A gene editing technology based on CRISPR was able to slow the progression of amyotrophic lateral sclerosis in a mouse model, a new study showed, demonstrating the approach’s potential for gene therapy in people with ALS.
The study, “Treatment of a Mouse Model of ALS by In Vivo Base Editing,” was published in Molecular Therapy.
Around a fifth of familial ALS cases, and 1% to 2% of sporadic ALS cases, involve mutations in the gene SOD1 (Cu-Zn superoxide dismutase 1). While it is not fully understood how these mutations contribute to ALS, reducing levels of mutant SOD1 protein has shown benefit in animal models of the disease.
Yet most current ways of doing this are burdensome, requiring routine administration of a therapeutic, often by direct injection into the fluid around the brain and spinal cord.
An alternate approach is to remove, or turn off, the mutant SOD1 gene by gene editing. Researchers here tested the use of a type of gene editing technology, called cytidine base editors (CBEs), for this purpose.
CBEs are a type of CRISPR single-base editor. Traditional CRISPR gene editing involves making a cut to both strands of DNA, disrupting the gene. While theoretically suitable for the removal of mutant SOD1, this process risks introducing other, unwanted genetic changes, so it is not really being considered for wide use.
Single-base editors like CBEs change just one nucleotide base (one ‘letter’) of the DNA sequence. Specifically, CBEs change cytosines (C) to thymines (T).
The researchers designed a CBE-based system that would make a change that induces a ‘stop’ signal early in the SOD1 gene. Functionally, such a mutation would prevent the production of mutant SOD1 protein by stopping the cell’s machinery from reading its RNA in full. (RNA is the intermediate molecule that gives rise to a protein from a gene.)
This CBE worked reasonably well in cells in dishes, but using it in living animals posed practical challenges. To get the CBE into cells, the researchers had to give the cells genes that code for the CBE; the cell would then make the CBE protein, and the CBE would then do its job.
In animals — whether mice or humans — getting genetic material into cells is typically done using a viral vector. In gene therapy, the vector most commonly used for this purpose is the adeno-associated virus (AAV), which is favored for safety reasons, as this virus is not known to cause disease in people.
But AAV is a relatively small virus, and a lot of genetic information is needed to encode a full CBE — more than can be fit in a single AAV. To circumvent this problem, the researchers developed a system in which the full CBE-encoding genetic material is split between two AAVs, each encoding half of the CBE. Then, when the two protein halves are manufactured in cells, they can work together as a single, functional CBE.
After confirming that this approach worked in ways that compared favorably to the original CBE in cell cultures, the researchers tested this two-part system in a mouse model of ALS with a mutant SOD1 gene. They then measured disease progression, primarily by assessing changes in the animals’ weight.
Mice treated with the SOD1-editing CBE lived about 11% longer (139.4 vs. 127.0 days) than untreated mice serving as controls. No difference was seen in disease onset, but the length of time with disease was extended by an average of about 39%. In particular, there was a roughly 85% increase in the duration of late disease.
Treated mice also had significantly greater grip strength than controls at 17 weeks (119 days) after treatment, and over the course of their disease, treated mice lost weight about 43% more slowly than control mice.
“We were excited to find that many of the improvements happened well after the onset of the disease,” study co-author Michael Gapinske, a graduate student at the University of Illinois, said in a press release. “This told us that we were slowing the progression of the disorder.”
Additional tissue analysis suggested an increase in motor neuron survival in treated mice, and a decrease in the number of aggregates containing a misfolded SOD1 protein in the cells of the nervous system.
“Base editing reduced the accumulation of SOD1 immunoreactive inclusions in certain areas of the spinal cord by up to 40% and decreased the rate of muscle atrophy and muscle denervation,” the researchers wrote. “This work thus establishes that CRISPR base editors can be used to treat a neurodegenerative disorder.”
Future work will be needed to optimize the editing and to minimize off-target edits, which remain a concern. This strategy might also be applicable in other conditions, such as Duchenne muscular dystrophy and spinal muscular atrophy.
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