Lesson 12

Question 1

what is one of the most potent erythroid transcription factors that we see expressed in the erythroid lineage?

Answer 1

GATA1

Question 2

on the beta globin gene, all biding sites besides the one for GATA1 are activators of transcription - what does this mean?

Answer 2

the basal transcription of the genes is not at a very high level, it is just very highly enhanced

Question 3

in addition to the minimal promotor that each gene has, what is upstream of all the loci?

Answer 3

locus control region (LCR)

Question 4

what is the locus control region composed of?

Answer 4

composed of a lot of transcription binding sites for activators

Question 5

what are hypersensitive sites?

Answer 5

different sequences of DNA from one to 5 that represent the LCR → recognized by activators

Question 6

how does the LCR interact?

Answer 6

though the bending of DNA with the single promotor of the gene that must be activated in that phase of development

Question 7

during fetal development, which genes are expressed?

Answer 7

gamma genes

Question 8

during adult life in the bone marrow, what promotor/s is the LCR engaged with?

Answer 8

only the β promotor

Question 9

the expression of a gene is influenced by what two things?

Answer 9

both activation and repression

Question 10

what two repressors act on the gamma genes to suppress them during adult life?

Answer 10

BCL11A and LRF

Question 11

what are pathologies associated with hemoglobin synthesis?

Answer 11

hemoglobinopathies

Question 12

what causes sickle cell disease?

Answer 12

there isn’t an absence of hemoglobin, but we have a single mutation → the substitution of glutamic acid with valine in position 6

Question 13

what does the reduction of hemoglobin causing severe anemia lead to?

Answer 13

β-thalassemia

Question 14

describe a patient with thalassemia major:

Answer 14

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

Question 15

give 7 examples of single point mutations that can happen in any of the relevant parts of the gene:

Answer 15

1. Mutations in the promoter: the gene is not expressed
2. Mutation in the 3’ UTR region
3. Mutation in the CAP site
4. Mutation in the initiation codon
5. 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.
6. 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
7. Mutation in the 3’ of the gene, also poly(A) can be site of mutation

Question 16

what is it called when there is some hemoglobin but it is not compatible with everyday life?

Answer 16

thalassemia intermedial

Question 17

β-thalassemia is one of the most common diseases in terms of what?

Answer 17

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

Question 18

how can β-thalassemia be cured in some cases?

Answer 18

give the patient a normal hematopoietic system → bone marrow transplant

Question 19

what area of the world is β-thalassemia most diffuse?

Answer 19

in the areas where malaria was endemic

Question 20

describe the vicious circle of β-thalassemia:

Answer 20

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)

Question 21

describe another detrimental mechanism besides the vicious circle making β-thalassemia so severe:

Answer 21

if we do not have the β-globulin there is an imbalance between the ⍺-chain and the β-chain

Question 22

describe the ⍺-chain and the β-chain in β-thalassemia:

Answer 22

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

Question 23

when there is an imbalance between the ⍺-chain and the β-chain, what occurs?

Answer 23

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

Question 24

when the red blood cells in the marrow and circulation are dying, what does this cause physiologically?

Answer 24

• splenomegaly
• marrow expansion
• bone deformities
• hyper metabolic state
• iron accumulation

Question 25

how are β-thalassemia patients treated?

Answer 25

give blood as well as iron chelation

Question 26

what is the target for all genetic modifications to treat β-thalassemia?

Answer 26

the long-term hematopoietic stem cells (HSC)

Question 27

what happens if there is the transfer of a normal copy of the gene?

Answer 27

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.

Question 28

in the case of hematopoietic stem cells, what is the only efficient way to transfer a gene?

Answer 28

through the use of viral vectors

Question 29

what are the viral vectors derived from?

Answer 29

retroviruses (ex vivo gene therapy)

Question 30

what two viruses are viral vectors derived from?

Answer 30

Moloney Murine Leukemia Virus (Mo-MLV) and HIV-1

Question 31

what does it mean if something is a retrovirus?

Answer 31

its genome is composed of RNA

Question 32

what was the first application of gene transfer in humans?

Answer 32

“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

Question 33

which of the two virus vectors is superior?

Answer 33

HIV-1 → only one so far efficient to transfer genes, particularly in quiescent stem cells as the hematopoietic ones

Question 34

when engineering the HIV virus as a vector, what is maintained in the final product?

Answer 34

we only keep some sequences, such as the ones present in the long terminal repeat and the part of gag

Question 35

what are the 5 steps for creating a new clinical therapy?

Answer 35

1. study basic molecular biology / develop a good vector
2. develop protocols of culture
3. perform pre-clinical studies / proof of concept studies
4. ensure it can be transferred into the larger organism
5. confer with clinicians to help create treatment

Question 36

what are the characteristics of a good vector?

Answer 36

high titer, efficiency to transfer the gene, no rearrangements, and very high levels of transgene expression

Question 37

what is a big difference between mouse model and humans in terms of virus vector research and genes?

Answer 37

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

Question 38

what does the fact that mice only have one switch mean?

Answer 38

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

Question 39

what is the transduction phase?

Answer 39

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

Question 40

what occurs after the transduction phase and the changes have been made?

Answer 40

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

Question 41

after transplantation, how long must the animal model stay alive in order to to study beta globulin expression?

Answer 41

one year, analyze results through hemoglobin levels