Page 120 - SRPSKO DRUŠTVO ISTRAŽIVAČA RAKA

Serbian Association for Cancer Research SDIRSACR

P23

Design and functional validation of self-deliverable siRNA for highly efficient suppression of PD-1
expression

Alexey Bogdanov, Anton Kornev, Andrey Bogdanov, Vladimir Moiseyenko

N.P. Napalkov Saint Petersburg Clinical Research and Practical Center of Specialized Types of Medical Care (Oncological), Saint
Petersburg, Russia

Keywords: immunotherapy, PD-1, siRNA

Background: The unique ability of small interfering RNAs (siRNAs) to suppress the expression of target genes
makes them a promising tool in oncology, offering effective and safe competition to both low-molecular-weight
targeted drugs and monoclonal antibodies. The possibility of targeting siRNAs to specific genes, even in the
presence of individual mutations, opens up new opportunities for personalized oncology. However, the main
and still unresolved challenge regarding siRNA application is the difficulty of delivering them into target cells,
which hinders their adoption into clinical practice. Therefore, the aim of this study was to develop a unique
cholesterol-modified siRNA molecule to silence the gene encoding the immune checkpoint protein PD-1.
Materials and Methods: A standard protocol was used for isolating T-lymphocytes from peripheral blood
by density gradient centrifugation (e.g., using Ficoll-Paque), after which T-lymphocytes were isolated
from the mononuclear fraction using immunomagnetic separation. The passenger strand of a unique
siRNA designed through bioinformatic methods was conjugated to a cholesterol molecule via a TEG linker.
Results: Flow cytometric analysis of standard-activated T-lymphocytes showed that CD3-positive cells comprised 95
± 6% of the samples. Flow cytometry results also demonstrated that within 6 hours after adding antiPD-1 siRNA-FAM
at a concentration of 2 µM into the culture medium, more than 80% of T-lymphocytes contained the FAM label. This
percentage decreased over time, likely due to siRNA degradation followed by the release and/or fading of the fluorescent
label. Additionally, changes in the mRNA levels of the PDCD1 gene in activated T-lymphocytes were evaluated by qPCR at
24 and 48 hours after the addition of antiPD-1 siRNA (2 µM) to the culture medium. Twenty-four hours after treatment,
the level of PDCD1 mRNA decreased by more than 80%, and after 48 hours it remained suppressed by more than 60%.
Conclusions: The obtained results demonstrate the high efficiency of the developed self-deliverable antiPD-1 siRNA.
The transfection efficiency and gene expression suppression levels either match or exceed those reported previously
for PD-1-targeting siRNAs in published studies.

Acknowledgments and funding: This work was supported by the Health Committee of Saint Petersburg state
assignment for N.P. Napalkov Saint Petersburg Clinical Research and Practical Center of Specialized Types of Medical
Care (Oncological).

P24

The impact of TGFB1 and IFNG gene expression on progression of metastatic melanoma patients
undergoing anti-PD-1 therapy

Luka Lisica1, Emina Mališić1, Milica Nedeljković1, Ana Vuletić1, Nina Petrović , Suzana Matković , Katarina Mirjačić
3
1,2
Martinović 1
1Department of Experimental Oncology, Institute for Oncology and Radiology of Serbia, Belgrade, Serbia
2Laboratory for Radiobiology and Molecular Genetics, “Vinča“ Institute of Nuclear Sciences-National Institute of the Republic of
Serbia, University of Belgrade, Belgrade, Serbia
3Department of Medical Oncology, Institute of Oncology and Radiology of Serbia, Belgrade, Serbia.

Keywords: Interferon-gamma, Melanoma, Pembrolizumab, Transforming Growth Factor beta 1

Background: PD-1 checkpoint inhibitors like Pembrolizumab have improved outcomes in patients with metastatic
melanoma (MM), but some patients still have disease progression, indicating the need for biomarkers of treatment
response. Cytokines like transforming growth factor beta-1 (TGFB1) and interferon-gamma (IFNG) are implicated in
immune regulation and tumor progression. Their predictive role in immunotherapy deserves further exploration.

105