Biology subjects

Philipp, J.

Publications and source records attributed to Philipp, J..

8 recordsLinked to original sources

bioRxiv2025

Buffer specificity of ionizable lipid nanoparticle transfection efficiency and bulk phase transition

Lipid nanoparticles (LNPs) are efficient and safe carriers for mRNA vaccines based on advanced ionizable lipids. It is understood that the pH dependent structural transition of the mesoscopic LNP core phase plays a key role in mRNA transfer. However, buffer specific variations in transfection efficiency remain obscure. Here we analyze the effect of buffer type on the structure and transfection efficiency of LNPs. We find that LNPs formulated with the cationic ionizable lipid DLin-MC3-DMA (MC3) in citrate compared to phosphate and acetate buffers exhibit earlier onset and stronger mRNA-GFP expression in-vitro. Using synchrotron small angle X-ray scattering (SAXS) we determine the pH dependent mesophases of ionizable lipid MC3/cholesterol/water dialyzed against the various buffers. The results show that the phase transition with decreasing pH from inverse micellar cubic to inverse hexagonal (Fd3m-HII) is shifted by one unit to a lower transition pH for acetate and phosphate compared to citrate buffer. Based on continuum theory and ion specific adsorption obtained from all-atom MD simulations, we propose a mechanism for buffer specificity. Citrate stabilizes the inverse hexagonal phase thus shifting the formation of HII to a higher pH. By contrast, phosphate and acetate stabilize LII. We propose that the Fd3m-to-HII transition, which is facilitated in citrate buffer, is responsible for a sensitized pH-response of the LNP core phase. This, in turn, enhances endosomal release efficiency and accounts for the earlier onset of gene expression observed in LNPs prepared with citrate buffer.

biophysics↗

bioRxiv2025

Lymphoid-tissue-on-chip recapitulates human antibody responses in vitro

In the past decades, vaccine development has made great strides. Nevertheless, more often than not, vaccine candidates fail in advanced stages of development and clinical trials. A key reason is the poor predictive value of non-clinical in vivo and in vitro models, due to either species-specific differences in the immune response or insufficient reflection of physiological vaccine mechanisms. Reliable modeling of human adaptive immune responses is a prerequisite to understand processes leading to vaccine-induced protective immunization and to drive informed decisions in vaccine development pipelines. Here, we present a centrifugal microfluidics based organ-on-chip approach to generate an organotypic high density lymphoid tissue on-chip. The model enables long-term culture of lymphoid tissue and raised antigen-specific antibody responses against influenza vaccines even after four weeks on-chip. Antibody response of different magnitude and quality could be induced both by direct antigen exposure as well as by recruitment of antigen-presenting cells from the periphery. The model represents an attractive approach to evaluate the impact of the mode of antigen delivery on adaptive immune responses. Beyond applications in vaccine development, the lymphoid-tissue-on-chip provides a platform to study cellular interactions during homeostasis, immune responses and long-term impact of immunomodulators over several weeks.

bioengineering↗

bioRxiv2023

Combining molecular dynamics simulations and X-ray scattering techniques for the accurate treatment of protonation degree and packing of ionizable lipids in monolayers

The pH-dependent change in protonation of ionizable lipids is crucial for the success of lipid-based nanoparticles as mRNA delivery systems. Despite their widespread application in vaccines, the structural changes upon acidification are not well understood. Molecular dynamics simulations support structure prediction but require an a-priori knowledge of the lipid packing and protonation degree. The presetting of the protonation degree is a challenging task in the case of ionizable lipids since it depends on pH and on the local lipid environment and often lacks experimental validation. Here, we introduce a methodology of combining all-atom molecular dynamics simulations with experimental total-reflection X-ray fluorescence and scattering measurements for the ionizable lipid Dlin-MC3-DMA (MC3) in POPC monolayers. This joint approach allows us to simultaneously determine the lipid packing and the protonation degree of MC3. The consistent parameterization is expected to be useful for further predictive modeling of the action of MC3-based lipid nanoparticles.

biophysics↗

bioRxiv2023

Isoform-specific translational control is evolutionarily conserved in primates

Alternative splicing (AS) alters messenger RNA (mRNA) coding capacity, localization, stability, and translation. Here we use comparative transcriptomics to identify cis-acting elements coupling AS to translational control (AS-TC). We sequenced total cytosolic and polyribosome-associated mRNA from human, chimpanzee, and orangutan induced pluripotent stem cells (iPSCs), revealing thousands of transcripts with splicing differences between subcellular fractions. We found both conserved and species-specific polyribosome association patterns for orthologous splicing events. Intriguingly, alternative exons with similar polyribosome profiles between species have stronger sequence conservation than exons with lineage-specific ribosome association. These data suggest that sequence variation underlies differences in the polyribosome association. Accordingly, single nucleotide substitutions in luciferase reporters designed to model exons with divergent polyribosome profiles are sufficient to regulate translational efficiency. We used position specific weight matrixes to interpret exons with species-specific polyribosome association profiles, finding that polymorphic sites frequently alter recognition motifs for trans-acting RNA binding proteins. Together, our results show that AS can regulate translation by remodeling the cis-regulatory landscape of mRNA isoforms.

