Fazirsiran for Liver Disease Associated with Alpha1-Antitrypsin Deficiency.

BACKGROUND: Alpha1-antitrypsin (AAT) deficiency results from carriage of a homozygous SERPINA1 "Z" mutation (proteinase inhibitor [PI] ZZ). The Z allele produces a mutant AAT protein called Z-AAT, which accumulates in hepatocytes and can lead to progressive liver disease and fibrosis. This open-label, phase 2 trial investigated the safety and efficacy of fazirsiran, an RNA interference therapeutic, in patients with liver disease associated with AAT deficiency. METHODS: We assigned adults with the PI ZZ genotype and liver fibrosis to receive fazirsiran at a dose of 200 mg (cohorts 1 [4 patients] and 2 [8 patients]) or 100 mg (cohort 1b [4 patients]) subcutaneously on day 1 and week 4 and then every 12 weeks. The primary end point was the change from baseline to week 24 (cohorts 1 and 1b) or week 48 (cohort 2) in liver Z-AAT concentrations, which were measured by means of liquid chromatography-mass spectrometry. RESULTS: All the patients had reduced accumulation of Z-AAT in the liver (median reduction, 83% at week 24 or 48). The nadir in serum was a reduction of approximately 90%, and treatment was also associated with a reduction in histologic globule burden (from a mean score of 7.4 [scores range from 0 to 9, with higher scores indicating a greater globule burden] at baseline to 2.3 at week 24 or 48). All cohorts had reductions in liver enzyme concentrations. Fibrosis regression was observed in 7 of 15 patients and fibrosis progression in 2 of 15 patients after 24 or 48 weeks. There were no adverse events leading to trial or drug discontinuation. Four serious adverse events (viral myocarditis, diverticulitis, dyspnea, and vestibular neuronitis) resolved. CONCLUSIONS: In this small trial, fazirsiran was associated with a strong reduction of Z-AAT concentrations in the serum and liver and concurrent improvements in liver enzyme concentrations. (Funded by Arrowhead Pharmaceuticals; AROAAT-2002 ClinicalTrials.gov number, NCT03946449.).

Kidney Allograft Fibrosis: Diagnostic and Therapeutic Strategies.

Interstitial fibrosis with tubule atrophy (IF/TA) is the response to virtually any sustained kidney injury and correlates inversely with kidney function and allograft survival. IF/TA is driven by various pathways that include hypoxia, renin-angiotensin-aldosterone system, transforming growth factor-ß signaling, cellular rejection, inflammation, and others. In this review, we will focus on key pathways in the progress of renal fibrosis, diagnosis and therapy of allograft fibrosis. This review discusses the role and origin of myofibroblasts as matrix producing cells and therapeutic targets in renal fibrosis with a particular focus on renal allografts. We summarize current trends to use multiomic approaches to identify new biomarkers for IF/TA detection and to predict allograft survival. Furthermore, we review current imaging strategies that might help to identify and follow-up IF/TA complementary or as alternative to invasive biopsies. We further discuss current clinical trials and therapeutic strategies to treat kidney fibrosis.

siRNA Design and GalNAc-Empowered Hepatic Targeted Delivery.

Small interfering RNA (siRNA) is a clinically approved therapeutic modality, which has attracted widespread attention not only from basic research but also from pharmaceutical industry. As siRNA can theoretically modulate any disease-related gene's expression, plenty of siRNA therapeutic pipelines have been established by tens of biotechnology companies. The drug performance of siRNA heavily depends on the sequence, the chemical modification, and the delivery of siRNA. Here, we describe the rational design protocol of siRNA, and provide some modification patterns that can enhance siRNA's stability and reduce its off-target effect. Also, the delivery method based on N-acetylgalactosamine (GalNAc)-siRNA conjugate that is widely employed to develop therapeutic regimens for liver-related diseases is also recapitulated.

