Wednesday, April 16, 2008

Update on AAV-based RNAi for Huntington’s Disease: Time to Move on from U6-shRNAs?

Huntington’s disease (HD) is a dominant genetic disease caused by a triplett expansion in one of the two copies of the huntingtin (htt) gene. So far there is no treatment that appropriately addresses this disease. As with other dominant diseases, suppressing the insulting gene copy by RNAi should bring a therapeutic benefit, hopefully even delay its progression. A recent paper from Beverly Davidson’s group in Iowa reports on their latest experience in developing an DNA-directed RNAi Therapeutic for the treatment of Huntington’s disease [HD; Mc Bride et al (2008): PNAS 105:5868).

Previous studies from the Davidson lab showed that AAV-delivered shRNAs were able to knockdown target genes in mouse brains and subsequently improve the behavioral deficits and pathology of polyglutamine-repeat diseases such as Huntington’s. These promising results led to a collaboration with the AAV gene therapy company Targeted Genetics and Sirna Therapeutics (pre-Merck acquisition) with the goal of advancing an AAV-based RNAi Therapeutic for HD into the clinic.

Their prior work on Huntington’s was based on a transgenic mouse model which expressed a human fragment of the huntingtin gene containing a triplet expansion. However, as the shRNAs in those studies were directed specifically against the human htt gene, but left the mice’s own huntingtin gene copies untargeted, the present studies were designed to evaluate the safety of knocking down all the huntingtin gene copies in a mouse. This is important for the development of an RNAi Therapeutic for HD in man since targeting a small RNA towards the highly structured triplet expansion is unlikely to be effective, and since the mutant htt allele is not associated with a common polymorphism in the htt mRNA which would facilitate a discrimination between mutant and wildtype huntingtin. This issue is also of particular concern since the huntingtin gene is considered to be essential during early mammalian development and little is known about its requirement in the adult brain.

For that reason, McBride and coworkers chose a different mouse model where a triplet expansion had been inserted into one of the existing mouse htt alleles which has the added advantage that it also preserves the natural expression pattern of htt.

Encouragingly, a 50-60% knockdown of mouse htt was observed over a period of 4 months without any obvious target-specific side-effects. Transduction of this particular AAV administered to the striatum, a region in the brain particularly affected in HD, was fairly broad and specific for neurons, the cell type critically affected during HD. Given that not every neuron was transduced and that non-neuronal cells may also express some htt, this 50-60% knockdown implies that very efficient knockdown of htt can be achieved in a subset of neurons and (based on previous studies) should be therapeutic and safe. Longer term studies are ongoing.

The unpleasant surprise, however, was that as previously reported by a number of other groups, some of the tested U6-driven shRNAs were toxic. This is likely caused by the very high levels of small hairpin RNAs from the strong U6 promoter which may jam up the endogenous microRNA gene regulation which is harnessed by RNAi. Whether it is just the level of the hairpins or also the specific biogenesis of the shRNAs, which only resemble microRNA precursors, but actually undergo a slightly different biogenesis involving an unusual set of enzymes and RNA structures, is an open question. In any case, to make a long story short, it is probably advisable that future DNA-directed RNAi Therapeutics programs avoid U6-driven shRNAs altogether and instead use other Pol III-driven systems or Pol II promoters. The latter have the added safety advantage as they can be chosen to be tissue specific so as to minimize side-effects in non-target cells. Since efficient RNAi does not require high small RNA levels, vector transduction and long-term expression should in many applications be more important considerations than mere promoter strength.

Whether it is advisable to go ahead with the particular U6-shRNA that was found to be non-toxic and efficient in knockdown, or instead go back and re-design all of the vectors and repeat 2-3 years of pre-clinical studies, must be a tough decision to make, a situation similar to that faced by other RNAi Therapeutics programs currently in development given the steep learning curve the field is still undergoing.

It is maybe for these reasons that Merck’s subsidiary Sirna Therapeutics decided not to continue with this program and fully hand over the commercial rights to Targeted Genetics. In some way, the AAV-RNAi program always stood out as being an awkward fit for Sirna Therapeutics as it seemed at odds with their siRNA modification focus. Maybe at the time there were not that many advanced RNAi Therapeutics programs to chose from so that Dr. Davidson’s studies seemed like an opportunity to fill the early pipeline. Given the unpredictability of early studies and the number of RNAi Therapeutics programs under evaluation, it is best to let pipeline decisions be driven not by history, but by where the science is progressing swiftest. AAV RNAi for HD still has much promise and the path forward fairly obvious, but as many things in drug development it will be a long and arduous one.

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By Dirk Haussecker. All rights reserved.

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