Maganin Halitta don EBS da RDEB (2022)

Duk da wahalar samun samfuran fata a lokacin ƙuntatawa na Covid, masu bincike sun nuna cewa maganin su na iya ba da damar canjin kwayoyin halitta wanda ke haifar da rasa RDEB ('exon skipping') a cikin fata da kuma a cikin sel a cikin tasa. Aiwatar da maganin cikin samfuran fata da ya rage daga hanyoyin tiyata, ya haifar da raguwar matakin tsalle-tsalle na exon a matakin kafin a samar da furotin collagen. Masu binciken sun ba da shawarar cewa za a buƙaci ƙarin aiki don nuna ko wannan ya ba da izinin samar da isasshen furotin collagen aiki don rage alamun RDEB.

Yin amfani da ƙananan ƙwayoyin nucleic acid (kamar DNA) don taimakawa wajen yin aikin collagen da furotin keratin daga karyewar RDEB ko kwayoyin EBS sun nuna wasu alkawura a cikin sel. Ya kasance da wahala a nuna tasirin a cikin fata inda waɗannan ƙananan ƙwayoyin nucleic acid suka bayyana suna aiki daban. Masu bincike sun buga a review na cigaba a wannan fanni a 2021. 

Wannan aikin ya gina kan aikin da masu binciken suka buga a cikin 2019:

• Exon Skipping Na Halitta Yana Sanya Matsayi don Tsallakewar Exon azaman Farfasa don dystrophic epidermolysis bullosa: Magungunan ƙwayoyin cuta - Nucleic Acids (cell.com)
• Magunguna don epidermolysis bullosa: isarwa shine mabuɗin - Bremer - 2019 - Jaridar Burtaniya na Dermatology - Wiley Online Library

Jami'ar Dundee ruwaito akan aikin a shekarar 2019.

Manyan masu bincike:

Dr Robyn Hickerson Babban Mai bincike ne a cikin Makarantar Kimiyyar Rayuwa tare da ƙungiyar bincike mai aiki tare da fifikon fifiko kan haɓaka hanyoyin warkewa don cututtukan fata masu ƙarancin gaske.
Dr Peter van den Akker, DEBRA Clinical Research Fellow, ƙwararren masanin ilimin likitanci ne kuma ya mayar da hankali ga aikin bincikensa akan hanyoyin kwantar da hankali na RNA don RDEB da sauran genodermatoses.

Co-bincike:

Dr Aileen Sandilands yana da kwarewa mai yawa daga shekaru 19 a cikin rukuni a cikin cututtukan fata na kwayoyin halitta da haɓaka tsarin tsalle-tsalle na exon tare da manufar magance cututtukan fata na kwayoyin halitta.

"Manufarmu ta ƙarshe ita ce haɓaka hanyoyin kwantar da hankali ga kowane nau'in EB… Gina kan sakamako mai ban sha'awa da ƙarfafawa daga aikinmu na baya, yanzu muna shirin haɓaka haɓaka haɓakar haɓakar exon a cikin tsohon vivo da samfuran fata na ɗan adam, don kawo mu kusa da mu. zuwa gwajin asibiti”

Dr Robyn Hickerson da Dr Peter Van den Akker

Taken Grant: Haɓaka Fasahar Novel Gene don Magance epidermolysis bullosa simplex (EBS) da recessive dystrophic epidermolysis bullosa (RDEB)

Kimanin kashi 70% na cututtukan epidermolysis bullosa (EB) an rarraba su da EB simplex (EBS), wanda ke haifar da maye gurbi (kuskure) a cikin kwayoyin halittar da ke kera sunadaran da ake kira keratin 5 da keratin 14 (KRT5 da KRT14). Keratins suna da mahimmanci don tabbatar da fata mai ƙarfi da lafiya. Babu wasu ingantattun jiyya na EBS, wanda ke da alaƙa da ƙumburi mai ɗorewa da rashin warkar da fata a ciki da waje. An gaji kwayoyin halitta, kwafi ɗaya daga kowane iyaye. Kwafi ɗaya kawai na kwayoyin halitta yana buƙatar ƙunshi maye gurbi don haifar da EBS - waɗannan ana kiran su manyan kwayoyin halitta. Ta hanyar zaɓin danne bayanin kuskuren kwafin kwayoyin halitta, wannan yana ba da damar kwafin kwayoyin halitta na yau da kullun suyi aiki da kyau, dabarun da aka yi imani za a iya haɓaka su zuwa hanyar da ta dace don EBS.
Manufar farko na wannan aikin shine haɓaka sabuwar fasaha don yin shiru na jiyya a cikin EBS. Lokacin da aka karanta jerin kwayoyin halitta ko DNA na kwayar halitta, daidai da girke-girke, an fassara shi ta hanyar matsakaicin mataki (manzo RNA) zuwa samar da furotin - a cikin wannan yanayin keratin da aka samu a saman Layer na fata, epidermis. Sabbin abubuwan da suka faru a fasahar kwayoyin halitta suna nufin cewa a yanzu ana iya hada wani dan karamin sinadarin nucleic acid wanda zai daure ga manzo RNA kuma ya hana shi aiki. Ana kiran wannan fasahar shiru ta kwayoyin halitta. Antisense oligonucleotides (ASOs) ƙananan guda ne na nucleic acid waɗanda za a iya ƙera su don ɗaure musamman kwafin RNA na wani kwayar halitta don lalata waɗannan.

