# Patent

## Title
Methods and products for expressing proteins in cells

## Abstract

The present invention relates in part to nucleic acids encoding proteins, therapeutics comprising nucleic acids encoding proteins, methods for inducing cells to express proteins using nucleic acids, methods, kits and devices for transfecting, gene editing, and reprogramming cells, and cells, organisms, and therapeutics 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. Therapeutics comprising nucleic acids encoding gene-editing proteins are also described.

## Background
PRIORITY
The present application is a continuation of U.S. application Ser. No. 15/270,469, filed Sep. 20, 2016 (now U.S. Pat. No. 9,657,282), which is a continuation of U.S. application Ser. No. 15/156,829, filed May 17, 2016 (now U.S. Pat. No. 9,487,768), which is a continuation of U.S. application Ser. No. 14/735,603, filed Jun. 10, 2015 (now U.S. Pat. No. 9,376,669), which is a continuation of U.S. application Ser. No. 14/701,199, filed Apr. 30, 2015 (now U.S. Pat. No. 9,447,395), which is a continuation of International Application No. PCT/US2013/068118, filed Nov. 1, 2013, which claims priority to U.S. Provisional Application No. 61/721,302, filed on Nov. 1, 2012, U.S. Provisional Application No. 61/785,404, filed on Mar. 14, 2013, and U.S. Provisional Application No. 61/842,874, filed on Jul. 3, 2013, the contents of which are herein incorporated by reference in their entireties. The present application is related to U.S. application Ser. No. 13/465,490, filed on May 7, 2012, International Application No. PCT/US2012/067966, filed on Dec. 5, 2012, and U.S. application Ser. No. 13/931,251, filed on Jun. 28, 2013, the contents of which are herein incorporated by reference in their entireties.


FIELD OF THE INVENTION
The present invention relates in part to nucleic acids encoding proteins, therapeutics comprising nucleic acids encoding proteins, methods for inducing cells to express proteins using nucleic acids, methods, kits and devices for transfecting, gene editing, and reprogramming cells, and cells, organisms, and therapeutics produced using these methods, kits, and devices.
DESCRIPTION OF THE TEXT FILE SUBMITTED ELECTRONICALLY
The contents of the text file submitted electronically herewith are incorporated herein by reference in their entirety: A computer readable format copy of the Sequence Listing (filename: FAB-005C3SL.txt; date recorded: May 16, 2016; file size: 410 KB).
BACKGROUND
Synthetic RNA and RNA Therapeutics
Ribonucleic acid (RNA) is ubiquitous in both prokaryotic and eukaryotic cells, where it encodes genetic information in the form of messenger RNA, binds and transports amino acids in the form of transfer RNA, assembles amino acids into proteins in the form of ribosomal RNA, and performs numerous other functions including gene expression regulation in the forms of microRNA and long non-coding RNA. RNA can be produced synthetically by methods including direct chemical synthesis and in vitro transcription, and can be administered to patients for therapeutic use.
Cell Reprogramming and Cell-Based Therapies
Cells can be reprogrammed by exposing them to specific extracellular cues and/or by ectopic expression of specific proteins, microRNAs, etc. While several reprogramming methods have been previously described, most that rely on ectopic expression require the introduction of exogenous DNA, which can carry mutation risks. DNA-free reprogramming methods based on direct delivery of reprogramming proteins have been reported. However, these methods are too inefficient and unreliable for commercial use. In addition, RNA-based reprogramming methods have been described (See, e.g., Angel. MIT Thesis. 2008. 1-56; Angel et al. PLoS ONE. 2010. 5, 107; Warren et al. Cell Stem Cell. 2010. 7, 618-630; Angel. MIT Thesis. 2011. 1-89; and Lee et al. Cell. 2012. 151, 547-558; the contents of all of which are hereby incorporated by reference). However, existing RNA-based reprogramming methods are slow, unreliable, and inefficient when performed on adult cells, require many transfections (resulting in significant expense and opportunity for error), can reprogram only a limited number of cell types, can reprogram cells to only a limited number of cell types, require the use of immunosuppressants, and require the use of multiple human-derived components, including blood-derived HSA and human fibroblast feeders. The many drawbacks of previously disclosed RNA-based reprogramming methods make them undesirable for both research and therapeutic use.
Gene Editing
Several naturally occurring proteins contain DNA-binding domains that can recognize specific DNA sequences, for example, zinc fingers (ZFs) and transcription activator-like effectors (TALEs). Fusion proteins containing one or more of these DNA-binding domains and the cleavage domain of FokI endonuclease can be used to create a double-strand break in a desired region of DNA in a cell (See, e.g., US Patent Appl. Pub. No. US 2012/0064620, US Patent Appl. Pub. No. US 2011/0239315, U.S. Pat. No. 8,470,973, US Patent Appl. Pub. No. US 2013/0217119, U.S. Pat. No. 8,420,782, US Patent Appl. Pub. No. US 2011/0301073, US Patent Appl. Pub. No. US 2011/0145940, U.S. Pat. No. 8,450,471, U.S. Pat. No. 8,440,431, U.S. Pat. No. 8,440,432, and US Patent Appl. Pub. No. 2013/0122581, the contents of all of which are hereby incorporated by reference). However, current methods for gene editing cells are inefficient and carry a risk of uncontrolled mutagenesis, making them undesirable for both research and therapeutic use. Methods for DNA-free gene editing of somatic cells have not been previously explored, nor have methods for simultaneous or sequential gene editing and reprogramming of somatic cells. In addition, methods for directly gene editing cells in patients (i.e., in vivo) have not been previously explored, and the development of such methods has been limited by a lack of acceptable targets, inefficient delivery, inefficient expression of the gene-editing protein/proteins, inefficient gene editing by the expressed gene-editing protein/proteins, due in part to poor binding of DNA-binding domains, excessive off-target effects, due in part to non-directed dimerization of the FokI cleavage domain and poor specificity of DNA-binding domains, and other factors. Finally, the use of gene editing in anti-bacterial, anti-viral, and anti-cancer treatments has not been previously explored.
Accordingly, there remains a need for improved compositions and methods for the expression of proteins in cells.
SUMMARY OF THE INVENTION
The present invention provides, in part, compositions, methods, articles, and devices for inducing cells to express proteins, methods, articles, and devices for producing these compositions, methods, articles, and devices, and compositions and articles, including cells, organisms, and therapeutics, produced using these compositions, methods, articles, and devices. Unlike previously reported methods, certain embodiments of the present invention do not involve exposing cells to exogenous DNA or to allogeneic or animal-derived materials, making products produced according to the methods of the present invention useful for therapeutic applications.
In some aspects, synthetic RNA molecules with low toxicity and high translation efficiency are provided. In one aspect, a cell-culture medium for high-efficiency transfection, reprogramming, and gene editing of cells is provided. Other aspects pertain to methods for producing synthetic RNA molecules encoding reprogramming proteins. Still further aspects pertain to methods for producing synthetic RNA molecules encoding gene-editing proteins.
In one aspect, the invention provides high-efficiency gene-editing proteins comprising engineered nuclease cleavage domains. In another aspect, the invention provides high-fidelity gene-editing proteins comprising engineered nuclease cleavage domains. Other aspects relate to high-efficiency gene-editing proteins comprising engineered DNA-binding domains. Still further aspects pertain to high-fidelity gene-editing proteins comprising engineered DNA-binding domains. Still further aspects relate to gene-editing proteins comprising engineered repeat sequences. Some aspects relate to methods for altering the DNA sequence of a cell by transfecting the cell with or inducing the cell to express a gene-editing protein. Other aspects relate to methods for altering the DNA sequence of a cell that is present in an in vitro culture. Still further aspects relate to methods for altering the DNA sequence of a cell that is present in vivo.
In some aspects, the invention provides methods for treating cancer comprising administering to a patient a therapeutically effective amount of a gene-editing protein or a nucleic-acid encoding a gene-editing protein. In one aspect, the gene-editing protein is capable of altering the DNA sequence of a cancer associated gene. In another aspect, the cancer-associated gene is the BIRC5 gene. Still other aspects relate to therapeutics comprising nucleic acids and/or cells and methods of using therapeutics comprising nucleic acids and/or cells for the treatment of, for example, type 1 diabetes, heart disease, including ischemic and dilated cardiomyopathy, macular degeneration, Parkinson's disease, cystic fibrosis, sickle-cell anemia, thalassemia, Fanconi anemia, severe combined immunodeficiency, hereditary sensory neuropathy, xeroderma pigmentosum, Huntington's disease, muscular dystrophy, amyotrophic lateral sclerosis, Alzheimer's disease, cancer, and infectious diseases including hepatitis and HIV/AIDS. In some aspects, the nucleic acids comprise synthetic RNA. In other aspects, the nucleic acids are delivered to cells using a virus. In some aspects, the virus is a replication-competent virus. In other aspects, the virus is a replication-incompetent virus.
The details of the invention are set forth in the accompanying description below. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, illustrative methods and materials are now described. Other features, objects, and advantages of the invention will be apparent from the description and from the claims. In the specification and the appended claims, the singular forms also include the plural unless the context clearly dictates otherwise. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.



