Using CRISPR to Fight ALS

ALS (Amyotrophic Lateral Sclerosis), otherwise known as Lou Gehrig’s disease, is a fatal neurodegenerative disease that involves the loss of motor neurons in the spinal cord, brainstem, and motor cortex. This disease, estimated to be afflicting around 20,000 Americans, leads to progressive muscle weakness throughout the body. Paralysis and death occur within 3-5 years of the onset of symptoms. Currently, there is no cure for the disease and there is only one medication, riluzole, that has been shown to increase lifespan by only 2 months.

One of the prominent genes associated with the disease is SOD1. Dominant mutations in this gene account for about 20% of inherited forms of the disease and about 2% of all cases worldwide. This gene encodes a metalloenzyme that converts superoxide anions into oxygen and hydrogen peroxide and is therefore important in cellular antioxidant defense. Transgenic animals that express the mutant form of this gene develop the same symptoms of ALS, including motor neuron degradation, muscle wasting, and paralysis.

Recently, researchers at the University of California in Berkeley used the gene-editing technology, CRISPR-Cas9, to disrupt the mutant SOD1 gene expression in the spinal cord of mice with ALS, effectively extending their lifespan by 25%.

CRISPR-Cas9 is a genome-editing tool that is more effective and – perhaps surprisingly – cheaper than previous techniques. It was developed after discovering that some forms of bacteria have a similar system of gene editing to fight invading pathogens or viruses, acting as a sort of immune system. The system was adapted to be used in animals and has two main features, Cas9 and guide RNA. The Cas9 protein acts as molecular scissors, cutting two strands of DNA at a specific location so that nucleotide bases can be added or removed. Cas9 finds this specific location thanks to a piece of guide RNA, which is a small pre-designed strand of RNA with bases that are complementary to the target DNA sequence that is to be edited. Hypothetically, the guide will only bind to the target sequence, making this system particularly accurate.

The research team engineered a viral vector containing the Cas9 gene. The mice were injected with the viral genomes; upon entrance of the virus into the nucleus of cells, the Cas9 gene is transcribed into its protein code (mRNA), then after leaving the nucleus it is translated into its protein, molecular scissors, which cut and disable the mutant SOD1 gene. They specifically found that the start of the disease was delayed about five weeks when treated using the CRISPR-Cas9 technology, and they also lived about a month longer than the usual four-month lifespan of a mouse with ALS. They also found that the only surviving motor neuron cells in the mice at disease end stage (determined by the point when the mice could no longer turn themselves over within 10 seconds of being placed on their back or after full paralysis of the hind limbs)were those who had the treatment.

This edited virus is only programmed to cut and disable the mutated SOD1 gene in the spinal cord, so one of the next steps is eliminating the mutation in other brain cells. The research team is currently working on a version of the virus that delivers the gene to astrocytes and oligodendrocytes, glial cells important for the maintenance of the central nervous system. They are also working on a “self-destruct” mechanism for the Cas9 protein so that once it does its job of cutting the SOD1 gene, it can be eliminated from the cell to prevent accidental modification of other genes or immune response activation.

The researchers stress that this treatment is by no means a cure, and it doesn’t make mice normal, but it provides strong proof that CRISPR-Cas9 could be a therapeutic molecule for ALS. “Being able to rescue motor neurons and motor neuron control over muscle function, especially the diaphragm, is critically important to being able to not only save patients but also maintain their quality of life,” said the head researcher, David Schaffer. He says that he would be very optimistic if they are able to eliminate SOD1 within not just neurons but also the astrocytes and supporting glia, because then, long extensions of lifespan may be observed.