Harvard researchers have improved the gene-editing process to study and treat genetic disorders. The primary editing method is effective in human cells, targeting single-nucleotide variants with the ability to precisely correct pathogenic mutations or install more protective variants. While success in the laboratory has been promising, translating these effects into live systems where the therapeutic potential can be unlocked has proven challenging.
In a new paper, “Efficient Baseline Editing in Mouse Brain, Liver and Heart Using Dual AAVs,” published in Nature BiotechnologyIn this study, we detail the identification of bottlenecks that limit the efficacy of AAV upstream editing in vivo and the development of vectors with increased upstream editing expression, RNA stability and DNA repair modulation.
Prime Editing is an ingenious editing strategy that, in its core configuration, utilizes Cas9 from CRISPR and scaffolding RNA and a reverse transcriptase enzyme to create a precise and accurate editing system.
One advantage of pre-editing is that the system causes double breaks, and instead scratches one side to open the DNA. This approach reduces unwanted indels that can occur with other editing systems.
The method works well enough in a lab environment where you can place the editor close to its target, but the vivo has the added difficulty of having to navigate a complex environment. In the current round of improvement, the researchers looked to get the editing system to the right location in vivo.
The currently preferred method for targeted gene delivery is adeno-associated virus (AAV) vectors, which can deliver a small payload to a specific locus on the chromosome. This payload capacity is, unfortunately, too small to contain the primary release mechanism.
Some assembly required
The solution to the vector capacity problem was to split the main editor into smaller pieces that could fit within the AAV’s charge limit and could self-assemble when they were close together. With the vector case somewhat resolved, the team moved on to the next series of hurdles.
In a series of efforts, the teams took advantage of a hepatitis C virus regulatory element, strengthened the RNA scaffolds, transduced promoters, ran out the chamber, added a third AAV vector, improved the reverse transcriptase enzyme, modified Cas9 with a mutation, and made sure everything was working. , then remove any elements they can shrink back into two AAV vector systems.
The resulting prototype for enhanced primary editing, v3em PE-AAVs, was tested in mice. Therapeutically relevant levels of upstream editing have been achieved in the mouse brain (up to 42% efficiency), liver (up to 46%), and heart (up to 11%). In vivo, upstream editing resulted in no detection of off-target editing. As a proof of concept, a genetic alteration in the brain was specific for resistance to Alzheimer’s disease and a gene associated with liver cholesterol-induced coronary artery disease was targeted.
Kind of a big deal
These levels of mutation-correction and variant-rewriting warrant an often unflattering set of phrases in the scientific community because they really are considered groundbreaking, monumental, landmark, breakthrough, game-changing, unprecedented, milestone, yes, even paradigm shifting moment.
The genetic mechanisms that have been discovered in the past few decades and all the knowledge accumulated about the causal relationships between cellular machinery and disease pathogenesis means that one day we will be able to repair or replace defective components of our genome.
The latest version of the Enhanced ProtoEdit isn’t quite ready to patch the human genome for errors, but it is the closest thing on the planet to a future where we can change the genetic cards dealt at birth. A future in which cancer risk factors can be eliminated, and neurological disorders routinely prevented or reversed — a future in which the long list of known diseases is simply forgotten. This is a future in the testing phase right now.
Jessie R. Davis et al, Efficient baseline editing in mouse brain, liver, and heart using dual AAVs, Nature Biotechnology(2023). DOI: 10.1038/s41587-023-01758-z
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the quote: Enhanced Prime Editing Alteres Genes of Live Mice, Representing a Major Advance (2023, May 18) Retrieved May 18, 2023 from https://phys.org/news/2023-05-optimized-prime-genes-mice-major. html
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