DETAILED DESCRIPTION OF THE FIGURES
FIG. 1A depicts RNA encoding the indicated proteins and containing adenosine, 50% guanosine, 50% 7-deazaguanosine, 70% uridine, 30% 5-methyluridine, and 5-methylcytidine, resolved on a denaturing formaldehyde-agarose gel.
FIG. 1B depicts RNA encoding the indicated proteins and containing adenosine, 50% guanosine, 50% 7-deazaguanosine, 50% uridine, 50% 5-methyluridine, and 5-methylcytidine, resolved on a denaturing formaldehyde-agarose gel.
FIG. 2 depicts primary human neonatal fibroblasts reprogrammed by five transfections with RNA encoding reprogramming proteins. Cells were fixed and stained for Oct4 protein. Nuclei were counterstained with Hoechst 33342.
FIG. 3A depicts primary human adult fibroblasts.
FIG. 3B depicts the primary human adult fibroblasts shown in FIG. 3A, reprogrammed by seven transfections with RNA encoding reprogramming proteins. Arrows indicate colonies of reprogrammed cells.
FIG. 3C depicts a large colony of reprogrammed primary human adult fibroblasts.
FIG. 4A depicts the location of a TALEN pair targeting the human CCR5 gene (SEQ ID NO: 649 and 650). Single-lines indicate the TALEN binding sites. Double-lines indicate the location of the Δ32 mutation.
FIG. 4B depicts synthetic RNA encoding the TALEN pair of FIG. 4A, resolved on a denaturing formaldehyde-agarose gel.
FIG. 4C depicts the results of a SURVEYOR assay testing the functionality of the RNA of FIG. 4B on human dermal fibroblasts (GM00609). The appearance of the 760 bp and 200 bp bands in the sample generated from cells transfected with RNA indicates successful gene editing. The percentage below each lane indicates the efficiency of gene editing (percentage of edited alleles).
FIG. 4D depicts a line-profile graph of the “Neg” and “TALENs” lanes of FIG. 4C. Numbers indicate the integrated intensity of the three bands, relative to the total integrated intensity.
FIG. 4E depicts the results of a SURVEYOR assay performed as in FIG. 4C, and also including a sample generated from cells that were transfected twice with RNA (the lane labeled “2×”).
FIG. 4F depicts simultaneous gene editing and reprogramming of primary human cells (GM00609) using synthetic RNA. Images show representative colonies of reprogrammed cells.
FIG. 4G depicts the results of direct sequencing of the CCR5 gene in gene-edited, reprogrammed cells generated as in FIG. 4F. Four of the nine lines tested contained a deletion between the TALEN binding sites, indicating efficient gene editing (SEQ ID NOS: 651-655, 676, and 656-663).
FIG. 5 depicts the results of a SURVEYOR assay performed as in FIG. 4C, except using RNA targeting the human MYC gene, and containing either canonical nucleotides (“A,G,U,C”) or non-canonical nucleotides (“A,7dG,5mU,5mC”). The dark bands at 470 bp and 500 bp indicate high-efficiency gene editing.
FIG. 6 depicts the results of a SURVEYOR assay performed as in FIG. 4C, except using RNA targeting the human BIRC5 gene, and containing either canonical nucleotides (“A,G,U,C”) or non-canonical nucleotides (“A,7dG,5mU,5mC”). The dark band at 710 bp indicates high-efficiency gene editing.
FIG. 7A depicts HeLa cells (cervical carcinoma) transfected with RNA targeting the human BIRC5 gene (RiboSlice). Cells were transfected with either a single RNA (“2× Survivin L”) or equal amounts of each member of an RNA pair (“Survivin L+R”), with the same total amount of RNA delivered in each case. As shown in the right panel, cells transfected with the RNA pair became enlarged, and exhibited fragmented nuclei and markedly reduced proliferation, demonstrating the potent anti-cancer activity of RiboSlice.
FIG. 7B depicts HeLa cells transfected with RNA targeting the human BIRC5 gene as in FIG. 7A. Cells were subsequently fixed and stained for survivin protein. Nuclei were counterstained with Hoechst 33342. The large, fragmented nuclei of cells transfected with RiboSlice are indicated with arrows.
FIG. 8 depicts primary human adult fibroblasts reprogrammed using synthetic RNA. Arrows indicate compact colonies of cells that exhibit a morphology indicative of reprogramming.
FIG. 9 depicts synthetic RNA encoding the indicated gene-editing proteins, resolved on a denaturing formaldehyde-agarose gel.
FIG. 10A depicts the results of a SURVEYOR assay testing the effectiveness of the RNA of FIG. 9 on human dermal fibroblasts. Cells were lysed approximately 48 h after transfection. Bands corresponding to digestion products resulting from successful gene editing are indicated with asterisks. Lane labels are of the form “X.Y”, where X refers to the exon from which DNA was amplified, and Y refers to the gene-editing protein pair. For example, “1.1” refers to the gene-editing protein pair targeting the region of exon 1 closest to the start codon. “X.N” refers to untransfected cells.
FIG. 10B depicts the results of a SURVEYOR assay testing the toxicity of the RNA of FIG. 9 on human dermal fibroblasts. Cells were lysed 11 days after transfection. Lanes and bands are labeled as in FIG. 10A. The appearance of the bands indicated with asterisks demonstrates that the transfected cells retained high viability.
FIG. 11 depicts the results of a study designed to test the safety of RNA encoding gene-editing proteins in vivo. The graph shows the mean body weight of four groups of mice (10 animals in each group), including one untreated group, one vehicle-only group, one group treated with RiboSlice via intratumoral injection, and one group treated with RiboSlice via intravenous injection. For all treated groups, animals were given 5 doses, every other day, from day 1 to day 9. Animals were followed until day 17. The lack of a statistically significant difference between the mean body weights of the four groups demonstrates the in vivo safety of RiboSlice.
FIG. 12A depicts the results of a SURVEYOR assay testing the effectiveness of gene-editing proteins comprising various 36 amino-acid-long repeat sequences. Human dermal fibroblasts were lysed approximately 48 h after transfection with RNA encoding gene-editing proteins containing the indicated repeat sequence. The band corresponding to the digestion product resulting from successful gene editing is indicated with an asterisk. Lane labels refer to the amino acids at the C-terminus of the repeat sequence (SEQ ID Nos: 677-679, respectively, in order of appearance). “Neg.” refers to untransfected cells.
FIG. 12B depicts the results of a SURVEYOR assay testing the effectiveness of gene-editing proteins in which every other repeat sequence is 36 amino acids long. Human dermal fibroblasts were lysed approximately 48 h after transfection with RNA encoding gene-editing proteins containing the indicated repeat sequence. The band corresponding to the digestion product resulting from successful gene editing is indicated with an asterisk. Lane labels refer to the amino acids at the C-terminus of the repeat sequences (“AGHGG” disclosed as SEQ ID NO: 678). “Neg.” refers to untransfected cells.
FIG. 13A depicts the results of a study designed to test the safety and efficacy of RiboSlice AAV replication-incompetent virus carrying nucleic acids encoding gene-editing proteins in vivo. The graph shows the mean body weight of three groups of mice carrying subcutaneous tumors comprising human glioma cells, including one untreated group (no treatment control, “NTC”, n=6), one group treated with AAV encoding GFP (“GFP”, n=2) via intratumoral injection, and one group treated with RiboSlice AAV encoding gene-editing proteins targeting the BIRC5 gene (“RiboSlice”, n=2) via intratumoral injection. Animals were dosed on day 1 for the GFP group, and days 1 and 15 for the RiboSlice group. Animals were followed until day 25. The lack of a statistically significant difference between the mean body weights of the three groups demonstrates the in vivo safety of RiboSlice AAV.
FIG. 13B depicts the normalized tumor volumes of the animals in the study shown in FIG. 13A. The slower increase in normalized tumor volume in the group treated with RiboSlice AAV compared to both the NTC and GFP groups demonstrates the in vivo efficacy of RiboSlice AAV.
FIG. 14 depicts the results of a SURVEYOR assay testing the effectiveness of gene-editing proteins, as in FIG. 12B. “RiboSlice” refers to gene-editing proteins in which every other repeat sequence is 36 amino acids long. “w.t.” refers to untransfected cells.
FIG. 15 depicts RNA encoding the indicated proteins and containing adenosine, 50% guanosine, 50% 7-deazaguanosine, 60% uridine, 40% 5-methyluridine, and 5-methylcytidine, resolved on a denaturing formaldehyde-agarose gel.
FIG. 16 depicts the results of an assay testing the integration of a repair template into the APP gene. The appearance of the 562 bp and 385 bp bands in the sample generated from cells transfected with RNA and a repair template indicates successful integration of a PstI restriction site. “−” refers to an undigested sample, “+” refers to a sample treated with PstI restriction nuclease.



