Mutations in USH2A are among the most common causes of syndromic and non-syndromic retinitis pigmentosa (RP). The two most recurrent mutations in USH2A, c.2299delG and c.2276G > T, both reside in exon 13. Skipping exon 13 from the USH2A transcript presents a potential treatment modality in which the resulting transcript is predicted to encode a slightly shortened usherin protein. Morpholino-induced skipping of ush2a exon 13 in zebrafish ush2a rmc1 mutants resulted in the production of usherinDexon 13 protein and a completely restored retinal function. Antisense oligonucleotides were investigated for their potential to selectively induce human USH2A exon 13 skipping. Lead candidate QR-421a induced a concentration-dependent exon 13 skipping in induced pluripotent stem cell (iPSC)-derived photoreceptor precursors from an Usher syndrome patient homozygous for the c.2299delG mutation. Mouse surrogate mQR-421a reached the retinal outer nuclear layer after a single intravitreal injection and induced a detectable level of exon skipping until at least 6 months post-injection. In conclusion, QR-421a-induced exon skipping proves to be a highly promising treatment option for RP caused by mutations in USH2A exon 13.

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This white paper, which is the 10th in a series intended to address issues associated with the development of therapeutic oligonucleotides, examines the subject of product-related impurities. The authors consider chemistry and safety aspects and advance arguments in favor of platform approaches to impurity identification and qualification. Reporting, identification, and qualification thresholds suitable for product-related impurities of therapeutic oligonucleotides are proposed.

Chronic administration of drisapersen, a 2 0 -OMe phosphorothioate antisense oligonucleotide (AON) to mice and monkeys resulted in renal tubular accumulation, with secondary tubular degeneration. Glomerulopathy occurred in both species with species-specific characteristics. Glomerular lesions in mice were characterized by progressive hyaline matrix accumulation, accompanied by the presence of renal amyloid and with subsequent papillary necrosis. Early changes involved glomerular endothelial hypertrophy and degeneration, but the chronic glomerular amyloid and hyaline alterations in mice appeared to be species specific. An immune-mediated mechanism for the glomerular lesions in mice was supported by early inflammatory changes including increased expression of inflammatory cytokines and other immunomodulatory genes within the renal cortex, increased stimulation of CD68 protein, and systemic elevation of monocyte chemotactic protein 1. In contrast, kidneys from monkeys given drisapersen chronically showed less severe glomerular changes characterized by increased mesangial and inflammatory cells, endothelial cell hypertrophy, and subepithelial and membranous electron-dense deposits, with ultrastructural and immunohistochemical characteristics of complement and complement-related fragments. Lesions in monkeys resembled typical features of C3 glomerulopathy, a condition described in man and experimental animals to be linked to dysregulation of the alternative complement pathway. Thus, inflammatory/immune mechanisms appear critical to glomerular injury with species-specific sensitivities for mouse and monkey. The lower observed proinflammatory activity in humans as compared to mice and monkeys may reflect a lower risk of glomerular injury in patients receiving AON therapy.

Eluforsen (previously known as QR-010) is a 33-mer 2′- O -methyl modified phosphorothioate antisense oligonucleotide targeting the F508del mutation in the gene encoding CFTR protein of cystic fibrosis patients. In this study, eluforsen was incubated with endo- and exonucleases and mouse liver homogenates to elucidate its in vitro metabolism. Mice and monkeys were used to determine in vivo liver and lung metabolism of eluforsen following inhalation. We developed a liquid chromatography-mass spectrometry method for the identification and semi-quantitation of the metabolites of eluforsen and then applied the method for in vitro and in vivo metabolism studies. Solid-phase extraction was used following proteinase K digestion for sample preparation. Chain-shortened metabolites of eluforsen by 3′ exonuclease were observed in mouse liver in an in vitro incubation system and by either 3′ exonuclease or 5′ exonuclease in liver and lung samples from an in vivo mouse and monkey study. This study provides approaches for further metabolite characterization of 2′-ribose-modified phosphorothioate oligonucleotides in in vitro and in vivo studies to support the development of oligonucleotide therapeutics.

In the present study the oxidative dehalogenation of a para-halogenated phenol was studied using pentafluorophenol and its non-para-halogenated analogue 2,3,5,6-tetrafluorophenol as model compounds. 19F NMR was used to characterize the metabolite patterns. In order to study the primary oxidation products of the microsomal cytochrome P450-catalyzed conversion, the alternative oxygen donors cumene hydroperoxide (CumOOH) and iodosobenzene (IOB) were used in addition to the use of NADPH and molecular oxygen. In a NADPH/oxygen-driven reaction, but also in a CumOOH- or IOB-driven cytochrome P450 reaction, tetrafluorophenol was converted to tetrafluorohydroquinone. However, for pentafluorophenol, the formation of tetrafluorohydroquinone as a product of its cytochrome P450-mediated conversion was only observed in the NADPH-driven system. Addition of reducing equivalents such as NADH to the CumOOH or IOB incubations resulted in the formation of tetrafluorohydroquinone. From these data it was concluded that the primary reaction product of the cytochrome P450-catalyzed conversion of pentafluorophenol is a reactive species that can be reduced to tetrafluorohydroquinone by NAD(P)H and, thus, must be tetrafluorobenzoquinone. Additional experiments with tetrafluorobenzoquinone, incubated in vitro with either microsomal protein or glutathione in the presence or absence of reducing equivalents, demonstrated that the tetrafluorobenzoquinone ends up bound to proteins, losing its fluorine atoms as fluoride anions. Thus, while cytochrome P450-mediated conversion of the 2,3,5,6-tetrafluorophenol results in the formation of tetrafluorohydroquinone as the primary reaction product, monooxygenation at a fluorinated para position, such as in pentafluorophenol, results in the formation of the reactive tetrafluorobenzoquinone derivative as the primary reaction product.(ABSTRACT TRUNCATED AT 250 WORDS)

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