CRISPR-Cas13d reduced viability of cultured uveal melanoma cells to below 5%
Uveal melanoma study targeting RNA instead of DNA
Targeting RASGRP3 RNA with CRISPR-Cas13d reduced the viability of all three cultured human uveal melanoma cell lines to below 5% 48 hours after treatment. The researchers delivered Cas13d mRNA and guide RNA together in lipid nanoparticles (LNPs). These results constitute a proof of concept demonstrated in cell culture. Tumor elimination and preservation of vision in animals or patients were not assessed. The study was published in Molecular Therapy Oncology.
Uveal melanoma arises from melanocytes in the iris, ciliary body, and choroid, and is the most common primary intraocular malignancy in adults. Local treatments include radiation and surgery, but treatment options for metastatic disease are limited. To target intracellular proteins that are difficult to target with existing drugs, the research team tested a strategy to degrade the RNA that encodes those proteins.
Exploiting both cancer-cell dependency and high expression
The researchers analyzed DepMap CRISPR gene-knockout data covering more than 1,000 cancer cell lines, along with TCGA and GTEx expression data, and selected RASGRP3 as a candidate. This gene showed high expression in uveal melanoma, whose cells exhibited a selective dependency on it for survival. The RasGRP3 protein helps relay RAS–MAPK signaling involved in cancer-cell proliferation, but it is difficult to target with existing approaches because it is located inside cells and lacks a clear drug-binding site.
Cas13d is an enzyme that cleaves RNA specified by a guide RNA. The research team tested 42 guides targeting RASGRP3 and selected g4. Separately, they compared the delivery efficiency of 30 LNP formulations using mRNA encoding the fluorescent protein GFP, then loaded Cas13d mRNA and g4 together into the selected LNP formulation. The final formulation was designed to act at the RNA level rather than permanently edit DNA.
Responses diverged between cancer and control cell lines after 48 hours
After Cas13d-LNP treatment, mean viability measured by flow cytometry was 4.73% in the uveal melanoma cell line 92-1, 3.90% in MP-41, and 2.80% in MP-46. The control cell lines HEK293T and HeLa, neither of which is a uveal melanoma cell line, had viabilities of 86.33% and 84.34%, respectively; the non-cancerous retinal pigment epithelium model ARPE-19 had a viability of 90.73%. Control-cell viability was similar to that of untreated controls. However, the controls also included cell lines derived from other cancers, so these results cannot be generalized to the safety of normal tissues overall.
In a separate comparison using 92-1 cells, Cas13d reduced viability more than Cas9 gene knockout and siRNA suppression. However, the delivery methods and assessment time points differed, and the researchers noted that the Cas9 and siRNA used in the comparison may not have been optimized. The result is difficult to interpret as evidence of general superiority over other technologies.
Target suppression and collateral RNA degradation acted together
The researchers interpreted the killing effect as arising from both a reduction in RASGRP3 mRNA and “collateral RNA cleavage.” After cleaving abundant target RNA, Cas13d can also degrade non-target RNA. At 12 hours after treatment, the non-target GAPDH RNA had also declined markedly in some cancer cell lines, but changed little in ARPE-19.
In a rescue experiment, the researchers added a copy of RASGRP3 that encodes the same protein but is not recognized by the guide. Viability partially recovered, from about 3% to about 46%. This supports the interpretation that collateral RNA degradation, in addition to suppression of the target gene, contributes to cell death.
Further studies need to examine differences in RASGRP3 expression among tumors, resistance arising from evasion of target recognition, biodistribution, and immune responses to repeated dosing. Delivery to the eye or to liver metastases also remains to be tested. The study design and results are available in the full paper and the bibliographic and abstract record.
Uveal melanoma, the most common eye cancer in adults, remains limited to surgical intervention and radiotherapy, with a dismal survival rate that has not improved in over 50 years. To address this therapeutic impasse, we systematically analyzed public gene expression and CRISPR knockout datasets, identifying
This study demonstrated, in a uveal melanoma cell model, a strategy for targeting at the RNA level a protein that is difficult for drugs to bind. In particular, it used both cancer-cell gene dependency and the abundance of the target RNA to harness Cas13d’s collateral RNA degradation for an anticancer effect. The linked process—from target discovery to guide selection and delivery-vehicle optimization—also offers a research framework for testing other cancer targets. The strategy’s value, however, depends on whether its strong killing effect and selectivity are maintained in complex living systems. Efficacy, tissue toxicity, and delivery feasibility need to be tested in primary cells, tumor models with heterogeneous target expression, and animals before the rationale for clinical development can be assessed.