Ferrari: Lecture XIV

Question 1

Why is the proof of concept needed?

Answer 1

to test human cells

Question 2

What kind of cells are the target cells in thalassemia?

Answer 2

HSC

Question 3

Why is it hard to isolated HSC?

Answer 3

rven though they are present in blood, we can only obtain them through the bone marrow (from donors)

Question 4

How are bone marrow cells withdrawn?

Answer 4

aspiration with a big syringe from illiac crest

Question 5

What happens before bone marow transplantation?

Answer 5

compatible donor is found and bone marrow from the donor is withdrawn and transplanted in oder to ensure the bone marrow does not have abnormalities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

Question 6

What is another method used to treat patients with thalassemia?

Answer 6

peripheral blood mobilization

Question 7

Describe peripheral blood mobilized:

Answer 7

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

Question 8

What is the vector used in peripheral blood mobilized expressing?

Answer 8

transgene of erythroid progeny

Question 9

How can the transgene expression in peripheral blood mobilized be checked?

Answer 9

using capillary electrophoresis that can distinguish different types of hemoglobin

Question 10

What all can be done in regards to human cells?

Answer 10

transduction

efficiency of transduction analysis

transgene expression analysis

Question 11

What are 2 proofs of concepts related to β-thalassemia?

Answer 11

thalassemia mice → correction of the pathology

human cells → synthesis at normal levels of the transgene

Question 12

Are the 2 proof of concepts enough proof to conduct clinical trials?

Why not?

Answer 12

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

Question 13

What is GLP?

Answer 13

Good Laboratory Practice

*studies conducted under this umbrella have a very high quality standard

Question 14

Who approves clinical trials?

Answer 14

Regulatory Authority

Question 15

What labs can perform clinical trials?

Answer 15

research labs

Question 16

What is being used in San Raffaele (in GLP testing facility) for gene therapy of BTHAL?

Answer 16

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)

Question 17

How long the do thalamic mice have to be alive?

Answer 17

1 year: interim analysis at 4 months and terminal analysis at 1 year

Question 18

What kind of monitoring is done in thalamic mice?

Answer 18

daily monitoring to ensure they do not have clinical signs, they are not suffering, they are not losing weight

Question 19

Describe chemical chemistry:

Answer 19

AST, ALT, GLU, and MLP (liver enzymes) are measured in order to evaluate absence of toxicity

Question 20

Describe reconstitution of normal hematopoiesis:

Answer 20

CD3 (T-lymphocytes), CD11b (myeloid cells), and B220 (B-lymphocytes) are measured

myeloid shows a difference, which could be an indication of infection

Question 21

Describe correction of anemia:

Answer 21

graph shows the mice were corrected for anemia

Question 22

Describe vector copy number:

Answer 22

treated group had a very high corrected copy number (VCN)

Question 23

Describe the survival curve:

Answer 23

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

Question 24

Describe tumor incidence:

Answer 24

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)

Question 25

What were the pathological findings and conclusion of the San Raffaele BTHAL experiment?

Answer 25

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)

Question 26

Where do lentiviral vectors integrate?

Answer 26

randomly, so it is important to map them to check if they go to risky genes before moving to humans

Question 27

What is the technique used to detect the integration site of lentiviral vectors?

Answer 27

link-mediated PCR and then sequencing

Question 28

What gives us an idea of clonal dominance?

Answer 28

percentage of sequence reads

Question 29

What is the percentage of sequence reads?

Answer 29

high reads → abnormal clone is expanding

Question 30

What does this graph infer?

Answer 30

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

Question 31

What can we see looking at the targeted genes in the bone marrow and peripheral blood?

Answer 31

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)

Question 32

What does the integration of human cells do?

Answer 32

gives level of potential toxicity

Question 33

What is the molecular mechanism for genotoxicity? Why might an integrating vector be toxic for the cell?

Answer 33

gene disruption, if the integration disrupts the coding sequence or other important sequences transactivation

Question 34

THE LCR (locus control region) has the ___ binding site adn this TF is expressed only in ___ lineage.

Answer 34

GATA1 erythroid

Question 35

When is transactivation a problem in genotoxicity?

Answer 35

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

Question 36

What is tested in phase 1/2 in clinical trials?

Answer 36

safety

Question 37

What is tested in phase 3 of clinical trials?

Answer 37

efficacy

Question 38

What was the goal of the 1st trial for gene therapy for BTHAL patients phase I study in UniSR?

Answer 38

demonstrate safety, tolerability, and polyclonal craft condition (demonstrates cells are not clones because clones can easily become tumor cells)

Question 39

What strategy was used to tested the BTHAlL patients from UniSR?

Answer 39

staggered strategy (first adults then children)

Question 40

Why is the staggered approach used?

Answer 40

to ensure no toxic effects are seen in adults before moving to younger patients

Question 41

Who produces cells at a very high standard of quality?

Answer 41

specific companies with Good Manufacturing Practice (GMP)

Question 42

Describe the study that was conducted by UniSR:

Answer 42

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

Question 43

If the cells pass quality control, what is done next?

Answer 43

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)

Question 44

Describe the process of BTHAL gene therapy transplantation:

Answer 44

chemotherapy cells are transplanted back through autologous transplantation (injection in illiac crest aka intrabone (I.B.) infusion)

Question 45

How do we know intrabone injection is better than intravenous injection?

Answer 45

demonstration was shown in mice

Question 46

What was used to track where the cells had travelled to in mice in the intrabone injection experiment?

Answer 46

Luciferase (natural marker)

Question 47

Why is intrabone injection better than intravenous injection?

Answer 47

cells from intravenous injection are trapped in filter organs and die since they cannot find a niche to home, engraft, and differentiate

Question 48

When does the follow-up of the 1st trial of gene therapy usually end?

Answer 48

two years after gene therapy

Question 49

What is long-term follow-up?

Answer 49

2nd follow-up phase which is finished 8 years after gene therapy

Question 50

What lab tests are performed during follow up?

Answer 50

bone marrow analysis vector copy analysis integration site analysis clinical exams like liver and cardiac function analysis

Question 51

Describe the clinical results of the BTHAL patients from UniSR:

Answer 51

the best sample reached 80% transduction and the lowest received 40% transduction

Question 52

Are the lentiviral vectors safe?

Answer 52

yes as thousands of patients have been treated and none have developed cancer when using the ex-vivo technique

Question 53

What is the ex-vivo technique?

Answer 53

engineered cells transduced with lentiviral vectors