EHA Library - The official digital education library of European Hematology Association (EHA)

IN VITRO VALIDATION OF DENDRIMER-BASED APPROACH FOR MICRO-RNA DELIVERY INTO LEUKEMIA CELLS
Author(s): ,
Nadezhda Knauer
Affiliations:
Laboratory of clinical immunopathology,Research Institute of fundamental and clinical immunology,Novosibirsk,Russie, Fédération De;Laboratory of clinical immunopathology,Research Institute of fundamental and clinical immunology,Novosibirsk,Russia;Laboratory of clinical immunopathology,Research Institute of fundamental and clinical immunology,Novosibirsk,Russia;Laboratory of clinical immunopathology,Research Institute of fundamental and clinical immunology,Novosibirsk,Russische Federatie;Laboratory of clinical immunopathology,Research Institute of fundamental and clinical immunology,Novosibirsk,Rússia;Laboratory of clinical immunopathology,Research Institute of fundamental and clinical immunology,Novosibirsk,Россия;Laboratory of RNA chemistry,Insitute of chemical biology and fundamental medicine ,Novosibirsk,Russie, Fédération De;Laboratory of RNA chemistry,Insitute of chemical biology and fundamental medicine ,Novosibirsk,Russia;Laboratory of RNA chemistry,Insitute of chemical biology and fundamental medicine ,Novosibirsk,Russia;Laboratory of RNA chemistry,Insitute of chemical biology and fundamental medicine ,Novosibirsk,Russische Federatie;Laboratory of RNA chemistry,Insitute of chemical biology and fundamental medicine ,Novosibirsk,Rússia;Laboratory of RNA chemistry,Insitute of chemical biology and fundamental medicine ,Novosibirsk,Россия;Department of preclinical studies,Heinrich Heine University,Düsseldorf,Allemagne;Department of preclinical studies,Heinrich Heine University,Düsseldorf,Deutschland;Department of preclinical studies,Heinrich Heine University,Düsseldorf,Germania;Department of preclinical studies,Heinrich Heine University,Düsseldorf,Germany;Department of preclinical studies,Heinrich Heine University,Düsseldorf,Alemania;Department of preclinical studies,Heinrich Heine University,Düsseldorf,Duitsland;Department of preclinical studies,Heinrich Heine University,Düsseldorf,Alemanha;Department of preclinical studies,Heinrich Heine University,Düsseldorf,Германия;Department of preclinical studies,Heinrich Heine University,Düsseldorf,Tyskland
,
Ekaterina Pashkina
Affiliations:
Laboratory of clinical immunopathology,Research Institute of fundamental and clinical immunology,Novosibirsk,Russie, Fédération De;Laboratory of clinical immunopathology,Research Institute of fundamental and clinical immunology,Novosibirsk,Russia;Laboratory of clinical immunopathology,Research Institute of fundamental and clinical immunology,Novosibirsk,Russia;Laboratory of clinical immunopathology,Research Institute of fundamental and clinical immunology,Novosibirsk,Russische Federatie;Laboratory of clinical immunopathology,Research Institute of fundamental and clinical immunology,Novosibirsk,Rússia;Laboratory of clinical immunopathology,Research Institute of fundamental and clinical immunology,Novosibirsk,Россия
,
Olga Boeva
Affiliations:
Faculty of medicine,Novosibirsk State University,Novosibirsk,Russie, Fédération De;Faculty of medicine,Novosibirsk State University,Novosibirsk,Russia;Faculty of medicine,Novosibirsk State University,Novosibirsk,Russia;Faculty of medicine,Novosibirsk State University,Novosibirsk,Russische Federatie;Faculty of medicine,Novosibirsk State University,Novosibirsk,Rússia;Faculty of medicine,Novosibirsk State University,Novosibirsk,Россия
,
Alina Aktanova
Affiliations:
Laboratory of clinical immunopathology,Research Institute of fundamental and clinical immunology,Novosibirsk,Russie, Fédération De;Laboratory of clinical immunopathology,Research Institute of fundamental and clinical immunology,Novosibirsk,Russia;Laboratory of clinical immunopathology,Research Institute of fundamental and clinical immunology,Novosibirsk,Russia;Laboratory of clinical immunopathology,Research Institute of fundamental and clinical immunology,Novosibirsk,Russische Federatie;Laboratory of clinical immunopathology,Research Institute of fundamental and clinical immunology,Novosibirsk,Rússia;Laboratory of clinical immunopathology,Research Institute of fundamental and clinical immunology,Novosibirsk,Россия
