Kev hnov ​​​​lus (HL) yog kab mob uas muaj feem cuam tshuam rau tib neeg.Hauv cov teb chaws tsim kho, kwv yees li 80% ntawm cov neeg lag ntseg ua ntej ua ntej ntawm cov menyuam yaus yog tshwm sim los ntawm caj ces.Feem ntau yog ib leeg-gene tsis xws luag (raws li qhia hauv daim duab 1), 124 noob kev hloov pauv tau pom tias muaj feem cuam tshuam nrog kev hnov ​​​​lus tsis zoo hauv tib neeg, tus so yog tshwm sim los ntawm ib puag ncig yam.Cochlear implant (ib qho khoom siv hluav taws xob tso rau hauv pob ntseg uas muab hluav taws xob ncaj qha mus rau lub paj hlwb) yog qhov kev xaiv zoo tshaj plaws rau kev kho mob hnyav HL, thaum lub pob ntseg (ib qho khoom siv hluav taws xob sab nraud uas hloov pauv thiab ua kom lub suab nrov) tuaj yeem pab cov neeg mob uas muaj HL.Txawm li cas los xij, tam sim no tsis muaj cov tshuaj muaj los kho cov kab mob HL (GHL).Nyob rau hauv xyoo tas los no, kev kho noob tau txais kev saib xyuas ntau ntxiv raws li kev cog lus los kho lub pob ntseg sab hauv.

Fig1.Kev faib tawm ntawm qhov lag ntseg cuam tshuam nrog kev hloov pauv yam[1]

