In 2016, at his home RANZCO scientific branch meeting in Tasmania, preeminent ophthalmic genetics expert Professor Alex Hewitt presented on the transformative impact of CRISPR/Cas9 gene editing for inherited retinal diseases (IRDs).
It was a particularly exciting time for ocular genetics in Australia. Hewitt, of the Menzies Institute for Medical Research, University of Tasmania, and his Australian research peers had just published a breakthrough paper a few months earlier describing the world’s first ‘in vivo’ viral mediated CRISPR/Cas gene editing in the retina.
This meant he and his team – including researchers from the Centre for Eye Research Australia, University of Melbourne, University of Queensland, Monash University and Jinan University, Guangzhou – had overcome the tall order of delivering gene editing machinery into the retinal cells of a live mouse using viral vectors. Altering cells while in the body (in vivo) is an incredibly complex challenge, but Hewitt and his team overcame this by splitting the load between two viruses before disabling the target mutant genes.
Ultimately, Hewitt’s team recorded an 84% reduction in retinal cells containing a specific protein that was being produced by the target gene.
“By demonstrating that CRISPR/Cas can also cause substantial gene modification activity when introduced by a viral delivery method in the retina, we are closer to translating gene editing technology for therapeutic purposes,” the researchers concluded.
Clinical Professor Brendan Vote, who established the charitable organisation the Tasmanian Eye Institute (TEI) in 2008, was in the audience when Hewitt delivered his 2016 presentation in Hobart. That day, he committed to supporting Hewitt’s vision of translating CRISPR/Cas gene editing from the lab into real world therapies by building what he describes as Australia’s first dedicated Ophthalmic Gene Therapy Centre (OGTC).
With the construction phase now almost complete, Vote hopes it will allow Australian ophthalmic enterprises to develop CRISPR treatments and join the world’s most valuable companies in the era of personalised medicine. The next decade will be truly exciting with more than 50 companies searching for treatments to the world’s biggest diseases using CRISPR technology in what he believes will be the next trillion-dollar industry.
Vote believes establishing the OGTC would ensure Australia remains globally competitive in the synthetic biology revolution, while negating the effects of global supply chain disruption or the at-times slow pace of pharmaceutical companies to register products here.
“Aside from cures that Alex and his team develop, this facility would make available critical biotechnology infrastructure for all ophthalmic researchers around Australia to translate their laboratory research into Therapeutic Goods Administration (TGA)-certified clinically deliverable treatments,” Vote says.
Establishing the centre
After the 2016 meeting, it took just one year for Vote to begin fulfilling his commitment to the OGTC through the Tasmanian Eye Institute, whose philanthropic efforts are dedicated to research, education and service in ophthalmology.
Vote was flying to a conference in 2017 when he spotted a derelict heritage building advertised in a Hobart real estate magazine. Located in Lenah Valley – only 3km from Hobart CBD on over 4,000sqm – Colebrook house was built in 1866 and had fallen into disrepair as a deceased estate without power, water or sewer connection while Hobart city had grown around it.
What also caught his eye was a similarly derelict water tank adjacent to the rundown heritage property. Built in the 1930s as infrastructure for the drought that never came, the water tank had never been used, except as a canvas for amateur graffiti artists.
“Here were two buildings part of Hobart’s history and DNA, and the opportunity to give them a new life seemed fitting for our vision of creating Australia’s first dedicated OGTC,” Vote says.
“Funding for purchase of both buildings came via Tasmanian Eye Institute, which in turn has been funded largely through private donations. Sadly, though ’supportive’ of the project, to date no federal or state government funding has been forthcoming. To have such important sovereign biotechnology infrastructure within Australia will be critical in delivering therapies currently being developed not just by Professor Alex Hewitt but also other ophthalmic researchers.”
After securing development approval from Hobart City Council, the buildings have undergone a significant facelift. The asphalt for the carpark has been laid, the ventilation systems will soon be commissioned, and indigenous artwork by local artist Mr Reuben Oates will connect the OGTC with past, present and future through vision.
More than $100,000 has been raised from community donations (in addition to TEI’s own contributions), but there is still some way to go to reaching the $2 million target. Major regulatory hoops also remain. Another $1 million is needed for the highly specialised equipment to obtain TGA approval, of which around $71,000 has been raised.
(Fully tax-deductible donations can be made at www.tasmanianeye.org).
The fundraising drive has also seen Vote’s hair change from bleached to blue before shaving it off entirely. While CRISPR has exponentially fast-tracked the ability to develop gene editing cures, Vote says progress towards any ‘personalised medicine’ requires standard Phase 1, 2 and 3 human studies. Therapies for human trials must be manufactured under strict aseptic conditions.
“Probably the best way of thinking about gene therapy is an amalgam of pathology, medicine and pharmacy roles. An individual’s specific genetic defect must be diagnosed (doctors, genetic screening services, DNA databanks), as the therapy must be delivered ultimately into someone’s body,” Vote explains.
