Delivery of gene editing systems and methods of use thereof

Abstract

The present disclosure describes improved LNP-based nucleobase editing systems and therapeutics for use in treating a disease. In particular, the disclosure describes improved LNPs, including novel and improved ionic lipids for making LNPs, that enhance the targeted delivery of LNP-based nucleobase editing systems and therapeutics based on linear and / or circular mRNAs. The improved LNPs protect linear and / or circular mRNA payloads from degradation and clearance while achieving targeted systemic or local delivery for use as enhanced nucleobase editing systems and / or therapeutic agents.

Description

This disclosure describes improved lipid nanoparticle (LNP)-based nucleobase editing systems and therapies for treating diseases. Specifically, this disclosure describes improved LNPs, including novel and improved ionic lipids for preparing LNPs, which enhance the targeted delivery of linear and / or circular mRNA-based nucleobase editing systems and therapies. The improved LNPs can protect linear and / or circular mRNA payloads from degradation and clearance while enabling targeted systemic or local delivery, thus serving as enhanced nucleobase editing systems and / or therapeutic agents. Abstract

Claims

CLAIMS1. A pharmaceutical composition comprising: a) at least one lipid nanoparticle comprising: i) at least one ionizable lipid selected from a compound of Formula (CY-VI”); Formula (X) or Formula (IC); and b) at least one nucleobase editing system.

2. The pharmaceutical composition of claim 1 , wherein the nucleobase editing system comprises a CRISPR-Cas gene editing system.

3. The pharmaceutical composition of claim 2, wherein the nucleobase editing system comprises a Type V CRISPR-Cas gene editing system.

4. The pharmaceutical composition of claim 1, wherein the nucleobase editing system comprises a prime editing system or components thereof.

5. The pharmaceutical composition of claim 1, wherein the nucleobase editing system comprises a retron editing system.

6. The pharmaceutical composition of claim 1 , wherein the nucleobase editing system comprises a TnpB editing system.

7. The pharmaceutical composition of claim 1, wherein the nucleobase editing system comprises an integrase editing system.

8. The pharmaceutical composition of claim 1, wherein the nucleobase editing system comprises a base editing system.

9. The pharmaceutical composition of claim 1 , wherein the nucleobase editing system comprises an epigenetic editing system.

10. The pharmaceutical composition of claim 1, wherein the nucleobase editing system comprises a gene writing system.

11. The pharmaceutical composition of claim 1 , wherein the nucleobase editing system comprises a gene inactivating system.

12. The pharmaceutical composition of claim 1, wherein the nucleobase editing system comprises zinc finger nuclease.

13. The pharmaceutical composition of claim 1, wherein the nucleobase editing system comprises a TALE Nuclease, a TALE nickase, Zinc Finger (ZF) Nuclease, ZF Nickase, meganuclease, or a combination thereof.

14. The pharmaceutical composition of claim 1, wherein the nucleobase editing system comprises a meganuclease.

15. The pharmaceutical composition of any one of claims 1-14, wherein the at least one lipid nanoparticle further comprises: i) at least one structural lipid; ii) at least one phospholipid; and iii) at least one PEGylated lipid.

16. The pharmaceutical composition of any of one of claims 1-15, wherein the at least one structural lipid is selected from cholesterol, fecosterol, fucosterol, beta sitosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatidine, cholic acid, sitostanol, litocholic acid, tomatine, ursolic acid, alpha-tocopherol, Vitamin D3, Vitamin D2, Calcipotriol, botulin, lupeol, oleanolic acid, beta-sitosterol-acetate and any combinations thereof.

