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1.
Hum Gene Ther ; 33(1-2): 37-60, 2022 01.
Artigo em Inglês | MEDLINE | ID: mdl-34806402

RESUMO

Huntington's disease is a fatal neurodegenerative disorder caused by an inherited mutation in the huntingtin (HTT) gene comprising an expanded cytosine-adenine-guanine (CAG) trinucleotide repeat sequence that results in a pathogenic huntingtin protein. Adeno-associated viral (AAV) gene therapy containing a primary artificial microRNA (pri-amiRNA) specifically targeting HTT messenger RNA (mRNA) has the potential to provide long-lasting therapeutic benefit, through durable reduction of mutant HTT expression after a single administration. The efficiency and precision of processing of the pri-amiRNA precursor to the mature guide (G) strand by transduced cells are critical for specific and potent HTT mRNA lowering. The selection of the optimized pri-amiRNA comprised a series of in vitro studies followed by in vivo studies in small and then large mammals. Our studies demonstrate the predictivity of certain cell culture systems and rodent models for nonhuman primates with respect to some, but not all key features of pri-amiRNA processing. In addition, our results show that the processing of pri-amiRNAs to the mature guide strand can differ greatly across different scaffolds and sequences while providing the same levels of target lowering. Importantly, our data demonstrate that there is a combinatorial effect of guide and passenger (P) strand sequences, together with the scaffold, on pri-amiRNA processing, with different guide and passenger strand sequences within the same scaffold dramatically altering pri-amiRNA processing. Taken together, our results highlight the importance of optimizing not only target lowering but also the efficiency and precision of pri-amiRNA processing in vitro, in rodents and in large mammals to identify the most potent and selective AAV gene therapy that harnesses the endogenous microRNA (miRNA) biogenesis pathway for target lowering without perturbing the endogenous cellular miRNA profile. The optimized pri-amiRNA was selected with this focus on efficiency and precision of pri-amiRNA processing in addition to its pharmacological activity on HTT mRNA lowering and general tolerability in vivo.


Assuntos
Doença de Huntington , MicroRNAs , Animais , Terapia Genética , Vetores Genéticos/genética , Proteína Huntingtina/genética , Doença de Huntington/genética , Doença de Huntington/terapia , Camundongos , MicroRNAs/genética , Primatas/genética
2.
Nat Rev Drug Discov ; 17(10): 767, 2018 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-30206384

RESUMO

This corrects the article DOI: 10.1038/nrd.2018.110.

3.
Nat Rev Drug Discov ; 17(9): 641-659, 2018 09.
Artigo em Inglês | MEDLINE | ID: mdl-30093643

RESUMO

Adeno-associated viral (AAV) vectors are a rapidly emerging gene therapy platform for the treatment of neurological diseases. In preclinical studies, transgenes encoding therapeutic proteins, microRNAs, antibodies or gene-editing machinery have been successfully delivered to the central nervous system with natural or engineered viral capsids via various routes of administration. Importantly, initial clinical studies have demonstrated encouraging safety and efficacy in diseases such as Parkinson disease and spinal muscular atrophy, as well as durability of transgene expression. Here, we discuss key considerations and challenges in the future design and development of therapeutic AAV vectors, highlighting the most promising targets and recent clinical advances.

4.
Nat Biotechnol ; 35(9): 845-851, 2017 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-28829437

RESUMO

Whereas stereochemical purity in drugs has become the standard for small molecules, stereoisomeric mixtures containing as many as a half million components persist in antisense oligonucleotide (ASO) therapeutics because it has been feasible neither to separate the individual stereoisomers, nor to synthesize stereochemically pure ASOs. Here we report the development of a scalable synthetic process that yields therapeutic ASOs having high stereochemical and chemical purity. Using this method, we synthesized rationally designed stereopure components of mipomersen, a drug comprising 524,288 stereoisomers. We demonstrate that phosphorothioate (PS) stereochemistry substantially affects the pharmacologic properties of ASOs. We report that Sp-configured PS linkages are stabilized relative to Rp, providing stereochemical protection from pharmacologic inactivation of the drug. Further, we elucidated a triplet stereochemical code in the stereopure ASOs, 3'-SpSpRp, that promotes target RNA cleavage by RNase H1 in vitro and provides a more durable response in mice than stereorandom ASOs.