Tsis ntev los no, cov kws tshawb fawb los ntawm Salk lub koom haum thiab University of Sheffield tau luam tawm cov kev tshawb fawb hauv Molecular Therapy - Methods & Clinical Development [2], uas tau qhia dav dav rau daim ntawv thov kev cia siab rau hauv vivo gene therapy ntawm kev lag ntseg ntawm caj ces.Uri Manor, tus kws tshawb fawb pabcuam ntawm lub koom haum Salk thiab tus thawj coj ntawm Waitt Center for Advanced Biophotonics, tau hais tias nws tau yug los nrog kev hnov ​​​​lus tsis zoo thiab xav tias kev kho lub rooj sib hais yuav yog ib qho khoom plig zoo.Nws txoj kev tshawb fawb yav dhau los pom tias Eps8 yog ib qho kev tswj hwm cov protein nrog actin khi thiab capping kev ua ub no;nyob rau hauv cochlear cov plaub hau hlwb, cov protein complex tsim los ntawm Eps8 nrog MYO15A, WHIRLIN, GPSM2 thiab GNAI3 tsuas yog muaj nyob rau hauv feem ntau Cov lus qhia ntawm ntev stereocilia, uas ua ke nrog MYO15A localize BAIAP2L2 ntawm cov lus qhia ntawm luv luv stereocilia, yuav tsum tau rau kev saib xyuas ntawm cov plaub hau bundles.Yog li ntawd, Eps8 tuaj yeem tswj qhov ntev ntawm stereocilia ntawm cov plaub hau hlwb, uas yog qhov tseem ceeb rau kev hnov ​​​​lus zoo;Eps8 tshem tawm lossis hloov pauv yuav ua rau luv luv stereocilia, uas ua rau nws tsis tuaj yeem hloov suab mus rau hauv hluav taws xob teeb liab rau lub hlwb kev xav, uas ua rau lag ntseg..Nyob rau tib lub sijhawm, tus neeg koom tes Walter Marcotti, tus xibfwb ntawm University of Sheffield, pom tias cov plaub hau tsis tuaj yeem txhim kho ib txwm thaum tsis muaj Eps8.Hauv txoj kev tshawb no, Manor thiab Marcotti tau sib koom ua ke los tshawb xyuas seb puas ntxiv Eps8 rau cov kab mob stereociliary tuaj yeem rov qab los ntawm lawv txoj haujlwm thiab, dhau los, txhim kho kev hnov ​​lus hauv nas.Pab neeg tshawb fawb tau siv tus kab mob adeno-associated virus (AAV) vector Anc80L65 los xa cov coding ib ntus uas muaj cov tsiaj qus EPS8 rau hauv cochlea ntawm Eps8-/- cov nas me nyuam mos P1-P2 los ntawm kev txhaj tshuaj puag ncig lub qhov rais;nyob rau hauv nas cochlear plaub hau hlwb Cov kev ua ntawm stereocilia tau kho ua ntej lawv matured;thiab cov nyhuv kho tau tus yam ntxwv los ntawm kev yees duab technology thiab kev ntsuas ntawm stereocilia.Cov txiaj ntsig tau pom tias Eps8 nce qhov ntev ntawm stereocilia thiab rov ua haujlwm ntawm cov plaub hau hauv cov hlwb tsawg.Lawv kuj pom tias, dhau sijhawm, cov hlwb zoo li poob lawv lub peev xwm los cawm tau los ntawm kev kho cov noob no.Qhov cuam tshuam yog tias qhov kev kho mob no yuav tsum tau muab coj los siv rau hauv lub tsev menyuam, vim Eps8-/- cov hlwb cov plaub hau tuaj yeem loj hlob lossis tsim kev puas tsuaj ntau dhau los kho tom qab cov nas tau yug los."Eps8 yog cov protein uas muaj ntau yam haujlwm sib txawv, thiab tseem muaj ntau yam los tshawb nrhiav," Manor hais.Kev tshawb fawb yav tom ntej yuav suav nrog kev tshawb xyuas cov txiaj ntsig ntawm Eps8 gene therapy nyob rau hauv kev kho cov kev hnov ​​​​lus ntawm ntau theem kev loj hlob, thiab seb nws puas tuaj yeem ua rau ncua sijhawm kho.Zoo sib xws, thaum lub Kaum Ib Hlis 2020, xibfwb KarenB Avraham ntawm Tel Aviv University hauv Ixayees tau luam tawm nws cov txiaj ntsig hauv phau ntawv journal EMBO Molecular Medicine [3], siv cov thev naus laus zis thev naus laus zis thev naus laus zis los tsim cov kab mob sib txuas tsis muaj teeb meem adeno-txuas AAV9-PHP.B, Cov noob tsis xws luag hauv cov plaub hau hlwb ntawm Syne4-/- nas tau kho los ntawm kev txhaj tshuaj tiv thaiv kab mob uas nqa cov txheej txheem ntawm Syne4 mus rau hauv pob ntseg ntawm nas, tso cai rau nws nkag mus rau hauv cov plaub hau hlwb thiab tso cov khoom siv caj ces, tso cai rau lawv kom loj hlob thiab ua haujlwm ib txwm (raws li hauv daim duab 2).

Fig 2.Schematic sawv cev ntawm lub pob ntseg sab hauv lub cev, nrog rau kev tsom mus rau lub cev ntawm Corti thiab cellular muaj nuj nqi ntawm nesprin-4.

Nws tuaj yeem pom tau tias kev siv cov noob kho kom ua tiav lub hom phiaj ntawm kev kho cov kab mob ntawm cov noob caj noob ces los ntawm kev ntxig, tshem tawm lossis kho cov noob hloov pauv rau kev kho mob (uas yog, tswj cov caj ces hloov hauv tus kab mob) muaj txiaj ntsig zoo hauv kev kho mob.thov kev cia siab.Txoj kev kho noob tam sim no rau kev lag ntseg tsis zoo tuaj yeem muab faib ua pawg hauv qab no:

noob hloov

Kev hloov noob caj noob ces yog qhov sib cav tshaj plaws ntawm "qhia ncaj nraim" daim ntawv ntawm kev kho noob, raws li kev txheeb xyuas thiab hloov cov noob tsis zoo nrog cov qauv qub lossis hom tsiaj qus.Thawj qhov kev vam meej hauv pob ntseg gene kho txoj kev kawm rau kev hnov ​​​​lus tsis zoo los ntawm kev tshem tawm ntawm vesicular glutamate transporter 3 (VGLUT3) gene;AAV1-mediated xa tawm ntawm exogenous VGLUT3 overexpression nyob rau hauv pob ntseg pob ntseg hlwb (IHCs) tuaj yeem ua rau muaj kev hnov ​​​​lus zoo, ib feem ntawm ribbon synaptic morphology rov qab, thiab cov lus teb convulsive [4].Txawm li cas los xij, hauv cov piv txwv suav nrog ob AAV-xa cov noob hloov pauv tau piav qhia hauv cov lus qhia saum toj no, nws yog ib qho tseem ceeb uas yuav tsum nco ntsoov tias cov qauv nas siv rau qee hom kev tshem tawm cov noob tsis hnov ​​​​lus txawv ntawm tib neeg lub cev, thiab hauv P1 nas, pob ntseg sab hauv yog nyob rau theem ntawm kev loj hlob.Piv txwv li, tib neeg yug los nrog lub pob ntseg paub tab.Qhov sib txawv no txwv tsis pub siv cov txiaj ntsig nas mus rau kev kho mob ntawm tib neeg kev lag ntseg lag ntseg tshwj tsis yog kev kho noob xa mus rau cov pob ntseg pob ntseg.

Gene Editing: CRISPR/Cas9

Piv nrog rau "gene hloov", txoj kev loj hlob ntawm noob kho tshuab tau coj kaj ntug ntawm kev kho cov kab mob caj ces los ntawm cov hauv paus hniav.Qhov tseem ceeb, txoj kev kho cov noob caj noob ces ua rau qhov tsis txaus ntawm cov txheej txheem kev kho cov noob caj noob ces uas tsis tsim nyog rau cov kab mob cuam tshuam txog kev lag ntseg, thiab qhov teeb meem uas txoj kev overexpression tsis kav ntev.Tom qab Suav cov kws tshawb fawb tshwj xeeb tau tshem tawm Myo6C442Y mutant allele hauv Myo6WT/C442Y nas siv AAV-SaCas9-KKH-Myo6-g2 noob kho, thiab nyob rau hauv 5 lub hlis ntawm knockout, nas Cov kev ua haujlwm ntawm tus qauv tau rov qab los;Nyob rau tib lub sijhawm, nws kuj tau pom tias qhov ciaj sia ntawm cov plaub hau hlwb hauv pob ntseg sab hauv tau txhim kho, cov duab cilia tau ua tsis tu ncua, thiab cov ntsuas electrophysiological tau kho [5].Qhov no yog thawj txoj kev tshawb fawb hauv ntiaj teb siv CRISPR/Cas9 thev naus laus zis los kho cov lag ntseg lag ntseg uas tshwm sim los ntawm Myo6 gene mutation, thiab nws yog ib qho kev tshawb fawb tseem ceeb ntawm cov noob kho cov thev naus laus zis rau kev kho cov lag ntseg ntawm caj ces.Lub chaw kho mob txhais lus ntawm kev kho mob muab lub hauv paus ruaj khov scientific.

