WEEK 3 - Sickle cell disease, thalassaemia and CRISPR

Question 1

Red Blood Cells
overview

Answer 1

make up one third of all cells in human body

99.9% of haematocrit

give blood its red colour (contains red pigment haemoglobin)

100 rbc : 100 platelets : 1 wbc
(in blood)

Question 2

red blood cells
a.k.a

Answer 2

erythrocytes

Question 3

red blood cells
properties

Answer 3

biconcave disc

large surface area to volume ratio

form stacks like dinner plates through narrow vessels (rouleaux)

bend and flex when entering small capillaries

Question 4

red blood cell
composition

Answer 4

lose most organelles during differentiation

retain only the cytoskeleton

no cell division or protein synthesis

lifespan less than 120 days

obtain energy by anaerobic metabolism of glucose absorbed from the plasma

haemoglobin >95% of intracellular protein (280 million molecules per cell)

Question 5

haemoglobin transports respiratory gases

Answer 5

carbon dioxide can bind to the globin polypeptides, not the heme, changing the conformation and reducing the affinity for oxygen

most of the carbon dioxide travels from tissues to the lungs as bicarbonate ions in the blood

Question 6

haemoglobinopathies

Answer 6

diseases caused by reduced or abnormal synthesis of globin

mutations in globin gene are the most common genetic disorders worldwide, affecting 7% of the world’s population

occur primarily in tropical and sub-tropical areas
- carrier state affords some protection against malaria
–> Malaria parasite cannot proliferate as well in the presence of this mutated haemoglobin

Question 7

globin gene clusters and expression

Answer 7

globin gene clusters
- two different alpha gene on chromosome 16
- only one beta genes in chromosome 11

different globin genes are expressed during different life phases

fetus –> alpha2, gamma2 –> higher affinity for oxygen
haemoglobin is our basis for CRISPR Therapy

Question 8

sickle cell disease

Answer 8

a group of haemoglobin disorders resulting from inheritance of the sickle b-globin gene (Hb S) –> single amino acid change

Question 9

sickle cell disease
what causes Hb S

Answer 9

Hb S is caused by mutation of glutamic acid to valine at position 6 of the globin B chain

Hb S forms insoluble crystals at low oxygen tension

Question 10

sickle cell disease
what do rbcs containing Hb S experience

Answer 10

experience membrane damage and form sickle shapes, blocking blood vessels which lead to organ damage

Question 11

sickle cell disease
symptoms

Answer 11

haemolytic anaemia
- red cells destroyed faster than then can be made

vaso-occlusive crises
- rigid cells block blood vessels

haemolytic crises
- cells can burst

aplastic crises
- bone marrow stops producing red cells (occurs as a result of parvovirus infection)

tiredness, increased susceptibility to infection, and potentially organ failure

Question 12

sickle cell disease
treatments

Answer 12

prevention and treatment of infections

pain relief, hydration for acute crises

blood transfusions

hydroxycarbamide to increase Hb F

new gene therapy

Question 13

sickle cell trait

Answer 13

a genetic condition where a person inherits one sickle cell gene (Hb S) and one normal haemoglobin gene (the mutation is in the beta chain)

Hb S varies from 25-45% of the total haemoglobin

most people affected do not exhibit symptoms of sickle cell disease

in rare cases they might experience complications under extreme conditions
- e.g. severe dehydration, high altitudes, or intense physical activity

this carrier state is found in up to 30% of west African people

Question 14

thalassaemias

Answer 14

heterogeneous group of genetic disorders resulting from reduced alpha- or beta- globin chain expression

Question 15

thalassaemias
three main syndromes:

Answer 15

thalassaemia major
- transfusion-dependent

thalassaemia intermedia
- non-transfusion-dependent

thalassaemia minor
- mild or no anaemia and red cell microcytosis, i.e. smaller red cells

