Permanent gene therapy kwa DEB

Gawo loyamba la njira yoyika jini yogwira ntchito yakwaniritsidwa m'maselo 'osavuta kugwiritsa ntchito' mu labotale. Ofufuza tsopano akugwiritsa ntchito njira yotetezeka komanso yothandiza yochitira izi m'maselo a khungu la odwala. Ofufuza akhala akugwiritsa ntchito nanoparticles kunyamula jini yogwira ntchito m'maselo m'mbale ndipo tsopano akuwayesa pakhungu lachitsanzo.

Zotsatira zidasindikizidwa mu 2024 mu Briteni Journal of Dermatology ndi mu Journal of Investigative Dermatology.

Dr Jacków adapereka zosintha za polojekitiyi pa Loweruka Lamlungu la Members 2024:

Wofufuza wamkulu:

Dr Joanna Jacków ali ndi chidziwitso chambiri komanso mbiri yotsimikizika mu epidermolysis bullosa gene therapy ndi ma gene editing applications. Dr Jacków adawonetsa kusintha kwa majini koyenera pogwiritsa ntchito zida zosiyanasiyana zosinthira ma gene, kuphatikiza CRISPR-Cas9, base and prime editing, mu DEB kukonza bwino masinthidwe a keratinocytes, fibroblasts ndi inducible pluripotent stem cell (iPSCs).

Ofufuza nawo:

Prof John McGrath wapanga chithandizo chapamwamba cha DEB cell kutengera jakisoni wa COL7A1-corrected fibroblasts, kuwonetsa chitetezo komanso kuthandizira koyambirira pamayesero azachipatala. Adzayang'ana pa kusankha kwa ma cell ndi mawonekedwe ake ndipo athandizira kupanga payipi yomasulira mwachangu zamankhwala atsopano amtundu wa COL7A1 mwa odwala.

Prof Stephen Hart (UCL, GOSH, London) ali ndi chidziwitso chambiri pakupanga zolemba zatsopano komanso njira zochizira ma nucleic acid okhala ndi ma nanoparticles osagwiritsa ntchito ma virus komanso kugwiritsa ntchito kwawo pochiza matenda monga cystic fibrosis, neuroblastoma, primary ciliary dyskinesia, ndi congenital melanocytic naevi. .

"Kumvetsera kwa anthu omwe ali ndi DEB, tikudziwa kuti maloto a "gene cream" ali pa mndandanda wa zofuna za aliyense… Ntchito yathu yatsopano yofufuza ndi yokonza mtundu wamuyaya wa mankhwala amtundu wa COL7A1. Pakadali pano, tikufuna kupanga ukadaulo watsopano woti tiyike kwanthawi zonse jini ya COL7A1 mu genome ya munthu yemwe ali ndi DEB. "

– Dr Joanna Jacków

Mutu wa zopereka: PASTE-Mediated Superexon Replacement of COL7A1 monga Chithandizo cha Dystrophic Epidermolysis Bullosa

Pazaka 30 zapitazi taphunzira kuti DEB imayambitsidwa ndi mitundu yosiyanasiyana ya DNA mumtundu wa VII collagen gene (COL7A1). Popanda jini yogwira ntchito ya COL7A1, khungu silingapange puloteni yokwanira ya VII ya collagen zomwe zikutanthauza kuti khungu silingathe kuvulala ndi matuza. Vuto la ofufuza lakhala momwe angasinthire kapena kukonza jini ya COL7A1. Kumvetsera kwa anthu omwe ali ndi DEB, tikudziwa kuti maloto a "gene cream" ali pamwamba pa mndandanda wa zofuna za aliyense.

Ndife okondwanso ndi kupita patsogolo kwaposachedwa popanga jini yatsopano ya COL7A1 yopangidwa ndi anzathu a ku yunivesite ya Stanford mogwirizana ndi Krystal Biotech, ngakhale kuti njira imeneyi ikufunika kubwerezedwa mobwerezabwereza kuti ikhale ndi zotsatira zokhalitsa. Ntchito yathu yatsopano yofufuza ndi yokhudza kupanga mtundu wanthawi zonse wa mankhwala amtundu wa COL7A1. Pakadali pano, tikufuna kupanga ukadaulo watsopano woti tiyike kwanthawi zonse jini ya COL7A1 mu genome ya munthu yemwe ali ndi DEB. Dongosolo latsopano la gene therapy limatchedwa PASTE, lomwe limayimira "Programmable Addition via Site-specific Targeting Elements". Pantchitoyi, tidzagwiritsa ntchito PASTE kuyika jini ya COL7A1 m'maselo akhungu a DEB. Kenako tifufuza kuti tiwone ngati tingabwezeretse mtundu wa VII collagen. Kenako tiyesetsa kukonza njira zoperekera lipid kuti tiwonetsetse kuti titha kupeza chithandizocho pakhungu osati ma cell okha. Pakadali pano, sitikupanga mayeso azachipatala, koma chimenecho chikhala dongosolo lathu lotsatira.

