THERAPEUTIC GENE EDITING WEEK 1

Question 1

What does gene modification involve?

Answer 1

• Introduction of ds breaks in DNA via NHEJ or HR

Question 2

What occurs in NHEJ to introduce double stranded breaks?

Answer 2

• 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

Question 3

What occurs in HR (homologous recombination) to introduce ds breaks?

Answer 3

• 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.

Question 4

What are the three different types of therapeutic gene editing?

Answer 4

1. Gene dsiruption (Silence a pathogenic gene)
2. NHEJ correction ( Deletion of a pathogenic insertion)
3. HDR (homology directed repair) –> correction of a deleterious insertion

Question 5

What is the first mechanism by which we need to modify genes and incorporate desired changes?

Answer 5

• Make ds breaks

Question 6

What are the three types of site directed nucleases used to target specific genes for ds breaks?

Answer 6

• Zinc Finger nucleases
• TALENs (Transcrption Activator Like effector nucleases)
• CRISPR/Cas9 (RNA guided engineered nucleases.

Question 7

What is involved in Zinc Finger Nucleases being used to target the specific genes for ds breaks?

Answer 7

• 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

Question 8

What is involved in TALENs? (i.e. what are they)

Answer 8

• Protein modules that bind the INDIVIDUAL nucleotides and bring Fok1 nuclease to the site (dimer becomes active)
• This process is easier than ZFNs

Question 9

What is involved in the recognition of RNA guided engineered nucleases?

Answer 9

• RNA guided engineered nucleases (CRISPR/Cas9) binds NUCLEOTIDES and targets Cas9 to site

Question 10

Why is it good that the RNA molecules binds the nucleotides and targets the Cas9 to the site?

Answer 10

• Because it is not dependent on proteins binding to the DNA –> it is only dependent on the RNA

Question 11

What are the advantages of CRISPR/Cas9 compared to TALENs and ZFNs?

Answer 11

• 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)

Question 12

What are the details of the CRISPR/Cas9 complex?

Answer 12

• It has a PAM sequence and guide RNA which allows for recognition -
• The sequence must ALREADY have a PAM site

Question 13

What are two ways that guide RNA can be incorporated into cells?

Answer 13

• Can be PREMADE and injected OR incorporate DNA encoding these genes

Question 14

What is genome editing used for? (4 things)

Answer 14

• Basic research: Making cell and animal models (knocking out/modifying genes)
• Screens to identify new therapeutic targets
• Gene drives
• Potential therapeutics to treat disease

Question 15

What is CCR5 and what does it have to do with HIV infection?

Answer 15

• A T cell co receptor for HIV-1

- Some individuals have a loss of 32aa in CCR5 making them resistant to HIV infection?

Question 16

What was the use of ZFNs in patients with HIV?

Answer 16

• 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)

Question 17

How are TALENs involved in B cell acute lymphoblastic leukemia therapy?

Answer 17

• 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

Question 18

Does having fetal haemoglobin protect against sickle cell disease?

Answer 18

• YES

- Some people with asymptomatic sickle cell disease have INHERITED fetal persistence (benign phenotype_

Question 19

What causes HPFH?

Answer 19

• Large deletions in beta globin gene cluster or pt mutations of gamma-globin genes –> person will have HIGH levels of Hb throughout life

Question 20

What is it about the fetal hb that is so protective?

Answer 20

• 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

Question 21

What is BCL11A?

Answer 21

• A TF that encodes a ZF TF which is a -ve regulator of gamma globin gene expression

Question 22

What happens in patients with BCL11A haploinsufficiency?

Answer 22

• 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.

Question 23

what is an ex vivo approach genetic therapy for sickle cell disease?

Answer 23

• Isolation of haematopoetic stem cells (CD34+) from patietns
• The genome tech. disrupts the BCL11A enhancer –> then autologous RE-INFUSION of modified cells

Question 24

How can the size of AAVs be overcome to package Cas9?

Answer 24

-The use of inteins –> split Cas9 to package into AAVs

Question 25

What are inteins?

Answer 25

• Protein introns that splice out autocatalytically from host polypeptides to generate functional protein

Question 26

How is Cas9 packaged into AAVs?

Answer 26

• 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