Viral Transduction KEY POINTS

What is viral transduction?

A method used to introduce genetic material into cells using viruses as vectors

The difference between viruses and viral vectors?

Viruses are natural infectious agents; they infect a host cell and use that cell’s machinery to replicate themselves.

Viral vectors are modified viruses engineered by scientists to deliver genetic material into cells. They are genetically modified to limit or eliminate their replicative ability

What are the most commonly used viral vectors in research?

Adeno-Associated Virus (AAV), Adenovirus (Ad), Lentivirus (LV), Retrovirus (RV)

What are the key features of Lentiviral/Retroviral vectors?

• RNA genome

• Integrate into host genome

• Stable expression

• Low immunogenicity

• 8kb capacity

Which virus delivers to dividing and non dividing cells?

Lentivirus, AAV, Ad

What are the key features of Adeno-Associated Viral vectors?

• ssDNA genome

• Sustained expression as episome 

• Low immunogenicity 

• 5kb capacity 

What are the key features of Adenoviral vectors?

• dsDNA genome

• Does not integrate into genome

• Transient expression 

• High immunogenicity 

• 7kb capacity, 30kb HDAs

What are the steps of viral vector production?

1. Clone transgene with promoter into transfer plasmid

2. Transfect packaging cells with all necessary plasmids

3. Harvest cells

4. Purify viral particles

5. QC testing

What do we need for Lentiviral vector production?

1. Plasmids (packaging, envelope, transfer)

2. Producer/packaging cell line 293T

3. Transfection

4. Harvest media after 48-72 hrs

5. Purify by ultracentrifugation

What do we need for AAV vector production?

1. Plasmids (packaging, helper, transfer) 

2. 293 T 

3. Transfection

4. Harvest cells/media after 72 hrs

5. Purify by density gradient ultracentrifugation 

What do we need for Adenoviral vector production?

1. Plasmid (adenoviral backbone containing the transgene and promoter)

2. Producer/packaging cell line: HEK293

3. Transfection

4. Several steps of viral expansion

5. Harvest cells

6. Purify by double cesium chloride gradient

What are the LTRs and ITRs?

LTR = Long terminal repeats, they play a role in integration of viral DNA into host genome

ITR = Inverted terminal repeats, viral sequence required for packaging DNA into capsids

What are some of the Quality Control of viral vectors production?

• Physical Titer 

• Infectious Titer 

• Check for integrity of particles 

• Test for replication competency (adenovirus) 

• Endotoxin test 

• Sterility test 

Physical titer

Concentration as it is expressed as a number of particles per milliliter (pt/ml) or number of genome copies per milliliter (gc/ml)

Infectious titer

Tell us how many particles are infectious, expressed as pfu/ml (plaque-forming unit per milliliter) or iu/ml (infectious unit per milliliter)

How to check for integrity of particles? 

Look for a Full versus Empty genome with (Transmission Electron Microscopy)

Multiplicity of Infection (MOI)

The number of infectious particles per cell.

Ex. If we aim to infect our target cells at a MOI of 10 we will use 10 infectious particles per cell.

If stock virus is 5×10^10 and there are 1×10^8 cells to infect the amount of virus to use to achieve a MOI of 10 is (1×10^8 / 5×10^10) = 0.02 ml or 20 ul