,
Valeria Arkhipova
Affiliations:
Laboratory of RNA chemistry,Insitute of chemical biology and fundamental medicine,Novosibirsk,Russie, Fédération De;Laboratory of RNA chemistry,Insitute of chemical biology and fundamental medicine,Novosibirsk,Russia;Laboratory of RNA chemistry,Insitute of chemical biology and fundamental medicine,Novosibirsk,Russia;Laboratory of RNA chemistry,Insitute of chemical biology and fundamental medicine,Novosibirsk,Russische Federatie;Laboratory of RNA chemistry,Insitute of chemical biology and fundamental medicine,Novosibirsk,Rússia;Laboratory of RNA chemistry,Insitute of chemical biology and fundamental medicine,Novosibirsk,Россия
,
Maria Meschaninova
Affiliations:
Laboratory of RNA chemistry,Insitute of chemical biology and fundamental medicine,Novosibirsk,Russie, Fédération De;Laboratory of RNA chemistry,Insitute of chemical biology and fundamental medicine,Novosibirsk,Russia;Laboratory of RNA chemistry,Insitute of chemical biology and fundamental medicine,Novosibirsk,Russia;Laboratory of RNA chemistry,Insitute of chemical biology and fundamental medicine,Novosibirsk,Russische Federatie;Laboratory of RNA chemistry,Insitute of chemical biology and fundamental medicine,Novosibirsk,Rússia;Laboratory of RNA chemistry,Insitute of chemical biology and fundamental medicine,Novosibirsk,Россия
,
Raphael Gomez
Affiliations:
Department of Organic and Inorganic Chemistry,University of Alcala,Alcala de Henares, Madrid,Espagne;Department of Organic and Inorganic Chemistry,University of Alcala,Alcala de Henares, Madrid,Spanien;Department of Organic and Inorganic Chemistry,University of Alcala,Alcala de Henares, Madrid,Spagna;Department of Organic and Inorganic Chemistry,University of Alcala,Alcala de Henares, Madrid,Spain;Department of Organic and Inorganic Chemistry,University of Alcala,Alcala de Henares, Madrid,España;Department of Organic and Inorganic Chemistry,University of Alcala,Alcala de Henares, Madrid,Spanje;Department of Organic and Inorganic Chemistry,University of Alcala,Alcala de Henares, Madrid,Espanha;Department of Organic and Inorganic Chemistry,University of Alcala,Alcala de Henares, Madrid,Испания;Department of Organic and Inorganic Chemistry,University of Alcala,Alcala de Henares, Madrid,Spanien
,
Javier Sánchez-Nieves
Affiliations:
Department of Organic and Inorganic Chemistry,University of Alcala,Alcala de Henares, Madrid,Espagne;Department of Organic and Inorganic Chemistry,University of Alcala,Alcala de Henares, Madrid,Spanien;Department of Organic and Inorganic Chemistry,University of Alcala,Alcala de Henares, Madrid,Spagna;Department of Organic and Inorganic Chemistry,University of Alcala,Alcala de Henares, Madrid,Spain;Department of Organic and Inorganic Chemistry,University of Alcala,Alcala de Henares, Madrid,España;Department of Organic and Inorganic Chemistry,University of Alcala,Alcala de Henares, Madrid,Spanje;Department of Organic and Inorganic Chemistry,University of Alcala,Alcala de Henares, Madrid,Espanha;Department of Organic and Inorganic Chemistry,University of Alcala,Alcala de Henares, Madrid,Испания;Department of Organic and Inorganic Chemistry,University of Alcala,Alcala de Henares, Madrid,Spanien
,
Ann-Christin Nickel
Affiliations:
Department of preclinical studies,Heinrich Heine University,Düsseldorf,Allemagne;Department of preclinical studies,Heinrich Heine University,Düsseldorf,Deutschland;Department of preclinical studies,Heinrich Heine University,Düsseldorf,Germania;Department of preclinical studies,Heinrich Heine University,Düsseldorf,Germany;Department of preclinical studies,Heinrich Heine University,Düsseldorf,Alemania;Department of preclinical studies,Heinrich Heine University,Düsseldorf,Duitsland;Department of preclinical studies,Heinrich Heine University,Düsseldorf,Alemanha;Department of preclinical studies,Heinrich Heine University,Düsseldorf,Германия;Department of preclinical studies,Heinrich Heine University,Düsseldorf,Tyskland
,
Ulf Kahlert
Affiliations:
Molecular and Experimental Surgery, Clinic for General-, Visceral-, Vascular-, and Transplant Surgery,Medical Faculty and University Hospital Magdeburg,Magdeburg,Allemagne;Molecular and Experimental Surgery, Clinic for General-, Visceral-, Vascular-, and Transplant Surgery,Medical Faculty and University Hospital Magdeburg,Magdeburg,Deutschland;Molecular and Experimental Surgery, Clinic for General-, Visceral-, Vascular-, and Transplant Surgery,Medical Faculty and University Hospital Magdeburg,Magdeburg,Germania;Molecular and Experimental Surgery, Clinic for General-, Visceral-, Vascular-, and Transplant Surgery,Medical Faculty and University Hospital Magdeburg,Magdeburg,Germany;Molecular and Experimental Surgery, Clinic for General-, Visceral-, Vascular-, and Transplant Surgery,Medical Faculty and University Hospital Magdeburg,Magdeburg,Alemania;Molecular and Experimental Surgery, Clinic for General-, Visceral-, Vascular-, and Transplant Surgery,Medical Faculty and University Hospital Magdeburg,Magdeburg,Duitsland;Molecular and Experimental Surgery, Clinic for General-, Visceral-, Vascular-, and Transplant Surgery,Medical Faculty and University Hospital Magdeburg,Magdeburg,Alemanha;Molecular and Experimental Surgery, Clinic for General-, Visceral-, Vascular-, and Transplant Surgery,Medical Faculty and University Hospital Magdeburg,Magdeburg,Германия;Molecular and Experimental Surgery, Clinic for General-, Visceral-, Vascular-, and Transplant Surgery,Medical Faculty and University Hospital Magdeburg,Magdeburg,Tyskland
,
Jean-Pierre Majoral
Affiliations:
Laboratory of coordination chemistry,CNRS,Toulouse,France;Laboratory of coordination chemistry,CNRS,Toulouse,Frankreich;Laboratory of coordination chemistry,CNRS,Toulouse,Francia;Laboratory of coordination chemistry,CNRS,Toulouse,France;Laboratory of coordination chemistry,CNRS,Toulouse,Francia;Laboratory of coordination chemistry,CNRS,Toulouse,Frankrijk;Laboratory of coordination chemistry,CNRS,Toulouse,França;Laboratory of coordination chemistry,CNRS,Toulouse,Франция ;Laboratory of coordination chemistry,CNRS,Toulouse,Frankrike
,
Vladimir Kozlov
Affiliations:
Laboratory of clinical immunopathology,Research Institute of fundamental and clinical immunology,Novosibirsk,Russie, Fédération De;Laboratory of clinical immunopathology,Research Institute of fundamental and clinical immunology,Novosibirsk,Russia;Laboratory of clinical immunopathology,Research Institute of fundamental and clinical immunology,Novosibirsk,Russia;Laboratory of clinical immunopathology,Research Institute of fundamental and clinical immunology,Novosibirsk,Russische Federatie;Laboratory of clinical immunopathology,Research Institute of fundamental and clinical immunology,Novosibirsk,Rússia;Laboratory of clinical immunopathology,Research Institute of fundamental and clinical immunology,Novosibirsk,Россия
Evgeny Apartsin
Affiliations:
Laboratory of coordination chemistry,CNRS,Toulouse,France;Laboratory of coordination chemistry,CNRS,Toulouse,Frankreich;Laboratory of coordination chemistry,CNRS,Toulouse,Francia;Laboratory of coordination chemistry,CNRS,Toulouse,France;Laboratory of coordination chemistry,CNRS,Toulouse,Francia;Laboratory of coordination chemistry,CNRS,Toulouse,Frankrijk;Laboratory of coordination chemistry,CNRS,Toulouse,França;Laboratory of coordination chemistry,CNRS,Toulouse,Франция ;Laboratory of coordination chemistry,CNRS,Toulouse,Frankrike
EHA Library. Knauer N. 06/10/22; 359059; PB2207
Nadezhda Knauer
Nadezhda Knauer
Contributions
Abstract
References