Effect of inclisiran, the small-interfering RNA against proprotein convertase subtilisin/kexin type 9, on platelets, immune cells, and immunological biomarkers: a pre-specified analysis from ORION-1.

AIMS: Small-interfering RNA (siRNA)-based targeting of proprotein convertase subtilisin/kexin type 9 (PCSK9) represents a novel therapeutic approach that may provide a convenient, infrequent, and safe dosing schedule to robustly lower low-density lipoprotein cholesterol (LDL-C). Given the long duration of action, however, establishing safety in particular with respect to immunogenicity is of paramount importance. In earlier clinical studies of other RNA-targeted treatment approaches (antisense oligonucleotide therapy) immunological and haematological adverse effects, in particular thrombocytopenia and pro-inflammatory effects, have been reported. Here, we present the pre-specified safety analysis from ORION-1 evaluating platelets, immune cells, immunological markers, antidrug antibodies, and clinical immunogenicity adverse events (AEs) under PCSK9 siRNA treatment with inclisiran. METHODS AND RESULTS: The pre-specified safety analysis from ORION-1 was performed in six different inclisiran dosing regimens in patients at high risk of cardiovascular disease with elevated LDL-C levels. Patients received either a single dose (SD: 200 mg, n = 60; 300 mg, n = 62 or 500 mg, n = 66) or double-dose starting regimen (DD: 100 mg, n = 62; 200 mg, n = 63; or 300 mg, n = 61 on days 1 and 90) of inclisiran or placebo (SD: n = 65; DD: n = 62). The effects of inclisiran on haematological parameters including platelet counts, lymphocytes, and monocytes as well as on the immune markers interleukin 6 (IL-6) and tumour necrosis factor-α (TNF-α) were examined after 180 days. Immunogenicity was further evaluated by analysis of anti-drug-antibodies (ADAs) towards inclisiran in 6068 study samples and by careful analysis of immunogenicity AEs as part of the pharmacovigilance strategy. At day 180, no significant alterations of platelet counts were observed in any of the dosing groups (change from baseline, SD: 200 mg: 0.8%; 300 mg: -0.5%; 500 mg: -1.8%; DD: 100 mg: 1.3%; 200 mg: -0.5%; 300 mg: 1.0%; no significant difference for any group as compared with placebo). No significant effects on other immune cells, including leucocytes, monocytes, or neutrophils were detected. Notably, no significant increase of inflammatory biomarkers (IL-6 or TNF-α) with either the SD or DD regimen became evident. There was no evidence for immunogenicity based on ADA level analysis and careful review of clinical immunogenicity AEs in none of the treatment regimens. CONCLUSION: In this pre-specified safety analysis of ORION-1 for the siRNA therapeutic inclisiran, no adverse effects on measures of inflammation or immune activation nor adverse effects on platelets or clinical immunogenicity AEs were observed over at least 6-month treatment. These safety findings in the largest analysis of an RNAi study in humans to date provide strong reassurance about the safety of inclisiran and the potential of cardiovascular RNA-targeted therapies.

Phase 3 Multicenter Study of Revusiran in Patients with Hereditary Transthyretin-Mediated (hATTR) Amyloidosis with Cardiomyopathy (ENDEAVOUR).

