RESUMO
RNAi technology is taking strong position among the key therapeutic modalities, with dozens of siRNA-based programs entering and successfully progressing through clinical stages of drug development. To further explore potentials of RNAi technology as therapeutics, we engineered and tested VEGFR2 siRNA molecules specifically targeted to tumors through covalently conjugated cyclo(Arg-Gly-Asp-d-Phe-Lys[PEG-MAL]) (cRGD) peptide, known to bind αvß3 integrin receptors. cRGD-siRNAs were demonstrated to specifically enter and silence targeted genes in cultured αvß3 positive human cells (HUVEC). Microinjection of zebrafish blastocysts with VEGFR2 cRGD-siRNA resulted in specific inhibition of blood vessel growth. In tumor-bearing mice, intravenously injected cRGD-siRNA molecules generated no innate immune response and bio-distributed to tumor tissues. Continuous systemic delivery of two different VEGFR2 cRGD-siRNAs resulted in down-regulation of corresponding mRNA (55 and 45%) and protein (65 and 45%) in tumors, as well as in overall reduction of tumor volume (90 and 70%). These findings demonstrate strong potential of cRGD-siRNA molecules as anti-tumor therapy.
Assuntos
Neoplasias/terapia , Peptídeos Cíclicos/administração & dosagem , Interferência de RNA , RNA Interferente Pequeno/administração & dosagem , Animais , Linhagem Celular Tumoral , Células Cultivadas , Feminino , Técnicas de Silenciamento de Genes , Células HeLa , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Camundongos Endogâmicos BALB C , Camundongos Nus , Neoplasias/irrigação sanguínea , Neovascularização Fisiológica , Peptídeos Cíclicos/análise , Peptídeos Cíclicos/química , RNA Interferente Pequeno/química , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/antagonistas & inibidores , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/genética , Peixe-Zebra/embriologiaRESUMO
RNA interference (RNAi) has been established as an important tool for functional genomics studies and has great promise as a therapeutic intervention for human diseases. In mammalian cells, RNAi is conventionally induced by 19-27-bp RNA duplexes generated by hybridization of two complementary oligonucleotide strands (oligos). Here we describe a novel class of RNAi molecules composed of a single 25-28-nucleotide (nt) oligo. The oligo has a 16-nt mRNA targeting region, followed by an additional 8-10 nt to enable self-dimerization into a partially complementary duplex. Analysis of numerous diverse structures demonstrates that molecules composed of two short helices separated by a loop can efficiently enter and activate the RNA-induced silencing complex (RISC). This finding enables the design of highly effective single-oligo compounds for any mRNA target.
Assuntos
Oligonucleotídeos/química , Interferência de RNA , RNA Interferente Pequeno/química , Complexo de Inativação Induzido por RNA/metabolismo , Células Cultivadas , Inativação Gênica , Células HeLa , Humanos , Conformação de Ácido Nucleico , RNA/química , RNA/metabolismo , RNA Mensageiro/química , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/metabolismo , Complexo de Inativação Induzido por RNA/químicaRESUMO
Nucleolar localization of vertebrate box C/D snoRNA involves transit through Cajal bodies, but the significance of this event is unknown. To define better the function of this compartment, we analyzed here the maturation pathway of mammalian U3. We show that 3'-extended U3 precursors possess a mono-methylated cap, and are not associated with fibrillarin and hNop58. Importantly, these precursors are detected at both their transcription sites and in Cajal bodies. In addition, mature U3, the core box C/D proteins and the human homolog of the methyltransferase responsible for U3 cap tri-methylation, hTgs1, are all present in Cajal bodies. In yeast, U3 follows a similar maturation pathway, and equivalent 3'-extended precursors are enriched in the nucleolus and in the nucleolar body, a nucleolar domain that concentrates Tgs1p under certain growth conditions. Thus, spatial organization of U3 maturation appears to be conserved across evolution, and involves specialized and related nuclear compartments, the nucleolus/nucleolar body in yeast and Cajal bodies in higher eukaryotes. These are likely places for snoRNP assembly, 3' end maturation and cap modification.