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Using ‘Human Knockouts’ to Ensure RNAi Safety

RNA interference or RNAi is a Nobel Prize winning technique that can silence almost any gene in the genome by binding and cleaving RNA transcripts. The first RNAi based therapeutic, called Onpattro,  was approved in 2018 for the treatment of peripheral nerve disease (polyneuropathy). This disease is caused by hereditary transthyretin-mediated amyloidosis (hATTR) in adult patients.

At the Festival of Genomics and Biodata 2022 Paul Nioi, Vice President, Discovery and Translational Research at Alnylam Pharmaceuticals explained how ‘human knockouts’ are helping jump the hurdles that RNAi therapeutics face.

R&D failures

From the 1950s until the 2010s, the number of drugs approved for clinical use per $1billion had fallen dramatically. Between 2007 to 2010, safety and efficacy issues accounted for 91% of phase three submission failures. Paul suggests that one of the reasons for this is that most drug target discovery efforts have been studied in animal models, like mice, which often do not easily transfer to Phase 3 clinical trials in humans.

Therefore, being able to provide evidence for efficacy and safety using data from humans rather than animals is likely to increase the chance of Phase 3 successes for new therapeutics. Because of the unique mechanisms by which RNAi operates, it is possible to acquire this evidence from genetic databases through human gene loss-of-function (LOF) mutations.

Human gene LOF data

Naturally occurring LOF mutations provide real world in vivo human models of gene inactivation, which is how RNAi therapy works. LOF mutations result in little to no mRNA and protein production from the expressed gene.

Human ‘knockouts’ can take a few forms. They can be heterozygous knockouts, where one allele is affected, homozygous which has both affected, and compound heterozygous, where both alleles are affected but in different ways.

An example Paul used was targeting the HAO1 gene in Primary Hyperoxaluria Type 1 (PH1) which is a rare genetic disorder characterised  by increased endogenous oxalate synthesis. PH1 currently has no approved treatments. An siRNA construct called Lumasiran developed by Paul’s group to treat PH1 is able to target the mRNA for HAO1 and decrease production ofhepatic oxalate production. But the risk of knocking down a gene completely is that the side-effects are sometimes unknown.

Fortunately, Paul’s collaboration with the East London Genes and Health Cohort revealed one individual who was HAO1 null. This individual was a female in her 50s with no adverse clinical phenotype. There was clear biological evidence to support that she lacked HAO1 as her plasma glycolate levels were 12 times higher than normal. With the only cure for PH1 being a double kidney transplant, this real-world example provides a good bit of evidence for HAO1 target safety.

Prevalence of human knockouts

The UK Biobank Exome Sequencing Consortium (UKB-ESC) is aiming to sequence the exome of 500,000 participants aged 40-69. Paul’s group worked to analyse UKB genomes, and the number of LOF genes both homozygous and heterozygous was revealed.

The team found 16,406 genes that were heterozygous LOF, and 2,262 homozygous LOF genes. For homozygous LOF mutations, 43% of the mutations were found in only a single individual, and 75% were found in fewer than 10 carriers. The point here is, it’s very rare to find a homozygous LOF carrier!

While these ‘Human Knockouts’ are extremely rare, they are also extremely valuable. Hopefully as the data available for analysis grows, novel RNAi therapies will have less difficulty being approved for clinical use

Image Credit: Canva

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Biobank / RNAi / safety / siRNA