siRNA used for PD-1

silencing in tumor-specific
T-cells
Anayet Chowdhury
Advanced Science Research Program
Year 3
Manhattan Center for Science and Mathematics
January 25 ,2011

Dr. Michel Sadelain

Somatic Cell Engineering and Gene Transfer Lab Director
Memorial Sloan-Kettering Cancer Center 1
Apoptosis
Many forms of
regulation are
present in the human
body to maintain
homeostasis.
Apoptosis is one of
the regulations.
Source: Youtube
Date of Retrieval:
January 20, 2011

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 Prostate Cancer
• Prostate cancer is the second
most common cancer in
American men.

• Symptoms: urinary and bowel

dysfunctions

• Treatments: surgery and/or

radiation therapy

Figure 1: Cancer Statistics

Source: American Cancer Society
Date of Retrieval: January 20, 2011
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 Introduction
Previous Studies:
Lisa Borkner and her team discover tumor cells some how activate the
Programmed Death-1 (PD-1) receptor on the T-cells. Therefore
causing the T-cell to undergo an accelerated death. As the level of
PD-1 was high, the T-cell was very inefficient.
Research Question : How efficient is siRNA when used for PD-1
silencing in tumor cells?
Hypothesis: Short Interfering RNA signals will enhance the efficiency
of T-cells.
Purpose: To down-regulate the expression of PD-1 to give T-cells their
normal life-cycle. The immune system will be in a stable shape and
the T-cells have a chance to fight the tumor cells without having its
cell death receptor activated.

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 What are siRNA’s?
Small interfering RNA (siRNA), is a class of double-stranded RNA
molecules,20-25 nucleotides in length. siRNA is involved in the RNA
interference(RNAi) pathway, where it interferes with the expression of a
specific gene. 

Figure 2: RNA Induced

Silencing Complex
Source: Google
Date of Retrieval: January 21,
2011

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 Introduction Cont’d
Materials:
 Centrifuge
 Oligonucleotides
 Microfuge tubes
 Pipette
 NanoDrop 2000

Figure 3: Biological Safety Cabinet

Source: Google
Date of Retrieval: January 21, 2011

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Procedure

Figure 4: Creating a Retrovirus Soup in

H29 cell line
Source: Anayet Chowdhury
Date of Retrieval: January 11, 2011
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 Results
Graph 1: The graph above shows a negative
population of transduction and PD-1 expression.
The sample contained untransduced T-cells;
therefore, nothing was added or modified.

Graph 2: The KanR target serves as the control for

this experiment, since a “KanR” region does not
exist in a T-cell. The purpose of this is to get a
baseline of the amount of PD-1 expression being
produced in a normal T-cell to compare it to a
siRNA knockdown of PD-1. T-cells with the siRNA
targeting the KanR region showed an 11.5%
expression of PD-1 with in a positive population of
26.8%. The positive population is the cells that have
the siRNA integrated in them. 8
 Results
Graph 3: T-cells with the siRNA targeting the hu3 region
showed a 4.17% expression of PD-1 with in a positive
population of 50.07%. The siRNA was successful in down-
regulating the amount of PD-1 expression being produced
by three times the original amount.

Graph 4: Expression of PD-1 in the

KanR sample and hu3 are compared.
There is a significant shift between the
two, with a mean fluorescent intensity of
390 for KanR and 141 for Hu3.

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 Discussion
 The results show that the siRNA’s did indeed down-regulate the
expression of PD-1. The siRNA down-regulated the PD-1 expression
up to 3 times its normal expression (Graph 4).
 In the control group, the KanR was a region that did not target the
PD-1 region. After doing a flow cytometry, there was 11.5%
expression of PD-1 within the positive cell population (Graph 2),
which showed that the viruses were integrated in the T-cells.
 The hu3 targeted T-cells showed a 4.17% expression of PD-1
(Graph 3).
 I now have re-engineered T-cells that have the ability to function
normally in a cancer patient. Since PD-1 has been shown to directly
cause apoptosis in mice, the next step for this project will be to move
on to mice and then clinical trials for patients.

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 Conclusion
 High levels of PD-1 have been shown to cause a lack of efficiency
in the T-cells. By reducing down the expression of PD-1, the T-cells
are now genetically re-engineered and have a better chance to fight
off the tumor cells. The results from my experiment supported my
hypothesis. The PD-1 expression in normal T-cells was 11.5% and
then down-regulated with the siRNA’s to a 4.16% expression. PD-1
has been extensively correlated with tumor cells and patients with
various types of cancer, such as breast cancer, ovarian cancer,
prostate cancer and etc. The approach to remove the PD-1 pathway
in T-cells can be an effective therapy for cancer patients. If the
introduction of a vaccine with re-engineered T-cells in mice show
positive results, this experiment can give rise to a new genetic
treatment for cancer patients.

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 References
 McIntyre G, Fanning G (2006). “Design and cloning strageties for
constructing shRNA expression vectors”. BMC Biotechnol. 6:
 Harper S, Staber P, He X, Eliason S, Martins I, Mao Q, Yang L, Kotin R,
Paulson H, Davidson B (2005). “RNA interference improves motor and
neuropathological abnormalities in a Huntington;s disease mouse
model. . Proc. Natl. Acad. Sci. U.S.A. 102 (16):
 Nielsen M, Pedersen F, Kjems J (2005).” Molecular strageies to inhibit
HIV-1 replication”. Retrovirology 2: 10. 
 Paddison P, Caudy A, Bernstein E, Hannon G, Conklin D (2002). "Short
hairpin RNAs (shRNAs) induce sequence-specific silencing in mammalian
cells". Genes Dev. 16 (8): 948–58. 
 Cao W, Hunter R, Strnatka D, McQueen C, Erickson R (2005). "DNA
constructs designed to produce short hairpin, interfering RNAs in
transgenic mice sometimes show early lethality and an interferon
response“. J. Appl. Genet. 46 (2): 217–25. 

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 Acknowledgment
Ms. Charlene Chan-Lee
Science Research Director
Manhattan Center for Science and Mathematics

Dr. Michel Sadelain

Somatic Cell Engineering and Gene Transfer Lab Director
Memorial Sloan-Kettering Cancer Center

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