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. An in vivo method for treating epidermolysis bullosa, comprising
delivering a synthetic RNA encoding a gene-editing protein that targets a COL7 gene to a patient in need thereof and
inducing a single-strand or double-strand break in the COL7 gene of the patient's keratinocytes,
thereby eliminating a mutation that is at least partially responsible for a disease phenotype, wherein:
the synthetic RNA is delivered to the patient's keratinocytes by injection to the epidermis and
the gene-editing protein comprises a DNA-binding domain and a nuclease domain.
2. The method of claim 1 , wherein the gene-editing protein is capable of targeting a nucleic acid sequence that encodes the amino acid sequence of SEQ ID NO: 78.
3. The method of claim 1 , 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.
4. The method of claim 1 , wherein the synthetic RNA further comprises one or both of a 5′-cap structure and a 3′-poly(A) tail.
5. The method of claim 1 , wherein the synthetic RNA further comprises one or both of a 5′-cap 1 structure and a 3′-poly(A) tail.
6. The method of claim 1 , wherein the DNA-binding domain and the nuclease domain are separated by a linker.