RESUMO
This unit attempts to provide a reasonably complete inventory of over 280 solid supports available to oligonucleotide chemists for preparation of natural and 3'-modified oligonucleotides. Emphasis is placed on non-nucleosidic solid supports. The relationship between the structural features of linkers and their behavior in oligonucleotide synthesis and deprotection is discussed wherever the relevant observations are available.
Assuntos
Ácidos Nucleicos/química , Oligonucleotídeos/síntese química , Técnicas de Síntese em Fase Sólida , Oligonucleotídeos/químicaRESUMO
Specific step-by-step instructions for conversion of 5'-O-(4,4'-dimethoxytrityl)- and base-protected nucleosides and other mono-O-(4,4'-dimethoxytrityl)-protected diols to their hemisuccinate esters and their coupling to CPG (controlled-pore glass) supports bearing aminopropyl or long chain aminoalkyl groups are presented. Additional guidelines are provided for selecting a coupling protocol and performing in-process control.
Assuntos
Nucleosídeos/química , Oligonucleotídeos/síntese química , Técnicas de Síntese em Fase Sólida/métodos , Vidro/química , Oligonucleotídeos/químicaRESUMO
Huntington's disease is an autosomal dominant neurodegenerative disease caused by a toxic gain of function mutation in the huntingtin gene (Htt). Silencing of Htt with RNA interference using direct CNS delivery in rodent models of Huntington's disease has been shown to reduce pathology and promote neuronal recovery. A key translational step for this approach is extension to the larger non-human primate brain, achieving sufficient distribution of small interfering RNA targeting Htt (siHtt) and levels of Htt suppression that may have therapeutic benefit. We evaluated the potential for convection enhanced delivery (CED) of siHtt to provide widespread and robust suppression of Htt in nonhuman primates. siHtt was infused continuously for 7 or 28 days into the nonhuman primate putamen to analyze effects of infusion rate and drug concentration on the volume of effective suppression. Distribution of radiolabeled siHtt and Htt suppression were quantified by autoradiography and PCR, respectively, in tissue punches. Histopathology was evaluated and Htt suppression was also visualized in animals treated for 28 days. Seven days of CED led to widespread distribution of siHtt and significant Htt silencing throughout the nonhuman primate striatum in an infusion rate and dose dependent manner. Htt suppression at therapeutic dose levels was well tolerated by the brain. A model developed from these results predicts that continuous CED of siHtt can achieve significant coverage of the striatum of Huntington's disease patients. These findings suggest that this approach may provide an important therapeutic strategy for treating Huntington's disease.
Assuntos
Convecção , Corpo Estriado/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , RNA Interferente Pequeno/administração & dosagem , Análise de Variância , Animais , Isótopos de Carbono/metabolismo , Corpo Estriado/diagnóstico por imagem , Relação Dose-Resposta a Droga , Feminino , Regulação da Expressão Gênica/genética , Regulação da Expressão Gênica/fisiologia , Técnicas de Transferência de Genes , Humanos , Proteína Huntingtina , Macaca mulatta , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Cintilografia , Fatores de TempoRESUMO
A novel, conformationally preorganized nonnucleosidic universal solid support for oligonucleotide synthesis was developed. The solid support featured two chemically equivalent hydroxy groups locked in syn-periplanar orientation and orthogonally protected with 4,4'-dimethoxytrityl and acetyl groups. The solid support was extensively tested in the preparation of oligonucleotides and their phosphorothioate analogues containing 2'-deoxy, 2'-O-methyl, and 2'-O-methoxyethylnucleoside residues at the 3'-terminus. Upon completion of oligonucleotide chain assembly, the support-bound oligonucleotide material was treated with concentrated ammonium hydroxide, which removed the O-acetyl protection. The deprotected hydroxy group then effected the transesterification of a phosphate linkage between the solid support and the 3'-terminal nucleoside residue to result in a facile release of the oligonucleotide to solution. The kinetics of the release process was studied in a continuous flow of concentrated aqueous ammonium hydroxide at a temperature of 300.15 K. Optimal conditions for the release of oligonucleotides depending on the chemistry of the backbone and 3'-terminal nucleoside residue were formulated.