L16: Gene Therapy

Question 1

Somatic gene therapy

Answer 1

genetic defect is corrected only in the somatic cells of a person affected by the disease where any genetic changes are restricted to the lifetime of the person treated

Question 2

Germ-line gene therapy

Answer 2

genetic modification is made to a gamete, fertilized egg or embryo before the germline has split off from the cells that will make the rest of the body where any change is passed on to subsequent generations

Question 3

In vivo vs Ex vivo gene therapy

Answer 3

In ex vivo gene therapy, transgene is introduced in isolate cells so that they can be manipulated in vitro. These cell clones would then be reintroduced into the patient’s body.

In in vivo gene therapy, transgene could be directly introduced into the cells affected by the disease

Question 4

Gene therapy relies upon methods of introducing transgenes into target cells. This means a vehicle is needed for its delivery.
The utility of the vehicle will depend on a number of factors:

Answer 4

Efficiency of delivery to the target cell
Specificity of delivery for the target cell (tropism)
Whether target cell needs to be dividing
Whether vehicle will provoke an immune response
Size of DNA that can be carried
Stability and longevity of gene in target cell
Expression of the introduced gene

Question 5

Gene Augmentation Therapy

Answer 5

transfer a cloned working gene copy into diseased cells of the patient in order to make some (increase) gene product that is lacking

Question 6

Gene Mutation Correction

Answer 6

transverse of diseased mutant allele with correct endogenous gene to correct mutation

Question 7

Gene Expression Inhibition

Answer 7

selectively blocking transcription or by targeting transcripts of disease cells with mutant allele (making harmful product) to be destroyed

Question 8

Direct Killing of Diseased Cells

Answer 8

1) Inserting toxin gene into diseased cell will kill cells expressing this toxin
2) Inserting prodrug metabolizing gene will kill cells expressing the toxic prodrug metabolites

Question 9

Assisted Killing of Diseased Cells

Answer 9

Inserting 1) foreign antigen gene into diseased cells
or 2) cytokine gene into diseased/non-diseased cells can kill diseased cells by stimulating an enhanced immune response directed at tumours

Question 10

Ex vivo gene therapy is suitable for genetic defects
that affect blood cells. Why?

Answer 10

1) All blood cells are derived from pluripotent stem cells in the bone marrow.
2) They can be isolated, cultured in vitro and reintroduced in vivo

Question 11

4 main vectors (summary)

Answer 11

Retrovirus: ssRNA, 7-8kb, integrating, only target dividing cells, long-lasting
Lentivirus: ssRNA, 8kb, integrating, target dividing/non-dividing cells, long-lasting & high level expression
Adenovirus: dsDNA, 7.5-35kb, non-integrating, target dividing/non-dividing cells, transient but high level expression
AAV: ssDNA, 4.5kb, non-integrating, target dividing/non-dividing cells, high level expression for a yr

Question 12

Retrovirus-based gene transfer

Answer 12

Retroviruses have two identical RNA genomes
Upon infection, RNA is copied into DNA by a virally encoded reverse transcriptase
This DNA copy is then integrated into host genome in a process that depends upon long terminal repeats (LTRs) at each end of genome

Question 13

Retroviruses packaging

Answer 13

The retroviral genes are transcribed from a promoter within the 5’ LTR regions.
The resulting RNA molecules are packaged into the virus particles.
This packaging process requires:
1. Packaging sequence
2. pol gene encoding reverse transcriptase
3. gag gene encodes a viral core protein
4. env gene that encodes a viral envelope protein

This packaging cell line produces retrovirus particles containing the gene of interest that can infect the target cell.
Once inside the target cell, the pol protein contained within the virus particle will ensure that the gene is integrated into the host genome.
The LTRs are necessary for integration and also act to express the gene once integrated.

Question 14

Retroviruses can be oncogenic, but it is essential that there is no contamination by replication-competent viruses. How it this achieved?

Answer 14

This is achieved by using a packaging cell line in which essential retroviral genes are divided b/w 2 diff DNA molecules, neither of which contains the packaging sequence.
A third DNA molecule carries the gene to be transferred together with a functioning packaging sequence sandwiched between LTRs.