Abstract: PB2207

Type: Publication Only

Session title: Gene therapy, cellular immunotherapy and vaccination - Biology & Translational Research

Background
microRNAs are known to be important players in the epigenetic regulation of many cell processes. Some of
them demonstrate anti-tumor properties (e.g. miR-34), others are associated with the enhancing of the tumor growth (e.g. miR-21), therefore being prospective effectors and targets for the gene therapy. For this aim, synthetic microRNAs or microRNA inhibitors can be used. We suggest to use dendrimers - highly symmetric hyperbranched polymers - as a novel type of nanocarriers for therapeutic RNAs delivery and gene therapy.

Aims
In this work we assessed the effects of microRNA-34a (miR-34a) and microRNA-21 inhibitor (anti-miR-21) on
Jurkat and 1301 cells (human T-cell leukemia) upon the dendrimer-mediated delivery.

Methods
As carriers for microRNAs, amphiphilic second- and third generation triazine-carbosilane dendrons (DG2
and DG3 respectively) and fully symmetrical 3rd generation carbosilane (BDEF33) and phosphorus (AE2G3) dendrimers have been taken. Oligonucleotides were complexed onto the surface of dendrimer vesicles by means of electrostatic interactions, forming dendriplexes. To prove the dendriplexes can be sufficiently internalized, we performed FACS analysis after 3 h of incubating cells with FITC-labelled microRNA.
Cells were cultivated with free dendrimers or complexes (25, 50, 100, 150 nM RNA, 10-fold excess of cations) in the complete cell medium for 72 h. We investigated cell viability (MTT assay, Annexin V/PI apoptosis assay) in comparison with non-tumor cells (PBMCs), IL-10 secretion and expression of several markers, characterizing interaction of tumor with immune microenvironment - PD-L1, TIM-3, CD47. Statistical analysis was done by using Mann-Whitney and Wilcoxon tests, differences between groups were considered significant if p<0.05.
This work was supported by RFBR grant No.18-33-20109, by the grant of the President of the Russian Federation No.2278.2019.4, by the HHU STIBET Scholarship grant support and based upon work from COST Action CA 17140 "Cancer Nanomedicine from the Bench to the Bedside" supported by COST (European Cooperation in Science and Technology).

Results
Free dendrimers have their own dose-dependent toxic effects, which is slightly higher for tumor cells, than for PBMCs. Treatment by free dendritic molecules led to increasing of expression of PD-L1, TIM-3 and CD47 on Jurkat cells, but decreased the IL-10 secretion.
Dendriplexes can be efficiently internalized into tumor cells, transport activity of dendrimers was higher than for Lipofectamine 3000 or was comparable. Dendron-based dendriplexes (DG2, DG3) had pro-apoptotic effect on leukemia cells. PD-L1 expression did not change after treatment by dendriplexes.

