PERIPHERIN-2 (PRPH2)

PERIPHERIN-2 (PRPH2) associated retinal degeneration

We are proud to be part of an exciting collaboration between the University of California San Diego and The Nixon Visions Foundation. This collaboration focuses on increasing the understanding and the development of breakthrough novel treatments for PRPH2 associated retinal degeneration (PARD).

Retinal disease caused by mutations in the gene PRPH2 is one of the commonest causes of inherited retinal disease. PARD is complex as it causes a wide spectrum of different retinal diseases. However, all these diseases are progressive and can lead to severe visual impairment (Seff Figure 1).

Figure 1 Autofluorescence images of different types of PRPH2 associated disease – (A) Pattern dystrophy, (B) Multifocal pattern dystrophy, (C) Cone dystrophy, (D) Macular dystrophy with atrophy, (E) Central areoalar chorioretinal dystrophy and (F) Retinitis pigmentosa
Figure 1 Autofluorescence images of different types of PRPH2 associated disease – (A) Pattern dystrophy, (B) Multifocal pattern dystrophy, (C) Cone dystrophy, (D) Macular dystrophy with atrophy, (E) Central areoalar chorioretinal dystrophy and (F) Retinitis pigmentosa

PARD is usually inherited in an autosomal dominant manner. This means that there is a 1 in 2 chance of affected individuals passing the disease on to children. As a result, these sight-threatening diseases can afflict multiple generations of families.

At present there are no treatments for PRPH2-associated retinal degeneration (PARD). The understanding of PARD and the development of new treatments has been hampered by the relative lack of models which replicate clinical disease and so help clinical translation of treatments. This highlights a critical need to develop new clinically relevant models to better understand human disease and to leverage these insights to develop novel sight saving treatments.

The Borooah lab specializes in modelling inherited retinal disease and testing gene modifying treatments. We have previously developed, characterized and treated both animal and humanized retinal disease models.

As part of this collaboration our scientists first generated pluripotent stem cells from patients’ blood samples. Pluripotent stem cells allow the growing of almost all tissues within the body with the right guidance (See Figure 2).

Figure 2 Demonstration of pluripotency – Induced pluripotent stem cells expressing pluripotency markers SSEA4 and Nanog, suggesting that these cells can differentiate into most cells found in the body.

Our scientists then differentiated these cells using our in-house protocols to generate ‘mini-eyes’, known as retinal organoids. (See Figure 3)

Figure 3 Microscope images of retinal organoids showing photoreceptors with outer segments – Image on the left is a light microscope image showing a 40-week old retinal organoid with a shaggy coat of photoreceptors. The image on the right is a ultra-thin slice of the retinal organoid with photoreceptors which have clear outer segments (Inset)

Retinal organoids develop most of the cells found in the adult human retina. Importantly, this includes photoreceptors, the main cells affected in PARD and so this is a good platform with which to study this disease. PRPH2 protein is mainly found in a specialized region known as the outer segment, in photoreceptors (See Figure 5).

The initial studies will initially focus on one particular mutation in PRPH2. This mutation mainly results in disease affecting the macula, the central part of the retina and the region of the retina important for central vision.

The studies have three main objectives:

Objective 1:

To understand the clinical effect of the specific mutation in patients with PRPH2 (See Figure 4)

Figure 4 Ultra-widefield fundus autofluorescence images of the right and left eye – Areas at baseline (A, B) and at 45 months follow-up (C, D) showing progressive retinal atrophy (black areas) as measured using semi-automated software.​
Objective 2:

To understand the cone dominant/macular phenotype of this mutation and to investigate the causes of phenotypic variability using human retinal organoids.

Figure 4 Antibody staining confirming the presence of cone photoreceptors expressing PRPH2 – (A, A’) Cone- Arrestin immunostaining demonstrating clear cone staining with photoreceptor morphology on the magnified image A’. (B,B’) PRPH2 immunostaining confirming strong PRPH2 expression, associated with outer segments, in the same organoid, more clearly seen in the magnified image B’.
Objective 3:

To test novel treatments using this organoid model

Figure 6 Molecular protein separation studies for PRPH2 and ROM1 from control organoids – The image on the left represents the PRPH2 protein separation and the image on the right the separation of the closely associated protein, ROM1 from control retinal organoids. ROM1 is thought to contribute to some of the variability in PRPH2 disease. It is hoped that the mutant retinal organoids will have a different pattern. We can then use this as a marker of correction using our treatment strategies.

This collaboration is likely to benefit not only those with the mutation of interest but will also likely help the understanding of disease in general. It is expected that the studies will provide clinical and basic science tools to help clinical translation and ultimately assist the development of new treatments to prevent blindness from PARD.