Question 15

Advantages of retrovirus-mediated gene transfer

Answer 15

Its efficiency and stability of the introduced gene.
It is generally used for ex vivo gene therapy which is based on replicating cells.

Question 16

Problems with the use of retroviruses for gene therapy

Answer 16

Can only infect proliferating cells
Limited in size of DNA molecule that can be accommodated, which mostly limits its use to cDNA.
Does not specifically infect one cell type.
Integrate at random sites in the genome.

Question 17

Lentivirus

Answer 17

Related to retroviruses but have evolved the capacity to replicate in non-dividing cells
The tropism has been changed by substituting the HIV env gene for the env gene of human vesicular stomatitis.
HIV vectors have been engineered to remove all viral genes except those necessary for infection of non-dividing cells. Such vectors contain only 25% of the original HIV genome.

Question 18

Adenovirus-based Gene Transfer

Answer 18

Adenoviruses are double-stranded DNA viruses
Naturally infect non-dividing cells of respiratory and gastrointestinal tracts
Evolved to evade host immune mechanisms and to be highly efficient at infecting their target cells
Once inside target cell, they replicate as episomes and so avoid dangers of uncontrolled integration
There are at least 50 different serotypes

Question 19

How would you disable the adenovirus to prevent it replicating and killing the host cell?

Answer 19

Gene expression can be divided into early and late phases.
DNA replication and expression of the genes whose products constitute the virion depend upon prior expression of early genes.
The infective cycle can therefore be halted by deleting E1 (essential early gene). At the same time this makes room for the insertion of foreign DNA, up to a size of about 8 kb.

Question 20

First Generation Adenovirus

Answer 20

First generation Adenovirus expression is transient so that the therapy would need to be repeated. Repeated therapy results in the immune response becoming more severe.
With deletions of other early genes such as E2, E3 and E4 further reduction of late gene expression reduced the immune response.

Question 21

Advantages of Adenoviruses as Gene Therapy Vectors

Answer 21

Well characterized.
Easily manipulated and propagated.
Produce high titer stocks.
Easily purified.
Highly stable particles.
Have the potential to carry large segments of foreign DNA.

Question 22

Disadvantages of Adenoviruses as Gene Therapy Vectors

Answer 22

Pre-exposure of most individuals to adenovirus.
1. Short term expression of transgenes.
2. Sub-optimal bio-distribution in vivo.
Underscores the need to design vectors with targeted tropism.

Question 23

Helper-Dependent “Gutless” Adenoviral Vectors

Answer 23

293Cre –used to excise
Co-infect with helper virus –packaging of that helper virus is flocked with loxP (remove packaging signal of helper virus)
Helper virus not packaged b/c excised by Cre
All left is ITR and target sequence to package

Question 24

Advantages of Helper-Dependent Adenoviruses

Answer 24

1. Greater longevity of transgene expression.
2. HD adenoviruses are devoid of virus genes and as a result may be less inflammatory.
3. Incorporating gene regulation elements is essential for long term expression.

Question 25

4) Adeno-associated viruses (AAV)

Answer 25

Naturally replication-defective viruses that depend on other helper viruses, such as adenovirus, to provide essential functions.

Single-stranded DNA molecule containing two genes, rep and cap.
Rep protein mediates viral integration into the specific site on chromosome 19. Cap is the structural protein of the capsid. These genes are flanked by 2 inverted terminal repeats that are essential to pack DNA into virus capsid.
For gene therapy the cap and rep genes are replaced by the transgene. This construct is introduced into a packaging cell line that supplies the cap and rep proteins in trans.

Question 26

Advantages of AAV

Answer 26

Not naturally pathogenic
Tropic for airway cells
Infect non-dividing cells
Show longer-lasting expression (up to 6 months in animal models)
Can show chromosome-specific integration in chromosome 19

Question 27

Disadvantages of AAV

Answer 27

Specific targeting to chromosome 19 is lost as the Rep protein is not present.
In animal models, there has been long-lasting expression of transgenes from such tissues as muscle, liver and brain.

Question 28

Ex vivo gene therapy for X-linked severe combined immunodeficiency disease (X-SCID)

Answer 28

Take bone marrow
Look for CD34 stem cells through magnetic bead-antibody and cultivate
Infect with viral vector containing IL2RG gene
Transduce cells to multiply and infuse cells before returning back to body