METHODS AND PRODUCTS FOR NUCLEIC ACID PRODUCTION AND DELIVERY
The present invention relates in part to nucleic acids, including nucleic acids encoding proteins, therapeutics and cosmetics comprising nucleic acids, methods for delivering nucleic acids to cells, tissues, organs, and patients, methods for inducing cells to express proteins using nucleic acids, methods, kits and devices for transfecting, gene editing, and reprogramming cells, and cells, organisms, therapeutics, and cosmetics produced using these methods, kits, and devices. Methods and products for altering the DNA sequence of a cell are described, as are methods and products for inducing cells to express proteins using synthetic RNA molecules, including cells present in vivo. Therapeutics comprising nucleic acids encoding gene-editing proteins are also described.
1 .- 202 . (canceled)
203 . An ex vivo method for treating dystrophic epidermolysis bullosa comprising:
(a) providing an isolated cell of the epidermis;
(b) delivering a synthetic RNA encoding a gene-editing protein that targets a COL7 gene to the isolated cell of the epidermis and which induces a single-strand or double-strand break in the COL7 gene of the isolated cell of the epidermis, thereby eliminating a mutation that is at least partially responsible for a dystrophic epidermolysis bullosa phenotype and producing a gene-edited cell,
wherein the gene-editing protein comprises a DNA-binding domain and a nuclease domain; and
(c) administering the gene-edited cell to a dystrophic epidermolysis bullosa patient to result in the amelioration of one or more of the patient's symptoms associated with dystrophic epidermolysis bullosa.
204 . The method of claim 203 , wherein the gene-editing protein is selected from the group consisting of a nuclease, a transcription activator-like effector nuclease (TALEN), a zinc-finger nuclease, a meganuclease, a nickase, and a clustered regularly interspaced short palindromic repeat (CRISPR)-associated protein.
205 . The method of claim 203 , wherein the gene-editing protein is capable of targeting a nucleic acid sequence that encodes the amino acid sequence of SEQ ID NO: 78.
206 . The method of claim 203 , wherein the synthetic RNA further comprises one or both of a 5′-cap structure and a 3′-poly(A) tail.
207 . The method of claim 203 , wherein the synthetic RNA further comprises one or both of a 5′-cap 1 structure and a 3′-poly(A) tail.
208 . The method of claim 203 , wherein the DNA-binding domain and the nuclease domain are separated by a linker.
209 . The method of claim 203 , wherein the isolated cell of the epidermis is a keratinocyte.
210 . An ex vivo method for treating dystrophic epidermolysis bullosa comprising:
(a) providing an isolated cell of the epidermis;
(b) delivering a synthetic RNA encoding a gene-editing protein that targets a COL7 gene to the isolated cell of the epidermis and delivering a COL7 repair template to the isolated cell of the epidermis, thereby editing the COL7 gene and producing a gene-edited cell,
wherein the gene-editing protein comprises a DNA-binding domain and a nuclease domain and causes a single-strand or double-strand break in the COL7 gene of the isolated cell of the epidermis; and
(c) administering the gene-edited cell to a dystrophic epidermolysis bullosa patient to result in the amelioration of one or more of the patient's symptoms associated with dystrophic epidermolysis bullosa.
211 . The method of claim 210 , wherein the COL7 repair template is a single-stranded DNA molecule or a double-stranded DNA molecule.
212 . The method of claim 210 , wherein the COL7 repair template does not contain a binding site of the gene-editing protein.
213 . The method of claim 210 , wherein the gene-editing protein is selected from the group consisting of a nuclease, a transcription activator-like effector nuclease (TALEN), a zinc-finger nuclease, a meganuclease, a nickase, and a clustered regularly interspaced short palindromic repeat (CRISPR)-associated protein.
214 . The method of claim 210 , wherein the gene-editing protein targets a nucleic acid sequence that encodes the amino acid sequence of SEQ ID NO: 78.
215 . The method of claim 210 , wherein the synthetic RNA further comprises one or both of a 5′-cap structure and a 3′-poly(A) tail.
216 . The method of claim 210 , wherein the synthetic RNA further comprises one or both of a 5′-cap 1 structure and a 3′-poly(A) tail.
217 . The method of claim 210 , wherein the DNA-binding domain and the nuclease domain are separated by a linker.
218 . The method of claim 210 , wherein the isolated cell of the epidermis is a keratinocyte.
219 . A method for treating dystrophic epidermolysis bullosa comprising:
(a) providing a non-pluripotent cell;
(b) delivering a first synthetic RNA encoding a gene-editing protein that targets a COL7 gene to the non-pluripotent cell and delivering a second synthetic RNA encoding a reprogramming factor to the non-pluripotent cell, thereby producing a gene-edited and reprogrammed cell;
wherein:
(i) the gene-editing protein comprises a DNA-binding domain and a nuclease domain and causes a single-strand or double-strand break in the COL7 gene of the cell; and
(ii) the cell expresses the reprogramming factor to result in the cell being reprogrammed; and
(c) administering the gene-edited and reprogrammed cell to a dystrophic epidermolysis bullosa patient to result in the amelioration of one or more of the patient's symptoms associated with dystrophic epidermolysis bullosa.
220 . The method of claim 219 , wherein the first synthetic RNA is delivered to the cell before the second synthetic RNA is delivered to the cell.
221 . The method of claim 220 , wherein the non-pluripotent cell is a skin cell.