E. Viruses As Gene Delivery Agents - Adeno And Vaccinia

Front 1

To review: what is the genome structure of Adenovirus?

Back 1

Unenveloped dsDNA

Front 2

Is Adeno linear or circular?

Back 2

Linear

Front 3

Where does genome replication occur in Adenoviruses?

Back 3

In the nucleus

Front 4

Can Adenovirus integrate into the host genome? Why not?

Back 4

No - no integrase

Front 5

What is the gene transcription scheme of adenovirus like in general?

Back 5

Regulated with IE, E, and L classes of mRNAs

Front 6

What are the viral proteins produced in Early gene synthesis?

Back 6

E1A and E1B

Front 7

What are E1A/E1B essential for?

Back 7

Genome replication

Front 8

So what were the E1A/B genes used for in early vaccines?

Back 8

Deleting out because you don't need genome replication in viral vectors

Front 9

What were the 293 cells?

Back 9

The cells that expressed E1 constitutively and in trans for making the viral vectors particles.

Front 10

What was the other gene region that was commonly deleted in early adeno virus vectors? Why was it used as a deletion site?

Back 10

E3 - because it was thought to be not necessary for replication and final virion packaging.

Front 11

So when you transfect a cell line like 293 with an adenoviral vector that has a therapeutic gene replacing the genes being produced in trans, what results?

Back 11

A producer cell that makes vector particles

Front 12

What are these vector particles NOT able to do? Why?

Back 12

They don't replicte - because you've deleted out essential genes for replication.

Front 13

What is the major difference when adenovirus vector particles are put into target patient cells, compared to retroviral?

Back 13

They do NOT integrate into the genome; they are simply expressed as a therapeutic product.

Front 14

What do the viral vectors do in the target patient cells?

Back 14

They float around as episomes and get translated.

Front 15

So what are adenovirus gene vectors NOT useful for?

Back 15

Long term gene therapy - they are transient and get diluted out as time goes on after the treatment.

Front 16

What needs to be done if you want to achieve long term therapy with Adenovirus vectors?

Back 16

Re-treatment, multiple times.

Front 17

For what disorders have adenovirus vectors been used to treat with gene therapy?

Back 17

Hereditary Lung Disorders
-Emphysema
-Cystic fibrosis

Front 18

Why is adenovirus used as the viral vector for treating lung diseases?

Back 18

Bc it is tropic for lung epithelial cells

Front 19

What are the advantages of Adenovirus vectors?

Back 19

-Not dependent on target cell for replication
-Able to produce high titer recombinant virus
-High rate of infectivity

Front 20

What does the viral vector contain when used for treating CF?

Back 20

The CFTR gene

Front 21

How is the gene cassette inserted into the Adenovirus genome?

Back 21

Via homologous recombination

Front 22

What type of cells are used to propagate the Adenovirus-CFTR virus?

Back 22

293 cells

Front 23

What do 293 cells provide?

Back 23

The E1a and other proteins in trans

Front 24

What is the final product of the 293 cells?

Back 24

High titer stoks of AD-CFTR vector particles

Front 25

How was the expression of the CFTR gene from the adenovirus vectors tested? (3 ways)

Back 25

-Cell culture
-Animal models
-Human trials

Front 26

What are the 3 main problems with adenovirus vectors?

Back 26

1. Limited short term gene expression bc no integration
2. Immune response
3. Size restrictions on the genes that can be inserted

Front 27

Why is immune response an issue in adenovirus vectors?

Back 27

Because the E3 region is functional in suppressing the immune response; when deleted, there is an increased immune response to the virus.

Front 28

What is the implication of inducing a vigorous immune response to E3 deleted adenovirus vectors?

Back 28

It limits the success of being able to transfect repeatedly - so longterm therapy is not as doable.

Front 29

What have been the 2 solutions to Adenovirus vector problems?

Back 29

-Better packaging cell lines that don't gut the vectors
-Better understanding of the immune response to adenovirus

Front 30

So what is the function of the E3 region in wild type adeno that causes human infections?

Back 30

Evasion of the immune system

Front 31

What was seen in the treatment of Jesse Gelsinger with an adenovirus gene vector for his OTC deficiency?

