RESUMO
Successful implementation of mRNA gene therapy is facing many hurdles, for example poor expression levels of the exogenously delivered mRNA transcripts. Herein we describe the synthesis of various 3'-modified RNA oligonucleotides, and we show that 3'-modification drastically stabilizes these oligonucleotides in cell extracts. Modification of the 3'-terminus of gaussia luciferase mRNA results in 3-fold increased and extended (>48â¯h) translation of the mRNA. Our findings suggest 3'-modification of RNA-transcripts as a valid approach to increase expression levels for application in mRNA gene therapy.
Assuntos
Terapia Genética , RNA Mensageiro/genética , Transcrição Gênica/genética , Animais , Copépodes/enzimologia , Relação Dose-Resposta a Droga , Células HeLa , Humanos , Luciferases/genética , Luciferases/metabolismo , Estrutura Molecular , Oligonucleotídeos/síntese química , Oligonucleotídeos/química , Oligonucleotídeos/genética , RNA Mensageiro/química , RNA Mensageiro/metabolismo , Relação Estrutura-AtividadeRESUMO
UNLABELLED: RNA interference (RNAi) is a potent and specific mechanism for regulating gene expression. Harnessing RNAi to silence genes involved in disease holds promise for the development of a new class of therapeutics. Delivery is key to realizing the potential of RNAi, and lipid nanoparticles (LNP) have proved effective in delivery of siRNAs to the liver and to tumors in animals. To examine the activity and safety of LNP-formulated siRNAs in humans, we initiated a trial of ALN-VSP, an LNP formulation of siRNAs targeting VEGF and kinesin spindle protein (KSP), in patients with cancer. Here, we show detection of drug in tumor biopsies, siRNA-mediated mRNA cleavage in the liver, pharmacodynamics suggestive of target downregulation, and antitumor activity, including complete regression of liver metastases in endometrial cancer. In addition, we show that biweekly intravenous administration of ALN-VSP was safe and well tolerated. These data provide proof-of-concept for RNAi therapeutics in humans and form the basis for further development in cancer. SIGNIFICANCE: The fi ndings in this report show safety, pharmacokinetics, RNAi mechanism of action, and clinical activity with a novel fi rst-in-class LNP-formulated RNAi therapeutic in patients with cancer. The ability to harness RNAi to facilitate specifi c multitargeting, as well as increase the number of druggable targets, has important implications for future drug development in oncology.
Assuntos
Cinesinas/genética , Neoplasias Hepáticas/terapia , Nanopartículas/administração & dosagem , Interferência de RNA , RNA Interferente Pequeno/administração & dosagem , Fator A de Crescimento do Endotélio Vascular/genética , Adulto , Idoso , Animais , Linhagem Celular Tumoral , Citocinas/sangue , Feminino , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Masculino , Camundongos , Camundongos SCID , Pessoa de Meia-Idade , RNA Mensageiro/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Since the discovery of RNA interference (RNAi), researchers have identified a variety of small interfering RNA (siRNA) structures that demonstrate the ability to silence gene expression through the classical RISC-mediated mechanism. One such structure, termed "Dicer-substrate siRNA" (dsiRNA), was proposed to have enhanced potency via RISC-mediated gene silencing, although a comprehensive comparison of canonical siRNAs and dsiRNAs remains to be described. The present study evaluates the in vitro and in vivo activities of siRNAs and dsiRNAs targeting Phosphatase and Tensin Homolog (PTEN) and Factor VII (FVII). More than 250 compounds representing both siRNA and dsiRNA structures were evaluated for silencing efficacy. Lead compounds were assessed for duration of silencing and other key parameters such as cytokine induction. We identified highly active compounds from both canonical siRNAs and 25/27 dsiRNAs. Lead compounds were comparable in potency both in vitro and in vivo as well as duration of silencing in vivo. Duplexes from both structural classes tolerated 2'-OMe chemical modifications well with respect to target silencing, although some modified dsiRNAs demonstrated reduced activity. On the other hand, dsiRNAs were more immunostimulatory as compared with the shorter siRNAs, both in vitro and in vivo. Because the dsiRNA structure does not confer any appreciable benefits in vitro or in vivo while demonstrating specific liabilities, further studies are required to support their applications in RNAi therapeutics.
Assuntos
Interferência de RNA , RNA Interferente Pequeno/metabolismo , Ribonuclease III/metabolismo , Animais , Sequência de Bases , Fator VII/genética , Feminino , Células HeLa , Humanos , Camundongos , Camundongos Endogâmicos C57BL , PTEN Fosfo-Hidrolase/genética , Complexo de Inativação Induzido por RNA/metabolismo , RatosRESUMO
Mir-290 through mir-295 (mir-290-295) is a mammalian-specific microRNA (miRNA) cluster that, in mice, is expressed specifically in early embryos and embryonic germ cells. Here, we show that mir-290-295 plays important roles in embryonic development as indicated by the partially penetrant lethality of mutant embryos. In addition, we show that in surviving mir-290-295-deficient embryos, female but not male fertility is compromised. This impairment in fertility arises from a defect in migrating primordial germ cells and occurs equally in male and female mutant animals. Male mir-290-295(-/-) mice, due to the extended proliferative lifespan of their germ cells, are able to recover from this initial germ cell loss and are fertile. Female mir-290-295(-/-) mice are unable to recover and are sterile, due to premature ovarian failure.
