Ukulahlekelwa kwindlebe (HL) sesona sifo sixhaphakileyo sokukhubazeka kwengqondo ebantwini.Kumazwe ahambele phambili kwezoqoqosho, malunga nama-80% eemeko zokungeva ngolwimi lwangaphambili ebantwaneni zibangelwa yimiba yemfuza.Ezona ziqhelekileyo ziziphene zohlobo olulodwa (njengoko kuboniswe kumfanekiso we-1), iinguqu ze-124 zofuzo zifunyenwe zidibene nokulahlekelwa kwindlebe ye-nonsyndromic ebantwini, ezinye zibangelwa yimeko yendalo.I-cochlear implant (isixhobo sombane esifakwe kwindlebe yangaphakathi enika ukuvuselela kombane ngokuthe ngqo kwi-nerve yokuva) yiyona ndlela isebenzayo kakhulu yokunyanga i-HL enzima, ngelixa uncedo lokuva (isixhobo sombane sangaphandle esiguqula kwaye sikhulise amaza omsindo) sinokunceda izigulane ezine-HL ephakathi.Nangona kunjalo, okwangoku akukho machiza akhoyo okunyanga i-HL yelifa (GHL).Kwiminyaka yakutshanje, unyango lwemfuza lufumene ingqwalasela eyongeziweyo njengendlela ethembisayo yokunyanga ukungasebenzi kakuhle kwendlebe.

Isazobe soku1.Ukusasazwa kohlobo lokwahluka olunxulumene nobuthulu.[1]

