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. 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.

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. 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.).

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. 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.

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 0.01 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.

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. These effects occurred after a 2-week schedule of intermittent, systemic administration of artemin and persisted for at least 6 months following treatment, suggesting a substantial translational advantage. Systemic artemin administration produced essentially complete and persistent restoration of nociceptive and sensorimotor functions, and could represent a promising therapy that may effectively promote sensory neuronal regeneration and functional recovery after injury.

Efficient and Precise Processing of the Optimized Primary Artificial MicroRNA in a Huntingtin-Lowering Adeno-Associated Viral Gene Therapy In Vitro and in Mice and Nonhuman Primates.

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.

Multiple actions of systemic artemin in experimental neuropathy.

The clinical management of neuropathic pain is particularly challenging. Current therapies for neuropathic pain modulate nerve impulse propagation or synaptic transmission; these therapies are of limited benefit and have undesirable side effects. Injuries to peripheral nerves result in a host of pathophysiological changes associated with the sustained expression of abnormal pain. Here we show that systemic, intermittent administration of artemin produces dose- and time-related reversal of nerve injury-induced pain behavior, together with partial to complete normalization of multiple morphological and neurochemical features of the injury state. These effects of artemin were sustained for at least 28 days. Higher doses of artemin than those completely reversing experimental neuropathic pain did not elicit sensory or motor abnormalities. Our results indicate that the behavioral symptoms of neuropathic pain states can be treated successfully, and that partial to complete reversal of associated morphological and neurochemical changes is achievable with artemin.

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. 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.

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. In this study, small interfering RNA (siRNA) oligonucleotides were identified, which downregulate the levels of torsinA or torsinADeltaE mRNA and protein by over 65% following transfection. Transfection of siRNA for torsinA message in control fibroblasts expressing Gluc reduced levels of luciferase secretion compared with the same cells non-transfected or transfected with a non-specific siRNA. Transfection of siRNA selectively inhibiting torsinADeltaE message in DYT fibroblasts increased luciferase secretion when compared with cells non-transfected or transfected with a non-specific siRNA. Further, transduction of DYT1 cells with a lentivirus vector expressing torsinA, but not torsinB, also increased secretion. These studies are consistent with a role for torsinA as an ER chaperone affecting processing of proteins through the secretory pathway and indicate that torsinADeltaE acts to inhibit this torsinA activity. The ability of allele-specific siRNA for torsinADeltaE to normalize secretory function in DYT1 patient cells supports its potential role as a therapeutic agent in early onset torsion dystonia.

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. 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.

Gene therapy for neurological disorders: progress and prospects.

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

Gene therapy for neurological disorders: progress and prospects.

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.

Neurotrophic factors as novel therapeutics for neuropathic pain.

Neuropathic pain is a chronic condition that is caused by injury to the nervous system. Unlike acute pain, which is protective, neuropathic pain persists and serves no useful purpose, and severely affects quality of life. However, present therapies have modest efficacy in most patients, are palliative rather than curative, and their side effects represent significant limitations. Tremendous progress has been made over the past decade in our understanding of the biology of pain sensory neurons. The recent discovery that neurotrophic factors play an important role in neuropathic pain indicates that these pathways could serve as novel intervention points for therapy. Moreover, neurotrophic factors have the potential to address the underlying pathophysiology of neuropathic pain, thereby halting or reversing the disease process.

Control of phosphorothioate stereochemistry substantially increases the efficacy of antisense oligonucleotides.

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.

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. RNA interference represents a therapeutic approach applicable to such non-druggable targets. Both non-viral and viral delivery strategies are being undertaken for in vivo silencing of molecular targets by RNA interference, which has resulted in robust efficacy in animal models of Alzheimer's disease, ALS, Huntington's disease, spinocerebellar ataxia, anxiety, depression, neuropathic pain, encephalitis and glioblastoma. These proof-of-concept data in animal models, together with the commencement of clinical trials using RNA interference for macular degeneration and respiratory syncytial virus infection, point to the potential of direct RNA interference for neurological disorders and neurodegenerative diseases.

Blockade of Nogo-66, myelin-associated glycoprotein, and oligodendrocyte myelin glycoprotein by soluble Nogo-66 receptor promotes axonal sprouting and recovery after spinal injury.

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.

New approaches for the treatment of pain: the GDNF family of neurotrophic growth factors.

This article focuses on the GDNF family of neurotrophic factors as a potential new class of therapeutics for neuropathic pain, with a particular emphasis on the ligands, artemin and GDNF. In vivo activity of the ligands, expression of ligands and receptors after peripheral nerve injury, and modulation of nerve injury-induced changes by the ligands are reviewed in detail. Structural considerations, particularly with regard to implications for binding interactions and biological activity are discussed.

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. 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.

Artemin induced functional recovery and reinnervation after partial nerve injury.

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.

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. 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.