Lesson 12 Flashcards
what is one of the most potent erythroid transcription factors that we see expressed in the erythroid lineage?
GATA1
on the beta globin gene, all biding sites besides the one for GATA1 are activators of transcription - what does this mean?
the basal transcription of the genes is not at a very high level, it is just very highly enhanced
in addition to the minimal promotor that each gene has, what is upstream of all the loci?
locus control region (LCR)
what is the locus control region composed of?
composed of a lot of transcription binding sites for activators
what are hypersensitive sites?
different sequences of DNA from one to 5 that represent the LCR → recognized by activators
how does the LCR interact?
though the bending of DNA with the single promotor of the gene that must be activated in that phase of development
during fetal development, which genes are expressed?
gamma genes
during adult life in the bone marrow, what promotor/s is the LCR engaged with?
only the β promotor
the expression of a gene is influenced by what two things?
both activation and repression
what two repressors act on the gamma genes to suppress them during adult life?
BCL11A and LRF
what are pathologies associated with hemoglobin synthesis?
hemoglobinopathies
what mutation causes sickle cell disease?
there isn’t an absence of hemoglobin, but we have a single mutation → the substitution of glutamic acid with valine in position 6
what does the reduction of hemoglobin causing severe anemia lead to?
β-thalassemia
describe a patient with thalassemia major:
the embryos and the fetus are normally developing because there are the other two globulins that are perfectly expressed, but then at the time of birth, when the second switching take place there’s not any hemoglobin
give 7 examples of single point mutations that can happen in any of the relevant parts of the gene:
- Mutations in the promoter: the gene is not expressed
- Mutation in the 3’ UTR region
- Mutation in the CAP site
- Mutation in the initiation codon
- A lot of mutations are in the splice sites, because globin needs to be spliced, and there are many mutations associated with the abolishment of the splice sites or creation of cryptic splices site.
- Mutation in the codon, it is possible to have both the frameshift mutation: the protein is not expressed, and a premature termination codon is formed
- Mutation in the 3’ of the gene, also poly(A) can be site of mutation
what is it called when there is some hemoglobin but it is not compatible with everyday life?
thalassemia intermedial
β-thalassemia is one of the most common diseases in terms of what?
one of the most common monogenic disorders that caused mortality in the most severe cases, but also morbidity if the patient is not appropriately treated
how can β-thalassemia be cured in some cases?
give the patient a normal hematopoietic system → bone marrow transplant
what area of the world is β-thalassemia most diffuse?
in the areas where malaria was endemic
describe the vicious circle of β-thalassemia:
When a subject become anemic, the very first trigger signal is that if hemoglobin goes down, EPO goes up and it brings to a very high stimulation for red blood cell production. But even if the stimulation of red blood cells production increase, they undergo apoptosis and hemolysis (don’t have sufficient hemoglobin, so they are never able differentiate to mature red blood cell)
describe another detrimental mechanism besides the vicious circle making β-thalassemia so severe:
if we do not have the β-globulin there is an imbalance between the ⍺-chain and the β-chain
describe the ⍺-chain and the β-chain in β-thalassemia:
the ⍺-chains are completely normal, but they are not able to bind with the β-chains, so all those chains undergo precipitations in the red blood cells → causing death
when there is an imbalance between the ⍺-chain and the β-chain, what occurs?
the hematopoietic progenitors are stimulated to make more red blood cells, but all these cells are going to die in the bone marrow or in circulation
when the red blood cells in the marrow and circulation are dying, what does this cause physiologically?
- splenomegaly
- marrow expansion
- bone deformities
- hypermetabolic state
- iron accumulation
how are β-thalassemia patients treated?
give blood as well as iron chelation
what is the target for all genetic modifications to treat β-thalassemia?
the long-term hematopoietic stem cells (HSC)
what happens if there is the transfer of a normal copy of the gene?
there will be production of beta chain, the imbalance between alfa and beta chains will reduce, so they do not precipitate, erythroid progenitors do not die and they express normal hemoglobin → if you transplant a genetically modified HSC from that cell all the lineages will be originated and will bring the modification.
in the case of hematopoietic stem cells, what is the only efficient way to transfer a gene?
through the use of viral vectors
what are the viral vectors derived from?
retroviruses (ex vivo gene therapy)
what two viruses are viral vectors derived from?
Moloney Murine Leukemia Virus (Mo-MLV) and HIV-1
what does it mean if something is a retrovirus?
its genome is composed of RNA
what was the first application of gene transfer in humans?
“bubble boy” → SCID-X1 (X-linked severe combined immunodeficiency), ADA-SCID (adenosine deaminase deficiency), and WAS (Wiskott-Aldrich Syndrome). In all these patients,
cells from the immune system are very compromised and they need to live in a sterile room, because any type of infection can be lethal for these children
which of the two virus vectors is superior?
HIV-1 → only one so far efficient to transfer genes, particularly in quiescent stem cells as the hematopoietic ones
when engineering the HIV virus as a vector, what is maintained in the final product?
we only keep some sequences, such as the ones present in the long terminal repeat and the part of gag
what are the 5 steps for creating a new clinical therapy?
- study basic molecular biology / develop a good vector
- develop protocols of culture
- perform pre-clinical studies / proof of concept studies
- ensure it can be transferred into the larger organism
- confer with clinicians to help create treatment
what are the characteristics of a good vector?
high titer, efficiency to transfer the gene, no rearrangements, and very high levels of transgene expression
what is a big difference between mouse model and humans in terms of virus vector research and genes?
mice do not have fetal genes → in the mouse we have only one switching:
we go from the embryonic to the adult which are already expressed in the fetus
what does the fact that mice only have one switch mean?
if we have homozygous mutation of the beta genes, the mice are dead in uterus when the switch occurs. That’s why we have to keep those mice heterozygous: one allele with the deletion of beta major and beta minor, while the other allele is wild type
what is the transduction phase?
once the mouse is euthanized, we take the hematopoietic stem cells and put our cells in vitro with our vector → in this phase we want to correct virtually all the cells
what occurs after the transduction phase and the changes have been made?
the engineered stem cells are then translated back into a live animal, which is treated with irradiation therapy to deplete the pre-existing marrow cells in order to favor the new transplanted cells
after transplantation, how long must the animal model stay alive in order to to study beta globulin expression?
one year, analyze results through hemoglobin levels
