Biology subjects

Schiffelers, R. M.

Publications and source records attributed to Schiffelers, R. M..

4 recordsLinked to original sources

bioRxiv2026

NEO-AAV: an engineered extracellular vesicle-enveloped AAV platform for activation-coupled T cell transduction and CAR-T cell generation

Efficient delivery of genetic cargo to primary human T cells remains a critical barrier for cell-based immunotherapies. AAVs are widely used but limited by immune neutralization and poor T cell transduction efficiency. We present NEO-AAV, an engineered fusion protein (PH-ALG2-PKD12) that recruits AAV capsids into endogenous extracellular vesicles (EVs) via PI(4,5)P2-directed membrane targeting, multivalent PKD12-capsid clustering, and ALG2-mediated ESCRT machinery recruitment, yielding EV-enveloped particles with improved AAV loading compared with passive EV-AAV controls. NEO-AAV displayed enhanced resistance to an anti-AAV6 neutralizing antibody (ADK6), maintaining transduction at concentrations that neutralized naked AAV6 and outperformed passively formed EV-AAV6. Surface display of a CD7/CD3/CD28 tri-chimera enabled single-step activation and transduction of CD7 T cells within PBMCs, reaching [~]38% eGFP cells without exogenous pre-activation. As a proof-of-concept, NEO-AAV generated functional CAR-T cells from primary human PBMCs, exhibiting antigen-specific IFN-{gamma} release and cytotoxicity against CD19+ target cells. CAR expression peaked at day 5 and declined by day 15, consistent with episomal AAV kinetics, framing NEO-AAV as an activation-coupled delivery module rather than a durable solution. Together, NEO-AAV provides a programmable EV-enveloped AAV platform with improved immune shielding and single-step T cell transduction, offering a building block for immune cell engineering with in vivo potential.

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↗

bioRxiv2020

EVQuant; high-throughput quantification and characterization of extracellular vesicle (sub)populations

Extracellular vesicles (EVs) reflect the cell of origin in terms of nucleic acids and protein content. They are found in biofluids and represent an ideal liquid biopsy biomarker source for many diseases. Unfortunately, clinical implementation is limited by available technologies for EV analysis. We have developed a simple, robust and sensitive microscopy-based high-throughput assay (EVQuant) to overcome these limitations and allow widespread use in the EV community. The EVQuant assay can detect individual immobilized EVs as small as 35 nm and determine their concentration in biofluids without extensive EV isolation or purification procedures. It can also identify specific EV subpopulations based on combinations of biomarkers and is used here to identify prostate-derived urinary EVs as CD9-/CD63+. Moreover, characterization of individual EVs allows analysis of their size distribution. The ability to identify, quantify and characterize EV (sub-)populations in high-throughput substantially extents the applicability of the EVQuant assay over most current EV quantification assays.

bioengineering↗

bioRxiv2020

Modular lipid nanoparticle platform technology for siRNA and lipophilic prodrug delivery

Successfully employing therapeutic nucleic acids, such as small interfering RNA (siRNA), requires chemical modifications or the use of nanocarrier technology to prevent their degradation in the circulation and to facilitate intracellular delivery. Lipid nanoparticles (LNP) are among the most advanced nanocarriers culminating in the first siRNA therapeutics clinical translation and approval. At the same time, their applicability as modular platform technology due to the interchangeable building blocks and siRNA payload hallmarks one of LNPs major advantages. In addition, drug derivatization approaches to synthesize lipophilic small molecule prodrugs enable stable incorporation in LNPs. This provides ample opportunities to develop combination therapies by co-encapsulating multiple therapeutic agents in a single formulation. Here, we describe how the modular LNP platform can be applied for combined gene silencing and chemotherapy to induce additive anti-cancer effects. We show that various lipophilic taxane prodrug derivatives and siRNA against the androgen receptor, a prostate cancer driver, can be efficiently and stably co-encapsulated in LNPs. In addition, we demonstrate that prodrug incorporation does not affect LNPs gene silencing ability and that the combination therapy induces additive therapeutic effects in vitro. Using a double-radiolabeling approach, we quantitively determined the LNPs and prodrugs pharmacokinetic properties and biodistribution following systemic administration in tumor-bearing mice. Our results indicate that co-encapsulation of siRNA and lipophilic prodrugs into LNPs is an attractive and straightforward approach for combination therapy development. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=159 SRC="FIGDIR/small/907394v1_ufig1.gif" ALT="Figure 1"> View larger version (41K): org.highwire.dtl.DTLVardef@b61be4org.highwire.dtl.DTLVardef@1e130c5org.highwire.dtl.DTLVardef@148e5dcorg.highwire.dtl.DTLVardef@eb0cb9_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