HRP20200529T1 - Postupci i pripravci za modificiranje ciljnog lokusa - Google Patents

Postupci i pripravci za modificiranje ciljnog lokusa Download PDF

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HRP20200529T1
HRP20200529T1 HRP20200529TT HRP20200529T HRP20200529T1 HR P20200529 T1 HRP20200529 T1 HR P20200529T1 HR P20200529T T HRP20200529T T HR P20200529TT HR P20200529 T HRP20200529 T HR P20200529T HR P20200529 T1 HRP20200529 T1 HR P20200529T1
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cell
polynucleotide
nucleic acid
nuclease
acid sequence
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HRP20200529TT
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Wojtek Auerbach
David Frendewey
Gustavo Droguett
Anthony Gagliardi
Junko Kuno
David M. Valenzuela
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Regeneron Pharmaceuticals, Inc.
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Claims (18)

1. Postupak za serijsku modifikaciju ciljnog lokusa u ćeliji, naznačen time što sadrži: (a) osiguravanje ćelije koja sadrži ciljni lokus, pri čemu ciljni lokus sadrži polinukleotid koji kodira prvi selekcijski marker koji je operativno povezan sa prvim promotorom i koji sadrži prvo mjesto za prepoznavanje za prvo sredstvo nukleaze, pri čemu je prvo mjesto za prepoznavanje nukleaze smješteno u kodirajućoj regiji prvog selekcijskog markera ili bilo kojoj ne-protein-kodirajućoj regiji prvog selekcijskog markera, izborno pri čemu je ciljni lokus u genomu ćelije ili je smješten u vektoru u ćeliji; (b) uvođenje u ćeliju: (i) prvog sredstva nukleaze, pri čemu prvo sredstvo nukleaze izaziva zarez ili dvolančani prekid na prvom mjestu za prepoznavanje nukleaze, čime se narušava ekspresija ili aktivnost prvog selekcijskog markera; i (ii) prvog vektora koji ciljno djeluje koji sadrži prvi umetnuti polinukleotid omeđen sa bočnih strana prvom homolognom granom koja odgovara prvom ciljnom mjestu smještenom u ciljnom lokusu i drugom homolognom granom koja odgovara drugom ciljnom mjestu smještenom u ciljnom lokusu, pri čemu prvi umetnuti polinukleotid sadrži: (I) prvi polinukleotid od interesa; i (II) polinukleotid koji kodira drugi selekcijski marker koji je operativno povezan sa drugim promotorom i koji sadrži drugo mjesto za prepoznavanje nukleaze za drugo sredstvo nukleaze, pri čemu su prvi selekcijski marker i drugi selekcijski marker različiti, pri čemu je prvo sredstvo nukleaze različito od drugog sredstva nukleaze, i pri čemu je drugo mjesto za prepoznavanje nukleaze smješteno u kodirajućoj regiji drugog selekcijskog markera ili bilo kojoj ne-protein-kodirajućoj regiji drugog selekcijskog markera; (c) identificiranje modificirane ćelije koja sadrži prvi umetnuti polinukleotid na ciljnom lokusu, pri čemu modificirana ćelija ima aktivnost drugog selekcijskog markera, ali nema aktivnost prvog selekcijskog markera, izborno pri čemu se identificiranje vrši putem testa modifikacije alela (MOA); (d) uvođenje u modificiranu ćeliju: (i) drugog sredstva nukleaze, pri čemu drugo sredstvo nukleaze izaziva zarez ili dvolančani prekid na drugom mjestu za prepoznavanje nukleaze, čime se narušava ekspresija ili aktivnost drugog selekcijskog markera; i (ii) drugog vektora koji ciljno djeluje koji sadrži drugi umetnuti polinukleotid omeđen sa bočnih strana trećom homolognom granom koja odgovara trećem ciljnom mjestu smještenom u ciljnom lokusu i četvrtom homolognom granom koja odgovara četvrtom ciljnom mjestu smještenom u ciljnom lokusu, pri čemu drugi umetnuti polinukleotid sadrži: (I) drugi polinukleotid od interesa; i (II) polinukleotid koji kodira treći selekcijski marker koji je operativno povezan sa trećim promotorom koji je aktivan u ćeliji i koji sadrži treće mjesto za prepoznavanje nukleaze za treće sredstvo nukleaze, pri čemu su prvi selekcijski marker i treći selekcijski marker identični, i pri čemu je treće mjesto za prepoznavanje nukleaze identično prvom mjestu za prepoznavanje nukleaze i različito je od drugog mjesta za prepoznavanje nukleaze, i prvo sredstvo nukleaze i treće sredstvo nukleaze identični su jedno drugome i različiti su od drugog sredstva nukleaze; i (e) identificiranje najmanje jedne ćelije koja sadrži drugi umetnuti polinukleotid integriran u ciljni lokus, izborno pri čemu se identificiranje vrši putem testa modifikacije alela (MOA).