Assuntos
Terapia Genética/métodos , Oligonucleotídeos Antissenso/química , Oligonucleotídeos Antissenso/farmacocinética , Oligonucleotídeos Fosforotioatos/química , Animais , Estabilidade de Medicamentos , Feminino , Humanos , Interações Hidrofóbicas e Hidrofílicas , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Oligonucleotídeos , Oligonucleotídeos Antissenso/uso terapêutico , Ratos , Ratos Sprague-Dawley , Ribonuclease H/metabolismo , Estereoisomerismo
5.
Mol Ther Nucleic Acids ; 4: e245, 2015 Jun 30.
Artigo em Inglês | MEDLINE | ID: mdl-26125484

RESUMO

One possible treatment for Huntington's disease involves direct infusion of a small, interfering RNA (siRNA) designed to reduce huntingtin expression into brain tissue from a chronically implanted programmable pump. Here, we studied the suppression of huntingtin mRNA achievable with short infusion times, and investigated how long suppression may persist after infusion ceases. Rhesus monkeys received 3 days of infusion of Magnevist into the putamen to confirm catheter patency and fluid distribution. After a 1-week washout period, monkeys received radiolabeled siRNA targeting huntingtin. After 1 or 3 days of siRNA delivery, monkeys were either terminated, or their pumps were shut off and they were terminated 10 or 24 days later. Results indicate that the onset of huntingtin mRNA suppression in the rhesus putamen occurs rapidly, achieving a plateau throughout the putamen within 4 days. Conversely, loss of huntingtin suppression progresses slowly, persisting an estimated 27-39 days in the putamen and surrounding white matter. These findings indicate the rapid onset and durability of siRNA-mediated target gene suppression observed in other organs also occurs in the brain, and support the use of episodic delivery of siRNA into the brain for treatment of Huntington's disease and possibly other neurodegenerative diseases.

6.
Pain ; 155(3): 476-484, 2014 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-24269493

RESUMO

Systemic artemin promotes regeneration of dorsal roots to the spinal cord after crush injury. However, it is unclear whether systemic artemin can also promote peripheral nerve regeneration, and functional recovery after partial lesions distal to the dorsal root ganglion (DRG) remains unknown. In the present investigation, male Sprague Dawley rats received axotomy, ligation, or crush of the L5 spinal nerve or sham surgery. Starting the day of injury, animals received intermittent subcutaneous artemin or vehicle across 2weeks. Sensory thresholds to tactile or thermal stimuli were monitored for 6weeks after injury. Immunohistochemical analyses of the DRG and nerve regeneration were performed at the 6-week time point. Artemin transiently reversed tactile and thermal hypersensitivity after axotomy, ligation, or crush injury. Thermal and tactile hypersensitivity reemerged within 1week of treatment termination. However, artemin-treated rats with nerve crush, but not axotomy or ligation, subsequently showed gradual return of sensory thresholds to preinjury baseline levels by 6weeks after injury. Artemin normalized labeling for NF200, IB4, and CGRP in nerve fibers distal to the crush injury, suggesting persistent normalization of nerve crush-induced neurochemical changes. Sciatic and intradermal administration of dextran or cholera toxin B distal to the crush injury site resulted in labeling of neuronal profiles in the L5 DRG, suggesting regeneration functional restoration of nonmyelinated and myelinated fibers across the injury site into cutaneous tissue. Artemin also diminished ATF3 and caspase 3 expression in the L5 DRG, suggesting persistent neuroprotective actions. A limited period of artemin treatment elicits disease modification by promoting sensory reinnervation of distal territories and restoring preinjury sensory thresholds.