17. The pharmaceutical composition of any one of claims 1-16, wherein the at least one phospholipid is selected from l,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2- dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1 ,2-dilinoleoyLsn-glycero-3- phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-phosphocholine (DMPC), 1.2-dioleoyL sn-glycero-3-phosphocholine (DOPC), 1 ,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-diundecanoyl-sn-glycero-phosphocholine (DUPC), l-palmitoyl-2-oleoyl-sn- glycero-3-phosphocho line (POPC), l ,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0Diether PC), l-oleoyl-2-cholesterylhemisuc cinoyl-sn-glycero-3-phosphocholine (OChemsPC), l-hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC), 1,2-dilinolenoyl-sn- glycero-3 -phosphocholine, 1 ,2-diarachidonoyl-sn-glycero-3-phosphocholine, 1 ,2- didocosahexaenoyl-sn-glycero-3-phosphocholine, l,2-diphytanoylsn-glycero-3- phosphoethanolamine (ME 16.0 PE), l,2-distearoyl-sn-glycero-3-phosphoethanolamine, 1,2- dilinoleoyl-sn-glycero-3-phosphoethanolamine, l,2-dilinolenoyl-sn-glycero-3- phosphoethanolamine, 1 ,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine, 1 ,2- didocosahexaenoyl-sn-glycero-3-phosphoethanolamine, l,2-dioleoyl-sn-glycero-3-phospho- rac-(l -glycerol) sodium salt (DOPG), sodium (S)-2-ammonio-3-((((R)-2-(oleoyloxy)-3- (stearoyloxy)propoxy)oxidophosphoryl)oxy)propanoate (L-a-phosphatidylserine; Brain PS), dimyristoyl phosphatidylcholine (DMPC), dimyristoyl phosphoethanolamine (DMPE), dimyristoylphosphatidylglycerol (DMPG), dioleoyl-phosphatidylethanolamine4-(N- maleimidomethyl)-cyclohexane- 1 -carboxylate (DOPE-mal), dioleoylphosphatidylglycerol (DOPG), l,2-dioleoyl-sn-glycero-3-(phospho-L-serine) (DOPS), acell-fusogenicphospholipid (DPhPE), dipalmitoylphosphatidylethanolamine (DPPE), 1 ,2-Dielaidoyl-sn- phosphatidylethanolamine (DEPE), dipalmitoylphosphatidylglycerol (DPPG), dipalmitoylphosphatidylserine (DPPS), distearoylphosphatidylcholine (DSPC), distearoyl- phosphatidyl-ethanolamine (DSPE), distearoyl phosphoethanolamineimidazole (DSPEI), 1,2- diundecanoyl-sn-glycero-phosphocholine (DUPC), egg phosphatidylcholine (EPC), 1,2- dioleoyl-sn-glycero-3 -phosphate (18: 1 PA; DOPA), ammonium bis((S)-2-hydroxy-3- (oleoyloxy)propyl) phosphate (18: 1 DMP; LBPA), l ,2-dioleoy]-sn-glycero-3-phospho-(r- myo-inositol) (DOPI; 18:1 PI), l,2-distearoyl-sn-glycero-3-phospho-L-serine (18:0 PS), 1,2- dilinoleoyl-sn-glycero-3-phospho-L-serine (18:2 PS), l-palmitoyl-2-oleoyl-sn-glycero-3- phospho-L-serine (16:0-18: 1 PS; POPS), l-stearoyl-2-oleoyl-sn-glycero-3-phospho-L-serine (18:0-18: 1 PS), l-stearoyl-2-linoleoyl-sn-glycero-3-phospho-L-serine (18:0-18:2 PS), 1- oleoyl-2-hydroxy-sn-glycero-3-phospho-L-serine (18:1 Lyso PS), l-stearoyl-2-hydroxy-sn- glycero-3-phospho-L-serine (18:0 Lyso PS), and sphingomyelin.