2. Postupak prema patentnom zahtjevu 1, naznačen time što korak identificiranja (c) sadrži: (i) uzgajanje ćelije pod uvjetima koji omogućavaju identificiranje ćelija koje nemaju aktivnost prvog selekcijskog markera; ili (ii) identificiranje najmanje jedne ćelije koja sadrži prvi umetnuti polinukleotid integriran u prvo i drugo ciljno mjesto; i/ili pri čemu korak identificiranja (e) sadrži: (i) uzgajanje ćelije pod uvjetima koji omogućavaju identificiranje ćelija koje nemaju aktivnost drugog selekcijskog markera; ili (ii) identificiranje najmanje jedne ćelije koja sadrži drugi umetnuti polinukleotid integriran u treće i četvrto ciljno mjesto.
3. Postupak prema bilo kojem prethodnom patentnom zahtjevu, naznačen time što je polinukleotid koji kodira drugi selekcijski marker u modificiranoj ćeliji u koraku (c) omeđen sa bočnih strana trećim ciljnim mjestom i četvrtim ciljnim mjestom.
4. Postupak prema bilo kojem prethodnom patentnom zahtjevu, naznačen time što prvi, drugi, ili treći selekcijski marker daje rezistenciju na antibiotik, izborno pri čemu antibiotik sadrži G418, higromicin, blasticidin, neomicin, ili puromicin, ili pri čemu je prvi, drugi, ili treći selekcijski marker operativno povezan sa inducibilnim promotorom, i ekspresija selekcijskog markera je toksična za ćeliju, izborno pri čemu prvi, drugi, ili treći selekcijski marker sadrži hipoksantin-guanin fosforiboziltransferazu (HGPRT) ili timidin kinazu Herpes simplex virusa (HSV-TK).
5. Postupak prema bilo kojem prethodnom patentnom zahtjevu, naznačen time što je ćelija eukariotska ćelija, izborno pri čemu je eukariotska ćelija ćelija sisavca, izborno pri čemu je ćelija sisavca: (a) ćelija ne-ljudskog sisavca; (b) pluripotentna ćelija; (c) ljudski izazvana pluripotentna matična ćelija; (d) ljudski fibroblast; ili (e) ćelija glodavca.
6. Postupak prema bilo kojem prethodnom patentnom zahtjevu, naznačen time što je ćelija embrionalna matična (ES) ćelija miša ili ES ćelija štakora.
7. Postupak prema bilo kojem prethodnom patentnom zahtjevu, naznačen time što kombinirana uporaba prvog vektora koji ciljno djeluje sa prvim sredstvom nukleaze rezultira povećanom efikasnošću ciljnog djelovanja u usporedbi sa uporabom samo prvog vektora koji ciljno djeluje, izborno pri čemu je efikasnost ciljnog djelovanja prvog vektora koji ciljno djeluje povećana najmanje dvostruko u usporedbi sa uporabom samo prvog vektora koji ciljno djeluje.
8. Postupak prema bilo kojem prethodnom patentnom zahtjevu, naznačen time što prvo sredstvo nukleaze, drugo sredstvo nukleaze, ili treće sredstvo nukleaze: (a) sadrži polinukleotid koji kodira sredstvo nukleaze, pri čemu je polinukleotid sadržan u kaseti ekspresije i operativno je povezan sa kondicionalnim promotorom, inducibilnim promotorom, konstitutivnim promotorom, ili tkivno-specifičnim promotorom; (b) je iRNK koja kodira nukleazu; (c) je cinčani prst nukleaze (ZFN); (d) je efektorska nukleaza slična aktivatoru transkripcije (TALEN); (e) je meganukleaza; ili (f) je okupljena kratkim palindromskim ponavljanjima na jednakim razmacima (CRISPR)-povezan (Cas) protein i vodeća RNK (gRNA).
