Transfection Efficiencies

Gene Therapy - the reason why transfection effiencies are important

Gene therapy is being heralded as the most disruptive advancement to medicine in our entire history which could result in the eradication of infectious diseases such as malaria, Yellow Fever, Dengue virus or Zika; the elimination of genetically inherited conditions such as cystic fibrosis or Epidermolysis bullosa (“butterfly children”) and amazingly…. curing cancer.

Gene therapy is a technique for correcting defective genes that are responsible for disease development.  Approaches include inserting a normal gene to compensate for a non-functional gene or trading a normal gene for an abnormal gene.  Gene therapy should not be confused with genetically modified organisms (GMOs) which contain ‘foreign DNA’ or ‘transgenes’ from another species.  Gene therapy will change the established healthcare market in profound ways, as many current treatments are replaced by more effective genetic treatments and even cures.  

However, while clinical trials show exciting promise, there are significant challenges to be overcome; the (cell) manufacturing of these gene therapies on a production scale is no easy task and differs significantly from traditional drug manufacturing where thousands of batches of one drug, manufactured in well-established and controlled processes, result in millions of doses to treat millions of patients.

Trying to improve your transfection efficiency? So are we. 

All researchers working with cell transfection methods, whether in academia or in industry, know about the pains involved in optimising cell transfection. Sometimes some cells just seem to be more resistant to transfection than others, often for frustratingly vague reasons.  Is poor transfection efficiency wasting your time?  We can help.

Inish Solutions:

1. Increase overall transfection efficiency

2. Increase quality of gene transfection data

3. Fast-track successful gene transfection methods and quickly optimise experimental conditions

 

What is Transfection?

Transfection is the process by with the genetic material is delivered into the cell.  Broadly speaking, there are two main methods:  Transfection (non-viral) vs. Transduction (viral).  Up to now, the most common method of gene editing was via the use of viral vectors (i.e. using viruses to deliver the DNA inside the cell), otherwise known as transduction.  Viral vectors run the risk of causing severe immune responses in patients and a number of deaths have been attributed to this in several clinical trials in the past.  However, viral vectors also pose significant manufacturing challenges, as in addition to the cells as starting material, the production process has to take into account the raw materials of viral vectors which are highly complex.  Many industry players now recognise that long-term, successful non-viral transfection methods will be less challenging for manufacturing. 

Thus, the move to non-viral transfection methods, in particular electroporation (where an electrical field is applied to cells increasing the permeability of the cell membrane, allowing DNA to be introduced into the cell), is gathering steam in terms of manufacturing capability:  increased reproducibility and quality control.  Electroporation has become increasingly popular in recent times as it is more effective than viral transduction when transfecting larger gene edits, which is also more common when using CRISPR techniques.  One of the most popular electrporators on the market is the Amaxa Nucleofector from Lonza.  However, there are still challenges with electroporation, in particular, primary cells such as T-cells, B-cells or stem cells are notoriously hard to transfect.

Problems with sorting succesfully transfected cells

Following electroporation, cells are typically plated in 96-well plates.  1-2 days later, successfully transfected cells begin to express the receptors of interest.  These cell receptors are labelled with a fluorescent dye/stain or magnetic bead which enables Fluorescense or Magnetic Activated Cell Sorters (FACS or MACS) to sort the succesfully transfected (i.e. genetically engineered) cells.  Aside from waiting for the expression of the receptors over 1-2 days, the process of preparing the samples for FACS or MACS is laborious and time-consuming.  

Inish Solutions

In practical terms; Inish Solutions will enable you to observe, in real-time:

  • The state of the cell membrane before transfection

  • The different cell sub-populations after transfection:  

    • Live cells with closed membranes - in other words, unsuccessfully transfected cells as the membrane failed to open in order to receive the vector/DNA.​

    • Live cells with open membranes - this is your pool of successfully transfected cells.  As the cell membrane has been successfully opened, these cells can uptake the vector.  The ability to analyse and sort cells of this type is totally unique to Cellix!

    • Damaged cells - unlikely to recover.

    • Dead cells.

  • The recovery of the cell membrane after various treatments, such as electroporation, cell squeezing (French press), Microinjection, and many other physical transfection methods.

  • Different cell population profiles - Inish Solutions are also capable of cell sorting based on size, membrane integrity, granule size....

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