Zentrum für
Gen- und RNA-
Therapie

Nukleinsäuren (DNA und RNA) sind die “Schlüsselmoleküle des Lebens” und bilden eine wichtige Grundlage für die Regulation von Gesundheit und Krankheit. Die Entdeckung der Rolle von Nukleinsäuren in Gesundheit und Krankheit begann 1869 in Tübingen, als Friedrich Miescher eine neue Substanz namens "Nuklein" in den Kernen von Immunzellen identifizierte. Diese Entdeckung löste eine Revolution in den Biowissenschaften und neuestens auch in der Medizin aus, die zu bedeutenden Innovationen in den Bereichen Diagnostik und Therapie führte. Die Gen- (DNA-) und RNA-Therapie bietet Behandlungsmöglichkeiten für Krankheiten, die traditionell als "unbehandelbar" galten. Es wird erwartet, dass sich die Zahl der behandelbaren Krankheiten drastisch erhöhen wird, was wiederum zu einer ähnlichen medizinischen Revolution führen wird wie die der monoklonalen Antikörper in den letzten 40 Jahren. Die Gen- und RNA-Therapie bietet auch sich schnell entwickelnde, fortschrittliche Tools zur Behandlung häufiger und seltener Krankheiten, die rasche und bahnbrechende Fortschritte aufweisen und die Medizin bereits jetzt und in naher Zukunft revolutionieren werden.  

Prof. Dr. Ludger Schöls
Department of Neurology and Hertie-Institute for Clinical Brain Research, University of Tübingen

AG Schöls - Clinical Neurogenetics

Hereditary spastic paraplegia type 5 (SPG) is a rare neurodegenerative disease that leads to progressive spastic gait disturbance and wheelchair dependency. SPG5 is caused by mutations in the enzyme CYP7B1 that is involved bile acid synthesis in the liver. Lack of CYP7B1 leads to increased concentrations of its substrates 25-hydroxycholesterol and 27-hydroxycholesterol in blood but also in brain of SPG5 patients. As 25- and 27-hydroxycholesterol are neurotoxic, these oxysterols are considered the primary factor causing neurodegeneration in SPG5. 

In our previous work we aimed to restore CYP7B1 in the liver by injections of mRNA. As RNA supplementation shows only to short term effects, we developed a gene replacement approach via an adeno associated virus type 8 (AAV8) which has been used earlier for gene supplementation in other human diseases. AAV8-CYP7b1 resulted in the normalization of hydroxycholesterols in blood and liver but unfortunately had only limited effect in brain. This suggests that CYP7B1 needs also to be replaced in the central nervous system (CNS) to prevent its neurotoxic effects in SPG5. 

Therefore, this flagship project aims to develop novel AAV vectors like AAV-PHP.eB and AAV.CAP-B22 which are able to cross the blood-brain barrier in mice as well as in non-human primates. Intravenous injection of these vectors is supposed to result in expression of its “cargo gene” not only in the liver but also in the CNS. We will investigate therapeutic efficacy and potential toxicity of the new vectors in cyp7b1 knockout mice. Oxysterol levels in blood, liver, and brain will be determined as the primary readout parameter by mass spectrometry. Vectors will be evaluated for liver and CNS toxicity. In addition, neural tropism and potential neural toxicity of the new vectors will be tested in vitro human neuronal cell cultures and brain organoids derived from induced pluripotent stem cells (iPSC).

Prof. Dr. Matthis Synofzik
Division Translational Genomics of Neurodegenerative Diseases, Center of Neurology and Hertie-Institute of Clinical Brain Research, University of Tübingen

AG Synofzik - Translational Genomics of Neurodegenerative Diseases

Gene-targeted therapies hold promise for treating many currently untreatable diseases.  In particular for manifold rare neurological diseases (RND) - for which treatments had seemed out of reach for long. However, existing drug development and regulatory processes are inadequate for very rare RNDs affecting only a few or even just one patient globally. To overcome this gap, we here propose a completely new drug-development paradigm in neurology: development of individual patient-customised molecular therapies tailored to each single patient’s very individual mutation.  For this we will leverage a highly-programmable RNA therapy - antisense oligonucleotide (ASO)-mediated splice modulation (Figure 1) - that allows to target various nano-rare or private mutations across different RNDs, enabling personalized therapies tailored to individual patients' mutations; but which is at the same time - by targeting the same mutation types shared across many RNDs- scalable to multiple RNDs alike (Figure 2)

To test this novel RNA therapy paradigm, this project will establish a systematic platform for developing patient-customized ASOs. As a demonstration showcase, we have selected particularly  severe childhood-onset brain disease: ataxia telangiectasia (A-T). As an end-to-end platform, the platform starts off with the individual patient, develops an individualised ASO through all pertinent preclinical and clinical steps, and then brings the individual ASO back into clinics to treat that very individual patient. The preclinical ASO platform module hereby follows a comprehensive approach from patient identification to ASO design, RNA and protein analysis, functional assessments, and various toxicity assays (Figure 3). Our platform will simultaneously explore four different ASO target strategies, each addressing specific clusters of shared mutation types in the A-T gene. If successful, this comprehensive approach could revolutionize genomic medicine for RND patients. The concept of individualized genomic therapeutics could become a groundbreaking paradigm, impacting a significant number of patients with rare neurological diseases.

