Abstract

Cell and tissue engineering based approaches have garnered significant interest for treating intervertebral disc degeneration and associated low back pain due to the substantial limitations of currently available clinical treatments. Herein we present a clustered regularly interspaced short palindromic repeats (CRISPR)-guided gene modulation strategy to improve the therapeutic potential of cell and tissue engineering therapies for treating intervertebral disc disease. Recently, we discovered a zinc finger (ZNF) protein, ZNF865 (BLST), which is associated with no in-depth publications and has not been functionally characterized. Utilizing CRISPR-guided gene modulation, we show that ZNF865 regulates cell cycle progression and protein processing. As a result, regulating this gene acts as a primary titratable regulator of cell activity. We also demonstrate that targeted ZNF865 regulation can enhance protein production and fibrocartilage-like matrix deposition in human adipose-derived stem cells (hASCs). Furthermore, we demonstrate CRISPR-engineered hASCs ability to increase GAG and collagen II matrix deposition in human-size tissue-engineered discs by 8.5-fold and 88.6-fold, respectively, while not increasing collagen X expression compared to naive hASCs dosed with growth factors. With this increased tissue deposition, we observe significant improvements in compressive mechanical properties, generating a stiffer and more robust tissue. Overall, we present novel biology on ZNF865 and display the power of CRISPR-cell engineering to enhance strategies treating musculoskeletal disease. STATEMENT OF SIGNIFICANCE: This work reports on a novel gene, ZNF865 (also known as BLST), that when regulated with CRISPRa, improves cartilagenous tissue deposition in human sized tissue engineering constructs. Producing tissue engineering constructs at human scale has proven difficult, and this strategy presents a broadly applicable tool to enhance a cells ability to produce tissue at these scales, as we saw an ∼8-88 fold increase in tissue deposition and significantly improved biomechanics in large tissue engineered intervertebral disc compared to traditional growth factor differentiation methods. Additionally, this work begins to elucidate the biology of this novel zinc finger protein, which appears to be critical in regulating cell function and activity.

Talk to us

Join us for a 30 min session where you can share your feedback and ask us any queries you have

Schedule a call

Disclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.