ALLELE-SELECTIVE RNA-FOCUSED THERAPEUTIC APPROACHES FOR REPEAT EXPANSION DISORDERS

Tandem repeats, or repeat expansion diseases (RED), are polymorphic nucleotide sequences distributed throughout the human genome. More than fifty neurological disorders are attributed to tandem repeats, which are an essential cause of human neurological disorders. These short repeat expansions come in various sizes, from dinucleotides to longer, and can be found in coding and non-coding sequences of different genes, resulting in diverse groups of diseases. The general mechanism of the tandem repeat expansion disease pathologies associated with bidirectional transcription, intranuclear RNA foci, and repeat-associated non-AUG (RAN) translation are main highlights for the introduction chapter. Our primary focus on REDs is on molecular mechanisms driving disease pathogenesis, which vary depending on repeat size, location, and the phenomena of clinical anticipation. Additionally, we discuss the emerging therapeutic approaches for several known repeat expansion diseases and their potential clinical applications. Throughout the chapter, our primary focus is on the molecular mechanisms and RNA biology of mainly two REDs, Huntington\u27s disease and C9ORF72-associated Frontotemporal dementia and Amyotrophic lateral Sclerosis. In the later chapters, we study the allele-selective potential RNA-centric therapeutic approaches for the mutant HTT gene in Huntington’s disease and the mutant C9ORF72 gene for FTD/ALS. Huntington’s disease (HD) is an incurable neurodegenerative disorder caused by genetic expansion of a CAG repeat sequence in one allele of the huntingtin (HTT) gene. Reducing expression of the mutant HTT (mutHTT) protein has remained a clear therapeutic goal but reduction of wild-type HTT (wtHTT) is undesirable as it compromises gene function and potential therapeutic efficacy. One promising allele-selective approach involves targeting the CAG repeat expansion with steric binding small RNAs bearing central mismatches. However, successful genetic encoding requires consistent placement of mismatches to the target within the small RNA guide sequence, which involves 5\u27 processing precision by cellular enzymes. Here, we used small RNA sequencing to monitor the processing precision of a limited set of CAG repeat-targeted small RNAs expressed from multiple scaffold contexts. Small RNA sequencing identified expression constructs with high guide strand 5\u27 processing precision that also conferred promising allele-selective inhibition of mutHTT. However, mRNA-seq revealed varying degrees of transcriptome-wide off-target effects, including certain CAG repeat-containing mRNAs. These results support the continued investigation and optimization of genetically encoded repeat-targeted small RNAs for allele-selective HD gene therapy and underscore the value of sequencing methods to balance specificity with allele selectivity during the design and selection process. The following two chapters describe the two potential RNA-focused therapeutics for C9FTD/ALS, an incurable neurological disorder. Amyotrophic Lateral Sclerosis (ALS) is a progressive neurological disease characterized by degeneration of motor neurons in the brain and spinal cord. The leading genetic cause of ALS is the hexanucleotide repeat expansion in chromosome 9 open reading frame 72 (C9ORF72). The molecular pathology cascade is primarily driven by the production of repeat expansion RNAs from that locus, which are subsequently translated into poly-dipeptide repeat (poly-DPR) proteins. Gaps in the underlying mechanism persist, which can aid in identifying promising therapeutic approaches. Selective and safe reduction of transcription across the mutant C9ORF72 locus could reduce repeat RNA and poly-DPR production and potentially offer a therapeutic window. Our first RNA focused approach is the low or moderate inhibition of guanosine triphosphate (GTP) and cytidine triphosphate (CTP) synthesis via targeting purine and pyrimidine pathways can serve as a treatment for C9ORF72-mediated amyotrophic lateral sclerosis (C9-FTD/ALS) by achieving the safe and selective reduction of the (G4C2)n repeat expansion RNA transcription. Pharmacological or genetic inhibition of enzymes involved in GTP and CTP biosynthesis represent successful antiviral therapies and treatments for organ transplant rejection. Our in-vitro screen gave us some potential small molecule inhibitors with specific doses that induce low toxicity on expanded and non-expanded reporter cells. We demonstrated that small molecule inhibitors targeting GTP and CTP depletion by targeting purine and pyrimidine biosynthesis reduced both sense and antisense repeat expansion RNA, and selectively preserved C9ORF72 protein at low and safe doses in patient-derived neural stem cells. We also genetically depleted the enzymes involved in the GTP and CTP biosynthesis pathway, which selectively reduced G4C2 repeat expansion RNA expression in our cell-based experiments, strengthening our proof of concept. Taken together, the findings provide a novel approach that ensures safety in assessing its efficacy and molecular mechanisms of action in cellular models of C9FTD/ALS. In the second RNA focused approach, we propose utilizing CRISPRoff, a highly precise and effective CRISPR-based dCas9-methyltransferase fusion system, to target the methylation of the C9ORF72 promoter and repeat expansion sequence. CRISPRoff enables precise CpG methylation for targeted and long-term gene silencing with minimal off-target effects and does not induce DNA cleavage. We investigated the impact of CRISPRoff in C9FTD/ALS models and patient-derived induced pluripotent stem cells (iPSCs). Our results demonstrated that transiently transfection with CRISPRoff and specific sgRNAs targeting the TetON promoter and CCCCGG repeat expansion in model cells led to allele-selective methylation, substantially reducing mCherry reporter expression and poly-(GP) in expanded poly-(GP)88-mCherry reporter cells. Additionally, in patient-iPSCs, transient targeting of the C9ORF72 promoter and repeat expansion resulted in a notable and significant decrease in repeat RNA. Nanopore sequencing confirmed the increased methylation at the repeat expansion sequences for reporter and patient-iPSCs cells. These findings suggest that multiple methylation events are occurring, and inducing silencing and targeting the repeat sequence could be selectively applied to the expanded allele. Given the relative rarity of G4C2 repeat tracts in the human genome, CRISPRoff offers a promising safety profile for C9FTD/ALS therapy. This two study not only provides a novel therapeutic approach for slowing down the progression of C9FTD/ALS but also highlights the potential of targeting nucleoside/nucleotide depletion and using CRISPRoff for epigenetic regulation of repeat expansion loci in multiple repeat expansion disorders. Taken all together, allele-selective RNA focused therapeutics provides therapeutic approaches for Huntington and C9FTD/ALS disease

