COMPOSITIONS COMPRISING SYNTHETIC POLYNUCLEOTIDES ENCODING CRISPR RELATED PROTEINS AND SYNTHETIC SGRNAs AND METHODS OF USE

Abstract

The present invention relates to compositions comprising and methods of using synthetic polynucleotides, e.g., modified mRNA, encoding CRISPR related proteins including dCAS9 and synthetic sgRNAs targeting a gene of interest.

1 - 92 . (canceled) 
     
     
         93 . An mRNA comprising:
 (i) a 5′-cap structure;   (ii) a 5′-UTR;   (iii) an open reading frame encoding a CRISPR-associated (CAS) polypeptide, wherein the open reading frame consists of nucleotides selected from the group consisting of 1-methylpseudouridine, cytidine, adenosine, and guanosine; and   (iv) a 3′-UTR.   
     
     
         94 . The mRNA of  claim 93 , wherein the CAS polypeptide comprises Cas9, Csy4, or dCas9. 
     
     
         95 . The mRNA of  claim 94 , wherein the CAS polypeptide comprises dCas9. 
     
     
         96 . The mRNA of  claim 93 , wherein the CAS polypeptide comprises a sequence having at least 80% identity to SEQ ID NO: 7, 8, 9, 61, 62, 63, 64, or 65. 
     
     
         97 . The mRNA of  claim 93 , wherein the open reading frame further encodes an effector domain. 
     
     
         98 . The mRNA of  claim 93 , wherein the mRNA further comprises a poly-A tail. 
     
     
         99 . A composition comprising a plurality of lipid nanoparticles that encapsulate the mRNA of  claim 93 . 
     
     
         100 . The composition of  claim 99 , wherein the lipid nanoparticles comprise a cationic lipid, a PEGylated lipid, a non-cationic lipid, and a sterol. 
     
     
         101 . The composition of  claim 99 , wherein the composition further comprises an sgRNA. 
     
     
         102 . The composition of  claim 101 , wherein the sgRNA comprises a chemically modified nucleotide. 
     
     
         103 . The composition of  claim 102 , wherein the sgRNA comprises 1-methylpseudouridine. 
     
     
         104 . The composition of  claim 101 , wherein the sgRNA comprises:
 (i) a first region of 20-25 linked nucleosides complementary to either strand of a 5′-UTR of the gene of interest; and   (ii) a second region located at the 3′-terminus of the first region comprising a guide RNA scaffold sequence.   
     
     
         105 . The composition of  claim 99 , further comprising a pharmaceutically acceptable excipient. 
     
     
         106 . A method of producing a CAS polypeptide in a subject, comprising administering the mRNA of  claim 93  to the subject. 
     
     
         107 . The method of  claim 106 , wherein the mRNA is administered to the subject at a total daily dose of between 1 pg and 1 mg. 
     
     
         108 . The method of  claim 106 , wherein the mRNA is administered to the subject in a single dose. 
     
     
         109 . The method of  claim 106 , wherein the mRNA is administered to the subject in more than a single dose. 
     
     
         110 . The method of  claim 106 , wherein the mRNA is administered to the subject orally, by injection, by ophthalmic administration, or by intranasal administration. 
     
     
         111 . The method of  claim 110 , wherein the mRNA is administered to the subject by an injection selected from the group consisting of intravenous, intraarterial, intraperitoneal, intradermal, subcutaneous, and intramuscular injection. 
     
     
         112 . A method of modulating transcription of a gene of interest in a cell, the method comprising contacting the cell with the mRNA of  claim 93  under conditions sufficient to modulate transcription of the gene of interest.