PURPOSE: The Phase 3 ENDEAVOUR study evaluated revusiran, an investigational RNA interference therapeutic targeting hepatic transthyretin (TTR) production, for treating cardiomyopathy caused by hereditary transthyretin-mediated (hATTR) amyloidosis. METHODS: Patients with hATTR amyloidosis with cardiomyopathy were randomized 2:1 to receive subcutaneous daily revusiran 500 mg (n = 140) or placebo (n = 66) for 5 days over a week followed by weekly doses. Co-primary endpoints were 6-min walk test distance and serum TTR reduction. RESULTS: Revusiran treatment was stopped after a median of 6.71 months; the study Sponsor prematurely discontinued dosing due to an observed mortality imbalance between treatment arms. Eighteen (12.9%) patients on revusiran and 2 (3.0%) on placebo died during the on-treatment period. Most deaths in both treatment arms were adjudicated as cardiovascular due to heart failure (HF), consistent with the natural history of the disease. A post hoc safety investigation of patients treated with revusiran found that, at baseline, a greater proportion of those who died were ≥ 75 years and showed clinical evidence of more advanced HF compared with those who were alive throughout treatment. Revusiran pharmacokinetic exposures and TTR lowering did not show meaningful differences between patients who died and who were alive. Revusiran did not deleteriously affect echocardiographic parameters, cardiac biomarkers, or frequency of cardiovascular and HF hospitalization events. CONCLUSIONS: Causes for the observed mortality imbalance associated with revusiran were thoroughly investigated and no clear causative mechanism could be identified. Although the results suggest similar progression of cardiac parameters in both treatment arms, a role for revusiran cannot be excluded. CLINICAL TRIAL REGISTRATION: NCT02319005.

Time-dependent p53 inhibition determines senescence attenuation and long-term outcome after renal ischemia-reperfusion.

Inhibition of p53 has been shown to be an efficient strategy for ameliorating kidney ischemia-reperfusion (I/R) injury in experimental models. The therapeutic value of p53 siRNA-based inhibition for I/R in renal transplantation is currently being evaluated in clinical studies. While the major rationale for these studies is the suppression of proapoptotic properties, there are more equally important injury response pathways regulated by p53. A p53-dependent pathway shown to be crucial for renal long-term outcome is cellular senescence. In this study, we tested the hypothesis that p53 siRNA reduces I/R-induced senescence and thereby improves kidney outcome. By comparing the impact of different treatment durations in a mouse model of renal I/R, we found that repetitive administration of p53 siRNA during the first 14 days after I/R reduced the senescence load and ameliorated the postischemic phenotype. Prolonged application of p53 siRNA over a 26-day period after I/R, however, did not provide any additional benefit for senescence reduction but reversed some of the renoprotective effects of the early treatment. These data suggest a time-dependent role of p53 activity supporting the current therapeutic concept of a short-term inhibition, while advocating against a prolonged treatment after I/R.

Effects of Patisiran, an RNA Interference Therapeutic, on Cardiac Parameters in Patients With Hereditary Transthyretin-Mediated Amyloidosis.

BACKGROUND: Hereditary transthyretin-mediated (hATTR) amyloidosis is a rapidly progressive, multisystem disease that presents with cardiomyopathy or polyneuropathy. The APOLLO study assessed the efficacy and tolerability of patisiran in patients with hATTR amyloidosis. The effects of patisiran on cardiac structure and function in a prespecified subpopulation of patients with evidence of cardiac amyloid involvement at baseline were assessed. METHODS: APOLLO was an international, randomized, double-blind, placebo-controlled phase 3 trial in patients with hATTR amyloidosis. Patients were randomized 2:1 to receive 0.3 mg/kg patisiran or placebo via intravenous infusion once every 3 weeks for 18 months. The prespecified cardiac subpopulation comprised patients with a baseline left ventricular wall thickness ≥13 mm and no history of hypertension or aortic valve disease. Prespecified exploratory cardiac end points included mean left ventricular wall thickness, global longitudinal strain, and N-terminal prohormone of brain natriuretic peptide. Cardiac parameters in the overall APOLLO patient population were also evaluated. A composite end point of cardiac hospitalizations and all-cause mortality was assessed in a post hoc analysis. RESULTS: In the cardiac subpopulation (n=126; 56% of total population), patisiran reduced mean left ventricular wall thickness (least-squares mean difference ± SEM: -0.9±0.4 mm, P=0.017), interventricular septal wall thickness, posterior wall thickness, and relative wall thickness at month 18 compared with placebo. Patisiran also led to increased end-diastolic volume (8.3±3.9 mL, P=0.036), decreased global longitudinal strain (-1.4±0.6%, P=0.015), and increased cardiac output (0.38±0.19 L/min, P=0.044) compared with placebo at month 18. Patisiran lowered N-terminal prohormone of brain natriuretic peptide at 9 and 18 months (at 18 months, ratio of fold-change patisiran/placebo 0.45, P<0.001). A consistent effect on N-terminal prohormone of brain natriuretic peptide at 18 months was observed in the overall APOLLO patient population (n=225). Median follow-up duration was 18.7 months. The exposure-adjusted rates of cardiac hospitalizations and all-cause death were 18.7 and 10.1 per 100 patient-years in the placebo and patisiran groups, respectively (Andersen-Gill hazard ratio, 0.54; 95% CI, 0.28-1.01). CONCLUSIONS: Patisiran decreased mean left ventricular wall thickness, global longitudinal strain, N-terminal prohormone of brain natriuretic peptide, and adverse cardiac outcomes compared with placebo at month 18, suggesting that patisiran may halt or reverse the progression of the cardiac manifestations of hATTR amyloidosis. CLINICAL TRIAL REGISTRATION: URL: https://www.clinicaltrials.gov . Unique identifier: NCT01960348.