Question 16

a-thalassaemia syndromes

Answer 16

causes by a-globin gene deletion (usually) or mutation

loss of three genes leads to moderately severe microcytic, hypochromic anaemia (cells appear pale due to lack of haemoglobin)

loss of all four genes leads to death in utero (hydrops fetalis)

Question 17

B-thalassaemia
caused by:

Answer 17

typically caused by B-globin gene mutations (over 400 identified)

b-thalassaemia major patients have either no B chain or small amounts are synthesised

Question 17

B-thalassaemia
symptoms

Answer 17

excess unpaired a-chains precipitate resulting in ineffective erythropoiesis and chronic haemolysis
–> because Not enough b chains for a chains to pair with

b thalassaemia major is associated with transfusion-dependent anaemia and iron overload (body accumulates too much iron) with liver, endocrine and cardiac damage

tiredness, increased susceptibility to infection and potential organ failure

Question 18

treatments for b-thalassaemia

Answer 18

b-thalassaemia major
- regular blood transfusions
- iron chelation therapy (To reduce iron overload that happens in the body) has greatly increased life expectancy

b-thalassaemia trait (minor)
- usually symptomless

Question 19

CRISPR-Cas9
what

Answer 19

emerged in 2013

Cas9 is an RNA-guided nuclease from the CRISPR bacterial adaptive immune system

Cas 9 links up with guide RNA
This guide RNA directs where Cas 9 opens up the sequence
if match Cas cuts both strands of DNA
- see slide for diagram

Can induce the cell to introduce or modify genes

Question 20

CRISPR-Cas9 genome editing

Answer 20

1. sgRNA (single guide RNA)
2. sgRNA + Cas9 protein
   Cas9 is a bacterial endonuclease
   stops at every PAM (protospacer adjacent motif) and looks for an exact match to the guide RNA
3. target specific cleavage
4. cellular error-prone repair “knocks out” gene
   (non-homologous end joining)

Question 21

CRISPR-Cas9 genome editing
generation of cell line KOs in the lab

Answer 21

chicken B actin promoter drives expression of Cas9
U6 RNA polymerase III promoter drives expression of small guide RNA

1. introduce sgRNA into plasmid vector using standard DNA cloning
2. transfect plasmid into cultured cells
3. select cells in which the target gene is deleted
4. test the function of the targeted cells

Question 22

CRISPR-Cas9 genome editing
specific genome changes can be made by…

Answer 22

providing a repair template

Cas9 + gRNA –> complex fromation and target binding (Target+PAM)
–> target cleavage (DSV formation

two options:
non homologous end joining
WT, insertion, deletion, frameshift
OR
homology directed repair
repair template with homology arms desired genomic edit and PAM mutation (So cell includes the wanted mutation when repairing)

Question 23

CRIAPR-based treatment for haemoglobinopathies
what

Answer 23

casgevy mechanism of action to treat sickle cell disease and B-thalassaemia major

BCL11A shuts down production of Hb F
So in theory if stop this action of BCL11A (knock it out) this will cause in increase of production of Hb F

BCL11A knocked out of genome using CRISPR therapy
- therefore have enough rbc to alleviate symptoms of sickle cell anemia

Question 24

Casvegy - mechanism summary

Answer 24

the first licensed therapy to utilise CRISPR/Cas9

deletes the BCL11A gene in haematopoietic stem cells (harvested from patients’ blood) to increase expression of fetal y-globin, thus compensating for loss of adult B-globin in sickle cell and B-thalassaemia diseases

the gene edited haematopoietic stem cells are transfused back into the patient after the patient had first received chemotherapy to destroy the diseased bone marrow

Question 25

Casvegy - ethical considerations

Answer 25

the cost $2.2 million per patient

patients have marked reductions in vaso-occlusive events (The blockage of blood vessels), but the efficacy and safety of the treatment beyond 1-2 years is unknown

serious adverse effects are related to the bone marrow ablation
e.g. low platelet and white blood cell counts
- Patients have to undergo chemo before the transfusion because need to ablate the bone marrow
Serious adverse consequences and patients have to stay in hospital during