molecular biology↗

bioRxiv2022

3D printed SAXS chamber for controlled in-situ dialysis and optical characterization

3D printing changes the scope of how samples can be mounted for small angle X-ray scattering (SAXS). In this paper we present a 3D printed X-ray chamber, which allows for in-situ exchange of buffer and in-situ optical transmission spectroscopy. The chamber is made of cyclic olefin copolymers (COC), including COC X-ray windows providing ultra low SAXS background. The design integrates a membrane insert for in-situ dialysis of the 100 {micro}l sample volume against a reservoir, which enables measurements of the same sample under multiple conditions using an in-house X-ray setup equipped with a 17.4 keV molybdenum source. We demonstrate the designs capabilities by measuring reversible structural changes in lipid and polymer systems as a function of salt concentration and pH. In the same chambers optical light transmission spectroscopy was carried out measuring optical turbidity of the mesophases and local pH values using pH-responsive dyes. Microfluidic exchange and optical spectroscopy combined with in-situ X-ray scattering enables vast applications for the study of responsive materials.

biophysics↗

bioRxiv2020

Post-transcriptional gene regulation by the RNA binding protein IGF2BP3 is critical for MLL-AF4 mediated leukemogenesis

Despite recent advances in therapeutic approaches, patients with MLL-rearranged leukemia still have poor outcomes and a high risk of relapse. Here, we found that MLL-AF4, the most common MLL fusion protein in patients, transcriptionally induces IGF2BP3 and that IGF2BP3 strongly amplifies MLL-Af4 mediated leukemogenesis. Deletion of Igf2bp3 significantly increases the survival of mice with MLL-Af4 driven leukemia and greatly attenuates disease, with a minimal impact on baseline hematopoiesis. At the cellular level, MLL-Af4 leukemia-initiating cells require Igf2bp3 for their function in leukemogenesis. eCLIP and transcriptome analysis of MLL-Af4 transformed stem and progenitor cells and MLL-Af4 bulk leukemia cells reveals a complex IGF2BP3-regulated post-transcriptional operon governing leukemia cell survival and proliferation. Regulated mRNA targets include important leukemogenic genes such as those in the Hoxa locus and numerous genes within the Ras signaling pathway. Together, our findings show that IGF2BP3 is an essential positive regulator of MLL-AF4 mediated leukemogenesis and represents an attractive therapeutic target in this disease.

cancer biology↗

bioRxiv2020

Splicing factor SRSF1 expands the regulatory logic of microRNA expression

The serine and arginine-rich splicing factor SRSF1 is an evolutionarily conserved, essential pre-mRNA splicing factor. Through a global protein-RNA interaction survey we discovered SRSF1 binding sites 25-50nt upstream from hundreds of pre-miRNAs. Using primary miRNA-10b as a model we demonstrate that SRSF1 directly regulates microRNA biogenesis both in vitro and in vivo. Selective 2 hydroxyl acylation analyzed by primer extension (SHAPE) defined a structured RNA element located upstream of the precursor miRNA-10b stem loop. Our data support a model where SRSF1 promotes initial steps of microRNA biogenesis by relieving the repressive effects of cis-regulatory elements within the leader sequence.

molecular biology↗

bioRxiv2018

Body size-dependent energy storage causes Kleibers law scaling of the metabolic rate in planarians

Kleibers law, or the [3/4]-power law scaling of the metabolic rate with body mass, is considered one of the few quantitative laws in biology, yet its physiological basis remains unknown. Here, we report Kleibers law scaling in the planarian Schmidtea mediterranea. Its reversible and life history-independent changes in adult body size over 2 orders of magnitude reveal that Kleibers law does not emerge from the size-dependent decrease in cellular metabolic rate, but from a size-dependent increase in mass per cell. Through a combination of experiment and theoretical analysis of the organismal energy balance, we further show that the mass allometry is caused by body size dependent energy storage. Our results reveal the physiological origins of Kleibers law in planarians and thus have general implications for understanding a fundamental scaling law in biology.

biophysics↗