Lec 5 Flashcards
What are the 2 types of gene therapy?
Gene transfer - in vivo and in vitro transfer
Gene correction - in vitro
what is the difference between in vivo and in vitro
in vivo is within living beings
in vitro is a controlled lab environment
What is nature’s gene therapy?
Revertant mosaicism
What must a successful vector be able to do?
- Target the right cells
- Integrate the desired gene in the cells
- Activate the gene
- Avoid adverse S/E
Explain Non viral vectors in gene therapy
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.
How do we increase efficiency of uptake?
The process of electroporation and sonoporation are used to increase efficiency of uptake.
Define transection
the process of deliberately introducing naked or purified nucleic acids into eukaryotic cells.
pros and cons of non viral vectors
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.
explain viral vectors in general
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.
What are the integrating viral vectors?
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
Non-integrating vector??
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.
How to do ex vivo gene transfer
- Take stem cells from BM
- Modifiy the cells with gene transfer
- Screen the cells to make sure you are only using cells that have safe gene insertions
- ** You can condition the patient beforehand by ablating some BM (So to decrease competition from the shitty BM)
- Transfer the cells back into the patient
in vivo transfers in the eye
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.
ex vivo treatment of ADA-SCID
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
Gene correction when is it performed?
Gene correction is usually perfermed using programmable nucleases that can produce site specific cleavages and repair.