Kunanirwa kwumva (HL) nindwara ikunze kwibasira ubumuga bwabantu.Mu bihugu byateye imbere, hafi 80% by’abana bafite ubumuga bwo kutumva mbere y’abana biterwa n’impamvu zishingiye ku ngirabuzima fatizo.Ibikunze kugaragara cyane ni inenge ya gene imwe (nkuko bigaragara ku gishushanyo 1), ihinduka rya gene 124 ryagaragaye ko rifitanye isano no kutumva neza kwa muntu, ibisigaye biterwa n’ibidukikije.Kwimika cochlear (igikoresho cya elegitoronike gishyirwa mumatwi yimbere gitanga imbaraga zamashanyarazi kumitsi yumva) kugeza ubu nuburyo bwiza cyane bwo kuvura HL ikabije, mugihe infashanyo yo kumva (igikoresho cya elegitoroniki yo hanze ihindura kandi ikongerera imbaraga amajwi) irashobora gufasha abarwayi bafite HL igereranije.Ariko, kuri ubu nta muti uhari wo kuvura umurage HL (GHL).Mu myaka yashize, ubuvuzi bwa gene bwarushijeho kwitabwaho nkuburyo butanga ikizere cyo kuvura imikorere mibi yamatwi.

Igishushanyo1.Ikwirakwizwa ryubwoko butandukanye bwo kutumva. [1]

Vuba aha, abahanga bo mu kigo cya Salk Institute na kaminuza ya Sheffield basohoye ibyavuye mu bushakashatsi bwakozwe na Molecular Therapy - Methods & Clinical Development [2], bwerekanye ibyifuzo byinshi byo kuvura vivo gene yo kuvura ubumuga bwo kutumva.Uri Manor, umwarimu wungirije w’ubushakashatsi mu kigo cya Salk akaba n’umuyobozi w’ikigo cya Waitt gishinzwe iterambere ry’ibinyabuzima, yavuze ko yavutse afite ikibazo cyo kutumva cyane kandi yumva ko kugarura kumva byaba ari impano nziza.Ubushakashatsi bwe bwambere bwerekanye ko Eps8 ari poroteyine igenga poroteyine ifite ibikorwa byo guhuza no gufata ibintu;muri selile cochlear, uruganda rwa poroteyine rwakozwe na Eps8 hamwe na MYO15A, WHIRLIN, GPSM2 na GNAI3 rubaho cyane cyane Inama za stereocilia ndende, hamwe na MYO15A zihindura BAIAP2L2 kumpanuro ya stereocilia ngufi, zirakenewe kugirango habeho imisatsi.Kubwibyo, Eps8 irashobora kugena uburebure bwa stereocilia yingirangingo zumusatsi, zikenewe mumikorere isanzwe yo kumva;Gusiba cyangwa guhinduka kwa Eps8 bizaganisha kuri stereocilia ngufi, ituma idashobora guhindura neza amajwi mubimenyetso byamashanyarazi kugirango imyumvire yubwonko, nayo iganisha ku kutumva..Muri icyo gihe, umufatanyabikorwa Walter Marcotti, umwarimu muri kaminuza ya Sheffield, yasanze ingirabuzimafatizo zidashobora gukura bisanzwe mu gihe Eps8 idahari.Muri ubu bushakashatsi, Manor na Marcotti bishyize hamwe kugira ngo bakore iperereza niba kongeramo Eps8 mu ngirabuzimafatizo zishobora kugarura imikorere yabo, ndetse no kunoza kumva mu mbeba.Itsinda ry’ubushakashatsi ryakoresheje virusi ya adeno (AAV) vector Anc80L65 kugirango itange urutonde rwa code ikubiyemo ubwoko bwa EPS8 bwo mu gasozi muri cochlea ya Eps8 - / - imbeba zavutse P1-P2 zatewe inshinge zuzuye idirishya;muri selile cochlear yimisatsi Imikorere ya stereocilia yasanwe mbere yuko ikura;n'ingaruka zo gusana zaranzwe no gukoresha amashusho no gupima stereocilia.Ibisubizo byerekanaga ko Eps8 yongereye uburebure bwa stereocilia kandi igarura imikorere yimisatsi mumasemburo make.Basanze kandi ko, igihe, ingirabuzimafatizo zasaga nkizatakaje ubushobozi bwo gutabarwa nubu buvuzi bwa gene.Ikigaragara ni uko ubu buvuzi bushobora gukenerwa gutangwa muri utero, kuko Eps8 - / - imisatsi yimisatsi ishobora kuba yarakuze cyangwa ikarundanya ibyangiritse birenze gusanwa nyuma yimbeba zavutse.Manor yagize ati: "Eps8 ni poroteyine ifite imirimo myinshi itandukanye, kandi haracyari byinshi byo gucukumbura."Ubushakashatsi bw'ejo hazaza buzaba bukubiyemo gukora ubushakashatsi ku ngaruka zo kuvura gene ya Eps8 mu kugarura kumva mu byiciro bitandukanye by'iterambere, kandi niba bishoboka ko ushobora kongera amahirwe yo kwivuza.Ku bw'amahirwe, mu Gushyingo 2020, Porofeseri KarenB Avraham wo muri kaminuza ya Tel Aviv muri Isiraheli yasohoye ibisubizo bye mu kinyamakuru EMBO Molecular Medicine [3], akoresheje ikoranabuhanga rishya ryo kuvura gene kugira ngo akore virusi AAV9-PHP itagira ingaruka mbi.B.