Conclusion
Dendrimers not onle have their own toxic effect per se, but also they can be used as effective
nanocarriers for therapeutic microRNAs. Using of complexes, containing microRNAs with anti-tumor activity, leads to induction of apoptosis of leukemia cells. Treatment by free dendrimers, but not by dendriplexes, increases expression of PD-L1, TIM-3, CD47 on Jurkat cells, but IL-10 secretion was found to be decreased.

We suppose, that dendrimer-based approach can be useful and perspective tool for delivery of therapeuthic microRNAs into tumor cells. At the same moment, characterization of their effects on interactions between tumor and immune microenvironment demands the further studies.

Keyword(s): Gene therapy, Leukemia cell line, Nanoparticle

Abstract: PB2207

Type: Publication Only

Session title: Gene therapy, cellular immunotherapy and vaccination - Biology & Translational Research

Background
microRNAs are known to be important players in the epigenetic regulation of many cell processes. Some of
them demonstrate anti-tumor properties (e.g. miR-34), others are associated with the enhancing of the tumor growth (e.g. miR-21), therefore being prospective effectors and targets for the gene therapy. For this aim, synthetic microRNAs or microRNA inhibitors can be used. We suggest to use dendrimers - highly symmetric hyperbranched polymers - as a novel type of nanocarriers for therapeutic RNAs delivery and gene therapy.

Aims
In this work we assessed the effects of microRNA-34a (miR-34a) and microRNA-21 inhibitor (anti-miR-21) on
Jurkat and 1301 cells (human T-cell leukemia) upon the dendrimer-mediated delivery.

Methods
As carriers for microRNAs, amphiphilic second- and third generation triazine-carbosilane dendrons (DG2
and DG3 respectively) and fully symmetrical 3rd generation carbosilane (BDEF33) and phosphorus (AE2G3) dendrimers have been taken. Oligonucleotides were complexed onto the surface of dendrimer vesicles by means of electrostatic interactions, forming dendriplexes. To prove the dendriplexes can be sufficiently internalized, we performed FACS analysis after 3 h of incubating cells with FITC-labelled microRNA.
Cells were cultivated with free dendrimers or complexes (25, 50, 100, 150 nM RNA, 10-fold excess of cations) in the complete cell medium for 72 h. We investigated cell viability (MTT assay, Annexin V/PI apoptosis assay) in comparison with non-tumor cells (PBMCs), IL-10 secretion and expression of several markers, characterizing interaction of tumor with immune microenvironment - PD-L1, TIM-3, CD47. Statistical analysis was done by using Mann-Whitney and Wilcoxon tests, differences between groups were considered significant if p<0.05.
This work was supported by RFBR grant No.18-33-20109, by the grant of the President of the Russian Federation No.2278.2019.4, by the HHU STIBET Scholarship grant support and based upon work from COST Action CA 17140 "Cancer Nanomedicine from the Bench to the Bedside" supported by COST (European Cooperation in Science and Technology).

Results
Free dendrimers have their own dose-dependent toxic effects, which is slightly higher for tumor cells, than for PBMCs. Treatment by free dendritic molecules led to increasing of expression of PD-L1, TIM-3 and CD47 on Jurkat cells, but decreased the IL-10 secretion.
Dendriplexes can be efficiently internalized into tumor cells, transport activity of dendrimers was higher than for Lipofectamine 3000 or was comparable. Dendron-based dendriplexes (DG2, DG3) had pro-apoptotic effect on leukemia cells. PD-L1 expression did not change after treatment by dendriplexes.

Conclusion
Dendrimers not onle have their own toxic effect per se, but also they can be used as effective
nanocarriers for therapeutic microRNAs. Using of complexes, containing microRNAs with anti-tumor activity, leads to induction of apoptosis of leukemia cells. Treatment by free dendrimers, but not by dendriplexes, increases expression of PD-L1, TIM-3, CD47 on Jurkat cells, but IL-10 secretion was found to be decreased.

