Biology subjects

de Jong, O. G.

Publications and source records attributed to de Jong, O. G..

8 recordsLinked to original sources

bioRxiv2025

THE NSL COMPLEX PROMOTES NEURAL DEVELOPMENT BY PREVENTING R-LOOP INDUCED REPLICATION STRESS

Early neuronal development relies on the proliferation of neural progenitor cells, making this developmental stage particularly vulnerable to DNA replication impediments. Here, we identify a non-canonical role for the non-specific lethal (NSL) complex in safeguarding DNA replication during neurodevelopment. The NSL complex, which acetylates histone 4 at gene promoters and is mutated in Koolen-de Vries syndrome (KdVS), prevents unscheduled R-loop accumulation at weakly transcribed promoters that are typically devoid of R-loops. Single-cell sequencing reveals that loss of NSL causes replisome stalling and delayed S-phase progression. Neural organoids derived from KdVS patients exhibit impaired DNA replication, developmental abnormalities, and reduced synapse formation, driven entirely by unscheduled R-loop accumulation. These findings reveal that faithful DNA replication is critical for early neurodevelopment.

genomics↗

bioRxiv2024

Advanced Peptide Nanoparticles Enable Robust and Efficient delivery of gene editors across cell types

Efficient delivery of the CRISPR/Cas9 system and its larger derivatives, base editors, and prime editors remain a significant challenge, particularly in tissue-specific stem cells and induced pluripotent stem cells (iPSCs). This study optimized a novel family of cell-penetrating peptides, hPep, to deliver gene-editing ribonucleoproteins. The hPep-based nanoparticles enable highly efficient and biocompatible delivery of Cre recombinase, Cas9, base-, and prime editors. Using base editors, robust and nearly complete genome editing was achieved in the human cells: HEK293T (96%), iPSCs (74%), and muscle stem cells (80%). This strategy opens promising avenues for ex vivo and, potentially, in vivo applications. Incorporating silica particles enhanced the systems versatility, facilitating cargo-agnostic delivery. Notably, the nanoparticles can be synthesized quickly on a benchtop and stored as lyophilized powder without compromising functionality. This represents a significant advancement in the feasibility and scalability of gene-editing delivery technologies.

bioengineering↗

bioRxiv2024

Tissue-specific modulation of CRISPR activity by miRNA-sensing guide RNAs

Nucleic acid nanostructures offer unique opportunities for biomedical applications due to their sequence-programmable structures and functions, which enable the design of complex responses to molecular cues. Control of the biological activity of therapeutic cargoes based on endogenous molecular signatures holds the potential to overcome major hurdles in translational research: cell specificity and off-target effects. Endogenous microRNAs can be used to profile cell type and cell state and are ideal inputs for RNA nanodevices. Here we present CRISPR MiRAGE (miRNA-activated genome editing), a tool comprising a dynamic single-guide RNA that senses miRNA complexed with Argonaute proteins and controls downstream CRISPR activity based on the detected miRNA signature. We study the operation of the miRNA-sensing single-guide RNA and attain muscle-specific activation of gene editing through CRISPR MiRAGE in models of Duchenne muscular dystrophy. By enabling RNA-controlled gene editing activity, this technology creates opportunities to advance tissue-specific CRISPR treatments for human diseases.

bioengineering↗

bioRxiv2024

A modular strategy for extracellular vesicle-mediated CRISPR-Cas9 delivery through aptamer-based loading and UV-activated cargo release