“This requires very strict pharmaceutical production standards, much like chemotherapy or a vaccine might be produced, with clean and ultraclean areas. Consequently, there were a lot of ‘regulatory’ hoops to jump through, not least of which addressing council development application and design planning. Ultimately the OGTC must meet PC-2 good manufacturing practice (GMP) facility, TGA-certified standards and all of our planning, design and construction has been towards this aim.
“This OGTC will be the first such PC-2 GMP facility, specifically dedicated to meeting Australia’s ophthalmic needs. TGA certification should be complete by the end of the year and the facility ready to produce its first ‘CRISPR cures for blindness’ next year.”
While gene therapy techniques have existed as lab experiments for several years, the TGA’s approval of Luxturna was a major milestone for Australia in August 2020. It marked the country’s first true, ‘in-vivo’ gene therapy for any disease. The therapy helps to restore vision for patients with IRD caused by pathogenic biallelic RPE65 gene mutations and is expected to be the first of many brought to market for previously untreatable IRDs.
Such approaches can be described as gene augmentation, which involves flooding the relevant cells with abundant functional copies of a healthy gene, in the hope the cell will incorporate it.
While impressive, it’s not considered the most sophisticated approach. It can be hit and miss, with even successful gene therapies only about 10- 15% effective, Vote says.
CRISPR/Cas holds much more promise because it can enter the cell and remove the incorrect DNA sequence and replace it with the correct sequence, thus permanently altering the genome. It can also simply ‘knock out’ a mutation, allowing the cell to make normal proteins.
“CRISPR/Cas9 is faster, cheaper, more accurate and more efficient and we will see an exponential increase in these personalised medicine cures,” Vote says.
Explaining this further, Vote says CRISPR has two components – a guide RNA and the Cas9 enzyme. Having diagnosed a patient’s exact genetic defect, the guide RNA acts as a messenger to find the patient’s DNA with the affected gene. Once found and bound to the gene, the Cas9 enzyme precisely cuts out the affected gene. By providing the manufactured repair (correct gene), this enhances restoration of normal gene function in 60-70% of cases. This is compared to a one-in-a-million chance if the DNA is left to repair itself naturally or 10-15% with existing gene augmentation techniques, as above.
While Hewitt is among the first to apply CRISPR/Cas to eye disease in Australia (in animal models), pharma giants Allergan and Editas have already advanced their CRISPR/Cas9 therapy to human trials, with the first patient with Leber congenital amaurosis type 10 treated in March last year.
Using the gene ‘knock out’ approach, it took place at the Oregon Health & Science University, Casey Eye Institute, and was reportedly the first ‘in vivo’ CRISPR medicine of any kind to be administered to a patient.
Speaking to Insight, Hewitt says there is a long road before the production of clinical-grade CRISPR therapies in Hobart, but once operational, he and his team may initially focus on a rare form of Usher syndrome. Other IRDs such as retinitis pigmentosa, cone-rod dystrophy, and Stargardt disease would also be considered.
These are all monogenetic diseases, typically caused by one or two ‘spelling mistakes’ in the single gene. However, it’s likely such therapies would only be useful for patients with a good degree of functional retinal cells.
“It means you could potentially correct any disease-causing variant that isn’t large. Obviously major structural genomic changes wouldn’t be readily amenable to CRISPR/Cas therapy, but small changes, which would account for the majority of IRDs would potentially be amenable,” he says.
CRISPR/Cas also doesn’t run into the ‘cargo size’ issue often seen in gene augmentation. The gene in Luxturna was chosen because it fit nicely into the viral vector, but some IRDs require larger genes that don’t fit commonly used viruses.
“The other potential advantage is on the regulatory front. The first therapy will need full pre-clinical modelling and testing in animal models, but after it is proven to be safe, I think the bar would be lowered for other modifications because you’re essentially redirecting where that gene editing machinery needs to go,” Hewitt explains.
“In the cell line you could show you are not giving off inadvertent off-targeting effects which is different to a gene augmentation approach where you might always have to do the rigorous pre-clinical screening because it is a major change in the composition of your therapy – redirecting the CRISPR machinery isn’t a major change in the machinery.”
In future, Vote anticipates most CRISPR applications will be biomedical (>90%) but won’t be limited to gene therapy. He says CRISPR already plays a role in regenerative medicine such as stem cells and pharmaceuticals, including drug development and mRNA vaccines for COVID-19.
“Aside from eyes and the variety of blinding genetic diseases, CRISPR cures will be produced for cardiovascular diseases, diabetes, Alzheimer’s, genetic haemoglobin disorders (anaemias like sickle
cell and thalassaemia, and cancer (CRISPR-edited immune cells to attack tumours).”
NOTE: Fully tax-deductible donations towards the Tasmanian Eye Institute’s Ocular Gene Therapy Centre (OGTC) can be made at www.tasmanianeye.org.