Nonviral Gene Therapy Platform Delivers CRISPR/Cas9 to Tumors

January 22, 2018

A platform for gene delivery and tumor therapy has been introduced that harnesses the power of the CRISPR/Cas9 gene-editing system. At the same time, the platform avoids some of the drawbacks of the CRISPR-Cas9 system. Specifically, the platform can cope with CRISPR-Cas9’s sheer bulk, achieving highly efficient and targeted delivery to tumor cells. At the same time, it provides a multifunctional bonus: the new system incorporates gold nanoparticles that can serve as thermotherapeutic agents.

The multifunctional gold-nanoparticle-based delivery vehicle was developed by a team of scientists based at the National Center for NanoScience and Technology, Beijing, China. In a paper (“Thermo-triggered Release of CRISPR-Cas9 System by Lipid-Encapsulated Gold Nanoparticles for Tumor Therapy”) that appeared January 15 in the journal Angewandte Chemie, the scientists described their system, which consists of Cas9-sgPlk-1 plasmids (CPs).

“We condensed CPs on TAT peptide-modified Au nanoparticles (AuNPs/CPs, ACPs) via electrostatic interactions, and coated lipids (DOTAP, DOPE, cholesterol, PEG2000-DSPE) on the ACPs to form lipid-encapsulated, AuNPs-condensed CPs (LACPs),” wrote the article’s authors. “LACPs can enter tumor cells and release CPs into the cytosol by laser-triggered thermo-effects of the AuNPs; the CPs can enter nuclei by TAT guidance, enabling effective knock-outs of target gene (Plk-1) of tumor (melanoma) and inhibition of the tumor both in vitro and in vivo.”

Laser irradiation was used to disassemble the lipid–nanogold vehicle after its entry in the tumor cells and enable the CRISPR-Cas9 gene editing. The knockout of the targeted gene then led to apoptosis and tumor growth inhibition.

Gold nanoparticles are especially attractive carriers for various biological molecules because of their easy modification, stability, and light-irradiation response. To convert them into a versatile biological transport and delivery vehicle, the scientists first attached Tat peptides—which facilitate the crossing of the cell nucleus membrane—to gold nanoparticles.

Then, the CRISPR-Cas9 plasmid containing the RNA targeting the Plk-1 gene—the knockout of which would severely impair tumor cell function—was attached to the Tat peptides through electrostatic interactions so that they would release their load right after entering the nucleus. Finally, the nanoparticular system was coated with a formulation of lipids to improve cellular uptake.

To test the system, cells and tumor-bearing mice were both administrated with the CRISPR-Cas9-plasmid-carrying nanogold vehicle, and the release of the gene-editing machine was triggered by a laser.

“In this study, light irradiation caused the release of the Tat peptide from the gold nanoparticles in a time- and laser-intensity-dependent manner,” the authors explained.

Nanoparticle-based systems are an attractive alternative to viral vectors, which can deliver CRISPR-Cas9 components efficiently but raise safety concerns such as off-target effects and, potentially, mutagenesis and carcinogenesis. Recently, a nanoparticle-based system developed by scientists based at the University of California, Berkeley, scientists who used it to repair the Duchenne muscular dystrophy gene in a mouse model.

In the current study, the focus was on delivering CRISPR/Cas9 to tumor cells: “The synergistic effects of the photothermo-mediated intracellular release of the CP and the subsequent CP-induced cell apoptosis render LACP a powerful tool for tumor therapy,” reported the study’s authors. “Furthermore, by combining interventional therapeutic techniques, LACP could extend its applications to tumors in deep tissues. Thus, our strategy provides a novel carrier for delivering various Cas9-sgRNAs and treating a wide spectrum of diseases by gene editing.”

This story originally appeared in Clinical OMICs' sister pulbication Genetic Engineering & Biotechnology News