Biology subjects

Levis, H.

Publications and source records attributed to Levis, H..

3 recordsLinked to original sources

bioRxiv2023

ZNF865 Regulates Senescence and Cell Cycle for Applications to Cell Engineering and Gene Therapy

Zinc finger (ZNF) proteins represent the largest group of regulatory proteins within eukaryotic genomes. However, despite their broad regulatory function, the majority of ZNF protein function remains unknown. Recently, we discovered ZNF865, which has no in-depth publications and has not been functionally characterized. Utilizing CRISPR-guided gene modulation, we show that ZNF865 regulates key cellular and molecular processes associated with healthy cell function by primarily regulating cellular senescence, cell cycle progression, and protein processing. As a result, regulating this gene acts as a primary titratable regulator of cell activity, and we demonstrate the potential of targeted ZNF865 regulation as a tool to control senescence and protein production in multiple clinically relevant cell types for cell engineering/tissue engineering/gene therapy applications. We demonstrate its ability to rescue human senescent cell populations, boost T-cell activity, and dramatically deposit more cartilaginous tissue in a whole organ tissue-engineered intervertebral disc. Overall, we present novel biology and regulatory mechanisms of senescence and cell cycle that were previously unknown and display the power of CRISPR-cell engineering to enhance cell engineering strategies treating disease.

bioengineering↗

bioRxiv2023

Therapeutic TNF-alpha Delivery After CRISPR Receptor Modulation in the Intervertebral Disc

Low back pain (LBP) ranks among the leading causes of disability worldwide and generates a tremendous socioeconomic cost. Disc degeneration, a leading contributor to LBP, can be characterized by the breakdown of the extracellular matrix of the intervertebral disc (IVD), disc height loss, and inflammation. The inflammatory cytokine TNF- has multiple pathways and has been implicated as a primary mediator of disc degeneration. We tested our ability to regulate the multiple TNF- inflammatory signaling pathways in vivo utilizing CRISPR receptor modulation to slow the progression of disc degeneration in rats. Sprague-Dawley rats were treated with CRISPRi-based epigenome-editing therapeutics targeting TNFR1 and showed a decrease in behavioral pain in a disc degeneration model. Surprisingly, while treatment with the vectors alone was therapeutic, TNF- injection itself became therapeutic after TNFR1 modulation. These results suggest direct inflammatory receptor modulation, to harness beneficial inflammatory signaling pathways, as a potent strategy for treating disc degeneration.

bioengineering↗

bioRxiv2022

CRISPRi-Driven Osteogenesis in Adipose-Derived Stem Cells for Bone Healing and Tissue Engineering

Engineered bone tissue synthesized from mesenchymal stem cell progenitors has numerous applications throughout the fields of regenerative medicine and tissue engineering. However, these multipotent cells offer little tissue-building assistance without differentiation direction from environmental cues such as bone morphogenetic proteins (BMPs). Unfortunately, BMP dosing and environmental cues can be difficult to control both in vitro and after in vivo delivery. Several BMP antagonists are expressed by cells in response to BMP dosing that bind extracellular BMPs and reduce their effective concentration. Here, we use CRISPR-guided gene-modulation technology to downregulate the expression of three BMP antagonists, noggin, gremlin-1, and gremlin-2, in adipose-derived stem cells (ASCs). We show that regulating noggin using this method results in ASC osteogenesis without the need for exogenous growth factors. To demonstrate the versatility and the precision capabilities of these engineered cells, we employ them with CRISPRa multiplex-engineered chondrogenic cells as a proof-of-concept tissue engineering application by creating a tissue gradient similar to the fibrocartilage-to-mineralized-fibrocartilage gradient in the tendon/ligament enthesis or intervertebral disc attachment. In doing so, we show that multiple CRISPR multiplex engineered cell types can be utilized in concert to provide a high degree of tissue developmental control without the use of exogenous growth factors.

bioengineering↗