Genomic studies of cancer patients have revealed thousands of mutations linked to tumor development, but researchers are unsure how the vast majority of these mutations contribute to cancer because there’s no easy way to study them in animal models. Massachusetts Institute of Technology researchers have now developed a way to easily engineer specific cancer-linked mutations into mouse models, and used the CRISPR genome editing-based approach to generate models of several different mutations of the cancer-causing Kras gene in different organs.

They believe that their prime editing method could help scientists start to understand many unexplored mutations, and generate models that could be used to help identify test new drugs. The researchers have made mice with the prime editing system engineered into their genome available through a repository at the Jackson Laboratory, and they hope that other labs will begin to use this technique for their own studies of cancer mutations.

“This is a remarkably powerful tool for examining the effects of essentially any mutation of interest in an intact animal, and in a fraction of the time required for earlier methods,” said Tyler Jacks, PhD, the David H. Koch Professor of Biology, a member of the Koch Institute for Integrative Cancer Research at MIT.

Jacks is co-senior author of the team’s published paper in Nature Biotechnology, which is titled “A prime editor mouse to model a broad spectrum of somatic mutations in vivo.” Francisco Sánchez-Rivera, PhD, an assistant professor of biology at MIT and member of the Koch Institute, and David Liu, PhD, a professor in the Harvard University Department of Chemistry and Chemical Biology and a core institute member of the Broad Institute, are co-senior authors of the study.

Cancer is driven by somatic mutations that accumulate throughout disease progression, and which can occur in thousands of different combinations across a human cancer, the authors noted. “The precise nature of driver mutations and their combinations can profoundly influence how cancers initiate, progress and respond to therapy, establishing tumor genotype as a critical determinant of disease outcome.” Genetically engineered mouse models (GEMMs) have proven “invaluable” for elucidating the mechanisms by which cancer drivers promote tumor development and progression in vivo, the team continued. And testing cancer drugs in such models is an important step in determining whether they are safe and effective enough to go into human clinical trials.

Over the past 20 years, researchers have used genetic engineering to create mouse models by deleting tumor suppressor genes or activating cancer-promoting genes. However, this approach is expensive, labor intensive and requires several months or even years to produce and analyze mice with a single cancer-linked mutation. “Established GEMMs can also take months for investigators to acquire and often require laborious breeding programs to combine multiple alleles of interest and to establish a colony of sufficient size for experimental cohorts,” the investigators noted.

“A graduate student can build a whole PhD around building a model for one mutation,” said co-first author Zack Ely, PhD, a former MIT graduate student who is now a visiting scientist at MIT. “With traditional models, it would take the field decades to catch up to all of the mutations we’ve discovered with the Cancer Genome Atlas.”

In the mid-2010s, researchers began exploring the possibility of using CRISPR genome editing to make cancerous mutations more easily. Some of this work occurred in Jacks’ lab, where Sánchez-Rivera (then an MIT graduate student) and his colleagues showed that they could use CRISPR to quickly and easily knock out genes that are often lost in tumors. However, while this approach makes it easy to knock out genes, it doesn’t lend itself to inserting new mutations into a gene because it relies on the cell’s DNA repair mechanisms, which tend to introduce errors. “Advances in genome editing technologies have accelerated functional genetic studies, yet most approaches to model cancer mutations have relied on Cas9-mediated gene disruption via non-homologous end joining, failing to recapitulate many genetic lesions observed in human cancer,” the team noted.

Inspired by research from Liu’s lab at the Broad Institute, the MIT team wanted to come up with a way to perform more precise gene-editing that would allow them to make very targeted mutations to either oncogenes (genes that drive cancer) or tumor suppressors.

In 2019, Liu and colleagues reported a new version of CRISPR genome editing, known as prime editing. Unlike the original version of CRISPR, which uses the Cas9 enzyme to create double-stranded breaks in DNA, prime editing uses a modified Cas9 nickase enzyme, which is fused to a reverse transcriptase. This fusion enzyme cuts only one strand of the DNA helix, and so avoids introducing double-stranded DNA breaks that can lead to errors when the cell repairs the DNA.

