Poster U19 - Targeted Multiplex CRISPRa Upregulation of ACAN, Col2a1, and Novel Gene, ZNF865, Enhances Cell Infiltration, Cartilage Deposition and Mechanical Properties of Human-sized Engineered Discs

Introduction: Recently, a CRISPR-activation (CRISPRa) screen led to our lab's discovery of a previously unknown zinc finger protein, ZNF865. ZNF865 regulates cell senescence, activity, and protein processing which has shown potential as a cell engineering tool. Disc-like angle ply structures (DAPS) are artificial total intervertebral disc (IVD) replacements that have shown promise as therapeutic interventions to treat severe cases of degenerative disc disease (DDD). Here we are testing ZNF865’s ability to amplify cell phenotype using CRISPRa. Utilizing targeted CRISPRa upregulation of aggrecan (ACAN), collagen-II (Col2a1), and ZNF865 in human adipose-derived stem cells (hASCs), we hypothesize that we can improve cartilaginous ECM deposition and the mechanical properties of large human-sized DAPS.

Materials and

Methods: RNAseq was utilized to evaluate global changes in gene expression by comparing dCas9-VPR ACAN/Col2a1/ZNF865 (Figure 1A) and ACAN/Col2a1/NTC-edited hASCs. Large-sized DAPS were seeded with naïve hASCs+GFs or ACAN/Col2/ZNF865-edited hASCs. Briefly, seeded electrospun polycaprolactone (PCL) annulus fibrosus (AF) and agarose nucleus pulposus (NP) scaffolds were cultured for 2.5 weeks before combining scaffolds and culturing for a total of 10 weeks (n=3-5), after which DAPS were submitted for biochemical, mechanical, or histological evaluation.

Results, Conclusions, and Discussions: The top GO Biological Processes and KEGG Molecular Processes affected by ZNF865 include processes associated with mRNA processing, gene expression, translation, cell cycle, DNA replication, and cellular senescence (Figure 1B/C). Additionally, genes highly associated with healthy cartilage deposition show high levels of expression whereas genes associated with chondrogenic hypertrophy do not (Figure 1D). After 10 weeks of culture, large-sized DAPS show robust cartilage matrix deposition. Representative images show cell morphology in the NP (Figure 1E) and cell infiltration into the PCL fibers in AF of large-sized DAPS (Figure 1F). Representative images showing human-sized DAPS after seeding (Figure 1G). ZNF865-edited DAPS show increases in cell survival compared to unedited hASCs+GFs (Figure 1H). CRISPR-engineered DAPS show increased GAG and collagen deposition in both NP and AF of DAPS (Figure 1I-N), which results in enhanced mechanical properties compared to hASCs+GFs (Figure 1O). Taken together, our results suggest that ACAN/Col2/ZNF865-edited hASCs enhance cartilage deposition, cell infiltration and integration, and the mechanical properties of DAPS compared to hASCs+GFs. Overall, our data presents targeted regulation of ZNF865 as a powerful tool to enhance cell engineering outcomes for use in cell and tissue engineering therapies.

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