Cns delivery of mrna and uses thereof

Abstract

The present invention provides, among other things, methods and compositions for effective delivery of messenger RNA (mRNA) to the central nervous system (CNS). In particular, the present invention provides methods and compositions for administering intrathecally to a subject in need of delivery a composition comprising an mRNA encoding a protein, encapsulated within a liposome, such that the administering of the composition results in the intracellular delivery of mRNA in neurons in the brain and/or spinal cord. The present invention is particularly useful for the treatment of CNS diseases, disorders or conditions, such as spinal muscular atrophy.

1 . A method of delivery of messenger RNA (mRNA) to the central nervous system (CNS), comprising
 administering intrathecally to a subject in need of delivery a composition comprising an mRNA encoding a protein, encapsulated within a liposome such that the administering of the composition results in the intracellular delivery of mRNA in neurons in the brain and/or spinal cord;   wherein the liposome comprises cationic or non-cationic lipid, cholesterol-based lipid and PEG-modified lipid.   
     
     
         2 - 4 . (canceled) 
     
     
         5 . The method of  claim 1 , wherein the liposome comprises one or more cationic lipids, one or more neutral lipids, one or more cholesterol-based lipids and one or more PEG-modified lipids. 
     
     
         6 . The method of  claim 5 , wherein the one or more cationic lipids are selected from the group consisting of C12-200, MC3, DLinDMA, DLinkC2DMA, cKK-E12, ICE (Imidazol-based), HGT5000, HGT5001, DODAC, DDAB, DMRIE, DOSPA, DOGS, DODAP, DODMA and DMDMA, DODAC, DLenDMA, DMRIE, CLinDMA, CpLinDMA, DMOBA, DOcarbDAP, DLinDAP, DLincarbDAP, DLinCDAP, KLin-K-DMA, DLin-K-XTC2-DMA, HGT4003, and combination thereof. 
     
     
         7 . The method of  claim 6 , wherein the one or more cationic lipids comprise C12-200. 
     
     
         8 . The method of  claim 6 , wherein the one or more cationic lipids comprise DLinkC2DMA. 
     
     
         9 . The method of  claim 6 , wherein the cationic lipid is cKK-E12: 
       
         
           
           
               
               
           
         
       
     
     
         10 - 12 . (canceled) 
     
     
         13 . The method of  claim 5 , wherein the one or more PEG-modified lipids constitute about 1-10% by molar ratio of the total lipid composition. 
     
     
         14 - 15 . (canceled) 
     
     
         16 . The method  claim 1 , wherein the liposome comprises a combination selected from
 C12-200, sphingomyelin, DOPE, Cholesterol, and DMG PEG;   C12-200, DOPE, cholesterol and DMG-PEG2K;   cKK-E12, DOPE, cholesterol and DMG-PEG2K;   cKK-E12, sphingomyelin, DOPE, cholesterol and DMG-PEG2K;   HGT5001, DOPE, cholesterol and DMG-PEG2K;   HGT4003, DOPE, cholesterol and DMG-PEG2K;   DLinKC2DMA, DOPE, cholesterol and DMG-PEG2K;   ICE, DOPE, cholesterol and DMG-PEG2K;   DODMA, DOPE, cholesterol and DMG-PEG2K; or   DODMA, sphingomyelin, DOPE, cholesterol and DMG-PEG2K.   
     
     
         17 . The method of  claim 1 , wherein the liposome has a size ranging from about 40-100 nm. 
     
     
         18 . The method of  claim 1 , wherein the mRNA has a length of or greater than about 0.5 kb, 1 kb, 1.5 kb, 2 kb, 2.5 kb, 3 kb, 3.5 kb, 4 kb, 4.5 kb, or 5 kb. 
     
     
         19 . The method of  claim 1 , wherein the protein encoded by the mRNA normally functions in the neurons in the brain and/or spinal cord. 
     
     
         20 . (canceled) 
     
     
         21 . The method of  claim 1 , wherein the protein encoded by the mRNA is the Survival of Motor Neuron (SMN) protein. 
     
     
         22 . The method of  claim 1 , wherein the mRNA is codon optimized. 
     
     
         23 . The method of  claim 22 , wherein the codon-optimized mRNA comprises SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:10 or SEQ ID NO:11. 
     
     
         24 - 29 . (canceled) 
     
     
         30 . The method of  claim 1 , wherein the protein encoded by the mRNA is an enzyme. 
     
     
         31 . (canceled) 
     
     
         32 . The method of  claim 1 , wherein the intracellular delivery of mRNA results in intracellular expression of the protein encoded by the mRNA within the cytosol of the neurons. 
     