Architecture to Increase Parallelism in Multisite Testing by Reducing I/O Scan Pins

Performing quality control on any system-on-a-chip (SoC) requires testing wafers and packages at multiple sites to save testing time and costs. Currently, the parallelism feasible with multisite testing is limited by the number of Input/Outputs (IOs) required because typical testers provide only a limited number of IO channels. This disclosure describes a circuit architecture to reduce the number of scan IN and scan OUT pins for multisite SoC testing by providing scan INs and scan OUTS from the same input port. The scan OUT comparison can then be performed internally within the SoC via one status bit. The status bit can be implemented as dedicated IO, or can be captured in a register that is read via the Joint Test Action Group (JTAG) interface on Test Data Output (TDO). The techniques can be implemented to support any type of multisite testing within any SoC. Implementation of the techniques can help achieve higher parallelism in multisite testing. The increase in parallelism can yield substantial time and cost savings, providing higher throughput and lower time to market

Effects of In Vivo Hepatic Ischemia-Reperfusion Injury on the Hepatobiliary Disposition of Rhodamine 123 and its Metabolites in Isolated Perfused Rat Livers

Purpose. A few studies have shown that normothermic hepatic ischemia-reperfusion (IR) injury may affect the mRNA and/or protein levels of canalicular transporters P-glycoprotein (P-gp) and multidrug resistance-associated protein 2 (Mrp2). However, the effects of the injury on the functions of these canalicular transporters with respect to the biliary excretion of drugs remain largely unknown. Therefore, the purpose of this study was to investigate the effects of warm hepatic IR on the hepatobiliary disposition of rhodamine 123 (RH-123), a P-gp substrate, and its glucuronidated metabolite (RH-Glu), an Mrp2 substrate, in rats. Methods. Twenty four or 72 h following a 60-min partial ischemia or sham operation in rats, livers were isolated and perfused ex vivo with a constant concentration (~100 ng/mL) of RH-123. The concentration of RH-123 and its glucuronidated (RH-Glu) and deacylated (RH-110) metabolites were determined in the outlet perfusate, bile, and the liver tissue using HPLC, and relevant pharmacokinetic parameters were estimated. Results. Twenty-four-h IR caused a significant reduction in the hepatic extraction ratio of RH-123 (IR: 0.857 ± 0.078; Sham: 0.980 ± 0.017) and the biliary recovery of the parent drug and RH-Glu by 43% and 44%, respectively. The reductions in the biliary recovery were associated with significant reductions in the apparent biliary clearance of RH-123 and RH-Glu. Mass balance data showed that the formation of the glucuronidated or deacylated metabolite was not significantly affected by the 24-h IR injury. In contrast to the 24-h IR, the injury did not have any effect on the hepatobiliary disposition of RH-123 or its metabolites following 72 h of reperfusion. Conclusions. It is concluded that the pharmacokinetics of drugs that are subject to biliary excretion by the canalicular P-gp and Mrp2 transporters may be altered shortly after hepatic IR injury

