Delivery of siRNA to the Mouse Lung via a Functionalized Lipopolyamine

Abstract

We have designed a series of versatile lipopolyamines which are amenable to chemical modification for in vivo delivery of small interfering RNA (siRNA). This report focuses on one such lipopolyamine (Staramine), its functionalized derivatives and the lipid nanocomplexes it forms with siRNA. Intravenous (i.v.) administration of Staramine/siRNA nanocomplexes modified with methoxypolyethylene glycol (mPEG) provides safe and effective delivery of siRNA and significant target gene knockdown in the lungs of normal mice, with much lower knockdown in liver, spleen, and kidney. Although siRNA delivered via Staramine is initially distributed across all these organs, the observed clearance rate from the lung tissue is considerably slower than in other tissues resulting in prolonged siRNA accumulation on the timescale of RNA interference (RNAi)-mediated transcript depletion. Complete blood count (CBC) analysis, serum chemistry analysis, and histopathology results are all consistent with minimal toxicity. An in vivo screen of mPEG modified Staramine nanocomplexes-containing siRNAs targeting lung cell-specific marker proteins reveal exclusive transfection of endothelial cells. Safe and effective delivery of siRNA to the lung with chemically versatile lipopolyamine systems provides opportunities for investigation of pulmonary cell function in vivo as well as potential treatments of pulmonary disease with RNAi-based therapeutics.

A High-Throughput Method for Direct Detection of Therapeutic Oligonucleotide-Induced Gene SilencingIn Vivo

“…1) [17,27]. It has been routinely used as an alternative for detection of mRNA expression and siRNA efficacy in cell culture assays for the past decade [15,[17][18][19][20][21]. Here, we evaluated the merit of using the bDNA assay for detecting mRNA expression directly from tissue biopsies, by using the automatic TissueLyser II (Qiagen) for tissue homogenization, followed with direct detection of mRNA levels by bDNA (see Materials and Methods for experimental details).…”

“…This method utilizes a plate-based, luminescent assay to directly assess the amount of mRNA in 96 samples simultaneously, without the need to isolate RNA or include a standard curve [17]. It has been used routinely for diagnostics, and to a lesser extent, to assess the efficacy of oligonucleotides in vivo [15,[17][18][19][20][21]. A previous report compared qRT-PCR to the QuantiGene assay and determined that QuantiGene has a better linear range and relative accuracy versus qRT-PCR [22].…”

siRNA Delivery to the lung: What's new?

“…To determine the biodistribution, the authors made use of a stem-loop qPCR protocol, which is a sophisticated, label-free method but is not able to detect degraded siRNA in contrast to label-based methods that may overestimate the presence of siRNA in tissues due to detecting the label only. While the GFP knockdown was only modest in the lung, CD31 was knocked down by 70%, which reflects the passive targeting of endothelial cells [69]. While the staramine lioplexes were PEGylated, Schlegel et al decided to PEGylate half of their formulations and to decrease the surface charge of their 2-{3-[Bis-(3-amino-propyl)-amino]-propylamino}-N-ditetradecyl carbamoyl methyl-acetamide (DMAPAP) lipoplexes by adding biodegradable anionic polymers of varying nature, namely alginic acid (AA), poly-L-glutamic, acid (PG), dextran sulfate (DS), polyacrylic acid (PAA), heparan sulfate (HS), sodium carboxymethyl cellulose (CMC) and hyaluronic acid (HA).…”

“…Since the establishment of tumor and metastasis models requires very sophisticated knowledge and handling, many studies performed simpler proof-of-principle experiments in which they knocked down the expression of the reporter genes (Enhanced) Green Fluorescent Protein ((e)GFP) [63, 69, 88, 108, 123] or Luciferase [31, 117]. These modes are especially helpful for in vitro studies in which the performance of a carrier is determined or optimized [117, 123], or for in vivo experiments that are interested in determining the biodistribution [31, 69, 88, 108, 148] or optimizing the formulation [148].…”

“…These modes are especially helpful for in vitro studies in which the performance of a carrier is determined or optimized [117, 123], or for in vivo experiments that are interested in determining the biodistribution [31, 69, 88, 108, 148] or optimizing the formulation [148]. …”