RNA Therapeutics in Cardiovascular Disease.

Noncoding RNAs have been shown to exert important physiological and pathophysiological functions. Various studies suggest that modulating noncoding RNAs may provide a therapeutic option. Noncoding RNAs comprise small RNAs, mainly microRNAs, and long noncoding RNAs. MicroRNAs postranscriptionally regulate gene expression pattern by binding to the 3'untranslated region of a given target mRNA, thereby blocking protein translation or inducing its degradation. Long noncoding RNAs on the contrary have more diverse functions acting as epigenetic regulators, molecular scaffolds, or decoys. In this article, we summarize examples of microRNAs and long noncoding RNAs, which might be promising novel targets for treatment of cardiovascular diseases, such as heart failure, acute myocardial infarction, fibrosis, as well as atherosclerosis. Furthermore, we give insights into the available tools to inhibit or overexpress noncoding RNAs and discuss the challenges for translation. Strategies for improving RNA therapeutics and reducing toxicity, for example, by augmenting tissue specificity or cellular uptake will be discussed.

Effect of an siRNA Therapeutic Targeting PCSK9 on Atherogenic Lipoproteins: Prespecified Secondary End Points in ORION 1.

BACKGROUND: The ORION-1 trial (Trial to Evaluate the Effect of ALN-PCSSC Treatment on Low Density Lipoprotein Cholesterol [LDL-C]) demonstrated that inclisiran, an siRNA therapeutic that targets protease proprotein convertase subtilisin/kexin type 9 mRNA within hepatocytes, produces significant low-density lipoprotein cholesterol reduction. The effects of inclisiran on other lipids are less well described. METHODS: ORION-1 was a phase 2 trial assessing 6 different inclisiran dosing regimens versus placebo. Participants with elevated low-density lipoprotein cholesterol despite receiving maximally tolerated statin therapy received a single-dose (200, 300, or 500 mg) or 2-dose starting regimen (100, 200, or 300 mg on days 1 and 90) of inclisiran or placebo. This prespecified analysis reports the percentage reductions in non-high-density lipoprotein cholesterol (non-HDL-C), apolipoprotein (apo) B, very-low-density lipoprotein cholesterol, lipoprotein(a), triglycerides, HDL-C, and apo A1 at the primary efficacy time point (day 180) with mixed-effect models for repeated measures. Additional prespecified analyses report time course of changes from baseline at each visit to day 210, interindividual variation in response, and lipid goal attainment. RESULTS: The mean age of the 501 participants was 63 years, 65% were male, 69% had atherosclerotic cardiovascular disease, 73% used statins, and mean low-density lipoprotein cholesterol was 128 mg/dL. A single dose of inclisiran reduced apo B, non-HDL-C, and very-low-density lipoprotein cholesterol over 210 days. A second dose of inclisiran provided additional lowering of these lipids. At day 180, non-HDL-C was lowered dose-dependently: by 25% from 148±43 to 110±45 mg/dL in the 200-mg single-dose group and by 46% from 161±58 to 91±58 mg/dL in the 2-dose 300-mg group. For the same dosing regimens, apo B was reduced by 23% from 101±23 to 78±29 mg/dL and by 41% from 106±31 to 65±33 mg/dL ( P<0.001 for all groups versus placebo). In the 300-mg 2-dose group, all individuals experienced apo B and non-HDL-C reductions. There was larger interindividual variation in very-low-density lipoprotein cholesterol, triglycerides, and lipoprotein(a) reductions. In the 300-mg 2-dose group, the percentages of patients achieving guideline-recommended apo B goals for high- and very-high-risk patients at day 180 were 78% and 90%; 68% and 83% of participants achieved non-HDL-C <100 and <130 mg/dL. CONCLUSIONS: Inclisiran produces significant and prolonged reductions in atherogenic lipoproteins, suggesting that inhibiting the synthesis of protease proprotein convertase subtilisin/kexin type 9 through siRNA may be a viable alternative to other approaches that target protease proprotein convertase subtilisin/kexin type 9. CLINICAL TRIAL REGISTRATION: URL: https://www.clinicaltrials.gov . Unique identifier: NCT02597127.

