Questions for Our Scientific Partners?
mRNA delivery to neurons is difficult because neurons and NPCs are sensitive to membrane perturbation, often post-mitotic, and prone to rapid loss of viability under electrical or chemical stress. The most common delivery methods (AAV, lentivirus, lipid nanoparticles, and electroporation) each carry tradeoffs in safety, payload, viability, or transient control.
The result is that most neuronal mRNA delivery still depends on viral vectors. AAV and lentivirus achieve high expression, but they bring packaging size limits, manufacturing complexity, immunogenicity considerations, and, in the case of integrating vectors, insertional risk. Lipid nanoparticles avoid integration but typically require modified nucleotides to manage innate immune sensing, and electroporation often costs a substantial portion of the starting population in viability.
For groups studying neurodevelopment, neurodegeneration, and CNS-directed therapeutics, this leaves a gap: a need for transient, controllable, virus-free protein expression in neuronal cells, delivered in a single workflow step that preserves viability.
Portal mechanoporation delivers mRNA into NPCs through controlled mechanical membrane disruption. There is no viral vector, no lipid nanoparticle, no electrical current, and no modified nucleotides.
Cells pass through precisely engineered pores in a MicroBooster™ cartridge. The controlled mechanical stress creates transient openings in the cell membrane, mRNA in the surrounding solution diffuses into the cytosol through those openings, and the membrane self-heals within seconds. Because the cargo enters directly into the cytosol rather than through endosomal uptake, no chemical base modifications are required to evade endosomal RNA sensors.
The platform is cargo-agnostic. The same instrument and workflow used here for mRNA also delivers circRNA, proteins, CRISPR ribonucleoproteins, and small molecules across T cells, NK cells, B cells, PBMCs, monocytes, neutrophils, and now neuronal progenitor cells.
Mechanoporation is non-viral, integration-free, and does not require modified nucleotides, while preserving high viability in NPCs (up to 80%+ post-boost). AAV and lentivirus achieve high neural expression but carry packaging size limits and, for lentivirus, integration risk. Lipid nanoparticles avoid integration but typically rely on modified nucleotides to manage innate immune sensing. Electroporation often costs a substantial fraction of the starting population in viability and induces broad transcriptomic perturbation.
Method | Viral / Integrating | Modified Bases Needed | Cargo Flexibility | Viability in NPCs |
|---|---|---|---|---|
AAV | Viral, non-integrating | No | Limited (~4.7 kb packaging) | Generally high |
Lentivirus | Viral, integrating | No | Moderate | Generally high |
Lipid nanoparticles (LNP) | Non-viral | Often required | RNA only | Variable |
Electroporation | Non-viral | Often required for RNA | Broad | Often reduced |
Portal | Non-viral, non-integrating | No | mRNA, circRNA, RNPs, proteins, small molecules | High |
Portal tested GFP mRNA delivery to neuronal progenitor cells across a matrix of chip sizes and pressures, with three controls (untreated, no-boost, and no-cargo) and dual readouts of same-day flow cytometry and next-day fluorescence microscopy.
A single mechanoporation step delivered GFP mRNA to 75%+ of NPCs at Day 1, with viability up to 80%+ post-boost, while untreated controls remained at 0% GFP-positive and near-zero dextran-positive.
Metric | Untreated Control | Boosted (Best Condition) |
|---|---|---|
GFP-positive (Day 1, attached cells) | 0% | 75%+ |
Dextran-positive (Day 0, live cells) | Near zero | 70%+ |
Viability post-boost | 85%+ | Up to 80%+ |
The untreated and no-boost controls were essentially zero for both GFP and dextran, confirming that delivery was driven by mechanoporation rather than passive uptake. No-cargo controls showed reduced viability, while boosted conditions retained viability up to 80%+.
Portal mRNA delivery to NPCs is a single-step workflow: suspend cells with mRNA, load the cartridge, run at the chosen chip size and pressure, allow membrane recovery, plate, and read out by flow cytometry and microscopy. Total active processing takes seconds to minutes.
Single-step delivery, no viral vector: A single pass through the cartridge produced 75%+ GFP-positive NPCs by Day 1, compared to 0% in untreated control. No virus, no LNP, no electroporation buffer, and no modified nucleotides were used.
High-efficiency, virus-free mRNA delivery to neuronal progenitor cells opens a set of workflows that were previously bottlenecked on viral preparation or constrained by the side effects of chemical and electrical delivery.
Can you deliver mRNA to neurons without using a virus?
Yes. Portal mechanoporation delivers mRNA into neuronal progenitor cells through controlled mechanical membrane disruption, with no viral vector, no lipid nanoparticle, no electrical current, and no modified nucleotides. A single processing step produced 75%+ GFP-positive NPCs at Day 1 versus 0% in untreated controls.
Do you need modified nucleotides like pseudouridine?
No. Mechanoporation delivers mRNA directly into the cytosol and bypasses endosomal RNA sensors, so unmodified mRNA can be used. Chemical base modifications are not required.
What efficiency does Portal achieve for mRNA delivery to NPCs?
75%+ of attached NPCs were GFP-positive on Day 1 after a single mechanoporation step. The co-delivered fluorescent dextran tracer reached 70%+ of live cells at Day 0 at the strongest condition. Viability was preserved up to 80%+ post-boost.
What chip size and pressure should you use?
The validated matrix is 7.5, 8, and 9 µm chip sizes crossed with 5, 7, and 10 psi pressures. Stronger conditions maximize the cargo-positive fraction. Milder conditions maximize per-cell cargo dose and viability. Match the operating point to the experimental priority.
How does mechanoporation compare to AAV, LNP, and electroporation?
Mechanoporation is non-viral, integration-free, and modification-free. AAV and lentivirus achieve high neural expression but carry packaging limits and, for lentivirus, integration risk. LNPs typically require modified nucleotides. Electroporation often costs viability and induces transcriptomic perturbation.
Can the same workflow deliver other cargos to neuronal cells?
Yes. The platform is cargo-agnostic. The same instrument and workflow delivers mRNA, circRNA, proteins, CRISPR ribonucleoproteins, and small molecules.
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