Onset Time and Durability of Huntingtin Suppression in Rhesus Putamen After Direct Infusion of Antihuntingtin siRNA.

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.

Tissue-specific gene silencing monitored in circulating RNA.

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.

Safety and efficacy of RNAi therapy for transthyretin amyloidosis.

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.

First-in-humans trial of an RNA interference therapeutic targeting VEGF and KSP in cancer patients with liver involvement.

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.

Focused ultrasound for targeted delivery of siRNA and efficient knockdown of Htt expression.

RNA interference is a promising strategy for the treatment of Huntington's disease (HD) as it can specifically decrease the expression of the mutant Huntingtin protein (Htt). However, siRNA does not cross the blood-brain barrier and therefore delivery to the brain is limited to direct CNS delivery. Non-invasive delivery of siRNA through the blood-brain barrier (BBB) would be a significant advantage for translating this therapy to HD patients.

Harnessing a physiologic mechanism for siRNA delivery with mimetic lipoprotein particles.

Therapeutics based on RNA interference (RNAi) have emerged as a potential new class of drugs for treating human disease by silencing the target messenger RNA (mRNA), thereby reducing levels of the corresponding pathogenic protein. The major challenge for RNAi therapeutics is the development of safe delivery vehicles for small interfering RNAs (siRNAs). We previously showed that cholesterol-conjugated siRNAs (chol-siRNA) associate with plasma lipoprotein particles and distribute primarily to the liver after systemic administration to mice.

Systemic delivery of transthyretin siRNA mediated by lactosylated dendrimer/α-cyclodextrin conjugates into hepatocyte for familial amyloidotic polyneuropathy therapy.

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.

Widespread suppression of huntingtin with convection-enhanced delivery of siRNA.

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.

Oligonucleotide therapeutic approaches for Huntington disease.

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.

Tumor heterogeneity is an active process maintained by a mutant EGFR-induced cytokine circuit in glioblastoma.

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.

Targeted delivery of RNAi therapeutics with endogenous and exogenous ligand-based mechanisms.

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.

Lipophilic siRNAs mediate efficient gene silencing in oligodendrocytes with direct CNS delivery.

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.

Rational design of cationic lipids for siRNA delivery.

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.

Lipid-like materials for low-dose, in vivo gene silencing.

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.

FAPP2 gene downregulation increases tumor cell sensitivity to Fas-induced apoptosis.

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.

Direct CNS delivery of siRNA mediates robust silencing in oligodendrocytes.

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.

siRNA Down-regulation of the PATZ1 Gene in Human Glioma Cells Increases Their Sensitivity to Apoptotic Stimuli.

The PATZ1 gene encodes a transcription factor that belongs to the BTB/POZ group of transcriptional regulators and has been implicated as a transcriptional repressor. We cloned cDNA from glioma cell lines and found they expressed transcript variant 2 of PATZ1. We designed a specific siRNA against PATZ1 and showed that this siRNA, but not a control randomized siRNA, reduced PATZ1 expression in glioma cells as determined by quantitative PCR.

Novel therapeutic modalities to address nondrugable protein interaction targets.

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.

A combinatorial library of lipid-like materials for delivery of RNAi therapeutics.

The safe and effective delivery of RNA interference (RNAi) therapeutics remains an important challenge for clinical development. The diversity of current delivery materials remains limited, in part because of their slow, multi-step syntheses. Here we describe a new class of lipid-like delivery molecules, termed lipidoids, as delivery agents for RNAi therapeutics.

Persistent restoration of sensory function by immediate or delayed systemic artemin after dorsal root injury.

Dorsal root injury results in substantial and often irreversible loss of sensory functions as a result of the limited regenerative capacity of sensory axons and the inhibitory barriers that prevent both axonal entry into and regeneration in the spinal cord. Here, we describe previously unknown effects of the growth factor artemin after crush injury of the dorsal spinal nerve roots in rats. Artemin not only promoted re-entry of multiple classes of sensory fibers into the spinal cord and re-establishment of synaptic function and simple behavior, but it also, surprisingly, promoted the recovery of complex behavior.

siRNA knock-down of mutant torsinA restores processing through secretory pathway in DYT1 dystonia cells.

Most cases of the dominantly inherited movement disorder, early onset torsion dystonia (DYT1) are caused by a mutant form of torsinA lacking a glutamic acid residue in the C-terminal region (torsinADeltaE). TorsinA is an AAA+ protein located predominantly in the lumen of the endoplasmic reticulum (ER) and nuclear envelope apparently involved in membrane structure/movement and processing of proteins through the secretory pathway. A reporter protein Gaussia luciferase (Gluc) shows a reduced rate of secretion in primary fibroblasts from DYT1 patients expressing endogenous levels of torsinA and torsinADeltaE when compared with control fibroblasts expressing only torsinA.

RNAi therapeutics: a potential new class of pharmaceutical drugs.

The rapid identification of highly specific and potent drug candidates continues to be a substantial challenge with traditional pharmaceutical approaches. Moreover, many targets have proven to be intractable to traditional small-molecule and protein approaches. Therapeutics based on RNA interference (RNAi) offer a powerful method for rapidly identifying specific and potent inhibitors of disease targets from all molecular classes.

Therapeutic potential of RNA interference for neurological disorders.

During the past decade, numerous molecular mediators of neurodegenerative diseases and neurological disorders have been identified and validated, yet few novel therapies have emerged and the unmet medical needs remain high. These molecular mediators belong to target classes such as ion channels, neurotransmitters and neurotransmitter receptors, cytokines, growth factors, enzymes and other proteins. In some cases, substantial pre-clinical validation exists, but the molecular target has not been readily druggable with small molecules, proteins or antibodies.

Distribution of GDNF family receptor alpha3 and RET in rat and human non-neural tissues.

The neurotrophic growth factor artemin binds selectively to GDNF family receptor alpha3 (GFRalpha3), forming a molecular complex with the co-receptor RET which mediates downstream signaling. This signaling pathway has been demonstrated to play an important role in the survival and maintenance of nociceptive sensory neurons and in the development of sympathetic neurons. However, the presence and potential role of this artemin-responsive pathway in non-neural tissues has not been fully explored to-date.