1 PhD project offered in the IPP winter call Molecular Biomedicine & Ageing

Scientific Background

Human Usher syndrome (USH) is a complex disease and the most common form of inherited deaf-blindness (1). Of the four clinical types, USH1 is the most severe, characterized by congenital profound hearing and vestibular areflexia, and prepubertal onset of vision loss, named Retinitis pigmentosa (RP), which progresses with age. There is no ocular therapy for USH, which is why most cases of USH1 lead to severe visual impairment and even complete blindness in the third quarter of life.
To date, 6 distinct gene loci are known to be associated with USH1, including USH1C, which is associated with mutations in the USH1C gene. USH1C encodes for the scaffold protein harmonin which is expressed in numerous isoforms, with harmonin a1 as the most abundant isoform in the human retina (2). We and others identified the abundance of USH1C transcripts and harmonin protein not only in photoreceptor cells but surprisingly also in Müller glial cells (2, 3, Wenck et al. in prep). The lack of suitable disease models for the ocular component of USH1 has so far prevented the elucidation of the mechanisms that lead to visual impairment in USH and hindered the development of appropriate ocular therapies for USH (4).
To overcome this dilemma, we have established a human cellular model system, namely retinal 3D organoids (ROs) developed from human induced pluripotent stem cells (iPSCs) derived from dermal fibroblasts of USH1C patients and healthy individuals (Wenck et al. in prep) to model USH1C. single cell RNA-sequencing (scRNA-seq) and subsequent bioinformatics analysis revealed significant differences between healthy and USH1C ROs. For example, in USH1C ROs, we detected a decrease of rod precursors and rod photoreceptors and in contrast, we observed an increase of Müller glia cells (MGCs), compared to ROs of healthy donors. GO term analysis and KEGG-pathway also revealed differentially expressed genes (DEGs) related to e.g. rod and cone photoreceptor function and “canonical Wnt singaling” in Müller glial cells confirming our previous data on harmonin acting as cWnt suppressor (5). The identified abundance of USH1C transcripts and harmonin protein in MGCs, combined with the significant molecular changes observed in MGCs of USH1C ROs, challenges the hypothesis of a photoreceptor-only pathophysiology of USH1C (2, 3, Wenck et al. in prep) currently held in the research field. Overall, the initial results obtained in USH1C-ROs already form the basis for the evaluation of gene-based therapeutic strategies in this model system.
Adeno-associated virus (AAV) gene augmentation is the gold standard among gene-based retinal therapies that is clinically approved for a subset of RP patients with hereditary vision loss (Luxturna®) (6). Determining the efficacy AAV-mediated gene augmentation in our USH1C models will provide hope for curing vision loss in USH1C-patients.

PhD project: USH1C patient-derived retinal organoids to model retinal degeneration and evaluate therapeutic strategies

The overall goals of the present project are to determine the mechanisms underlying the retinal phenotype in USH1C and to conduct a preclinical evaluation of AAV-mediated gene augmentation in iPSC-derived retinal organoids for proof of concept. To achieve these goals, we have compiled the following work packages (WP):

WP1: Generate retinal organoids from patient-derived, isogenic-corrected patient-derived controls and healthy-derived iPSCs and evaluate pathomechanisms through in-depth molecular, physiologic, and morphologic phenotype analysis.
WP2: Determine efficacy of AAV-gene augmentation by analyzing different promotors to drive USH1C-expression in mitigating the disease phenotypes in USH1C retinal organoid model.
 