     
         33 . The method of  claim 1 , wherein the intracellular delivery of mRNA results in expression of the protein encoded by the mRNA and secretion extracellularly from the neurons after expression. 
     
     
         34 . The method of  claim 1 , wherein the mRNA comprises one or more modified nucleotides. 
     
     
         35 . (canceled) 
     
     
         36 . The method of  claim 1 , wherein the mRNA is unmodified. 
     
     
         37 . The method of  claim 1 , wherein the mRNA is delivered at an amount ranging from about 0.01 mg/kg to about 10 mg/kg body weight. 
     
     
         38 - 39 . (canceled) 
     
     
         40 . A method of treating a disease, disorder or condition associated with deficiency of a protein in the central nervous system (CNS), comprising delivering a messenger RNA (mRNA) encoding the protein that is deficient to the CNS using a method according to  claim 1 . 
     
     
         41 . A method of treating spinal muscular atrophy, comprising
 administering intrathecally to a subject in need of treatment a composition comprising an mRNA encoding the Survival of Motor Neuron (SMN) protein, encapsulated within a liposome such that the administering of the composition results in the intracellular delivery of mRNA in neurons in the brain and/or spinal cord;   wherein the liposome comprises cationic or non-cationic lipid, cholesterol-based lipid and PEG-modified lipid.   
     
     
         42 - 43 . (canceled) 
     
     
         44 . The method of  claim 41 , wherein the codon-optimized mRNA comprises SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:10 or SEQ ID NO:11. 
     
     
         45 - 48 . (canceled) 
     
     
         49 . The method of  claim 41 , wherein the liposome comprises a combination selected from
 C12-200, sphingomyelin, DOPE, Cholesterol, and DMG PEG;   C12-200, DOPE, cholesterol and DMG-PEG2K;   cKK-E12, DOPE, cholesterol and DMG-PEG2K;   cKK-E12, sphingomyelin, DOPE, cholesterol and DMG-PEG2K;   HGT5001, DOPE, cholesterol and DMG-PEG2K;   HGT4003, DOPE, cholesterol and DMG-PEG2K;   DLinKC2DMA, DOPE, cholesterol and DMG-PEG2K;   ICE, DOPE, cholesterol and DMG-PEG2K;   DODMA, DOPE, cholesterol and DMG-PEG2K; or   DODMA, sphingomyelin, DOPE, cholesterol and DMG-PEG2K.   
     
     
         50 . A composition for treating spinal muscular atrophy, comprising
 an mRNA encoding the Survival of Motor Neuron (SMN) protein, encapsulated within a liposome; wherein the codon optimized mRNA comprises SEQ ID NO:3, SEQ ID NO: 4, SEQ ID NO: 10 or SEQ ID NO:11, and   further wherein the liposome comprises cationic or non-cationic lipid, cholesterol-based lipid and PEG-modified lipid.   
     
     
         51 . A composition for treating spinal muscular atrophy, comprising
 an mRNA encoding the Survival of Motor Neuron (SMN) protein, encapsulated within a liposome, wherein the liposome comprises a cationic lipid of formula I-c1-a:   
       
         
           
           
               
               
           
         
         or a pharmaceutically acceptable salt thereof, wherein:
 each R 2  independently is hydrogen or C 1-3  alkyl; 
 each q independently is 2 to 6; 
 each R′ independently is hydrogen or C 1-3  alkyl; 
 and each R 1  independently is C 8-12  alkyl. 
 
       
     
     
         52 . The composition of  claim 51 , wherein the cationic lipid is cKK-E12: 
       
         
           
           
               
               
           
         
       
     
     
         53 . A composition for treating spinal muscular atrophy, comprising
 an mRNA encoding the Survival of Motor Neuron (SMN) protein, encapsulated within a liposome; wherein the liposome comprises a combination selected from   C12-200, sphingomyelin, DOPE, Cholesterol, and DMG PEG;   C12-200, DOPE, cholesterol and DMG-PEG2K;   cKK-E12, DOPE, cholesterol and DMG-PEG2K;   cKK-E12, sphingomyelin, DOPE, cholesterol and DMG-PEG2K;   HGT5001, DOPE, cholesterol and DMG-PEG2K;   HGT4003, DOPE, cholesterol and DMG-PEG2K;   DLinKC2DMA, DOPE, cholesterol and DMG-PEG2K;   ICE, DOPE, cholesterol and DMG-PEG2K;   DODMA, DOPE, cholesterol and DMG-PEG2K; or   DODMA, sphingomyelin, DOPE, cholesterol and DMG-PEG2K.