Assuntos
Fator de Crescimento Neural/farmacologia , Regeneração Nervosa/efeitos dos fármacos , Proteínas do Tecido Nervoso/farmacologia , Recuperação de Função Fisiológica/efeitos dos fármacos , Nervos Espinhais/efeitos dos fármacos , Nervos Espinhais/lesões , Animais , Masculino , Regeneração Nervosa/fisiologia , Ratos , Ratos Sprague-Dawley , Recuperação de Função Fisiológica/fisiologia , Nervos Espinhais/metabolismo
7.
RNA ; 20(2): 143-9, 2014 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-24355758

RESUMO

Pharmacologic target gene modulation is the primary objective for RNA antagonist strategies and gene therapy. Here we show that mRNAs encoding tissue-specific gene transcripts can be detected in biological fluids and that RNAi-mediated target gene silencing in the liver and brain results in quantitative reductions in serum and cerebrospinal fluid mRNA levels, respectively. Further, administration of an anti-miRNA oligonucleotide resulted in decreased levels of the miRNA in circulation. Moreover, ectopic expression of an adenoviral transgene in the liver was quantified based on measurement of serum mRNA levels. This noninvasive method for monitoring tissue-specific RNA modulation could greatly advance the clinical development of RNA-based therapeutics.


Assuntos
Técnicas de Silenciamento de Genes , Interferência de RNA , RNA Mensageiro/sangue , Idoso , Animais , Biomarcadores Tumorais/sangue , Biomarcadores Tumorais/genética , Expressão Gênica , Glipicanas/genética , Humanos , Fígado/metabolismo , Macaca fascicularis , Masculino , Especificidade de Órgãos , RNA Mensageiro/genética , RNA Interferente Pequeno/genética , Ratos , Ratos Sprague-Dawley , Especificidade da Espécie , alfa-Fetoproteínas/genética
8.
N Engl J Med ; 369(9): 819-29, 2013 Aug 29.
Artigo em Inglês | MEDLINE | ID: mdl-23984729

RESUMO

BACKGROUND: Transthyretin amyloidosis is caused by the deposition of hepatocyte-derived transthyretin amyloid in peripheral nerves and the heart. A therapeutic approach mediated by RNA interference (RNAi) could reduce the production of transthyretin. METHODS: We identified a potent antitransthyretin small interfering RNA, which was encapsulated in two distinct first- and second-generation formulations of lipid nanoparticles, generating ALN-TTR01 and ALN-TTR02, respectively. Each formulation was studied in a single-dose, placebo-controlled phase 1 trial to assess safety and effect on transthyretin levels. We first evaluated ALN-TTR01 (at doses of 0.01 to 1.0 mg per kilogram of body weight) in 32 patients with transthyretin amyloidosis and then evaluated ALN-TTR02 (at doses of 0.01 to 0.5 mg per kilogram) in 17 healthy volunteers. RESULTS: Rapid, dose-dependent, and durable lowering of transthyretin levels was observed in the two trials. At a dose of 1.0 mg per kilogram, ALN-TTR01 suppressed transthyretin, with a mean reduction at day 7 of 38%, as compared with placebo (P=0.01); levels of mutant and nonmutant forms of transthyretin were lowered to a similar extent. For ALN-TTR02, the mean reductions in transthyretin levels at doses of 0.15 to 0.3 mg per kilogram ranged from 82.3 to 86.8%, with reductions of 56.6 to 67.1% at 28 days (P<0.001 for all comparisons). These reductions were shown to be RNAi-mediated. Mild-to-moderate infusion-related reactions occurred in 20.8% and 7.7% of participants receiving ALN-TTR01 and ALN-TTR02, respectively. CONCLUSIONS: ALN-TTR01 and ALN-TTR02 suppressed the production of both mutant and nonmutant forms of transthyretin, establishing proof of concept for RNAi therapy targeting messenger RNA transcribed from a disease-causing gene. (Funded by Alnylam Pharmaceuticals; ClinicalTrials.gov numbers, NCT01148953 and NCT01559077.).


