Multidisciplinary Vesicle Program (MVP) Courses

Planned for Summer 2026 – Exact Date TBA

Syllabus

Advancing molecular biology research and gene therapy with nucleic acid delivery by Lipid Nanoparticles (LNPs) and Extracellular Vesicles (EVs)

Course organizers

• Avraham Dayan, PhD - Senior Staff Scientist, Multidisciplinary Vesicle Program, Life Sciences Core Facilities, Weizmann Institute of Science
• Dmitri Tentler Ph.D. - The Nancy and Stephen Grand Israel National Center for Personalized Medicine (G-INCPM), HTS Unit, Weizmann Institute of Science

Instructors

• Dr. Avraham Dayan – Extracellular Vesicles & Nano-Analytical Platforms
• Dr. Dmitri Tentler – Lipid Nanoparticles & IVT-mRNA Synthesis
• Dr. Karin Broennimann – Lipid Nanoparticles & IVT-mRNA Synthesis

Course Overview

Lipid Nanoparticles (LNPs) and Extracellular Vesicles (EVs) are crucial tools in delivery of nucleic acids into cells and tissues and also have been used in pharmacologically centered applications. Thus, LNPs efficiently encapsulate and protect sensitive nucleic acids (mRNA, siRNA, plasmid DNA) and facilitate their delivery into cells, making them essential for molecular biology experiments and also for therapy. The mRNA COVID-19 vaccines are a prime clinical example of this technology.

In addition to the increased use of LNPs to deliver RNA for modulating gene expression, they have also been used to deliver gene-editing components, such as CRISPR-Cas modules, enabling molecular biologists to study gene functions and even develop the next wave of treatments for genetic diseases. LNPs are also versatile carriers for various hydrophobic therapeutic agents, improving drug stability, circulation time, and targeted delivery to specific tissues.

Extracellular Vesicles (EVs) are naturally secreted nanoscale particles that mediate intercellular communication by transferring proteins, lipids, and nucleic acids between cells. Derived from endosomal or plasma membrane pathways, EVs, including exosomes and microvesicles, reflect the physiological state of their parent cells and can be engineered or selectively enriched for therapeutic delivery. Their endogenous origin confers exceptional biocompatibility and targeting potential, positioning EVs as a biologically inspired alternative to synthetic delivery systems in molecular biology, diagnostic and gene therapy.

Overall, EVs and LNPs are superior to conventional transfection methods as they combine the high stability and large cargo capacity with the natural targeting capabilities and biocompatibility of cell-derived EVs, aiming for superior delivery efficiency and reduced toxicity in classical molecular biology experiments as well as for therapeutic applications.

Our laboratory course will provide a comprehensive theoretical and practical framework for understanding and implementing these two major delivery systems.  The course combines lectures, protocol-focused discussions, and hands-on laboratory sessions, enabling participants to design and execute complete workflow, from mRNA synthesis via in vitro transcription (IVT) to nanoparticle production, biological enrichment, purification, single-particle characterization, and functional gene expression/silencing assays.

Finally, we will be comparing EVs to LNPs-based translational approaches, highlighting aspects such as biogenesis, scalability, and functional efficiency in gene delivery applications.

Learning Objectives

Upon completion, participants will be able to:

1. Explain the molecular basis of EV biogenesis and LNP assembly as delivery systems for molecular biology experiments and therapeutic RNA.
2. Perform in vitro transcription (IVT) for generating transfection-grade mRNA.
   Students who would like to produce their own IVT RNA of interest are encouraged to contact us in advance.
3. Produce EVs and LNPs using ultracentrifugation, microfluidic assembly, and controlled formulation techniques.
4. Conduct functional gene delivery experiments using mRNA (expression) and siRNA (silencing) assays.
5. Apply EV/LNP purification and enrichment strategies.
6. Perform nanoparticle characterization using various platforms.
7. Critically evaluate the advantages and limitations of EVs versus LNPs in clinical translation and regulatory contexts.

Prerequisites

Participants are expected to have:

• Basic background in molecular biology, genetics, or biotechnology.
• Previous hands-on experience with mammalian cell culture.
• Familiarity with qPCR and fluorescence microscopy (optional).