DEFINITIONS
By “molecule” is meant a molecular entity (molecule, ion, complex, etc.).
By “RNA molecule” is meant a molecule that comprises RNA.
By “synthetic RNA molecule” is meant an RNA molecule that is produced outside of a cell or that is produced inside of a cell using bioengineering, by way of non-limiting example, an RNA molecule that is produced in an in vitro-transcription reaction, an RNA molecule that is produced by direct chemical synthesis or an RNA molecule that is produced in a genetically-engineered E. coli cell.
By “transfection” is meant contacting a cell with a molecule, wherein the molecule is internalized by the cell.
By “upon transfection” is meant during or after transfection.
By “transfection reagent” is meant a substance or mixture of substances that associates with a molecule and facilitates the delivery of the molecule to and/or internalization of the molecule by a cell, by way of non-limiting example, a cationic lipid, a charged polymer or a cell-penetrating peptide.
By “reagent-based transfection” is meant transfection using a transfection reagent.
By “cell-culture medium” is meant a medium that can be used for cell culture, by way of non-limiting example, Dulbecco's Modified Eagle's Medium (DMEM) or DMEM+10% fetal bovine serum (FBS).
By “complexation medium” is meant a medium to which a transfection reagent and a molecule to be transfected are added and in which the transfection reagent associates with the molecule to be transfected.
By “transfection medium” is meant a medium that can be used for transfection, by way of non-limiting example, Dulbecco's Modified Eagle's Medium (DMEM) or DMEM/F12.
By “recombinant protein” is meant a protein or peptide that is not produced in animals or humans. Non-limiting examples include human transferrin that is produced in bacteria, human fibronectin that is produced in an in vitro culture of mouse cells, and human serum albumin that is produced in a rice plant.
By “lipid carrier” is meant a substance that can increase the solubility of a lipid or lipid-soluble molecule in an aqueous solution, by way of non-limiting example, human serum albumin or methyl-beta-cyclodextrin.
By “Oct4 protein” is meant a protein that is encoded by the POU5F1 gene, or a natural or engineered variant, family-member, orthologue, fragment or fusion construct thereof, by way of non-limiting example, human Oct4 protein (SEQ ID NO: 8), mouse Oct4 protein, Oct1 protein, a protein encoded by POU5F1 pseudogene 2, a DNA-binding domain of Oct4 protein or an Oct4-GFP fusion protein. In some embodiments the Oct4 protein comprises an amino acid sequence that has at least 70% identity with SEQ ID NO: 8, or in other embodiments, at least 75%, 80%, 85%, 90%, or 95% identity with SEQ ID NO: 8. In some embodiments, the Oct4 protein comprises an amino acid sequence having from 1 to 20 amino acid insertions, deletions, or substitutions (collectively) with respect to SEQ ID NO: 8. Or in other embodiments, the Oct4 protein comprises an amino acid sequence having from 1 to 15 or from 1 to 10 amino acid insertions, deletions, or substitutions (collectively) with respect to SEQ ID NO: 8.
By “Sox2 protein” is meant a protein that is encoded by the SOX2 gene, or a natural or engineered variant, family-member, orthologue, fragment or fusion construct thereof, by way of non-limiting example, human Sox2 protein (SEQ ID NO: 9), mouse Sox2 protein, a DNA-binding domain of Sox2 protein or a Sox2-GFP fusion protein. In some embodiments the Sox2 protein comprises an amino acid sequence that has at least 70% identity with SEQ ID NO: 9, or in other embodiments, at least 75%, 80%, 85%, 90%, or 95% identity with SEQ ID NO: 9. In some embodiments, the Sox2 protein comprises an amino acid sequence having from 1 to 20 amino acid insertions, deletions, or substitutions (collectively) with respect to SEQ ID NO: 9. Or in other embodiments, the Sox2 protein comprises an amino acid sequence having from 1 to 15 or from 1 to 10 amino acid insertions, deletions, or substitutions (collectively) with respect to SEQ ID NO: 9.
By “Klf4 protein” is meant a protein that is encoded by the KLF4 gene, or a natural or engineered variant, family-member, orthologue, fragment or fusion construct thereof, by way of non-limiting example, human Klf4 protein (SEQ ID NO: 10), mouse Klf4 protein, a DNA-binding domain of Klf4 protein or a Klf4-GFP fusion protein. In some embodiments the Klf4 protein comprises an amino acid sequence that has at least 70% identity with SEQ ID NO: 10, or in other embodiments, at least 75%, 80%, 85%, 90%, or 95% identity with SEQ ID NO: 10. In some embodiments, the Klf4 protein comprises an amino acid sequence having from 1 to 20 amino acid insertions, deletions, or substitutions (collectively) with respect to SEQ ID NO: 10. Or in other embodiments, the Klf4 protein comprises an amino acid sequence having from 1 to 15 or from 1 to 10 amino acid insertions, deletions, or substitutions (collectively) with respect to SEQ ID NO: 10.
By “c-Myc protein” is meant a protein that is encoded by the MYC gene, or a natural or engineered variant, family-member, orthologue, fragment or fusion construct thereof, by way of non-limiting example, human c-Myc protein (SEQ ID NO: 11), mouse c-Myc protein, 1-Myc protein, c-Myc (T58A) protein, a DNA-binding domain of c-Myc protein or a c-Myc-GFP fusion protein. In some embodiments the c-Myc protein comprises an amino acid sequence that has at least 70% identity with SEQ ID NO: 11, or in other embodiments, at least 75%, 80%, 85%, 90%, or 95% identity with SEQ ID NO: 11. In some embodiments, the c-Myc protein comprises an amino acid having from 1 to 20 amino acid insertions, deletions, or substitutions (collectively) with respect to SEQ ID NO: 11. Or in other embodiments, the c-Myc protein comprises an amino acid sequence having from 1 to 15 or from 1 to 10 amino acid insertions, deletions, or substitutions (collectively) with respect to SEQ ID NO: 11.
By “reprogramming” is meant causing a change in the phenotype of a cell, by way of non-limiting example, causing a β-cell progenitor to differentiate into a mature β-cell, causing a fibroblast to dedifferentiate into a pluripotent stem cell, causing a keratinocyte to transdifferentiate into a cardiac stem cell or causing the axon of a neuron to grow.
By “reprogramming factor” is meant a molecule that, when a cell is contacted with the molecule and/or the cell expresses the molecule, can, either alone or in combination with other molecules, cause reprogramming, by way of non-limiting example, Oct4 protein.
By “feeder” is meant a cell that can be used to condition medium or to otherwise support the growth of other cells in culture.
By “conditioning” is meant contacting one or more feeders with a medium.
By “fatty acid” is meant a molecule that comprises an aliphatic chain of at least two carbon atoms, by way of non-limiting example, linoleic acid, α-linolenic acid, octanoic acid, a leukotriene, a prostaglandin, cholesterol, a glucocorticoid, a resolvin, a protectin, a thromboxane, a lipoxin, a maresin, a sphingolipid, tryptophan, N-acetyl tryptophan or a salt, methyl ester or derivative thereof.
By “short-chain fatty acid” is meant a fatty acid that comprises an aliphatic chain of between two and 30 carbon atoms.
By “albumin” is meant a protein that is highly soluble in water, by way of non-limiting example, human serum albumin.
By “associated molecule” is meant a molecule that is non-covalently bound to another molecule.
By “associated-molecule-component of albumin” is meant one or more molecules that are bound to an albumin polypeptide, by way of non-limiting example, lipids, hormones, cholesterol, calcium ions, etc. that are bound to an albumin polypeptide.
By “treated albumin” is meant albumin that is treated to reduce, remove, replace or otherwise inactivate the associated-molecule-component of the albumin, by way of non-limiting example, human serum albumin that is incubated at an elevated temperature, human serum albumin that is contacted with sodium octanoate or human serum albumin that is contacted with a porous material.
By “ion-exchange resin” is meant a material that, when contacted with a solution containing ions, can replace one or more of the ions with one or more different ions, by way of non-limiting example, a material that can replace one or more calcium ions with one or more sodium ions.
By “germ cell” is meant a sperm cell or an egg cell.
By “pluripotent stem cell” is meant a cell that can differentiate into cells of all three germ layers (endoderm, mesoderm, and ectoderm) in vivo.
By “somatic cell” is meant a cell that is not a pluripotent stem cell or a germ cell, by way of non-limiting example, a skin cell.
By “glucose-responsive insulin-producing cell” is meant a cell that, when exposed to a certain concentration of glucose, can produce and/or secrete an amount of insulin that is different from (either less than or more than) the amount of insulin that the cell produces and/or secretes when the cell is exposed to a different concentration of glucose, by way of non-limiting example, a β-cell.
By “hematopoietic cell” is meant a blood cell or a cell that can differentiate into a blood cell, by way of non-limiting example, a hematopoietic stem cell or a white blood cell.
By “cardiac cell” is meant a heart cell or a cell that can differentiate into a heart cell, by way of non-limiting example, a cardiac stem cell or a cardiomyocyte.
By “retinal cell” is meant a cell of the retina or a cell that can differentiate into a cell of the retina, by way of non-limiting example, a retinal pigmented epithelial cell.
By “skin cell” is meant a cell that is normally found in the skin, by way of non-limiting example, a fibroblast, a keratinocyte, a melanocyte, an adipocyte, a mesenchymal stem cell, an adipose stem cell or a blood cell.
By “Wnt signaling agonist” is meant a molecule that can perform one or more of the biological functions of one or more members of the Wnt family of proteins, by way of non-limiting example, Wnt1, Wnt2, Wnt3, Wnt3a or 2-amino-4-[3,4-(methylenedioxy)benzylamino]-6-(3-methoxyphenyl)pyrimidine.
By “IL-6 signaling agonist” is meant a molecule that can perform one or more of the biological functions of IL-6 protein, by way of non-limiting example, IL-6 protein or IL-6 receptor (also known as soluble IL-6 receptor, IL-6R, IL-6R alpha, etc.).
By “TGF-β signaling agonist” is meant a molecule that can perform one or more of the biological functions of one or more members of the TGF-β superfamily of proteins, by way of non-limiting example, TGF-β1, TGF-β3, Activin A, BMP-4 or Nodal.
By “immunosuppressant” is meant a substance that can suppress one or more aspects of an immune system, and that is not normally present in a mammal, by way of non-limiting example, B18R or dexamethasone.
By “single-strand break” is meant a region of single-stranded or double-stranded DNA in which one or more of the covalent bonds linking the nucleotides has been broken in one of the one or two strands.
By “double-strand break” is meant a region of double-stranded DNA in which one or more of the covalent bonds linking the nucleotides has been broken in each of the two strands.
By “nucleotide” is meant a nucleotide or a fragment or derivative thereof, by way of non-limiting example, a nucleobase, a nucleoside, a nucleotide-triphosphate, etc.
By “nucleoside” is meant a nucleotide or a fragment or derivative thereof, by way of non-limiting example, a nucleobase, a nucleoside, a nucleotide-triphosphate, etc.
By “gene editing” is meant altering the DNA sequence of a cell, by way of non-limiting example, by transfecting the cell with a protein that causes a mutation in the DNA of the cell.
By “gene-editing protein” is meant a protein that can, either alone or in combination with one or more other molecules, alter the DNA sequence of a cell, by way of non-limiting example, a nuclease, a transcription activator-like effector nuclease (TALEN), a zinc-finger nuclease, a meganuclease, a nickase, a clustered regularly interspaced short palindromic repeat (CRISPR)-associated protein or a natural or engineered variant, family-member, orthologue, fragment or fusion construct thereof.
By “repair template” is meant a nucleic acid containing a region of at least about 70% homology with a sequence that is within 10 kb of a target site of a gene-editing protein.
By “repeat sequence” is meant an amino-acid sequence that is present in more than one copy in a protein, to within at least about 10% homology, by way of non-limiting example, a monomer repeat of a transcription activator-like effector.
By “DNA-binding domain” is meant a region of a molecule that is capable of binding to a DNA molecule, by way of non-limiting example, a protein domain comprising one or more zinc fingers, a protein domain comprising one or more transcription activator-like (TAL) effector repeat sequences or a binding pocket of a small molecule that is capable of binding to a DNA molecule.
By “binding site” is meant a nucleic-acid sequence that is capable of being recognized by a gene-editing protein, DNA-binding protein, DNA-binding domain or a biologically active fragment or variant thereof or a nucleic-acid sequence for which a gene-editing protein, DNA-binding protein, DNA-binding domain or a biologically active fragment or variant thereof has high affinity, by way of non-limiting example, an about 20-base-pair sequence of DNA in exon 1 of the human BIRC5 gene.
By “target” is meant a nucleic acid that contains a binding site.
Other definitions are set forth in U.S. application Ser. No. 13/465,490, U.S. Provisional Application No. 61/664,494, U.S. Provisional Application No. 61/721,302, International Application No. PCT/US12/67966, U.S. Provisional Application No. 61/785,404, and U.S. Provisional Application No. 61/842,874, the contents of which are hereby incorporated by reference in their entireties.
It has now been discovered that the non-canonical nucleotide members of the 5-methylcytidine de-methylation pathway, when incorporated into synthetic RNA, can increase the efficiency with which the synthetic RNA can be translated into protein, and can decrease the toxicity of the synthetic RNA. These non-canonical nucleotides include, for example: 5-methylcytidine, 5-hydroxymethylcytidine, 5-formylcytidine, and 5-carboxycytidine (a.k.a. “cytidine-5-carboxylic acid”). Certain embodiments are therefore directed to a nucleic acid. In one embodiment, the nucleic acid is a synthetic RNA molecule. In another embodiment, the nucleic acid comprises one or more non-canonical nucleotides. In one embodiment, the nucleic acid comprises one or more non-canonical nucleotide members of the 5-methylcytidine de-methylation pathway. In another embodiment, the nucleic acid comprises at least one of: 5-methylcytidine, 5-hydroxymethylcytidine, 5-formylcytidine, and 5-carboxycytidine or a derivative thereof. In a further embodiment, the nucleic acid comprises at least one of: pseudouridine, 5-methylpseudouridine, 5-methyluridine, 5-methylcytidine, 5-hydroxymethylcytidine, N4-methylcytidine, N4-acetylcytidine, and 7-deazaguanosine or a derivative thereof.
5-Methylcytidine De-Methylation Pathway