Assuntos
Neuropatias Amiloides Familiares/terapia , Pré-Albumina/genética , RNA Interferente Pequeno/uso terapêutico , Adolescente , Adulto , Neuropatias Amiloides Familiares/genética , Animais , Relação Dose-Resposta a Droga , Feminino , Humanos , Lipossomos , Macaca fascicularis , Masculino , Nanocápsulas , Pré-Albumina/metabolismo , RNA Interferente Pequeno/administração & dosagem , Adulto Jovem
9.
Cancer Discov ; 3(4): 406-17, 2013 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-23358650

RESUMO

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 Xenoenxerto
10.
Amyloid ; 19 Suppl 1: 47-9, 2012 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-22519861

RESUMO

RNA interference (RNAi) is a sequence-specific gene-silencing mechanism triggered by double-stranded RNA and powerful tools for a gene function study and RNAi therapy. Although siRNAs offer several advantages as potential new drugs to treat various diseases, the efficient delivery system of siRNAs in vivo remains a crucial challenge for achieving the desired RNAi effect in clinical development. In particular, when considering the siRNA therapeutics for familial amyloidotic polyneuropathy (FAP) caused by the deposition of variant transthyretin (TTR) in various organs, hepatocyte-selective siRNA delivery is desired because TTR is predominantly synthesized by hepatocytes. In this study, to reveal the potential use of lactosylated dendrimer (G3)/α-cyclodextrin conjugate (Lac-α-CDE (G3)) as novel hepatocyte-selective siRNA carriers in order to treat FAP, we evaluated the RNAi effect of siRNA complex with Lac-α-CDE (G3) both in vitro and in vivo.


Assuntos
Neuropatias Amiloides Familiares/terapia , Dendrímeros/química , Hepatócitos/metabolismo , Pré-Albumina/genética , RNA Interferente Pequeno/genética , alfa-Ciclodextrinas/química , Animais , Humanos , Interferência de RNA
11.
Exp Neurol ; 233(1): 463-71, 2012 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-22119622

RESUMO

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 Tempo
12.
J Clin Invest ; 121(2): 500-7, 2011 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-21285523

RESUMO

Huntington disease is an autosomal dominant neurodegenerative disorder caused by a toxic expansion in the CAG repeat region of the huntingtin gene. Oligonucleotide approaches based on RNAi and antisense oligonucleotides provide promising new therapeutic strategies for direct intervention through reduced production of the causative mutant protein. Allele-specific and simultaneous mutant and wild-type allele-lowering strategies are being pursued with local delivery to the brain, each with relative merits. Delivery remains a key challenge for translational success, especially with chronic therapy. The potential of disease-modifying oligonucleotide approaches for Huntington disease will be revealed as they progress into clinical trials.


Assuntos
Doença de Huntington/tratamento farmacológico , Oligonucleotídeos/uso terapêutico , Ensaios Clínicos como Assunto , Inativação Gênica , Humanos , Proteína Huntingtina , Doença de Huntington/genética , Mutação , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Neurônios/patologia , Neurônios/fisiologia , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Oligonucleotídeos Antissenso/uso terapêutico , Interferência de RNA , Expansão das Repetições de Trinucleotídeos
13.
Genes Dev ; 24(16): 1731-45, 2010 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-20713517

RESUMO

Human solid tumors frequently have pronounced heterogeneity of both neoplastic and normal cells on the histological, genetic, and gene expression levels. While current efforts are focused on understanding heterotypic interactions between tumor cells and surrounding normal cells, much less is known about the interactions between and among heterogeneous tumor cells within a neoplasm. In glioblastoma multiforme (GBM), epidermal growth factor receptor gene (EGFR) amplification and mutation (EGFRvIII/DeltaEGFR) are signature pathogenetic events that are invariably expressed in a heterogeneous manner. Strikingly, despite its greater biological activity than wild-type EGFR (wtEGFR), individual GBM tumors expressing both amplified receptors typically express wtEGFR in far greater abundance than the DeltaEGFR lesion. We hypothesized that the minor DeltaEGFR-expressing subpopulation enhances tumorigenicity of the entire tumor cell population, and thereby maintains heterogeneity of expression of the two receptor forms in different cells. Using mixtures of glioma cells as well as immortalized murine astrocytes, we demonstrate that a paracrine mechanism driven by DeltaEGFR is the primary means for recruiting wtEGFR-expressing cells into accelerated proliferation in vivo. We determined that human glioma tissues, glioma cell lines, glioma stem cells, and immortalized mouse Ink4a/Arf(-/-) astrocytes that express DeltaEGFR each also express IL-6 and/or leukemia inhibitory factor (LIF) cytokines. These cytokines activate gp130, which in turn activates wtEGFR in neighboring cells, leading to enhanced rates of tumor growth. Ablating IL-6, LIF, or gp130 uncouples this cellular cross-talk, and potently attenuates tumor growth enhancement. These findings support the view that a minor tumor cell population can potently drive accelerated growth of the entire tumor mass, and thereby actively maintain tumor cell heterogeneity within a tumor mass. Such interactions between genetically dissimilar cancer cells could provide novel points of therapeutic intervention.


