WEEK 3 - Sickle cell disease, thalassaemia and CRISPR Flashcards
Red Blood Cells
overview
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)
red blood cells
a.k.a
erythrocytes
red blood cells
properties
biconcave disc
large surface area to volume ratio
form stacks like dinner plates through narrow vessels (rouleaux)
bend and flex when entering small capillaries
red blood cell
composition
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)
haemoglobin transports respiratory gases
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
haemoglobinopathies
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
globin gene clusters and expression
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
sickle cell disease
a group of haemoglobin disorders resulting from inheritance of the sickle b-globin gene (Hb S) –> single amino acid change
sickle cell disease
what causes Hb S
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
sickle cell disease
what do rbcs containing Hb S experience
experience membrane damage and form sickle shapes, blocking blood vessels which lead to organ damage
sickle cell disease
symptoms
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
sickle cell disease
treatments
prevention and treatment of infections
pain relief, hydration for acute crises
blood transfusions
hydroxycarbamide to increase Hb F
new gene therapy
sickle cell trait
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
thalassaemias
heterogeneous group of genetic disorders resulting from reduced alpha- or beta- globin chain expression
thalassaemias
three main syndromes:
thalassaemia major
- transfusion-dependent
thalassaemia intermedia
- non-transfusion-dependent
thalassaemia minor
- mild or no anaemia and red cell microcytosis, i.e. smaller red cells
a-thalassaemia syndromes
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)
B-thalassaemia
caused by:
typically caused by B-globin gene mutations (over 400 identified)
b-thalassaemia major patients have either no B chain or small amounts are synthesised
B-thalassaemia
symptoms
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
treatments for b-thalassaemia
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
CRISPR-Cas9
what
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
CRISPR-Cas9 genome editing
- sgRNA (single guide RNA)
- 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 - target specific cleavage
- cellular error-prone repair “knocks out” gene
(non-homologous end joining)
CRISPR-Cas9 genome editing
generation of cell line KOs in the lab
chicken B actin promoter drives expression of Cas9
U6 RNA polymerase III promoter drives expression of small guide RNA
- introduce sgRNA into plasmid vector using standard DNA cloning
- transfect plasmid into cultured cells
- select cells in which the target gene is deleted
- test the function of the targeted cells
CRISPR-Cas9 genome editing
specific genome changes can be made by…
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)
CRIAPR-based treatment for haemoglobinopathies
what
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
