Compositions for Transfecting Resistant Cell Types

Abstract

A transfection reagent composition comprising: 30-60 MOL % of an cationic lipid, or pharmaceutical acceptable salt thereof; 10-60 MOL % structural lipid; a sterol and 0.1 to about 10 MOL % of a stabilizing agent is provided. The reagent is particularly adapted for neuron and related cell types. A method of manufacturing LNP including nucleic acid for selective uptake into either neurons or astrocytes or neural progenitor cells is also provided.

What is claimed is: 
     
         1 . A transfection reagent composition comprising:
 (a) 30-60 MOL % of a cationic lipid, or a pharmaceutically acceptable salt thereof;   (b) 10-60 MOL % structural lipid;   (c) a sterol   (d) 0.1 to about 10 MOL % stabilizing agent.   
     
     
         2 . The composition of  claim 1 , wherein the cationic lipid is an amino lipid or a pharmaceutically acceptable salt thereof. 
     
     
         3 . The composition of  claim 1  or  2 , wherein the cationic lipid is selected from the group consisting of 1,17-bis(2-octylcyclopropyl)heptadecan-9-yl-4-(dimethylamino)butanoate, DODAC, DOTMA, DDAB, DOTAP, DOTAP⋅Cl, DC-Chol, DOSPA, DOGS, DODAP, DODMA, DMRIE, C12-200, and pharmaceutically acceptable salts thereof. 
     
     
         4 . The composition of  claim 1 , wherein the cationic lipid has the formula: 
       
         
           
           
               
               
           
         
         or a pharmaceutically acceptable salt thereof, wherein: 
         R 1  and R 2  are each independently H, alkyl, akenyl, alkynyl, cycloalkyl, aryl, heteroaryl, and heterocyclyl, 
         wherein each of alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, and heterocyclyl is optionally substituted by H, halo, hydroxy, cyano, oxo, C 1 -C 6  alkyl optionally substituted by halo, hydroxy, or alkoxy; 
         or R 1  and R 2  are taken together with the N atom to which they are both attached to form a 3-8 member heteroaryl or heterocyclyl; 
         wherein each of the heteroaryl and heterocyclyl is optionally substituted by H, halo, hydroxy, cyano, oxo, nitro, C 1 -C 6  alkyl optionally substituted by halo, hydroxyl, or alkoxy; 
         R 3  is absent, H, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, or heterocyclyl; 
         R 4  and R 5  are each independently H, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, or heterocyclyl; 
         wherein each of alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, and heterocyclyl is optionally substituted by H, halo, hydroxy; cyano; oxo; C 1 -C 6  alkyl optionally substituted by halo, hydroxy, or alkoxy; 
         X is O, S, —NR 4 —, —S—S—, —OC(═O)—, —C(═O)O—, —OC(═O)O—, —NR 4 C(═O)—, —C(═O)NR 4 —, —NR 4 C(═O)O—, —OC(═O)NR 4 —, —NR 4 C NR 4 , —NR 4 C(═S)O—, —OC(═S)NR 4 —, —NR 4 C(═S)NR 4 —, or —CR 4 R 5 —; 
         Y and Z are independently C 10  to C 30  groups having the formula L 1 -(CR 6 R 7 )a-[L 2 -(CR 6 R 7 ) P ] y -L 3 -Re, wherein: 
         L 1  is a bond, —(CR 6 Ry)-, —O—, —CO—, —NR 8 —, —S—, or a combination thereof; each R 5  and R 7 , independently, is H, halo, hydroxyl, cyano, C 1 -C 6  alkyl optionally substituted by halo, hydroxyl, or alkoxy; 
         L 2  is a bond, —(CR 6 R 7 )—, —O—, —CO—, —NR 8 —, —S—, 
       
       
         
           
           
               
               
           
         
         or a combination thereof, or has the formula 
       
       
         
           
           
               
               
           
         
         wherein b, c, and d are each independently 0, 1, 2, or 3, given the sum of b, c, and d is at least 1 and no greater than 8; and R 9  and R 10  are each independently R 7 , or adjacent R 9  and R 10 , taken together, are optionally a bond; 
         L 3  is a bond, —(CR 6 R 7 )—, —O—, —CO—, —NR 8 —, —S—, 
       
       
         
           
           
               
               
           
         
         or a combination thereof; 
         R 8  is independently H, halo, hydroxy, cyano, alkoxy, aryl, heteroaryl, or C 1 -C 6  alkyl optionally substituted by halo, hydroxy, or heterocyclyl, or R 8  has the formula: 
       
       
         
           
           
               
               
           
         
         a is 0, 1, 2, 3, or 4; 
         a is 0-6; 
         each β, independently, is 0-6; and 
         γ is 0-6. 
       
     
     
         5 . The composition of any one of  claims 1 - 4 , wherein the cationic lipid is 1,17-bis(2-octylcyclopropyl)heptadecan-9-yl-4-(dimethylamino)butanoate or a pharmaceutically acceptable salt thereof. 
     
     
         6 . The composition of any one of  claims 1 - 5 , wherein the structural lipid is selected from the group consisting of diacylphosphatidylcholines, diacylphosphatidylethanolamines, sterols, ceramides, sphingomyelins, dihydrosphingomyelins, cephalins, and cerebrosides. 
     
