KENYA JOSEPH

Interactions of siRNA Functionalized Therapeutic RNA Nanoparticles with Whole Blood & Isolated Lymphocytes

• Introduction p. 3-5• Research Objective p. 6• What is siRNA? p. 7• siRNAs as Nanoparticles p. 8• siRNA Nanoparticle Production p. 9• Process Advantages p. 10• Methods p. 11• Results p. 12-15• Conclusions p. 16• Discussion p. 17• References p. 18• Acknowledgments p. 19• Questions p. 20

Table of Contents

www.home.ccr.cancer.gov

• Newest frontier of drug delivery and disease treatment

• Targets

• Cancers• CNS and Brain Disorders• Potential for infectious disease

• Why nucleic acids?

• fully customizable• programmable • can carry multiple functionalities

Introduction

• Nucleic acid based nanoparticles functionalized with multiple short interference RNAs (siRNAs)

• Can be other therapeutic oligonucleotides

• Formulated with lipid-like carriers

• Can perform efficient intracellular delivery OR • Can act as the active pharmaceutical ingredient itself

Introduction

• It is important to study how siRNA nanoparticles interact with blood and lymphocytes

• to elucidate efficacy

• to examine blood stability

• Identify possible undesirable side-effects

Introduction

• To examine the cellular uptake by whole blood and by lymphocyte isolations from human donors of several fluorescently tagged functional RNA nanoparticles selected from the laboratory library.

• Observing three different siRNA nanoparticle designs, how well do lymphocytes take up these particles?

• Are there differences in uptake between two different transfection agents, Lipofectamine and PgP?

Research Objective

• Small or short interfering RNA• Double-stranded RNA• 20-25 base pairs• Synthetic, micro RNA

molecule• Works within the RNA

interference (RNAi) pathway• Interferes with expression of

specific genes with complementary nucleotide sequences by degrading mRNA after transcription

What is siRNA?

www.scbt.com

siRNA as Nanoparticles• Requirements for the nanoparticle carrier• Protection of siRNA from degradation• Enrichment of the siRNA in target tissue• Facilitate cellular uptake of siRNA

• Why use siRNA?• DNA is too large to diffuse across the cell membrane• Effective targeting of specific tissue or cells

siRNA Nanoparticle Production• Co-transcriptional One-Pot

Assembly• Start with DNA templates for the

RNA nanoparticle of interest in a transcription mixture

• Add T7 RNA Polymerase• In vitro one-pot transcription• RNA is formed and self-assembles

into functionalized RNA nanoparticles

• The particles are purified• The siRNAs are released by a

cell’s Dicer molecule

• Co-transcriptional One-Pot Assembly Advantages

• Programmability (different modules at different ratios)

• High one-pot yields of assembly (>90%)

• Relatively small sizes (Rh(nanocube)~6 nm, Rh(nanoring)~8nm)

• Relatively short sequences (nanocube 52 nts, nanoring 44 nts)

• Tunable thermodynamic and chemical stabilities

• Ability to introduce multiple different functionalities

• Can be formed co-transcriptionally

Process Advantages

• Co-transcriptional assembly of functionalized siRNA nanoparticles with Alexa fluorescent tags using T7 polymerase

• Healthy adults were brought into the lab between 7:30 and 8:30a

• Whole Blood was drawn and Lymphocytes isolated from each sample using a Histopaque isolation assay – 10 donors

• siRNA nanoparticles were transfected using Lipofectamine and PgP and incubated for 30 minutes at room temperature

• Whole Blood and isolated Lymphocytes were plated and diluted twice at 50 ul:1 ml of PBS

• Transfected nanoparticles were introduced to the blood and lymphocyte samples, comparing samples with transfection added prior to or after dilution

• Samples were analyzed using BD Accuri C6 flow cytometer to observe fluorescent shift

Methods

• Figure 1 shows 2 fluorescent shifts from the Whole Blood Control• Using Lipofectamine as

transfection agent• shift 1 in red from the

introduction of the Nanoring • shift 2 in blue and green

from the introduction of the Cube and Duplex

• The Cube and Duplex experience greater uptake with a larger shift

Results

• Figure 2 shows a fluorescent shift from the Whole Blood Control in Black• Red - shift 1 from the Duplex

transfected with Lipofectamine

• Duplex transfected with PgP does not show a shift (Blue)

• Pgp may not be effective with duplex OR Pgp may be killing cells

Results

• Figure 3 shows a fluorescent shift from the Isolated Lymphocytes control in Pink• Blue - shift 1 from the

Nanoring transfected with Lipofectamine

Results

• Figure 4 shows a fluorescent shift from the Isolated Lymphocytes Control in Black• Blue - shift 1 from the Duplex

transfected with Lipofectamine.

• Duplex transfected with PgP does not show a shift (Red)

• Pgp may not be effective with duplex OR Pgp may be killing cells

Results

• Marked difference in nanoparticle uptake when the formulation is introduced after the samples are serially diluted as opposed to introduction to undiluted human blood samples

• Lymphocytes did not function as competently when isolated from whole blood

• Construct design affects nanoparticle uptake but all 3 constructs had uptake

• Nanoring & Cube design showed most effective uptake

• Lipofectamine worked better for transfection of these particles than PgP – which may be killing cells

Conclusions

• The results indicate that the currently used experimental protocol may affect apoptosis and cell morphology of lymphocytes thus promoting their interaction with nanoparticles.

• Further experimentation aims to examine • which constructs and lymphocyte cells have the most efficient

cellular uptake by blood cells• Ideal concentrations of nanoparticle administration in whole

blood• which lipid-like carrier works best for uptake • mechanisms for cellular entry.

Discussion

This research was funded via internal funds from UNC Charlotte. Thank you to Dr. K. Afonin and the collaboration formed with Dr. J. Bennett of the StressWAVES Biobehavioral Research Lab.

Acknowledgements

1. Afonin, K. A., Bindewald, E., Yaghoubian, A. J., Voss, N., Jacovetty, E., Shapiro, B. A., & Jaeger, L. (2010). In vitro assembly of cubic RNA-based scaffolds designed in silico. Nature Nanotechnology, 5(9), 676–682. http://doi.org/10.1038/nnano.2010.160

2. Afonin, K. A., Grabow, W. W., Walker, F. M., Bindewald, E., Dobrovolskaia, M. A., Shapiro, B. A., & Jaeger, L. (2011). Design and self-assembly of siRNA-functionalized RNA nanoparticles for use in automated nanomedicine. Nature Protocols, 6(12), 2022–2034. http://doi.org/10.1038/nprot.2011.418

3. Afonin, K. A., Kireeva, M., Grabow, W. W., Kashlev, M., Jaeger, L., & Shapiro, B. A. (2012). Co-transcriptional Assembly of Chemically Modified RNA Nanoparticles Functionalized with siRNAs. Nano Letters, 12(10), 5192–5195. http://doi.org/10.1021/nl302302e

4. Afonin, K. A., Viard, M., Kagiampakis, I., Case, C. L., Dobrovolskaia, M. A., Hofmann, J., … Shapiro, B. A. (2015). Triggering of RNA Interference with RNA–RNA, RNA–DNA, and DNA–RNA Nanoparticles. ACS Nano, 9(1), 251–259. http://doi.org/10.1021/nn504508s

5. Grabow, W. W., Zakrevsky, P., Afonin, K. A., Chworos, A., Shapiro, B. A., & Jaeger, L. (2011). Self-Assembling RNA Nanorings Based on RNAI/II Inverse Kissing Complexes. Nano Letters, 11(2), 878–887. http://doi.org/10.1021/nl104271s

References

Questions?