Gene therapy and RNA therapeutics 1 Flashcards
What is gene therapy?
Delivery of genetic material into a patinets cells to treat diseases which ar asscoiated with genetic mutation and chnages in gene expression
Ethical considerations
You are actually changing someones genome which can be a major ethical issue.
Basics and undertanding
DNA (stores genetic information)-double stranded which is copied to form RNA.(Transcriptions)
RNA then undrgoes translation to form a protein.
Human gene expression: transciption and translation.
Introns and exons
Somewhere there will be a promoter and this is where transcriptions factors start.
Also have enhancers which help strong transcription of paticular genes.
Exons shorter and introns longer.
When transcription is occuring a cap gets added on to 5’ end which stabilises mRNA and helps translation.
When transcription is occuring splicing happens which removes introns.
Polyadenylation also takes place where transcription terminates at the last exon.
This forms mRNA which you have a stop and a start codon 5’ end to 3’ end, bit between two codons is the coding sequenecr that add on amino acids to add on the proteins via connecting to codons. which is translation.
How can a gene mutation lead to disease? 1
Direct mutation in the coding sequence that incorporates the wrong amino acid.
This affects splicing, how much mRNA is made, untranslated regions impacts translation or mRNA stability.
This happens all the time, but in this case this swap of positions is da
How can a gene mutation lead to disease? SCD
Mutation in coding seq of DNA chnage samino acid seq of protien affecting production.
For example in SCD(sickle) which is an autosomal recessive disease causeed by mutations to both copies of beta-globin gene.
Haemoglobin S forms instead of haemoglobin A Leads to RBCs forming sickle cell shapes- anemia, pain and reduced life span.
Muation of GAG to GTG in coding, leads to glutamic acid being replaced with valine at position 6.
Del/sub,premature termination.
Other mutations
Deletion or subsit can occur.
Leads to incorrect sequence downstream.
Known as a FRAMESHIFT muatation, wouldnt happennin multiples of 3.
Premature termination codon, changes Arg into stop codonm (stops making proteins at this point/ downstream pathway affetcted) which can lead to a trunctuated protein.
Mutations in the promoter
Deletion in DNA sequence can remove binding sites from a transcription factor that would activate transcription of gene which will make less protein.
What diseases can be treated with gene therapies>
Single gene disorders (cystic fibrosis)
Infectious diseases (HIV)
Cancer: multiple genetic chnages required, increased activity of oncogene products.
You can make the immune system more effective by the use of gene therapy.
Glybera
How it works and must work/challenges?
Transfer of genetic info into cells.
Must be able to targte correct cells and act for the correct amount of time.
Glybera treats rare metabolic disease that causes pancreatitis.
Challenges
* Delivery to correct target cell
* Maintence of delivered gene in target cell
* High cost of drugs development
* Only suitable for diseases caused by reduced production of protein product
* Cant recieve a precise level of expression of delivered gene, only practical if expression level doesnt matter too much
Ex vivo
Take target cells out of patinets
Add gene and return to patient
You can also screen the genes to see if is the right one, but not suitable for most cells in our body, anything outsie of the blood it would have to be via bone marrow and stem cells.
In-vivo
Introduce gene directly into the patient by method appropriate target tissue (aerosol delivery in lungs)
Viral vs non-viral
Viral
* Deliver genetic material into host cells.
* Modify viral genome and include gene of interest.
* Highly selective, efficient uptake, limit of size to incorporate, immune response, can persit in cells
Non-viral
* Naked DS DNA which cn have nanoparticles
* Less efficient, hard specifity, larger DNA delivery, low immunogenicity, transient delivery.
Retroviruses
Virus encodes reverse trasncriptase which copies RNA to DNA which is then intergrayed into host genome.
Mutates the virus to remove all pathogenic elements, just leaving the genes necessary to reverse transcribe and intergrate DNA.
It allows incorporation of delivered gene into host genome and thus maintentennace during cell division.
Cant control site of integration (Insertional mutagenis) Could interact with a tumour promoter and cause cancer.
Viral vector for gene delivery.
Ability to attach to and enter target cell.
Transport of genetic material to the nucleys and maintenance as DNA
Lack of toxicity and immunigenicty
Lentiviruses
Highly modded versisons of viruses such as HIV
Similar to retro
Integrates into non-dividing cells
Modded for less risk of insertional mutagenesis and lower immunogenicity.
AAV
Low immunogrnicity but has been a problem in recent trials.
Infects dividing and non cells
Needs helper virus to replicate
Make own separte DNA which is separate to the genome, less risk of insert-muta
But when rapidly dividing you can lose DNA.
Higher specifity but quite small, so not much gene can be inserted.
CAR-T immunotherapy
Take T cells from patient and produce chimeric antigen receptors which recognise antigens on canver
Return cells to patient where they multiply and induce cell death.
Can develop CAR for specific tumours but can cause cytokine release syndrome
Solid tumours are harder to target
Gene therapy in retina
Injected into the retina
Hughly expensive over £600K
Haemophilia
Blood clotting disorders
X-linked to recessive inhertiance
Phase 1/2 clinical trials for haemophilia B led to 9/10 patients having no bleeds over 18 months
Hepatocytes were damaged in patients but was treated with steroids
Has been approved by the FDA, highly effective but again expensive.
mRNA therapeutics
Onky needs to reach the cytoplasm, immediatley translated to protein.
This transient delivery is good (short term delivery)
Rapidly developed.
Acoid immune response and mRNA needs to be stable
VEGF probably the most advanced
Covid-19 was an example of this.
