THERAPEUTIC GENE EDITING WEEK 1 Flashcards
What does gene modification involve?
- Introduction of ds breaks in DNA via NHEJ or HR
What occurs in NHEJ to introduce double stranded breaks?
- The formation of INDELS (insertions/deletions of 5-20 nucleotides)
- The reading frame can be disrupted and it is useful if you have to get rid of a pathogenic gene
What occurs in HR (homologous recombination) to introduce ds breaks?
- More elegant method
- Template DNA added with homologous arms (must have homologous regions flanking the DNA
- Can have variable sequences (mutations or insertions) in between.
What are the three different types of therapeutic gene editing?
- Gene dsiruption (Silence a pathogenic gene)
- NHEJ correction ( Deletion of a pathogenic insertion)
- HDR (homology directed repair) –> correction of a deleterious insertion
What is the first mechanism by which we need to modify genes and incorporate desired changes?
- Make ds breaks
What are the three types of site directed nucleases used to target specific genes for ds breaks?
- Zinc Finger nucleases
- TALENs (Transcrption Activator Like effector nucleases)
- CRISPR/Cas9 (RNA guided engineered nucleases.
What is involved in Zinc Finger Nucleases being used to target the specific genes for ds breaks?
- They are protein modules (zinc fingers fused together) that BIND 3 nucleotides–> bring Fok1 nuclease to site
- this then turns into a dimer and becomes active to cut the DNA
What is involved in TALENs? (i.e. what are they)
- Protein modules that bind the INDIVIDUAL nucleotides and bring Fok1 nuclease to the site (dimer becomes active)
- This process is easier than ZFNs
What is involved in the recognition of RNA guided engineered nucleases?
- RNA guided engineered nucleases (CRISPR/Cas9) binds NUCLEOTIDES and targets Cas9 to site
Why is it good that the RNA molecules binds the nucleotides and targets the Cas9 to the site?
- Because it is not dependent on proteins binding to the DNA –> it is only dependent on the RNA
What are the advantages of CRISPR/Cas9 compared to TALENs and ZFNs?
- CRISPR/Cas9 has DNA binding by classic base pair rules (not protein to DNA interactions)
- Making a custom gRNA used by CRISPR is MUCH easier, cheaper, quicker (just simple mol bio)
What are the details of the CRISPR/Cas9 complex?
- It has a PAM sequence and guide RNA which allows for recognition -
- The sequence must ALREADY have a PAM site
What are two ways that guide RNA can be incorporated into cells?
- Can be PREMADE and injected OR incorporate DNA encoding these genes
What is genome editing used for? (4 things)
- Basic research: Making cell and animal models (knocking out/modifying genes)
- Screens to identify new therapeutic targets
- Gene drives
- Potential therapeutics to treat disease
What is CCR5 and what does it have to do with HIV infection?
- A T cell co receptor for HIV-1
- Some individuals have a loss of 32aa in CCR5 making them resistant to HIV infection?
What was the use of ZFNs in patients with HIV?
- Caused the CCR5 gene to be dyfunctional in CD4 T cells that were injected into patients
- It was safe and the progression of HIV slowed in patients with elevated T cell levels (bc. virus couldn’t destroy those T cells)
How are TALENs involved in B cell acute lymphoblastic leukemia therapy?
- Girl with leukemia in remission after TALENs were used to allow T cells to express CAR (Chimeric Antigen Receptor) against a B cell antigen CD19
Does having fetal haemoglobin protect against sickle cell disease?
- YES
- Some people with asymptomatic sickle cell disease have INHERITED fetal persistence (benign phenotype_
What causes HPFH?
- Large deletions in beta globin gene cluster or pt mutations of gamma-globin genes –> person will have HIGH levels of Hb throughout life
What is it about the fetal hb that is so protective?
- The gamma chains have a LESS hydrophobic patch for making LATERAL inrteractions with Val6 of the beta-subunit
- The aim is to turn fetal hb on in sickle cell people
What is BCL11A?
- A TF that encodes a ZF TF which is a -ve regulator of gamma globin gene expression
What happens in patients with BCL11A haploinsufficiency?
- They have a HPFH phenotpye (fetal hb persistance)
- BCL11A has an enhancer in an intron that drives its expression in erythroid cells –> so it can be used to increase fetal haemoglobin in people with sicke cell anaemia.
what is an ex vivo approach genetic therapy for sickle cell disease?
- Isolation of haematopoetic stem cells (CD34+) from patietns
- The genome tech. disrupts the BCL11A enhancer –> then autologous RE-INFUSION of modified cells
How can the size of AAVs be overcome to package Cas9?
-The use of inteins –> split Cas9 to package into AAVs
What are inteins?
- Protein introns that splice out autocatalytically from host polypeptides to generate functional protein
How is Cas9 packaged into AAVs?
- N terminal and C terminal half of Cas9 packages into two AAVs
- When these are expressed together, inteins undergo Post Translational autocatalytic excision while LIGATING the two Cas9 portions together.
- N and C intein come together, knock off, and allow Cas9 to be joined to make a functional Cas9.
e. g. DMD in pigs
