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U Utah Researcher Aims to Enable Cancer Research with Inducible CRISPR Mouse Model


NEW YORK (GenomeWeb) – While cancer therapies that target the genetic drivers of a particular mutation hold great promise, identifying targets remains a challenge. To address this issue and with the support of a National Cancer Institute grant, a University of Utah scientist is developing a mouse melanoma model in which CRISPR/Cas9 gene editing can be induced to knock out currently undruggable genes after tumor formation.

If successful, the mouse model will allow cancer researchers to study the effects of inhibiting genes that are pharmacologically out of reach to identify new up- and downstream targets of known oncogenes.

Personalized medicines are helping usher in a new era in cancer treatment, with drugs like Genentech's Zelboraf and GlaxoSmithKline's Tafinlar, both of which target mutant BRAF to combat melanoma, receiving US Food and Drug Administration approval in recent years.

But other oncogenic drivers — such as NRAS, which is mutated in an estimated 20 percent of melanoma cases — remain elusive, leading drug developers to focus on other players in the oncogenic pathways.

As part of his efforts to develop new therapeutic strategies for melanoma, the University of Utah's Matthew VanBrocklin and colleagues at the Nevada Cancer Institute where he previously worked developed a mouse model in which genetic alterations implicated in the disease could be validated.

The model involves a viral vector, RCASBP(A), that is derived from the avian leukosis virus (ALV), according to a 2010 paper. The receptor for RCASBP(A) is encoded by the tumor virus A (TVA) gene, which is normally expressed in avian cells, and infection with ALV results in the stable integration of the virus into the genome of replicating cells.

When introduced into mammalian cells that express TVA, the viral vector can stably integrate into the cellular DNA and express any gene that has been inserted. Because the virus is replication-defective, multiple rounds of infection are possible.

"The ability of TVA-expressing mammalian cells to be infected by multiple ALV-derived viruses allows efficient modeling of human melanoma because multiple oncogenic alterations can be introduced into the same cell or animal without the expense or time associated with creating multiple strains of transgenic mice," the investigators noted in that paper.

This mouse model "is great for discovery" since different genetic mutations can be studied for their potential to trigger tumor formation, VanBrocklin told GenomeWeb. But he and his lab want to be able to look at undruggable genes involved in tumor maintenance, which are more relevant targets in terms of treating human disease.

"That's where CRISPR comes in," he said.

CRISPR involves the use of a nuclease known as Cas9 to induce double-strand DNA breaks. These breaks are targeted to specific locations in the genome using a synthetic RNA that guides Cas9.

Because the RCASBP(A) vector is too small to carry the components of a CRISPR/Cas9, VanBrocklin's team has modified lentiviral vectors. With the changes, they can be loaded with a Cre recombinase cassette, so that specific tumor suppressor genes can be silenced; a tet-on inducible expression system; and Cas9 guide RNAs targeting a gene of interest. A separate vector is loaded with Cas9 under the control of a tet-response element and an oncogene to induce tumor formation.

"The two vectors … integrate into the genome," VanBrocklin explained. "After the tumor has formed … we can turn on Cas9 [using the tet system] … and we can have loss of gene function. That's the hope."

With the NCI grant, VanBrocklin and his lab will first test their inducible CRISPR/Cas9 system in the previously developed mouse model, first going after NRAS. Should that effort work out, they will then move in to other genes implicated in NRAS tumor maintenance including CCND1 and SOS1.

He is also exploring the potential of the inducible CRISPR/Cas9 approach beyond melanoma, including in lung cancer using a KRAS mouse model, although that work is not covered under the NCI grant.

VanBrocklin's grant began on Jan. 1 and runs until Dec. 31, 2016. It is worth $194,445 in its first year.