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.
