Liquid chromatography electrospray ionization mass spectrometry analysis of the ocular metabolites from a short interfering RNA duplex

Beverly, Michael B.; Hartsough, Kim; Machemer, Lynn; Pavco, Pam A.; Lockridge, Jennifer A.

doi:10.1016/j.jchromb.2006.03.008

Cited by 52 publications

(62 citation statements)

References 38 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…This route of administration is also currently being used in clinical trials with siRNAs targeting VEGF (Brucker, 2006), VEGFR1 (Kaiser et al, 2010) and hypoxia-inducible RTP801 genes (Nguyen et al, 2012b). As chemically modified siRNAs can also be degraded by vitreous endonucleases (Beverly et al, 2006), siRNA-based treatments of posterior segment diseases could require multiple intravitreal injections, similar to conventional treatments. This need for multiple injections increases the potential for cataract, retinal detachment, vitreous haemorrhage and endophthalmitis (Kurz and Ciulla, 2002).…”

Section: Figurementioning

confidence: 99%

“…These modifications include the introduction of phosphorothioate backbone linkages at the 3′-end of the RNA strands (Gaglione and Messere, 2010), boranophosphate backbone modifications (Hall et al, 2004) or the incorporation of chemical modifications at the 2′ position of the ribose such as 2′-O-methyl or 2′-fluoro (Chiu and Rana, 2003). However, even chemically modified siRNAs can be susceptible to degradation as some endonucleases present in the extracellular environment, such as the vitreous humour, are able to avoid the terminal protection by bypassing them (Beverly et al, 2006). …”

mentioning

confidence: 99%

See 1 more Smart Citation

Small‐interfering RNAs (siRNAs) as a promising tool for ocular therapy

Guzmán-Aránguez

Loma

Pintor

2013

British J Pharmacology

109

View full text Add to dashboard Cite

RNA interference (RNAi) can be used to inhibit the expression of specific genes in vitro and in vivo, thereby providing an extremely useful tool for investigating gene function. Progress in the understanding of RNAi-based mechanisms has opened up new perspectives in therapeutics for the treatment of several diseases including ocular disorders. The eye is currently considered a good target for RNAi therapy mainly because it is a confined compartment and, therefore, enables local delivery of small-interfering RNAs (siRNAs) by topical instillation or direct injection. However, delivery strategies that protect the siRNAs from degradation and are suitable for long-term treatment would be help to improve the efficacy of RNAi-based therapies for ocular pathologies. siRNAs targeting critical molecules involved in the pathogenesis of glaucoma, retinitis pigmentosa and neovascular eye diseases (age-related macular degeneration, diabetic retinopathy and corneal neovascularization) have been tested in experimental animal models, and clinical trials have been conducted with some of them. This review provides an update on the progress of RNAi in ocular therapeutics, discussing the advantages and drawbacks of RNAi-based therapeutics compared to previous treatments. AbbreviationsAMD, age-related macular degeneration; Apaf-1, apoptotic protease-activating factor-1; CNV, choroidal neovascularization; CTGF, connective tissue growth factor; DR, diabetic retinopathy; dsRNA, double-stranded RNA; ECM, extracellular matrix; HIF-1α, hypoxia-inducible factor-1α; IOP, intraocular pressure; ONC, optic nerve crush; POAG, open-angle glaucoma; RGC, retinal ganglion cell; RISC, RNA-induced silencing complex; RNAi, RNA interference; RP, retinitis pigmentosa; Smad, signalling mathers against decapentaplegic; SPARC, secreted protein acidic and rich in cysteine; TLR3, toll-like receptor 3; TM, trabecular meshwork; TXNIP, thioredoxin interacting protein IntroductionRNA interference (RNAi) technology has been used to elucidate gene function, to generate model systems and to identify new molecular targets. In addition, RNAi is currently progressing from basic research to potential therapeutic applications. Because any gene that causes or contributes to a disease is susceptible to suppression by RNAi, RNAi therapy represents a promising biomedical strategy for treating a diverse range of diseases including cancer, cardiovascular diseases, neurodegenerative diseases, inflammatory conditions, viral infections and ocular diseases (Guo et al., 2010). In particular, the accessibility of the eye facilitates small-interfering RNA (siRNA) delivery, and naked siRNA has been efficiently applied by topical administration to the anterior segment or by intravitreal injection to the posterior segment (de Fougerolles, 2008). As the localized delivery of siRNA to the eye is less challenging than for other tissues, there has been significant progress made towards its use as a therapeutic procedure for eye diseases. In fact, several siRNA-based therapeutic agents for o...

show abstract

Section: Figurementioning

confidence: 99%

mentioning

confidence: 99%

Small‐interfering RNAs (siRNAs) as a promising tool for ocular therapy

Guzmán-Aránguez

Loma

Pintor

2013

British J Pharmacology

109

View full text Add to dashboard Cite

show abstract

“…Chromatographic separation of duplex from such truncated duplex species has been shown to be possible, yet difficult [33]. Unfortunately, liquid chromatography-mass spectrometry (LC-MS) analysis of the intact duplex has demonstrated a decrease in sensitivity compared with the denatured individual strands due to increased cation adduction [34]. MALDI-time-of flight (TOF) MS has demonstrated the ability to detect intact siRNA duplexes using 6-aza2-thiothymine (ATT) containing diammonium hydrogen citrate (DAHC) as the matrix for quality control purposes [35], yet to date there have been no reports of MALDI being applied to analysis of intact siRNA duplexes isolated from biological matrices.…”

Section: Double-stranded Characteristic Of Sirnamentioning

confidence: 99%

“…The primary depots for siRNAs delivered in vivo with a delivery vehicle, such as a clinically acceptable lipid nanoparticle (LNP), are to organs such as the liver and spleen [46], thus requiring tissue extraction protocols in order to identify and quantitate the minor amounts of siRNA in these matrices. Isolating siRNAs from plasma and tissue for MS analysis have proven to be difficult and laborious, usually requiring a two-step liquid-liquid, solid-phase extraction (SPE) [34,47]. The high protein binding of oligonucleotides requires an extraction with phenol : chloroform to disrupt protein-RNA interactions, which impedes using automation and high-throughput sample preparation.…”

Section: Difficult Isolation From Biomatricesmentioning

confidence: 99%

“…Beverly et al used IP-LC-MS to characterize metabolites of highly modified siRNA duplexes from rabbit ocular vitreous humor and retinal/choroid tissue after intravitreal injection [34,45]. The vitreous samples were prepared using phenol/ chloroform LLE followed by evaporation of the aqueous layer.…”

Section: Quantitative and Qualitative Analysis Of Sirna From Biologicmentioning

confidence: 99%

See 1 more Smart Citation