Back 31

A rapid and extreme immune reaction

Front 32

What was the cause of death?

Back 32

A buildup of IL-6 which caused ARDS.

Front 33

What is the MAIN difference regarding Vaccinia vectors compared to Retroviral and Adenovirus vectors?

Back 33

They are REPLICATION COMPETENT because they are live!

Front 34

What type of genome does the vaccinia virus have?

Back 34

Large dsDNA genome

Front 35

What does the fact that the genome is large allow for Vaccinia vectors?

Back 35

Flexibility in the size of DNA that can be packaged into it

Front 36

Where in the cell does Vaccinia replicate?

Back 36

In the cytoplasm

Front 37

What does the vaccinia virus vector construct have to encode for a productive vaccine to be made?

Back 37

-RNA polymerase
-Capping/methylating/polyadenylating enzymes
-Viral RNA polymerase promoter

Front 38

Why does there have to be a specific viral RNA pol promoter in the vector construct?

Back 38

Because host cellular promoters are not recognized by the vaccinia polymerase

Front 39

So what are the 2 components of the Plasmid construct used to create Vaccinia vectors?

Back 39

-Foreign gene
-Vaccinia promoter to control the expression of the foreign gene
(a chimera)

Front 40

How is the Chimeric gene put into the vaccinia genome?

Back 40

By homologous recombination

Front 41

What is important about where you put the foreign gene into the vaccinia genome?

Back 41

It can't be in a site that is essential for vaccinia replication and synthesis.

Front 42

What is the most popular site for placing a foreign gene in the vaccinia genome? Why?

Back 42

The Thymidine Kinase gene - because this is nonessential for replication and it's easy to select for viruses that retain the gene.

Front 43

What selects for TK+ viruses that didn't undergo successful gene recombination?

Back 43

BUdR

Front 44

How can you potentiate the recombination of the chimeric plasmid construct with the vaccinia virus?

Back 44

By flanking the promoter-gene chimera with Vaccinia DNA

Front 45

What is achieved by homologous recombination of the chimeric gene/vaccinia promoter with Vaccinia virus genome?

Back 45

The genome acquires the gene of interest

Front 46

What is the whole purpose of putting a foreign gene into the vaccinia virus?

Back 46

So that you can create vaccines against a variety of diseases using immunogenic epitopes from those organisms that would be too strong as a vaccine by themselves.

Front 47

What is a very old example of how vaccinia virus was used as a vaccine against a different disease?

Back 47

Hepatitis B vaccine

Front 48

What hep B gene was put into the vaccinia genome, and what did it replace?

Back 48

HBsAg - replaced TK

Front 49

What was placed just upstream the HBsAg?

Back 49

P2 - the viral promoter

Front 50

And then what was the Chimeric construct placed into?

Back 50

Cell culture containing wild type vaccinia to undergo homologous recombination

Front 51

And the product of the homologous recombination was:

Back 51

Vaccinia virus that expressed HBsAg

Front 52

What was the first test to see whether the Vaccinia vectors indeed expressed HBsAg? Result?

Back 52

Cell culture - yes cell culture with media expressed HepBsAg

Front 53

What was the 2nd test to see whether the Vaccinia vectors indeed expressed HBsAg? Result upon rechallenge with Hep B virus?

Back 53

Chimpanzee testing
-Evidence of a vigorous immune response due to an anamnestic response

Front 54

What is so great about the fact that the vaccinia virus DNA genome is so large?

Back 54

You can put in MULTIPLE foreign genes and immunize against SEVERAL diseases - as many as 10 different antigens!

Front 55

How can vaccinia be used as a replication competent virus?

Back 55

It doesn't cause severe disease in human hosts.

Front 56

What is a situation in which Vaccinia COULD cause disease?

Back 56

Immunocompromised patients

Front 57

Why is it unfortunate that vaccinia is less useful for immunocompromised patients?

Back 57

We'd like to be able to use it as a vector for an HIV vaccine

Front 58

What are 2 other poxviruses that can be used in immunocompromised patients, but work by the exact same strategy as vaccinia?

Back 58

-Canarypox
-Modified vaccinia ankara

Front 59

How can Canarypox and MVA be used in immunocompromised?

Back 59

They don't replicate but you do get expression of genes.