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

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

Elevated expression of M1 and M2 components and drug-induced posttranscriptional modulation of ribonucleotide reductase in a hydroxyurea-resistant mouse cell line

Ribonucleotide reductase, a rate-limiting enzyme in the synthesis of DNA, consists of two nonidentical subunits, proteins M1 and M2. Hydroxyurea, a specific inhibitor of DNA synthesis, acts by destroying the unique tyrosyl free radical of protein M2. In the past, we have described a mouse L cell line which exhibited a stable resistance to high concentrations of hydroxyurea [McClarty, G. A., Chan, A., & Wright, J.A. (1986) Somat. Cell Mol. Genet. 12, 121-131]. When this line was grown in the absence of hydroxyurea, the cells contained a modest but stable elevation in ribonucleotide reductase activity. However, the activity was further increased on the addition of drug to the culture medium. This was accompanied by an increase in protein M2 activity as shown by activity titration experiments. Likewise, removal of hydroxyurea resulted in a decrease in M2 activity. In the present study, we make use of recently isolated cDNAs and monoclonal antibodies for both the M1 and M2 proteins to further our understanding of the mechanism of hydroxyurea resistance at the molecular level in a subclone of this cell line. Our results indicated that protein M1 levels were elevated 2-3-fold and protein M2 levels were increased about 50-fold in the mutant cells when they were grown in the absence of hydroxyurea, compared to wild-type cells. These protein increases were accompanied by corresponding elevations in the levels of mRNAs for both subunits and increased rates of transcription of both genes. There was a 6-fold amplification in the gene copy number for protein M2.(ABSTRACT TRUNCATED AT 250 WORDS