Periodic Report Summary - RNAI IN HSC VIA NP (Delivery of siRNAs to hematopoietic stem cells using nanoparticles)
A summary description of the project objectives:
The goal of this proposal is to explore the hypothesis that targeted and stabilised nanoparticles (tsNP) entrapping siRNAs can be developed to induce in vitro and in vivo gene silencing in hematopoietic stem cells. Utilising this system, we plan to identify the major players that are responsible for self-renewal properties of pHSC in physiological settings and under environmental stress. In particular, our aims are to:
1. develop siRNA delivery strategies to target pHSC;
2. investigate ex vivo and in vivo the ability of the siRNAs-targeted nanoparticles to efficiently silence gene expression specifically in pHSC;
3. identify and validate key genes responsible for self-renewal properties in pHSC.
A description of the work performed since the beginning of the project and the main results obtained:
During the first two years of the project (1 January 2010 - 31 December 2011), we worked in parallel to achieve aim 1 of the project and start (in parallel aim 2 and 3).
We created nano-scale (about 100 nm) unilamellar vesicle (ULV) coated with hyaluronan (HA). HA is stabilising the particles, both during subsequent RNAi entrapment, and during systemic circulation in vivo. The resulting stabilised nanoparticles (tsNP) were equipped with a targeting capacity by covalently attaching the hyaluronan with monoclonal antibody (mAb) against a cell surface marker Sca-1. The mAb was purified from a unique hybridoma that we received from Prof. Irving Weissmann from Stanford University, we optimised the culturing conditions and purified the mAb against Sca-1.
We have prepared and characterised in different methods (dynamic light scattering, different electron microscopy techniques, surface charge) the particles.
We then, purify and facs sorted heamtopoitic stem and progenitors cells (HSPC) and tested the binding of our sca-1-tageted and stabilised nanoparticles (sca-1-tsNPs) to HSPC compared to control particles that do not recognise the receptor (IgG-sNPs).
We show that the biding of the Sca-1-tsNPs to primary HSPC is as good as free mAb (and even better). We then wanted to see if we could observe internalisation of the Sca-1-tsNPs into primary HSPC. In order to observe internalisation, we pre-labeled the Sca-1 or its isotype control mAb with Alexa 488 dye. We then immobilised the mAb on the surface of the NPs using an amine coupling chemistry (Sulfo-NHS / EDC). We next, loaded tsNPs with RNAi by rehydrating lyophilised particles in the presence of protamine condensed Cy3-siRNAs as we previously demonstrated (1) and improve the chemistry and purification steps as reported recently (1 - 3). We observed full internalisation in primary HSPC using confocal microscopy and live cell imaging techniques.
Next, we tested for gene silencing of a reference gene is expressed on HSPC. We chose CD45, leukocytes marker, as a surrogate marker to examine ex vivo knockdown using the Sca-1-tsNPs. We observed a robust and specific knockdown of CD45 both at the mRNA level and the protein level 48 h (mRNA) and 72-96 h (protein level) of the CD45 in a Sca-1 dependent manner. When incubated with IgG sNPs (control particles) loaded with CD45-siRNAs no silencing was observed ex vivo, as well as when we used Sca-1-tsNPs with control siRNA (luciferase).
Next, we asked if indeed the Sca-1-tsNPs can reach the bone marrow HSPC. We labelled the tsNPs with Rhodamine and the Sca-1 mAb or its isotype control with Alexa 647 and injected the particles into the tail vein of healthy C57BL/6 mice.
With two hours post administration, BM was isolated, HSPC were FACS sorted and purify and then the cells that were positive for Sca-1 and the NPs (double positive) were detected. We could not detect any of the following control in the bone marrow: IgG-sNPs (control particles) or NPs coated with hyaluronan without any mAb.
It is obvious that we need to understand the kinetics of the particles in vivo and more time points are needed (1, 3, 6 and 12 h post injection). These experiments are underway.
We then, start using a GFP mice which is brightest in its HSPC. Next, we loaded the Sca-1-tsNPs with siRNAs against EGFP and administrated into the GFP mice. 72 h post administration, we observed knockdown of the GFP from isolated HSPC compare to control particles (IgG sNPs).
1. Peer D., Park E. J., Morishita Y., Carman C. V., Shimaoka M. Systemic leukocyte-directed siRNA delivery revealing cyclin D1 as an anti-inflammatory target. Science. 1 February 2008;319(5863):627 - 30.
2. Kim S. S., Peer D., Kumar P., Subramanya S., Wu H., Asthana D., et al. RNAi-mediated CCR5 silencing by LFA-1-targeted nanoparticles prevents HIV infection in BLT mice. Mol Ther. February 2010;18(2):370 - 376.
3. Ben-Arie N., Kedmi R., Peer D. Integrin-targeted nanoparticles for siRNA delivery. Methods Mol Biol. 2012;757:497 - 507.
