel gene, BLST, which amplifies cartilage deposition in vitro showcases the potential of BLST as a tool to amplify cell phenotype and ECM deposition. These results show that the increased cartilage deposition seen in DAPS can improve the therapeutic potential of total IVD replacements utilizing DAPS and ACAN/Col2/BLST ASCs.­





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