Understanding the Workflow for Viral Gene Delivery

Researchers use viral vectors for gene delivery because of their infectious nature and ability to introduce specific genes into a cell. A virus must first be packaged with the gene of interest to be introduced. The virus is then quantified, purified, and finally transduced into the target cell.

In addition to a vector containing the gene of interest, a packaging signal and certain structural genes are required for proper viral packaging; these may be provided by cell lines that express the required genes, transfecting plasmids into a cell, or a combination of these. After viral packaging, the recombinant viruses are harvested; the method depends on how the virus matures. For example, adenovirus and adeno-associated virus (AAV) mature within the cell, so the medium is discarded and the viruses are collected by disrupting the cells. Retrovirus and lentivirus, however, mature through a membrane budding mechanism and are therefore found not in the cells but rather in the conditional medium, which can then be concentrated.

Once you have your virus prep, measure its titer. Knowing your viral titer at each step of the workflow will help later if you don’t see the gene expression you expect. There are two main types of viral titer:

1.    Physical titer is a measurement of how much virus is present, and is expressed as the number of viral particles per mL (VP/mL), or for AAV as genome copies per mL (GC/mL).

2.    Functional titer, or infectious titer, is the measurement of how much virus actually infects a target cell and is expressed in the form of transduction units per mL (TU/mL), or for adenovirus as plaque-forming units per mL (pfu/mL) or infectious units per mL (ifu/mL).

Functional titer is more accurate because it measures how much virus actually gets into the target cell. However, functional titer takes longer to determine and is sometimes not practical. In such cases physical titer may be measured, and functional titer is calculated from physical titer. Functional titer will always be lower than physical titer, usually by a factor of 10 to 100-fold.

Once you have your viral titer you may purify the viral prep to remove serum proteins and contaminants that might interfere with viral transduction of target cells. It is also possible to further concentrate your virus during this stage. Purification usually involves column chromatography, filtration or ultracentrifugation, although ultracentrifugation provides only partial purification. Following any purification procedure, it is always a good practice to measure the titer again. It is also wise to reserve a small part of your unpurified viral prep where practical.

The final step involves viral transduction of your target cells. The efficiency of transduction can vary widely depending on the target cell and the viral vector used. Various reagent cocktails are available to help increase transduction rate, and they are typically tailored to use with a specific viral vector.