Development and characterization of mouse models of RP59 DHDDS deficiency

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7. Project Summary/Abstract Within the family of Congenital Disorders of Glycosylation (CDGs), defects in the dehydrodolichyl diphosphate synthase (DHDDS) gene cause a recessive form of retinitis pigmentosa (RP59; OMIM #613861). Patients with a K42E mutation in DHDDS exhibit progressive loss of both rod and cone function, as well as macular changes, suggestive of RPE involvement. The DHDDS enzyme is ubiquitously required in all cells for protein N- glycosylation. We wish to understand the basis for selective ocular pathology associated with ubiquitous DHDDS mutation and the contribution of specific ocular cell types to the pathology of mutant Dhdds-mediated retinal degeneration. As a first step, we devised a selective knockout scheme to study the importance of the enzyme in specific retinal cell types. To circumvent known embryonic lethality associated with DHDDS knockout, we generated a Cre-dependent knockout allele of murine Dhdds (Dhddsflx/flx). Additionally, we used CRISPR/Cas technology to generate a knock-in K42E mouse model of RP59. We propose to use these novel mouse lines to examine the mechanism of disease induced by DHDDS enzyme deficiency and to identify the primary site(s) of ocular pathology. In Aim 1, using rod-specific Cre expression, we will address the prevailing hypothesis that defective rhodopsin glycosylation is the major cause of Dhdds mutation-mediated pathology. In Aim 2, using retinal pigment epithelium (RPE)-specific Cre expression, we will examine the effects of perturbation of DHDDS activity on RPE structure and function. In Aim 3, we will selectively perturb DHDDS activity in Müller glia, and examine their contribution to Dhdds-dependent retinal degeneration. In Aim 4, we will characterize a newly generated K42E knock-in RP mouse model and compare the resulting structural and functional changes with those observed in the three knockout models to assess the pathological importance of each cell type. These four Aims will utilize a combination of state-of-the-art methodologies, including Cre-lox technology, CRISPR-Cas9- mediated genome editing, electroretinography (ERG), ultrahigh resolution spectral domain-optical coherence tomography (UHR SD-OCT), optokinetic reflex (OKR), light and electron microscopy, immunohistochemistry and lectin histochemistry, Western blot analysis, HPLC, and MALDI imaging mass spectrometry (IMS). Each stand- alone aim will provide significant new insights into the importance of DHDDS activity in the neural retina and RPE, and collectively will provide information that can guide rational treatment design for specific intervention in DHDDS-mediated RP. The overarching hypothesis is that, contrary to conclusions from initial reports, RP59 is not a simple disorder of altered rhodopsin glycosylation; rather, it is a complex disorder involving primary pathology in more than one cell type that may require a more global approach for effective therapeutic intervention. Upon completion of the proposed studies, we will be well positioned to pursue future proof-of- principle preclinical gene therapy studies to correct the genetic defect that otherwise would result in blindness.