Using CRISPR to Remove Mutated Sequences of Nuclear DNA Required by Cancerous Cells

Fusion genes feature in many cancers, a form of mutation in which two genes are joined together, such as through deletion of the DNA sequences that normally separate the two genes. The resulting mutant fusion gene sequence encodes a fusion protein that can have novel effects, or in which both portions remain functional, but are now produced in at inappropriate times and in inappropriate amounts. This change in cell biochemistry can be important in driving cancerous behavior, and this appears to be the case in a meaningful fraction of cancer types.

Today's research materials discuss a clever use of CRISPR DNA editing techniques. CRISPR is used to induce targeted breaks in nuclear DNA at specific points relative to two well known fusion genes, with the result that the gene, if present, is skipped over and removed by the DNA repair mechanisms responsible for reassembling the broken chromosome. This same strategy could well be applied to a range of fusion genes in cancer. The most promising part of this approach is that it is very specific to the cells that exhibit this fusion gene mutation. Thus gene therapy vectors can be used deliver the CRISPR tools into tissues quite generally, with no detrimental effect on normal cells.

Scientists succeed in reprogramming the CRISPR system in mice to eliminate tumour cells without affecting healthy cells

Fusion genes are the abnormal result of an incorrect joining of DNA fragments that come from two different genes, an event that occurs by accident during the process of cell division. If the cell cannot benefit from this error, it will die and the fusion genes will be eliminated. But when the error results in a reproductive or survival advantage, the carrier cell will multiply and the fusion genes and the proteins they encode thus become an event triggering tumour formation. Many chromosomal rearrangements and the fusion genes they produce are at the origin of childhood sarcomas and leukaemias. Fusion genes are also found in among others prostate, breast, lung and brain tumours: in total, in up to 20% of all cancers.

Because they are only present in tumour cells, fusion genes attract a great deal of interest among the scientific community because they are highly specific therapeutic targets, and attacking them only affects the tumour and has no effect on healthy cells. And this is where the CRISPR technology comes into play. With this technology, researchers can target specific sequences of the genome and, as if using molecular scissors, cut and paste DNA fragments and thus modify the genome in a controlled way. In a new study, researchers worked with cell lines and mouse models of Ewing's sarcoma and chronic myeloid leukaemia, in which they managed to eliminate the tumour cells by cutting out the fusion genes causing the tumour.

"Our strategy was to make two cuts in introns, non-coding regions of a gene, located at both ends of the fusion gene. In that way, in trying to repair those breaks on its own, the cell will join the cut ends which will result in the complete elimination of the fusion gene located in the middle." As this gene is essential for the survival of the cell, this repair automatically causes the death of the tumour cell.

In vivo CRISPR/Cas9 targeting of fusion oncogenes for selective elimination of cancer cells

In the context of cancer gene therapy, it is clear that targeting a single gene is often insufficient to eliminate cancer cells - yet, many types of cancers are addicted to the presence of a single oncogenic event that can reprogram cells and initiate tumorigenesis. This is the case for the so-called fusion oncogenes (FOs), which are chimeric genes resulting from in-frame fusions of the coding sequences of two genes involved in a chromosomal rearrangement. While the nature of the FOs may be diverse, they are primarily classified as involving transcription factors or tyrosine kinases. Silencing of FO transcripts has been shown to inhibit tumor cell growth in vitro and in vivo, demonstrating FO addiction in many human cancers.

FOs are ideal therapeutic targets for the development of new directed cancer treatments, owing to their cancer-driving roles, their restriction to cancer cells and the reliance of tumors on them. Unfortunately, FOs are challenging to target directly with candidate drugs. The ability to precisely manipulate cancer cell genomes to correct or eliminate cancer-causing aberrations by highly-efficient CRISPR/Cas9 genome editing opens new possibilities to develop FO-targeted options to eliminate cancer cells. In the present study, we describe a simple and efficient genome editing strategy specifically targeting FOs in cancer cells. Our CRISPR/Cas9-based approach induces two targeted intronic double strand breaks in both genes involved in a FO that, importantly, produces a cancer cell-specific genomic deletion that is dependent on the presence of the FO, and has no effect on wild-type gene expression in non-cancer cells.

Comments

Last year there was the discovery that cancer cells secrete PD-L1 containing exosomes that mess with immune memory formation in the lymph nodes. Transplanting CRISPR edited cancer cells that can no longer produce PD-L1 containing exosomes (by slashing two genes needed to produce exosomes) seemed to act as a vaccine, no longer allowing PD-L1 competent cancers cells to hide from the mouse immune system. I could definitely see in vivo intra tumor injection of CRISPR as a form of "in situ vaccination" going foward:

https://www.genengnews.com/news/cancer-immunotherapy-may-run-afoul-of-exosomal-checkpoint-proteins/

"In a surprising result from the new paper, the researchers found that they could use CRISPR-edited, exosome-deficient cancer cells to induce an anti-cancer immune response that targeted tumors that normally resist immune attack.

The researchers first transplanted CRISPR-edited cancer cells unable to produce exosomes into normal mice and waited 90 days. They then transplanted unedited-and presumably immune-evading-cancer cells into the same mice. After having exposed the immune system to the CRISPR-edited, exosome-deficient cancer cells, the unedited cells were no longer invisible. Instead of ignoring these cells, the immune system mounted a vigorous response that targeted these formerly immune-evading cancer cells and prevented them from proliferating.

"The immune system develops an anti-tumor memory after being exposed to cancer cells that can't produce exosomal PD-L1. Once the immune system has developed memory, it is no longer sensitive to this form of PD-L1 and thus targets exosomal PD-L1-producing cancer cells as well," Blelloch said.

Another surprising result was achieved when both unedited and CRISPR-edited, exosome-deficient cancer cells were simultaneously transplanted into opposite sides of the same mouse. Though they were introduced at the same time, the CRISPR-edited cells proved dominant - they were able to activate the immune system, which then launched an attack that destroyed the unedited, supposedly immune-resistant tumors growing on the other side."

Posted by: jimofoz at October 17th, 2020 5:42 AM

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