Daily Schedule Format

Weekly Structure Overview

Detailed Course Description by Day

Day 1 – Introduction, Scale-Up and Scale-Down Strategies

Lead: Dr. Avraham Dayan

Lecture Topics:

• Biological EV biogenesis pathways (exosomes, microvesicles)
• Synthetic nanoparticle assembly (microfluidic LNP formation mechanics)
• Scale-up vs. scale-down strategies in translational manufacturing
• Comparative regulatory considerations: natural vesicles vs. synthetic nanocarriers

Laboratory Session:

• EV-compatible cell culture handling
• Pre-clearing and differential centrifugation
• Ultracentrifugation-based EV isolation
• Sterile workflow discipline and buffer preparation

Key Instruments: Biosafety Cabinet, CO₂ Incubator, Refrigerated Centrifuge, Ultracentrifuge

Day 2 – IVT-mRNA Production for Encapsulation

Lead: Dr. Karin Broennimann

Lecture Topics:

• IVT enzymatic workflow: template design, cap analogues, poly-A tail considerations
• RNase-free handling, quality parameters for transfection-grade RNA
• Purity and integrity criteria prior to nanoparticle assembly

Laboratory Session:

• Setup of IVT reaction for fluorescent reporter mRNA (e.g., mCherry or EGFP)
• RNA purification steps and concentration adjustment
• RNA quality control using spectrophotometry and basic gel or integrity check if applicable

Key Instruments: Heat Block/Thermocycler, RNA Purification Columns, Nanodrop

Day 3 – LNP Assembly and EV Enrichment via SEC/TFF

Lead: Dr. Dmitri Tentler & Dr. Avraham Dayan

Lecture Topics:

• Lipid component selection: ionizable lipids, cholesterol, helper phospholipids, PEGylation
• Microfluidic control of particle size and encapsulation parameters
• EV enrichment using SEC vs. TFF: trade-offs between purity and yield

Laboratory Session:

• LNP assembly using NanoAssemblr microfluidic system - encapsulate mRNA into LNPs
• SEC-based EV fractionation and/or TFF concentration
• Buffer exchange and sample preparation for analytical characterization

Key Instruments: NanoAssemblr, SEC Columns (qEV), TFF System, Amicon Filters

Day 4 – Nanoparticle Characterization and Single-Particle Analytics

Lead: Dr. Avraham Dayan

Lecture Topics:

• Analytical methods: DLS (hydrodynamic diameter), NTA (particle concentration), zeta potential (colloidal stability)
• Single-particle fluorescence detection via flow cytometry (ImageStream)
• Discussion on fluorescence thresholds, swarm detection, bead calibration, and publication standards (MISEV, clinical LNP metrics)

Laboratory Session:

• Measurement of EV and LNP samples using Zetasizer (DLS + Zeta) and NanoSight NTA
• Fluorescent flow cytometry of EV/LNP preparations using ImageStream
• Threshold calibration using reference particle standards
• Loading siRNA or mRNA into EVs (if electroporation or passive loading)
• Transfection of reporter cells with EV and LNP formulations

Key Instruments: Malvern Zetasizer, NanoSight NS300 or ZetaView, ImageStream, Calibration Beads

Day 5 – Functional Gene Delivery: Expression and Silencing

Lead: Co-Led by Dr. Dmitri Tentler & Dr. Avraham Dayan

Lecture Topics:

• Biological vs. synthetic delivery mechanisms: endocytosis, fusion, endosomal escape
• Experimental design for dose-response analysis and silencing kinetics
• Troubleshooting inefficiencies: degradation, heterogeneity, suboptimal internalization

Laboratory Session:

• Optional qRT-PCR or plate reader quantification of gene expression/silencing outcomes
• Comparative analysis and interpretation
• Fluorescence microscopy imaging, ImageStream

Key Instruments: Electroporator (optional), qRT-PCR, Fluorescence Microscope, Plate Reader

Assessment and Final Project

Each participant will design a complete translational delivery workflow including:

• Selection and justification of EV or LNP-based system
• Defined production and purification plan
• Analytical QC checklist
• Functional assay design with expected data interpretation parameters

Final project may be submitted as a workflow poster or concise protocol manuscript format, suitable for core facility or academic documentation.

Closing Statement

This course delivers a unique comparative framework between biological and synthetic nanocarriers for gene therapy applications. By integrating EV and LNP systems within a single structured curriculum, participants gain a dual technical and analytical skill set aligned with current academic and translational standards. The methodological alignment with MISEV guidelines and clinical nanoparticle release criteria ensures that participants leave with a perspective applicable to both research and regulated development environments.