Methods and products for transfecting cells

Abstract

The present invention relates in part to nucleic acids encoding proteins, nucleic acids containing non-canonical nucleotides, therapeutics comprising nucleic acids, methods, kits, and devices for inducing cells to express proteins, methods, kits, and devices for transfecting, gene editing, and reprogramming cells, and cells, organisms, and therapeutics produced using these methods, kits, and devices. Methods for inducing cells to express proteins and for reprogramming and gene-editing cells using RNA are disclosed. Methods for producing cells from patient samples, cells produced using these methods, and therapeutics comprising cells produced using these methods are also disclosed.

1 - 61 . (canceled) 
     
     
         62 . A composition comprising an in vitro transcribed synthetic RNA molecule comprising at least one non-canonical nucleotide having a substitution at the pyrimidine 5C position and encoding a gene-editing protein for translation in a mammalian cell, wherein:
 the non-canonical nucleotide having a substitution at the pyrimidine 5C position is selected from the group consisting of 5-methyluridine, 5-hydroxyuridine, pseudouridine, 5-methylpseudouridine, 5-hydroxypseudouridine, 5-methylcytidine, and 5-hydroxycytidine and   the gene-editing protein is selected from the group consisting of a nuclease, a zinc-finger nuclease, a meganuclease, a nickase, and a transcription activator-like effector nucleases (TALEN).   
     
     
         63 . The composition of  claim 62 , wherein the synthetic RNA molecule comprises at least two non-canonical nucleotides having a substitution at the pyrimidine 5C position wherein,
 the first non-canonical nucleotide is at least one of 5-methyluridine, 5-hydroxyuridine, pseudouridine, 5-methylpseudouridine, and 5-hydroxypseudouridine, and   the second non-canonical nucleotide is at least one of 5-methylcytidine and 5-hydroxycytidine.   
     
     
         64 . The composition of  claim 62 , wherein the synthetic RNA comprises at least one of pseudouridine, 5-methylpseudouridine, 5-hydroxypseudouridine, and 5-hydroxyuridine at about 50%-100% of the uridine residues. 
     
     
         65 . The composition of  claim 62 , wherein the synthetic RNA comprises 5-methyluridine at about 20%-60% of the uridine residues. 
     
     
         66 . The composition of  claim 62 , wherein the synthetic RNA comprises at least one of 5-methylcytidine and 5-hydroxycytidine at about 50%-100% of the cytidine residues. 
     
     
         67 . The composition of  claim 62 , wherein the synthetic RNA comprises at least three non-canonical nucleotides having a substitution at the pyrimidine 5C position. 
     
     
         68 . The composition of  claim 62 , wherein the synthetic RNA improves translation of the gene-editing protein in a mammalian cell relative to a synthetic RNA molecule containing only canonical nucleotides. 
     
     
         69 . The composition of  claim 62 , wherein the synthetic RNA does not increase toxicity in the mammalian cell relative to a synthetic RNA molecule containing only canonical nucleotides. 
     
     
         70 . The composition of  claim 62 , wherein the synthetic RNA molecule further comprises one or more of a 5′-cap, a 5′-cap 1 structure, and a 3′-poly(A) tail. 
     
     
         71 . The composition of  claim 62 , wherein:
 the non-canonical nucleotide having a substitution at the pyrimidine 5C position is both of pseudouridine and 5-methylcytidine; and   the gene-editing protein is a TALEN.