CRISPR/Cas9 don gyara halittar DEB (2025)

Wannan aikin na shekara guda ya ba da shaida ta farko don amfani da hanyar CRISPR/Cas9 jiyya na jiyya mai suna homology directed gyara (HDR) don magance EB. Masu binciken sunyi amfani da HDR a cikin sel da aka ɗauka daga biopsies na fata daga mutane biyar tare da EB dystrophic. Tsarin su ya sadar da kwayoyin gyara kwayoyin halitta zuwa cikin sel a cikin dakin gwaje-gwaje ta amfani da bugun wutar lantarki (electroporation) maimakon ƙwayoyin cuta da aka canza (Viral vectors). Sun gano cewa za su iya gyara canje-canjen kwayoyin halitta yadda ya kamata a lokuta biyu, zuwa wani matsayi a daya, kuma a sauran biyun ba su da tasiri. Wannan aikin ya nuna cewa ana iya amfani da HDR a cikin jiyya na EB amma yana buƙatar ingantawa ga kowane mai haƙuri daban-daban.

An gabatar da ci gaban aikin ga ESGCT/SITGEC Collaborative Congress (Rome, Italiya) 22-25 Oktoba 2024. Saukewa: P1008. Duba fosta.

Rabin aikin, masu bincike sun bayar da rahoton cewa, sun sami nasarar kai rabin matsayinsu. 'Patches' na kwayoyin halittarsu suna aiki da kyau fiye da yadda ake tsammani amma sun fi girma. Wannan yana nufin za su buƙaci ƙarin su kuma za su mayar da hankali kan wannan don rabin na biyu na aikin.

Jagoran bincike: Dokta Sergio López-Manzaneda mai bincike ne a CIEMAT tare da ingantaccen tushe a gyaran genome.

Masu bincike: Dr Fernando Larcher mataimakin farfesa ne a fannin ilimin halittu a Universidad Carlos III de Madrid kuma shugaban sashen a CIEMAT (Epithelial Biomedicine Division).

Alex Bassons Bascuñana dalibin PhD ne a cikin rukunin Epithelial Biomedicine na Sashin Innovation na Biomedical a CIEMAT.

Haɗin kai: Dokta Paula Rio kwararre ce ta duniya da aka santa a fannin gyaran kwayoyin halitta da maganin kwayoyin cututtukan da aka gada, daga kimiyyar asali zuwa gwaje-gwajen asibiti.

"Wannan tsarin gyare-gyaren kwayoyin halitta ba na kwayar cutar ba yana neman wani tsari mai rahusa da na duniya don samar da ɗakin karatu na kayan aikin da zai iya magance kowane maye gurbi da aka sani ... A cikin binciken da aka rigaya a gaba, za a yi amfani da kwayoyin "patched" edita keratinocytes da fibroblasts daga marasa lafiya. don samar da daidaitattun fata na bioengineered."

– Dr Sergio López-Manzaneda

Sunan bayar: Facin genome ba na hoto ba na COL7A1.

A cikin wannan aikin, muna ba da shawarar dabarun gyara kwayoyin halitta da ke mai da hankali kan maganin ba kawai maye gurbin maki ɗaya ba amma ƙungiyoyin maye gurbi da ke haifar da RDEB, ta hanyar gyara cikakkun exons da ke ɗauke da su. Wannan “patching-genetic-patching” ya dogara ne akan tsarin gyaran DNA na Homologous Recombination (HR) wanda tsarin CRISPR/Cas9 ya sauƙaƙe. Alamar "faci" suna wakiltar kananan (300-400 tushe nau'i-nau'i) sau biyu secterning hedr Donor don rufe 'yan kwastomomin Col7a1 na ColXNUMXaXNUMX Nau'in VII Colagen. Manufarmu ita ce siffata ( inganci, aminci) da daidaita amfani da waɗannan fasaha tare da ra'ayin samun takamaiman faci a shirye don magance ƙungiyoyin maye gurbi. Fasahar da ba ta hoto ta bidiyo da aka tsara don tsohuwar maganin tantanin halitta na RDEB za ta sauƙaƙe fassararsa zuwa asibitin a farashi mai rahusa.

Ɗaya daga cikin mafi kyawun hanyoyin warkewa don EB shine gyaran kwayoyin halitta ta amfani da fasahar CRISPR/Cas9. A cikin shekaru 7 da suka gabata dakin gwaje-gwajenmu yana aiki tuƙuru a cikin wannan filin yana ba da ƙwaƙƙwaran bayanan hujja da nufin fassara sakamakonsa zuwa asibiti. Yayin da muke gab da aiwatar da aikace-aikacen asibiti tare da ɗayan waɗannan hanyoyin da suka sami sunan Marayu Drug ta Hukumar Kula da Magunguna ta Turai. (EU/3/20/2253), muna ci gaba da neman mafi inganci, mafi aminci da dabarun gyaran genome masu dacewa.