Assuntos
Receptores ErbB/genética , Receptores ErbB/metabolismo , Regulação Neoplásica da Expressão Gênica , Glioblastoma/fisiopatologia , Mutação/genética , Animais , Linhagem Celular Tumoral , Proliferação de Células , Sobrevivência Celular/fisiologia , Receptor gp130 de Citocina/metabolismo , Citocinas/metabolismo , Glioblastoma/genética , Glioma/fisiopatologia , Humanos , Interleucina-6/genética , Interleucina-6/metabolismo , Fator Inibidor de Leucemia/genética , Fator Inibidor de Leucemia/metabolismo , Ligantes , Camundongos , Camundongos Nus , Células-Tronco Neoplásicas/patologia , Regulação para Cima
14.
Mol Ther ; 18(7): 1357-64, 2010 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-20461061

RESUMO

Lipid nanoparticles (LNPs) have proven to be highly efficient carriers of short-interfering RNAs (siRNAs) to hepatocytes in vivo; however, the precise mechanism by which this efficient delivery occurs has yet to be elucidated. We found that apolipoprotein E (apoE), which plays a major role in the clearance and hepatocellular uptake of physiological lipoproteins, also acts as an endogenous targeting ligand for ionizable LNPs (iLNPs), but not cationic LNPs (cLNPs). The role of apoE was investigated using both in vitro studies employing recombinant apoE and in vivo studies in wild-type and apoE(-/-) mice. Receptor dependence was explored in vitro and in vivo using low-density lipoprotein receptor (LDLR(-/-))-deficient mice. As an alternative to endogenous apoE-based targeting, we developed a targeting approach using an exogenous ligand containing a multivalent N-acetylgalactosamine (GalNAc)-cluster, which binds with high affinity to the asialoglycoprotein receptor (ASGPR) expressed on hepatocytes. Both apoE-based endogenous and GalNAc-based exogenous targeting appear to be highly effective strategies for the delivery of iLNPs to liver.


Assuntos
Interferência de RNA/fisiologia , Animais , Apolipoproteínas E/genética , Apolipoproteínas E/metabolismo , Receptor de Asialoglicoproteína/metabolismo , Feminino , Células HeLa , Hepatócitos/metabolismo , Humanos , Ligantes , Camundongos , Camundongos Endogâmicos C57BL , Nanopartículas/química , Receptores de LDL/genética , Receptores de LDL/metabolismo
15.
J Control Release ; 144(2): 227-32, 2010 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-20170694

RESUMO

Conjugation of small interfering RNA (siRNA) with lipophilic molecules has been demonstrated to enhance cellular uptake in cell culture and to produce efficient endogenous gene silencing in the liver after systemic administration and in neurons after direct local injection. Here, we evaluated the in vivo delivery of siRNAs conjugated with different linkers to cholesterol by targeting CNPase (2'-3'-cyclic nucleotide 3'-phosphodiesterase) in oligodendrocytes. Cholesterol-conjugated siRNAs administered to the rat corpus callosum by intraparenchymal central nervous system (CNS) infusion show improved silencing ability compared with unconjugated siRNA. Furthermore, conjugation of siRNA to cholesterol with a cleavable disulfide linker appears to be beneficial for improving the potency of silencing of CNPase mRNA in oligodendrocytes in vivo. Taken together, these findings indicate that cholesterol-conjugated siRNAs are effective for direct CNS delivery to oligodendrocytes, and that the biocleavable disulfide linker appears to be beneficial for improving the potency of silencing of target mRNA in vivo.


