Lesson 14

Question 1

how do patients undergo peripheral blood mobilization?

Answer 1

1. samples were obtained from apheresis or bone marrow harvest
2. 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+
3. cells are put into the culture with the vector for two weeks
4. all cells remaining are only cells differentiated in the erythroid lineage
5. assess the expression of the transgene

Question 2

where is the vector expressing the transgene located?

Answer 2

erythroid progeny → from these cells you obtain the erythroid lineages

Question 3

how can you check transgene expression?

Answer 3

capillary electrophoresis → distinguished different types of hemoglobin

Question 4

what were the studies done as San Raffaele standardized by?

Answer 4

the umbrella of GLP (good laboratory practice) meaning a very high quality standard

Question 5

in the gene therapy of BTHAL study, what were the three control groups?

Answer 5

1. group of mice who received HSCs transduced with the vector
2. thalassemic mice untreated
3. mice transplanted with the HSCs, but without the vector because you have to demonstrate the toxicity induced by the vector

Question 6

how to you test for the chemical chemistry?

Answer 6

measure liver enzymes (AST, ALT, GLU, MLP) to evaluate the absence of toxicity

Question 7

what were the pathological findings of the gene therapy of BTHAL mouse trials?

Answer 7

• 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)

Question 8

how to Lentiviral vectors integrate?

Answer 8

randomly → they land in different parts of the genome so its important to map the integration

Question 9

what technique is used to detect integration sites?

Answer 9

linked-mediated PCR followed by sequencing

Question 10

if we look at the percentage of sequencing reads from the linked-mediated PCR analysis, what result does this give us?

Answer 10

an idea of the clonal dominance

Question 11

if you have a high percentage of reads, what does this mean?

Answer 11

this could mean you might have a clone expanding (abnormal clone)

Question 12

what are you looking for when testing for biodistribution?

Answer 12

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

Question 13

when transplanting the human cells into mice, what were the three groups used?

Answer 13

• Control mice, untreated
• Control mice receiving HSCs cultured in vitro without the vector
• Control mice receiving HSCs with the vectorwher

Question 13

when transplanting the human cells into mice, what were the three groups used?

Answer 13

• Control mice, untreated
• Control mice receiving HSCs cultured in vitro without the vector
• Control mice receiving HSCs with the vectorwher

Question 14

when transplanting the human cells into mice, what were the three groups used?

Answer 14

• Control mice, untreated
• Control mice receiving HSCs cultured in vitro without the vector
• Control mice receiving HSCs with the vectorwher

Question 14

when transplanting the human cells into mice, what were the three groups used?

Answer 14

• Control mice, untreated
• Control mice receiving HSCs cultured in vitro without the vector
• Control mice receiving HSCs with the vector

Question 15

where is the bio distribution of human cells observed?

Answer 15

in peripheral blood, bone, bone marrow, ad the spleen with no significant difference between the control and treated groups

Question 16

what are the two molecular mechanisms for gene toxicity?

Answer 16

gene disruption and transactivation

Question 17

when is transactivation a problem in genotoxicity?

Answer 17

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.

Question 18

what was the object of the phase I β-thalassemia clinical trial performed at UniSR?

Answer 18

to demonstrate safety

Question 19

what are some safety endpoints wanting to be reached in a phase I clinical trial?

Answer 19

• 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

Question 20

what is the efficacy endpoint of the β-thalassemia clinical trial?

Answer 20

to reduce transfusions → patients being treated for β-thalassemia need to have repeated transfusions

Question 21

how many patients were tested for the β-thalassemia clinical trial?

Answer 21

9 - three adults, three adolescents (8-17) and three small children (3-7)

Question 22

what two ways were cells taken from the patients?

Answer 22

through mobilization and harvesting

Question 23

after blood collection, what occurs?

Answer 23

they had to select the cells using a specific machine that allowed a clinical grade sterile selection of CD34+ cells (HSC)

Question 24

after the sterile CD34+ cells were collected, what happens?

Answer 24

the transduction is then done in big bioreactors, using a closed system, after which cells will be conserved in sterile conditions through cryopreservation.

Question 25

why were patients recommended to undergo fertility preservation before the implantation of cells?

Answer 25

they were required to undergo chemotherapy for conditioning

Question 26

how were the cells transplanted back into the patients?

Answer 26

through autologous transplantation into the iliac crest (intrabone infusion)

Question 27

what is the downside to transplanting the cell intravenously?

Answer 27

many cells become trapped in the lungs and spleen, never making it to the marrow so you would lose a lot of cells

Question 28

how long after the transplantation does the follow up phase last?

Answer 28

2 years

Question 29

how long does the long-term follow up phase last?

Answer 29

8 years

Question 30

during the follow up phase what is tested for?

Answer 30

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

Question 31

what were the transduction results between all 9 patients tested?

Answer 31

the best sample reached 80% of transduction, and the lowest transduction was about 40%, meaning that not all the cells were corrected in vitro and then transplanted in vivo → high variability between patients

Question 32

Can you follow the cells differentiating from CD34 cells in vivo in the peripheral blood of patients?

Answer 32

Yes, because we have a marker and we can do PCR: you put primers matching with vector sequence, you extract the DNA from monocytes, granulocytes, and erythroid cells from the lymphocytes at different timepoints in each patient. We can calculate now how many copies of the transgene are present in these cells

Question 33

what is one of the biggest issues with transduction?

Answer 33

we could also have transduction of 100% in vitro, but when we go in vivo, after transplantation, there’s no
possibility to predict if those cells will be able to repopulate the mutated lineages.