A cikin wannan aikin, muna ba da shawara don gwada sabon tsarin gyara kwayoyin halitta wanda baya buƙatar amfani da ƙwayoyin cuta (duk kayan da aka gabatar da su ta hanyar lantarki guda ɗaya). Manufar ita ce a yi amfani da waɗannan "faci na kwayoyin halitta" don gyara yankunan da suka canza bayan sun yanke wannan yanki na kwayoyin halitta tare da fasahar CRISPR/Cas9. Mun tsara “faci” guda huɗu daban-daban, wannan yana nufin, samfuran masu ba da gudummawa na dsDNA musamman waɗanda aka canza su a ƙarshensu ta hanyar fasahar mallaka ta mai ba da mu (Haɗin fasahar DNA, IDT) don fifita HDR. Waɗannan samfuran sun ƙunshi exons biyu da kewaye (73 da 80, “faci” biyu kowanne) da maki huɗu daban-daban (biyu don Exon 73 da biyu don exon 80). Wannan samfuri (kimanin 300-400 bp) kuma yana rufe maƙwabta exons.

Muna nufin kimanta girman gyare-gyare a cikin "faci" wanda zai ba mu damar magance ƙungiyoyin maye gurbi ko, idan tsarin ya yi aiki sosai, don daidaitawa ga exons a cikin "patches". Mun yi imanin cewa wannan labari, babu hoto ko bidiyo mai zagaya yanar gizo da sauri, dabarun gyara kwayoyin halitta na keɓaɓɓen da za a iya fassara shi cikin sauƙi zuwa asibiti tare da ƙarancin tsadar tattalin arziki.

Mun samu nasarar kai rabin matakan tallafin. Da farko, dabarunmu da nufin sake rubuta kwayoyin halitta ta hanyar amfani da "faci" DNA don rufe nau'i-nau'i na exons (musamman niyya maye gurbi a cikin nau'i-nau'i na exon: 72-73, 73-74, 79-80, da 80-81). Koyaya, facin DNA ɗin da muka yi amfani da shi ya zama ɗan bambanta da abin da muke tsammani. Waɗannan facin na iya sake rubuta kwayar halitta tare da inganci sama da 80%, wanda ya fi yadda aka ruwaito a baya, amma aikinsu yana iyakance ga kunkuntar taga. Idan aka ba wannan, dabarun mu na zamani na 2 yanzu za su mai da hankali kan facin DNA na kowane exon. Don tabbatar da cewa ba mu canza jerin DNA masu lafiya ba, CRISPR/Cas9 “yanke kwayoyin halitta” za su yi niyya ne kawai ga jerin abubuwan da aka canza. Wannan zai buƙaci takamaiman ƙananan RNA don kowane maye gurbi, yayin da ana iya amfani da facin DNA na kowa ga kowane exon. (Rahoto daga Yuli 2024).

Da farko, dabarunmu da nufin sake rubuta kwayoyin halitta ta hanyar amfani da DNA "patches" don rufe nau'i-nau'i na exons, musamman ma'anar maye gurbi a cikin exon nau'i-nau'i 72-73, 73-74, 79-80, da 80-81. Koyaya, facin DNA ɗin da aka yi amfani da shi ya ɗan bambanta da tsammaninmu. Ba kamar dabarun HDR na gargajiya ba, wannan sabon tsarin IDT (Integrated DNA Technologies) ba zai iya sake rubuta jerin abubuwan da suka wuce 30 nucleotides ba, yana iyakance aikinsa zuwa kunkuntar taga.

Ganin wannan, dabararmu ta 2 ta mayar da hankali kan facin DNA na kowane exon. Don tabbatar da cewa jerin DNA masu lafiya sun kasance ba a gyaggyarawa ba, CRISPR/Cas9 “yanke kwayoyin halitta” yana nufin jerin sauye-sauye ne kawai. Wannan yana buƙatar takamaiman ƙananan RNA don kowane maye gurbi, yayin da ana iya amfani da facin DNA na kowa ga kowane exon. Mun sami nasarar sake rubuta DNA a babban inganci (> 70%) a cikin 2 daga cikin maye gurbi na 5 (masu haƙuri 2 da haƙuri 73.4), sun sami matsakaicin matsakaici (> 25%) a cikin wasu 2 (majinyata 73.2 da haƙuri 2), kuma da kyar aka gyara ɗaya (5%) (E73.3). Ko da yake warware gyare-gyaren clones dabara ce mai yuwuwa, yin amfani da waɗannan clones don dasawa zai gabatar da ƙalubale na fasaha a cikin gwaje-gwajen asibiti idan an fara daga ƙananan ƙimar inganci. Duk da haka, wannan aikin yana ba da aƙalla hanyoyin warkewa guda biyu masu ban sha'awa don maye gurbin c.6041_6042delAG da c.6100G>A, ta yin amfani da tsarin HDR mara hoto na hoto maimakon tsarin isar da ƙwayar cuta ta gargajiya. (Daga rahoton ƙarshe na Maris 2025.)