Advances in lipid-lowering therapy through gene-silencing technologies.

New treatment opportunities are emerging in the field of lipid-lowering therapy through gene-silencing approaches. Both antisense oligonucleotide inhibition and small interfering RNA technology aim to degrade gene mRNA transcripts to reduce protein production and plasma lipoprotein levels. Elevated levels of LDL, remnant lipoproteins, and lipoprotein(a) all cause cardiovascular disease, whereas elevated levels of triglyceride-rich lipoproteins in some patients can cause acute pancreatitis. The levels of each of these lipoproteins can be reduced using gene-silencing therapies by targeting proteins that have an important role in lipoprotein production or removal (for example, the protein products of ANGPTL3, APOB, APOC3, LPA, and PCSK9). Using this technology, plasma levels of these lipoproteins can be reduced by 50-90% with 2-12 injections per year; such dramatic reductions are likely to reduce the incidence of cardiovascular disease or acute pancreatitis in at-risk patients. The reported adverse effects of these new therapies include injection-site reactions, flu-like symptoms, and low blood platelet counts. However, newer-generation drugs are more efficiently delivered to liver cells, requiring lower drug doses, which leads to fewer adverse effects. Although these findings are promising, robust evidence of cardiovascular disease reduction and long-term safety is needed before these gene-silencing technologies can have widespread implementation. Before the availability of such evidence, these drugs might have roles in patients with unmet medical needs through orphan indications.

Control the intracellular NF-κB activity by a sensor consisting of miRNA and decoy.

Many diseases are associated with the abnormal activation of NF-κB and its signaling pathway. NF-κB has become an important target for disease treatment and development of new drugs. Many various NF-κB inhibitors were therefore developed; however, they have difficulties to become clinical drugs due to their adverse side effects resulted from the affected normal physiological functions of this transcription factor. To overcome this limitation, this study construct a transgenic vector that can express an artificial miRNA targeting NF-κB RelA under the control of a NF-κB-specific promoter. The promoter consists of a NF-κB decoy and a minimal promoter. The vector was named as decoy minimal promoter-artificial microRNA (DMP-amiRNA). This study verified that this vector can sense and control the intracellular NF-κB activity upon transfection. Working of the vector forms a perfect feedback loop that realizes the NF-κB self-control. With the vector in cells, the higher NF-κB activity, the higher DMP transcriptional activity, and the more amiR533 expression. DMP-amiRNA can moderately inhibit the intracellular NF-κB activity but exert no significant effect on cell viability. This study therefore develops a new strategy for inhibiting over activity of NF-κB, which should has great potential in clinical application.

PCSK9 in context: A contemporary review of an important biological target for the prevention and treatment of atherosclerotic cardiovascular disease.

