Past Paper Qs Flashcards
Advantages of viruses for gene therapy
1) Efficient entry into cells:
They have surface proteins that interact with receptors on the surface of the cells
2) Many cell-surface receptors:
That the virus can attach on to, higher efficiency uptake system
3) Often tissue-specific entry:
Receptors are expressed in a tissue-specific manner, accounting for tissue-specific nature of virus infection (adenoviruses and respiratory tract – cell surface receptors it uses are highly specific to the respiratory tract)
4) Can integrate or persist:
For some viruses, only one administration needed; retroviruses, herpesviruses
5) Can create disabled viruses that are replication-deficient for gene therapy:
We need to ensure that by using the virus we don’t cause another disease that is going to be at least as bad at the disease that the patient is suffering from. It is possible and necessary to make disabled viruses that are replication-deficient (they cant cause a viral disease).
Qualified for cancer applications as people are often trying to make viruses that replicate selectively in cancer cells and not in normal cells.
Disadvantages of viruses for gene therapy
1) Inflammation due to viral proteins:
The immune system recognises viral proteins and eliminates cells that are expressing viral proteins
2) Pre-existing immunity against the virus:
If we use human viruses there could be ore-existing immunity. There may already be an immune response, limiting their effectiveness
3) Repeat dosing required if expression not sustained:
Leads to an immune response against the vector. If its transient, we may have to repeat the dose which could lead to an immune response
4) Integrating viruses (e.g. retroviruses) may insert into critical genes in host DNA, altering gene function:
Critical genes such as genes that regulate cell cycle which could generate cells that grow rapidly and form into cancer.
5) Expensive to produce
Unlike liposomes.
Advantages of Retrovirus for gene therapy
Same as virus but w…
1) Their ability to transform their single-stranded RNA genome into a double-stranded DNA molecule that stably integrates into the target cell genome.
This means that retroviral vectors can be used to permanently modify the host cell nuclear genome.
2) Infect non-cycling cells.
3) Can integrate into the host cell chromosome
Raises the possibility of insertional mutagenesis and oncogene activation
Disadvantages of Retrovirus for gene therapy
Same as virus w/
1) Integrating virus
Problems when integrating their genetic information into genomes causing damage.
2) T- Cell Leukemia:
https://gvt-journal.biomedcentral.com/articles/10.1186/1479-0556-2-9
A recent clinical trial of gammac mediated gene therapy for X-linked severe combined immunodeficiency (X-SCID) = proven the potential of retroviral mediated gene transfer for the treatment of inherited metabolic disease.
BUT 2 out of 10 patients developing T cell leukaemia as a consequence of the treatment - use vectors from non-oncogenic retroviruses
Advantages of Liposome vectors for use in human gene therapy
1) Easy to manufacture and quality control
Already in use in drug delivery
2) A pharmaceutical product
Heavily used in cosmetics and OTC products. Also used for drug delivery and can enhance the delivery of cancer drugs. Cancer drugs are formulated into liposomes – efficient form of delivery.
Simple to form plasmid- liposome complexes
3) Non-immunogenic
Although some people have autoimmune diseases where people make antibodies against DNA or proteins, this autoimmune type disease not exist against liposomes. So they can be injected/ administered a number of times without the immune response compromising their effectiveness
4) Repeat administrations possible
5) No limit on the size of therapeutic transgene
In principle. This is because often we run into problems with viral vectors because there is no capacity to put in bigger and bigger segments of DNA or RNA due to their being constraints to how much nucleic acid they can package. This constraint does not exist in liposomes, so we can in theory package up very large genes
Disadvantages to using Liposomes in human gene therapy
1) Non-specific
There is no targeting mechanism built into them that will target particular cells. They will just be taken up by most cells
2) The transgene of complexed DNA often poorly and transiently expressed
Due to the entry issue. They can culture in laboratory systems and can be expressed for a few days, but the expression will go down as there is no mechanism to maintain the DNA in the cells so it is not particularly efficient. Repeated administrations can compensate.
