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Toni Cathomen

Publications and source records attributed to Toni Cathomen.

7 recordsLinked to original sources
PubMed2026

Targeted insertion of an optimized donor DNA is effective in a humanized mouse model of dominant retinitis pigmentosa.

Retinitis pigmentosa (RP) affects 1 in 3,000 individuals worldwide, with 30%-40% of cases inherited as autosomal dominant (AD). Mutations in RHO (RP4) are the most common cause of ADRP. Because most RHO mutations exert gain-of-function or dominant-negative effects, conventional gene supplementation is insufficient, requiring mutant allele inactivation. Allele-specific editing is impractical, as each mutation requires a unique therapeutic strategy. We present a mutation-agnostic, RHO-specific approach using adeno-associated viral vector-mediated homology-independent targeted integration (AAV-HITI). Optimized donor DNA design enables targeted integration and efficient transgene expression from the endogenous RHO locus. In a humanized RP4 mouse model harboring the RHO P23H mutant allele alongside an endogenous wild-type mouse Rho allele, AAV-HITI significantly improves retinal structure, function, and visual acuity up to 1 year post-treatment. Comprehensive molecular analyses characterize on-target editing in mouse retina and off-target editing in a human cell line. These findings establish an effective, human-centric AAV-HITI platform for RP4 and support its evaluation in this and other dominant genetic conditions.

AAV↗
PubMed2012

The nontoxic cell cycle modulator indirubin augments transduction of adeno-associated viral vectors and zinc-finger nuclease-mediated gene targeting.

Parameters that regulate or affect the cell cycle or the DNA repair choice between non-homologous end-joining and homology-directed repair (HDR) are excellent targets to enhance therapeutic gene targeting. Here, we have evaluated the impact of five cell-cycle modulating drugs on targeted genome engineering mediated by DNA double-strand break (DSB)-inducing nucleases, such as zinc-finger nucleases (ZFNs). For a side-by-side comparison, we have established four reporter cell lines by integrating a mutated EGFP gene into either three transformed human cell lines or primary umbilical cord-derived mesenchymal stromal cells (UC-MSCs). After treatment with different cytostatic drugs, cells were transduced with adeno-associated virus (AAV) vectors that encode a nuclease or a repair donor to rescue EGFP expression through DSB-induced HDR. We show that transient cell-cycle arrest increased AAV transduction and AAV-mediated HDR up to six-fold in human cell lines and ten-fold in UC-MSCs, respectively. Targeted gene correction was observed in up to 34% of transduced cells. Both the absolute and the relative gene-targeting frequencies were dependent on the cell type, the cytostatic drug, the vector dose, and the nuclease. Treatment of cells with the cyclin-dependent kinase inhibitor indirubin-3'-monoxime was especially promising as this compound combined high stimulatory effects with minimal cytotoxicity. In conclusion, indirubin-3'-monoxime significantly improved AAV transduction and the efficiency of AAV/ZFN-mediated gene targeting and may thus represent a promising compound to enhance DSB-mediated genome engineering in human stem cells, such as UC-MSCs, which hold great promise for future clinical applications.

Blotting, Western↗
PubMed2006

Targeted chromosomal gene modification in human cells by single-stranded oligodeoxynucleotides in the presence of a DNA double-strand break.

A DNA double-strand break (DSB) cannot be tolerated by a cell and is dealt with by several pathways. Here, it was hypothesized that DSB induction close to a targeted mutation in the genome of a mammalian cell might attract oligodeoxynucleotide (ODN)-directed gene repair. A HEK-293-derived cell line had been engineered harboring a single target locus with open reading frames encoding the living-cell reporter proteins LacZ and EGFP, the latter translationally decoupled by a DNA spacer with a unique I-SceI recognition site for defined DSB induction. To enable expression of a fluorescent LacZ-EGFP fusion protein, single-stranded (ss) ODNs (80 or 96 nucleotides long) spanning the DSB were designed to fuse both reading frames by altering a few base-pair positions, deleting 59 bp or introducing a 10-bp fragment. The ssODNs alone generated few EGFP-positive cells. With I-SceI transiently expressed, more than 0.3% of cells revealed EGFP expression 7 days after transfection, with up to 96% of the loci faithfully corrected, depending on the ssODN used. During these correction events, the ssODN did not become physically incorporated into the chromosome, but served only as information template. Unwanted insertional mutagenesis also occurred. Both observations have important implications for gene therapy.

Base Sequence↗
PubMed2005

Custom zinc-finger nucleases for use in human cells.

Genome engineering through homologous recombination (HR) is a powerful instrument for studying biological pathways or creating treatment options for genetic disorders. In mammalian cells HR is rare but the creation of targeted DNA double-strand breaks stimulates HR significantly. Here, we present a method to generate, evaluate, and optimize rationally designed endonucleases that promote HR. The DNA-binding domains were synthesized by assembling predefined zinc-finger modules selected by phage display. Attachment of a transcriptional activation domain allowed assessment of DNA binding in reporter assays, while fusion with an endonuclease domain created custom nucleases that were tested for their ability to stimulate HR in episomal and chromosomal gene repair assays. We demonstrate that specificity, expression kinetics, and protein design are crucial parameters for efficient gene repair and that our two-step assay allows one to go quickly from design to testing to successful employment of the custom nucleases in human cells.

Base Sequence↗
PubMed2004

AAV vectors for gene correction.

Permanent correction of genetic mutations is the ultimate goal in the treatment of inherited disorders. Although gene therapy protocols have proven successful in several clinical trials that focused on monogenetic diseases, alternative approaches have become the subject of growing interest since two cases of insertional mutagenesis were reported in one of these trials. Gene correction is an alternative technology that aims to repair a mutated gene rather than complement the mutation by adding a wild-type copy of the affected gene. This review focuses on viral approaches to gene correction, especially the use of vectors based on adeno-associated viruses. The pitfalls and prospects, as well as the steps needed to improve and advance the technology for clinical applications, are discussed.

Animals↗
PubMed2003

Efficient gene targeting mediated by adeno-associated virus and DNA double-strand breaks.

Gene targeting is the in situ manipulation of the sequence of an endogenous gene by the introduction of homologous exogenous DNA. Presently, the rate of gene targeting is too low for it to be broadly used in mammalian somatic cell genetics or to cure genetic diseases. Recently, it has been demonstrated that infection with recombinant adeno-associated virus (rAAV) vectors can mediate gene targeting in somatic cells, but the mechanism is unclear. This paper explores the balance between random integration and gene targeting with rAAV. Both random integration and spontaneous gene targeting are dependent on the multiplicity of infection (MOI) of rAAV. It has previously been shown that the introduction of a DNA double-stranded break (DSB) in a target gene can stimulate gene targeting by several-thousand-fold in somatic cells. Creation of a DSB stimulates the frequency of rAAV-mediated gene targeting by over 100-fold, suggesting that the mechanism of rAAV-mediated gene targeting involves, at least in part, the repair of DSBs by homologous recombination. Absolute gene targeting frequencies reach 0.8% with a dual vector system in which one rAAV vector provides a gene targeting substrate and a second vector expresses the nuclease that creates a DSB in the target gene. The frequencies of gene targeting that we achieved with relatively low MOIs suggest that combining rAAV vectors with DSBs is a promising strategy to broaden the application of gene targeting.

Cell Line↗