Kutshanje, izazinzulu ezivela kwi-Salk Institute kunye neYunivesithi yaseSheffield zipapashe isiphumo sophando kwi-Molecular Therapy - Iindlela kunye noPhuhliso lweKliniki [2], ebonise amathuba okusebenza okubanzi kwi-vivo gene therapy ye-hereditary deafness.U-Uri Manor, unjingalwazi oncedisayo wophando kwi-Salk Institute kunye nomlawuli we-Waitt Centre ye-Advanced Biophotonics, uthe wazalwa eneendlebe ezinzulu kwaye wayevakalelwa kukuba ukubuyisela ukuva kuya kuba sisipho esihle.Uphando lwakhe lwangaphambili lufumene ukuba i-Eps8 yiprotheni yokulawula i-actin ene-actin yokubopha kunye nemisebenzi ye-capping;kwiiseli zeenwele ze-cochlear, i-protein complex eyenziwe yi-Eps8 kunye ne-MYO15A, i-WHIRLIN, i-GPSM2 kunye ne-GNAI3 ikakhulu ikhona kakhulu Iingcebiso ze-stereocilia ezide, ezihlangene ne-MYO15A zendawo ye-BAIAP2L2 kwiingcebiso ze-stereocilia ezimfutshane, ziyafuneka ekugcinweni kweenwele zeenwele.Ngoko ke, i-Eps8 inokulawula ubude be-stereocilia yeeseli zeenwele, eziyimfuneko kumsebenzi oqhelekileyo wokuva;Ukucinywa kwe-Eps8 okanye ukuguqulwa kwenguqu kuya kukhokelela kwi-stereocilia emfutshane, eyenza ukuba ingakwazi ukuguqula ngokufanelekileyo isandi kwiimpawu zombane zokuqonda kwengqondo, okukhokelela ekungevani..Kwangaxeshanye, umdibanisi uWalter Marcotti, unjingalwazi kwiYunivesithi yaseSheffield, wafumanisa ukuba iiseli zeenwele azikwazi ukukhula ngokuqhelekileyo ngokungabikho kwe-Eps8.Kolu phononongo, uManor kunye noMarcotti badibane ukuba baphande ukuba ukongeza i-Eps8 kwiiseli ze-stereociliary kunokubuyisela umsebenzi wabo kwaye, ngokulandelayo, kuphuculwe ukuva kwiimpuku.Iqela lophando lisebenzise i-adeno-associated virus (AAV) vector Anc80L65 ukuhambisa ulandelelwano lwekhowudi oluqulethe uhlobo lwasendle lwe-EPS8 kwi-cochlea ye-Eps8-/- iigundane ezisanda kuzalwa ze-P1-P2 nge-injection ye-membrane yefestile ejikelezayo;kwiiseli zeenwele zemouse cochlear Umsebenzi we-stereocilia walungiswa ngaphambi kokuba zivuthwe;kwaye umphumo wokulungisa ubonakaliswe ngeteknoloji yokucinga kunye nokulinganisa kwe-stereocilia.Iziphumo zibonise ukuba i-Eps8 yonyusa ubude be-stereocilia kunye nokubuyisela umsebenzi weseli yeenwele kwiiseli eziphantsi-frequency.Kwakhona bafumanisa ukuba, ekuhambeni kwexesha, iiseli zabonakala zingakwazi ukuhlangulwa ngolu nyango lwemfuza.Intsingiselo kukuba olu nyango lunokufuneka luqhutywe kwi-utero, njengoko iiseli ze-Eps8-/- zeenwele zinokuthi zikhulile okanye ziqokelele umonakalo ongaphaya kokulungiswa emva kokuba iigundane zizalwe.“I-Eps8 yiprotheyini enemisebenzi emininzi eyahlukeneyo, kwaye kusekuninzi ekufuneka kujongwe kuyo,” utshilo uManor.Uphando lwexesha elizayo luya kubandakanya ukuphanda isiphumo sonyango lwemfuza ye-Eps8 ekubuyiseleni ukuva kumanqanaba ophuhliso ahlukeneyo, kunye nokuba kunokwenzeka na ukwandisa amathuba onyango.Ngokuzenzekelayo, ngoNovemba ka-2020, uNjingalwazi uKarenB Avraham weYunivesithi yaseTel Aviv kwa-Israel wapapasha iziphumo zakhe kwijenali ye-EMBO Molecular Medicine [3], esebenzisa iteknoloji yonyango lofuzo olutsha ukwenza intsholongwane engenabungozi yokwenziwa kwe-adeno ehambelana ne-AAV9-PHP.B, I-gene defect kwiiseli zeenwele ze-Syne4-/- iigundane zalungiswa ngokujova intsholongwane ephethe ukulandelelana kwekhowudi ye-Syne4 kwindlebe yangaphakathi yeegundane, ezivumela ukuba zingene kwiiseli zeenwele kwaye zikhulule izinto ezithwele imfuzo, ezivumela ukuba zikhule kwaye zisebenze ngokuqhelekileyo (njengoko kuMfanekiso 2).

Isazobe 2.Ukubonakaliswa kweSchematic ye-anatomy yangaphakathi yendlebe, ngokugxininisa kwi-organ yeCorti kunye nomsebenzi weselula we-nesprin-4.

Ingabonwa ukuba ukusetyenziswa konyango lwemfuza ukufezekisa injongo yonyango lwezifo zemfuza kwinqanaba lemfuza ngokufaka, ukususa okanye ukulungisa nayiphi na i-gene eguqulweyo yonyango (oko kukuthi, ukulawula utshintsho lwemfuza kwisifo) kunempembelelo ephezulu yeklinikhi.izicelo ezilindelekileyo.Iindlela zangoku zonyango lwemfuza yesithulu ngokwemfuza zinokwahlulwa ngokwezi ndidi zilandelayo:

ukutshintshwa kofuzo

Ukutshintshwa kweGene ngokungathandabuzekiyo lolona hlobo “luthe ngqo” lonyango lwemfuza, olusekwe ekuchongeni nasekutshintsheni ijini enesiphene ngekopi eqhelekileyo okanye yasendle yejini.Uphononongo lokuqala oluyimpumelelo lwe-ear ear gene gene therapy ngenxa yokulahlekelwa kwindlebe okubangelwa kucinywa kwe-vesicular glutamate transporter 3 (VGLUT3) gene;Ukuhanjiswa kwe-AAV1-mediated exogenous VGLUT3 overexpression kwiiseli zeenwele zendlebe yangaphakathi (IHCs) Inokubangela ukubuyiswa kokuva okuzinzileyo, i-ribbon ye-synaptic morphology recovery, kunye neempendulo ezixubileyo [4].Nangona kunjalo, kwimizekelo ebandakanya ii-genes ezimbini ezinikezelweyo ze-AAV ezinikezelweyo ezichazwe kwintshayelelo engentla, kubalulekile ukuqaphela ukuba iimodeli zemouse ezisetyenziselwa iintlobo ezithile zokususwa kwemfuza ukuphazamiseka kwelahleko yokulahlekelwa kokuva zihluke okwethutyana kubantu, kwaye kwiimpuku ze-P1, indlebe yangaphakathi ikwinqanaba eliqolileyo lophuhliso.Ngokwahlukileyo koko, abantu bazalwa benendlebe engaphakathi ekhulileyo.Lo mahluko unqanda usetyenziso olunokwenzeka lweziphumo zempuku kunyango lwezithulu ezizuzwa njengelifa ngaphandle kokuba unyango lwemfuza lunikezelwe kwiindlebe ezivuthiweyo zempuku.

UHlelo lweGene: CRISPR/Cas9

Xa kuthelekiswa ne "gene replacement", uphuhliso lwetekhnoloji yokuhlela imfuza iye yazisa isifingo sokunyanga izifo zofuzo kwingcambu.Okubalulekileyo, indlela yokuhlela imfuza yenza iintsilelo zeendlela zonyango zemfuza ezingafanelekanga ezingafanelekanga kwizifo ezilawulayo zokungeva, kunye nengxaki yokuba indlela yokuchaza ngokugqithisileyo ayihlali ixesha elide.Emva kokuba abaphandi baseTshayina babethe ngokuthe ngqo i-Myo6C442Y i-mutant allele kwi-Myo6WT / C442Y iigundane usebenzisa i-AAV-SaCas9-KKH-Myo6-g2 inkqubo yokuhlela i-gene, kwaye kwiinyanga ezi-5 zokunkqonkqoza, iigundane Umsebenzi wokuvalelwa kwemodeli wabuyiselwa;kwangaxeshanye, kwaphawulwa kwakhona ukuba izinga lokusinda iiseli iinwele kwindlebe yangaphakathi yaphuculwa, imilo cilia waba rhoqo, kwaye izikhombisi electrophysiological zalungiswa [5].Olu luphononongo lokuqala ehlabathini ukusebenzisa itekhnoloji yeCRISPR/Cas9 kunyango lobuthulu belifa obubangelwa kukuguqulwa kofuzo lweMyo6, kwaye yinkqubela phambili yophando olubalulekileyo lwetekhnoloji yokuhlelwa kofuzo kunyango lobuthulu belifa.Ukuguqulelwa kweklinikhi yonyango kunika isiseko esiqinileyo sesayensi.

