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.

Genable Finds EUR5M for ddRNAi-Gene Replacement Approach

5 million Euros these days is serious money in RNAi Therapeutics. Last week, Irish biotechnology company Genable Technologies announced that it succeeded in raising such funds for its pre-clinical DNA-directed RNAi Therapeutics-Gene Replacement combo for the treatment of rhodopsin-linked autosomal dominant retinitis pigmentosa (RHO-adRP), an inherited retinal degenerative condition leading to blindness.

To see it flow into DNA-directed RNAi Therapeutics at that makes it particularly noteworthy. It illustrates that ddRNAi is somewhat uncoupled from the current depression in the synthetic RNAi Therapeutics arena due to some technical differences which make it in many ways more similar to traditional gene therapy. Gene therapy, of course, is experiencing a revival, fueled by more and more clinical validation. Incidentally, it which follows a similar boom-bust cycle as RNAi Therapeutics is going through now.

One of the great benefits of ddRNAi Therapeutics is that it can be combined with other DNA-directed modalities, especially the co-expression of protein-encoding genes from the same DNA template. As such, it is particularly amenable to treat autosomal dominant-negative diseases in which the affected gene is important for cellular function thereby precluding approaches that eliminate both the mutated and the healthy copies of a gene. Accordingly, one could use a single DNA molecule from which to direct the suppression of endogenous versions of the affected gene (wildtype and mutant forms) and then replace gene function by providing a version of the gene that is immune to the RNAi.

In Genable’s case, AAV vector capacity limitations meant that a mix of two AAV vectors had to be co-administered by subretinal injection, one carrying the ‘immunized’ rhodopsin gene, the other the shRNA against the endogeneous rhodopsin copies. This apparently did not affect much treatment efficacy. Indeed, it was argued that the co-administration approach would actually increase dosing flexibility by being able to adjust the amount of each component independently. On the other hand, it is possible that the use of vectors with larger capacities, such as the lentiviral system that Oxford Biomedica and Sanofi-Aventis are busy exploring for ocular gene therapy, may have its advantages here. Importantly, it was shown that therapeutic efficacy in the retinitis pigmentosa animal models strictly depended on the presence of both gene replacement and gene knockdown components.

Although the concern with causing harm by knocking down the wild-type allele is often only a theoretical one, as is for example the case with Huntington’s Disease (HD), and depends on the degree of knockdown, other applications for which this approach holds promise are sickle cell anemia, alpha-1 antitrypsin, pachyonychia congenita (PC), amonst many others.

The PC project by TransDerm and collaborators is a good example in the RNAi Therapeutics space for the challenges faced by the alternative approach to treating such autosomal dominant diseases, namely by specifically directing the drug candidate towards the mutated gene/gene product so as to leave the wild-type copy untouched. The problem here is that because the potential mutations can be broadly distributed over a gene, restricting a therapeutic to targeting only one such genetic lesion (or associated gene polymorphisms) can dramatically reduce the number of patients eligible for such a targeted therapeutic. This also explains why the first clinical trial by TransDerm was a placebo-controlled, split-body dose-escalation trial in a single patient (!) as there may not be more than a handful of known patients in the world with the applicable mutation.

The 5 million Euros will be used to bring Genable’s lead candidate, GT038, into clinical development. RHO-adRP seems like a good ddRNAi application as even the correction of only a fraction, e.g. 1/3 to 1/2 of the affected photoreceptors, may be enough to support vision. It is good to finally see a number of ddRNAi Therapeutics candidates based on sound pre-clinical data move into the clinic.

(For another promising late preclinical candidate, see Calimmune's HIV approach)