LMP299Y1 Lecture Notes - Viral Vector, Ex Vivo, Liposome

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Actual gene therapy use a gene as a therapeutic in genetic disease or acquired genetic disease
Introducing gene into somatic cells for treatment
Correct the gene directing therapy at the ROOT cause
Duration of effect for genetic disease, long-term effect
No other conventional treatment
To achieve gene transfer generally, two roots one is the idea of ex vivo gene therapy; other is in vivo
In vivo directly insert the gene into the individual; hope that approach gets to the right kinds of cells
- Good idea, difficult to accomplish
Ex vivo gene therapy good delivery vehicle ex. Exploit viruses
Target gene has optimal gene expression
What is best target cell? Turnover best target are stem cells, that can give rise to multiple other cells
Do not take tissue samples repeatedly from patient not desired
These issues apply to all types of gene therapy, all strategies
Most used method of delivery type of viral vector multiple kinds idea is that vector takes
advantage of the very efficient gene transfer that tends to happen with viral infection
Oncoretroviral vectors viruses that are RNA viruses to get DNA, transfer RNA into DNA, using reverse
Adenovirus turn down gene expression so gives short term expression; DNA does not integrate (good
thing) but limited term of effect
Adenoviruses large viruses produce lots of proteins unwanted immune responses are concerns
Gutless vectors less immunogenic large payload
Adeno-associated virus vectors element of safety
Herpes simplex can infect central nervous system establish latent infection interest property
have good insert capacity do not integrate some notable ability for long-term expression
Integration is an issue if the integrated virus does not stay put; they may be able to move about, using
transposition machinery of the cell
Nonviral vectors
Developed primarily to get around concern of immune response of individuals
Receptor-mediated endocytosis DNA coupled to targeting molecule, which can bind to cell receptor so
cell may uptake the DNA
Direct injection gene gun technique may get local injury
Liposome smallest representation of what can fuse with the cell membrane synthetic lipid bilayer
that is cell compatible stimulate to fuse with membrane variety of liposome that bind negatively
charged DNA, package nicely around DNA fairly easily can get into cells simply because have
property that is similar to cell membrane calcium, stress, to allow lipids to fuse quite effective
Liposomes work best for liver gene therapry, but not as effective for other organs
Liposomes artificial viruses
Rationale for gene therapy approach
Recessive more than 50% loss of gene product
Dominant haplosufficient boost with more
Nature of mutation and disease features
Approaches to gene therapy take particular gene put in disease cell increase in gene product for
recessive disease, where there is loss of function
Variations on idea of adding gene and giving function ex, treatment for cancer
Add to disease cell that is toxic gives off toxin that causes cells to die toxic genes
Or, add prodrug metabolizing gene can metabolize product that once you add it some control about
so control toxicity one such drug some drugs developed to combat retroviruses what genes there
are some genes metabolized killing at particular time to produce toxic product that kills cells
How to target just the diseased cells? For example, cancer cells grow much rapidly than neighbouring
normal cells exploited to use to target healthy cells idea of these types of methods
Other types of gene therapy can we teach our immune system to work better?
Can add foreign gene that makes the cell more sensitive to immune system and attracts an immune
More theoretical approaches:
How do you dispose of mutant gene that gives rise to harmful product?
Gene expression inhibition variety of antisense type oligonucleotide siRNA type this should be
fairly gene specific event have opportunity to be quite gene specific make antisense that targets only
undesirable product gained interest because can be gene specific variety of approaches where they
would come to do ways around that do antisense RNA used to change the splicing of a particular
gene product to get rid of something detrimental
Gene therapy in somatic cells only
Gene therapy in stem cells have issues with respect to ethics
Some successes for treatment of inherited diseases
Administration of ADA disease of purine metabolism immunological disorder try to provide this
gene patients who got gene therapy got ADA gene replacement therapy have to give standard of
care to the patients
Example 2
SCID patients detected when child small cannot sustain any kind of infections immune systems so
This treatment only if no other options
Believed that if both LMO2 and IL2RG activated, tendency to leukemia bizzare finding because thought
that retroviral integration was random NOT RANDOM integrate into open regions of chromatin, that
are transcribed chance that gave such high viral dose that hit many genes in the genome and
consequently, the integration event was selected for by the hematopoietic system
This leukemia not seen in the ADA patients, even using the same virus
Some successes for treatment of inherited disease
Not invasive cells can hone back into bone marrow comparable to bone marrow transplant
Example 3 gene therapy for eye diseases immune response in eye almost no immune response
there - some interesting success cases with patients treating them gene to restore sight or vision
restore low levels quite helpful and encouraging for the patient, gives benefits other examples
where have seen successful gene therapy rare, not many examples
Example 4 X-linked ADL example 5 almost immune type disease
Other examples where retroviral vectors were used some issues with silencing of gene mixed success
One example of using the idea can you develop a strategy due to toxicity due to gene? Duchenne
Muscular Dystrophy this gene has portion of repeating units multiple exons - can have restored
function if take out a few units build antisense oligo use RNA molecule that had- that was very
stable, mimic to oligo, hybridizes to RNA make antisense oligo, block splicing of the particular exon
some interesting success
Dystrophin is a gene that sits on surface of muscle green is where gene should be; in the DMD patient,
no dystrophin see restoration
Strategies TBC