CRISPR Regulation of ZNF865 Rescues Human Nucleus Pulposus Cell Populations from Senescence

Introduction:

Introduction: Pathology of the intervertebral disc (IVD) and associated back pain is a major healthcare concern in the US with health care costs exceeding $100 billion annually. About 40% of this back pain can be attributed to degeneration of the IVD. Degeneration of the IVD has been closely associated with IVD cell senescence, with many cells present in degenerative IVD tissue demonstrating a senescent state. Current treatments for IVD degeneration are mainly palliative, aimed at reducing pain, providing a critical need for the development of novel treatments for IVD degeneration. Cell engineering has provided the opportunity to tune cell function to address therapeutic needs and discover novel biology. Recently, in a set of CRISPRa genome wide screens, our lab identified a novel zinc finger protein previously unpublished on, ZNF865, referred to here as BLST, that produces robust cell-engineering phenotypes relating to cell cycle, protein processing and cellular senescence. Preliminary data from publicly available RNAseq datasets suggested that BLST is differentially regulated within degenerating human IVDs. Here we investigate BLST within primary human IVD samples to determine relative levels of gene expression within degenerative IVDs and examine the ability for CRISPR regulation of BLST to regulate senescence within these human primary IVD cells.

Materials and

Methods: Materials and

Methods: Human healthy IVD samples were obtained from surgical tissue waste from deidentified trauma patients. Degenerative IVD samples were obtained from surgical tissue waste from deidentified patients undergoing surgery for axial back pain with degenerative disc disease. Nucleus pulposus (NP) from IVD samples was macroscopically dissected and digested with pronase and collagenase to release cells from native ECM, then plated onto tissue culture plastic. NP cells were then cultured to 90% confluency and then passaged into new plates for processing. Relative gene expression in native degenerative samples was measured by qRT-PCR after one passage, normalized to the youngest patient (n=4). Degenerative NP samples were transduced with lentiviral vectors targeting BLST for upregulation (CRISRPa) at passage 2 (n=3). Proliferation was measured by fluorescent cell counting for 4 days starting 1-week post-transduction (n=4). Senescence was evaluated using SA-β-gal staining, p16 and p21 immunofluorescence staining 1-week post-transduction (n=4). Immunofluorescence staining for BLST was performed 10 days post-transduction of lentiviral vectors. ATAC-seq was performed on degenerative (n=2), healthy (n=2) and degenerative CRISPRa-BLST (n=2).

Results, Conclusions, and Discussions:

Results: Fold difference in BLST expression showed that BLST expression decreases with increased age (1A). Fold change gene expression showed significant activation by VPR (1B). Upregulation of BLST within degenerative IVD cells showed increased BLST staining per cell (1C) and increased proliferation (1D) compared to the same naive degenerative cells. Upregulation of BLST also showed decreased levels of senescence staining in SA-β-gal, p16 and p21 compared to naïve degenerative cells, while not significantly different from healthy cells after BLST upregulation (1E,1F). Upregulation of BLST in degenerative IVD cells also rescued the chromatin structure to more closely match healthy cells by reducing chromatin differences by ~93% compared to the healthy IVD cells (1G, H). One-way (qRT-PCR, SA-β-gal immunofluorescence) and Two-way (proliferation) ANOVA were used to determine significance ( = 0.05).

Discussion: This research examines the novel BLST gene within degenerative IVD tissue and further investigates the effects of CRISPR regulation of the BLST gene within IVD samples in vitro. Overall, our results suggest that BLST may be linked to aging and degenerative disc disease of the IVD, as we saw that decreased BLST expression was correlated with increased patient age. Moreover, we show that targeted upregulation of BLST can be used to increase the proliferation rate of degenerative primary cells and eliminate senescent markers within these degenerative IVD cells. Our results also suggest that CRISPR regulation of BLST may be used as a tool to restore degenerative cells to a healthy state more broadly as we saw that by upregulation of BLST, the degenerative cells were rescued from a senescent phenotype and began to resemble the healthy phenotype more closely as shown by the number of differential chromatin sites dropping by 93% between the degenerative vs healthy and the degenerative VPR BLST vs healthy samples.

Acknowledgements (Optional):