Assuntos
Inativação Gênica , RNA Interferente Pequeno/genética , Animais , Sistema Nervoso Central , Colesterol/genética , Masculino , Neurônios , Oligodendroglia , RNA Mensageiro/genética , RNA Interferente Pequeno/administração & dosagem , Ratos , Ratos Sprague-Dawley
16.
Proc Natl Acad Sci U S A ; 107(5): 1864-9, 2010 Feb 02.
Artigo em Inglês | MEDLINE | ID: mdl-20080679

RESUMO

Significant effort has been applied to discover and develop vehicles which can guide small interfering RNAs (siRNA) through the many barriers guarding the interior of target cells. While studies have demonstrated the potential of gene silencing in vivo, improvements in delivery efficacy are required to fulfill the broadest potential of RNA interference therapeutics. Through the combinatorial synthesis and screening of a different class of materials, a formulation has been identified that enables siRNA-directed liver gene silencing in mice at doses below 0.01 mg/kg. This formulation was also shown to specifically inhibit expression of five hepatic genes simultaneously, after a single injection. The potential of this formulation was further validated in nonhuman primates, where high levels of knockdown of the clinically relevant gene transthyretin was observed at doses as low as 0.03 mg/kg. To our knowledge, this formulation facilitates gene silencing at orders-of-magnitude lower doses than required by any previously described siRNA liver delivery system.


Assuntos
Materiais Biocompatíveis/química , Inativação Gênica , Lipídeos/química , RNA Interferente Pequeno/administração & dosagem , RNA Interferente Pequeno/genética , Animais , Materiais Biocompatíveis/síntese química , Sistemas de Liberação de Medicamentos , Fator VII/antagonistas & inibidores , Fator VII/genética , Células HeLa , Hepatócitos/metabolismo , Humanos , Lipídeos/síntese química , Macaca fascicularis , Camundongos , Camundongos Endogâmicos C57BL , Estrutura Molecular , Interferência de RNA
17.
Nat Biotechnol ; 28(2): 172-6, 2010 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-20081866

RESUMO

We adopted a rational approach to design cationic lipids for use in formulations to deliver small interfering RNA (siRNA). Starting with the ionizable cationic lipid 1,2-dilinoleyloxy-3-dimethylaminopropane (DLinDMA), a key lipid component of stable nucleic acid lipid particles (SNALP) as a benchmark, we used the proposed in vivo mechanism of action of ionizable cationic lipids to guide the design of DLinDMA-based lipids with superior delivery capacity. The best-performing lipid recovered after screening (DLin-KC2-DMA) was formulated and characterized in SNALP and demonstrated to have in vivo activity at siRNA doses as low as 0.01 mg/kg in rodents and 0.1 mg/kg in nonhuman primates. To our knowledge, this represents a substantial improvement over previous reports of in vivo endogenous hepatic gene silencing.


Assuntos
Portadores de Fármacos/química , Composição de Medicamentos/métodos , Desenho de Fármacos , Lipídeos/química , RNA Interferente Pequeno/química , Transfecção/métodos , Cátions , RNA Interferente Pequeno/administração & dosagem
18.
Biochem Biophys Res Commun ; 383(2): 167-71, 2009 May 29.
Artigo em Inglês | MEDLINE | ID: mdl-19341712

