Ferrari: Lecture XIV Flashcards
Gene Therapy for Hemoglobinopathies
Why is the proof of concept needed?
to test human cells
What kind of cells are the target cells in thalassemia?
HSC
Why is it hard to isolated HSC?
rven though they are present in blood, we can only obtain them through the bone marrow (from donors)
How are bone marrow cells withdrawn?
aspiration with a big syringe from illiac crest
What happens before bone marow transplantation?
compatible donor is found and bone marrow from the donor is withdrawn and transplanted in oder to ensure the bone marrow does not have abnormalities
if validated, chemotherapy is done
What is another method used to treat patients with thalassemia?
peripheral blood mobilization
Describe peripheral blood mobilized:
samples are obtained from apheresis or bone marrow harvest
enriched fraction of HSC is selected (means cells express CD34+ on the surface)
cells are separated through flow cytometry
cells are placed in culture with the vector expressing the human globin gene
after 2 weeks, the cells remaining are the ones differentiated in erythroid lineage, and they can be followed due to specific markers using cytometry
What is the vector used in peripheral blood mobilized expressing?
transgene of erythroid progeny
How can the transgene expression in peripheral blood mobilized be checked?
using capillary electrophoresis that can distinguish different types of hemoglobin
What all can be done in regards to human cells?
transduction
efficiency of transduction analysis
transgene expression analysis
What are 2 proofs of concepts related to β-thalassemia?
thalassemia mice → correction of the pathology
human cells → synthesis at normal levels of the transgene
Are the 2 proof of concepts enough proof to conduct clinical trials?
Why not?
No because all procedures must be deemed safe before starting a clinical trial, and it must be proven that the human cells are retaining all of their features after the transduction with the vector
What is GLP?
Good Laboratory Practice
*studies conducted under this umbrella have a very high quality standard
Who approves clinical trials?
Regulatory Authority
What labs can perform clinical trials?
research labs
What is being used in San Raffaele (in GLP testing facility) for gene therapy of BTHAL?
mice who received HSCs tranduced with the vector
1st control: untreated thalamic mice
2nd control: mice transplanted with HSCs, but without the vector (used to demonstrate toxicity induced by vector)
How long the do thalamic mice have to be alive?
1 year: interim analysis at 4 months and terminal analysis at 1 year
What kind of monitoring is done in thalamic mice?
daily monitoring to ensure they do not have clinical signs, they are not suffering, they are not losing weight
Describe chemical chemistry:
AST, ALT, GLU, and MLP (liver enzymes) are measured in order to evaluate absence of toxicity
Describe reconstitution of normal hematopoiesis:
CD3 (T-lymphocytes), CD11b (myeloid cells), and B220 (B-lymphocytes) are measured
myeloid shows a difference, which could be an indication of infection
Describe correction of anemia:
graph shows the mice were corrected for anemia