Certain embodiments are directed to a protein. Other embodiments are directed to a nucleic acid that encodes a protein. In one embodiment, the protein is a protein of interest. In another embodiment, the protein is selected from: a reprogramming protein and a gene-editing protein. In one embodiment, the nucleic acid is a plasmid. In another embodiment, the nucleic acid is present in a virus or viral vector. In a further embodiment, the virus or viral vector is replication incompetent. In a still further embodiment, the virus or viral vector is replication competent. In one embodiment, the virus or viral vector includes at least one of: an adenovirus, a retrovirus, a lentivirus, a herpes virus, an adeno-associated virus or a natural or engineered variant thereof, and an engineered virus.
It has also been discovered that certain combinations of non-canonical nucleotides can be particularly effective at increasing the efficiency with which synthetic RNA can be translated into protein, and decreasing the toxicity of synthetic RNA, for example, the combinations: 5-methyluridine and 5-methylcytidine, 5-methyluridine and 7-deazaguanosine, 5-methylcytidine and 7-deazaguanosine, 5-methyluridine, 5-methylcytidine, and 7-deazaguanosine, and 5-methyluridine, 5-hydroxymethylcytidine, and 7-deazaguanosine. Certain embodiments are therefore directed to a nucleic acid comprising at least two of: 5-methyluridine, 5-methylcytidine, 5-hydroxymethylcytidine, and 7-deazaguanosine or one or more derivatives thereof. Other embodiments are directed to a nucleic acid comprising at least three of: 5-methyluridine, 5-methylcytidine, 5-hydroxymethylcytidine, and 7-deazaguanosine or one or more derivatives thereof. Other embodiments are directed to a nucleic acid comprising all of: 5-methyluridine, 5-methylcytidine, 5-hydroxymethylcytidine, and 7-deazaguanosine or one or more derivatives thereof. In one embodiment, the nucleic acid comprises one or more 5-methyluridine residues, one or more 5-methylcytidine residues, and one or more 7-deazaguanosine residues or one or more 5-methyluridine residues, one or more 5-hydroxymethylcytidine residues, and one or more 7-deazaguanosine residues.
It has been further discovered that synthetic RNA molecules containing certain fractions of certain non-canonical nucleotides and combinations thereof can exhibit particularly high translation efficiency and low toxicity. Certain embodiments are therefore directed to a nucleic acid comprising at least one of: one or more uridine residues, one or more cytidine residues, and one or more guanosine residues, and comprising one or more non-canonical nucleotides. In one embodiment, between about 20% and about 80% of the uridine residues are 5-methyluridine residues. In another embodiment, between about 30% and about 50% of the uridine residues are 5-methyluridine residues. In a further embodiment, about 40% of the uridine residues are 5-methyluridine residues. In one embodiment, between about 60% and about 80% of the cytidine residues are 5-methylcytidine residues. In another embodiment, between about 80% and about 100% of the cytidine residues are 5-methylcytidine residues. In a further embodiment, about 100% of the cytidine residues are 5-methylcytidine residues. In a still further embodiment, between about 20% and about 100% of the cytidine residues are 5-hydroxymethylcytidine residues. In one embodiment, between about 20% and about 80% of the guanosine residues are 7-deazaguanosine residues. In another embodiment, between about 40% and about 60% of the guanosine residues are 7-deazaguanosine residues. In a further embodiment, about 50% of the guanosine residues are 7-deazaguanosine residues. In one embodiment, between about 20% and about 80% or between about 30% and about 60% or about 40% of the cytidine residues are N4-methylcytidine and/or N4-acetylcytidine residues. In another embodiment, each cytidine residue is a 5-methylcytidine residue. In a further embodiment, about 100% of the cytidine residues are 5-methylcytidine residues and/or 5-hydroxymethylcytidine residues and/or N4-methylcytidine residues and/or N4-acetylcytidine residues and/or one or more derivatives thereof. In a still further embodiment, about 40% of the uridine residues are 5-methyluridine residues, between about 20% and about 100% of the cytidine residues are N4-methylcytidine and/or N4-acetylcytidine residues, and about 50% of the guanosine residues are 7-deazaguanosine residues. In one embodiment, about 40% of the uridine residues are 5-methyluridine residues and about 100% of the cytidine residues are 5-methylcytidine residues. In another embodiment, about 40% of the uridine residues are 5-methyluridine residues and about 50% of the guanosine residues are 7-deazaguanosine residues. In a further embodiment, about 100% of the cytidine residues are 5-methylcytidine residues and about 50% of the guanosine residues are 7-deazaguanosine residues. In one embodiment, about 40% of the uridine residues are 5-methyluridine residues, about 100% of the cytidine residues are 5-methylcytidine residues, and about 50% of the guanosine residues are 7-deazaguanosine residues. In another embodiment, about 40% of the uridine residues are 5-methyluridine residues, between about 20% and about 100% of the cytidine residues are 5-hydroxymethylcytidine residues, and about 50% of the guanosine residues are 7-deazaguanosine residues. In some embodiments, less than 100% of the cytidine residues are 5-methylcytidine residues. In other embodiments, less than 100% of the cytidine residues are 5-hydroxymethylcytidine residues. In one embodiment, each uridine residue in the synthetic RNA molecule is a pseudouridine residue or a 5-methylpseudouridine residue. In another embodiment, about 100% of the uridine residues are pseudouridine residues and/or 5-methylpseudouridine residues. In a further embodiment, about 100% of the uridine residues are pseudouridine residues and/or 5-methylpseudouridine residues, about 100% of the cytidine residues are 5-methylcytidine residues, and about 50% of the guanosine residues are 7-deazaguanosine residues.
Other non-canonical nucleotides that can be used in place of or in combination with 5-methyluridine include, but are not limited to: pseudouridine and 5-methylpseudouridine (a.k.a. “1-methylpseudouridine”, a.k.a. “N1-methylpseudouridine”) or one or more derivatives thereof. Other non-canonical nucleotides that can be used in place of or in combination with 5-methylcytidine and/or 5-hydroxymethylcytidine include, but are not limited to: pseudoisocytidine, 5-methylpseudoisocytidine, 5-hydroxymethylcytidine, 5-formylcytidine, 5-carboxycytidine, N4-methylcytidine, N4-acetylcytidine or one or more derivatives thereof. In certain embodiments, for example, when performing only a single transfection or when the cells being transfected are not particularly sensitive to transfection-associated toxicity or innate-immune signaling, the fractions of non-canonical nucleotides can be reduced. Reducing the fraction of non-canonical nucleotides can be beneficial, in part, because reducing the fraction of non-canonical nucleotides can reduce the cost of the nucleic acid. In certain situations, for example, when minimal immunogenicity of the nucleic acid is desired, the fractions of non-canonical nucleotides can be increased.
Enzymes such as T7 RNA polymerase may preferentially incorporate canonical nucleotides in an in vitro-transcription reaction containing both canonical and non-canonical nucleotides. As a result, an in vitro-transcription reaction containing a certain fraction of a non-canonical nucleotide may yield RNA containing a different, often lower, fraction of the non-canonical nucleotide than the fraction at which the non-canonical nucleotide was present in the reaction. In certain embodiments, references to nucleotide incorporation fractions (for example, “50% 5-methyluridine”) therefore can refer both to nucleic acids containing the stated fraction of the nucleotide, and to nucleic acids synthesized in a reaction containing the stated fraction of the nucleotide (or nucleotide derivative, for example, nucleotide-triphosphate), even though such a reaction may yield a nucleic acid containing a different fraction of the nucleotide than the fraction at which the non-canonical nucleotide was present in the reaction. In addition, different nucleotide sequences can encode the same protein by utilizing alternative codons. In certain embodiments, references to nucleotide incorporation fractions therefore can refer both to nucleic acids containing the stated fraction of the nucleotide, and to nucleic acids encoding the same protein as a different nucleic acid, wherein the different nucleic acid contains the stated fraction of the nucleotide.
The DNA sequence of a cell can be altered by contacting the cell with a gene-editing protein or by inducing the cell to express a gene-editing protein. However, previously disclosed gene-editing proteins suffer from low binding efficiency and excessive off-target activity, which can introduce undesired mutations in the DNA of the cell, severely limiting their use in therapeutic applications, in which the introduction of undesired mutations in a patient's cells could lead to the development of cancer. It has now been discovered that gene-editing proteins that comprise the StsI endonuclease cleavage domain (SEQ ID NO: 1) can exhibit substantially lower off-target activity than previously disclosed gene-editing proteins, while maintaining a high level of on-target activity. Other novel engineered proteins have also been discovered that can exhibit high on-target activity, low off-target activity, small size, solubility, and other desirable characteristics when they are used as the nuclease domain of a gene-editing protein: StsI-HA (SEQ ID NO: 2), StsI-HA2 (SEQ ID NO: 3), StsI-UHA (SEQ ID NO: 4), StsI-UHA2 (SEQ ID NO: 5), StsI-HF (SEQ ID NO: 6), and StsI-UHF (SEQ ID NO: 7). StsI-HA, StsI-HA2 (high activity), StsI-UHA, and StsI-UHA2 (ultra-high activity) can exhibit higher on-target activity than both wild-type StsI and wild-type FokI, due in part to specific amino-acid substitutions within the N-terminal region at the 34 and 61 positions, while StsI-HF (high fidelity) and StsI-UHF (ultra-high fidelity) can exhibit lower off-target activity than both wild-type StsI and wild-type FokI, due in part to specific amino-acid substitutions within the C-terminal region at the 141 and 152 positions. Certain embodiments are therefore directed to a protein that comprises a nuclease domain. In one embodiment, the nuclease domain comprises one or more of: the cleavage domain of FokI endonuclease (SEQ ID NO: 53), the cleavage domain of StsI endonuclease (SEQ ID NO: 1), StsI-HA (SEQ ID NO: 2), StsI-HA2 (SEQ ID NO: 3), StsI-UHA (SEQ ID NO: 4), StsI-UHA2 (SEQ ID NO: 5), StsI-HF (SEQ ID NO: 6), and StsI-UHF (SEQ ID NO: 7) or a biologically active fragment or variant thereof.