“Prime editors employ a Cas9 nickase coupled with a reverse transcriptase that complexes with prime editing guide RNAs (pegRNAs), the scientists explained. These pegRNAs encode mutations of interest within a reverse transcriptase template (RTT), to enable highly precise and programmable editing. “Prime editing thus offers a versatile approach to study the full spectrum of cancer driver mutations, their combinations and the growing catalog of secondary mutations that confer resistance to targeted therapies,” the team stated.

The MIT researchers designed their new prime editing GEMMS (PE GEMM) by engineering the gene for the prime editor enzyme into the germline cells of the mice, meaning that it would be present in every cell of the organism. “Encoding the prime-editing machinery within the mouse germline also minimizes confounding acute or chronic antitumor immune responses that could be induced by exogenous delivery of a Cas9-based fusion protein,” they stated. The encoded prime editor enzyme allows cells to copy an RNA sequence into DNA that is incorporated into the genome. However, the prime editor gene remains silent until activated by the delivery of a specific protein called Cre recombinase.

Since the prime editing system is installed in the mouse genome, researchers can initiate tumor growth by injecting Cre recombinase into the tissue where they want a cancer mutation to be expressed, along with the guide RNA that directs Cas9 nickase to make a specific edit in the cells’ genome. The RNA guide can be designed to induce single DNA base substitutions, deletions, or additions in a specified gene, allowing the researchers to create any cancer mutation they wish.

“In conjunction with the development of PE GEMMs, we also developed a range of DNA vectors and engineered pegRNAs (epegRNAs) that promote efficient prime editing in a variety of cell lines and organoids derived from these mice.,” the investigators explained.

To demonstrate the potential of their technology, the researchers engineered several different mutations into the Kras gene, which drives about 30% of all human cancers, including nearly all pancreatic adenocarcinomas. However, not all Kras mutations are identical. Many Kras mutations occur at a location known as G12, where the amino acid glycine is found. Depending on the mutation, this glycine can be changed for one of several different amino acids.

The researchers developed models of four different types of Kras mutations found in lung cancer: G12C, G12D, G12R, and G12A. To their surprise, they found that the tumors generated in each of these models had very different traits. For example, G12R mutations produced large, aggressive lung tumors, while G12A tumors were smaller and progressed more slowly. “… we observed that different Kras mutations exhibit variable in vivo tumor-initiating potential, consistent with previous study comparing KrasG12C and KrasG12D autochthonous models in the pancreas,” they stated. “In the lung, we found that KrasG12A, KrasG12D and KrasG12R promote efficient but variable tumor formation.’

Learning more about how these mutations affect tumor development differently could help researchers develop drugs that target each of the different mutations. Currently, there are only two FDA-approved drugs that target Kras mutations, and they are both specific to the G12C mutation, which accounts for about 30% of the Kras mutations seen in lung cancer.

The researchers also used their technique to create pancreatic organoids with several different types of mutations in the tumor suppressor gene p53, and they are now developing mouse models of these mutations. They are in addition working on generating models of additional Kras mutations, along with other mutations that help to confer resistance to Kras inhibitors.

“One thing that we’re excited about is looking at combinations of mutations including Kras mutations that drives tumorigenesis, along with resistance associated mutations,” said co-first author Nicolas Mathey-Andrews. “We hope that will give us a handle on not just whether the mutation causes resistance, but what does a resistant tumor look like?” The authors further stated in their paper, “While we focused on installing somatic cancer driver mutations, we anticipate that PE GEMMs could be employed for broader applications … We believe that this approach will accelerate functional studies of cancer-associated mutations and complex genetic combinations that are challenging to construct with traditional models.” And with further developments, they suggested “… PE GEMMs can provide a rapid preclinical avenue to complement both fundamental and clinical investigations aimed at treating cancer with precision treatment paradigms.”

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