HPV Oncogene Manipulation Using Nonvirally Delivered CRISPR/Cas9 or Natronobacterium gregoryi Argonaute

Abstract

CRISPR/Cas9 technology enables targeted gene editing; yet, the efficiency and specificity remain unsatisfactory, particularly for the nonvirally delivered, plasmid‐based CRISPR/Cas9 system. To tackle this, a self‐assembled micelle is developed and evaluated for human papillomavirus (HPV) E7 oncogene disruption. The optimized micelle enables effective delivery of Cas9 plasmid with a transient transgene expression profile, benefiting the specificity of Cas9 recognition. Furthermore, the feasibility of using the micelle is explored for another nucleic acid‐guided nuclease system, Natronobacterium gregoryi Argonaute (NgAgo). Both systems are tested in vitro and in vivo to evaluate their therapeutic potential. Cas9‐mediated E7 knockout leads to significant inhibition of HPV‐induced cancerous activity both in vitro and in vivo, while NgAgo does not show significant E7 inhibition on the xenograft mouse model. Collectively, this micelle represents an efficient delivery system for nonviral gene editing, adding to the armamentarium of gene editing tools to advance safe and effective precision medicine‐based therapeutics.

Delivery of CRISPR/Cas9 for therapeutic genome editing

“…This strategy was used to target the HPV18‐E7 oncogene associated with human papillomavirus pathogenesis. Proteasome activity can be decreased by 31.9% after transfection for 72 hours, whereas the cells showed 68.1% viability for the Cas9 control . Recently, a facile method to construct a multifunctional vector that can deliver CRISPR/Cas9 plasmids into cancer cells was also developed.…”

Delivery of CRISPR/Cas9 by Novel Strategies for Gene Therapy

“…Recently, researchers have proposed a self‐assembled micelle composed of quaternary ammonium terminated poly(propylene oxide) (PPO‐NMe 3 ) and amphiphilic Pluronic F127 that enables effective delivery of the Cas9 plasmid targeting the human papillomavirus (HPV) E7 oncogene and inhibits cancerous activity both in vitro and in vivo. These micelles represent an efficient delivery system for nonviral gene editing and add to the armamentarium of gene‐editing tools to advance safe and effective precision medicine‐based therapeutics …”

“…Quaternary ammonium terminated poly-(propylene oxide) (PPO-NMe 3 )a nd amphiphilic Pluronic F127 form al ower charge density,s elf-assembled micelle as the delivery platform for the plasmid-based CRISPR/Cas9 system, and it efficiently delivers Cas9 plasmida nd disrupts the HPV18-E7 gene to suppress cancer progression. [38] Other interesting research reportst he self-assembly of nucleic acids to construct a ChemBioChem 2019, 20,634 -643 www.chembiochem.org vector to deliver the CRISPR/Cas9s ystem. [39] However,c onsidering the essential drawback of nucleic acids, this strategy needs to combine other approaches to avoid their degradation and enhance their intracellulardelivery.Lately,nanoscale zeolitic imidazole frameworks (ZIFs) have been utilized to encapsulate CRISPR RNPs and to enhance the endosomal escape of payloads.…”

The Impact of CRISPR-Cas System on Antiviral Therapy

“…Applying CRISPR-Cas9 technology to target virus genomes and to find signaling pathways that are involved in virus infections Virus Gene target site in virus/Human Model Delivery Reference EBV EBNA1, LMP1, EBNA3CBurkitt’s lymphoma cell lines Raji cellTransfection 41 BVRF1Gastric Cancer Cell line, SUN719 and YCCEL1Transfection 42 BART5, BART6, or BART16gastric carcinoma cell line SNU-719Transduction 43 HTLV1 pX regionED T-CellTransduction 44 RNF8HeLa cellsElectroporation 45 JCV T-antigenHuman oligodendroglioma cell line, primary human fetal glial cellsTransfection 46 NCCR-a and VP1-bglial derived SVG-A cells and human fetal kidney derived hTERT transformed HuK(i)G10 cellsTransduction 47 HPV E6SiHa and CaSki cells (cervical carcinoma cell lines)Transfection 48 E7SiHa and Caski cellsTransfection 49 E7HeLa cellsNano-micelle 50 HSV UL8, UL29, and UL52Vero cellsTransduction 43 ICP0HEK293T cellsTransduction 51 UL7HEK293T cells, Vero cells and BALB/c mice…”