It has also been discovered that engineered gene-editing proteins that comprise DNA-binding domains comprising certain novel repeat sequences can exhibit lower off-target activity than previously disclosed gene-editing proteins, while maintaining a high level of on-target activity. Certain of these engineered gene-editing proteins can provide several advantages over previously disclosed gene-editing proteins, including, for example, increased flexibility of the linker region connecting repeat sequences, which can result in increased binding efficiency. Certain embodiments are therefore directed to a protein comprising a plurality of repeat sequences. In one embodiment, at least one of the repeat sequences contains the amino-acid sequence: GabG (SEQ ID NO: 674), where “a” and “b” each represent any amino acid. In one embodiment, the protein is a gene-editing protein. In another embodiment, one or more of the repeat sequences are present in a DNA-binding domain. In a further embodiment, “a” and “b” are each independently selected from the group: H and G. In a still further embodiment, “a” and “b” are H and G, respectively. In one embodiment, the amino-acid sequence is present within about 5 amino acids of the C-terminus of the repeat sequence. In another embodiment, the amino-acid sequence is present at the C-terminus of the repeat sequence. In some embodiments, one or more G in the amino-acid sequence GabG is replaced with one or more amino acids other than G, for example A, H or GG. In one embodiment, the repeat sequence has a length of between about 32 and about 40 amino acids or between about 33 and about 39 amino acids or between about 34 and 38 amino acids or between about 35 and about 37 amino acids or about 36 amino acids or greater than about 32 amino acids or greater than about 33 amino acids or greater than about 34 amino acids or greater than about 35 amino acids. Other embodiments are directed to a protein comprising one or more transcription activator-like effector domains. In one embodiment, at least one of the transcription activator-like effector domains comprises a repeat sequence. Other embodiments are directed to a protein comprising a plurality of repeat sequences generated by inserting one or more amino acids between at least two of the repeat sequences of a transcription activator-like effector domain. In one embodiment, one or more amino acids is inserted about 1 or about 2 or about 3 or about 4 or about 5 amino acids from the C-terminus of at least one repeat sequence. Still other embodiments are directed to a protein comprising a plurality of repeat sequences, wherein about every other repeat sequence has a different length than the repeat sequence immediately preceding or following the repeat sequence. In one embodiment, every other repeat sequence is about 36 amino acids long. In another embodiment, every other repeat sequence is 36 amino acids long. Still other embodiments are directed to a protein comprising a plurality of repeat sequences, wherein the plurality of repeat sequences comprises at least two repeat sequences that are each at least 36 amino acids long, and wherein at least two of the repeat sequences that are at least 36 amino acids long are separated by at least one repeat sequence that is less than 36 amino acids long. Some embodiments are directed to a protein that comprises one or more sequences selected from, for example, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, and SEQ ID NO: 60.
Other embodiments are directed to a protein that comprises a DNA-binding domain. In some embodiments, the DNA-binding domain comprises a plurality of repeat sequences. In one embodiment, the plurality of repeat sequences enables high-specificity recognition of a binding site in a target DNA molecule. In another embodiment, at least two of the repeat sequences have at least about 50%, or about 60%, or about 70%, or about 80%, or about 90%, or about 95%, or about 98%, or about 99% homology to each other. In a further embodiment, at least one of the repeat sequences comprises one or more regions capable of binding to a binding site in a target DNA molecule. In a still further embodiment, the binding site comprises a defined sequence of between about 1 to about 5 bases in length. In one embodiment, the DNA-binding domain comprises a zinc finger. In another embodiment, the DNA-binding domain comprises a transcription activator-like effector (TALE). In a further embodiment, the plurality of repeat sequences includes at least one repeat sequence having at least about 50% or about 60% or about 70% or about 80% or about 90% or about 95% or about 98%, or about 99% homology to a TALE. In a still further embodiment, the gene-editing protein comprises a clustered regularly interspaced short palindromic repeat (CRISPR)-associated protein. In one embodiment, the gene-editing protein comprises a nuclear-localization sequence. In another embodiment, the nuclear-localization sequence comprises the amino-acid sequence: PKKKRKV (SEQ ID NO: 61). In one embodiment, the gene-editing protein comprises a mitochondrial-localization sequence. In another embodiment, the mitochondrial-localization sequence comprises the amino-acid sequence: LGRVIPRKIASRASLM (SEQ ID NO: 62). In one embodiment, the gene-editing protein comprises a linker. In another embodiment, the linker connects a DNA-binding domain to a nuclease domain. In a further embodiment, the linker is between about 1 and about 10 amino acids long. In some embodiments, the linker is about 1, about 2, or about 3, or about 4, or about 5, or about 6, or about 7, or about 8, or about 9, or about 10 amino acids long. In one embodiment, the gene-editing protein is capable of generating a nick or a double-strand break in a target DNA molecule.
Certain embodiments are directed to a method for modifying the genome of a cell, the method comprising introducing into the cell a nucleic acid molecule encoding a non-naturally occurring fusion protein comprising an artificial transcription activator-like (TAL) effector repeat domain comprising one or more repeat units 36 amino acids in length and an endonuclease domain, wherein the repeat domain is engineered for recognition of a predetermined nucleotide sequence, and wherein the fusion protein recognizes the predetermined nucleotide sequence. In one embodiment, the cell is a eukaryotic cell. In another embodiment, the cell is an animal cell. In a further embodiment, the cell is a mammalian cell. In a still further embodiment, the cell is a human cell. In one embodiment, the cell is a plant cell. In another embodiment, the cell is a prokaryotic cell. In some embodiments, the fusion protein introduces an endonucleolytic cleavage in a nucleic acid of the cell, whereby the genome of the cell is modified.
Other embodiments are directed to a nucleic acid molecule encoding a non-naturally occurring fusion protein comprising an artificial transcription activator-like (TAL) effector repeat domain comprising one or more repeat units 36 amino acids in length and restriction endonuclease activity, wherein the repeat domain is engineered for recognition of a predetermined nucleotide sequence and wherein the fusion protein recognizes the predetermined nucleotide sequence. In one embodiment, the repeat units differ by no more than about seven amino acids. In another embodiment, each of the repeat units contains the amino acid sequence: LTPXQVVAIAS (SEQ ID NO: 63) where X can be either E or Q, and the amino acid sequence: LTPXQVVAIAS (SEQ ID NO: 64) is followed on the carboxyl terminus by either one or two amino acids that determine recognition for one of adenine, cytosine, guanine or thymine. In one embodiment, the nucleic acid encodes about 1.5 to about 28.5 repeat units. In another embodiment, the nucleic acid encodes about 11.5, about 14.5, about 17.5 or about 18.5 repeat units. In a further embodiment, the predetermined nucleotide sequence is a promoter region.
Some embodiments are directed to a vector comprising a nucleic acid molecule or sequence. In one embodiment, the vector is a viral vector. In another embodiment, the viral vector comprises one or more of: an adenovirus, a retrovirus, a lentivirus, a herpes virus, an adeno-associated virus or a natural or engineered variant thereof, and an engineered virus.
Certain embodiments are directed to a nucleic acid molecule encoding a non-naturally occurring fusion protein comprising a first region that recognizes a predetermined nucleotide sequence and a second region with endonuclease activity, wherein the first region contains an artificial TAL effector repeat domain comprising one or more repeat units about 36 amino acids in length which differ from each other by no more than seven amino acids, and wherein the repeat domain is engineered for recognition of the predetermined nucleotide sequence. In one embodiment, the first region contains the amino acid sequence: LTPXQVVAIAS (SEQ ID NO: 63) where X can be either E or Q. In another embodiment, the amino acid sequence LTPXQVVAIAS (SEQ ID NO: 64) of the encoded non-naturally occurring fusion protein is immediately followed by an amino acid sequence selected from: HD, NG, NS, NI, NN, and N. In a further embodiment, the fusion protein comprises restriction endonuclease activity. Some embodiments are directed to a nucleic acid molecule encoding a protein that comprises one or more sequences selected from: SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60.