Igishushanyo2.Igishushanyo cyerekana ugutwi kwimbere imbere, hamwe kwibanda kuri organorgan ya Corti nimikorere ya selire ya nesprin-4.

Birashobora kugaragara ko gukoresha imiti ivura gene kugirango ugere ku ntego yo kuvura indwara zishingiye ku murage ku rwego rwa gene winjizamo, ukuraho cyangwa ukosora ingirabuzima fatizo zose zahinduwe kugira ngo zivurwe (ni ukuvuga kugenzura ihinduka ry’imiterere y’indwara) rifite ingaruka zikomeye ku ivuriro.ibyifuzo.Uburyo bwa none bwo kuvura gene kubumuga bwo kutumva burashobora kugabanywamo ibyiciro bikurikira:

gusimbuza gene

Gusimbuza Gene ni uburyo "bworoshye" bwo kuvura gene, bushingiye ku kumenya no gusimbuza gene ifite inenge hamwe na kopi isanzwe cyangwa ubwoko bwa gasozi.Ubushakashatsi bwambere bwamatwi yimbere yo kuvura kunanirwa kwumva guterwa no gusiba gene glutamate itwara 3 (VGLUT3);Gutanga AAV1-yunganirwa no gukabya gukabije VGLUT3 gukabya gukabije kwingirangingo yimisatsi yimbere yimbere (IHCs) Birashobora gutuma umuntu yumva neza, kumva igice cya synaptic synaptic morphologie, hamwe nibisubizo bihungabana [4].Nyamara, mu ngero zirimo na AAV ebyiri zasimbuwe na gene zasobanuwe mu ntangiriro yavuzwe haruguru, ni ngombwa kumenya ko imiterere yimbeba zikoreshwa muburyo bumwe na bumwe bwo gusiba gene iterwa no kutumva neza gutandukana kwabantu bitandukanye nigihe gito, naho mu mbeba za P1, ugutwi kwimbere kurikuze.Ibinyuranye, abantu bavuka bafite ugutwi kwimbere.Iri tandukaniro ririnda uburyo bushoboka bwo gukoresha ibisubizo byimbeba kugirango bivure indwara ziterwa no kutumva kwa muntu keretse ubuvuzi bwa gene bwatanzwe mumatwi akuze.

Guhindura Gene: CRISPR / Cas9

Ugereranije na “gusimbuza gene”, iterambere rya tekinoroji yo guhindura gene yazanye umuseke wo kuvura indwara zikomoka kumuzi.Icy'ingenzi, uburyo bwo guhindura gene bugizwe nubusembwa bwuburyo gakondo bwo kuvura gene bikabije bidakwiranye nindwara yiganjemo abadafite ubumuga bwo kutumva, kandi ikibazo cyuko uburyo bukabije bwo kumara igihe kirekire.Nyuma yuko abashakashatsi b'Abashinwa bakuyeho Myo6C442Y mutant allele mu mbeba za Myo6WT / C442Y bakoresheje sisitemu yo guhindura gene AAV-SaCas9-KKH-Myo6-g2, kandi mu mezi 5 nyuma yo gukomanga, imbeba Imikorere yo kumva icyitegererezo yagaruwe;icyarimwe, byagaragaye kandi ko igipimo cyo kubaho kwingirangingo zumusatsi mumatwi yimbere cyarushijeho kuba cyiza, imiterere ya cilia iba isanzwe, kandi ibipimo bya electrophysiologique byakosowe [5].Ubu ni bwo bushakashatsi bwa mbere ku isi bwifashishije ikoranabuhanga rya CRISPR / Cas9 mu kuvura ubumuga bwo kutumva bwatewe na mutation ya Myo6, kandi ni intambwe ikomeye y’ubushakashatsi bwakozwe mu buhanga bwo guhindura gene mu kuvura ubumuga bwo kutumva.Ubuvuzi bwa clinique yubuvuzi butanga ishingiro ryubumenyi.