Gene therapy txoj kev xa khoom

Txhawm rau txhawm rau kho cov noob kom ua tiav, cov liab qab DNA molecules tsis tuaj yeem nkag mus rau cov hlwb tau zoo vim lawv cov dej tsis zoo thiab cov nqi tsis zoo ntawm phosphate pawg, thiab kom ntseeg tau tias kev ncaj ncees ntawm cov nucleic acid molecules ntxiv, yuav tsum xaiv txoj kev nyab xeeb thiab siv tau zoo.Cov DNA ntxiv yog xa mus rau lub hom phiaj ntawm tes lossis cov ntaub so ntswg.AAV tau dav siv los ua lub tsheb xa khoom rau kev kho mob vim nws cov kab mob sib kis tau zoo, tsis muaj kev tiv thaiv kab mob, thiab dav tropism rau ntau hom ntaub so ntswg.Tam sim no, lub cev loj ntawm kev tshawb fawb ua haujlwm tau txiav txim siab qhov tropism ntawm cov subtypes sib txawv ntawm AAV txheeb ze rau ntau hom cell hauv nas cochlea.Siv AAV tus yam ntxwv xa khoom ua ke nrog cov neeg txhawb nqa tshwj xeeb hauv xov tooj tuaj yeem ua tiav cov lus qhia tshwj xeeb ntawm tes, uas tuaj yeem txo cov teebmeem ntawm lub hom phiaj.Tsis tas li ntawd, raws li lwm txoj hauv kev rau AAV vectors, cov khoom siv hluav taws xob AAV tshiab tau tsim los tas li thiab pom muaj peev xwm hloov pauv tau zoo nyob rau hauv pob ntseg sab hauv, ntawm cov AAV2 / Anc80L65 yog qhov siv dav tshaj plaws.Txoj kev xa tsis yog kab mob tuaj yeem muab faib ntxiv rau hauv txoj kev siv lub cev (microinjection thiab electroporation) thiab cov txheej txheem tshuaj (lipid-based, polymer-based, thiab kub nanoparticles).Ob txoj hauv kev tau siv los kho cov kab mob lag ntseg ntawm caj ces thiab tau pom qhov zoo sib txawv thiab kev txwv.Ntxiv nrog rau lub tsheb xa mus rau kev kho noob raws li lub tsheb, ntau txoj hauv kev rau hauv vivo gene tswj tuaj yeem ua haujlwm raws li cov hom phiaj sib txawv ntawm tes, txoj hauv kev ntawm kev tswj hwm, thiab kev kho mob zoo.Cov qauv sib txawv ntawm pob ntseg sab hauv ua rau nws nyuaj rau ncav cuag lub hom phiaj ntawm lub hlwb thiab kev faib cov genome kho cov neeg ua haujlwm qeeb.Lub membranous labyrinth yog nyob rau hauv lub pob txha labyrinth ntawm lub cev nqaij daim tawv thiab muaj xws li cov cochlear duct, semicircular duct, utricle, thiab balloon.Nws cov txheeb ze sib cais, tsawg kawg nkaus lymphatic ncig, thiab kev sib cais los ntawm cov ntshav los ntawm cov ntshav-maze barrier txwv qhov kev siv tau zoo ntawm kev kho mob rau cov nas neonatal nkaus xwb.Yuav kom tau txais cov kab mob titers tsim nyog rau kev kho noob, kev txhaj tshuaj ncaj qha hauv zos ntawm cov kab mob kis mus rau hauv pob ntseg sab hauv yog qhov tsim nyog.Tsim txoj hauv kev ntawm kev txhaj tshuaj muaj xws li [6]: (1) round window membrane (RWM), (2) tracheostomy, (3) endolymphatic lossis perilymphatic cochleostomy, (4) round window membrane plus Tube fenestration (CF) (raws li hauv daim duab 3).

Fig 3.Kev kho pob ntseg sab hauv ntawm kev kho noob.

Txawm hais tias muaj ntau yam kev kho mob tau ua tiav hauv cov noob caj noob ces, raws li cov hom phiaj kho mob txhais lus, yuav tsum tau ua haujlwm ntau ntxiv ua ntej kev kho noob tuaj yeem dhau los ua thawj kab kev kho mob rau cov neeg mob uas muaj kab mob caj ces, tshwj xeeb tshaj yog nyob rau hauv kev txhim kho kev nyab xeeb thiab zoo vectors thiab kev xa khoom.Tab sis peb ntseeg tias yav tom ntej, cov kev kho mob no yuav dhau los ua ib qho tseem ceeb ntawm kev kho tus kheej thiab yuav muaj txiaj ntsig zoo rau lub neej ntawm cov neeg muaj kab mob caj ces thiab lawv tsev neeg.

Foregene kuj tau tsim cov khoom siv tshuaj ntsuam xyuas siab rau cov hom phiaj, uas yog ceev thiab tuaj yeem ua rov qab thiab qPCR cov tshuaj tiv thaiv yam tsis muaj RNA rho tawm.

Cov khoom txuas

Cell Direct RT-qPCR cov khoom siv—Taqman/SYBR GREEN I

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