Skip to content
Brilliant Blue Biosciences logoBrilliant BlueBiosciences
Gene therapy

Building the delivery backbone for cures

Non-viral delivery is usually discussed as the destination. In practice it is also the foundation: AAV and lentiviral vectors are manufactured by transfecting producer cells, transgenes are validated by transient transfection long before clinical use, and the same chemistry carries plasmid DNA and minicircles into the clinic.

3 plasmids
Typical AAV production system: capsid, rep/helper, transgene
10¹³–10¹⁴
Vector genomes per litre targeted in scalable AAV production
4 plasmid
Third-generation lentiviral systems split packaging functions for safety
01

Transfection as the first manufacturing step

AAV production in HEK293T cells requires co-delivery of a capsid plasmid, a rep/helper plasmid and the transgene flanked by inverted terminal repeats. The transfection reagent determines the fraction of producer cells that receive all three plasmids — a co-transfection efficiency problem, not a single-plasmid one — and therefore sets the vector titre.

Lentiviral production follows the same logic with third-generation systems that split gag-pol, rev and the transfer vector across four plasmids, reducing the chance of replication-competent recombinants. Here PEI is common at process scale because it is cheap, tolerates serum and scales predictably; PEIpro provides the same chemistry with a regulatory paper trail.

Titre correlates better with co-transfection balance than with total reagent dose. When one plasmid is limiting, the capsid-to-genome ratio in the harvested vector also shifts, which changes transduction behaviour downstream — a delivery problem that masquerades as a biology problem.

  • Hold the molar ratio of the three or four plasmids constant and vary only the total.
  • Harvest at 48–72 h — earlier loses titre, later increases empty capsids and cell lysis.
  • Clarify, concentrate and titrate with an orthogonal pair of assays (qPCR plus infectivity) before use.
  • Keep plasmid preparations endotoxin-free and at A260/A280 ≥ 1.8, especially for process-scale PEI.
02

Validating transgenes before packaging

Transient transfection is the fastest way to answer whether a construct works: whether a promoter drives expression in the target cell type, whether a fusion protein localises correctly, whether an shRNA knocks down its target. A two-day experiment replaces a two-week vector production cycle.

Promoter and enhancer screens rely on the same approach at scale. Library transfection in multi-well format, with a reporter readout and normalisation to a co-transfected control, allows hundreds of regulatory elements to be ranked in a single experiment.

03

Non-viral carriers in the clinic

Polymer and lipid carriers deliver plasmid DNA, minicircles and mRNA for diseases where viral vectors are impractical — hemophilia, cystic fibrosis, inherited metabolic disease. The advantages are the absence of viral immunogenicity, no packaging size limit and the ability to re-dose.

Clinical translation changes the requirement set. Endotoxin must be below regulatory limits, every batch must be characterised for size and encapsulation, and the manufacturing process must be reproducible at scale with a documented chain of custody from raw material to released product. The transfection chemistry that worked on a bench plate is usually the starting point — not the final process.

Workflow

How a working experiment is built

  1. 1

    Validate the construct transiently

    Confirm expression, localisation and function in the target cell type before committing to vector production.

  2. 2

    Choose the production chemistry

    PEI or PEIpro for scalable suspension production; lipid reagents where co-transfection of three plasmids needs to be maximised.

  3. 3

    Control the co-transfection ratio

    The balance between capsid, helper and transgene plasmids sets both titre and the empty-to-full capsid ratio.

  4. 4

    Harvest at the titre peak

    48–72 h for most systems; monitor viability to decide the exact window for each process.

  5. 5

    Titrate with two assays

    A genome-titre assay and an infectivity assay answer different questions; report both.

Failure patterns

What usually goes wrong, and the fix

Titre varies between runs

Fix: Endotoxin load, plasmid ratio drift and cell density at transfection are the usual causes — control all three

High particle count, low infectivity

Fix: Check the capsid-to-genome ratio and harvest timing — late harvests accumulate empty particles

Transient expression works, packaged vector does not

Fix: Verify the inverted terminal repeats are intact and the transgene size is within the packaging limit

Process will not scale from flask to bioreactor

Fix: Move to a process-grade reagent with documented toxicity and use the vendor's transfection medium for the scale-up study

The other two applications

Provenance

References for this application

Every protocol on this page was reconstructed from the manufacturer’s own documentation. Use these links to confirm concentrations, catalogue numbers and cell-line-specific variants before running the experiment.

  1. [1]Thermo Fisher — AAV production and transfection protocols
  2. [2]Polyplus — PEIpro for large-scale viral vector production
  3. [3]Promega — Transfection for bioproduction applications
  4. [4]Mirus Bio — Viral vector production transfection protocols

Let's engineer the next delivery breakthrough together

We co-develop nanocarrier and biosensing programmes with pharma, biotech and academic groups — from target selection through GMP supply.

ResearchersRequest a technical consultationTalk formulation, characterisation or assay design with one of our scientists.Industry partnersExplore co-developmentFeasibility studies, co-development programmes, licensing and GMP supply.InvestorsDownload our capabilities deckPlatform, pipeline milestones, manufacturing and partnership models in one PDF.