A5049223212- CRISPR and Genetic Engineering
- Virus-based gene therapy research
- CAR-T cell therapy research
- RNA Interference and Gene Delivery
- HIV Research and Treatment
- Pluripotent Stem Cells Research
- Advanced biosensing and bioanalysis techniques
- Retinal Development and Disorders
- RNA regulation and disease
- Immune Cell Function and Interaction
- Epigenetics and DNA Methylation
- Cytomegalovirus and herpesvirus research
- Hepatitis B Virus Studies
- Hemoglobinopathies and Related Disorders
- Plant Virus Research Studies
- Transgenic Plants and Applications
- Innovation and Socioeconomic Development
- Viral Infectious Diseases and Gene Expression in Insects
- Photoreceptor and optogenetics research
- Skin and Cellular Biology Research
- Animal Genetics and Reproduction
- T-cell and B-cell Immunology
- Immunotherapy and Immune Responses
- Ocular Disorders and Treatments
- DNA Repair Mechanisms
University Medical Center Freiburg
2016-2025
University of Freiburg
2016-2025
Institute for Transfusion Medicine
2020-2024
Gene Therapy Laboratory
2015
Medizinische Hochschule Hannover
2011-2014
University of Modena and Reggio Emilia
2012
Leiden University Medical Center
2012
Telethon Institute Of Genetics And Medicine
2005-2011
Telethon Foundation
2008-2009
European School of Molecular Medicine
2005-2007
Sequence-specific nucleases represent valuable tools for precision genome engineering. Traditionally, zinc-finger (ZFNs) and meganucleases have been used to specifically edit complex genomes. Recently, the DNA binding domains of transcription activator-like effectors (TALEs) from bacterial pathogen Xanthomonas harnessed direct nuclease desired genomic loci. In this study, we tested a panel truncation variants based on TALE protein AvrBs4 identify (TALENs) with high cleavage activity. The...
The array of genome editing strategies based on targeted double-stranded DNA break formation have recently been enriched through the introduction transcription activator-like type III effector (TALE) nucleases (TALENs). To advance testing TALE-based approaches, it will be crucial to deliver these custom-designed proteins not only into transformed cell types but also more relevant, chromosomally stable, primary cells. Viral vectors are among most effective gene transfer vehicles. Here, we...
RNA-guided nucleases (RGNs) based on the type II CRISPR-Cas9 system of Streptococcus pyogenes (Sp) have been widely used for genome editing in experimental models. However, nontrivial level off-target activity reported several human cells may hamper clinical translation. RGN specificity depends both guide RNA (gRNA) and protospacer adjacent motif (PAM) recognized by Cas9 protein. We hypothesized that more stringent PAM requirements reduce occurrence mutagenesis. To test this postulation, we...
Chronic hepatitis B virus (HBV) infection remains an important global health problem. Stability of the episomal covalently closed circular HBV DNA (cccDNA) is largely responsible for modest curative efficacy available therapy. Since licensed anti-HBV drugs have a post-transcriptional mechanism action, disabling cccDNA potentially therapeutic benefit. To develop this approach, we engineered mutagenic transcription activator-like effector nucleases (TALENs) that target four HBV-specific sites...
ABSTRACT Severe inherited retinal diseases, such as retinitis pigmentosa and Leber congenital amaurosis, are caused by mutations in genes preferentially expressed photoreceptors. While adeno-associated virus (AAV)-mediated gene transfer can correct pigment epithelium (RPE) defects animal models, approaches for the correction of photoreceptor-specific diseases less efficient. We evaluated ability novel AAV serotypes (AAV2/7, AAV2/8, AAV2/9, AAV2rh.43, AAV2rh.64R1, AAV2hu.29R) combination with...
Designer nucleases have been successfully employed to modify the genomes of various model organisms and human cell types. While specificity zinc-finger (ZFNs) RNA-guided endonucleases has assessed some extent, little data are available for transcription activator-like effector-based (TALENs). Here, we engineered TALEN pairs targeting three loci (CCR5, AAVS1 IL2RG) performed a detailed analysis their activity, toxicity specificity. The TALENs showed comparable activity benchmark ZFNs, with...
