Ukulahlekelwa kokuzwa (HL) yisifo esivame kakhulu sokukhubazeka kwezinzwa kubantu.Emazweni athuthukile, cishe amaphesenti angama-80 abantu abanenkinga yokungezwa ngaphambi kolimi ezinganeni abangelwa izici zofuzo.Okuvame kakhulu ukukhubazeka kofuzo olulodwa (njengoba kuboniswe ku-Fig. 1), ukuguqulwa kwezakhi zofuzo ze-124 kutholakale ukuthi kuhlotshaniswa nokulahlekelwa kokuzwa okungavamile kubantu, okunye kubangelwa izici zemvelo.Ukufakelwa kwe-cochlear (umshini we-elekthronikhi obekwe endlebeni yangaphakathi enikeza ukugqugquzelwa kukagesi ngqo ku-nerve yokuzwa) iyindlela ephumelela kakhulu yokwelapha i-HL enzima, kuyilapho insiza yokuzwa (umshini wangaphandle we-elekthronikhi oguqula futhi ukhulise amaza omsindo) ungasiza Iziguli ezine-HL emaphakathi.Nokho, okwamanje azikho izidakamizwa ezitholakalayo zokwelapha i-HL (GHL) yofuzo.Eminyakeni yamuva nje, ukwelashwa ngofuzo sekuthole ukunakwa okwandayo njengendlela ethembisayo yokwelapha ukungasebenzi kahle kwezindlebe zangaphakathi.

Umdwebo1.Ukusatshalaliswa kohlobo lokuhlukahluka oluhlobene nokungezwa.[1]

Muva nje, ososayensi abavela ku-Salk Institute kanye naseNyuvesi yaseSheffield bashicilele umphumela wocwaningo ku-Molecular Therapy - Izindlela & Nokuthuthukiswa Komtholampilo [2], obonise amathuba okusetshenziswa okubanzi kokwelashwa kwezakhi zofuzo ezitholakala ngofuzo.U-Uri Manor, umsizi kaprofesa wocwaningo e-Salk Institute kanye nomqondisi we-Waitt Center for Advanced Biophotonics, uthe wazalwa enenkinga yokungezwa futhi waba nomuzwa wokuthi ukubuyisela ukuzwa kungaba isipho esihle kakhulu.Ucwaningo lwakhe lwangaphambilini lwathola ukuthi i-Eps8 iyiphrotheni elawula i-actin enemisebenzi yokubopha kanye ne-capping;kumaseli ezinwele ze-cochlear, i-protein complex eyakhiwe i-Eps8 ene-MYO15A, i-WHIRLIN, i-GPSM2 kanye ne-GNAI3 ikakhulukazi ikhona kakhulu Amathiphu e-stereocilia amade, lawo kanye ne-MYO15A enza i-BAIAP2L2 ibe yasendaweni kumathiphu we-stereocilia emfushane, ayadingeka ukuze kugcinwe izinqwaba zezinwele.Ngakho-ke, i-Eps8 ingalawula ubude be-stereocilia yamangqamuzana ezinwele, okubalulekile ekusebenzeni okujwayelekile kokuzwa;Ukususwa noma ukuguqulwa kwe-Eps8 kuzoholela ku-stereocilia emfushane, okuyenza ingakwazi ukuguqula kahle umsindo ube amasignali kagesi