CRISPR-Cas9 gene editing technology offers the potential to permanently repair genes containing pathological mutations. However, efficient intracellular delivery of the Cas9 ribonucleoprotein complex remains one of the major hurdles in its therapeutic application. Extracellular vesicles (EVs) are biological nanosized membrane vesicles released by cells, that play an important role in intercellular communication. Due to their innate capability of intercellular transfer of proteins, RNA, and various other biological cargos, EVs have emerged as a novel promising strategy for the delivery of macromolecular biotherapeutics, including CRISPR-Cas9 ribonucleoproteins. Here, we present a versatile, modular strategy for the loading and delivery of Cas9. We leverage the high affinity binding of MS2 coat proteins (MCPs) fused to EV-enriched proteins to MS2 aptamers incorporated into single guide RNAs (sgRNAs), in combination with a UV-activated photocleavable linker domain, PhoCl. Combined with the Vesicular stomatitis virus G (VSV-G) protein this modular platform enables efficient loading and subsequent delivery of the Cas9 ribonucleoprotein complex, which shows critical dependence on the incorporation and activation of the photocleavable linker domain. As this approach does not require any direct fusion of Cas9 to EV-enriched proteins, we demonstrate that Cas9 can readily be exchanged for other variants, including transcriptional activator dCas9-VPR and adenine base editor ABE8e, as confirmed by various sensitive fluorescent reporter assays. Taken together, we describe a robust and modular strategy for successful Cas9 delivery, which can be applied for CRISPR-Cas9-based genetic engineering as well as transcriptional regulation, underlining the potential of EV-mediated strategies for the treatment of genetic diseases.

molecular biology↗

bioRxiv2023

Anti-cancer compound screening identifies Aurora Kinase A inhibition as a means to favor CRISPR/Cas9 gene correction over knock-out

CRISPR gene therapy holds the potential to cure a variety of genetic diseases by targeting causative mutations and introducing double stranded DNA breaks, subsequently allowing the host DNA repair mechanisms to introduce mutations. One option to introduce precise gene corrections is via the homology-directed repair (HDR) pathway. HDR can introduce a range of desired mutations dictated by a DNA template which holds a corrected DNA sequence which is written into the targeted gene. The problem in utilizing this pathway is that CRISPR-induced double stranded DNA breaks are repaired more often through the non-homologous end joining (NHEJ) pathway, which does not use a designed template and introduces random DNA damage in the form of insertions and deletions at the cut site. Since HDR activation depends on many interconnected processes in the cell, we aimed to screen a small library of drug compounds in clinical use or clinical development for cancer, to steer the DNA repair process towards preferential HDR activation. We included compounds in our screen based on three relevant mechanisms in CRISPR gene editing: the cell cycle, DNA repair processing and chromosomal packing. We included forty compounds, based on these criteria, screened their toxicity and dosed them in sub-toxic concentrations in cells during genome editing. Of these forty compounds we identified nine potential hits to have an effect on preferential activation of the HDR pathway over NHEJ. Alisertib, rucaparib and belinostat revealed a significant and major effect on gene editing pathway selection in further validation. Alisertib, an Aurora kinase A inhibitor, showed a particularly strong effect towards improving HDR over NHEJ. We subsequently investigated this effect at the genetic level and in a murine hepatoma cell line, which corroborated the initial findings. Alisertib led to an over 4-fold increase in preferential gene correction over gene knock-out, at a dose of 0.3 micromolar. However, the observations that Aurora kinase A inhibitors show considerable cytotoxicity (<50% cell viability) and can induce morphological changes at this concentration pose a limitation for the direct use of these inhibitors as HDR enhancers. However these findings do implicate that the pathways mediated by Aurora kinase A strongly influence HDR outcomes, which warrants further investigation into the downstream pathways driving this effect.

pharmacology and toxicology↗

bioRxiv2023

EV-Elute: a universal platform for enrichment of functional surface marker-defined extracellular vesicle subpopulations

Intercellular communication via extracellular vesicles (EVs) has been identified as a vital component of a steadily expanding number of physiological and pathological processes. To accommodate these roles, EVs are equipped with specific proteins, lipids, and RNA molecules by EV-secreting cells. Consequently, EVs have highly heterogeneous molecular compositions. Given that surface molecules on EVs determine their interactions with their environment, it is conceivable that EV functionality differs between subpopulations with varying surface compositions. However, it has been technically challenging to examine such functional heterogeneity due to a lack of non-destructive methods to separate EV subpopulations based on their surface markers. Here, we used Design-of-Experiments methodology to rapidly optimize a protocol, which we name EV-Elute, to elute intact EVs from commercially available Protein G-coated magnetic beads. We captured EVs from various cell types on these beads using antibodies against CD9, CD63, CD81 and a custom-made protein binding phosphatidylserine (PS). When applying EV-Elute, over 70% of bound EVs could be recovered from the beads in a pH- and incubation time-dependent fashion. EV subpopulations were found to be devoid of co-isolated protein contaminants observed in whole EV isolates and showed intact morphology by electron microscopy. Proteinase K protection assays showed a mild and reversible decrease of EV membrane integrity during elution. Depending on the type of capturing antibody used, some antibodies remained EV-associated after elution. EV subpopulations showed uptake patterns similar to whole EV isolates in co-cultures of peripheral blood mononuclear cells and endothelial cells. However, in Cas9/sgRNA delivery assays, CD63+ EVs showed a lower capacity to functionally deliver cargo as compared to CD9+, CD81+ and PS+ EVs. Taken together, we developed a novel, easy-to-use platform to isolate and functionally compare surface marker-defined EV subpopulations. Importantly, this platform does not require specialized equipment or reagents and is universally applicable to any capturing antibody and EV source. Hence, EV-Elute can open new opportunities to study EV functionality at the subpopulation level.