     
         7 . The composition of any one of  claims 1 - 6 , wherein the stabilizing agent is selected from the group consisting of polyethylene glycol, polyethylene glycol-DMG, polyoxyethylene alkyl ethers, diblock polyoxyethylene ether co-polymers, triblock polyoxyethylene alkyl ethers co-polymers, and amphiphilic branched polymers. 
     
     
         8 . The composition of any one of  claims 1 - 7  wherein the sterol is cholesterol. 
     
     
         9 . The composition of any one of  claims 1 - 8 , wherein the stabilizing agent is selected from the group consisting of polyoxyethylene (20) oleyl ether, polyoxyethylene (23) lauryl ether, polyoxyethylene (40) stearate, poly(propylene glycol)11-block-poly(ethylene glycol)16-block-poly(propylene glycol)11, poly(propylene glycol)12-block-poly(ethylene glycol)28-block-poly(propylene glycol)12. 
     
     
         10 . The composition of any one of  claims 1 - 9  wherein the stabilizing agent is PEG-conjugated lipid. 
     
     
         11 . The composition of  claim 10  wherein the stabilizing agent is PEG-DMG 
     
     
         12 . The composition of any one of  claims 1 - 11 , wherein the cationic lipid comprises about 40 MOL %. 
     
     
         13 . The composition of any one of  claims 1 - 12 , wherein the stabilizing agent comprises about 2.5 MOL % stabilizer. 
     
     
         14 . The composition of  claim 1 , wherein:
 (a) the cationic lipid is 1,17-bis(2-octylcyclopropyl)heptadecan-9-yl-4-(dimethylamino)butanoate or a pharmaceutically acceptable salt thereof;   (b) the structural lipid is 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE);   (c) the sterol is cholesterol, and   (d) the stabilizing agent is polyoxyethylene (40) stearate.   
     
     
         15 . The composition of  claim 1 , wherein:
 (a) the cationic lipid is 1,17-bis(2-octylcyclopropyl)heptadecan-9-yl-4-(dimethylamino)butanoate or a pharmaceutically acceptable salt thereof;   (b) the structural lipid is 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE); and   (c) the sterol is cholesterol, and   (d) the stabilizing agent is polyethylene glycol conjugated lipid.   
     
     
         16 . The composition  claim 1 , wherein the structural lipid comprises about 10 to about 40 MOL % 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE). 
     
     
         17 . The composition of of any one of  claims 1 - 16  wherein the sterol is present from 10 to 20 MOL %. 
     
     
         18 . The composition of any one of  claims 1 - 17 , further comprising a nucleic acid. 
     
     
         19 . The composition of  claim 18 , wherein the nucleic acid is a DNA, an RNA, a locked nucleic acid, a nucleic acid analog, or a plasmid capable of expressing an RNA. 
     
     
         20 . The composition of  claim 18 , wherein the nucleic acid is an antisense oligonucleotide, ribozyme, miRNA, rRNA, tRNA, siRNA, saRNA, snRNA, snoRNA, lncRNA, piRNA, tsRNA, srRNA, crRNA, tracrRNA, sgRNA, shRNA, ncRNA, miRNA, mRNA, pre-condensed DNA, pDNA, an aptamer, or a combination thereof. 
     
     
         21 . The composition of any one of  claims 1 - 20 , wherein the cell is a neuron. 
     
     
         22 . The composition of any one of  claims 1 - 20 , wherein the cell is astrocyte. 
     
     
         23 . The composition of any one of  claims 1 - 20 , wherein the cell is a progenitor cell. 
     
     
         24 . The composition of any one of  claims 1 - 20 , wherein the composition exists in the form of nanoparticles having a diameter of from about 15 nm to about 300 nm. 
     
     
         25 . The composition of any one of  claims 1 - 20  wherein the composition exists in the form of nanoparticles having a diameter of from about 80 nm to 150 nm. 
     
     
         26 . A method for introducing a nucleic acid into a cell, while maintaining activity of the nucleic acid and viability of the cell, comprising contacting the cell with the composition of any one of  claims 18 - 20 . 
     
     
         27 . A method for modulating the expression of a target polynucleotide or polypeptide in a cell, while maintaining cell viability, comprising contacting a cell with the composition of any one of  claims 18 - 20 , wherein the nucleic acid is capable of modulating the expression of a target polynucleotide or polypeptide. 
     
     
         28 . The method of  claim 26  or  27 , wherein the cell is a neuron. 
     
     
         29 . The method of  claim 26  or  27 , wherein the cell is astrocyte. 
     
     
         30 . The method of  claim 26  or  27 , wherein the cell is a progenitor cell. 
     
     
         31 . The method of  claims 26 - 30  wherein the cell is a mammalian cell. 
     
     
         32 . A method of manufacturing a lipid nanoparticle capable of targeting neurons, the method including increasing the ratio of cationic lipid to structural lipid in the lipid nanoparticle. 
     
     
         33 . A method of manufacturing a lipid nanoparticle capable of targeting astrocytes, the method including decreasing the ratio of cationic lipid to structural lipid in the lipid nanoparticle.