ukuze ubone ubuchopho, okubuye kuholele ekungezwani..Ngesikhathi esifanayo, umhlanganyeli u-Walter Marcotti, uprofesa eNyuvesi yaseSheffield, wathola ukuthi amangqamuzana ezinwele awakwazi ukukhula ngendlela evamile uma ingekho i-Eps8.Kulolu cwaningo, uManor noMarcotti bahlangane ukuze baphenye ukuthi ukungeza i-Eps8 kumaseli e-stereociliary kungabuyisela ukusebenza kwawo, futhi, kuthuthukise ukuzwa kumagundane.Ithimba labacwaningi lisebenzise i-adeno-associated virus (AAV) vector Anc80L65 ukuze ilethe ukulandelana kwekhodi equkethe uhlobo lwasendle lwe-EPS8 ku-cochlea ye-Eps8-/- amagundane e-P1-P2 asanda kuzalwa ngomjovo we-membrane yewindi eliyindilinga;kumaseli ezinwele ze-mouse cochlear Umsebenzi we-stereocilia walungiswa ngaphambi kokuba zivuthwe;futhi umphumela wokulungisa wawubonakala ngobuchwepheshe be-imaging kanye nesilinganiso se-stereocilia.Imiphumela yabonisa ukuthi i-Eps8 inyuse ubude be-stereocilia kanye nokusebenza kweseli lezinwele elibuyiselwe kumaseli anemvamisa ephansi.Baphinde bathola ukuthi, ngokuhamba kwesikhathi, amangqamuzana abonakala elahlekelwa amandla awo okutakulwa yilokhu kwelashwa kofuzo.Okushiwo ukuthi lokhu kwelashwa kungase kudingeke ukuthi kunikezwe esibelethweni, njengoba amaseli ezinwele e-Eps8-/- engase akhule noma aqongelele umonakalo ongenakulungiseka ngemva kokuzalwa kwamagundane.“I-Eps8 iyiphrotheni enezici eziningi ezihlukene, futhi kusekuningi okufanele kuhlolwe,” kusho uManor.Ucwaningo lwesikhathi esizayo luzobandakanya ukuphenya umthelela wokwelashwa kofuzo kwe-Eps8 ekubuyiseleni ukuzwa ezigabeni zokuthuthuka ezihlukene, kanye nokuthi kungenzeka yini ukwelula amathuba okwelashwa.Kwaqondana nje, ngoNovemba 2020, uSolwazi KarenB Avraham wase-Tel Aviv University kwa-Israel washicilela imiphumela yakhe kujenali i-EMBO Molecular Medicine [3], esebenzisa ubuchwepheshe obusha bokwelapha ngofuzo ukuze akhe igciwane elingenabungozi le-adeno-associated AAV9-PHP.B, Isici sofuzo emangqamuzaneni ezinwele ze-Syne4-/- amagundane salungiswa ngokujova igciwane elithwala ukulandelana kwekhodi ye-Syne4 endlebeni yangaphakathi yamagundane, evumela ukuthi ingene emangqamuzaneni ezinwele futhi ikhulule izinto ezithwala izakhi zofuzo, ezivumela ukuba zivuthwe futhi zisebenze ngokujwayelekile (njengakuFig. 2).

