Wednesday, April 2, 2025
4/2/2025
Novel Zinc-Finger Protein Regulator of Senescence and Cell Engineering - Cell engineering has provided the opportunity to tune cell function to address therapeutic needs and discover novel biology. We recently performed a set of experimental CRISPR gene perturbation screens that indicated a gene, ZNF865, which is only known through sequence analysis and does not have any in depth publications to date, has significant effects on cell function. Our preliminary data indicate that ZNF865 regulates many genes that modify the cell's overall activity regarding cell cycle, protein processing, senescence, and RNA transport in multiple unrelated cell types. ZNF865 appears to regulate these functions in all human cell types, implying a wide range of potential therapeutic applications and a need to understand the biology of this gene more broadly. Our group has utilized advancements in CRISPR-based cell engineering to engineer cells to tightly control their phenotype by modulating cell gene activation/repression in a highly targeted and multiplexed manner. Our preliminary data demonstrates that we can engineer cells that show dramatic increases in chondrogenic tissue deposition without growth factors after CRISPRa upregulation of ZNF865 in chondrogenic engineered cells. Additionally, our preliminary data demonstrate that CRISPRi targeting of ZNF865 drives a senescent phenotype in primary cells across multiple cell types. Finally, we are able to cause human degenerative IVD cells to escape senescence and the senescence-associated secretory phenotype by upregulating ZNF865 with CRISPRa. Our overall hypothesis is that ZNF865 acts as a transcription factor to regulate key processes in cell activity/senescence that can be regulated by CRISPRi/CRISPRa to control cell activity levels, improve cell therapeutic outcomes, and manipulate senescence. Aim 1: We will modulate ZNF865 with CRISPR in the disc to regulate cell activity and senescence in the IVD. (Aim 1a). CRISPRi vectors downregulating ZNF865 will be delivered to the healthy rat IVD to investigate its ability to induce disc degeneration through the induction of senescence within the IVD, potentially developing a senescence-driven model of disc degeneration. (Aim 1b). CRISPRa vectors upregulating ZNF865 will be delivered after induction of disc degeneration using the rat annular puncture model to observe the ability for enhanced cell activity to protect and/or recover from disc degeneration. (Aim 1c) We will investigate ZNF865s interactions with the genome using ChIP-seq, which can be combined with rapid immunoprecipitation mass spectrometry of endogenous protein (RIME). Aim 2: We will modulate ZNF865 upregulation in multiplex CRISPR-engineered chondrogenic cells to understand its ability to boost cell therapy outcomes. (Aim 2a). ZNF865 will be modulated in engineered chondrogenic cells and investigated in human-sized engineered IVDs (DAPS) to investigate their ability to boost the production and maintenance of functional engineered human IVD tissue. (Aim 2b). DAPS with ZNF865-modified cells will be challenged subcutaneously in the rat to investigate the ability of CRISPR-engineered cells to produce functional tissue in vivo in human-sized DAPS.
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