The identification of the critical role of proprotein convertase subtilisin/kexin type 9 (PCSK9) has rapidly led to the development of PCSK9 inhibition with monoclonal antibodies (mAbs). PCSK9 mAbs are already in limited clinical use and are the subject of major cardiovascular outcomes trials, which, if universally positive, could see much wider clinical application of these agents. Patients with familial hypercholesterolaemia are the most obvious candidates for these drugs, but other patients with elevated cardiovascular risk, statin intolerance or hyperlipoproteinaemia(a) may also benefit. PCSK9 mAbs, administered once or twice monthly, reduce LDL cholesterol levels by 50% to 70%, and appear to be safe and acceptable to patients over at least 2 years of treatment; however, treatment-emergent adverse effects are not always identified in clinical trials, as well-evidenced by statin myopathy. Inclisiran is a promising RNA-based therapy that promotes the degradation of PCSK9 mRNA transcripts and has similar efficacy to mAbs, but with a much longer duration of action. The cost-effectiveness and long-term safety of therapies targeted at inhibiting PCSK9 remain to be demonstrated if they are to be used widely in coronary prevention.

AAV5-miHTT gene therapy demonstrates suppression of mutant huntingtin aggregation and neuronal dysfunction in a rat model of Huntington's disease.

Huntington's disease (HD) is a fatal progressive neurodegenerative disorder caused by a mutation in the huntingtin (HTT) gene. To date, there is no treatment to halt or reverse the course of HD. Lowering of either total or only the mutant HTT expression is expected to have therapeutic benefit. This can be achieved by engineered micro (mi)RNAs targeting HTT transcripts and delivered by an adeno-associated viral (AAV) vector. We have previously showed a miHTT construct to induce total HTT knock-down in Hu128/21 HD mice, while miSNP50T and miSNP67T constructs induced allele-selective HTT knock-down in vitro. In the current preclinical study, the mechanistic efficacy and gene specificity of these selected constructs delivered by an AAV serotype 5 (AAV5) vector was addressed using an acute HD rat model. Our data demonstrated suppression of mutant HTT messenger RNA, which almost completely prevented mutant HTT aggregate formation, and ultimately resulted in suppression of DARPP-32-associated neuronal dysfunction. The AAV5-miHTT construct was found to be the most efficient, although AAV5-miSNP50T demonstrated the anticipated mutant HTT allele selectivity and no passenger strand expression. Ultimately, AAV5-delivered-miRNA-mediated HTT lowering did not cause activation of microglia or astrocytes suggesting no immune response to the AAV5 vector or therapeutic precursor sequences. These preclinical results suggest that using gene therapy to knock-down HTT may provide important therapeutic benefit for HD patients and raised no safety concerns, which supports our ongoing efforts for the development of an RNA interference-based gene therapy product for HD.

Cationic liquid crystalline nanoparticles for the delivery of synthetic RNAi-based therapeutics.

RNA interference (RNAi)-based therapeutics have been used to silence the expression of targeted pathological genes. Small interfering RNA (siRNAs) and microRNA (miRNAs) inhibitor have performed this function. However, short half-life, poor cellular uptake, and nonspecific distribution of small RNAs call for the development of novel delivery systems to facilitate the use of RNAi. We developed a novel cationic liquid crystalline nanoparticle (CLCN) to efficiently deliver synthetic siRNAs and miRNAs. CLCNs were prepared by using high-speed homogenization and assembled with synthetic siRNA or miRNA molecules in nuclease-free water to create CLCN/siRNA or miRNA complexes. The homogeneous and stable CLCNs and CLCN-siRNA complexes were about 100 nm in diameter, with positively charged surfaces. CLCNs are nontoxic and are taken up by human cells though endocytosis. Significant inhibition of gene expression was detected in transiently transfected lung cancer H1299 cells treated with CLCNs/anti-GFP complexes 24 hours after transfection. Biodistribution analysis showed that the CLCNs and CLCNs-RNAi complexes were successfully delivered to various organs and into the subcutaneous human lung cancer H1299 tumor xenografts in mice 24 hours after systemic administration. These results suggest that CLCNs are a unique and advanced delivery system capable of protecting RNAi from degradation and of efficiently delivering RNAi in vitro and in vivo.

