A new technique to correct pathogenic mutations | MIT News


Gene editing, or the deliberate alteration of a gene’s DNA sequence, is a powerful tool for studying how mutations cause disease and for making changes to an individual’s DNA for therapeutic purposes. . A new method of gene editing that can be used for both purposes has now been developed by a team led by Guoping Feng, Professor James W. (1963) and Patricia T. Poitras in Brain and Cognitive Sciences at MIT.

“This technical breakthrough can accelerate the production of disease models in animals and, most importantly, opens up a whole new methodology for correcting disease-causing mutations,” says Feng, who is also a fellow of the Broad Institute at Harvard and MIT and the associate director of the McGovern Institute for Brain Research at MIT. The new findings were published online May 26 in the journal Cell.

Genetic models of the disease

A major goal of the Feng lab is to precisely define what is wrong with neurodevelopmental and neuropsychiatric disorders by creating animal models that carry the genetic mutations that cause these disorders in humans. New patterns can be generated by injecting embryos with gene editing tools, as well as a piece of DNA carrying the desired mutation.

In one of these methods, the CRISPR gene editing tool is programmed to cut a targeted gene, thereby activating the natural DNA mechanisms that “repair” the broken gene with the injected template DNA. The modified cells are then used to generate offspring capable of passing the genetic change on to subsequent generations, thus creating a stable genetic line in which disease and therapies are tested.

Although CRISPR has sped up the process of generating such disease models, the process can still take months or years. The reasons for the ineffectiveness are that many treated cells do not undergo the desired DNA sequence change at all, and the change occurs only on one of the two copies of the gene (for most genes, each cell contains two versions, one from the father and one from the mother).

In an effort to increase the efficiency of the gene editing process, the Feng lab team initially hypothesized that adding a DNA repair protein called RAD51 to a standard mixture of CRISPR gene editing tools would increase the chances that a cell (in this case, a fertilized mouse) egg or unicellular embryo) would undergo the desired genetic change.

As a test case, they measured the speed at which they were able to insert (“knock-in”) a mutation in the gene. Chd2 which is associated with autism. The overall proportion of correctly modified embryos remained unchanged, but to their surprise, a significantly higher percentage carried the desired genetic modification on both chromosomes. Tests with a different gene gave the same unexpected result.

“The simultaneous modification of the two chromosomes is normally very rare,” explains postdoctoral fellow Jonathan Wilde. “The high rate of editing seen with RAD51 was really striking, and what started out as a simple attempt to make mutants Chd2 mouse quickly turned into a much larger project focused on RAD51 and its applications in genome editing, ”said Wilde, co-author of the Cell paper with researcher Tomomi Aida.

A molecular copier

The Feng lab team then set out to understand the mechanism by which RAD51 improves gene editing. They hypothesized that RAD51 initiates a process called interhomologous repair (IHR), whereby a break in DNA on one chromosome is repaired using the second copy of the chromosome (from the other parent) as a template.

To test this, they injected mouse embryos with RAD51 and CRISPR but left out the template DNA. They programmed CRISPR to cut only the sequence of the gene on one of the chromosomes, then tested if it was repaired to match the sequence of the uncut chromosome. For this experiment, they had to use mice whose sequences on the maternal and paternal chromosomes were different.

They found that control embryos injected with CRISPR alone rarely showed RSI repair. However, the addition of RAD51 significantly increased the number of embryos in which the gene targeted by CRISPR was altered to match the uncut chromosome.

“Previous studies on IHR have found it to be incredibly ineffective in most cells,” says Wilde. “Our finding that it occurs much more easily in embryonic cells and can be enhanced by RAD51 suggests that a deeper understanding of what makes the embryo permissive to this type of DNA repair could help us design. safer and more effective gene therapies. “

A new way to correct pathogenic mutations

Standard gene therapy strategies that rely on injecting a piece of corrective DNA to serve as a template for repairing the mutation initiates a process called homology-directed repair (HDR).

“HDR-based strategies still suffer from low efficiency and carry the risk of unwanted integration of donor DNA throughout the genome,” explains Feng. “IHR has the potential to overcome these problems because it relies on the natural cellular pathways and the patient’s own normal chromosome for the correction of the deleterious mutation.

Feng’s team went on to identify other proteins associated with DNA repair that can stimulate IHR, including several that not only promote high levels of IHR, but also suppress errors in the repair process. DNA. Additional experiments that allowed the team to examine the genomic characteristics of RSI events have provided insight into the mechanism of RSI and suggested ways to use the technique to make gene therapies safer.

“While there is still a lot to learn about this new application of IHR, our findings are the basis of a new approach to gene therapy that could help solve some of the big problems with current approaches,” says Aida .

This study was supported by the Hock E. Tan and K. Lisa Yang Center for Autism Research at MIT, the Poitras Center for Psychiatric Disorders Research at MIT, a grant from the NIH / NIMH Conte Center, and the Office of the Director of the NIH.


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