Brainscape's Knowledge GenomeTM

Browse over 1 million classes created by top students, professors, publishers, and experts.


See full index

Regulation of Gene Expression > Ferrari: Lecture XII > Flashcards

Ferrari: Lecture XII Flashcards

Gene Therapy of Hemoglobinopathies

1
Q

What is the beta globin locus composed of?

A

different genes that are expressed during development

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
2
Q

Describe the beta globin promoter:

A

eukaryotic promoter

TATA box

binding sites (GATA1, CP1, and NF1) located upstream

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
3
Q

What is important to know in regards to the GATA1 binding site?

A

one of the most potent erythroid TF expressed in the erythroid lineage

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
4
Q

Where is the locus control region (LCR) located?

A

upstream all the loci

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
5
Q

What is the representation of LCR made by?

A

different sequences of DNA from 1 to 5, which are called hypersensitive sites

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
6
Q

Describe how the LCR interacts?

A

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

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
7
Q

During adult life, LCR interact and activate ____

A

beta-globin gene

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
8
Q

What are the 2 main repressors of the the gamma gene?

A

BCL11A and LRF

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
9
Q

What are hemoglobinopathies?

A

pathologies associated with hemoglobin synthesis

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
10
Q

What are 2 mutations that affect the adult globin genes?

A

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)

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
11
Q

Review the structure of the human hemoglobin:

A
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
12
Q

List the locations where point mutations can occur:

A

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)

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
13
Q

All of the point mutations and mutations including deletion can lead to what disease?

A

β-thalessemia or Cooley’s anemia (no globin is produced)

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
14
Q

How can a disease like β-thalassemia or Cooley’s disease be cured?

A

giving the patient a normal hematopoietic system through procedures like a bone marrow transplant

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
15
Q

What is a major barrier in transplantation?

A

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

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
16
Q

What are areas where thalassemia is more prevalent?

A

areas with malaria

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
17
Q

What happens when a subject becomes anemic?

A

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

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
18
Q

What else can happen if we do not have β-globulin?

A

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

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
19
Q

What does the absence of β-globin cause?

A

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

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
20
Q

Review the death circle:

A
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
21
Q

What is the easiest thing to do to rescue someone from anemia?

A

give a transfusion

22
Q

What is an issue with transfusion to treat someone with anemia?

A

iron is accumulated, which is toxic for the liver and heart

23
Q

How are patients managed if chronic blood transfusions are dangerous?

A

blood is given along with iron chelations

24
Q

In regards to β-thalassemia, what can be done with gene therapy?

A

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

25
Q

What are the long-term hematopoietic stem cells (HSC)?

A

target for all genetic modifications

26
Q

What is a key concept for the clinical success of transplantation?

A

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

27
Q

What is one way to efficiently transfer genes in hematopoietic stem cells?

A

using viral vectors

28
Q

What can viral vectors be derived from?

A

ex-vivo gene therapy, which are derived from retroviruses: Moloney Murine Leukemia Virus (Mo-MLV) and HIV-1

29
Q

What are retroviruses?

A

genome is composed of RNA

30
Q

What is the lentiviral vector?

A

vector derived from the HIV1 retrovirus, and the vector has a human tropism

31
Q

What kind of tropism does Mo-MLV have?

A

murine

32
Q

What is special about the HIV1 vector?

A

it is the only vector that is efficient in transferring genes in quiescent stem cells as the hematopoietic ones

33
Q

Describe the engineering of a lethal virus:

A

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

34
Q

List the diseases that were treated with hematopoietic stem cells engineered with lentiviral vectors:

A

ALD
MLD
WAS
Bthal
SCID
SCID-X1
ADA-SCID
CGD
Fanconi anemia
MPSI
PKD

35
Q

What do all of the diseases that have been treated using a lentivector have in common?

A

structure of the vector: only need to change the promoter and the transgene

very rare disease

no cure

36
Q

What defines a good vector?

A

it should be able to express the transgene at very high levels (for ex: in RBC, hemoglobin is one of the most expressed proteins)

37
Q

What is important to keep in mind when we develop protocols for the culture?

A

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)

38
Q

Why do we do pre-clinical studies?

A

demonstrate our hypothesis is right (vector is able to correct the disease)

39
Q

What are the proof-of-concept studies?

A

pre-clinical studies

40
Q

What is important to demonstrate in new approaches?

A

they are safe

41
Q

What is a clinical consideration to take when demonstrating a new approach?

A

can the approach be manufactured in large scale?

42
Q

What should protocols contain?

A

kind of patient

where stem cells are derived from

how many cells are we giving the patients

safety

43
Q

What do we need in order to express the beta blobin?

A

locus control region

44
Q

How big is the locus control region?

A

15Kb

45
Q

What is the cloning capacity of the lentivector?

A

8-10Kb

46
Q

What happens if we exceed the vector cloning capacity?

A

we will never have good production of the vectors

47
Q

What are the main characteristics of a good vector?

A

high titer

efficiently transfers genes

no rearrangements

high levels of transgene expression

48
Q

Describe the vector:

A

2 LCR from the HIV1

RRE (Rev Response Element) from HIV1

transgene with promoter

beta promoter

exon1-2-3

poly(A)

49
Q

Where will the transgene be activated?

A

ONLY in the erythroid lineage (RBC)

50
Q

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?

A

cells are placed in vitro with vector

we want to correct all the cells

51
Q

If we don’t have an available animal model, how can we obtain an animal model?

A

using a CRISPR/Cas9 approach (if the disease of interest is a genetic disease)

Regulation of Gene Expression (28 decks)

Brainscape helps you reach your goals faster, through stronger study habits.
© 2024 Bold Learning Solutions. Terms and Conditions