Ferrari: Lecture XV Flashcards
Genome Editing: BTHAL and Sickle Cell Disease
What causes sickle cell disease (anemia)?
single point mutation of the β-globin gene
*β-thalassemia is a mutation of the β-globin gene
What does the point mutation in sickle cell disease do?
substitues glutamic acid with a valine in position 6, which gives rise to a mutant globin chain called HbS (S stands for sickle)
Describe the pahthophyiology of HbS
during deoxygenation, HbS forms fibers in a process called polymerization
the fibers are toxic and lead to the erythrocytes becoming dehydrated and assume an elongated sickle shape
What are the clinical symptoms of sickle cell disease?
haemolytic anemia (not severe)
vaso-occlusion (very painful, patients go to the ER and treated with opiods)
disease is associated with morbidity and mortality
List the pathway of sickle cell disease:
HbS is polymerized
RBC are sickled
vaso-occlusion
haemolysis is possible, which leads to heme group being released → induces endothelial dysfunction → cytokines are released → inflamed microenvironment → sterile inflammation
What is the epidemiology of sickle cell disease?
most common genetic disorder (almost 300,000 newborns are born with this condition)
What is a highly debated hypothesis of sickle cell disease’s correlation with malaria?
the mutation protects from malaria infection
What is the life expectancy of people with anemia?
less than 30 years
What does hemolytic anemia do? and leads to?
blocks small blood vessels leads to vaso-occlusion
What is acute stress syndrome?
symptom of anemia which requires immediate hospitalization
What are chronic complication associated with sickle cell disease?
general vasculopathy and progressive ischemic organ damage
What are the treatments available for sickle cell disease?
blood transfusions during vaso-occlusive crisis to dilate HbS with new blood and bone marrow transplantations
Why is it hard to find donors for sickle cell disease?
Majority of patients are not Caucasian so there is different major histocompatibility complexes (MHC) so it is more dificult to find suitable donors
also ethical/religious reasons can prevent bone marrow donation
Is there a cure for sickle cell disease?
no, there is not a pharmacological or definitive cure
What is the HPFH condition?
hereditary persistence of fetal haemoglobin
Why is it important to understand hereditary persistence of fetal hemoglobin?
it is important to understand gene editing for sickle cell disease or β-thalassemia
What is HPFH condition associated with?
high level of fetal hemoglobin in adults
What is the molecular cause of the reactivation of HbF?
point mutations in the HGB gene or locus (promoter of 𝜸 globin) → non-deletional HPFH: disruption of repressor binding site
deletions in the HBG gene or locus downstream the 𝜸 globin gene → deletion of HPFH: deletion of repressor binding sites
How do we correct defective β-globin gene cells?
homology direct repair (HDR), which only has 20%-40% efficiency if I am very good
How does BCL11A silence 𝜸-genes?
forms a complex with GATA1 (activator), FOG1 (activator), and NuRD complex 9repressor complex) to bind to DNA and have a negative effect on 𝜸-gene promoters → repression
List the 4 techniques that can be used to induce HPFH (treat β-hemoglobinopathies):
targeting the BCL11A erythroid enhancer
introducing non-deletional HPFH
introducing deletional HPFH
gene repair
Describe targeting the BCL11A erythroid enhancer:
done with zinc finger, Cas9…
enhancer is deleted to obtain less BCL11A expression → 𝜸-gene expression is reactivated
Describe introduction of non-deletional HPFH:
mutations are created on binding sites of the promoters of 𝜸-genes so the repressors do not bind
Describe the introduction of deletional HPFH:
zinc fingers, Cas9, NHEJ delete repressor binding sites, resulting in big deletions
Describe gene repair:
NHEJ is used (high efficiency) to introduce the repair of the mutated gene through HDR
*limitation is DR occurs at a low frequency