9. Postupak prema patentnom zahtjevu 8, naznačen time što je prvo sredstvo nukleaze, drugo sredstvo nukleaze, ili treće sredstvo nukleaze Cas protein i vodeća RNK, pri čemu je Cas protein Cas9, i pri čemu vodeća RNK (gRNA) sadrži: (a) CRISPR RNK (crRNA) koja cilja prvo, drugo, ili treće mjesto za prepoznavanje, pri čemu je prvo, drugo, ili treće mjesto za prepoznavanje neposredno omeđeno sa bočnih strana protorazmaknice susjednog motiva (PAM) sekvencom. (b) trans-aktivirajuću CRISPR RNK (tracrRNA); izborno pri čemu ciljni lokus sadrži nukleotidnu sekvencu SEQ ID NO: 1; i izborno pri čemu gRNA sadrži himernu RNK koja ima sekvencu nukleinske kiseline SEQ ID NO: 2 ili SEQ ID NO: 3 ili tracrRNA koja sadrži SEQ ID NO: 7 ili SEQ ID NO: 8.
10. Postupak prema bilo kojem prethodnom patentnom zahtjevu, naznačen time što: (a) prvo ciljno mjesto i drugo ciljno mjesto su neposredno pored prvog mjesta za prepoznavanje nukleaze; (b) prvo ciljno mjesto i drugo ciljno mjesto su oko 10 nukleotida do oko 14 kb od prvog mjesta za prepoznavanje nukleaze; (c) treće ciljno mjesto i četvrto ciljno mjesto su neposredno pored drugog mjesta za prepoznavanje nukleaze; ili (d) treće ciljno mjesto i četvrto ciljno mjesto su oko 10 nukleotida do oko 14 kb od drugog mjesta za prepoznavanje nukleaze.
11. Postupak prema bilo kojem prethodnom patentnom zahtjevu, naznačen time što: (a) ukupan zbroj prve homologne grane i druge homologne grane je najmanje oko 10 kb ili je svaka od prve i druge homologne grane u opsegu od oko 5 kb do oko 100 kb; i/ili (b) ukupan zbroj treće homologne grane i četvrte homologne grane je najmanje oko 10 kb ili je svaka od treće i četvrte homologne grane u opsegu od oko 5 kb do oko 100 kb; i/ili (c) prvi vektor koji ciljno djeluje je najmanje oko 10 kb ili je od oko 20 kb do oko 300 kb; i/ili (d) drugi vektor koji ciljno djeluje je najmanje oko 10 kb ili je od oko 20 kb do oko 300 kb; i/ili (e) prvi umetnuti polinukleotid dugačak je u opsegu od oko 5 kb do oko 300 kb; i/ili (f) drugi umetnuti polinukleotid dugačak je u opsegu od oko 5 kb do oko 300 kb.
12. Postupak prema bilo kojem prethodnom patentnom zahtjevu, naznačen time što: (a) integracija prvog umetnutog polinukleotida u ciljni lokus rezultira knockout-om, knock-in-om, točkastom mutacijom, zamjenom domena, zamjenom egzona, zamjenom introna, zamjenom regulatorne sekvence, zamjenom gena, ili njihovom kombinacijom; i/ili (b) integracija drugog umetnutog polinukleotida u ciljni lokus rezultira knockout-om, knock-in-om, točkastom mutacijom, zamjenom domena, zamjenom egzona, zamjenom introna, zamjenom regulatorne sekvence, zamjenom gena, ili njihovom kombinacijom.
