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some! benefit! with! respect! to! other! pathologies! or! strategies.! In! contrast,! RNAi!
systemic!delivery!requires!larger!amounts!of!product!and!favours!a!broad!delivery,!
sometimes!affecting,!nonYspecific!tissues!such!as!liver,!lung!and!spleen.!!

In!this!way,!one!important!point!to!be!considered!is!to!verify!that!the!topical!
application! of! the! siRNA! effectively! passes! through! the! cornea! to! reach! the!
targeted!tissues.!The!eGFP!mouse!has!been!a!useful!model!to!validate!the!delivery!
of! these! oligonucleotides! to! the! ciliary! body.! The! siRNA! silencing! GFP! are!
efficiently! taken! up! by! ocular! tissues,! reaching! the! ciliary! body! and! other!
structures,! and! effectively! silenced! endogenous! eGFP! expression! for! at! least! four!
days,!when!consecutive!administrations!were!applied.!SiRNAs!have!been!topically!
administered! and! it! is! presumed! that! it! can! be! delivered! by! either! transcellular!
transport!across!the!corneal!epithelium!and!stroma!(9)!or!by!the!paracellular!way!
(10).! The! corneal! epithelium! seems! to! be! the! barrier,! which! limits! transcorneal!
diffusion! although! the! existence! of! the! paracellular! way! would! be! a! mechanism!
that! allows! the! transport! of! siRNA! inside! the! eye.! The! spaces! between! cells,!
estimated! to! be! about! 60! Å,! permit! molecules! to! gain! access! to! the! anterior!
chamber! (10).! It! is! difficult! to! evaluate! quantitatively! how! much! of! the! applied!
siRNA!reaches!the!ciliary!processes,!nevertheless!and!by!means!of!the!experiments!
performed! with! the! eGFP! mouse! it! has! been! possible! to! demonstrate! that! siRNA!
penetrates! and! that! they! can! diminish! the! fluorescence! of! intracellular! ocular!
structures.!Therefore,!these!experiments!allowed!us!to!confirm!a!positive!delivery!
in!several!ocular!structures!and!to!establish!the!proper!protocol!for!each!of!them.!!

Once! the! in! vivo! ocular! approach! of! RNAi! technology! was! tried! out,! our!
main!aim!was!to!confirm!this!strategy!as!a!new!open!angle!glaucoma!treatment.!To!
this! end,! we! focused! our! attention! on! beta2! adrenergic! receptors,! since! their!
commercial! inhibitors! are! effective! in! lowering! IOP,! the! primary! indication! of!
glaucoma!(11).!These!receptors!are!mainly!located!in!the!trabecular!meshwork!in!
the!ciliary!body.!!

The!principal!finding!of!this!paper!is!the!development!of!therapeutic!siRNAs!
targeting! the! beta2! adrenergic! receptors,! which! are! involved! in! intraocular!
pressure.! The! use! of! siRNA! may! offer! advantages! and! may! be! preferable! to! DNAY
based! strategies! for! in! vivo! applications,! although! the! instability! and! the! poor!
uptake! into! target! tissues! remain! the! most! important! handicaps.! A! commercial!
sequence!was!tested!in!vivo!in!rabbits!with!very!interesting!results.!!

According! to! the! in! vivo! data! obtained! in! the! eGFP! mouse! model,! optimal!
delivery!time,!application!pattern!and!effective!RNAi!sequence!dose!was!estimated!
for! the! normotensive! New! Zealand! white! rabbit! model.! For! these! experiments,!
targeting! the! ciliary! body,! 0.265! mg! of! beta2! adrenergic! receptor! siRNA! were!
administered!daily!in!a!single!dose!on!four!consecutive!days.!The!results!confirmed!
the!reduction!of!the!IOP!in!rabbits,!in!a!marked!and!sustained!way.!!

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