Gene Therapeutics and Gene Therapy Flashcards
What are the two general approaches to gene therapy?
1) Gene supplementation (augmentation) a 2) Gene inhibition - silencing genes
What is the ethical issue surrounding germ line gene therapy?
Genetic alterations are heritable - therefore you could potentially be changing the evolution of the human species if you change the genetics - Though some may argue that you could potentially eliminate genetic diseases entirely
How can we incorporate a gene into the cell (two general ways)
Stable - integrated into the host cell chromosome transferred to daughter cells Transient - remains episcopal - separate from host cell chromosome- either degraded or expelled more quickly
explain the difference between integrated vs. episomal expression
An integrated gene actually incorporates itself into a genome and is therefore still present upon cell replication Episomal genes do not integrate into gene itself, but remains in the nucleus
In vivo vs. Ex vivo?
In vivo = directly transmitted into patient Ex vivo= cells removed from patient, manipulated in vitro and subsequently transplanted back into patient
Describe gene silencing by antisense
prevents translation of mRNA by speeding up RNA degradation - it does so by binding an oligonucleotide to an mRNA sequence - therefore making it a double stranded RNA- oligonucleotide hybrid- which is then degraded naturally by RNase H degradation
Describe Gene silencing by Ribozymes
We use RNA molecules to function as enzymes- we make an RNA sequence that will bind to a specific sequence and it will act as an enzyme to degrade the complementary sequence of an mRNA molecule.
Describe gene silencing by siRNA interference
siRNA pathway - *under normal conditions* when a cell detects double stranded RNA (typical in a viral infection) it targets this for destruction by Dicer. Dicer cleaves them and the short fragents get put into a series of proteins that make up a ‘RISC’ complex - where an antisense strand is encorporated into the proteins and finds complementarity in the RNA sequence - then the antisense strand is degraded by the enzyme and the sense strand is naturally degraded in the cytoplasm
What we can do is make synthetic fragment RNAs (siRNA) which are then inducing the breakdown of these mRNAs
Describe the three common mechanisms of post transcriptional gene silencing
all block protein synthesis (unlike normal drugs that target proteins post synthesis)
What is a gene vector?
It is a gene delivery stem used to deliver gene therapy to a patient
can be viral or non-viral
How do we choose a gene vector for a particular disease?
Consider the following
- how efficient is the drug vector? (viruses are very effective)
- is it cell specific? (viruses tend to target all cells)
- is it able to enter non-dividing cells?
- Can it cause persistent expression?
- Can it selectively integrate within the host genome?
- what is its toxicity or immune response profile? (could the virus vector cause virulence down the road? Or cross react with latent viruses? Could it cause an immune response?)
- Is it easily processed and quality controlled?
How do we alter viral vectors to make them safe?
We replace their virulence and replication genes with therapeutic genes
What is a retrovirus?
Retroviruses are RNA packed within viral proteins which is reverse transcribed into DNA
What are the advantages and Disadvantages of using retroviruses as vectors?
Advantages= stable expression and complete integration into host cell DNA, capable of entering most cells in the body
Disadvantages= non-specific integration into genome, and these viruses tend to (intrinsically) insert into the promotor regions and can upregulate oncogenes, cannot infect non-dividing cells, and can only accommodate up to 8kb of DNA
What disease have we ‘successfully’ treated with a retrovirus gene therapy?
Severe combined immunodeficiency Disease (SCID - X1)
patients with this disease lack the gamma C interleukin receptor protein which is critical for T, B and naturall killer cell development therefore they are significantly immune compromised
-scientists introduced retroviral vectors encoding their missing gene into their own cells and infused them back into the patients. The patients began to express Gamma C and thus the T,B, and NK cell counts were growing
But 3 years later - 2/9 patients developed leukaemia like disorder due to the placemnt of retrovirals into promotor reginos of the cell (mutagen)