13. Postupak prema bilo kojem prethodnom patentnom zahtjevu, naznačen time što: (a) prvi polinukleotid od interesa sadrži ljudski polinukleotid, sekvencu nukleinske kiseline koja je homologna ili ortologna sekvenci nukleinske kiseline u genomu ćelije, ili sekvencu egzogene nukleinske kiseline, izborno pri čemu prvi polinukleotid od interesa sadrži: (i) regiju alfa lokusa T ćelijskog receptora, izborno pri čemu prvi polinukleotid od interesa sadrži najmanje jedan segment gena varijabilne regije i/ili segment gena povezujuće regije alfa lokusa T ćelijskog receptora; ili (ii) nereorganizranu sekvencu nukleinske kiseline varijabilne regije teškog lanca ljudskog imunoglobulina koja je operativno povezana sa sekvencom nukleinske kiseline konstantne regije teškog lanca imunoglobulina; i/ili (b) drugi polinukleotid od interesa sadrži ljudski polinukleotid, sekvencu nukleinske kiseline koja je homologna ili ortologna sekvenci nukleinske kiseline u genomu ćelije, ili sekvencu egzogene nukleinske kiseline, izborno pri čemu drugi polinukleotid od interesa sadrži: (i) regiju alfa lokusa T ćelijskog receptora, izborno pri čemu drugi polinukleotid od interesa sadrži najmanje jedan segment gena varijabilne regije i/ili segment gena povezujuće regije alfa lokusa T ćelijskog receptora; ili (ii) nereorganiziranu sekvencu nukleinske kiseline varijabilne regije teškog lanca ljudskog imunoglobulina koja je operativno povezana sa sekvencom nukleinske kiseline konstantne regije teškog lanca imunoglobulina.
14. Postupak prema bilo kojem prethodnom patentnom zahtjevu, naznačen time što: (a) prvi polinukleotid od interesa i/ili drugi polinukleotid od interesa sadrži najmanje jedan alel bolesti; (b) prvi polinukleotid od interesa i/ili drugi polinukleotid od interesa sadrži sekvencu genske nukleinske kiseline koja kodira amino-kiselinsku sekvencu varijabilne regije teškog lanca ljudskog imunoglobulina; ili (c) prvi polinukleotid od interesa i/ili drugi polinukleotid od interesa sadrži sekvencu genske nukleinske kiseline koja kodira amino-kiselinsku sekvencu varijabilne regije lakog lanca ljudskog imunoglobulina, izborno pri čemu: (i) sekvenca genske nukleinske kiseline sadrži nereorganiziranu sekvencu nukleinske kiseline varijabilne regije λ i/ili κ lakog lanca; ili (ii) sekvenca genske nukleinske kiseline sadrži reorganiziranu sekvencu nukleinske kiseline varijabilne regije λ i/ili κ lakog lanca.
15. Postupak prema bilo kojem prethodnom patentnom zahtjevu, naznačen time što: (a) ciljni lokus sadrži lokus imunoglobulina; ili (b) ciljni lokus sadrži lokus T ćelijskog receptora, izborno pri čemu je lokus T ćelijskog receptora alfa lokus T ćelijskog receptora.
16. Postupak prema bilo kojem prethodnom patentnom zahtjevu, naznačen time što prvo sredstvo nukleaze, drugo sredstvo nukleaze, i treće sredstvo nukleaze su svaki Cas protein i vodeća RNK, i pri čemu je vodeća RNK (gRNA) specifična za gen za rezistenciju na higromicin ili neomicin.
17. Postupak prema patentnom zahtjevu 16, naznačen time što je gRNA specifična za gen za rezistenciju na neomicin kodirana nukleinskom kiselinom koja sadrži nukleotidnu sekvencu iznijetu u SEQ ID NO: 13, 14, 15, ili 16, ili pri čemu je gRNA specifična za gen za rezistenciju na higromicin kodirana nukleinskom kiselinom koja sadrži nukleotidnu sekvencu iznijetu u SEQ ID NO: 17, 18, 19, ili 20.
18. Postupak prema patentnom zahtjevu 17, naznačen time što: (a) prva gRNA kodirana je nukleinskom kiselinom koja sadrži nukleotidnu sekvencu iznijetu u SEQ ID NO: 13, 14, 15, ili 16, i druga gRNA kodirana je nukleinskom kiselinom koja sadrži nukleotidnu sekvencu iznijetu u SEQ ID NO: 17, 18, 19, ili 20; ili (b) prva gRNA kodirana je nukleinskom kiselinom koja sadrži nukleotidnu sekvencu iznijetu u SEQ ID NO: 17, 18, 19, ili 20, i druga gRNA kodirana je nukleinskom kiselinom koja sadrži nukleotidnu sekvencu iznijetu u SEQ ID NO: 13, 14, 15, ili 16.
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