The Retina within Sight of RNAi Therapeutics

 From the highly prevalent diseases such as the age-related macular degenerations (dry and wet) to the rarer, but more numerous diseases such as the retinitis pigmentosa, the eye is the subject of a significant and growing unmet medical need.  From a genetic point of view, the application of RNAi Therapeutics to these diseases is very attractive.  However- yes, you guessed it- delivery challenges have made it difficult to exploit its potential.  In 2013, I have come across at least two pieces of evidence, one in synthetic and one related to DNA-directed RNAi, which make me believe that the time for ocular RNAi Therapeutics is nigh.

A bit of history

Following an early rush into ocular diseases that saw 3 wet AMD RNAi candidates, one each by Opko Health, Quark Pharmaceuticals (partnered with Pfizer), and Sirna Therapeutics/Merck (partnered with Allergan), speed into phase II and III studies, considerable doubts about their scientific foundations killed off the enthusiasm.  

Firstly, it was thought that the preclinical validations of the wet AMD candidates rested on TLR3-activation artifacts.  While I have not seen this claim more widely supported and chemical modifications and short double-strandedness should readily get around this issue, the more vexing question to me has been how were these either unmodified or simply 2’-O-methyl modified, unformulated RNAi triggers supposed to enter their target cells?

Moreover, for ddRNAi where Genable Technologies is readying an AAV-based candidate for retinitis pigmentosa to commence clinical trials, vector distribution following needle administration represents serious safety and efficacy issues.  Because the AAV and lentiviral gene therapy workhorses do not diffuse on their own from intravitreal injection sites to the back of the eye, ocular gene therapy has largely involved subretinal injections which a) is a relatively dangerous procedure that can seriously harm retinal architecture and integrity, and b) limits vector diffusion and therefore therapeutic activity to a small area surrounding the subretinal administration site.

Smartly evolved AAV can penetrate from the vitreous into the retina

In order to develop viral vectors capable of overcoming the physical barriers between the vitreous and retina, Dalkara and colleaguespublished earlier this year a study on the selection of AAV2 variants capable of doing just that. 

The selection process of this now popular method of directing AAV to various tissues and cell types starts out with large libraries of more or less randomly mutated AAV variants.  In each selection round, the viral DNA is extracted from the target cells and re-amplified for the next round of selection such that the AAV variants that most efficiently transduce the target cells eventually become highly enriched.

 

The 7m8 AAV that they identified was thus capable of broadly transducing retinal cells following intravitreal administration in mice, from the inner ganglion cells to the outer retinal pigment epithelial cells.  In non-human primates, 7m8 also greatly enhanced retinal penetration from the vitreous compared to old gold standard AAV serotypes.  However, retinal cells were not transduced as broadly as in mice suggesting that either additional candidates from mouse evolution experiments need to be characterized in non-human primates or that the selection itself should be performed in non-human primates.

I view such progress exceptionally promising for retinal degenerative diseases in which rescue of only a fraction of cells should be therapeutic (e.g. by getting rid of a mutant mRNA that leads to the death of the cell expressing it), but potentially also for the more challenging disease settings where RNAi action would probably have to occur in the majority of cells in order to be therapeutic.

 

Along these lines, after deciding on the best AAV variant, an important task will be to determine the more precise percentage of cells of each cell type that can be transduced to then match them up with potential indications.

Sticky self-delivering RNAi triggers penetrate deeply into the retina

In the field of synthetic RNAi Therapeutics, a peer-reviewed paper came out by RXi Pharmaceuticals (Byrne et al.) which confirmed earlier claims that self-delivering RNAi triggers (sd-rxRNAs as they call their own versions) were able to fully penetrate the retinal cell layers in both the mouse and rabbit following intravitreal injections.  The rabbit is an important model because their eye volume, believe it or not, is close to ours (~1.5ml vs ~5.0ml).  Formal proof of RNAi-mediated gene silencing was then confirmed in the mouse.

The self-delivering RNAi triggers had guide strands of ~19 nucleotides and passenger strands of less than 15 nucleotides.  An extended phosphorothioate (PS) single-stranded tail on the guide and additional hydrophobic modifications such as sterol groups were supposed to enhance tissue penetration and functional cell uptake.  The 6-8nt PS tail obviously has been borrowed from standard antisense chemistry.  Therefore, a battery of tests was performed to exclude visual abnormalities resulting from these extensive modifications.  No notable tox findings were made.

Despite the similarities in chemistry, sd-rxRNAs appear to be superior to ISIS-type PS-ASOs (RNaseH mechanism).  This is because in the Byrne et al. study, ~3 microgram RNAi trigger was required for 50% gene knockdown, whereas 50 micrograms phosphorotioate antisense oligos were required for a 50% knockdown of MALAT as presented by ISIS at OTS 2013 in Naples (report still available).  To wit­, MALAT as a nuclear RNA can be expected to be far more susceptible to RNaseH ASOs than your average mRNA, so the potency difference could be even bigger.  Also, it would be interesting to determine the safety implications of administering 3 micrograms versus 50 micrograms or whatever the equivalent dosages in humans.

The potency difference would have been much bigger still if RXi were not so married to their less than 15bp double-strandedness, a misguided decision driven by old IP considerations (when the Kreutzer-Limmers were still a concern).  I therefore expect other RNAi companies with more active R&D to thank RXi for the validation and come up with more optimal RNAi reagents for ocular applications.

Ocular RNAi Therapeutics- keep an eye on it in 2014 and beyond.