3.6: Viral Mediated Delivery of Genes to Neurons
There are a number of ways to deliver DNA (or other genetic information) to neurons including transfection, electroporation, biolistic delivery, and nanoparticles, but within an intact biological system like the brain is challenging. Luckily, neurons are very easily infected by viruses (see Unit 1). Neurobiologists will therefore package genetic material that they want to be expressed within specific groups of neurons inside different types of viruses (Figure \(\PageIndex{1}\).).
Figure \(\PageIndex{1}\).
Schematic overview of how viruses (i.e. adenovirus) can be used to deliver recombinant genetic material to neurons, or other cell types within the nervous system. Steps include viral binding to cell membrane, endosomal packaging and breakdown, delivery and integration of vector into host DNA, and ending with the expression of the desired protein.
In fact, as molecular neurobiologists, we often have a number of different viral vectors that we can use to our benefit. Some viruses for example package only a small amount of genetic material (AAV for example) while others like HSV will be able to package up to 150 kb worth of genetic material (and this is a lot) into the virus. Speed of expression and how long the expression of the recombinant genetic material will persist for, are often considerations on the choice of viral vector (see Table \(\PageIndex{1}\)).
| Table \(\PageIndex{1}\). Properties of recombinant viral vectors useful for gene delivery in the adult nervous system | |||
|---|---|---|---|
| Adeno-Associated Virus (AAV) | Lentivirus | Herpes Simplex Virus (HSV) Amplicon | |
| Genetic material | Single-stranded DNA | RNA | Double-stranded DNA |
| Capacity for genetic material | ~5 kilobases | ~8 kilobases | ~150 kilobases |
| Speed of expression | Weeks | Weeks | Days |
| Duration of expression | Years | Years | Weeks to months, but elements can be added for persistent expression |
One technical consideration that is often overlooked – how are viruses containing the gene of interest that we hope to express in neurons generated? Viruses aren’t truly alive so how does this work? We often use a cell line like the HEK293 cell which is a non-neuronal cell line. We transfect 3 separate plasmids into the HEK293 cells in culture. 2 of the plasmids are viral (pVSV-G and packaging plasmids for example for lentiviruses, and the last one is the construct that we want to specifically deliver. Figure \(\PageIndex{2}\). shows the workflow for production of lentiviruses (upper panel) and AAV viruses (lower panel). In both cases, viral particles are released from the transfected HEK293 cells and the virus is collected from the cells and spun on a gradient using ultracentrifugation and a final purification step. The purified fluid then contains live virus that can infect neurons.
Figure \(\PageIndex{2}\).
Lentivirus (upper panel) – Part of the retroviridae family (viral sizes are 80-100 nm diameter) derived from HIV-1 virus. To produce lentiviruses with the gene of interest as the lentiviral DNA construct, first transfect cells with a packaging plasmid and the envelope vector (VSVG).
Adeno Associated Virus (AAV)
(lower panel) – Member of the parvoviridae family (20 nm diameter). To produce AAV, package a gene of interest into the AAV-ITR vector and transfect cells with a Helper vector and the Rep/Cap DNA integration vector.