Thinking of doing your PhD in the Life Sciences? The International PhD Programme (IPP) Mainz is offering talented scientists the chance to work on cutting edge research projects within the open call on “Molecular Biomedicine & Ageing”. As an IPP PhD student, you will join a community of exceptional scientists working on diverse topics ranging from how organisms age or how our DNA is repaired, to how epigenetics regulates cellular identity or neural memory. The research group of Uwe Wolfrum offers the following PhD project:
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 USH1G, which is associated with mutations in the USH1G gene. USH1G encodes for the scaffold protein SANS which has been related to the microtubule-based intracellular transport in the cytoplasm of the cell and ciliogenesis in primary ciliary cell models and retinal photoreceptor cells (2,3). More recently we have shown that SANS also regulates pre-mRNA splicing by interacting with several pre-mRNA splicing factors in the nucleus (4-5). We further demonstrated that SANS deficiency leads to mis-splicing of genes such as ciliary and other USH genes.
Since there are no suitable disease models for USH1G in the human eye, it has not yet been possible to clarify which cells of the retina, and which cellular functions of SANS described above are affected by USH1G defects and underlie the pathophysiology leading to visual impairment in USH1G. Until now, this lack of knowledge has also significantly hindered the development of suitable ocular therapies for the disease.
To overcome this dilemma, we have decided to make use of advanced stem-cell technology and have established over the last years robust protocols to generate human 3D retinal organoids (RO) by reprogramming induced pluripotent stem cells (iPSCs) derived from dermal fibroblast of patients and healthy individuals. As ROs contain all retinal cells and their complex organization reflects the characteristic layers of the human retina, they are well suited to model the healthy and diseased retina in a dish (6).
To achieve our research goals in the PhD project below we now plan to exploit the potential of 3D retinal organoid models in our research projects.
PhD project: USH1G patient-derived retinal organoids to model retinal degeneration and identification of splicing target genes of USH1G/SANS
The aims of the present project are to decipher the pathomechanisms leading to USH1G in the eye and to perform a preclinical evaluation of AAV-mediated gene propagation 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: Identification of differentially spliced genes and determination of retinal cells in which mis-splicing occurs in USH1G retinal organoids
WP3: Determine efficacy of AAV-gene augmentation for USH1G mitigating the disease phenotypes in USH1G retinal organoid model
In WP1, we will generate isogenic-corrected control lines by CRISPR-Cas9-mediated genome-editing (7) from our existing iPSC lines derived from three USH1G patients. Next, we will differentiate USH1G 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 morphological and biochemical methods as well as transcriptomic approaches at different time points during RO maturation. We expect robust phenotypes such as altered morphology and differentially spliced genes in USH1G which will be further specified in WP2 and can be applied as biomarkers for readout measures of the therapeutic treatments in WP3.
In WP2, we will identify genes that are not correctly spliced by comparisons of long-read RNAseq data of USH1G retinal organoids and controls (8). The specific retinal cell types in which the mis-splicing events occur are determined by spatial transcriptomics using antisense RNA probes specific to splicing events.
In WP3, we will generate different AAV (serotype: rAAV2.NN) vectors suitable to deliver USH1G/SANS into ROs (9). We will design AAVs with promotors specific to the retinal cell types identified in WP1/2. We will evaluate their capacity and efficacy for the restorage of the USH1G/SANS transgene expression and the rescue of the pathological phenotypes and mis-splicing events identified in WP1 and 2, respectively.
In conclusion, we will decipher the mechanisms underlying the physiological defects leading to the retinal degeneration in USH1G and will provide a proof-of-concept for ocular gene-based therapy of USH1G.
If you are interested in this project, please select Wolfrum your group preference in the IPP application platform. Are you an ambitious scientist looking to push the boundaries of research while interacting with colleagues from multiple disciplines and cultures? Then joining the IPP is your opportunity to give your scientific career a flying start!
All you need is:
Master or equivalent
Interactive personality & good command of English
2 letters of reference
Exciting, interdisciplinary projects in a lively international environment, with English as our working language
Advanced training in scientific techniques and professional skills
Access to our state-of-the-art Core Facilities and their technical expertise
Fully funded positions with financing until the completion of your thesis
A lively community of more than 200 PhD students from 44 different countries
For more details on the projects offered and how to apply via our online form, please visit www.imb.de/phd.