Antisense oligonucleotides (ASOs) and small interfering RNA (siRNA) promise specific correction of disease-causing gene expression. Therapeutic implementation, however, has been forestalled by poor ...delivery to the appropriate tissue, cell type, and subcellular compartment. Topical administration is considered to circumvent these issues. The availability of inhalation devices and unmet medical need in lung disease has focused efforts in this tissue. We report the development of a novel cell sorting method for quantitative, cell type-specific analysis of siRNA, and locked nucleic acid (LNA) ASO uptake and efficacy after intratracheal (i.t.) administration in mice. Through fluorescent dye labeling, we compare the utility of this approach to whole animal and whole tissue analysis, and examine the extent of tissue distribution. We detail rapid systemic access and renal clearance for both therapeutic classes and lack of efficacy at the protein level in lung macrophages, epithelia, or other cell types. We nevertheless observe efficient redirection of i.t. administered phosphorothioate (PS) LNA ASO to the liver and kidney leading to targeted gene knockdown. These data suggest delivery remains a key obstacle to topically administered, naked oligonucleotide efficacy in the lung and introduce inhalation as a potentially viable alternative to injection for antisense administration to the liver and kidneys.
TT-034 (PF-05095808) is a recombinant adeno-associated virus serotype 8 (AAV8) agent expressing three short hairpin RNA (shRNA) pro-drugs that target the hepatitis C virus (HCV) RNA genome. The ...cytosolic enzyme Dicer cleaves each shRNA into multiple, potentially active small interfering RNA (siRNA) drugs. Using next-generation sequencing (NGS) to identify and characterize active shRNAs maturation products, we observed that each TT-034–encoded shRNA could be processed into as many as 95 separate siRNA strands. Few of these appeared active as determined by Sanger 5′ RNA Ligase-Mediated Rapid Amplification of cDNA Ends (5-RACE) and through synthetic shRNA and siRNA analogue studies. Moreover, NGS scrutiny applied on 5-RACE products (RACE-seq) suggested that synthetic siRNAs could direct cleavage in not one, but up to five separate positions on targeted RNA, in a sequence-dependent manner. These data support an on-target mechanism of action for TT-034 without cytotoxicity and question the accepted precision of substrate processing by the key RNA interference (RNAi) enzymes Dicer and siRNA-induced silencing complex (siRISC).
A series of C–H functionalisation plate-based chemical screens and other C–H activation protocols were developed for the chemical diversification of drug molecules. In this Letter, metalloporphyrin ...and other catalytic oxidation systems are described in addition to chlorination. Mifepristone and antalarmin are used as substrates. The products obtained and the biological data demonstrate the potential utility of this approach.
Automation of a commercially available Fourier transform ion cyclotron resonance (FTICR) mass spectrometer for the routine analysis of the synthetic products from high-speed chemistry is described. ...The automation includes software written by the instrument manufacturer as well as in-house-developed software that allows electronic submission of samples by the chemist and e-mailing of results back to the chemist. The use of samples of relatively high concentration (approx. 1 mg mL−1) is possible due to the protocol that has been developed, which includes dilution by the autosampler during sample injection. Though high concentrations are used for speed and convenience, the amount of sample consumed is still small at approx. 15 μg per injection. The results from this method have been shown to be both accurate (typical error range – 2.29 to 2.36 ppm, average error 1.07 ppm) and precise (standard deviation of the order of 0.3 ppm). The system is capable of analysing up to 800 samples per 24 hours. As high-speed chemistry becomes more highly utilised within Pfizer Discovery, the number of samples requiring accurate mass analysis will rise but the method we have described will prevent high-resolution mass spectrometry becoming the bottleneck in new chemical entity production. The accuracy and precision demonstrated by this method allows high confidence levels in assigned molecular formulae for expected compounds and reduces the number of possible formulae to consider when working with a compound that is not the desired product of a given reaction.
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