Lec 5

Question 1

What are the 2 types of gene therapy?

Answer 1

Gene transfer - in vivo and in vitro transfer

Gene correction - in vitro

Question 2

what is the difference between in vivo and in vitro

Answer 2

in vivo is within living beings

in vitro is a controlled lab environment

Question 3

What is nature’s gene therapy?

Answer 3

Revertant mosaicism

Question 4

What must a successful vector be able to do?

Answer 4

• Target the right cells
  
  • Integrate the desired gene in the cells
  • Activate the gene
  • Avoid adverse S/E

Question 5

Explain Non viral vectors in gene therapy

Answer 5

Simplest method is to use naked DNA (circular piece of double stranded DNA - plasmid).
The plasmid would be directly inserted into the cell and into the nucleus resulting in the gene being transcribed.
The method widely used is cationic lipids to condense the DNA to facilitate encapsulation into liposomes. The liposomes are endocytosed into the cell resulting in formation of endosomes. The endosomes release genes into the cytoplasm of the cell.

Question 6

How do we increase efficiency of uptake?

Answer 6

The process of electroporation and sonoporation are used to increase efficiency of uptake.

Question 7

Define transection

Answer 7

the process of deliberately introducing naked or purified nucleic acids into eukaryotic cells.

Question 8

pros and cons of non viral vectors

Answer 8

Advantages: relatively low immune response/non-toxic

Disadvantages: greatest issue is getting the DNA into the cell. DNA doesn’t integrate into host genome therefore repeated doses required.

Question 9

explain viral vectors in general

Answer 9

To form viral vectors we use packaging cells.
Into the packaging cell we input the desired DNA, packaging mix (viral proteins, enzymes etc).
The packaging cell produces viral titre/viral supematant which can be used to transduce the cells.

There are two types of viral vectors:
Integrating: The DNA is incorporated into the host genome
Non-integrating: The DNA sits as a separate chromosomal part.

Question 10

What are the integrating viral vectors?

Answer 10

retrovirus:
modified from murine leukaemia virus and targets dividing cells so it is a permanent integration

uses integrase to add DNA to host DNA

Lentivirus:
Based on the HIV virus 
Targets dividing and non dividing cells 
Elicits a HUGE immune response 
However, you cannot control where the gene inserts if it is in vivo

Question 11

Non-integrating vector??

Answer 11

Adenovirus (dsDNA)
Common cold virus

Targets all cells - inputs double stranded DNA
Endocytosis into cell
Enters nucleus and forms extrachromasomal DNA next to existing.

SO during division the effects are lost.
Elicits a LARGE Immune response

Adeno-associated virus (ssDNA) -

Discovered as a contaminant in preparations of adenovirus.

Its used to increase the efficiency of transfer of adenovirus into cells.
Doesn’t give rise to an immune response, and can transduce it with a helper virus to become an integrating vector. The Integration is directed into a 2 base pair site on chromosome 19 known as AAVS1. BUT it has a low cloning capacity, and also can only carry small amounts of genetic code due to its size.

Question 12

How to do ex vivo gene transfer

Answer 12

1. Take stem cells from BM
   
   2. Modifiy the cells with gene transfer
   3. Screen the cells to make sure you are only using cells that have safe gene insertions
   4. ** You can condition the patient beforehand by ablating some BM (So to decrease competition from the shitty BM)
   5. Transfer the cells back into the patient

Question 13

in vivo transfers in the eye

Answer 13

The compartment anatomy of the eye permits localized delivery meaning:

1. Decreased doses needed
2. Minimising risk of systemic adverse effects

Retinal cells are post mitotic so vectors don’t need to integrate or target dividing cells.
The eye is an immune privileged site therefore there will be no immune response to the vectors.

In 2012:
15 patients, treated with modified adenovirus with RPR65 gene
Virus injected under retina
Restored vision within 1 week lasting a year.
Non-integrating vectors are ideal.

Question 14

ex vivo treatment of ADA-SCID

Answer 14

GSK developed strimvelis

1. Take stem cells from BM
2. Modifiy the cells with gene transfer 
3. Screen the cells to make sure you are only using cells that have safe gene insertions
4. ** You can condition the patient beforehand by ablating some BM (So to decrease competition from the shitty BM)
5. Transfer the cells back into the patient

Question 15

Gene correction when is it performed?

Answer 15

Gene correction is usually perfermed using programmable nucleases that can produce site specific cleavages and repair.

Question 16

3 methods of gene correction

Answer 16

3 methods include:

1. Zinc finger nucleases (ZHNs)
2. Transcription activator-like effector nucleases (TALENs)
3. RNA-guided engineered nucleases (RGENs) derived from bacterial clustered regularly interspaced short palindromic repeat (CRISPR)

Question 17

when does revertant mosaicism occur

Answer 17

It occurs when an inherited disease-causing mutation is corrected by a spontaneous genetic event within a somatic cell
This results in co-existence of cells carrying disease-causing mutations with other cells in which the inherited mutation is genetically corrected.