(68b) Delivery Materials to Induce RNAi in Bone Marrow to Control Hematopoietic Stem Cell Trafficking

Delivery Materials to Induce RNAi in Bone Marrow to Control
Hematopoietic Stem Cell Trafficking

Michael J. Mitchell¹_,
Robert Langer¹

¹Department of Chemical Engineering, Koch Institute for
Integrative Cancer Research, MIT, Cambridge, MA 02139

Introduction: Hematopoietic
stem cell (HSC) transplantations (HSCT) are essential for restoring a patientÕs
immune system destroyed by radiation or chemotherapy. However, HSCT remains a
dangerous procedure for patients with life-threatening diseases, with a major
complication being harvesting HSCs from blood to restore the recipientÕs immune
system. Here, we developed a nanoparticle (NP) platform to induce RNAi in bone
marrow (BM) in vivo to increase HSCs in
blood for harvesting and transplantation (Fig 1A,B).

Methods: siRNA targeting stromal-derived
factor-1 (SDF-1) and luciferase (Luc) were encapsulated in NPs consisting of
lipids and modified polyethylenimine via rapid microfluidic mixing. NPs were
characterized using DLS, SEM, and cryo-TEM. BM endothelial
cells (BMECs) were treated with NPs at siRNA dosages of 1-60 nM in vitro. C57/BL6
mice were treated with NPs at a siRNA dosage of 1.0
mg/kg to determine in vivo SDF-1
knockdown. HSCs were harvested from blood 1-week after initial NP treatment and
characterized via flow cytometry as Lin^-Sca-1^-c-Kit⁺
(LSK) cells.

Figure 1: (A) HSC homing into marrow via SDF-1. (B) SDF-1 knockdown
via in vivo NP-mediated RNAi reduces
homing and increase HSCs within blood. (C) Cryo-TEM
micrograph of lipid-polymer NPs encapsulated with siRNA. Scale bar: 100
nm.  (D) NP-mediated siRNA (red)
uptake in BMECs. Scale bar: 10 μm (E)
NP-mediated SDF-1 knockdown in BMECs. (F) Lin^-Sca-1^-c-Kit⁺
(LSK) HSCs isolated from blood per 10⁶ peripheral blood mononuclear
cells (PBMCs) 1-week post-SDF-1 knockdown. *P<0.05. **P<0.01.
***P<0.001. NS: not significant.

Results
and Discussion: 60
nm polymer-lipid NPs containing siSDF-1 were formulated via microfluidic mixing
(Fig 1C). NPs containing fluorescent siRNA were uptaken
by BMECs as per confocal micrographs (Fig 1D). NPs containing siSDF-1 induced
potent gene knockdown in BMECs, compared to siLuc
controls (Fig 1E). In vivo, silencing
of SDF-1 induced a nearly 300% increase in the number of HSCs harvested from
blood, compared to controls (Fig 1F).

Conclusions: We have
developed a NP platform that, potentially for the first time, induces potent
gene knockdown in the bone marrow microenvironment in vivo. Clinically, this platform can be used to silence BM factors to increase
the number of HSCs in blood available for harvesting and subsequent
transplantation.