Gene therapy and RNA therapeutics 2 Flashcards
What is the new technology that have been developed?
CRISPR/Cas9
Targets specific sites in the genome and allows a double strand break.
Thus gene modification can happen during repair processes
Potential to correct disease associated mutations (restoration of faulty genes)
What is CRISPR/Cas9
Prokaryotic immune system
Repurposed to allow site-specific genome modification.
Fleixible and easy to use.
Limitations
-Off target effects
-Less efficient at engineering in a precise nucleotide sequence change than introducing a non-specific deletion.
CRISPR adaptation for genome modification.
Two bacterial RNAs combined to give single guide RNA which targets sequence of interest
Cas9 (protein) is also expressed in cells with the endonuclease (cuts with in nucleic acid DNA molecueles-causes DS breaks)
NEED GUIDE RNA AND CAS9 for the system to work
sgRNA and Cas9 protein
Cas9 causes DS break into DNA when recruited by sgRNA
sgRNA is single guide RNA which has the ability to interact with the target DNA seq and Cas9
Yellow bit is has precise complemntary to the DNA where you want to target.
Green bit recognised by Cas9
PAM is 3 nucleotides at the end of guide RNA and cuts each strand of the DNA
How is it used to edit genes?
DSB caused by Cas9 cleavage
Thus this causes repairing due to the cell not be used to this.
Non-homologous end joining (NHEJ)
Small insertions or deletions
But causes errors, effcient imprecise
Homolgy directed repair (HDR)
Have to have a template matching with the cleaving site, and the desired mutation included
Mutation then incorporated as dsDNA break is repaired
Allows precise mutations but not very efficient
Expensive
When was the first effective CRISPR?
2020
This was used for beta-thalassemia and sickle cell disease.
Targets BCL11 leading to increased production of foetal haemoglobin, which replaces the faulty haemoglobin beta.
This just requires gene disruption which is NHEJ method instead of HDR mediated repair.
Ex vivo approach- relies on bone marrow ablation (high risk of infection) and transplant, requires autologus one which means it has to go back into the patient mutated.
They need to be tested and screened to see if they have been correctly edited.
Is tricky to deliver
How do we deliver genome editing?
saCas9 mRNA is small so can be encoded in a single AAV vector with gRNA.
Potentially a template if HDR is required.
Lipid nanoparticles is also a possibilty.
Ethical considerations of CRISPR?
- You are modifying someones genome.
- Genome mod: somatic (any other cells, such as bone marrow and wont be passed down) versus germline (cells to make next gen like egg/sperm so will be passed on)
Germ line doesnt really happen, reproduction has not happened yet due to lack of undertsanding.
Not practical:
High chance of off target effects
Inefficient
Disease causing mutations can already be avoided by preimplantation genetic diagnosis (IVF babies made, screened for without the disease carrying gene and then that embryo is used)
CRISPR gernline editing scandal
Edited the germline of two twin girls
Disrupted CCR5, with the aim of preventing HIV transmission form infected father.
Unregulated activity
Other consequences of CCR5 deletion
Can be avoided anyway with anti-retroviral drugs.
3 years in prison.
Antisense oligonucleotides.
Short nucleotide sequenvces thats is complementary to a sequence of mRNA and ilict a function.
Can cause steric block which prevents protein or RNA from binding to the site.
Induces RNA degradation by RNAse H recruitment.
Modifications to chain can improve RNA binding and have an impact what method it uses which is above.
How is antisense tech used to modulate gene expression?
Designed to mask a sequence that is recognised by a spicling factor
This in turn causes promotion of exon inclusiom or exon skipping.
RNase H
Cleaves RNA at RNA-DNA hybrids (only recognises sites where RNA-DNA are complemntary paired)
Gapmer ASOs, has DNA in a gap via LNA, strong binding to sequence which binds to DNA and then allows formation of RNA-DNA.
Therapeutic modulation of splicing by ASO:steric block
Many diseases associated genes contain multiple exons and intrins.
They are very gene specific, bind to splice sites or enhancers.
Spinal muscular atrophy
What is SMA?
This is the most common genetic cause of death in babies.
Caused by autosomal recessive mutaion in SMN1
Insufficient SMN protein production leads to loss of spinal motor neurons and gradual paralysis.
SMN2 gene is identical, so restore SMN2 so the baby with mutant SMN1 is make more protein from SMN2 which compensates for defct in SMN1.
Done via **steric block ASO **
Where does the oligonucleotide target for SNM2?
Targets hnRNP responsible for splicing on exon 7