Iindlela zokuhambisa unyango lwemfuza

Ukuze unyango lwemfuza luphumelele, iimolekyuli ze-DNA ze-naked azikwazi ukungena kwiiseli ngokufanelekileyo ngenxa ye-hydrophilicity kunye nentlawulo engafanelekanga yamaqela e-phosphate, kunye nokuqinisekisa ukunyaniseka kwee-molecule ze-nucleic acid ezongezelelweyo, kufuneka kukhethwe indlela ekhuselekileyo nesebenzayo.I-DNA eyongezelelweyo ihanjiswa kwiseli ekujoliswe kuyo okanye kwizicubu.I-AAV isetyenziswa ngokubanzi njengesithuthi sokuhambisa unyango lwezifo ngenxa yesiphumo sayo esiphakamileyo esosulelayo, ukugonywa okuphantsi, kunye ne-tropism ebanzi kwiintlobo ezahlukeneyo zeethishu.Okwangoku, iqela elikhulu lomsebenzi wophando limisele i-tropism yee-subtypes ezahlukeneyo ze-AAV ngokubhekiselele kwiintlobo zeeseli ezahlukeneyo kwi-cochlea ye-mouse.Ukusebenzisa iimpawu zokuhanjiswa kwe-AAV ezidityaniswe nabakhuthazi be-cell-specific bangakwazi ukufikelela kwintetho ye-cell-specific, enokunciphisa iziphumo ezijoliswe kuyo.Ukongeza, njengenye indlela kwiivektha ze-AAV zemveli, iivektha ze-AAV ezintsha zokwenziwa zihlala ziphuhliswa kwaye zibonisa isakhono esiphakamileyo sokuguqula kwindlebe yangaphakathi, apho i-AAV2/Anc80L65 yeyona isetyenziswa kakhulu.Izindlela zokuhanjiswa kwe-non-viral zingahlukaniswa ngakumbi kwiindlela ezibonakalayo (i-microinjection kunye ne-electroporation) kunye neendlela zamachiza (i-lipid-based, i-polymer-based, kunye ne-nanoparticles yegolide).Zombini iindlela zisetyenzisiwe kunyango lwezithulu zofuzo kwaye zibonise iingenelo ezahlukeneyo kunye nemida.Ukongeza kwisithuthi sokuhambisa i-gene yonyango njengesithuthi, iindlela ezahlukeneyo zokulawula i-vivo gene zingaqeshwa ngokusekelwe kwiindidi zeeseli ezijoliswe kuzo, iindlela zokulawula, kunye nokusebenza konyango.Ulwakhiwo oluntsonkothileyo lwendlebe yangaphakathi lwenza kube nzima ukufikelela kwiiseli ekujoliswe kuzo kwaye ukuhanjiswa kwee-agenome editing agents kucotha.I-membranous labyrinth ibekwe ngaphakathi kwethambo lethambo lethambo lexeshana kwaye ibandakanya i-cochlear duct, i-semicircular duct, utricle, kunye nebhaluni.Ukwahlukaniswa kwayo okunxulumeneyo, ukujikeleza okuncinci kwe-lymphatic, kunye nokwahlulwa kwegazi ngumqobo wegazi-maze kunciphisa ukuhanjiswa okusebenzayo konyango kwiimpuku ezisanda kuzalwa.Ukufumana ii-viral titers ezifanelekileyo kunyango lwemfuza, inaliti ethe ngqo yendawo yeentsholongwane zentsholongwane kwindlebe engaphakathi iyimfuneko.Iindlela ezimiselweyo zenaliti ziquka [6]: (1) i-membrane yefestile ejikelezayo (RWM), (2) i-tracheostomy, (3) i-endolymphatic okanye i-perilymphatic cochleostomy, (4) i-membrane yefestile ejikelezayo kunye ne-Tube fenestration (CF) (njengoko kumfanekiso we-3).

Isazobe 3.Ukuhanjiswa kwendlebe yangaphakathi yonyango lwemfuza.

Nangona uninzi lwenkqubela phambili lwenziwe kunyango lofuzo, olusekelwe kwiinjongo zokuguqulelwa kweklinikhi, umsebenzi omninzi kufuneka wenziwe ngaphambi kokuba unyango lwemfuza lube lukhetho lokuqala lonyango lwezigulane ezinezifo zofuzo, ngakumbi ekuphuhliseni ii-vectors ezikhuselekileyo nezisebenzayo kunye nendlela yokuhambisa.Kodwa siyakholelwa ukuba kwixesha elizayo elingekude, ezi ntlobo zonyango ziya kuba sisiseko sonyango lomntu kwaye ziya kuba nefuthe elihle kakhulu kubomi babantu abanokuphazamiseka kwemfuza kunye neentsapho zabo.

I-Foregene iphinde yaphehlelela ikhithi yokuhlola i-high-throughput screening ye-genenes ekujoliswe kuyo, ekhawulezayo kwaye inokwenza uguqulelo lokukhuphela kunye neempendulo ze-qPCR ngaphandle kokutsalwa kwe-RNA.

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