Myocardial depressant effects of interleukin 6 in meningococcal sepsis are regulated by p38 mitogen-activated protein kinase

Our findings demonstrate an integral role of the p38 mitogen-activated protein kinase pathway in interleukin 6-mediated cardiac contractile dysfunction and inotrope insensitivity. Dysregulation of the p38 mitogen-activated protein kinase pathway in meningococcal septicemia suggests that this pathway may be an important target for novel therapies to reverse myocardial dysfunction in patients with meningococcal septic shock who are not responsive to inotropic support

Effects of In Vivo Hepatic Ischemia-Reperfusion Injury on the Hepatobiliary Disposition of Rhodamine 123 and its Metabolites in Isolated Perfused Rat Livers

Purpose. A few studies have shown that normothermic hepatic ischemia-reperfusion (IR) injury may affect the mRNA and/or protein levels of canalicular transporters P-glycoprotein (P-gp) and multidrug resistance-associated protein 2 (Mrp2). However, the effects of the injury on the functions of these canalicular transporters with respect to the biliary excretion of drugs remain largely unknown. Therefore, the purpose of this study was to investigate the effects of warm hepatic IR on the hepatobiliary disposition of rhodamine 123 (RH-123), a P-gp substrate, and its glucuronidated metabolite (RH-Glu), an Mrp2 substrate, in rats. Methods. Twenty four or 72 h following a 60-min partial ischemia or sham operation in rats, livers were isolated and perfused ex vivo with a constant concentration (~100 ng/mL) of RH-123. The concentration of RH-123 and its glucuronidated (RH-Glu) and deacylated (RH-110) metabolites were determined in the outlet perfusate, bile, and the liver tissue using HPLC, and relevant pharmacokinetic parameters were estimated. Results. Twenty-four-h IR caused a significant reduction in the hepatic extraction ratio of RH-123 (IR: 0.857 ± 0.078; Sham: 0.980 ± 0.017) and the biliary recovery of the parent drug and RH-Glu by 43% and 44%, respectively. The reductions in the biliary recovery were associated with significant reductions in the apparent biliary clearance of RH-123 and RH-Glu. Mass balance data showed that the formation of the glucuronidated or deacylated metabolite was not significantly affected by the 24-h IR injury. In contrast to the 24-h IR, the injury did not have any effect on the hepatobiliary disposition of RH-123 or its metabolites following 72 h of reperfusion. Conclusions. It is concluded that the pharmacokinetics of drugs that are subject to biliary excretion by the canalicular P-gp and Mrp2 transporters may be altered shortly after hepatic IR injury

Exposure-Response and Population Pharmacokinetic Analyses of a Novel Subcutaneous Formulation of Daratumumab Administered to Multiple Myeloma Patients