An ba wa Ƙungiyar Bincike na Clinical alhakin haɓaka wannan sabuwar fasahar yin shiru ta hanyar da za a iya shigar da ita cikin asibiti. Tawagar a Dundee tana aiki tare da kamfanin harhada magunguna WAVE Life Sciences akan wannan aikin kuma sun gano ASOs da yawa waɗanda zasu iya yin shiru da manzon KRT14 RNA a cikin ƙwayoyin fata na ɗan adam wanda aka girma a cikin dakin gwaje-gwaje.

Yin amfani da ASOs don recessive dystrophic epidermolysis bullosa (wani nau'i mai tsanani na EB) daidai yake da kalubale. Recessive dystrophic epidermolysis bullosa (RDEB) yana haifar da kurakurai a cikin kwayar halittar COL7A1, girke-girke na kwayoyin halitta don nau'in furotin collagen 7. Kowa yana ɗaukar kwafin COL7A1 guda biyu, amma, sabanin EBS, akwai buƙatar samun maye gurbi a kan kwafin biyu na kwayoyin halitta don nuna alamun RDEB - waɗannan kwayoyin halitta ne. Hanyar lalata RNA manzo mara kyau ba zai yi aiki a nan ba. Koyaya, ana iya amfani da nau'in ASO daban-daban wanda zai iya yaudarar sel don cire ɓangaren RNA manzo inda maye gurbin yake. Ana kiran wannan hanyar 'exon skipping' kuma ko da yake wannan zai haifar da ɗan gajeren gajere RNA, ana iya amfani dashi don samar da nau'in collagen na 7 mai aiki (amma gajarta). A cikin binciken wallafe-wallafen, Fellow ya gano cewa mutanen da ke da tsalle-tsalle a cikin su yana faruwa ta halitta (ba tare da amfani da ASO ba amma saboda ƙarin bambancin DNA) har yanzu suna da nau'i na DEB, amma wannan ya fi sauƙi fiye da yadda aka saba. Wannan yana jaddada cewa tsalle-tsalle na exon wata dabara ce ta warkewa. Tawagar a Dundee sun tsara ASOs da yawa waɗanda zasu iya haifar da tsalle-tsalle na COL7A1 a cikin ƙwayoyin fata na ɗan adam waɗanda aka girma a cikin dakin gwaje-gwaje.

Novel antisense fasahar don EB - inganta exon skipping a cikin fata mutum (Clinical Research Fellowship Year 5).