RESUMO

The gene for phosphatidylinositol-4-phosphate adaptor-2 (FAPP2) encodes a cytoplasmic lipid transferase with a plekstrin homology domain that has been implicated in vesicle maturation and transport from trans-Golgi to the plasma membrane. The introduction of ribozymes targeting the FAPP2 gene in colon carcinoma cells induced their apoptosis in the presence of Fas agonistic antibody. Furthermore, by quantitative PCR we showed that a siRNA specific to FAPP2, but not a randomized siRNA control, reduced FAPP2 gene expression in tumor cells. Transfection of FAPP2 siRNA into human tumor cells then incubated with FasL resulted in reduction of viable cell numbers. Also, FAPP2 siRNA transfected glioma and breast tumor cells showed significant increases in apoptosis upon incubation with soluble FasL, but the apoptosis did not necessarily correlate with increased Fas expression. These data demonstrate a previously unknown role for FAPP2 in conferring resistance to apoptosis and indicate that FAPP2 may be a target for cancer therapy.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/fisiologia , Apoptose , Proteína Ligante Fas/agonistas , Neoplasias/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Anticorpos/imunologia , Apoptose/genética , Linhagem Celular Tumoral , Regulação para Baixo , Proteína Ligante Fas/imunologia , Proteína Ligante Fas/metabolismo , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias/patologia , RNA Interferente Pequeno/genética
19.
Oligonucleotides ; 19(1): 23-29, 2009 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-19093781

RESUMO

The most significant challenge remaining in the development of small interfering RNAs (siRNAs) as a new class of therapeutic drugs is successful delivery in vivo. The majority of reported studies describing delivery of siRNA or short hairpin RNA (shRNA) to the central nervous system (CNS) have focused on RNA interference (RNAi) in neurons. Here we show direct CNS delivery of siRNA to a different cell type-oligodendrocytes-using convection-enhanced delivery, and demonstrate robust silencing of an endogenous oligodendrocyte-specific gene, 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNPase) with siRNA formulated in saline. The silencing is not sequence-dependent as several different siRNAs are effective in inhibiting target gene expression. Furthermore, we show that CNPase mRNA reduction is dose-dependent, durable for up to 1 week, and mediated by an RNAi mechanism. Increasing the flow rate of siRNA infusion increased the distribution of mRNA suppression to encompass white matter regions distant from the infusion site. Finally, we demonstrate suppression of CNPase mRNA in the nonhuman primate CNS. Taken together, these results show for the first time robust RNAi within oligodendrocytes in vivo and demonstrate the important potential of siRNAs in the treatment of CNS disorders involving oligodendrocyte pathology.


Assuntos
Sistema Nervoso Central/efeitos dos fármacos , Oligodendroglia/efeitos dos fármacos , RNA Interferente Pequeno/administração & dosagem , Animais , Sequência de Bases , Sistema Nervoso Central/metabolismo , Imuno-Histoquímica , Masculino , Oligodendroglia/metabolismo , RNA Interferente Pequeno/farmacologia , Ratos , Ratos Sprague-Dawley
20.
Neuropsychopharmacology ; 34(1): 142-58, 2009 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-18754007

RESUMO

Small molecule drugs are relatively effective in working on 'drugable' targets such as GPCRs, ion channels, kinases, proteases, etc but ineffective at blocking protein-protein interactions that represent an emerging class of 'nondrugable' central nervous system (CNS) targets. This article provides an overview of novel therapeutic modalities such as biologics (in particular antibodies) and emerging oligonucleotide therapeutics such as antisense, small-interfering RNA, and aptamers. Their key properties, overall strengths and limitations, and their utility as tools for target validation are presented. In addition, issues with regard to CNS targets as it relates to the blood-brain barrier penetration are discussed. Finally, examples of their application as therapeutics for the treatment of pain and some neurological disorders such as Alzheimer's disease, multiple sclerosis, Huntington's disease, and Parkinson's disease are provided.


Assuntos
Fármacos do Sistema Nervoso Central/uso terapêutico , Doenças do Sistema Nervoso Central/tratamento farmacológico , Sistemas de Liberação de Medicamentos/métodos , Domínios e Motivos de Interação entre Proteínas/efeitos dos fármacos , Animais , Anticorpos/uso terapêutico , Elementos Antissenso (Genética)/uso terapêutico , Aptâmeros de Nucleotídeos/uso terapêutico , Barreira Hematoencefálica , Fármacos do Sistema Nervoso Central/farmacologia , Humanos , Ligação Proteica/efeitos dos fármacos , RNA Interferente Pequeno/uso terapêutico , Estudos de Validação como Assunto
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