In one embodiment, the repeat sequence comprises: LTPvQVVAIAwxyzHG (SEQ ID NO: 65), wherein “v” is D or E, “w” is S or N, “x” is N, H or I, “y” is any amino acid or no amino acid, and “z” is GGRPALE (SEQ ID NO: 664), GGKQALE (SEQ ID NO: 665), GGKQALETVQRLLPVLCQDHG (SEQ ID NO: 666), GGKQALETVQRLLPVLCQAHG (SEQ ID NO: 667), GKQALETVQRLLPVLCQDHG (SEQ ID NO: 668), or GKQALETVQRLLPVLCQAHG (SEQ ID NO: 669). In another embodiment, the repeat sequence comprises: LTPvQVVAIAwxyzHG (SEQ ID NO: 66), wherein “v” is D or E, “w” is S or N, “x” is N, H or I, “y” is selected from: D, A, I, N, H, K, S, and G, and “z” is GGRPALE (SEQ ID NO: 664), GGKQALE (SEQ ID NO: 665), GGKQALETVQRLLPVLCQDHG (SEQ ID NO: 666), GGKQALETVQRLLPVLCQAHG (SEQ ID NO: 667), GKQALETVQRLLPVLCQDHG (SEQ ID NO: 668), or GKQALETVQRLLPVLCQAHG (SEQ ID NO: 669). In yet another embodiment, the repeat sequence comprises: LTPvQVVAIAwxyzHG (SEQ ID NO: 67), wherein “v” is D or E, “w” is S or N, “x” is any amino acid other than N, H and I, “y” is any amino acid or no amino acid, and “z” is GGRPALE (SEQ ID NO: 664), GGKQALE (SEQ ID NO: 665), GGKQALETVQRLLPVLCQDHG (SEQ ID NO: 666), GGKQALETVQRLLPVLCQAHG (SEQ ID NO: 667), GKQALETVQRLLPVLCQDHG (SEQ ID NO: 668), or GKQALETVQRLLPVLCQAHG (SEQ ID NO: 669). In yet another embodiment, the repeat sequence comprises: LTPvQVVAIAwIyzHG (SEQ ID NO: 68), wherein “v” is D or E, “w” is S or N, “y” is any amino acid other than G, and “z” is GGRPALE (SEQ ID NO: 664), GGKQALE (SEQ ID NO: 665), GGKQALETVQRLLPVLCQDHG (SEQ ID NO: 666), GGKQALETVQRLLPVLCQAHG (SEQ ID NO: 667), GKQALETVQRLLPVLCQDHG (SEQ ID NO: 668), or GKQALETVQRLLPVLCQAHG (SEQ ID NO: 669). In yet another embodiment, the repeat sequence comprises: LTPvQVVAIAwIAzHG (SEQ ID NO: 69), wherein “v” is D or E, “w” is S or N, and “z” is GGRPALE (SEQ ID NO: 664), GGKQALE (SEQ ID NO: 665), GGKQALETVQRLLPVLCQDHG (SEQ ID NO: 666), GGKQALETVQRLLPVLCQAHG (SEQ ID NO: 667), GKQALETVQRLLPVLCQDHG (SEQ ID NO: 668), or GKQALETVQRLLPVLCQAHG (SEQ ID NO: 669). In yet another embodiment, the repeat sequence comprises: LTPvQVVAIAwxyzHG (SEQ ID NO: 70), wherein “v” is D or E, “w” is S or N, “x” is S, T or Q, “y” is any amino acid or no amino acid, and “z” is GGRPALE (SEQ ID NO: 664), GGKQALE (SEQ ID NO: 665), GGKQALETVQRLLPVLCQDHG (SEQ ID NO: 666), GGKQALETVQRLLPVLCQAHG (SEQ ID NO: 667), GKQALETVQRLLPVLCQDHG (SEQ ID NO: 668), or GKQALETVQRLLPVLCQAHG (SEQ ID NO: 669). In yet another embodiment, the repeat sequence comprises: LTPvQVVAIAwxyzHG (SEQ ID NO: 71), wherein “v” is D or E, “w” is S or N, “x” is S, T or Q, “y” is selected from: D, A, I, N, H, K, S, and G, and “z” is GGRPALE (SEQ ID NO: 664), GGKQALE (SEQ ID NO: 665), GGKQALETVQRLLPVLCQDHG (SEQ ID NO: 666), GGKQALETVQRLLPVLCQAHG (SEQ ID NO: 667), GKQALETVQRLLPVLCQDHG (SEQ ID NO: 668), or GKQALETVQRLLPVLCQAHG (SEQ ID NO: 669). In yet another embodiment, the repeat sequence comprises: LTPvQVVAIAwx (SEQ ID NO: 72), wherein “v” is D or E, “w” is S or N, and “x” is S, T or Q. In yet another embodiment, the repeat sequence comprises: LTPvQVVAIAwxy (SEQ ID NO: 73), wherein “v” is D or E, “w” is S or N, “x” is S, T or Q, and “y” is selected from: D, A, I, N, H, K, S, and G. In yet another embodiment, the repeat sequence comprises: LTPvQVVAIAwxyzGHGG (SEQ ID NO: 74), wherein “v” is Q, D or E, “w” is S or N, “x” is N, H or I, “y” is any amino acid or no amino acid, and “z” is GGRPALE (SEQ ID NO: 664), GGKQALE (SEQ ID NO: 665), GGKQALETVQRLLPVLCQD (SEQ ID NO: 670), GGKQALETVQRLLPVLCQA (SEQ ID NO: 671), GKQALETVQRLLPVLCQD (SEQ ID NO: 672) or GKQALETVQRLLPVLCQA (SEQ ID NO: 673). In yet another embodiment, the repeat sequence comprises: LTPvQVVAIAwxyzGHGG (SEQ ID NO: 75), wherein “v” is Q, D or E, “w” is S or N, “x” is N, H or I, “y” is selected from: D, A, I, N, H, K, S, and G, and “z” is GGRPALE (SEQ ID NO: 664), GGKQALE (SEQ ID NO: 665), GGKQALETVQRLLPVLCQD (SEQ ID NO: 670), GGKQALETVQRLLPVLCQA (SEQ ID NO: 671), GKQALETVQRLLPVLCQD (SEQ ID NO: 672) or GKQALETVQRLLPVLCQA (SEQ ID NO: 673). In yet another embodiment, the repeat sequence comprises: LTPvQVVAIAwxyzGHGG (SEQ ID NO: 76), wherein “v” is Q, D or E, “w” is S or N, “x” is any amino acid other than N, H and I, “y” is any amino acid or no amino acid, and “z” is GGRPALE (SEQ ID NO: 664), GGKQALE (SEQ ID NO: 665), GGKQALETVQRLLPVLCQD (SEQ ID NO: 670), GGKQALETVQRLLPVLCQA (SEQ ID NO: 671), GKQALETVQRLLPVLCQD (SEQ ID NO: 672) or GKQALETVQRLLPVLCQA (SEQ ID NO: 673). In yet another embodiment, the repeat sequence comprises: LTPvQVVAIAwIyzGHGG (SEQ ID NO: 77), wherein “v” is Q, D or E, “w” is S or N, “y” is any amino acid other than G, and “z” is GGRPALE (SEQ ID NO: 664), GGKQALE (SEQ ID NO: 665), GGKQALETVQRLLPVLCQD (SEQ ID NO: 670), GGKQALETVQRLLPVLCQA (SEQ ID NO: 671), GKQALETVQRLLPVLCQD (SEQ ID NO: 672) or GKQALETVQRLLPVLCQA (SEQ ID NO: 673). In yet another embodiment, the repeat sequence comprises: LTPvQVVAIAwIAzGHGG (SEQ ID NO: 78), wherein “v” is Q, D or E, “w” is S or N, and “z” is GGRPALE (SEQ ID NO: 664), GGKQALE (SEQ ID NO: 665), GGKQALETVQRLLPVLCQD (SEQ ID NO: 670), GGKQALETVQRLLPVLCQA (SEQ ID NO: 671), GKQALETVQRLLPVLCQD (SEQ ID NO: 672) or GKQALETVQRLLPVLCQA (SEQ ID NO: 673). In yet another embodiment, the repeat sequence comprises: LTPvQVVAIAwxyzGHGG (SEQ ID NO: 79), wherein “v” is Q, D or E, “w” is S or N, “x” is S, T or Q, “y” is any amino acid or no amino acid, and “z” is GGRPALE (SEQ ID NO: 664), GGKQALE (SEQ ID NO: 665), GGKQALETVQRLLPVLCQD (SEQ ID NO: 670), GGKQALETVQRLLPVLCQA (SEQ ID NO: 671), GKQALETVQRLLPVLCQD (SEQ ID NO: 672) or GKQALETVQRLLPVLCQA (SEQ ID NO: 673). In yet another embodiment, the repeat sequence comprises: LTPvQVVAIAwxyzGHGG (SEQ ID NO: 80), wherein “v” is Q, D or E, “w” is S or N, “x” is S, T or Q, “y” is selected from: D, A, I, N, H, K, S, and G, and “z” is GGRPALE (SEQ ID NO: 664), GGKQALE (SEQ ID NO: 665), GGKQALETVQRLLPVLCQD (SEQ ID NO: 670), GGKQALETVQRLLPVLCQA (SEQ ID NO: 671), GKQALETVQRLLPVLCQD (SEQ ID NO: 672) or GKQALETVQRLLPVLCQA (SEQ ID NO: 673). In yet another embodiment, the repeat sequence comprises: LTPvQVVAIAwx (SEQ ID NO: 81), wherein “v” is Q, D or E, “w” is S or N, and “x” is S, T or Q. In yet another embodiment, the repeat sequence comprises: LTPvQVVAIAwxy (SEQ ID NO: 82), wherein “v” is Q, D or E, “w” is S or N, “x” is S, T or Q, and “y” is selected from: D, A, I, N, H, K, S, and G.
Certain fragments of an endonuclease cleavage domain, including fragments that are truncated at the N-terminus, fragments that are truncated at the C-terminus, fragments that have internal deletions, and fragments that combine N-terminus, C-terminus, and/or internal deletions, can maintain part or all of the catalytic activity of the full endonuclease cleavage domain. Determining whether a fragment can maintain part or all of the catalytic activity of the full domain can be accomplished by, for example, synthesizing a gene-editing protein that contains the fragment according to the methods of the present invention, inducing cells to express the gene-editing protein according to the methods of the present invention, and measuring the efficiency of gene editing. In this way, a measurement of gene-editing efficiency can be used to ascertain whether any specific fragment can maintain part or all of the catalytic activity of the full endonuclease cleavage domain. Certain embodiments are therefore directed to a biologically active fragment of an endonuclease cleavage domain. In one embodiment, the endonuclease cleavage domain is selected from: FokI, StsI, StsI-HA, StsI-HA2, StsI-UHA, StsI-UHA2, StsI-HF, and StsI-UHF or a natural or engineered variant or biologically active fragment thereof.
Certain fragments of a DNA-binding domain or repeat sequence, including fragments that are truncated at the N-terminus, fragments that are truncated at the C-terminus, fragments that have internal deletions, and fragments that combine N-terminus, C-terminus, and/or internal deletions, can maintain part or all of the binding activity of the full DNA-binding domain or repeat sequence. Examples of fragments of DNA-binding domains or repeat sequences that can maintain part or all of the binding activity of the full repeat sequence include Ralstonia solanacearum TALE-like proteins (RTLs). Determining whether a fragment can maintain part or all of the binding activity of the full DNA-binding domain or repeat sequence can be accomplished by, for example, synthesizing a gene-editing protein that contains the fragment according to the methods of the present invention, inducing cells to express the gene-editing protein according to the methods of the present invention, and measuring the efficiency of gene editing. In this way, a measurement of gene-editing efficiency can be used to ascertain whether any specific fragment can maintain part or all of the binding activity of the full DNA-binding domain or repeat sequence. Certain embodiments are therefore directed to a biologically active fragment of a DNA-binding domain or repeat sequence. In one embodiment, the fragment enables high-specificity recognition of a binding site in a target DNA molecule. In another embodiment, the fragment comprises a sequence that encodes a Ralstonia solanacearum TALE-like protein or a biologically active fragment thereof.
Certain embodiments are directed to a composition for altering the DNA sequence of a cell comprising a nucleic acid, wherein the nucleic acid encodes a gene-editing protein. Other embodiments are directed to a composition for altering the DNA sequence of a cell comprising a nucleic-acid mixture, wherein the nucleic-acid mixture comprises: a first nucleic acid that encodes a first gene-editing protein, and a second nucleic acid that encodes a second gene-editing protein. In one embodiment, the binding site of the first gene-editing protein and the binding site of the second gene-editing protein are present in the same target DNA molecule. In another embodiment, the binding site of the first gene-editing protein and the binding site of the second gene-editing protein are separated by less than about 50 bases, or less than about 40 bases, or less than about 30 bases or less than about 20 bases, or less than about 10 bases, or between about 10 bases and about 25 bases or about 15 bases.
In one embodiment, the nuclease domain of the first gene-editing protein and the nuclease domain of the second gene-editing protein are capable of forming a dimer. In another embodiment, the dimer is capable of generating a nick or double-strand break in a target DNA molecule. In one embodiment, the composition is a therapeutic composition. In another embodiment, the composition comprises a repair template. In a further embodiment, the repair template is a single-stranded DNA molecule or a double-stranded DNA molecule.
Other embodiments are directed to an article of manufacture for synthesizing a protein or a nucleic acid encoding a protein. In one embodiment, the article is a nucleic acid. In another embodiment, the protein comprises a DNA-binding domain. In a further embodiment, the nucleic acid comprises a nucleotide sequence encoding a DNA-binding domain. In one embodiment, the protein comprises a nuclease domain. In another embodiment, the nucleic acid comprises a nucleotide sequence encoding a nuclease domain. In one embodiment, the protein comprises a plurality of repeat sequences. In another embodiment, the nucleic acid encodes a plurality of repeat sequences. In a further embodiment, the nuclease domain is selected from: FokI, StsI, StsI-HA, StsI-HA2, StsI-UHA, StsI-UHA2, StsI-HF, and StsI-UHF or a natural or engineered variant or biologically active fragment thereof. In one embodiment, the nucleic acid comprises an RNA-polymerase promoter. In another embodiment, the RNA-polymerase promoter is a T7 promoter or a SP6 promoter. In a further embodiment, the nucleic acid comprises a viral promoter. In one embodiment, the nucleic acid comprises an untranslated region. In another embodiment, the nucleic acid is an in vitro-transcription template.