Umdwebo 2.Ukumelwa okuhleliwe kwe-anatomy yendlebe yangaphakathi, ngokugxila ku-organ ye-Corti kanye nomsebenzi weselula we-nesprin-4.

Kungabonakala ukuthi ukusetshenziswa kokwelashwa kwezakhi zofuzo ukufeza injongo yokwelapha izifo zofuzo ezingeni lofuzo ngokufaka, ukususa noma ukulungisa noma yiziphi izakhi zofuzo eziguquliwe ukuze zelashwe (okungukuthi, ukulawula izinguquko zofuzo kulesi sifo) kunomphumela ophezulu womtholampilo.amathemba ohlelo lokusebenza.Izindlela zamanje zokwelapha izakhi zofuzo zokungezwa ezintula ngokofuzo zingahlukaniswa zibe izigaba ezilandelayo:

ukushintshwa kofuzo

Ukushintsha izakhi zofuzo ngokungangabazeki kuyindlela “eqondile” kakhulu yokwelapha izakhi zofuzo, okusekelwe ekuhlonzeni nasekumiseleni isakhi sofuzo esinesici ngekhophi evamile noma yasendle yofuzo.Ucwaningo lokuqala oluyimpumelelo lokwelapha isakhi sofuzo lwendlebe yangaphakathi ngenxa yokulahlekelwa ukuzwa okubangelwa ukususwa kofuzo lwe-vesicular glutamate transporter 3 (VGLUT3);Ukulethwa kwe-AAV1-mediated of exogenous VGLUT3 overexpression kumaseli ezinwele zendlebe yangaphakathi (IHCs) Kungabangela ukululama kokuzwa okuqhubekayo, ukubuyiswa kwe-ribbon synaptic morphology, kanye nezimpendulo eziphazamisayo [4].Kodwa-ke, ezibonelweni ezihlanganisa ukushintshwa kwezakhi zofuzo ezimbili ezilethiwe ze-AAV ezichazwe esingenisweni esingenhla, kubalulekile ukuqaphela ukuthi amamodeli egundane asetshenziselwa izinhlobo ezithile zokususwa kofuzo ukuphazamiseka kokuzwa okuzuzwa kwefa ahluke okwesikhashana kubantu, futhi kumagundane e-P1, indlebe yangaphakathi isesigabeni sokukhula esivuthiwe.Ngokuphambene, abantu bazalwa benendlebe yangaphakathi evuthiwe.Lo mehluko uvimbela ukusetshenziswa okungenzeka kwemiphumela yegundane ekwelapheni izifo zokungezwa ezizuzwa ngofuzo zofuzo ngaphandle uma ukwelapha ngofuzo kulethwa ezindlebeni zegundane ezikhulile.

Ukuhlela iGene: CRISPR/Cas9

Uma kuqhathaniswa “nokushintshwa kofuzo”, ukuthuthukiswa kobuchwepheshe bokuhlela izakhi zofuzo kulethe ukusa kokwelapha izifo zofuzo kusuka empandeni.Okubalulekile, indlela yokuhlela izakhi zofuzo yenza ukushiyeka kwezindlela zokwelashwa zofuzo zokukhuluma ngokweqile ezingafaneleki ezifweni ezibusayo zofuzo zobuthulu, kanye nenkinga yokuthi indlela yokukhuluma ngokweqile ayihlali isikhathi eside.Ngemuva kokuthi abacwaningi baseShayina bekhiphe ngokuqondile i-Myo6C442Y mutant allele ku-Myo6WT/C442Y amagundane besebenzisa i-AAV-SaCas9-KKH-Myo6-g2 uhlelo lokuhlela izakhi zofuzo, futhi phakathi nezinyanga ezingu-5 zokungqongqoza, amagundane Umsebenzi wokuzwa wemodeli wabuyiselwa;ngasikhathi sinye, kwaphawulwa nokuthi izinga lokusinda lamaseli ezinwele endlebeni yangaphakathi lithuthukisiwe, ukuma kwe-cilia kwaba njalo, futhi izinkomba ze-electrophysiological zalungiswa [5].Lolu ucwaningo lokuqala emhlabeni ukusebenzisa ubuchwepheshe be-CRISPR/Cas9 ekwelapheni ubuthulu obuzuzwa ufuzo olubangelwa ukuguqulwa kofuzo lwe-Myo6, futhi kuyinqubekelaphambili ebalulekile yocwaningo lobuchwepheshe bokuhlela izakhi zofuzo ekwelapheni ubuthulu obuzuzwa ngofuzo.Ukuhunyushwa komtholampilo kokwelashwa kunikeza isisekelo esiqinile sesayensi.