Targeting androgen receptor versus targeting androgens to suppress castration resistant prostate cancer.

Prostate cancer (PCa) is the 2nd leading cause of cancer-related death among men in the United States and its progression is tightly associated with the androgen/androgen receptor (AR) signals. Men castrated before puberty (eunuchs) or men with inherited deficiency of type II 5α-reductase (with failure to convert testosterone to the more potent dihydrotestosterone) (DHT) do not develop PCa. To date, androgen deprivation therapy (ADT) with anti-androgen treatments to reduce or prevent androgens from binding to the AR remains the main therapeutic option for advanced PCa since its discovery by Huggins and Hodges in 1941. Multiple strategies related to surgical/chemical castration with combinations of various anti-androgens, including Cyproterone Acetate, Flutamide, Nilutamide, Bicalutamide (Casodex) and Enzalutamide, as well as some androgen synthesis blockers, including Abiraterone, have been used to control PCa progression. However, patients on ADT with anti-androgen treatment eventually develop resistance, which might be accompanied with the unwanted side effects of enhanced metastasis. New therapeutic approaches via directly targeting the AR with ASC-J9®, Cisplatin, EPI-001, Niclosamide, and VPC compounds as well as silencing AR with siRNAs or non-coding RNAs have been developed to further suppress PCa at the castration resistant stages.

Therapeutic Targeting of Protein Kinase CK2 Gene Expression in Feline Oral Squamous Cell Carcinoma: A Naturally Occurring Large-Animal Model of Head and Neck Cancer.

Protein kinase CK2 (CK2) is a highly promising target for cancer therapy, and anti-CK2 gene expression therapy has shown effectiveness in rodent models of human head and neck cancer (HNC). To date, there has been no large-animal model of cancer in which to further explore anti-CK2 therapies. Feline oral squamous cell carcinoma (FOSCC) has been proposed as a large-animal model for human HNC, and we have previously shown that CK2 is a rational target in FOSCC. Here we have tested the hypothesis that a novel tenfibgen-coated tumor-specific nanocapsule carrying RNA interference (RNAi) oligonucleotides targeting feline CK2α and CK2α' (TBG-RNAi-fCK2αα') would be safe in cats with FOSCC; assessment of target inhibition and tumor response were secondary aims. Nine cats were enrolled and treated at two dose levels in a 3+3 escalation. Cats received a total of six treatments with TBG-RNAi-fCK2αα'. Pre- and posttreatment, tumor and normal oral mucosa biopsies were collected to assess CK2 expression, using immunohistochemistry (IHC) preparations evaluated by light microscopy. Toxicity and tumor response were assessed on the basis of standard criteria. The most common adverse events were grade 1 or 2 weight loss and anorexia. Grade 3 tissue necrosis was seen in association with tumor response in one cat, asymptomatic grade 4 elevations in aspartate transaminase and creatine phosphokinase in one cat, and asymptomatic grade 3 hypokalemia in one cat. Of six cats with evaluable biopsies, two had a reduction in CK2 IHC score in tumors after treatment. Four cats had progressive disease during the study period, three had stable disease, one had partial response, and response could not be evaluated in one cat. We conclude that the drug appeared safe and that there is some evidence of efficacy in FOSCC. Further investigation regarding dosing, schedule, target modulation, toxicity, and efficacy in a larger group of cats is warranted and may inform future clinical studies in human head and neck cancer.

Genetic pharmacology: progresses in siRNA delivery and therapeutic applications.