Certain embodiments are directed to a method for inducing a cell to express a protein. Other embodiments are directed to a method for altering the DNA sequence of a cell comprising transfecting the cell with a gene-editing protein or inducing the cell to express a gene-editing protein. Still other embodiments are directed to a method for reducing the expression of a protein of interest in a cell. In one embodiment, the cell is induced to express a gene-editing protein, wherein the gene-editing protein is capable of creating a nick or a double-strand break in a target DNA molecule. In another embodiment, the nick or double-strand break results in inactivation of a gene. Still other embodiments are directed to a method for generating an inactive, reduced-activity or dominant-negative form of a protein. In one embodiment, the protein is survivin. Still other embodiments are directed to a method for repairing one or more mutations in a cell. In one embodiment, the cell is contacted with a repair template. In another embodiment, the repair template is a DNA molecule. In a further embodiment, the repair template does not contain a binding site of the gene-editing protein. In a still further embodiment, the repair template encodes an amino-acid sequence that is encoded by a DNA sequence that comprises a binding site of the gene-editing protein.
Other embodiments are directed to a method for treating a patient comprising administering to the patient a therapeutically effective amount of a protein or a nucleic acid encoding a protein. In one embodiment, the treatment results in one or more of the patient's symptoms being ameliorated. Certain embodiments are directed to a method for treating a patient comprising: a. removing a cell from the patient, b. inducing the cell to express a gene-editing protein by transfecting the cell with a nucleic acid encoding a gene-editing protein, c. reprogramming the cell, and e. introducing the cell into the patient. In one embodiment, the cell is reprogrammed to a less differentiated state. In another embodiment, the cell is reprogrammed by transfecting the cell with one or more synthetic RNA molecules encoding one or more reprogramming proteins. In a further embodiment, the cell is differentiated. In a still further embodiment, the cell is differentiated into one of: a skin cell, a glucose-responsive insulin-producing cell, a hematopoietic cell, a cardiac cell, a retinal cell, a renal cell, a neural cell, a stromal cell, a fat cell, a bone cell, a muscle cell, an oocyte, and a sperm cell. Other embodiments are directed to a method for treating a patient comprising: a. removing a hematopoietic cell or a stem cell from the patient, b. inducing the cell to express a gene-editing protein by transfecting the cell with a nucleic acid encoding a gene-editing protein, and c. introducing the cell into the patient.
It has now been discovered that a cell-culture medium consisting essentially of or comprising: DMEM/F12, ascorbic acid, insulin, transferrin, sodium selenite, ethanolamine, basic fibroblast growth factor, and transforming growth factor-beta is sufficient to sustain pluripotent stem cells, including human pluripotent stem cells, in vitro. Certain embodiments are therefore directed to a cell-culture medium consisting essentially of or comprising: DMEM/F12, ascorbic acid, insulin, transferrin, sodium selenite, ethanolamine, basic fibroblast growth factor, and transforming growth factor-beta. In one embodiment, the ascorbic acid is present at about 50 μg/mL. In another embodiment, the insulin is present at about 10 μg/mL. In a further embodiment, the transferrin is present at about 5.5 μg/mL. In a still further embodiment, the sodium selenite is present at about 6.7 ng/mL. In a still further embodiment, the ethanolamine is present at about 2 μg/mL. In a still further embodiment, the basic fibroblast growth factor is present at about 20 ng/mL. In a still further embodiment, the transforming growth factor-beta is present at about 2 ng/mL. In one embodiment, the ascorbic acid is ascorbic acid-2-phosphate. In another embodiment, the transforming growth factor-beta is transforming growth factor-beta 1 or transforming growth factor-beta 3. In one embodiment, the cell-culture medium is used for the culture of pluripotent stem cells. In another embodiment, the pluripotent stem cells are human pluripotent stem cells. In a further embodiment, the cell-culture medium is used for the culture of cells during or after reprogramming. In one embodiment, the cell-culture medium contains no animal-derived components. In another embodiment, the cell-culture medium is manufactured according to a manufacturing standard. In a further embodiment, the manufacturing standard is GMP. In one embodiment, the cells are contacted with a cell-adhesion molecule. In another embodiment, the cell-adhesion molecule is selected from: fibronectin and vitronectin or a biologically active fragment thereof. In a further embodiment, the cells are contacted with fibronectin and vitronectin. In a still further embodiment, the cell-adhesion molecule is recombinant.
In certain situations, for example, when producing a therapeutic, it can be beneficial to replace animal-derived components with non-animal-derived components, in part to reduce the risk of contamination with viruses and/or other animal-borne pathogens. It has now been discovered that synthetic cholesterol, including semi-synthetic plant-derived cholesterol, can be substituted for animal-derived cholesterol in transfection medium without decreasing transfection efficiency or increasing transfection-associated toxicity. Certain embodiments are therefore directed to a transfection medium containing synthetic or semi-synthetic cholesterol. In one embodiment, the semi-synthetic cholesterol is plant-derived. In another embodiment, the transfection medium does not contain animal-derived cholesterol. In a further embodiment, the transfection medium is a reprogramming medium. Other embodiments are directed to a complexation medium. In one embodiment, the complexation medium has a pH greater than about 7, or greater than about 7.2, or greater than about 7.4, or greater than about 7.6, or greater than about 7.8, or greater than about 8.0, or greater than about 8.2, or greater than about 8.4, or greater than about 8.6, or greater than about 8.8, or greater than about 9.0. In another embodiment, the complexation medium comprises transferrin. In a further embodiment, the complexation medium comprises DMEM. In a still further embodiment, the complexation medium comprises DMEM/F12. Still other embodiments are directed to a method for forming nucleic-acid-transfection-reagent complexes. In one embodiment, the transfection reagent is incubated with a complexation medium. In another embodiment, the incubation occurs before a mixing step. In a further embodiment, the incubation step is between about 5 seconds and about 5 minutes or between about 10 seconds and about 2 minutes or between about 15 seconds and about 1 minute or between about 30 seconds and about 45 seconds. In one embodiment, the transfection reagent is selected from Table 1. In another embodiment, the transfection reagent is a lipid or lipidoid. In a further embodiment, the transfection reagent comprises a cation. In a still further embodiment, the cation is a multivalent cation. In a still further embodiment, the transfection reagent is N1-[2-((1S)-1-[(3-aminopropyl)amino]-4-[di(3-amino-propyl)amino]butylcarboxamido)ethyl]-3,4-di[oleyloxy]-benzamide (a.k.a. MVL5) or a derivative thereof.
Certain embodiments are directed to a method for inducing a cell to express a protein by contacting the cell with a nucleic acid. In one embodiment, the cell is a mammalian cell. In another embodiment, the cell is a human cell or a rodent cell. Other embodiments are directed to a cell produced using one or more of the methods of the present invention. In one embodiment, the cell is present in a patient. In another embodiment, the cell is isolated from a patient. Other embodiments are directed to a screening library comprising a cell produced using one or more of the methods of the present invention. In one embodiment, the screening library is used for at least one of: toxicity screening, including: cardiotoxicity screening, neurotoxicity screening, and hepatotoxicity screening, efficacy screening, high-throughput screening, high-content screening, and other screening.
Other embodiments are directed to a kit containing a nucleic acid. In one embodiment, the kit contains a delivery reagent (a.k.a. “transfection reagent”). In another embodiment, the kit is a reprogramming kit. In a further embodiment, the kit is a gene-editing kit. Other embodiments are directed to a kit for producing nucleic acids. In one embodiment, the kit contains at least two of: pseudouridine-triphosphate, 5-methyluridine triphosphate, 5-methylcytidine triphosphate, 5-hydroxymethylcytidine triphosphate, N4-methylcytidine triphosphate, N4-acetylcytidine triphosphate, and 7-deazaguanosine triphosphate or one or more derivatives thereof. Other embodiments are directed to a therapeutic comprising a nucleic acid. In one embodiment, the therapeutic is a pharmaceutical composition. In another embodiment, the pharmaceutical composition is formulated. In a further embodiment, the formulation comprises an aqueous suspension of liposomes. Example liposome components are set forth in Table 1, and are given by way of example, and not by way of limitation. In one embodiment, the liposomes include one or more polyethylene glycol (PEG) chains. In another embodiment, the PEG is PEG2000. In a further embodiment, the liposomes include 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE) or a derivative thereof. In one embodiment, the therapeutic comprises one or more ligands. In another embodiment, the therapeutic comprises at least one of: androgen, CD30 (TNFRSF8), a cell-penetrating peptide, CXCR, estrogen, epidermal growth factor, EGFR, HER2, folate, insulin, insulin-like growth factor-I, interleukin-13, integrin, progesterone, stromal-derived-factor-1, thrombin, vitamin D, and transferrin or a biologically active fragment or variant thereof. Still other embodiments are directed to a therapeutic comprising a cell generated using one or more of the methods of the present invention. In one embodiment, the therapeutic is administered to a patient for the treatment of at least one of: type 1 diabetes, heart disease, including ischemic and dilated cardiomyopathy, macular degeneration, Parkinson's disease, cystic fibrosis, sickle-cell anemia, thalassemia, Fanconi anemia, severe combined immunodeficiency, hereditary sensory neuropathy, xeroderma pigmentosum, Huntington's disease, muscular dystrophy, amyotrophic lateral sclerosis, Alzheimer's disease, cancer, and infectious diseases including: hepatitis and HIV/AIDS.