18. The pharmaceutical composition of any one of claims 1-17, wherein the at least one PEGylated lipid is selected from (R)-2,3-bis(octadecyloxy)propyl-l- (methoxypoly(ethyleneglycol)2000)propylcarbamate, PEG-S-DSG, PEG-S-DMG, PEG-PE, PEG-PAA, PEG-OH DSPE Cl 8, PEG-DSPE, PEG-DSG, PEG-DPG, PEG-DOMG, PEG- DMPE Na, PEG-DMPE, PEG-DMG2000, PEG-DMG C14, PEG-DMG 2000, PEG-DMG, PEG-DMA, PEG-Ceramide Cl 6, PEG-C-DOMG, PEG-c-DMOG, PEG-c-DMA, PEG-eDMA, PEGA, PEG750-C-DMA, PEG400, PEG2k-DMG, PEG2k-Cl 1, PEG2000-PE, PEG2000P, PEG2000-DSPE, PEG2000-DOMG, PEG2000-DMG, PEG2000-C-DMA, PEG2000, PEG200, PEG(2k)-DMG, PEG DSPE C18, PEG DMPE C14, PEG DLPE C12, PEG Click DMG C14, PEG Click C12, PEG Click CIO, N(Carbonyl- methoxypolyethylenglycol-2000)-l,2-distearoyl-sn-glycero3-phosphoethanolamine, Myrj52, mPEG-PLA, MPEG-DSPE, mPEG3000-DMPE, MPEG-2000-DSPE, MPEG2000-DSPE, mPEG2000-DPPE, mPEG2000-DMPE, mPEG2000-DMG, mDPPE-PEG2000, 1,2- distearoyl-sn-glycero-3-phosphoethanolamine-PEG2000, HPEG-2K-LIPD, Folate PEG- DSPE, DSPE-PEGMA 500, DSPE-PEGMA, DSPE-PEG6000, DSPE-PEG5000, DSPE- PEG2K-NAG, DSPE-PEG2k, DSPE-PEG2000maleimide, DSPE-PEG2000, DSPE-PEG, DSG-PEGMA, DSG-PEG5000, DPPE-PEG-2K, DPPE-PEG, DPPE-mPEG2000, DPPE- mPEG, DPG-PEGMA, DOPE-PEG2000, DMPE-PEGMA, DMPE-PEG2000, DMPE-Peg, DMPE-mPEG2000, DMG-PEGMA, DMG-PEG2000, DMG-PEG, distearoyl-glycerol- polyethyleneglycol, C18PEG750, CI8PEG5000, CI8PEG3OOO, CI8PEG2000, CI6PEG2000, CI4PEG2000, C18-PEG5000, C18PEG, C16PEG, C16 mPEG (polyethylene glycol) 2000 Ceramide, C14-PEG-DSPE200, C14-PEG2000, C14PEG2000, C14-PEG 2000, C14-PEG, C14PEG, 14:0-PEG2KPE, l,2-distearoyl-sn-glycero-3-phosphoethanolamine-PEG2000, (R)- 2,3-bis(octadecyloxy)propyl-l-(methoxypoly(ethyleneglycol)2000)propylcarbamate, (PEG)- C-DOMG, PEG-C-DMA, and DSPE-PEG-X.

