Subido por Mind Niramind

Script SRC proceedings

• Page 1
Good morning, committees and audiences. My name is Niramol Prombandit. Today I’m going to
present in the title of ‘Optimization of pLVX-TetOne-Puro-AcGFP1 Lentiviral Vector Transduction for
Hematopoietic Stem Cell Gene Therapy’. I will give some background of hematopoietic stem cells.
• Page 2
Hematopoietic stem cells or HSCs are the multipotent progenitor cells that have the ability to selfrenew and develop into all types of blood cells.
• Page 3
Hematopoietic stem cells can be obtained from primary tissue sources, such as adult bone marrow
(BM), mobilized peripheral blood (mPB) and umbilical cord blood (UCB), or from cultured cell sources, such
as human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs).
• Page 4
Hematopoietic stem cells-targeted gene therapy has the potential to cure a variety of hereditary
immunodeficiencies and are currently under development for the treatment of genetic blood disorders, such
as beta-thalassemia and sickle cell disease.
• Page 5
Keys to success in gene therapy are the efficiency of the gene delivery into target cells and the
maintenance of the biological functions of target cells.
• Page 6-7
However, hematopoietic stem cells are difficult to gene transfer because inherent characteristics of
hematopoietic stem cells, as most hematopoietic stem cells are in a quiescent state, and displayed
relatively delayed response to cytokine stimulation, and, when induced to divide, tend to lose the capacity
of long-term repopulating potential.
• Page 8
So these are reason why I tried to optimized protocol for lentiviral transduction of hematopoietic
stem cells that essential for successful gene transfer. And this slide show the overall of my project.
• Page 9
This slide show that the pLVX-TetOne-Puro-AcGFP1 vector that I obtained. This vector composed
of the TetOne systems which are inducible gene expression systems.
• Page 10
The Tet-On system allows activation of this gene expression by doxycycline. In the presence of
doxycycline, doxycycline binds to Tet-On 3G protein in order to promote this complex binds specifically to
TRE3G promoter (PTRE3GS) that activates transcription of the transgene.
• Page 11
The lentiviral vector titration was performed on HEK293T cells. At 24 hours post-seeding, cells were
transduced with serial dilutions of lentiviral vector: 10-1, 10-2, 10-3, and 10-4. After 72 hours, the lentiviral titer
was estimated on FACS based on GFP fluorescence.
• Page 12
For the calculation of the lentiviral titer, only dilutions yielding to 10% to 20% of GFP-positive cells
were considered for titer calculations.
When calculating functional titer, choose 1% to 20% fluorophore-positive (FP-positive) populations.
Below 1% FP-positive, FACS may not reliably determine the number of positive cells; above 20% FPpositive, the chance for each positive target cell to be transduced twice increases significantly, resulting in
an underestimation of the number of transducing particles.
The lentiviral vector titration was calculated using this equation (ชี้สมการ), and the titer of the
lentiviral stock is 4.0 × 107.
• Page 13
***What is the MOI?
- MOI is a ratio of infectious agents to infection targets. In many cases, It is the ratio of viral particles to
target cells in a defined space, such as a cell cultured well.
• Page 14
- For example, if you add 10 million viruses to 1 million cells, you would have an MOI of 10 and an average
probability of 10 viral particles infecting one cells
• Page 15
I evaluated conditions of MOI on hematopoietic stem cells from mobilized peripheral blood. The
cells were pre-stimulated for 24 hours and transduced with the lentiviral vector at MOIs of 0, 0.1, 1, 5, 10
and, 25. The vector used was derived from the pLVX-TetOne-Puro and contains the AcGFP1 reporter gene.
The transduction efficiency was monitored by flow cytometry analysis of eGFP at 72 hours.
• Page 16
The result showed that an increase in the percentage of GFP positive cells correlated well with the
increase in MOI, except MOI 25 (0%, 0.17%, 0.34%, 0.57%, 0.97% and 0%, respectively).
• Page 17
To assess the effects of virus concentration on cell viability and hematopoietic stem cell function in
this experiment, I analyzed the number of hematopoietic marker-expressing cells.
CD34 and CD38 were used as surface markers to identify hematopoietic stem cells. CD34 is a
transmembrane phosphoglycoprotein that is highly expressed in pluripotent stem cells and its expression
gradually reduces as the level of maturation of hematopoietic cell lineages increases. CD38 is a type II
glycoprotein that was described as a lymphoid cell surface differentiation marker. So the CD34+ cell
subpopulation with low CD38 expression (CD34+ CD38- cells) is primitive hematopoietic stem cells.
• Page 18
The result showed that the higher MOI resulted in more efficient lentiviral transduction while
decreasing the percentage of primitive hematopoietic stem cell. So the lentiviral transduction at MOI of 10 is
an optimal MOI due to this MOI give the highest lentiviral transduction efficiency and maintain the functional
characteristic of primitive hematopoietic stem cells.
• Page 19
To confirm the inducible expression of the pLVX-TetOne-Puro-AcGFP1 vector, I observed
tetracycline-dependent induction of GFP in hematopoietic stem cells carrying this lentiviral vector. GFP
expression was induced by doxycycline that had previously shown to be effective in inducing expression
and did not inhibit cell growth.
• Page 20
As shown in this picture, GFP expression was clearly induced when doxycycline was present (on)
compared to when it was absent (off). These results indicate that the controllable induction of transduced
hematopoietic stem cells can be seen by expression of GFP.
• Page 21
I found that human mobilized peripheral blood-derived CD34+ hematopoietic stem cells can be
transduced with lentiviral vectors at MOI 10. Moreover, our study also proved the efficiency of tetracyclineinducible system of the pLVX-TetOne-Puro-AcGFP1 vector..
• Page 21
I would expected that I will obtain the best lentiviral transduction protocol would contribute to highefficiency gene transfer, avoid the toxicity of lentiviral vector, and extend the length of transgene expression
in targeted cells. And I hope that this procedure can be useful for hematopoietic stem cell-gene therapy
which one of the approaches to cure the inherited blood diseases.