Marasa lafiya da ke fama da cututtukan fata recessive dystrophic epidermolysis bullosa (RDEB) suna fama da fata mai rauni sosai saboda wani mahimmin furotin (collagen 7) wanda ke riƙe sassan fata tare ya ɓace. A cikin marasa lafiya tare da RDEB, kurakuran rubutu ("maye gurbi") a cikin kwayar halittar da ke ƙididdige furotin na collagen 7 yana hana jiki yin shi. A Jami'ar Dundee, muna ƙoƙarin ƙetare kurakuran rubutu a cikin kwayar halitta don collagen 7 ta hanyar ƙaddamar da matakin da ke faruwa kafin a yi furotin. Kafin a yi furotin na collagen 7, kwayar halitta ta fara yin kwafin kanta mai ɗaukar hoto, ana kiran wannan kwafin Messenger RNA kuma ana amfani dashi azaman samfuri don yin furotin. A cikin marasa lafiya na RDEB, kwafin RNA na manzo ya ƙunshi kurakuran rubutun iri ɗaya kamar kwayar halittar collagen 7. Dabarar mu ita ce kawar da ɗan ƙaramin sashi na RNA manzo wanda ya ƙunshi kurakuran rubutun. Ana kiran wannan hanyar a matsayin "exon skipping". Ko da yake manzo RNA zai ɗan gajarta fiye da na al'ada amma ana iya amfani dashi don yin furotin na collagen 7. Tabbas, wannan furotin kuma zai ɗan ɗan gajarta fiye da na al'ada, amma wannan har yanzu ya fi samun furotin collagen 7 kwata-kwata.
Exon skipping yana amfani da ƙananan ƙwayoyin cuta da ake kira Antisense oligonucleotides (ASOs) waɗanda ke manne wa manzo RNA kwafin kwayar halittar collagen 7 kuma suna yaudarar sel don cire ɓangaren da aka samu kuskuren rubutun. Mun tsara ASOs da yawa kuma mun nuna cewa suna aiki a tsalle-tsalle lokacin da aka ƙara su cikin ƙwayoyin fatar jikin mutum da aka girma a cikin dakin gwaje-gwaje. Mun kuma sami damar nuna cewa ASOs suna aiki a cikin ainihin fata na mutum (fatar sharar aikin tiyata). Duk da haka, adadin tsalle-tsalle na exon wanda za mu iya ganowa a cikin fata yana da ƙananan kuma ASOs suna da alama suna aiki daban-daban a cikin fata idan aka kwatanta da kwayoyin da aka girma a cikin dakin gwaje-gwaje.
A cikin shekarar da ta gabata mun inganta hanyar da za ta ba mu damar bin diddigin yadda ake rarraba ASO lokacin da aka yi masa allura a cikin fata. Wannan hanya ta koya mana cewa ASO da aka yi masa allura a cikin fatar mutum hakika ya yi tafiya daga dermis (zurfin fatar fatar da aka yi wa ASO allurar), zuwa kwayoyin halitta a cikin epidermis (babban Layer na fata), inda exon ke tsallakewa. yana bukatar faruwa. Wannan sakamakon ya tabbatar mana da cewa maganin ASO na iya haifar da tsalle-tsalle a cikin fata na ainihi. Bugu da ƙari, wannan sakamako ne mai ƙarfafawa. Duk da haka, adadin tsalle-tsalle na exon da muka gano a cikin fata yana da ƙananan kuma ASOs suna da alama suna aiki daban-daban a cikin fata idan aka kwatanta da kwayoyin da aka girma a cikin dakin gwaje-gwaje. Don haka, yanzu mun mai da hankali kan yin tsalle-tsalle a cikin fata mafi inganci.
A bara mun kuma tsara kuma mun gwada sabbin ASO 37 akan exon 13 a cikin ƙwayoyin fata don ganin ko za mu iya gano ASO wanda ke aiki har ma fiye da waɗanda muke amfani da su a halin yanzu. Mun kuma tsara sabbin ASOs guda 33 akan exon 15 don gwadawa ta wannan hanya. Sakamakon farko ya gano ASOs da yawa waɗanda ke nuna ayyukan tsalle-tsalle na exon 13 don haka za a ƙara bincikar waɗannan. (Daga rahoton ci gaba na 2022.)

Novel antisense fasahar don EB - ciyar da exon skipping a cikin fata mutum (Shekaru 3-4).

Babban manufar yanzu shine yin nazarin ko ASOs masu aiki zasu iya haifar da tsallakewar kwayar halittar COL7A1 da yin shuru na kwafin KRT14 RNA na mutant lokacin amfani da fatar mutum (ta amfani da ragowar fata daga hanyoyin tiyata). Wannan ya haɗa da nazarin hanyoyin da za a sadar da waɗannan ASO zuwa wurin da ya dace a cikin fata. Saboda haka, Fellow yana aiki tare da ƙungiyar Dr Hickerson.
Babban makasudin haɗin gwiwar binciken asibiti da ke ci gaba shine:
• Ƙaddamar da "ƙasa" na mutant KRT14 mRNA don magance EBS (don yin aiki akan kawar da keratin messenger RNA wanda ke ɗauke da maye gurbin).
Tsallake exons wanda ke dauke da maye gurbi a cikin kwayar halittar COL7A1 don magance RDEB (don yaudarar sel na jiki don cirewa ko rashin karanta sashin kwayar halitta inda maye gurbin yake).

Marasa lafiya da ke fama da cututtukan fata recessive dystrophic epidermolysis bullosa (RDEB) suna fama da fata mai rauni sosai saboda wani mahimmin furotin (collagen 7) wanda ke riƙe sassan fata tare ya ɓace. A cikin marasa lafiya tare da RDEB, kurakuran rubutu ("maye gurbi") a cikin kwayar halittar da ke ƙididdige furotin na collagen 7 yana hana jiki yin shi.

