RNA Editing to Generate Protective Variants

Last week, ProQR participated at its first investor conference after a half year hiatus following a failed binary clinical read-out of a antisense splice modulator and corporate re-organisation to solely focus on RNA Editing.

To raise awareness of the company as a serious contender in this exciting field, the CEO detailed the new corporate strategy of growing the company as a major future biotechnology company.  This will be based on foundational IP, know-how and cash-generating partnerships as well as a broad internal pipeline reflecting the numerous ways RNA Editing can bring unique differentiation to addressing disease.  At the EuroTIDES two days later, the company provided scientific data illustrating the types of possible applications.

What caught my attention here is using RNA Editing for generating protective variants.  Protective variants are genetic variants in the human population that make carriers less likely to develop a disease.  PCSK9 and ApoCIII are well known in the cardiovascular field for those and have yielded promising therapeutics as a result.  Protective variants can also be found in other areas such as infectious disease (e.g. CCR5 and HIV) and Alzheimer's.

Not least because the story surprised and intrigued me for its translational potential, I will illustrate protective variant generation using RNA Editing taking PCSK9 as an example. 

 

Mimicking a PCSK9 mutation causing drop in bad cholesterol

With Inclisiran, an RNAi trigger suppressing the expression of PCSK9, RNA Therapeutics have become a commercial reality in cardiovascular disease aimed at very large populations.  A key attraction of this agent is the infrequent, semiannual dosing regimen that clamps down bad LDL cholesterol by ~-50%.

50%, however, somewhat lags the efficacy of the competitive PCSK9 monoclonal antibodies (~-60%) which have to be given at least monthly.  The fact that more robust LDL cholesterol lowering should therefore be possible also for RNA Therapeutics, the race for the most effective PCSK9 in this class is yet to be decided. 

Finding better RNA knockdown agents may be one, certainly attainable strategy, although AstraZeneca has just given up on an RNaseH antisense oligo that had looked promising in that regard.  Using an entirely new mechanism another.

Intriguingly, the sequencing of the PCSK9 gene in an individual with conspicuously low LDL cholesterol in Canadian Quebec province (Mayne et al. 2011) revealed that a Q152H variant in the heterozygous (!) state could lower circulating PCSK9 levels by ~-80% compared to the average, non-related population. This translated to a 60-70% LDLc lowering.  Subsequent cell culture experiments confirmed the causality of this mutation in regulating LDL-receptor levels via PCSK9 expression.  Curiously, while it was initially assumed that the mutation inhibited autocatalytic cleavage of pro-PCSK9, these experiments suggested that such mutant PCSK9s can also inhibit PCSK9 cleavage and subsequent export out of the cells in trans.  This dominant negative effect explains why the mutation in the heterozygous state could have such an outsized effect on serum PCSK9 and LDLc levels.

While other protective PCSK9 variants had been found before, this one was intriguing enough for ProQR to follow up on.  As histidine (H) is unlikely to be unique in messing up PCSK9 processing, ProQR chose QàR editing at the 152 site, possibly because glutamines cannot be converted into histidine via AàI editing, but also possibly because they suspected that the more highly charged arginine (R) residue could be even more impactful.

Impactful it was.  Even at only modest 25% editing (which certainly can, and has to be improved upon for further development), cleaved and secreted PCSK9 in cell culture was reduced by roughly -90%.   

It is important for the RNA Editing space to continue to advertise what the technology can do.  Because RNA Editing is not very useful as a genetic tool for general, academic molecular biology, the onus is on the companies to raise the awareness as some of the best translational ideas may come from scientists and physicians that have yet to hear about the technology.