We report the population pharmacokinetic (PK) and exposure-response analyses of a novel subcutaneous formulation of daratumumab (DARA) using data from 3 DARA subcutaneous monotherapy studies (PAVO Part 2, MMY1008, COLUMBA) and 1 combination therapy study (PLEIADES). Results were based on 5159 PK samples from 742 patients (DARA 1800 mg subcutaneously, n = 487 [monotherapy, n = 288; combination therapy, n = 199]; DARA 16 mg/kg intravenously, n = 255 [all monotherapy, in COLUMBA]; age, 33-92 years; weight, 28.6-147.6 kg). Subcutaneous and intravenous DARA monotherapies were administered once every week for cycles 1-2, once every 2 weeks for cycles 3-6, and once every 4 weeks thereafter (1 cycle is 28 days). The subcutaneous DARA combination therapy was administered with the adaptation of corresponding standard-of-care regimens. PK samples were collected between cycle 1 and cycle 12. Among monotherapy studies, throughout the treatment period, subcutaneous DARA provided similar/slightly higher trough concentrations (Ctrough) versus intravenous DARA, with lower maximum concentrations and smaller peak-to-trough fluctuations. The PK profile was consistent between subcutaneous DARA monotherapy and combination therapies. The exposure-response relationship between daratumumab PK and efficacy or safety end points was similar for subcutaneous and intravenous DARA. Although the ≤65-kg subgroup reported a higher incidence of neutropenia, no relationship was found between the incidence of neutropenia and exposure, which was attributed, in part, to the preexisting imbalance in neutropenia between subcutaneous DARA (45.5%) and intravenous DARA (19%) in patients ≤50 kg. A flat relationship was observed between body weight and any grade and at least grade 3 infections. The results support the DARA 1800-mg subcutaneous flat dose as an alternative to the approved intravenous DARA 16 mg/kg.The clinical studies and the analyses presented here were supported by research funding from Janssen Research & Development, LLC

Development and validation of stability indicating method for determination of sertraline following ICH guidlines and its determination in pharmaceuticals and biological fluids

<p>Abstract</p> <p>Background</p> <p>Sertraline is a well known antidepressant drug which belongs to a class called selective serotonin reuptake inhibitor. Most published methods do not enable studying the stability of this drug in different stress conditions.</p> <p>Results</p> <p>Two new methods were developed for the determination of sertraline (SER). Both methods are based on coupling with 4-chloro-7-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl) in borate buffer of pH 7.8 and measuring the reaction product spectrophotometrically at 395 nm (Method I) or spectrofluorimetrically at 530 nm upon excitation at 480 nm (Method II). The response-concentration plots were rectilinear over the range 2-24 μg/mL and 0.25-5 μg/mL for methods I and II respectively with LOD of 0.18 μg/mL and 0.07 μg/mL, and LOQ of 0.56 μg/mL and 0.21 μg/mL for methods I and II, respectively.</p> <p>Conclusion</p> <p>Both methods were applied to the analysis of commercial tablets and the results were in good agreement with those obtained using a reference method. The fluorimetric method was further applied to the in vivo determination of SER in human plasma. A proposal of the reaction pathway was presented. The spectrophotometric method was extended to stability study of SER. The drug was exposed to alkaline, acidic, oxidative and photolytic degradation according to ICH guidelines. Moreover, the method was utilized to investigate the kinetics of oxidative degradation of the drug. The apparent first order rate constant and t_1/2of the degradation reaction were determined.</p

Characterization of Rhodamine-123 as a Tracer Dye for Use In In vitro Drug Transport Assays

Fluorescent tracer dyes represent an important class of sub-cellular probes and allow the examination of cellular processes in real-time with minimal impact upon these processes. Such tracer dyes are becoming increasingly used for the examination of membrane transport processes, as they are easy-to-use, cost effective probe substrates for a number of membrane protein transporters. Rhodamine 123, a member of the rhodamine family of flurone dyes, has been used to examine membrane transport by the ABCB1 gene product, MDR1. MDR1 is viewed as the archetypal drug transport protein, and is able to efflux a large number of clinically relevant drugs. In addition, ectopic activity of MDR1 has been associated with the development of multiple drug resistance phenotype, which results in a poor patient response to therapeutic intervention. It is thus important to be able to examine the potential for novel compounds to be MDR1 substrates. Given the increasing use rhodamine 123 as a tracer dye for MDR1, a full characterisation of its spectral properties in a range of in vitro assay-relevant media is warranted. Herein, we determine λmax for excitation and emission or rhodamine 123 and its metabolite rhodamine 110 in commonly used solvents and extraction buffers, demonstrating that fluorescence is highly dependent on the chemical environment: Optimal parameters are 1% (v/v) methanol in HBSS, with λex = 505 nm, λem = 525 nm. We characterise the uptake of rhodamine 123 into cells, via both passive and active processes, and demonstrate that this occurs primarily through OATP1A2-mediated facilitated transport at concentrations below 2 µM, and via micelle-mediated passive diffusion above this. Finally, we quantify the intracellular sequestration and metabolism of rhodamine 123, demonstrating that these are both cell line-dependent factors that may influence the interpretation of transport assays