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 were the repressors of the 𝜸-globin gene identified?
using GWAS (genome-wide association studies) by which the correlation of SNPs (single-nucleotide polymorphisms) has been highlighted
What are the 3 regions that were identified as associated with high levels of HbF?
intergenic region between HBS1L and MYB on chr 6
β-locus: reactivates 𝜸-globin
erythroid enhancer on the promoter of BCL11A gene on chr 2:erythroid form of BCL11A is a silencer for the 𝜸-genes
What can silence 𝜸-genes?
BCL11A
LFR
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
Can 𝜸-gene reactivation be induced?
yes by silencing the 𝜸-gene repressors (BCL11A or LRF)
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
Why can HbF be therapeutic?
it has anti-sickling properties, and it increases the total levels of hemoglobin in β-thalassemia patients (not SCD)
HbF impedes the ____ of HbS → ____
polymerization
anti-sickling molecule
Describe the therapeutic approach for β-thalassemia:
!!
presence of HbF is induced → no polymerization → sickling of cells is prevented → HbS is diluted → formation of polymers is less → cells have both HbS and HbS → HbF repels HbS naturally
What was an advantage with the experiment using CRISPR/Cas9 β-globin gene targeting in HPSC?
it had the 1st use of ribonuclear particle (Rnp) was used, which allowed for a higher efficiency in indels (of cutting) → higher eficiency in tageting
BCL11A is an ____
enhancer disruptor
What is important when using zinc fingers?
detection of off-target sites because the system is very specific and does not cut undesired regions
BCL11A is an ____ ____ enhancer.
erythroid specific
If the ___ ___ is eliminated, there will not be expression of BCL11A in ALL the cells.
coding sequence
If we target the specific erythroid enhancer sequence, ___ is deleted only from erythroid cells.
BCL11A
What does a mutation on the 𝜸 promoter create?
e-box motif which composes a consensus sequence for an activator, TAL1 erythroid specific activator
Where is the TAL1 erythroid specific activator created?
antisense strand of the 𝜸-globin promoter
Why are we creating a site for an activator?
it is something that occurs naturally in patients with HPFH mutation
What 2 methods can be used to create a site for an activator?
mutation on the -175 nt in the promoter of the 𝜸-gene
through genome editing
How can we generate a deletional HPFH mutation? What cell line was used for this technique?
using 2 guides that simultaneously target the sequence and then have Cas9 cut and create small deletions
HUDEP cell line
What has shown the most efficient approach in the clinical phase?
elimination of the erythroid specific BCL11A expression
What was the aim of the 2 trials related to BTHAL and SCD?
elevate HbF levels to achieve a therapeutic effect
What did the trial conducted using ex-vivo technique with CRISPR-Cas9 demonstrate?
disrupting the GATA1 binding site in BCL11A promoter only in the erythroid lineage causes the % of HbF to increase by 30% with respect to the control…this provided proof of concept of reactivation of HbF in vitro
Why can we perform lentiviral vector gene addition to correct mice, but not perform the preclinical correction on β-thalassemia murine model or SCD murine model?
the mouse switch from HbF to HbA happens before birth
genome editing works well in humans, but not in mice (usually the opposite)
What was incredible after 20 weeks in the β-thalassemia patient?
no more transfusions were needed after 20 weeks
