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 a gene-edited cell, the gene-edited cell comprising:
 (a) a target DNA sequence; and   (b) a plurality of synthetic RNA molecules, wherein the synthetic RNA molecules include:
 i. a first synthetic RNA molecule encoding a first fusion protein comprising a DNA-binding domain and a catalytic domain of a nuclease; and 
 ii. a second synthetic RNA molecule encoding a second fusion protein comprising a DNA-binding domain and a catalytic domain of a nuclease; 
 wherein:
 the first fusion protein and the second fusion protein are independently a transcription activator-like effector nuclease (TALEN); and 
 the cell expresses the first fusion protein and the second fusion protein, which cause a double-strand break in the target DNA sequence. 
 
   
     
     
         63 . The composition of  claim 62 , wherein the first synthetic RNA molecule and the second synthetic RNA molecule independently encode a plurality of monomer repeats, wherein each monomer repeat comprises a repeat variable domain (RVD), and wherein the RVDs are selected to target a sequence within the target DNA sequence. 
     
     
         64 . The composition of  claim 62 , wherein the first synthetic RNA molecule, the second synthetic RNA molecule, or both the first synthetic RNA molecule and the second synthetic RNA molecule comprise at least one member of the group consisting of: a 5-methyluridine residue, a pseudouridine residue, a 5-methylpseudouridine residue, a 5-hydroxyuridine residue, a 5-hydroxypseudouridine residue, and a 5-methylcytidine residue. 
     
     
         65 . The composition of  claim 64 , wherein the first synthetic RNA molecule, the second synthetic RNA molecule, or both the first synthetic RNA molecule and the second synthetic RNA molecule comprise uridine residues, and wherein between 20% and 100% of the uridine residues are 5-hydroxyuridine residues. 
     
     
         66 . The composition of  claim 62 , wherein the first synthetic RNA molecule, the second synthetic RNA molecule, or both the first synthetic RNA molecule and the second synthetic RNA molecule further comprise one or more of a 5′-cap, a 5′-cap 1 structure, and a 3′-poly(A) tail. 
     
     
         67 . A composition comprising a gene-edited cell comprising an inserted DNA sequence, the gene-edited cell comprising an inserted DNA sequence comprising:
 (a) a target DNA sequence;   (b) a plurality of synthetic RNA molecules, wherein the synthetic RNA molecules include:
 i. a first synthetic RNA molecule encoding a first fusion protein comprising a DNA-binding domain and a catalytic domain of a nuclease; and 
 ii. a second synthetic RNA molecule encoding a second fusion protein comprising a DNA-binding domain and a catalytic domain of a nuclease; 
 wherein:
 the first fusion protein and the second fusion protein are independently a transcription activator-like effector nuclease (TALEN); and 
 the cell expresses the first fusion protein and the second fusion protein, which cause a double-strand break in the target DNA sequence; and 
 
   (c) a DNA repair template comprising a sequence for insertion and one or more regions of homology to the DNA of the cell, wherein the one or more regions of homology comprise regions upstream and/or downstream of the double-strand break and result in insertion of the sequence in the region of the double-strand break.   
     
     
         68 . The composition of  claim 67 , wherein the first synthetic RNA molecule and the second synthetic RNA molecule independently encode a plurality of monomer repeats, wherein each monomer repeat comprises a repeat variable domain (RVD), and wherein the RVDs are selected to target a sequence within the target DNA sequence. 
     
     
         69 . The composition of  claim 67 , wherein the first synthetic RNA molecule, the second synthetic RNA molecule, or both the first synthetic RNA molecule and the second synthetic RNA molecule comprise at least one member of the group consisting of: a 5-methyluridine residue, a pseudouridine residue, a 5-methylpseudouridine residue, a 5-hydroxyuridine residue, a 5-hydroxypseudouridine residue, and a 5-methylcytidine residue. 
     
     
         70 . The composition of  claim 69 , wherein the first synthetic RNA molecule, the second synthetic RNA molecule, or both the first synthetic RNA molecule and the second synthetic RNA molecule comprise uridine residues, and wherein between 20% and 100% of the uridine residues are 5-hydroxyuridine residues. 
     
     
         71 . The composition of  claim 67 , wherein the first synthetic RNA molecule, the second synthetic RNA molecule, or both the first synthetic RNA molecule and the second synthetic RNA molecule further comprise one or more of a 5′-cap, a 5′-cap 1 structure, and a 3′-poly(A) tail.