The goal of this proposal is to explore the hypothesis that targeted and stabilised nanoparticles (tsNP) entrapping siRNAs can be developed to induce in vitro and in vivo gene silencing in hematopoietic stem cells. Utilising this system, we plan to identify the major players that are responsible for self-renewal properties of pHSC in physiological settings and under environmental stress. In particular, our aims are to:
1. develop siRNA delivery strategies to target pHSC;
2. investigate ex vivo and in vivo the ability of the siRNAs-targeted nanoparticles to efficiently silence gene expression specifically in pHSC;
3. identify and validate key genes responsible for self-renewal properties in pHSC.
A description of the work performed since the beginning of the project and the main results obtained:
During the first two years of the project (1 January 2010 - 31 December 2011), we worked in parallel to achieve aim 1 of the project and start (in parallel aim 2 and 3).
We created nano-scale (about 100 nm) unilamellar vesicle (ULV) coated with hyaluronan (HA). HA is stabilising the particles, both during subsequent RNAi entrapment, and during systemic circulation in vivo. The resulting stabilised nanoparticles (tsNP) were equipped with a targeting capacity by covalently attaching the hyaluronan with monoclonal antibody (mAb) against a cell surface marker Sca-1. The mAb was purified from a unique hybridoma that we received from Prof. Irving Weissmann from Stanford University, we optimised the culturing conditions and purified the mAb against Sca-1.
We have prepared and characterised in different methods (dynamic light scattering, different electron microscopy techniques, surface charge) the particles.
We then, purify and facs sorted heamtopoitic stem and progenitors cells (HSPC) and tested the binding of our sca-1-tageted and stabilised nanoparticles (sca-1-tsNPs) to HSPC compared to control particles that do not recognise the receptor (IgG-sNPs).
We show that the biding of the Sca-1-tsNPs to primary HSPC is as good as free mAb (and even better). We then wanted to see if we could observe internalisation of the Sca-1-tsNPs into primary HSPC. In order to observe internalisation, we pre-labeled the Sca-1 or its isotype control mAb with Alexa 488 dye. We then immobilised the mAb on the surface of the NPs using an amine coupling chemistry (Sulfo-NHS / EDC). We next, loaded tsNPs with RNAi by rehydrating lyophilised particles in the presence of protamine condensed Cy3-siRNAs as we previously demonstrated (1) and improve the chemistry and purification steps as reported recently (1 - 3). We observed full internalisation in primary HSPC using confocal microscopy and live cell imaging techniques.
Next, we tested for gene silencing of a reference gene is expressed on HSPC. We chose CD45, leukocytes marker, as a surrogate marker to examine ex vivo knockdown using the Sca-1-tsNPs. We observed a robust and specific knockdown of CD45 both at the mRNA level and the protein level 48 h (mRNA) and 72-96 h (protein level) of the CD45 in a Sca-1 dependent manner. When incubated with IgG sNPs (control particles) loaded with CD45-siRNAs no silencing was observed ex vivo, as well as when we used Sca-1-tsNPs with control siRNA (luciferase).
Next, we asked if indeed the Sca-1-tsNPs can reach the bone marrow HSPC. We labelled the tsNPs with Rhodamine and the Sca-1 mAb or its isotype control with Alexa 647 and injected the particles into the tail vein of healthy C57BL/6 mice.
With two hours post administration, BM was isolated, HSPC were FACS sorted and purify and then the cells that were positive for Sca-1 and the NPs (double positive) were detected. We could not detect any of the following control in the bone marrow: IgG-sNPs (control particles) or NPs coated with hyaluronan without any mAb.
It is obvious that we need to understand the kinetics of the particles in vivo and more time points are needed (1, 3, 6 and 12 h post injection). These experiments are underway.
We then, start using a GFP mice which is brightest in its HSPC. Next, we loaded the Sca-1-tsNPs with siRNAs against EGFP and administrated into the GFP mice. 72 h post administration, we observed knockdown of the GFP from isolated HSPC compare to control particles (IgG sNPs).
1. Peer D., Park E. J., Morishita Y., Carman C. V., Shimaoka M. Systemic leukocyte-directed siRNA delivery revealing cyclin D1 as an anti-inflammatory target. Science. 1 February 2008;319(5863):627 - 30.
2. Kim S. S., Peer D., Kumar P., Subramanya S., Wu H., Asthana D., et al. RNAi-mediated CCR5 silencing by LFA-1-targeted nanoparticles prevents HIV infection in BLT mice. Mol Ther. February 2010;18(2):370 - 376.
3. Ben-Arie N., Kedmi R., Peer D. Integrin-targeted nanoparticles for siRNA delivery. Methods Mol Biol. 2012;757:497 - 507.