Ntchitoyi ikuyang'ana pakupanga cholinga chochiza chithandizo cha dystrophic epidermolysis bullosa (DEB). Kupita patsogolo kwakukulu kwachitika popanga mankhwala opangira ma jini a DEB pogwiritsa ntchito ma virus osaphatikizika kuti apereke COL7A1 pakhungu lovulala ngakhale kubwereza mobwerezabwereza ndikofunikira kuti apindule.

Monga njira inanso, tikufuna kugwiritsa ntchito kuphatikizika kosatha kwa jini ya COL7A1 yautali wonse pogwiritsa ntchito pulogalamu yowonjezeretsa kudzera pazida zomwe zalunjika pa tsamba (PASTE). PASTE imaphatikiza kutsimikizika ndi chitetezo chakusintha koyambirira ndi kuchuluka kwakukulu kwa serine integrases kuti iwonetse mpaka 36kb yazinthu pamasamba opangidwa mwapadera mu genome. Kuphatikizika kosatha kwa jini yautali wa COL7A1 kudzalola "kukula kumodzi kokwanira" njira yosinthira DNA.

Choyamba, tidzasankha ndikuwonetsa ma keratinocytes ndi/kapena fibroblasts kuchokera pakhungu la odwala DEB. Kenako tidzatsimikizira mapangidwe a PASTE pogwiritsa ntchito electroporation ya plasmids makonda. Kuphatikiza kwa ma gene COL7A1 kudzatsimikiziridwa pogwiritsa ntchito kutsatizana kwa PCR- ndi Sanger. Maselo osinthidwa bwino adzasiyanitsidwa ndikuzindikiridwanso kuti awone kupulumutsidwa kwa mtundu wa VII collagen. Pambuyo potsimikizira kapangidwe kake, m'malo mwa plasmid DNA, ma enzymes a PASTE adzaperekedwa kudzera mu in-vitro synthesized electroporated mRNA. Kenako, tidzayang'ana njira zoperekera, kupanga ma receptor-targeted lipid-based nanoparticles (LNPs) kuti apereke makina a PASTE ku maselo a chandamale. Chiwonetsero cha phage chidzazindikiritsa ma peptide omwe amamangiriza mwamphamvu komanso makamaka ku fibroblasts ndi keratinocytes. Zigawo za lipid zidzafufuzidwa kuti zikuyenera kugwiritsidwa ntchito pamawonekedwe akhungu a 3D. Mwachidule, polojekitiyi ikufuna kupanga njira yatsopano yothandizira jini ya DEB.

Ntchito yathu ili ndi zolinga ziwiri:

Choyamba, tikufuna kutenga kopi yonse ya jini yosasinthika ndikuyiyika mu DNA ya odwala kuti maselo awo ayambe kupanga mapuloteni ofunikira pakhungu lathanzi. Izi zidzatilola kupanga genotype-agnostic (ie imagwira ntchito mosasamala kanthu za masinthidwe omwe wodwala ali nawo) ndi kuchiritsa kosatha. M'miyezi isanu ndi umodzi yapitayi, tapeza sitepe yoyamba ya kusintha kwa DNA ikugwira ntchito bwino m'maselo osavuta kusintha, koma maselo a khungu la odwala akukhala ouma khosi. Tsopano tikuyesetsa kuwonetsetsa kuti tili ndi ndondomeko yotetezeka komanso yothandiza kuti tisinthe DNA m'maselo ofunika.

Kachiwiri, tikufuna kupanga ma nanoparticles omwe amatha kunyamula makina osintha a DNAwa m'maselo apakhungu okhala ndi mawonekedwe osavuta kugwiritsa ntchito topical cream. Tikuyesa mitundu yosiyanasiyana ya ma nanoparticles awa. Tapeza zotsatira zabwino poyika ma nanoparticles m'maselo omwe ali m'mbale, ndipo tsopano tikuwayesa pakhungu lachitsanzo. Izi zidzakhala zovuta kwambiri (khungu lasintha makamaka kuti zinthu zisamatuluke), koma mwa odwala EB, chotchinga cha khungu chayamba kale kufooka ndi kusokonezeka. Tikuyang'ana njira zochiritsira khungu lachitsanzo kuti lifanane ndi khungu la odwala EB, ndipo mwachiyembekezo izi zidzalola kuti nanoparticles alowe mkati mwa minofu.

Panopa tikukambilana ndi a Economist kuti mtolankhani abwere kudzaona ntchito yathu kuti athe kulemba nkhani ya momwe tikuyendera.