Non-viral methods for gene delivery to human cells
1) Liposomes:
2) Naked DNA
3) Synthetic Oligonucleotides
Important features of liposomes and comment on their use in gene therapy
1) Form lipid bilayers similar to the cell membrane
Because of hydrophobic interactions that are similar to cell membranes
2) Current liposomes very safe
Heavily used in cosmetics and OTC products. Also used for drug delivery and can enhance the delivery of cancer drugs. Cancer drugs are formulated into liposomes – efficient form of delivery.
3) Will accommodate large therapeutic genes
In principle. This is because often we run into problems with viral vectors because there is no capacity to put in bigger and bigger segments of DNA or RNA due to their being constraints to how much nucleic acid they can package. This constraint does not exist in liposomes, so we can in theory package up very large genes
4) Deliver DNA well to the cell cytoplasm, but transport to the nucleus inefficient
Types:
- Anionic Liposomes
Sometimes called pH-sensitive liposomes (because they can be modified by adjusting pH – because of these negative groups are usually in the neutral range)
There are negative groups sticking out on the outside. DNA phospholipid backbone is negatively charged at a physiological pH, so there is a mutual repulsion between the polar group of the lipid and the DNA backbone, which is an issue when encapsulating DNA into this structure. This is why most studies are done with cationic liposomes.
- Cationic Liposomes
Positively charged side chains/polar group.
Build up sheets called llamale, whereby the liposomes form electrostatic interactions with DNA that is built up into sheets
Important features of Naked DNA as a non-viral method of gene therapy
- Simplest method
- Clinical Trials for intramuscular injection of naked DNA with success BUT w/ low expression
Research into more efficient ways:
- electroporation
- gene gun (shoots DNA coated gold particles into cell using high pressure gas)
Synthetic Oligonucleotides as a non-viral method of gene therapy
Methods to inactivate genes involved in the disease process:
1) Uses antisense specific to target gene to disrupt transcription of faulty gene
How mammalian retroviruses have been modified for use as gene therapy vectors
Must disrupt lifecycle Plasmd 1 = remove essential viral genes (gag, pol, env) & replace w/ therapeutic genes
2 => need packaging cell - provide viral genes needed for capsid production & viron maturation (must contain gag,pol,env)
Following insertion of desired gene wt retroviral DNA vector & maintenence of proper packaing lines => must just prepare retroviral vectors
Key features of adenoviruses that make them useful as vectors for human gene therapy
1) Tissue-specific entry
adenoviruses and respiratory tract – cell surface receptors it uses are highly specific to the respiratory tract
2) Relatively safe
We know that people don’t usually die from an adenovirus infection (even children)
US military found that their recruits acquired respiratory diseases when in barracks, so they began routinely vaccinating recruits against adenoviruses, with no reported cancers or deaths from the vaccine.
3) Capacity for large inserts
~ 20% of the genome can be replaced, about 7kb
4) Infection of a variety of cells from different tissues
Limiting factors Adeni virus
1) The wide distribution of CAR receptor BUT loss of CAR receptor in cancer tissues.
Cancer cells lose contact inhibition, cell regulation and interaction. It could also be a selection mechanism for cells that have lost CAR and they would proliferate more actively and eliminate cells that are expressing CAR.
2) Immune response to adenovirus proteins
3) Inflammatory response
Particularly if we are deleting the e3 gene it could be particularly receptive to inflammation
4) Difficulty of re-administration
Adeno-Associated Viral Vectors
Small virus
o Single stranded DNA
o 4.6 kb
Integrates into a specific region of human genome in chromosome 19
o More predictable that retrovirus
Needs to be co-infected with adenovirus for growth
o Can enter is absence of adenovirus and establish latent infection
Infect non-dividing cells
Trials:
Treat muscle and eye diseases
- 117 clinical trials
Advantages of AAV
Non-pathogenic (most people carry this harmless virus)
o In contrast to adenovirus, most people treated with AVV will not build up an immune response to remove the cells
