Ferrari: Lecture XII Flashcards
Gene Therapy of Hemoglobinopathies
What is the beta globin locus composed of?
different genes that are expressed during development
Describe the beta globin promoter:
eukaryotic promoter
TATA box
binding sites (GATA1, CP1, and NF1) located upstream
What is important to know in regards to the GATA1 binding site?
one of the most potent erythroid TF expressed in the erythroid lineage
Where is the locus control region (LCR) located?
upstream all the loci
What is the representation of LCR made by?
different sequences of DNA from 1 to 5, which are called hypersensitive sites
Describe how the LCR interacts?
bending of the DNA with the single promoter of the gene that must be activated
in fetal: LCR element interacts with the transcription factor bound to the gamma gene, which is with the locus control
in adult: LCR is only engaged with the beta promoter
During adult life, LCR interact and activate ____
beta-globin gene
What are the 2 main repressors of the the gamma gene?
BCL11A and LRF
What are hemoglobinopathies?
pathologies associated with hemoglobin synthesis
What are 2 mutations that affect the adult globin genes?
mutant β-chain (valin is substituted with glutamic acid in position 6): sickle cell disease; the mutant hemoglobin can deform the RBC
β-thalessemia; hemoglobin production is reduced and it can be caused by many mutations (more than 300)
Review the structure of the human hemoglobin:
List the locations where point mutations can occur:
mutations in the promoter: gene is not expressed
mutation in 3’ region
mutation in CAP site
mutation in initiation codon
mutations in splice sites
mutation in codon
mutation of 3’ gene
mutation in poly(A)
All of the point mutations and mutations including deletion can lead to what disease?
β-thalessemia or Cooley’s anemia (no globin is produced)
How can a disease like β-thalassemia or Cooley’s disease be cured?
giving the patient a normal hematopoietic system through procedures like a bone marrow transplant
What is a major barrier in transplantation?
the compatibility of the major complex genes needs to be greater than 90%
less than 30% of patients find a donor with a match, so this is why there is a major medical need
What are areas where thalassemia is more prevalent?
areas with malaria
What happens when a subject becomes anemic?
trigger signal due to dropped hemoglobin levels
EPO increases
RBC are produced
RBC undergo apoptosis and hemolysis because of insufficient hemoglobin, so they never mature
What else can happen if we do not have β-globulin?
there is an imbalance between the alpha and beta chains
the alpha chain levels are normal, but they cannot bind with beta, so the chains undergo precipitations and this causes the death of these cells in the bone marrow or in circulation since they cannot differentiate
What does the absence of β-globin cause?
hematopoietic progenitors make more RBC, but the cells die in the bone marrow or in circulation → causes bone marrow expansion as it tries to cope with anemia→ erythroblasts die
Review the death circle:
What is the easiest thing to do to rescue someone from anemia?
give a transfusion
What is an issue with transfusion to treat someone with anemia?
iron is accumulated, which is toxic for the liver and heart
How are patients managed if chronic blood transfusions are dangerous?
blood is given along with iron chelations
In regards to β-thalassemia, what can be done with gene therapy?
gene addition: transfer a normal copy of the gene into somatic cells
gene editing
*genes must be modified in ex-vivo (in culture) and then transplanted back into patients
What are the long-term hematopoietic stem cells (HSC)?
target for all genetic modifications
What is a key concept for the clinical success of transplantation?
hematopoietic stem cells need to be in a good niche, and if too much time passes by, the niche can be in a disease-state, which is why it is important to act very early
children have less damage than adults
What is one way to efficiently transfer genes in hematopoietic stem cells?
using viral vectors
What can viral vectors be derived from?
ex-vivo gene therapy, which are derived from retroviruses: Moloney Murine Leukemia Virus (Mo-MLV) and HIV-1
What are retroviruses?
genome is composed of RNA
What is the lentiviral vector?
vector derived from the HIV1 retrovirus, and the vector has a human tropism
What kind of tropism does Mo-MLV have?
murine
What is special about the HIV1 vector?
it is the only vector that is efficient in transferring genes in quiescent stem cells as the hematopoietic ones
Describe the engineering of a lethal virus:
it is quite complex
from the original genome with all the genes, HIV1 genome is deleted
only sequences kept are the ones present in the long terminal sequence and gag
everything that is missing from the original genome is replaced by our gene and promoter
List the diseases that were treated with hematopoietic stem cells engineered with lentiviral vectors:
ALD
MLD
WAS
Bthal
SCID
SCID-X1
ADA-SCID
CGD
Fanconi anemia
MPSI
PKD
What do all of the diseases that have been treated using a lentivector have in common?
structure of the vector: only need to change the promoter and the transgene
very rare disease
no cure
What defines a good vector?
it should be able to express the transgene at very high levels (for ex: in RBC, hemoglobin is one of the most expressed proteins)
What is important to keep in mind when we develop protocols for the culture?
we want to transfer all the genes in all the HPSC at a high efficiency
we want cells to remain stem cells even after they are cultured (we do not want them to differentiate)
Why do we do pre-clinical studies?
demonstrate our hypothesis is right (vector is able to correct the disease)
What are the proof-of-concept studies?
pre-clinical studies
What is important to demonstrate in new approaches?
they are safe
What is a clinical consideration to take when demonstrating a new approach?
can the approach be manufactured in large scale?
What should protocols contain?
kind of patient
where stem cells are derived from
how many cells are we giving the patients
safety
What do we need in order to express the beta blobin?
locus control region
How big is the locus control region?
15Kb
What is the cloning capacity of the lentivector?
8-10Kb
What happens if we exceed the vector cloning capacity?
we will never have good production of the vectors
What are the main characteristics of a good vector?
high titer
efficiently transfers genes
no rearrangements
high levels of transgene expression
Describe the vector:
2 LCR from the HIV1
RRE (Rev Response Element) from HIV1
transgene with promoter
beta promoter
exon1-2-3
poly(A)
Where will the transgene be activated?
ONLY in the erythroid lineage (RBC)
What do we do in the tranduction phase, after the bone marrow has been extracted from the mouse and the hematopoietic stem cells have been taken?
cells are placed in vitro with vector
we want to correct all the cells
If we don’t have an available animal model, how can we obtain an animal model?
using a CRISPR/Cas9 approach (if the disease of interest is a genetic disease)