19. The pharmaceutical composition of any one of claims 1 -18, wherein the LNP further comprises at least one additional lipid component selected from 1,2-di-O-octadecenyl-sn- glycero-3 -phosphocholine (18:0 Diether PC), 1 ,2-dilinolenoyl-sn-glycero-3 -phosphocholine (18:3 PC), Acylcarnosine (AC), l-hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC), N-oleoyl-sphingomyelin (SPM) (Cl 8:1), N-lignoceryl SPM (C24:0), N- nervonoylshphingomyelin (C24:l), Cardiolipin (CL), l,2-bis(tricosa-10,12-diynoyl)-sn- glycero-3 -phosphocholine (DC8-9PC), dicetyl phosphate (DCP), dihexadecyl phosphate (DCP1), l,2-Dipalmitoylglycerol-3-hemisuccinate (DGSucc), short-chain bis -n- heptadecanoyl phosphatidylcholine (DHPC), dihexadecoyl-phosphoethanolamine (DHPE), l,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), l,2-dilauroyl-sn-glycero-3-PE (DLPE), dimyristoyl glycerol hemisuccinate (DMGS), dimyristoyl phosphatidylcholine (DMPC), dimyristoyl phosphoethanolamine (DMPE), dimyristoylphosphatidylglycerol (DMPG), dioleyloxybenzylalcohol (DOB A), l,2-dioleoylglyceryl-3-hemisuccinate (DOGHEMS), N- [2-(2-{2-[2-(2,3-Bis-octadec-9-enyloxy-propoxy)-ethoxy]-ethoxy}-ethoxy)-ethyl]-3-(3,4,5-lrihydroxy-6-hydroxymethyl- 1 etrahydro-pyran-2-ylsulfanyl)-propionamide (D0GP4aMan), dioleoylphosphatidylcholine (DOPC), dioleoylphosphatidylethanolamine (DOPE), dioleoyl- phosphatidylethanolamine4-(N-maleimidomethyl)-cyclohexane-l -carboxylate (DOPE-mal), dioleoylphosphatidylglycerol (DOPG), 1 ,2-dioleoyl-sn-glycero-3-(phospho-L-serine) (DOPS), acell-fusogenicphospholipid (DPhPE), dipalmitoylphosphatidylethanolamine (DPPE), dipalmitoylphosphatidylglycerol (DPPG), dipalmitoylphosphatidylserine (DPPS), distearoylphosphatidylcholine (DSPC), distearoyl-phosphatidyl-ethanolamine (DSPE), distearoyl phosphoethanolamineimidazole (DSPEI), 1,2-diundecanoyl-sn-glycero- phosphocholine (DUPC), egg phosphatidylcholine (EPC), histaminedistearoylglycerol (HDSG), 1,2-Dipalmitoylglycerol-hemisuccinate-Na-Histidinyl-Hemisuccinate (HistSuccDG), N-(5’-hydroxy-3’-oxypentyl)-10-12-pentacosadiynamide (h-Pegi-PCDA), 2-[l- hexyloxyethyl]-2-devinylpyropheophorbide-a (HPPH), hydrogenatedsoybeanphosphatidylcholine (HSPC), 1 ,2-Dipalmitoylglycerol-O-a-histidinyl- Na-hemisuccinate (IsohistsuccDG), mannosialized dipalmitoylphosphatidylethanolamine (ManDOG), l,2-Dioleoyl-sn-Glycero-3-Phosphoethanolamine-N-[4-(p- maleimidomethyl)cyclohexane-carbox amide] (MCC-PE), 1 ,2-diphytanoyl-sn-glycero-3- phosphoethanolamine (ME 16:0 PE), l-myristoyl-2-hydroxy-sn-glycero-phosphocholine (MHPC), a thiol-reactive maleimide headgroup lipid e.g.l,2-dioleoyl-sn-glycero-3- phosphoethanolamine-N-[4-(p-maleimidophenyl)but-yramid (MPB-PE), Nervonic Acid (NA), sodium cholate (NaChol), l,2-dioleoyl-sn-glycero-3-[phosphoethanolamine-N- dodecanoyl (NC12-DOPE), l-oleoyl-2-cholesteryl hemisuccinoyl-sn-glycero-3- phosphocholine (OChemsPC), phosphatidylethanolamine lipid (PE), PE lipid conjugated with polyethylene glycol(PEG) (e.g., polyethylene glycol-distearoylphosphatidylethanolamine lipid (PEG-PE)), phosphatidylglycerol (PG), partially hydrogenated soy phosphatidylchloline (PHSPC), phosphatidylinositol lipid (PI), phosphotidylinositol-4-phosphate (PIP), palmitoyloleoylphosphatidylcholine (POPC), phosphatidylethanolamine (POPE), palmitoyloleyolphosphatidylglycerol (POPG), phosphatidylserine (PS), lissamine rhodamineB-phosphatidylethanolamine lipid (Rh-PE), purifiedsoy- derivedmixtureofphospholipids (SIOO), phosphatidylcholine (SM), 18-l-trans-PE,l-stearoyl- 2-oleoyl-phosphatidyethanolamine (SOPE), soybean phosphatidylcholine (SPC), sphingomyelins (SPM), alpha, alpha-trehalose-6,6'-dibehenate (TDB), 1,2-dielaidoyl-sn- glycero-3-phophoethanolamine (transDOPE), ((23S,5R)-3-(bis(hexadecyloxy)methoxy)-5-(5- methyl-2,4-dioxo-3 ,4-dihydropyrimidin- 1 (2H)-yl)tetrahydrofuran-2- yl)methylmethylphosphate, 1 ,2-diarachidonoyl-sn-glycero-3-phosphocholine, 1 ,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine, l,2-didocosahexaenoyl-sn-glycero-3- phosphocholine, 1 ,2-didocosahexaenoyl-sn-glycero-3-phosphoethanolamine, 1 ,2- dilinolenoyl-sn-glycero-3-phosphocholine, l,2-dilinolenoyl-sn-glycero-3- phosphoethanolamine, 1 ,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1 ,2-dioleyl-sn- glycero-3-phosphoethanolamine, l,2-distearoyl-sn-glycero-3-phosphoethanolamine, 16-0- monomethyl PE, 16-0-dimethyl PE, and dioleylphosphatidylethanolamine.

20. A method of delivering a nucleobase editing system to a subject in need thereof, the method comprising administering to the subject the pharmaceutical composition of any one of claims 1-19.

21. The pharmaceutical composition of any of claims 1-19 for use as a medicament.

22. Use of a pharmaceutical composition of claims 1-19 for the manufacture of a medicament for delivery of a nucleobase editing system.