A yayin wannan aikin mun kasance muna ƙoƙarin yaudarar sel don ƙetare kurakuran rubutu a cikin kwayar halitta don collagen 7. Don yin wannan mun yi niyya wani muhimmin mataki da ke faruwa kafin a samar da furotin. Dole ne kwayar halitta ta fara yin kwafin kanta mai ɗaukar hoto, ana kiran wannan kwafin Messenger RNA kuma ƙwayoyin suna amfani da su azaman samfuri don yin furotin. A cikin marasa lafiya na RDEB, kwafin RNA na manzo ya ƙunshi kurakuran rubutun iri ɗaya kamar kwayar halittar collagen 7. Dabarar mu ita ce gyara ƙaramin ɓangaren manzo RNA wanda ke ɗauke da kuskuren rubutun. Muna kiran wannan hanyar "exon skipping". Ta hanyar gyara manzo RNA ta wannan hanyar da kawar da kuskuren rubutun kalmomin yanzu suna iya yin furotin na collagen 7. Tabbas, wannan furotin zai ɗan ɗan gajarta fiye da na al'ada, amma har yanzu yana da kyau fiye da rashin furotin collagen 7 kwata-kwata.

Exon skipping yana amfani da ƙananan ƙwayoyin da ake kira antisense oligonucleotides (ASOs), waɗannan suna manne wa manzo RNA kwafin kwayar halittar collagen 7 kuma suna yaudarar sel su cire ɓangaren da ke ɗauke da kuskuren rubutun. Mun tsara ASOs da yawa kuma mun nuna cewa suna iya haifar da tsalle-tsalle yayin da muke kula da ƙwayoyin fatar jikin mutum da aka girma a cikin dakin gwaje-gwaje. Mun kuma yi ƙoƙarin magance ƙwayoyin fata daga mai haƙuri na RDEB tare da ASOs kuma suna da alama suna aiki a cikin hanya ɗaya. Hakanan zamu iya gano furotin na collagen 7 a cikin waɗannan sel bayan an yi magani tare da ASOs, wanda ke gaya mana cewa ASOs suna aiki kamar yadda aka yi niyya.

An kashe adadi mai yawa na aikin don gano ko ASOs suna aiki a cikin fata na mutum. Manufarmu ta ƙarshe ita ce amfani da ASOs don kula da marasa lafiya don haka yana da mahimmanci don sanin idan suna aiki a cikin fata maimakon kawai a cikin sel a cikin tasa. Kafin mu fara wannan aikin, dole ne mu haɓaka hanyoyin ganowa sosai don ɗaukar tsalle-tsalle na exon saboda waɗannan kayan aikin bincike ba su wanzu ta kasuwanci. Don gwaje-gwajen fata mun allurar ASOs zuwa cikin lafiyayyen fata da aka bari daga hanyoyin tiyata sannan mu bincika fata don tsalle-tsalle. Mun gano cewa ASOs na iya haifar da tsalle-tsalle a cikin fata na mutum wanda ba shakka sakamako ne mai ƙarfafawa. Duk da haka, babban ƙalubalen da muke fuskanta shi ne cewa yawan tsalle-tsalle na exon da za mu iya ganowa a cikin fata yana da ƙasa. ASOs kuma suna da alama suna aiki da ɗan bambanta a cikin fata idan aka kwatanta da ƙwayoyin da aka girma a cikin dakin gwaje-gwaje. Gaskiya ba mu sani ba ko matakin tsalle-tsalle na exon da muke gani a cikin fata ya isa ya zama fa'ida ga marasa lafiya (gwajin asibiti kawai zai ba da cikakkiyar amsa) amma muna jin cewa har yanzu akwai sauran damar ingantawa. Saboda haka, a cikin sashin ƙarshe na aikin mun mayar da hankali kan hanyoyin da za a ƙara yawan tsalle-tsalle na exon a cikin fata. Mun tsara wani sabon tsari na ASOs don gwadawa da gano wanda ke aiki har ma fiye da waɗanda muke amfani da su a halin yanzu, haka kuma mun gwada haɗa ASOs tare da reagent wanda aka tsara don sauƙaƙe ɗaukar ASOs cikin sel bayan an yi musu allura a ciki. fata.

Gabaɗaya, wannan aikin ya nuna cewa yin amfani da ASOs don haifar da tsalle-tsalle a cikin fata na ainihi yana yiwuwa. Ƙaddamar da ASOs da ingantawa a cikin hanyar da aka ba da su a cikin fata zai zama dole don ƙarfafa tasirin su. Idan za'a iya shawo kan waɗannan cikas to exon tsallakewa azaman magani ga RDEB ya kasance zaɓi mai yuwuwa. (Daga rahoton ƙarshe na 2022.)