Finding cures for children's genetic diseases

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Genetic retinal diseases: functional variant characterization using retinal organoids

Genetic retinal diseases affect approximately 1 in 1,000 individuals, or more than five million people worldwide, and are the commonest cause of blindness in working-age people. They are caused by abnormality of photoreceptor (PR) cells, with retinal pigment epithelial (RPE) cells also affected. The photoreceptors contain outer segments which are specialised sensory cilia. Consistent with other studies, over two-thirds of the variants we detect are in genes encoding proteins involved in cilia structure or sensory functions of the PRs, and/or affect genes only expressed in the retina. The human retina is an inaccessible organ for functional biological assays, so human induced pluripotent stem cell (hiPSC)-derived retinal organoids and RPE cells provide an exciting solution system for improved diagnosis and for assessment of therapies in these conditions.

Using patient-derived hiPSC-retinal organoids and RPE and application of RNA-based analyses, we are investigating non-canonical splice site, as well as missense variants in RPGR, IQCB1, RPE65, PDE6B and other retinal disease genes. Control hiPSC lines are also used to engineer in variants of interest using CRISPR/Cas9 gene editing approaches. Single cell transcriptomic studies in retinal organoids are used to interrogate pathways affected in these retinal conditions. This facilitates clearcut variant interpretation and helps towards mechanistic understanding of disease and development of new therapies for these disorders.


Genetic therapies for retinal diseases

The eye is an ideal organ to apply advanced genome engineering, AAV, stem cell and other therapeutic technologies, due to its relatively small size (so less demand on the scale-up of the technology), and its ease of accessibility. We have selected two retinal dystrophies for development of novel gene replacement therapies, due to their suitable gene size for this approach. Gene replacement and promoter construct have been created for use in an AAV vector construct designed for subretinal delivery. We are testing these therapeutic constructs in our mouse models, and patient-derived retinal organoids. Gene-editing approaches are also in use for other variants where this is applicable. This work paves the way for genetic therapies for patients in NSW and other Australian states for genetic retinal diseases suitable for clinical trials.

While some AAV serotypes are in use for subretinal delivery in retinal diseases, there is a need for improved precision and efficacy through delivery of therapeutic constructs to the correct cells, and intravitreal delivery would add additional safety. We use a primary human retinal explant culture system for selection and investigation of novel retinal AAV capsids. All of the advances we make in AAV and other delivery technologies in this project, will be applicable for the various forms of retinal dystrophies present in affected patients.


Functional genomics for novel retinal disease pathway investigations

We recently identified a gain of function mutation in a novel genetic retinal disease gene, ALPK1, causative of the newly identified ROSAH (Retinopathy, Optic oedema, Splenomegaly, Anihidrosis, Headaches) syndrome. ALPK1 encodes an atypical protein kinase. ALPK1 is localised to the transition zone of the connecting cilium of the photoreceptors, and in the innate immunity system, acts as a pattern recognition receptor (PRR) impacting phospho-signalling and downstream NFkB mediated inflammation. In this project, further mechanistic studies will be pursued using a multi-omics approach for phenotype comparisons, which is powerful in revealing dynamics and controlling factors in biological pathways. Transcriptomic, proteomic and phosphoproteomic studies will be undertaken, along with evaluation of modulation of the ALPK1 pathway in our model systems using CRISPR/Cas9 gene editing approaches.


Genetics and natural history studies in preparation for retinal gene therapy trials

Our research genomic strategies have successfully led to availability of clinical genomic diagnostic testing in the genetic retinal diseases, with our collaborators at The Children’s Hospital at Westmead, Sydney Children’s Hospitals Network. These genomic diagnoses provide a strong foundation for accurate groupings in our natural history studies in preparation for clinical trials, with our collaborators at The Children’s Hospital at Westmead, Sydney Children’s Hospitals Network and Save Sight Institute, Sydney Eye Hospital.


Functional genomics for ocular developmental eye disorders

Congenital cataracts, disorders of the anterior segment of the eye, glaucoma and very small eyes are also debilitating ocular conditions, with previous low genetic diagnostic detection rates. Our research genomic studies in these disorders are proving successful in identifying the most useful strategies to maximise genetic diagnosis. Functional genomic studies are also used to increase the diagnostic yield in these disorders, and have potential for further therapy development for the associated visual impairment.