Engineered zinc finger nucleases (ZFNs) induce DNA double-strand breaks at specific recognition sequences and can promote efficient introduction of desired insertions, deletions or substitutions near the cut site via homology-directed repair (HDR) with a double- and/or single-stranded donor template. However, mutagenic events caused by error-prone non-homologous end-joining (NHEJ)-mediated are introduced equal higher frequency nuclease cleavage site. Furthermore, unintended mutations also...
Targeted modulation of gene expression represents a valuable approach to understand the mechanisms governing regulation. In therapeutic context, it can be exploited selectively modify aberrant disease-causing or provide target cells with new function. Here, we have established novel platform for achieving precision epigenome editing using designer modifiers (DEMs). DEMs combine in single molecule DNA binding domain based on highly specific transcription activator-like effectors (TALEs) and...
Recent success in clinical trials supports the use of adeno-associated viral (AAV) vectors for gene therapy retinal diseases caused by defects pigment epithelium (RPE). In contrast, evidence efficacy AAV-mediated transfer to photoreceptors, major site inherited diseases, is less robust. addition, although RPE transduction appears efficient, independently serotype used and species treated, photoreceptor has not been systematically investigated thus so far large animal models, which also may...
The potential of adoptive cell therapy can be extended when combined with genome editing. However, variation in the quality starting material and different manufacturing steps are associated production failure product contamination. Here, we present an automated T engineering process to produce off-the-shelf chimeric antigen receptor (CAR) cells on CliniMACS Prodigy platform containing in-line electroporation unit. This setup was used combine lentiviral delivery a CD19-targeting CAR transfer...
Disruption of CCR5 or CXCR4, the main human immunodeficiency virus type 1 (HIV-1) co-receptors, has been shown to protect primary CD4+ T cells from HIV-1 infection. Base editing can install targeted point mutations in cellular genomes, and thus efficiently inactivate genes by introducing stop codons eliminating start without double-stranded DNA break formation. Here, we applied base editors for individual simultaneous disruption both co-receptors cells. Using cytosine observed premature...
Abstract Precise genome editing requires the resolution of nuclease-induced DNA double strand breaks (DSBs) via homology-directed repair (HDR) pathway. In mammals, this is typically outcompeted by non-homologous end-joining (NHEJ) that can generate potentially genotoxic insertion/deletion mutations at DSB sites. Because higher efficacy, clinical has been restricted to imperfect but efficient NHEJ-based approaches. Hence, strategies promote HDR are essential facilitate transition HDR-based...
Human leucocyte antigen class I (HLA-I) molecules play a central role for both NK and T-cell responses that prevent serious human cytomegalovirus (HCMV) disease. To create opportunities viral spread, several HCMV-encoded immunoevasins employ diverse strategies to target HLA-I. Among these, the glycoprotein US10 is so far insufficiently studied. While it was reported interferes with HLA-G expression, its ability manipulate classical HLA-I presentation remains unknown. In this study, we...
Epidermolysis bullosa simplex (EBS) is a blistering skin disease caused by dominant-negative mutations in either KRT5 or KRT14, resulting impairment of keratin filament structure and epidermal fragility. Currently, nearly 200 distributed across the entire length these genes are known to cause EBS. Genome editing using programmable nucleases enables development ex vivo gene therapies for genetic diseases. A clinically feasible strategy involves disruption mutant allele while leaving wild-type...
Abstract Homology directed repair (HDR)-based genome editing via selectable long flanking arm donors can be hampered by local transgene silencing at transcriptionally silent loci. Here, we report efficient bi-allelic modification of a locus in patient-derived hiPSC using Cas9 nickase and silencing-resistant donor construct that contains an excisable selection/counter-selection cassette. To identify the most active single guide RNA (sgRNA)/nickase combinations, employed lentiviral...