Methods and products for transfecting cells
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. An in vitro or ex vivo method for producing a gene-edited cell, comprising:
(a) providing 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
(b) transfecting in vitro or ex vivo a cell with the plurality of synthetic RNA molecules, the cell comprising a target DNA sequence,
wherein the transfecting results in the cell expressing the first fusion protein and the second fusion protein to result in a double-strand break in the target DNA sequence.
2. The method of claim 1 , wherein the first synthetic RNA molecule and the second synthetic RNA molecule are independently synthesized by in vitro transcription from a DNA template.
3. The method of claim 2 , wherein the DNA template encodes 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.
4. The method of claim 1 , wherein the cell is a human cell.
5. The method of claim 1 , further comprising contacting the cell with at least one of poly-L-lysine, poly-L-ornithine, RGD peptide, fibronectin, vitronectin, collagen, and laminin.
6. The method of claim 1 , wherein the transfecting is in the presence of a medium which is substantially free of immunosuppressants.
7. The method of claim 1 , 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.
8. The method of claim 7 , 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.
9. The method of claim 1 , 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.
10. An in vitro or ex vivo method for producing a gene-edited cell comprising an inserted DNA sequence, comprising:
(a) providing:
(i) a plurality of synthetic RNA molecules, wherein the synthetic RNA molecules include:
(1) a first synthetic RNA molecule encoding a first fusion protein comprising a DNA-binding domain and a catalytic domain of a nuclease; and
(2) 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
(ii) a DNA repair template comprising a sequence for insertion and one or more regions of homology to the DNA of the cell, and
(b) transfecting in vitro or ex vivo a cell with the plurality of synthetic RNA molecules and the DNA repair template, the cell comprising a target DNA sequence, wherein:
the transfecting results in the cell expressing the first fusion protein and the second fusion protein to result in a double-strand break in the target DNA sequence, and insertion of the sequence in the region of the double-strand break.
11. The method of claim 10 , wherein the first synthetic RNA molecule and the second synthetic RNA molecule are independently synthesized by in vitro transcription from a DNA template.
12. The method of claim 11 , wherein the DNA template encodes 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.
13. The method of claim 10 , wherein the cell is a human cell.
14. The method of claim 10 , further comprising contacting the cell with at least one of poly-L-lysine, poly-L-ornithine, RGD peptide, fibronectin, vitronectin, collagen, and laminin.
15. The method of claim 10 , wherein the transfecting is in the presence of a medium which is substantially free of immunosuppressants.
16. The method of claim 10 , 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.
17. The method of claim 16 , 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.
18. The method of claim 10 , 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.
19. The method of claim 10 , wherein the DNA repair template comprises one or more regions of homology to the DNA of the cell upstream of the double-strand break.
20. The method of claim 10 , wherein the DNA repair template comprises one or more regions of homology to the DNA of the cell downstream of the double-strand break.
21. The method of claim 10 , wherein the DNA repair template comprises one or more regions of homology to the DNA of the cell upstream or downstream of the double-strand break.