We suppose, that dendrimer-based approach can be useful and perspective tool for delivery of therapeuthic microRNAs into tumor cells. At the same moment, characterization of their effects on interactions between tumor and immune microenvironment demands the further studies.

Keyword(s): Gene therapy, Leukemia cell line, Nanoparticle

Abstract: PB2207

Type: Publication Only

Session title: Gene therapy, cellular immunotherapy and vaccination - Biology & Translational Research

Background
microRNAs are known to be important players in the epigenetic regulation of many cell processes. Some of
them demonstrate anti-tumor properties (e.g. miR-34), others are associated with the enhancing of the tumor growth (e.g. miR-21), therefore being prospective effectors and targets for the gene therapy. For this aim, synthetic microRNAs or microRNA inhibitors can be used. We suggest to use dendrimers - highly symmetric hyperbranched polymers - as a novel type of nanocarriers for therapeutic RNAs delivery and gene therapy.

Aims
In this work we assessed the effects of microRNA-34a (miR-34a) and microRNA-21 inhibitor (anti-miR-21) on
Jurkat and 1301 cells (human T-cell leukemia) upon the dendrimer-mediated delivery.

Methods
As carriers for microRNAs, amphiphilic second- and third generation triazine-carbosilane dendrons (DG2
and DG3 respectively) and fully symmetrical 3rd generation carbosilane (BDEF33) and phosphorus (AE2G3) dendrimers have been taken. Oligonucleotides were complexed onto the surface of dendrimer vesicles by means of electrostatic interactions, forming dendriplexes. To prove the dendriplexes can be sufficiently internalized, we performed FACS analysis after 3 h of incubating cells with FITC-labelled microRNA.
Cells were cultivated with free dendrimers or complexes (25, 50, 100, 150 nM RNA, 10-fold excess of cations) in the complete cell medium for 72 h. We investigated cell viability (MTT assay, Annexin V/PI apoptosis assay) in comparison with non-tumor cells (PBMCs), IL-10 secretion and expression of several markers, characterizing interaction of tumor with immune microenvironment - PD-L1, TIM-3, CD47. Statistical analysis was done by using Mann-Whitney and Wilcoxon tests, differences between groups were considered significant if p<0.05.
This work was supported by RFBR grant No.18-33-20109, by the grant of the President of the Russian Federation No.2278.2019.4, by the HHU STIBET Scholarship grant support and based upon work from COST Action CA 17140 "Cancer Nanomedicine from the Bench to the Bedside" supported by COST (European Cooperation in Science and Technology).

Results
Free dendrimers have their own dose-dependent toxic effects, which is slightly higher for tumor cells, than for PBMCs. Treatment by free dendritic molecules led to increasing of expression of PD-L1, TIM-3 and CD47 on Jurkat cells, but decreased the IL-10 secretion.
Dendriplexes can be efficiently internalized into tumor cells, transport activity of dendrimers was higher than for Lipofectamine 3000 or was comparable. Dendron-based dendriplexes (DG2, DG3) had pro-apoptotic effect on leukemia cells. PD-L1 expression did not change after treatment by dendriplexes.

Conclusion
Dendrimers not onle have their own toxic effect per se, but also they can be used as effective
nanocarriers for therapeutic microRNAs. Using of complexes, containing microRNAs with anti-tumor activity, leads to induction of apoptosis of leukemia cells. Treatment by free dendrimers, but not by dendriplexes, increases expression of PD-L1, TIM-3, CD47 on Jurkat cells, but IL-10 secretion was found to be decreased.

We suppose, that dendrimer-based approach can be useful and perspective tool for delivery of therapeuthic microRNAs into tumor cells. At the same moment, characterization of their effects on interactions between tumor and immune microenvironment demands the further studies.

Keyword(s): Gene therapy, Leukemia cell line, Nanoparticle

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