In WP1, we will generate from our existing iPSC lines derived from USH1C patient and healthy individuals isogenic-corrected control lines by CRISPR-Cas9-mediated genome-editing (7). Next, we will differentiate USH1C patient-derived iPSC, isogenic controls and healthy controls into iPSC-derived 3D retinal organoids (ROs). For this we will use our newly introduced, refined protocol, which allows us to produce a higher number of retinal organoids per batch than with our previous protocol. This improved protocol, will enable us to analyze the generated ROs by complementary methods such as morphological, biochemical, electrophysiologic and proteomic approaches at different time points during RO maturation. The in-depth analysis will provide insights into the pathomechanisms underlying the retinal phenotype in USH1C and we expect to identify robust phenotypes which can be applied as biomarkers for readout measurements of the therapeutic treatments in WP2.
In WP2, we will generate different AAV (serotype: rAAV2.NN) vectors suitable to deliver USH1C/harmonin-a1 into ROs (8). Besides AAVs with a ubiquitous promoter, we will use two others that ensure cell specific expression of the USH1C/harmonin transgene in rod and cone photoreceptors and in MGCs, respectively. Applying the different vectors, we will evaluate their capacity and efficacy for the restorage of the USH1C/harmonin-a1 transgene expression and the rescue of the pathological phenotypes identified in WP1. The expected data will also allow us to narrow down the cell types (MGCs or photoreceptor cells) in which USH1C defects cause retinal degeneration. In addition, the data obtained will support the design of a preclinical gene therapy study in an existing large animal model (9) to evaluate the eventual toxicity and efficacy of AAV-mediated gene transfer in an organism.
In conclusion, we will decipher the mechanisms underlying the physiological defects leading to the retinal degeneration in USH1C and will provide a proof-of-concept for ocular gene-based therapy of USH1C.

If you are interested in this project, please select Wolfrum your group preference in the IPP application platform.

Publications relevant to this project

Fuster-García C, García-Bohórquez B, Rodríguez-Muñoz A, Aller E, Jaijo T, Millán JM, García-García G (2021) Usher Syndrome: Genetics of a Human Ciliopathy. Int J Mol Sci 22:6723. Link

Nagel-Wolfrum K, Fadl BR, Becker MM, Wunderlich KA, Schäfer J, Sturm D, Fritze J, Gür B, Kaplan L, Goldmann T, Brooks M, Starosk MR, Lokhande A, Apel M, Fath KR, Stingl K, Kohl S, DeAngelis MM, Schlötzer-Schrehardt U, Kim IK, Owen LA, Vetter JM, Pfeiffer N, Andrade-Navarro MA, Grosche A, Swaroop A, Wolfrum U (2023) Expression and subcellular localization of USH1C/harmonin in human retina provides insights into pathomechanisms and therapy. Hum Mol Genet 32:431-449. Link

Cowan CS et al. .... Scholl HPN, Roma G, Nigsch F, Roska B (2020) Cell Types of the Human Retina and Its Organoids at Single-Cell Resolution. Cell 182:1623-1640.e34. Link

Williams DS (2008) Usher syndrome: animal models, retinal function of Usher proteins, and prospects for gene therapy.Vision Res 48, 433-441. Link

Schäfer J, Wenck N, Janik K, Linnert, J, Stingl, K, Kohl S, Nagel-Wolfrum K, Wolfrum U (2023) The Usher syndrome 1C protein harmonin regulates canonical Wnt signaling. Front Cell Dev Biol 11:1130058. Link

Bennett J (2023) Overview of Retinal Gene Therapy: Current Status and Future Challenges. Cold Spring Harb Perspect Med 13:a041278. Link

Sanjurjo-Soriano C, Jimenez-Medina C, Erkilic N, Cappellino L, Lefevre A, Nagel-Wolfrum K, Wolfrum U, Van Wijk E, Roux AF, Meunier I, Kalatzis V (2023) USH2A variants causing retinitis pigmentosa or Usher syndrome provoke differential retinal phenotypes in disease-specific organoid. HGG Adv 4:100229. Link

Völkner M, Pavlou M, Büning H, Michalakis S, Karl MO (2021) Optimized adeno-associated virus vectors for efficient transduction of human retinal organoids. Gene Ther 32:694-706. Link

Grotz S*, Schäfer J* et al. ...... Vandenberghe LH, Wolf E, Nagel-Wolfrum K, Motlik J, Fischer MD, Wolfrum U*, Klymiuk N* (2022) Early disruption of photoreceptor cell architecture and loss of vision in a humanized pig model of Usher syndrome. EMBO Mol Med 4:e14817, *equal contribution. Link