Viral Vectors Applications

Key considerations in choosing a viral vector system

1. Target cell type: dividing vs non dividing 

2. Packaging capacity: how big the gene is you want to deliver

3. Expression: transient vs stable vs integrate into genome 

4. Usage: in vivo, in vitro 

5. Adverse effects: immune response? 

What is important about packaging capacity? 

There are different limits for each vector 

The main question of in vivo vs in vitro?

Are immune cells wanted at the site of injection or not

Which viruses will integrate into the genome?

Retroviruses or anti-viruses

Which viruses are best for long term experiments?

Retro and Lenti

Which virus is this? (2)

• Efficient gene transfer 

• Therapeutic high expression

• no integration

AAV & Ad

Which viruses?

• Host genome integration

• Long term correction of chronic disorders

RV/LV 

What are the characteristics of the ideal viral vector?

• large packaging capacity

• infect dividing and non dividing cells

• strong expression of transgene

• broad tissue tropism

• low immunogenicity

• can be produced in large scale

Advantages of AAV?

• non pathogenic

• low immunogenicity

• infects dividing and non diving cells

• long term expression

• broad tissue tropism 

Disadvantages of AAV?

• limited packaging ~4.7 kb

• low transduction efficiency

• difficult to produce large scale

What is AAV best for?

In vivo gene therapy, CNS, eye, muscle, and liver targeting

Is one viral vector suitable for all applications? 

No 

Advantages of Ad?

• high transduction efficiency in dividing and non dividing cells

• high level of transient expression

• large transgene capacity ~7.5kb - 36kb

• large scale production

Disadvantages of Ad?

• high immunogenicity

• low transient expression in vivo

• limited duration of expression

What is Ad best used for? 

Vaccines, cancer therapy, transient expression in many tissues 

Advantages of RV?

• stable genomic integration

• high long term expression

• simple production system

• low immunogenicity

Disadvantages of RV?

• Infect only dividing cells

• risk of insertional mutagenesis

• limited tropism

RV is best used for? 

Stable gene integration in dividing cells

Advantages of LV?

• stable genomic integration, long term expression

• infects both dividing and non dividing cells

• large cloning capacity ~8kb

• low immunogenicity

Disadvantages of LV?

• risk of insertional mutagenesis

• moderate titer and production yield

LV is best used for? 

Stable gene delivery to both dividing and non dividing cells 

If you want a virus to activate the immune system response, which should you use?

Adenovirus

Tropism

Natural affinity of a virus for a cell type or tissue

2 ways to alter viral vector tropism? Why would you do this?

Entry and Expression specificity 

Purpose: Enhance specificity for expression in the cell or tissue of interest

Entry Specificity

Structural modification in capsid (AAV), envelope (RV/LV), or fiber knob (Ad)

Expression specificity

Adjust tissue specific promoter and enhancers

LV structure modification (Entry Specificity)

Envelope (physical change)

Ad structure modification (Entry Specificity)

Fiber Knob (physical change)

Expression specificity is an ___

inner changes

Different types of constitutive promoters

1. CMV - very strong, robust transgene expression (AAV and Ad) 

2. CAG - very strong, hybrid promoter, stable

3. EF-1a - stable, long term expression (LV used in stem cells and neurons)

4. PGK - weaker, long term expression (LV) when lower expression level is beneficial

Types of tissue specific promoters that drive gene expression ONLY in a particular cell type or tissue

1. hSyn - strong, specific to neurons (brain targeting)

2. TBG - strong, specific to hepatocytes

3. Albumin - strong, liver specific (larger than TBG)

4. MCK - strong, muscle specific

One is one validation technique?

Re-express the deleted gene

Gene overexpression

Introducing a gene of interest in high quantities to study its effect on cell physiology and behavior 

Gene Silencing

Deliver shRNA or CRISPR/Cas9 components to silence or delete specific genes, helping to study their function 

How does Cre work in tissue specific gene delivery?

The transgenic mice with floxed DNA will express Cre in specific tissue based on the AAV tropism. In this specific tissue, Cre recombinase will bind and excise the loxP sites, and inactivate the GOI

How does inducible gene delivery with tet-on work? 

Doxycycline binds to rtTA, activates TRE promoter, expresses the GOI 

The expression is reversible and can be turned on/off by adding or removing doxycycline 

Inducible gene delivery with (Cre-ERT2-loxP)

Tamoxifen activates Cre > excision of the stop cassette > expression of GFP

• Expression is permanent, Cre excises or inverts DNA

• use for lineage tracing

*The viral vector delivers a gene that remains inactive until a certain drug is administered (Tamoxifen)

Lineage Tracing 

A technique that marks a single cell and tracks the cell’s progeny 

• Viral particles carrying reporter gene (such as GFP) infect cells, integrating the desired recombinant DNA into the host genome to allow cell tracing

Why are retroviruses largely used for cell tracing?

Because it integrates into the genome of dividing target cells and are transferred to all progenitor cells

Oncolytic and therapeutic vectors

Genetically engineered adenovirus designed to selectively infect, replicate in cancer cells and destroy them

• in normal cells virus cannot replicate because p53 stops it

• in tumor cells (mutated p53) virus replicates and causes lysis of cells

CAR-T therapy steps 

1. T cells are taken from the patients whole blood 

2. Viral vector delivers CAR encoding gene into T cells 

3. T cells express CAR on their surfaces and are known as CAR T cells 

4. CAR T cells infuse into the patients bloodstream 

5. Lymphodepleting chemotherapy 

6. CAR T cell infusion

Tropism of AAV and Ad

Broad

Tropism of RV?

Limited

Tropism of LV? 

Moderate 

Permanent expression systems

RV/LV