Biology subjects

Kreuz, S.

Publications and source records attributed to Kreuz, S..

4 recordsLinked to original sources

bioRxiv2025

STARR-CRAAVT: A platform to design cell type-specific regulatory elements for next-generation gene therapy

Precise control of transgene expression through novel enhancer-promoter combinations is a promising strategy for advancing gene therapy mediated by adeno-associated virus (AAV). We present STARR-CRAAVT, a novel STARR-Seq-derived platform to screen for enhancers in the AAV context. Using HepG2 and HaCaT cells as screening models, we applied STARR-CRAAVT for the identification of cell type-specific enhancers. We integrated epigenetic datasets into an in-silico library of putative HepG2 enhancers and captured corresponding fragments from genomic DNA. The fragments were processed into AAV libraries and applied to HepG2 and HaCaT cells. STARR-CRAAVT analysis revealed a selective activity of the libraries confirming the HepG2-directed in-silico design and the specificity of single enhancer-promoter combinations could be validated using luciferase reporter assays. In addition, we scrutinized the impact of key experimental parameters on enhancer identification and found that the used promoter type had significant influence on the ability of candidates to act as enhancers. Furthermore, switching the location of identified enhancers in reporter assays revealed that the level of enhancer activity is highly dependent on the position in the AAV genome. Taken together, our study yields novel insights into enhancer function and demonstrates that STARR-CRAAVT can be employed to identify cell type-specific enhancers, highlighting promoter preference and enhancer positioning as key considerations for enhancer screening campaigns.

genomics↗

bioRxiv2025

Small molecule-controlled gene expression: Design of drug-like high affinity small molecule modulators of a custom-made riboswitch

Riboswitches are regulatory RNA structures that modulate gene expression in response to a small molecule. Until now, efforts to design ligand analogs were motivated by their potential antibiotic activity. However, riboswitches are ideally suited as tools for gene therapy, enabling precise control of gene ex-pression without the need of potentially immunogenic regulatory proteins. Developing synthetic RNA switches starting from natural riboswitches will require engineering both, the ligand and the RNA sequence in order to achieve full orthogonality i.e., sensitivity to the designed small molecule modulator, but not to the natural ligand. We present the structure-based design of a drug-like small molecule ligand of the thiamine pyrophosphate (TPP) aptamer, BI-5232. BI-5232 is structurally highly diverse from the natural ligand TPP but rivals its binding affinity (KD = 1.0 nM). Importantly, in our design the pyrophosphate of TPP was replaced by an uncharged heterocycle that interacts with the PP helix in an unprecedented way, as revealed by Molecular Dynamics simulations. Subsequently, we altered the aptamer sequence to drastically reduce its affinity to TPP while retaining binding properties for our designed ligand. Based on the developed orthogonal small molecule/RNA aptamer interaction we finally constructed orthogonal ribozyme-based ON- and OFF-switches of gene expression in human cell lines. Such systems are valuable additions to the synthetic toolbox for conditionally controlling gene expression with potential applications in next-generation gene therapies.

synthetic biology↗

bioRxiv2021

Human stem cell-based retina-on-chip as new translational model for validation of AAV retinal gene therapy vectors

Gene therapies using adeno-associated viruses (AAVs) are amongst the most promising strategies to treat or even cure hereditary and acquired retinal diseases. However, the development of new efficient AAV vectors is slow and costly, largely because of the lack of suitable non-clinical models. By faithfully recreating structure and function of human tissues, human induced pluripotent stem cell (iPSC)-derived retinal organoids could become an essential part of the test cascade addressing translational aspects. Organ-on-Chip (OoC) technology further provides the capability to recapitulate microphysiological tissue environments as well as a precise control over structural and temporal parameters. By employing our recently developed Retina-on-chip that merges organoid and OoC-technology, we analyzed the efficacy, kinetics and cell tropism of seven first and second generation AAV vectors. The presented data demonstrate the potential of iPSC-based OoC models as the next generation of screening platforms for future gene therapeutic studies.

bioengineering↗

bioRxiv2020

The biphasic and age-dependent impact of Klotho on hallmarks of aging and skeletal muscle function

Aging is accompanied by a disrupted information flow, which results from accumulation of molecular mistakes. These mistakes ultimately give rise to debilitating disorders such as skeletal muscle wasting, or sarcopenia. To estimate the growing "disorderliness" of the aging muscle system, we employed a statistical physics approach to estimate the state parameter, entropy, as a function of genes associated with hallmarks of aging. Although the most prominent structural and functional alterations were observed in the oldest old mice (27-29 months), we found that the escalating network entropy reached an inflection point at old age (22-24 months). To probe the potential for restoration of molecular "order" and reversal of the sarcopenic phenotype, we overexpressed the longevity protein, -Klotho. Klotho overexpression modulated genes representing all hallmarks of aging in both old and oldest-old mice. However, whereas Klotho improved strength in old mice, intervention failed to induce a benefit beyond the entropic tipping point.

physiology↗