Viral Transduction Part 2

AAV Production (Transfection)

1. Use a human packaging cell line: 293 T

2. Use the 3 plasmids of transfection

• Expression plasmid: contains the promoter + transgene enclosed by ITR

• Rep/Cap plasmid: supplies the genes necessary for capsid formation and replication

• Helper plasmid: provides the required adenovirus helper genes

AAV Production Timeline

Day 1 - Prep cells, cells need to be 70% confluent on next day

Day 2 - Transfection

Day 5 - Harvesting, collect the cells using cell scraper

2 weeks until fully produced with QC

What needs to be harvested in AAV?

Both cells and media

• deoxycholate is needed to break down the lipids of the cell membrane and release the virus 

• DNAse and RNAse is needed to degrade unprotected nucleic acids 

• PEG is need for precipitation 

AAV Purification 

Works by creating a density gradient with Cesium Chloride or Iodixanol. 

1. Virus is added on top of density gradient 

2. Ultra speed centrifugation for 19-20 hrs 

3. AAV band at 40/60% interface 

4. Concentration is found using a molecular weight cutoff filter (contaminants at the wrong conc. will pass through) 

5. AAV is collected from the filter 

Adenovirus Characteristics

• non enveloped virus with linear, double stranded DNA, 36Kbp

• High transduction efficiency

• High level transgene expression

• does not integrate host genome, transient expression

• major immune response

Adenovirus Structure (Nucleocapsid components)

• Hexon - base, composes most of the viral capsid

• Fiber and Penton - receptor binding, internalization of Ad into host cells

Adenovirus Pathway 

1. Adenovirus binds to the CAR receptor via fiber protein 

2. Virus is taken into the cell by endocytosis 

3. The viral capsid is disassembled

4. DNA enters the nucleus but stays episomal 

5. Host cell reads the DNA and protein is released 

What genes make up the adenovirus genome?

1. E1A

2. E1B

3. E2

4. E3

5. E4

E1A & B are usually deleted and replaced with a promoter transgene

What is the cytopathic effect caused by viruses?

CPE is visible changes in cells caused by a viral infection

In adenovirus the cells usually burst and are released to infect neighboring cells, takes around 48hrs

How to purify adenovirus? 

Use double cesium chloride density gradient ultracentrifugation 

Use a Sephadex columnn to concentrate the viral particle and get rid of CsCl salt

The density gradient will be added first and then the cell lysate on top

Helper dependent adenovirus

“gutless adenovirus”

All viral coding genes are deleted, there is no toxicity or immunogenicity and a longer term transgene expression

What is a disadvantage of the helper dependent adenovirus?

They are more difficult to produce and require a helper virus which may be left behind

HDAd production

Transfection of 293 cre cells with HDAd plasmid carrying transgene and co-infected with the helper virus carrying all other Ad genes

Physical titer 

number of viral particle/ml

• measure of how much viral nucleic acid is present

Infectious titer

used to check if the viral vector is functional

#infectious particle/ml

What does quality control always include?

Physical and Infectious titer

What assay can be used for endotoxins?

Quantitative limulus amebocyte lysate (LAL) assay

What can be used to check if AAV has full genome packaging or no packaging? 

Transmission electron microscopy - can identify empty particles versus full particles 

What is the sterility control for?

Confirm that viral vector preparations are free from microbial contamination

• confirm by incubation in bacterial media, useful for mycoplasma detection

How to test for genome integrity?

Sequence the transgene

MOI formula

Virus Titer x Virus Volume / Total Cell Number 

What is the MOI needed for AAV?

1,000 - 100,000 vg per cell in vitro

MOI too high = 

cytotoxicity

MOI too low =

not 100% infection

Multiplicity of infection (MOI)

ratio of infectious particles to infection targets

ex. 10 millions viral particles for 1 million of cells the MOI = 10

An average of 10 viral particles infect one cell

Factors affecting how easily viruses can infect their target cell

• current state of cell (dividing or not)

• which virus used

• transduction efficiency

Process of selecting the best MOI?

• determine the optimal MOI for your purpose

• use a reporter virus

• transduce cells with a range of MOI

• Determine the % of efficiency

• select the lowest MOI that gives the highest efficiency