PD Dr. med. Dr. rer. nat. Dipl. Biol. Markus Mezger
Dr. rer. nat. Tahereh Mohammadian Gol
Dr. rer. nat. Justin Antony Selvaraj
Department of General Paediatrics, Cell & Gene Therapy group University Children's Hospital Tübingen, University of Tübingen

Pediatric Cell and Gene Therapy - AG Mezger

β-hemoglobinopathies are inherited hematological disorders caused by mutations in the human hemoglobin beta (HBB) gene that reduce or abrogate β-globin expression. Allogeneic hematopoietic stem cell transplantation (HSCT) using stem cells from a foreign donor is the standard therapy but has certain limitations such as finding an HLA matched donor and HSCT associated complications. In addition, previous gene therapy approaches have utilized lentiviral vectors (LVs) to introduce a functional copy of the β-globin gene into the patient's hematopoietic stem and progenitor cells (HSPCs). Alternatively, reactivating fetal hemoglobin (HbF) using CRISPR-Cas9 method can reduce or even prevent the symptoms of disease. These approaches have significant disadvantages including risk of insertional mutagenesis and continued expression of defective β-globin. 

To overcome these limitations, we have combined CRISPR-Cas9 technology with adeno-associated virus (AAV) based gene delivery method to apply a precise gene correction of mutated HBB gene (Lamsfus-Calle et al. The CRISPR Journal 2021). Using this approach, we attained high rates of gene correction in HSPCs of β-thalassemia patients, achieving a significant increase in adult hemoglobin (HbA) level. 

Following promising result in preclinical studies, in this project we aim to develop an optimized transfection protocol for correction of CD34⁺ HSPCs at a clinical and GMP production scale following a recently established protocol for CRISPR/Cas9 transfection on the CliniMACS Prodigy platform (Ureña-Bailén et al. The CRISPR Journal 2023). Moreover, we will implement our findings to conduct a Phase 1/2 clinical trial to assess safety and efficacy of this approach in patients with transfusion dependent β-Thalassemia.

Prof. Dr. Julia Skokowa¹, Dr. Masoud Nasri¹, Malte Ritter¹, Prof. Claudia Lengerke¹, Prof. Wolfgang Bethge^1,2, PD Dr. Dr. Markus Mezger³, Prof. Dr. Peter Lang³, Prof. Dr. Johannes Schulte³, Prof. Dr. Karl Welte³, Dr. Cornelia Zeidler¹.

¹Department of Oncology, Hematology, Clinical Immunology, and Rheumatology, University Hospital Tübingen, Germany
²Center for Clinical Trials, University Hospital Tübingen
³Department of Pediatric Hematology and Oncology, Children`s Hospital, University Hospital Tübingen, Germany

AG Skokowa

Congenital neutropenia, a condition caused by specific genetic mutations (ELANE-CN), leads to a significant decrease in the number of infection-fighting white blood cells, making individuals highly prone to severe bacterial infections. This condition also elevates the risk of developing myelodysplastic syndromes or acute myeloid leukemia. The conventional treatment involves daily injections of a substance called recombinant human granulocyte colony-stimulating factor (rhG-CSF) to boost white blood cell counts. However, this approach does not address the underlying genetic issue and may have side effects. Some patients don't respond to this treatment.

To address these challenges, we have developed a gene therapy called MILESTONE for ELANE-CN patients. This innovative approach utilizes CRISPR/Cas9 technology, employing a double Cas9 nickase strategy to target the genetic mutations and inhibit the expression of the faulty ELANE gene. This not only restores the production of infection-fighting white blood cells but also enhances precision and minimizes unintended effects. MILESTONE has the potential to revolutionize the treatment of ELANE-CN.

Our next step is to initiate an Investigator-Initiated Trial (IIT) for this gene therapy. The trial will focus on editing CD34+ cells derived from patients, incorporating MILESTONE technology, and utilizing autologous transplantation. This single-site trial will involve ELANE-CN patients from the European Registry, attracting participants not only from Germany but also from across Europe and potentially from other regions. It is worth noting that our innovative approach, protected by patents, has broader applications beyond ELANE-CN and can be adapted for other genetic disorders and even for inhibiting cancer-related genes in the future.

Furthermore, the establishment of a Good Manufacturing Practice (GMP)-grade gene editing facility for CD34+ cells at the University Hospital Tübingen (UKT) will be a significant development. This facility will not only benefit ELANE-CN patients but also attract interest from biotech companies and patients, given the current scarcity of such facilities in Germany and Europe at large.

Prof. Dr. Christopher Cederroth, 
Prof. Dr. Ellen Reisinger, 
Prof. Dr. Steffen Hage 
Department for Otolaryngology, University Hospital Tübingen

Gene Therapy for Hearing Impairment - AG Reisinger

Hearing impairment affects 1 to 2 newborns, and the same number becomes hearing impaired in childhood. At least half of the cases are due to mutations in certain genes. In affected people, more than 300 genes are screened for mutations to find the individual cause. For many forms of inherited hearing impairment, researchers aim to find out which cells of the inner ear require the respective protein encoded by this gene, and how a mutation leads to hearing loss. The long-term aim of many groups of researchers is to provide targeted gene therapies for as many forms of hearing impairment as possible. To transfer genetic information, we employ modified viruses, so-called recombinant adeno-associated viruses (AAVs), of which a number of naturally occurring and engineered variants are known. However, to date, only a limited number of these viral vector variants have been tested for the inner ear. As consequence, only few inner ear cell types can currently be targeted with gene therapy. 

In this project, we aim to inject a series of AAV variants into the inner ear of mice and marmoset monkeys. Each of the vector variant will contain a genetic “barcode” for later identification. We will compare two different injection routes: the direct injection into the bony cavity of the inner ear, and an indirect injection via the cisterna magna, a fluid-filled compartment of the hindbrain which is connected to the inner ear. By sequencing the genetic information of individual cells from the inner ear, the brain, the liver, and more tissues, we will find out which viral vectors transduced which cell types. This information is crucial to design targeted gene therapies for the inner ear, but potentially also for brain disorders.