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How are bone marrow cells withdrawn?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compatible donor is found and bone marrow from the donor is withdrawn and transplanted in oder to ensure the bone marrow does not have abnormalities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
Describe vector copy number:
treated group had a very high corrected copy number (VCN)
Describe the survival curve:
there were some deaths along the 1 year follow up, but there was no statistical significance, so the mice could have died for any reason, but there was no cause-effect
Describe tumor incidence:
occurs in gene therapy group and mock group meaning here is no statistical significance (mice develop tumors with aging, but they are not caused by the vectors)
What were the pathological findings and conclusion of the San Raffaele BTHAL experiment?
pathological findings were caused by the conditioning and not the treatment because mice underwent chemotherapy before transplant
no difference in the tumors between groups
no tumorgenicity
no background of spontaneous tumors
confirmation of gene therapy (reduction of EMH)
Where do lentiviral vectors integrate?
randomly, so it is important to map them to check if they go to risky genes before moving to humans
What is the technique used to detect the integration site of lentiviral vectors?
link-mediated PCR and then sequencing
What gives us an idea of clonal dominance?
percentage of sequence reads
What is the percentage of sequence reads?
high reads → abnormal clone is expanding
What does this graph infer?
in vitro, PB, BM, and tumors mice all show reads, but there is no statistical significance, so it shows the absence of clonal dominance
*population is polyclonal
What can we see looking at the targeted genes in the bone marrow and peripheral blood?
no risky genes are present, so the lentiviral vectors enter the genome with no specific preference for risky genes (there is also no enrichment in cancer related genes)
What does the integration of human cells do?
gives level of potential toxicity
What is the molecular mechanism for genotoxicity? Why might an integrating vector be toxic for the cell?
gene disruption, if the integration disrupts the coding sequence or other important sequences
transactivation
THE LCR (locus control region) has the ___ binding site adn this TF is expressed only in ___ lineage.
GATA1
erythroid
When is transactivation a problem in genotoxicity?
when there is a very strong promoter in the vector which can transactivate any gene in any cell
*if a protooncogene is transactivated, it gives rise to leukemia or lymphoma
What is tested in phase 1/2 in clinical trials?
safety
What is tested in phase 3 of clinical trials?
efficacy
What was the goal of the 1st trial for gene therapy for BTHAL patients phase I study in UniSR?
demonstrate safety, tolerability, and polyclonal craft condition (demonstrates cells are not clones because clones can easily become tumor cells)
What strategy was used to tested the BTHAlL patients from UniSR?
staggered strategy (first adults then children)
Why is the staggered approach used?
to ensure no toxic effects are seen in adults before moving to younger patients
Who produces cells at a very high standard of quality?
specific companies with Good Manufacturing Practice (GMP)
Describe the study that was conducted by UniSR:
cells are taken from patient (mobilization and harvesting)
patient is treated for 2-3 days before gene therapy and then the cells enter and reach peak
after blood collection, cells are selcted using clinical-grade sterile selection of CD34+ cells (HSC)
transduction is done in bioreactors, using a closed system where cells will be conserved in sterile conditions through cryoperservation
cells are now genetically modified, but quality control must be performed before transplantation
If the cells pass quality control, what is done next?
if patients can undergo fertility preservation, it should be done to give them the possibility of pregnancy (this is done because patients undergo chemotherapy for conditioning)
Describe the process of BTHAL gene therapy transplantation:
chemotherapy
cells are transplanted back through autologous transplantation (injection in illiac crest aka intrabone (I.B.) infusion)
How do we know intrabone injection is better than intravenous injection?
demonstration was shown in mice
What was used to track where the cells had travelled to in mice in the intrabone injection experiment?
Luciferase (natural marker)
Why is intrabone injection better than intravenous injection?
cells from intravenous injection are trapped in filter organs and die since they cannot find a niche to home, engraft, and differentiate
When does the follow-up of the 1st trial of gene therapy usually end?
two years after gene therapy
What is long-term follow-up?
2nd follow-up phase which is finished 8 years after gene therapy
What lab tests are performed during follow up?
bone marrow analysis
vector copy analysis
integration site analysis
clinical exams like liver and cardiac function analysis
Describe the clinical results of the BTHAL patients from UniSR:
the best sample reached 80% transduction and the lowest received 40% transduction
Are the lentiviral vectors safe?
yes as thousands of patients have been treated and none have developed cancer when using the ex-vivo technique
What is the ex-vivo technique?
engineered cells transduced with lentiviral vectors