Izindlela zokulethwa kokwelashwa kofuzo

Ukuze ukwelashwa kwezakhi zofuzo kuphumelele, ama-molecule e-DNA enqunu awakwazi ukungena kumaseli ngokuphumelelayo ngenxa ye-hydrophilicity yawo kanye nokukhokhiswa okungalungile kwamaqembu e-phosphate, nokuqinisekisa ubuqotho bama-nucleic acid angeziwe, indlela ephephile nephumelelayo kufanele ikhethwe.I-DNA engeziwe ihanjiswa kuseli eliqondiwe noma izicubu.I-AAV isetshenziswa kakhulu njengemoto yokulethwa ekwelapheni izifo ngenxa yomphumela wayo ophezulu othathelanayo, ukuzivikela komzimba okuphansi, kanye ne-tropism ebanzi ezinhlotsheni zezicubu ezihlukahlukene.Njengamanje, indikimba enkulu yomsebenzi wocwaningo inqume i-tropism yezinhlobo ezincane ezihlukene ze-AAV ngokuhlobene nezinhlobo zamaseli ezihlukene ku-cochlea yegundane.Ukusebenzisa izici zokulethwa kwe-AAV kuhlanganiswe nabathuthukisi beseli ethile kungafinyelela isisho esiqondene neseli, esinganciphisa imithelela engekho ekuhlosweni kwayo.Ngaphezu kwalokho, njengenye indlela yamavekhtha endabuko e-AAV, amavektha amasha e-AAV okwenziwa ahlala ethuthukiswa futhi abonisa ikhono eliphakeme lokuguqula endlebeni yangaphakathi, okuyi-AAV2/Anc80L65 esetshenziswa kakhulu.Izindlela zokulethwa okungezona igciwane zingaphinde zihlukaniswe zibe izindlela ezibonakalayo (i-microinjection kanye ne-electroporation) nezindlela zamakhemikhali (i-lipid-based, i-polymer-based, ne-nanoparticles yegolide).Zombili lezi zindlela zisetshenziswe ekwelapheni izifo zokungezwa zofuzo futhi zibonise izinzuzo nemikhawulo ehlukene.Ngaphezu kwemoto yokulethwa kokwelashwa kofuzo njengemoto, izindlela ezihlukene zokulawulwa kwezakhi zofuzo ze-vivo zingasetshenziswa ngokusekelwe ezinhlotsheni zamangqamuzana okuhloswe ngazo, izindlela zokuphatha, kanye nempumelelo yokwelapha.Isakhiwo esiyinkimbinkimbi sendlebe engaphakathi senza kube nzima ukufinyelela amaseli okuqondiwe futhi ukusatshalaliswa kwama-ejenti wokuhlela wofuzo kuhamba kancane.I-membranous labyrinth itholakala ngaphakathi kwe-labyrinth engamathambo yethambo lesikhashana futhi ihlanganisa i-cochlear duct, i-semicircular duct, utricle, nebhaluni.Ukuhlukaniswa kwayo okulinganiselwe, ukujikeleza kwe-lymphatic okuncane, kanye nokuhlukaniswa negazi ngomgoqo we-blood-maze kunciphisa ukulethwa okusebenzayo kwezindlela zokwelapha kumagundane asanda kuzalwa kuphela.Ukuze uthole ama-viral titers alungele ukwelashwa kofuzo, ukujova okuqondile kwendawo kwama-viral vectors endlebeni yangaphakathi kuyadingeka.Imizila emisiwe yomjovo ihlanganisa i- [6]: (1) i-membrane yewindi eliyindilinga (RWM), (2) i-tracheostomy, (3) i-endolymphatic noma i-perilymphatic cochleostomy, (4) i-membrane yefasitela eliyindilinga kanye ne-Tube fenestration (CF) (njengakuFig. 3).

Umfanekiso wesi-3.Ukulethwa kwendlebe yangaphakathi kokwelashwa kofuzo.

Nakuba kuye kwenziwa intuthuko eminingi ekwelashweni kwezakhi zofuzo, ngokusekelwe ezinhlosweni zokuhumusha zomtholampilo, umsebenzi omningi kufanele wenziwe ngaphambi kokuba ukwelashwa ngezakhi zofuzo kube indlela yokwelapha yomugqa wokuqala ezigulini ezinezifo zofuzo, ikakhulukazi ekuthuthukisweni kwama-vector aphephile futhi asebenzayo kanye nendlela yokulethwa.Kodwa sikholelwa ukuthi esikhathini esizayo esiseduze, lezi zinhlobo zokwelapha zizoba isisekelo sokwelashwa komuntu siqu futhi kuzoba nomthelela omuhle kakhulu ezimpilweni zabantu abanezinkinga zofuzo kanye nemindeni yabo.

I-Foregene iphinde yethula ikhithi yokuhlola izinga eliphezulu lezakhi zofuzo eziqondiwe, ezisheshayo futhi ezingenza ukuloba okuhlanekezelwe kanye nokusabela kwe-qPCR ngaphandle kokukhipha i-RNA.

Izixhumanisi Zomkhiqizo

Ikhithi ye-Cell Direct RT-qPCR—Taqman/SYBR GREEN I

Ukuze uthole ulwazi olwengeziwe lomkhiqizo, sicela uthinte:

overseas@foregene.com