Evidence of RNAi in humans from systemically administered siRNA via targeted nanoparticles

Abstract

Therapeutics that are designed to engage RNA interference (RNAi) pathways have the potential to provide new, major ways of imparting therapy to patients. Long, double-stranded RNAs were first shown to mediate RNAi in Caenorhabditis elegans, and the potential use of RNAi for human therapy has been demonstrated by the finding that small interfering RNAs (siRNAs; approximately 21-base-pair double-stranded RNA) can elicit RNAi in mammalian cells without producing an interferon response. We are at present conducting the first in-human phase I clinical trial involving the systemic administration of siRNA to patients with solid cancers using a targeted, nanoparticle delivery system. Here we provide evidence of inducing an RNAi mechanism of action in a human from the delivered siRNA. Tumour biopsies from melanoma patients obtained after treatment show the presence of intracellularly localized nanoparticles in amounts that correlate with dose levels of the nanoparticles administered (this is, to our knowledge, a first for systemically delivered nanoparticles of any kind). Furthermore, a reduction was found in both the specific messenger RNA (M2 subunit of ribonucleotide reductase (RRM2)) and the protein (RRM2) levels when compared to pre-dosing tissue. Most notably, we detect the presence of an mRNA fragment that demonstrates that siRNA-mediated mRNA cleavage occurs specifically at the site predicted for an RNAi mechanism from a patient who received the highest dose of the nanoparticles. Together, these data demonstrate that siRNA administered systemically to a human can produce a specific gene inhibition (reduction in mRNA and protein) by an RNAi mechanism of action.

Figure 1

Detection of targeted nanoparticles in human tumors. (a). Schematic representation of the targeted nanoparticles. The polyethyleneglycol (PEG) molecules are terminated with adamantane (AD) that form inclusion complexes with surface cyclodextrins in order to decorate the surface of the nanoparticle with PEG for steric stabilization and PEG-hTf for targeting. (b). Confocal images of post-treatment biopsy sections from patients A, B and C: Au-PEG-AD stain (left), DAPI stain (middle), merged images of the left and right panels with the bright field (right). Image labels: epi = epidermis, t = tumor side, s = skin side, m = melanophage.

Figure 2

RRM2 mRNA and protein expression in tumor tissue. (a). qRT-PCR analysis of RRM2 mRNA levels in samples from patients A and B before and after dosing. RRM2 mRNA levels are normalized to TBP mRNA levels. Results are presented as percentage of the pre-dosing RRM2/TBP mRNA levels for each patient. (b). qRT-PCR and Western blot analysis of RRM2 protein expression from patient samples C2_pre and C2_post. Bar graph is average volume of Western blot bands from two independent experiments; one representative blot is pictured. Archived samples are indicated by (*); samples obtained during the trial are indicated by (¶).

Figure 3

Ribonucleotide reductase (RRM2) and tranferrin receptor (TfR) protein expression in C1_pre and C1_post samples. Photomicrographs of malignant melanoma belonging to a, b, c, pre-treatment and d, e, f post-treatment samples. Protein expression is represented as brick-red (Nova Red) chromagen staining in immunohistochemically-treated slides (a, d: RRM2; b, e:TfR). c, f, The same tissues are stained with Hematoxylin and Eosin (H&E). d, e, f, Brown, diffuse, finely granular color seen in these images is the endogenous pigment of this lightly melanized tumor. Photomicrographs were captured using a 40× objective.

Figure 4

5’-RLM-RACE detection of siRNA induced mRNA cleavage fragment. (a) Schematic depicting the location of the predicted anti-RRM2 siRNA cleavage site and the primers used for PCR amplification of the cleavage fragment. (b) Agarose gel of 5’-RLM-RACE PCR amplification products from post treatment samples (A _post, B _post, C2_post) and in vitro positive control (cell culture). (c) The RRM2 mRNA sequence and siRNA antisense strand are illustrated to show where the cleavage occurs with an RNAi mechanism. The sequence chromatographs obtained from an in vitro cell culture experiment with HT-144 melanoma cells and the patient C2_post sample are illustrated.