Uburyo bwo gutanga imiti

Kugirango ubuvuzi bwa gene bugende neza, molekile ya ADN yambaye ubusa ntishobora kwinjira mu ngirabuzimafatizo neza bitewe na hydrophilique hamwe n’amafaranga mabi y’amatsinda ya fosifate, kandi kugira ngo habeho ubusugire bwa molekile ya acide nucleique yuzuye, hagomba gutoranywa uburyo bwizewe kandi bunoze.ADN yuzuzwa igezwa kuri selile cyangwa tissue.AAV ikoreshwa cyane nk'imodoka itanga uburyo bwo kuvura indwara bitewe n'ingaruka zayo zanduza, ubudahangarwa buke, hamwe na tropism yagutse ku bwoko butandukanye.Kugeza ubu, umubiri munini wubushakashatsi wagennye tropism yubwoko butandukanye bwa AAV ugereranije nubwoko butandukanye muri cochlea yimbeba.Gukoresha AAV itanga ibiranga bihujwe na selile yihariye ya porotokoro irashobora kugera kumagambo yihariye, ishobora kugabanya ingaruka zitari intego.Mubyongeyeho, nkuburyo busanzwe bwa AAV vectors, sisitemu nshya ya AAV ya syntetique ya AAV ihora itezwa imbere kandi ikerekana ubushobozi bwogukwirakwiza mumatwi yimbere, muri yo AAV2 / Anc80L65 niyo ikoreshwa cyane.Uburyo bwo gutanga virusi ntabwo bushobora kugabanywa muburyo bwumubiri (microinjection na electroporation) nuburyo bwa chimique (bishingiye kuri lipide, bishingiye kuri polymer, na nanoparticles ya zahabu).Ubwo buryo bwombi bwakoreshejwe mu kuvura indwara zo kutumva no kwerekana ibimenyetso bitandukanye.Usibye ibinyabiziga bitanga imiti ivura gene nk'ikinyabiziga, uburyo butandukanye bwo kuyobora vivo gene burashobora gukoreshwa hashingiwe ku bwoko butandukanye bw'utugari, inzira z'ubuyobozi, hamwe no kuvura neza.Imiterere igoye yugutwi kwimbere ituma bigora kugera kungingo zigenewe kandi ikwirakwizwa ryimikorere ya genome iratinda.Membrane labyrint iherereye muri bony labyrint yamagufa yigihe gito kandi ikubiyemo umuyoboro wa cochlear, umuyoboro wa semicircular, utricle, na ballon.Kwigereranya kwayo, kuzenguruka kwa lymphatike ntoya, no gutandukana n'amaraso n'inzitizi y'amaraso-maze bigabanya uburyo bwiza bwo gutanga imiti ku mbeba zivuka gusa.Kugirango ubone titereri ya virusi ikwiranye nubuvuzi bwa gene, birakenewe gutera inshinge za virusi mumatwi yimbere.Inzira zashyizweho zo gutera inshinge zirimo [6]: (1) uruziga ruzengurutse idirishya (RWM), (2) tracheostomy, (3) endolymphatic cyangwa perilymphatic cochleostomy, (4) idirishya ryizengurutsa ryongeyeho Tube fenestration (CF) (nkuko bigaragara ku gishushanyo 3).

Igishushanyo3.Gutanga amatwi yimbere yo kuvura gene.

Nubwo hari byinshi bimaze gutera imbere mu kuvura gene, hashingiwe ku ntego z’ubuhinduzi bw’amavuriro, hakenewe gukorwa byinshi mbere yo kuvura gene bishobora kuba uburyo bwa mbere bwo kuvura abarwayi bafite indwara zishingiye ku ngirabuzima fatizo, cyane cyane mu guteza imbere ibice byizewe kandi byiza ndetse n’uburyo bwo gutanga.Ariko twizera ko mugihe cya vuba, ubu buryo bwo kuvura buzahinduka urwego rwubuvuzi bwihariye kandi bizagira ingaruka nziza mubuzima bwabantu bafite ibibazo byubwoko bwabo nimiryango yabo.

Foregene yanatangije ibikoresho byinshi byinjira mugusuzuma gen zigenewe, byihuta kandi birashobora gukora transcription transcription hamwe na qPCR reaction nta gukuramo RNA.

Guhuza ibicuruzwa

Akagari ka Direct RT-qPCR kit-Taqman / SYBR GREEN I.

Kubindi bisobanuro byibicuruzwa, nyamuneka hamagara:

overseas@foregene.com