In the RNA interference process, the catalytic degradation of an endogenous mRNA results from the Watson-Crick complementary recognition by either a small silencing synthetic double-stranded ribonucleotide (siRNA) or by a small hairpin RNA (shRNA) produced in the cell by transcription from a DNA template. This interference process ideally results in an exquisitely specific mRNA suppression. The present review is dedicated to siRNAs. It describes the mechanism of RNA silencing and the main siRNA delivery techniques, with a focus on siRNA self-complexing to cationic lipids to form nanoparticles, which are called lipoplexes. The addition to lipoplexes of an anionic polymer leads to the ternary formulation APIRL (Anionic-Polymer-Interfering-RNA-Lipoplexes) with increased in vivo stability and biological efficacy. In terms of clinical development, the review focuses on therapeutic applications by intravenous delivery to the liver and inflammatory joints, and to localized siRNA delivery to the ocular sphere.

RNA interference-induced hepatotoxicity results from loss of the first synthesized isoform of microRNA-122 in mice.

Small RNAs can be engineered to target and eliminate expression of disease-causing genes or infectious viruses, resulting in the preclinical and clinical development of RNA interference (RNAi) therapeutics using these small RNAs. To ensure the success of RNAi therapeutics, small hairpin RNAs (shRNAs) must co-opt sufficient quantities of the endogenous microRNA machinery to elicit efficient gene knockdown without impeding normal cellular function. We previously observed liver toxicity-including hepatocyte turnover, loss of gene repression and lethality-in mice receiving high doses of a recombinant adeno-associated virus (rAAV) vector expressing shRNAs (rAAV-shRNAs); however the mechanism by which toxicity ensues has not been elucidated. Using rAAV-shRNAs we have now determined that hepatotoxicity arises when exogenous shRNAs exceed 12% of the total amount of liver microRNAs. After this threshold was surpassed, shRNAs specifically reduced the initially synthesized 22-nucleotide isoform of microRNA (miR)-122-5p without substantially affecting other microRNAs, resulting in functional de-repression of miR-122 target mRNAs. Delivery of a rAAV-shRNA vector expressing mature miR-122-5p could circumvent toxicity, despite the exogenous shRNA accounting for 70% of microRNAs. Toxicity was also not observed in Mir122-knockout mice regardless of the level or sequence of the shRNA. Our study establishes limits to the microRNA machinery that is available for therapeutic siRNAs and suggests new paradigms for the role of miR-122 in liver homeostasis in mice.

Potential and development of inhaled RNAi therapeutics for the treatment of pulmonary tuberculosis.

Tuberculosis (TB), caused by the infection of Mycobacterium tuberculosis (Mtb), continues to pose a serious threat to public health, and the situation is worsening with the rapid emergence of multidrug resistant (MDR) TB. Current TB regimens require long duration of treatment, and their toxic side effects often lead to poor adherence and low success rates. There is an urgent need for shorter and more effective treatment for TB. In recent years, RNA interference (RNAi) has become a powerful tool for studying gene function by silencing the target genes. The survival of Mtb in host macrophages involves the attenuation of the antimicrobial responses mounted by the host cells. RNAi technology has helped to improve our understanding of how these bacilli interferes with the bactericidal effect and host immunity during TB infection. It has been suggested that the host-directed intervention by modulation of host pathways can be employed as a novel and effective therapy against TB. This therapeutic approach could be achieved by RNAi, which holds enormous potential beyond a laboratory to the clinic. RNAi therapy targeting TB is being investigated for enhancing host antibacterial capacity or improving drug efficacy on drug resistance strains while minimizing the associated adverse effects. One of the key challenges of RNAi therapeutics arises from the delivery of the RNAi molecules into the target cells, and inhalation could serve as a direct administration route for the treatment of pulmonary TB in a non-invasive manner. However, there are still major obstacles that need to be overcome. This review focuses on the RNAi candidates that are currently explored for the treatment of TB and discusses the major barriers of pulmonary RNAi delivery. From this, we hope to stimulate further studies of local RNAi therapeutics for pulmonary TB treatment.