Transthyretin amyloidosis is largely caused by synthesis of mutant transthyretin in the liver and deposition of transthyretin in other organs. A therapeutic approach mediated by RNA interference ...resulted in reduced transthyretin levels in affected patients and in controls.
Transthyretin amyloidosis is a life-threatening disorder caused by the deposition of hepatocyte-derived transthyretin amyloid in various tissues and organs.
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,
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Circulating transthyretin is derived from the liver
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and can form amyloid deposits in peripheral nerves and in the gastrointestinal tract, heart, and kidneys. Transthyretin is also synthesized by the retina and choroid plexus,
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,
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which can lead to vitreal and leptomeningeal deposits. More than 100 genetic variants of the gene encoding transthyretin (
TTR
) are associated with autosomal dominant forms of the disease, known as familial amyloidotic polyneuropathy
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and familial amyloidotic cardiomyopathy.
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The most common mutation associated . . .
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.
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IJS, NUK, SBMB, UL, UM, UPUK
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. Numerous proof-of-concept studies in animal models of human disease demonstrate the broad potential application of RNAi therapeutics. The major challenge for successful drug development is identifying delivery strategies that can be translated to the clinic. With advances in this area and the commencement of multiple clinical trials with RNAi therapeutic candidates, a transformation in modern medicine may soon be realized.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
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.
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.
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BFBNIB, NMLJ, NUK, PNG, SAZU, UL, UM, UPUK
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.
The growth of injured axons in the adult mammalian CNS is limited after injury. Three myelin proteins, Nogo, MAG (myelin-associated glycoprotein), and OMgp (oligodendrocyte myelin glycoprotein), bind ...to the Nogo-66 receptor (NgR) and inhibit axonal growth in vitro. Transgenic or viral blockade of NgR function allows axonal sprouting in vivo. Here, we administered the soluble function-blocking NgR ectodomain aa 27-310; NgR(310)ecto to spinal-injured rats. Purified NgR(310)ecto-Fc protein was delivered intrathecally after midthoracic dorsal over-hemisection. Axonal sprouting of corticospinal and raphespinal fibers in NgR(310)ecto-Fc-treated animals correlates with improved spinal cord electrical conduction and improved locomotion. The ability of soluble NgR(310)ecto to promote axon growth and locomotor recovery demonstrates a therapeutic potential for NgR antagonism in traumatic spinal cord injury.
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. Chemical methods were developed to allow the rapid synthesis of a large library of over 1,200 structurally diverse lipidoids. From this library, we identified lipidoids that facilitate high levels of specific silencing of endogenous gene transcripts when formulated with either double-stranded small interfering RNA (siRNA) or single-stranded antisense 2'-O-methyl (2'-OMe) oligoribonucleotides targeting microRNA (miRNA). The safety and efficacy of lipidoids were evaluated in three animal models: mice, rats and nonhuman primates. The studies reported here suggest that these materials may have broad utility for both local and systemic delivery of RNA therapeutics.
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.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
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 28days 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 28days. 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.
► Chronic convection enhanced delivery of siRNA targeting Huntingtin in primate brain. ► Widespread drug distribution results in Huntingtin lowering throughout striatum. ► Empirical model of data enables scaling of siRNA delivery. ► Potential as key disease-modifying therapeutic approach for Huntington's disease.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
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