Lesson 14 Flashcards
how do patients undergo peripheral blood mobilization?
- samples were obtained from apheresis or bone marrow harvest
- The cells are separated from the others by using flow cytometry or through specific columns with beads conjugated antibodies against a specific antigen that allow you to select only the population of interest CD34+
- cells are put into the culture with the vector for two weeks
- all cells remaining are only cells differentiated in the erythroid lineage
- assess the expression of the transgene
where is the vector expressing the transgene located?
erythroid progeny → from these cells you obtain the erythroid lineages
how can you check transgene expression?
capillary electrophoresis → distinguished different types of hemoglobin
what were the studies done as San Raffaele standardized by?
the umbrella of GLP (good laboratory practice) meaning a very high quality standard
in the gene therapy of BTHAL study, what were the three control groups?
- group of mice who received HSCs transduced with the vector
- thalassemic mice untreated
- mice transplanted with the HSCs, but without the vector because you have to demonstrate the toxicity induced by the vector
how do you test for the chemical chemistry?
measure liver enzymes (AST, ALT, GLU, MLP) to evaluate the absence of toxicity
what were the pathological findings of the gene therapy of BTHAL mouse trials?
- Pathological findings were caused not by the treatment, but by the conditioning because the mice underwent chemotherapy before transplant.
- No difference in the tumors incidence or type between the groups.
- No tumorigenicity.
- There is a background of spontaneous tumors.
- Confirmation of efficacy of gene therapy (reduction of EMH)
how do Lentiviral vectors integrate?
randomly → they land in different parts of the genome so its important to map the integration
what technique is used to detect integration sites?
linked-mediated PCR followed by sequencing
if we look at the percentage of sequencing reads from the linked-mediated PCR analysis, what result does this give us?
an idea of the clonal dominance
if you have a high percentage of reads, what does this mean?
this could mean you might have a clone expanding (abnormal clone)
what are you looking for when testing for biodistribution?
you don’t test toxicity and tumorigenicity, but you just look that genetically modified cells are able to engraft in the chimera and developed normally
when transplanting the human cells into mice, what were the three groups used?
- Control mice, untreated
- Control mice receiving HSCs cultured in vitro without the vector
- Control mice receiving HSCs with the vector
where is the bio distribution of human cells observed?
in peripheral blood, bone, bone marrow, ad the spleen with no significant difference between the control and treated groups
what are the two molecular mechanisms for gene toxicity?
gene disruption and transactivation
when is transactivation a problem in genotoxicity?
When you have a very strong promoter in the vector which can transactivate any kind of gene in any kind of cells, included the primitive ones. At that point, if you transactivate a protooncogene (which is normally not expressed), you have leukemia or lymphoma.
what was the object of the phase I β-thalassemia clinical trial performed at UniSR?
to demonstrate safety
what are some safety endpoints wanting to be reached in a phase I clinical trial?
- Survival
- The reconstitution of the hematopoietic system, remember that patients are transplanted with their own genetically modified cells
- Tolerability
- Polyclonal graft condition, which is evaluated from the integration-site analysis. We need to demonstrate that the cells that we have transplanted into the patient are not clones, because a clone has a very high proliferative advantage and can easily become a tumoral cell
what is the efficacy endpoint of the β-thalassemia clinical trial?
to reduce transfusions → patients being treated for β-thalassemia need to have repeated transfusions
how many patients were tested for the β-thalassemia clinical trial?
9 - three adults, three adolescents (8-17) and three small children (3-7)
what two ways were cells taken from the patients?
through mobilization and harvesting
after blood collection, what occurs?
they had to select the cells using a specific machine that allowed a clinical grade sterile selection of CD34+ cells (HSC)
after the sterile CD34+ cells were collected, what happens?
the transduction is then done in big bioreactors, using a closed system, after which cells will be conserved in sterile conditions through cryopreservation.
why were patients recommended to undergo fertility preservation before the implantation of cells?
they were required to undergo chemotherapy for conditioning
