Discovery of siRNA Lipid Nanoparticles to Transfect Suspension Leukemia Cells and Provide In Vivo Delivery Capability

Abstract

As a powerful research tool, siRNA's therapeutic and target validation utility with leukemia cells and long-term gene knockdown is severely restricted by the lack of omnipotent, safe, stable, and convenient delivery. Here, we detail our discovery of siRNA-containing lipid nanoparticles (LNPs) able to effectively transfect several leukemia and difficult-to-transfect adherent cell lines also providing in vivo delivery to mouse spleen and bone marrow tissues through tail-vein administration. We disclose a series of novel structurally related lipids accounting for the superior transfection ability, and reveal a correlation between expression of Caveolins and successful transfection. These LNPs, bearing low toxicity and long stability of >6 months, are ideal for continuous long-term dosing. Our discovery represents the first effective siRNA-containing LNPs for leukemia cells, which not only enables high-throughput siRNA screening with leukemia cells and difficult-to-transfect adherent cells but also paves the way for the development of therapeutic siRNA for leukemia treatment.

Polymeric nanoparticle-mediated silencing of CD44 receptor in CD34+ acute myeloid leukemia cells

“…Furthermore, siRNA silencing was much more effective (∼63%) in K562 cells (a CML cell line) than in THP-1 cells and in LSPC using PEI1.2-PA but not PEI2-LA [39], indicating that siRNA delivery in leukemic cells is dependent on the nature of the lipid substituent. The different efficiencies of siRNA silencing in leukemic cells could be explained by the different targets used, endocytic activities or endosomal processing pathway gene expression (Caveolin-1 and -2) in the target cells, as recently claimed [38]. Our CD44 silencing strategy using polymeric nanoparticles is more efficient compared to silencing of Raf-1 and Bcl-2 proteins in AML cells using the synthetic carrier Oligofectamine, which required a very high siRNA concentration (400 nM) that is not clinically practical [32].…”

“…Moreover, it was recently reported that the same siRNA silencing efficiency in KG-1 cells was obtained with a 2.5-fold higher siRNA concentration using lipid nanoparticle-based delivery [38]. However, lipid nanoparticle-based siRNA silencing was highly efficient (∼90%) at low siRNA concentration (50 nM) in other leukemic cell lines, including THP-1, HEL and K562 cells [38], indicating a higher efficiency for this system [34,39].…”

“…Our CD44 silencing strategy using polymeric nanoparticles is more efficient compared to silencing of Raf-1 and Bcl-2 proteins in AML cells using the synthetic carrier Oligofectamine, which required a very high siRNA concentration (400 nM) that is not clinically practical [32]. Moreover, it was recently reported that the same siRNA silencing efficiency in KG-1 cells was obtained with a 2.5-fold higher siRNA concentration using lipid nanoparticle-based delivery [38]. However, lipid nanoparticle-based siRNA silencing was highly efficient (∼90%) at low siRNA concentration (50 nM) in other leukemic cell lines, including THP-1, HEL and K562 cells [38], indicating a higher efficiency for this system [34,39].…”

“…Use of RNAi in leukemic cells mainly relies on viral delivery [35,36], which is not amenable for clinical practice due to the undesirable consequences of the viral integration process and potential development of unwanted immune responses against vectors [37]. In fact, siRNA silencing in LSPC has been shown to be the most challenging [38], making siRNA-mediated anti-CD44 approaches a difficult therapeutic modality. To overcome this limitation, we recently developed functional lipid-substituted polymeric materials that could efficiently deliver siRNA in both adherent breast cancer cells and leukemic cells grown in suspension [25][26][27]34,39].…”

Harnessing RNAi-based nanomedicines for therapeutic gene silencing in B-cell malignancies

“…However, the clinical application of this approach is hindered by the lack of appropriate systems that could deliver RNAi payloads to MCL cells in an efficient and safe manner (10,11). RNAi therapeutics for B-cell malignancies is especially challenging because these cells are dispersed and are intrinsically resistant to transfection with nucleic acids (3,12,13). Therefore, to test the potential therapeutic effect of cycD1 inhibition in vivo and to demonstrate the feasibility of RNAi therapeutics in MCL, we needed to develop a suitable RNAidelivery platform for potent gene silencing.…”