Development of a small molecule combinatorial treatment for RGC survival and axon regeneration to restore sight after optic neuropathy

We plan to investigate the possibility of developing a new molecular treatment to promote the regeneration of nerves in the visual system following damage to the optic nerves. The optic nerve connects the eye to the brain and is essential for providing signals about visual stimuli for the brain to interpret as images. There are several conditions in which the optic nerve is damaged, including glaucoma, ischaemic damage and following head/eye traumatic injury. These conditions cause severe visual impairment and are common causes of blindness. Once optic nerve damage has taken place, the nerve cells die and there are currently no treatments available that can reverse the process and restore vision.

Recent laboratory research models suggest that there are molecular mechanisms that can be manipulated in order to promote the survival and regeneration of nerve tissue following disease or injury. It may also be possible to restore connections to the brain that are lost in the disease process. The research to be undertaken at the Department of Clinical and Experimental Medicine at the University of Birmingham aims to examine this in more detail and develop a new molecular treatment in order to protect and regenerate nerves and restore visual function. To investigate this we will construct a new molecule that acts in combination with others already shown to influence the nerve regeneration mechanisms. This will then be investigated in an animal model to assess whether nerve cells are able to survive and regenerate as we hypothesise, and then undertake further experiments to assess whether regeneration of nerve cells enables the restoration of visual function.

Through this research we hope to show that optic nerve tissue can be regenerated following disease/injury and that visual function can be restored. This may then be translated into new therapies and offer hope for patients who have lost vision from optic nerve conditions.

We aim to develop a treatment to promote retinal ganglion cell (RGC) survival and restoration of visual function in a rat model of optic nerve injury. RGC regeneration has been demonstrated in response to neurotrophin administration and ocular inflammation and genetic studies have implicated the mTOR signalling pathway in neuronal regeneration. Knockdown of SOCS3, a negative regulatory molecule may potentiate this mechanism.

The objectives are to develop a combinatorial molecule to a triple RGC axogenic/neuroprotective combination of siRNAcasp2/siRNARedd1/2/shRNASOCS3/AAV2cntf and test this in our rat model to monitor neuronal survival, regeneration and re-innervation of central targets with restoration of visual function.
Study 1: test for SOCS3 knockdown in RGC by transfecting retinal cultures with a synthesised siRNA for SOCS3 and performing Western blots and detection of relevant protein bands using specific antibodies against SOCS3.
Study 2: in vivo evaluation of SOCS3 knockdown, by constructing an AAV short hairpin RNA targeting SOCS3 mRNA. After intravitreal injection we will evaluate RGC transduction rates and knockdown of SOCS3 by RT-PCR and western blotting.
Study 3: development of pupillary constriction measurement and assessment of visually guided behaviour by standard water maze tasks.
Study 4: evaluation of anatomic restitution and return of visual function after intravitreal delivery of the combinatorial nucleic acid treatment in optic nerve transected rats. Treated rats will be compared to control groups at defined time periods following optic nerve crush. FluroGold backfilled RGC indicating RGC survival, visually guided behaviour, immunostaining for re-innervation of central targets and electrophysiological assessment of visual pathway function will be evaluated.

Results will have implication to axonal regeneration in the CNS as a whole and be translated into clinical treatments for patients with diseases that are presently untreatable.

The impact of the proposed research will be on the academic, military, industrial and patient based communities with interests in neurodegenerative conditions. If neuroprotection and neurorepartive therapies are successful in reducing retinal apoptosis and restoring visual function following ocular trauma, this could rapidly be translated into clinical trials in humans. The military population in particular would stand to benefit greatly in terms of preserved visual function after ocular trauma from the availability of a neuroprotective/neuroregenerative drug. Knowledge of the mechanisms of retinal ganglion cell repair and a working animal model will form the basis for further evaluation of protective and regenerative therapies. The addition of in vivo functional assessment gives these studies greater impact. In vivo functional assessment also adds significant value to future models of CNS injury in the University of Birmingham laboratories, thereby increasing the efficacy of research aimed at improving outcomes in brain injury, and - indirectly - in brain/spinal cord injured patients.
Intellectual property developed in the course of the project will be patented through Alta Innovations Ltd at the University of Birmingham and will therefore be available for commercial exploitation. Success with these pre-clinical experiments will enable us to engage pharmaceutical companies like Quark Pharmaceuticals (who have an established collaboration with Prof Logan) who may be interested in taking the drugs forward for clinical trial for this medical condition. This option are likely though licensing of patents or technologies for further development. It is through this route that the proposed research is ultimately likely to make a major economic and health impact.
Finally it is likely that regulatory and charitable organisations will also be direct beneficiaries of the research project. The development of RNAi therapies is in its infancy but is still likely to make a major impact on medical research and therapeutic development over the next 10-20 years. RNAi therapies for neurological diseases/conditions have special potential given the lack of therapies for major neurological conditions and the potential impact of RNAi. The ability to develop an RNAi neuroregenerative therapy for the retina will be impact the regulatory community since it will provide a unique opportunity to assess the efficacy of this kind of therapeutic approach for CNS neurons. The research will be of interest to charity and patient advocacy organisations by providing prospects of a new therapy that will impact on their members.