Developing Retinal Gene Therapy for Zellweger Spectrum Disorder (ZSD)

We selected the AAV8 serotype as it most efficiently transduces our primary target cell type (the photoreceptor cells), while still transducing our secondary target (the RPE cells). We chose a ubiquitous promoter as all cells in this model are PEX1-deficient, and we do not fear off-target effects of PEX1 expression. As this was our first proof-of-concept study, we elected to use highly-expressing promoter, and also included an HA tag to facilitate HsPEX1 protein detection in mice with endogenous MmPEX1-G844D. We have since moved to a more clinically-translatable vector design and have achieved more consistent and robust effects than this initial POC.

We selected subretinal delivery to best access our target cells – the outer retina, where the retinal phenotype originates in this model. We have not tried intravitreal injection.

The maximum dose is limited by vector concertation, and will differ depending on production batch. This was the highest dose we could administer without diluting the vector.

These injections were trans-scleral. Retinal detachment and vector delivery were performed in one step.

The HsPEX1.HA distribution pattern matches that of endogenous MmPEX1, but overexpressed at least 5-fold.

PEX1 is concentrated at the inner segment and outer plexiform layer in both mouse and human retina.

We have characterized the RPE defects in the mouse but cannot yet disclose the phenotype. In our more recent expanded study, gene therapy did correct the phenotype.

Total plasmalogens are not reduced in the PEX1-G844D mouse model, but DHA specifically can be mildly but significantly reduced. DHA levels do not recover with gene therapy.

There is no larger animal model of Pex1 disease.

Very long chain fatty acids are elevated in peroxisome disorders, and gene therapy corrects this. We have not tried supplementing this model with DHA, which is reduced. However, peroxisome biogenesis disorders result in multiple enzyme deficiencies and perturbations in many metabolic pathways. It is unknown if correcting one metabolite would improve the course of disease. Deeper phenotyping to study this pathophysiology is warranted.

There were no detectable structural or functional side effects post injection. The retina had re-attached when it was visualized histologically 2 months post injection.

The progression of retinal degeneration in this model supports it being a cone-rod dystrophy. We do not plan to target the cones exclusively as multiple cell types are affected quickly. However, it would indeed be interesting to test whether exclusive cone-rescue is sufficient to delay or prevent deterioration of other cell types.

Mice were tested once daily (i.e. one test type per day to determine threshold) in the morning hours. This prevented prolonged testing time and reduce the chance of testing fatigue in the mouse.

Both. We expect the eligibility criteria to be presence of photoreceptor cells. The rate of cell deterioration can vary depending on disease severity.

Yes. We have recently completed a retrospective analysis of vision in a large ZSD cohort as part of a natural history study. We will soon commence a funded prospective study assessing retinal pathology and functional vision in participants of various ages at 2-3 sites across North America. This will identify the optimal treatment window and clinical outcomes for this population. Assessments will be performed yearly for 5 years, but we expect informative results even after the first tests.

In clinical practice, more inflammation is detection post intravitreal AAV administration compared to subretinal. Inflammation is treatable and typically resolves.

We have seen striking improvements in retinal structure in our more recent studies, but cannot disclose details at this time.