bioengineering↗

bioRxiv2023

Multimodal engineering of extracellular vesicles for efficient intracellular protein delivery

Extracellular vesicles (EVs) are promising tools to transfer macromolecular therapeutic molecules to recipient cells, however, efficient functional intracellular protein delivery by EVs remains challenging. Here, we have developed novel and versatile systems that leverage selected molecular tools to engineer EVs for robust cytosolic protein delivery both in vitro and in vivo. These systems, termed VSV-G plus EV-sorting Domain-Intein-Cargo (VEDIC) and VSV-G-Foldon-Intein-Cargo (VFIC), exploit an engineered mini-intein (intein) protein with self-cleavage activity to link cargo to an EV-sorting domain and release it from the EV membrane inside the EV lumen. In addition, we utilize the fusogenic protein VSV-G to facilitate endosomal escape and cargo release from the endosomal system to the cytosol of recipient cells. Importantly, we demonstrate that the combination of the self-cleavage intein, fusogenic protein and EV-sorting domain are indispensable for efficient functional intracellular delivery of cargo proteins by engineered EVs. As such, nearly 100% recombination and close to 80% genome editing efficiency in reporter cells were observed by EV-transferred Cre recombinase and Cas9/sgRNA RNPs, respectively. Moreover, EV-mediated Cre delivery by VEDIC or VFIC engineered EVs resulted in significant in vivo recombination in Cre-LoxP R26-LSL-tdTomato reporter mice following both local and systemic injections. Finally, we applied these systems for improved treatment of LPS-induced systemic inflammation by delivering a super-repressor of NF-B activity. Altogether, this study describes a platform by which EVs can be utilized as a vehicle for the efficient intracellular delivery of macromolecular therapeutics for treatments of disease. Graphic summary: Development of VEDIC and VFIC systems for high-efficiency intracellular protein delivery in vitro and in vivo.Intein in tripartite fusion protein (EV-sorting Domain-Intein-Cargo) performs C-terminal cleavage during the process of EV-biogenesis, resulting in enriched free cargo proteins inside of vesicles. Together with fusogenic protein, VSV-G, these engineered EVs achieve high-efficiency intracellular delivery of cargo protein (Cre and super repressor of NF-B) or protein complex (Cas9/sgRNA RNPs) both in reporter cells and in mice models. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=142 SRC="FIGDIR/small/535834v4_ufig1.gif" ALT="Figure 1"> View larger version (41K): org.highwire.dtl.DTLVardef@d0da37org.highwire.dtl.DTLVardef@1a6514corg.highwire.dtl.DTLVardef@235c9aorg.highwire.dtl.DTLVardef@194bbfa_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

bioRxiv2019

Combining multi-omics and drug perturbation profiles to identify novel treatments that improve disease phenotypes in spinal muscular atrophy

Spinal muscular atrophy (SMA) is a neuromuscular disorder caused by loss of survival motor neuron (SMN) protein. While SMN restoration therapies are beneficial, they are not a cure. We aimed to identify novel treatments to alleviate muscle pathology combining transcriptomics, proteomics and perturbational datasets. This revealed potential drug candidates for repurposing in SMA. One of the lead candidates, harmine, was further investigated in cell and animal models, improving multiple disease phenotypes, including SMN expression and lifespan. Our work highlights the potential of multiple, parallel data driven approaches for development of novel treatments for use in combination with SMN restoration therapies.

neuroscience↗