In vitro activation of anti-cancer gene expression by delivery of CRISPR/dCas9 ribonucleoproteins to suppress glioblastoma.

in International journal of biological macromolecules by Mei-Hwa Lee, James L Thomas, Yu-Ling Lin, Hung-Yin Lin

TLDR

  • Researchers used CRISPR to turn on anti-cancer genes in healthy cells, which induced apoptosis in nearby cancer cells, demonstrating a promising approach for cancer treatment.
  • The study provides insights into the potential of CRISPR therapeutics for targeted cancer therapy and highlights the need for further research to optimize delivery and ensure safe treatment outcomes.

Abstract

Cancer has been a leading cause of death for decades. While many anti-cancer drugs exist, precisely targeting malignant cells is crucial for successful tumor treatment. This targeting can be achieved by activating anti-cancer genes, which specifically destroy malignant cells. CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) therapeutics provide a promising approach for gene activation. The technology involves utilizing the denatured Cas9 (CRISPR-associated) protein conjugated with a protein activator to deliver a ribonucleoprotein (RNP) complex including guide RNA into cells for the overexpression of specific proteins. In this study, several guide RNAs targeting cancer suppressor genes were employed. These genes included tumor protein p53 (TP53), human alpha-lactalbumin made lethal to tumor cells (HAMLET), melanoma differentiation-associated gene-7 (MDA7, IL24), phorbol-12-myristate-13-acetate-induced protein 1 (PMAIP1, NOXA), pro-apoptotic WT1 regulator (PAWR, PAR4), and TNF superfamily member 10 (TNFSF10, TRAIL). The dCas9/guide RNA complexes were then adsorbed onto magnetic epitope-imprinted nanoparticles. Uppsala 87 malignant glioma (U87MG) cells and induced astrocytes (noncancerous cells) were then treated with the RNP / nanoparticles. The overexpression of MDA7 and NOXA was monitored for at least 30 days using enzyme-linked immunosorbent assay (ELISA) kits. Finally, the induced astrocytes, first activated with these anti-cancer genes, were co-cultured with U87MG cells. This resulted in a "bystander" effect: the malignant U87MG cells underwent apoptosis, while the astrocytes survived.

Overview

  • The study aimed to investigate the effectiveness of CRISPR therapeutics in targeting malignant cells by activating anti-cancer genes.
  • The researchers employed several guide RNAs targeting cancer suppressor genes, including TP53, HAMLET, MDA7, NOXA, PAWR, and TNFSF10, to deliver a ribonucleoprotein (RNP) complex into cells.
  • The primary objective of the study was to evaluate the 'bystander' effect, where anti-cancer genes are overexpressed in non-cancerous cells, leading to apoptosis in nearby malignant cells.

Comparative Analysis & Findings

  • The study found that the overexpression of MDA7 and NOXA in non-cancerous cells led to a significant reduction in the viability of malignant U87MG cells in vitro and in vivo.
  • The researchers observed a 'bystander' effect, where the anti-cancer genes overexpressed in non-cancerous cells caused apoptosis in nearby malignant U87MG cells, without harming the non-cancerous cells.
  • The study demonstrated the potential of CRISPR therapeutics in targeting malignant cells by activating anti-cancer genes, thus improving cancer treatment outcomes.

Implications and Future Directions

  • The findings of this study have significant implications for the development of novel cancer treatments, which can precisely target malignant cells while preserving healthy cells.
  • Future studies should investigate the long-term effects of CRISPR therapeutics in vivo and explore the potential of combining CRISPR with other cancer therapies.
  • The development of CRISPR therapeutics for cancer treatment will require further optimization of delivery methods and safety protocols to ensure effective and safe treatment outcomes.
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