Editing the Genome with Cas9 mRNA
Danny Crawford, PhD, Director, Nucleic Acid Process Sciences, Intellia Therapeutics
Intellia Outlines Plans for mRNA and CRISPR
Combining CRISPR-Cas9 gene editing with the power of emerging mRNA could yield the next generation of therapies, according to Intellia.
The coronavirus pandemic firmly established the concept — and efficacy — of mRNA-based vaccines. Similarly, researchers have been working on using mRNA to replace missing proteins as a treatment for disease for decades.
But the medical applications of mRNA do not stop there, according to Danny Crawford, director of nucleic acid process sciences at Intellia Therapeutics, who set out the rationale for combining mRNA technology with therapeutic gene editing at TIDES USA.
“mRNA as a medicine is a really new concept. Just 60 years ago Sid Brenner and team identified mRNA. In the decades that followed, people understood that it had potential application as a medicine application, but there were a lot of challenges to overcome.”
Crawford cited immunogenicity, explaining that while efforts to make mRNA sequences safer from an immunological standpoint began in the 1980s, it was only relatively recently that a solution was found.
“In the mid-2000s, work by Drew Weissman and Katalin Karikó showed modified nucleotides could really dampen the impact of the innate immune response with mRNA. That, of course, earned them the Nobel Prize in collaboration with how all of their work was applied toward providing what really became the first idea of mRNA medicine that all of us came to know.”
And work on mRNA medicine — vaccines in particular — has continued to advance with, according to Crawford, COVID-19 being the obvious example.
“In 2020, the world went into lockdown. Nobody had heard of mRNA before, but by 2021, everyone in the world was getting dosed with mRNA. It’s kind of amazing to see what that how that happened,” he said
Therapeutic applications
As the pandemic ebbs, more therapeutic applications of mRNA are likely to emerge, Crawford said, citing his firm’s efforts to combine it with gene editing technology as an example.
“There’s a pretty diverse landscape of ways that you can apply mRNA. In general, you want to deliver it to a cell. Lipid nanoparticles are a very good way to deliver mRNA to a cell. Now what can that mRNA do? Well, one approach is protein replacement therapies.
“Of course, vaccines really came into everybody’s focus a few years ago, but there’s some really amazing work going on. And then what we’re doing, which is using gene editing tools with mRNA. Gene editing tools really are a great way to use mRNA.”
Mechanism of action
Intellia’s approach is based on the delivery of the Cas9 protein, which is used in the CRISPR-Cas9 gene editing system.
The basic idea is to introduce, by injection, a lipid nanoparticle that contains mRNA coding for the protein to induce changes in the cell’s genetic information such that a disease state is corrected, and the patient is cured.
Crawford told delegates, “An in vivo dose is an IV infusion of a lipid nanoparticle that contains an mRNA and a single guide RNA, and it gets recognized by the LDL receptor in a common pathway and via endocytosis gets taken into hepatocytes.
“At the point endosomal release occurs and two things are released into the cell - Cas9 mRNA and single guide RNA. The Cas9 mRNA is translated into the Cas9 protein, which complexes with the single guide RNAs and goes up into the nucleus where it can do the gene editing.
“The gene editing results in insertion or deletion events where we’re targeting insertion of a stop codon, a premature stop codon, such that when the mRNA for the target protein we’re trying to hit is expressed, it gets decayed and we get systemic knockdown of protein throughout the body,” Crawford said.
Pipeline
In March, Intellia announced it had administered the first dose of its most advanced mRNA-based gene editing therapy — NTLA-2001 — in a phase 3 clinical trial for the treatment of transthyretin amyloidosis (ATTR).
Crawford said, “ATTR is a rare disease, which is caused by accumulation of the TTR protein. It misfolds and generates amyloid plaques in peripheral nerve neurons or in cardiac tissue.
“Of course, there’s a lot of great work that has shown the knockdown of the TTR gene or the mRNA. Using siRNA or something can really change the outcome of patients with this. Our approach is to permanently knock it down using our in vivo platform.”
He added, “The hope is with a single dose, we can reduce the mutant protein and potentially be able to address both the polyneuropathy and the cardiomyopathy indications.”