TABLE 1





Exemplary Biocompatible Lipids
















1
3β-[N-(N′,N′-dimethylaminoethane)-carbamoyl]cholesterol (DC-



Cholesterol)


2
1,2-dioleoyl-3-trimethylammonium-propane (DOTAP/18:1 TAP)


3
N-(4-carboxybenzyl)-N,N-dimethyl-2,3-bis(oleoyloxy)propan-1-



aminium (DOBAQ)


4
1,2-dimyristoyl-3-trimethylammonium-propane (14:0 TAP)


5
1,2-dipalmitoyl-3-trimethylammonium-propane (16:0 TAP)


6
1,2-stearoyl-3-trimethylammonium-propane (18:0 TAP)


7
1,2-dioleoyl-3-dimethylammonium-propane (DODAP/18:1 DAP)


8
1,2-dimyristoyl-3-dimethylammonium-propane (14:0 DAP)


9
1,2-dipalmitoyl-3-dimethylammonium-propane (16:0 DAP)


10
1,2-distearoyl-3-dimethylammonium-propane (18:0 DAP)


11
dimethyldioctadecylammonium (18:0 DDAB)


12
1,2-dilauroyl-sn-glycero-3-ethylphosphocholine (12:0 EthylPC)


13
1,2-dimyristoyl-sn-glycero-3-ethylphosphocholine (14:0 EthylPC)


14
1,2-dimyristoleoyl-sn-glycero-3-ethylphosphocholine (14:1 EthylPC)


15
1,2-dipalmitoyl-sn-glycero-3-ethylphosphocholine (16:0 EthylPC)


16
1,2-distearoyl-sn-glycero-3-ethylphosphocholine (18:0 EthylPC)


17
1,2-dioleoyl-sn-glycero-3-ethylphosphocholine (18:1 EthylPC)


18
1-palmitoyl-2-oleoyl-sn-glycero-3-ethylphosphocholine (16:1-18:1



EthylPC)


19
1,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA)


20
N1-[2-((1S)-1-[(3-aminopropyl)amino]-4-[di(3-amino-



propyl)amino]butylcarboxamido)ethyl]-3,4-di[oleyloxy]-benzamide



(MVL5)


21
2,3-dioleyloxy-N-[2-spermine carboxamide]ethyl-N,N-dimethyl-1-



propanammonium trifluoroacetate (DOSPA)


22
1,3-di-oleoyloxy-2-(6-carboxy-spermyl)-propylamid (DOSPER)


23
N-[1-(2,3-dimyristyloxy)propyl]-N,N-dimethyl-N-(2-



hydroxyethyl)ammonium bromide (DMRIE)


24
dioctadecyl amidoglyceryl spermine (DOGS)


25
dioleoyl phosphatidyl ethanolamine (DOPE)









Certain embodiments are directed to a nucleic acid comprising a 5′-cap structure selected from Cap 0, Cap 1, Cap 2, and Cap 3 or a derivative thereof. In one embodiment, the nucleic acid comprises one or more UTRs. In another embodiment, the one or more UTRs increase the stability of the nucleic acid. In a further embodiment, the one or more UTRs comprise an alpha-globin or beta-globin 5′-UTR. In a still further embodiment, the one or more UTRs comprise an alpha-globin or beta-globin 3′-UTR. In a still further embodiment, the synthetic RNA molecule comprises an alpha-globin or beta-globin 5′-UTR and an alpha-globin or beta-globin 3′-UTR. In one embodiment, the 5′-UTR comprises a Kozak sequence that is substantially similar to the Kozak consensus sequence. In another embodiment, the nucleic acid comprises a 3′-poly(A) tail. In a further embodiment, the 3′-poly(A) tail is between about 20 nt and about 250 nt or between about 120 nt and about 150 nt long. In a further embodiment, the 3′-poly(A) tail is about 20 nt, or about 30 nt, or about 40 nt, or about 50 nt, or about 60 nt, or about 70 nt, or about 80 nt, or about 90 nt, or about 100 nt, or about 110 nt, or about 120 nt, or about 130 nt, or about 140 nt, or about 150 nt, or about 160 nt, or about 170 nt, or about 180 nt, or about 190 nt, or about 200 nt, or about 210 nt, or about 220 nt, or about 230 nt, or about 240 nt, or about 250 nt long.
Other embodiments are directed to a method for reprogramming a cell. In one embodiment, the cell is reprogrammed by contacting the cell with one or more nucleic acids. In one embodiment, the cell is contacted with a plurality of nucleic acids encoding at least one of: Oct4 protein, Sox2 protein, Klf4 protein, c-Myc protein, Lin28 protein or a biologically active fragment, variant or derivative thereof. In another embodiment, the cell is contacted with a plurality of nucleic acids encoding a plurality of proteins including: Oct4 protein, Sox2 protein, Klf4 protein, and c-Myc protein or one or more biologically active fragments, variants or derivatives thereof. Still other embodiments are directed to a method for gene editing a cell. In one embodiment, the cell is gene-edited by contacting the cell with one or more nucleic acids.
Animal models are routinely used to study the effects of biological processes. In certain situations, for example, when studying a human disease, an animal model containing a modified genome can be beneficial, in part because such an animal model may more closely mimic the human disease phenotype. Certain embodiments are therefore directed to a method for creating an organism containing one or more genetic modifications (a.k.a. “mutations”, a.k.a. “gene edits”). In one embodiment, the one or more genetic modifications is generated by transfecting a cell with one or more nucleic acids encoding one or more gene-editing proteins. In another embodiment, the one or more nucleic acids include a synthetic RNA molecule. In one embodiment, the one or more gene-editing proteins include at least one of: a zinc finger nuclease, a TALEN, a clustered regularly interspaced short palindromic repeat (CRISPR)-associated protein, a nuclease, a meganuclease, and a nickase or a biologically active fragment or variant thereof. In one embodiment, the cell is a pluripotent cell. In another embodiment, the cell is an embryonic stem cell. In a further embodiment, the cell is an embryo. In a still further embodiment, the cell is a member of: an animal cell, a plant cell, a yeast cell, and a bacterial cell. In one embodiment, the cell is a rodent cell. In another embodiment, the cell is a human cell. In certain embodiments, the cell is transfected with one or more nucleic acids encoding one or more gene-editing proteins and one or more nucleic acids encoding one or more repair templates. In one embodiment, the cell is introduced into a blastocyst. In another embodiment, the cell is introduced into a pseudopregnant female. In a further embodiment, the presence or absence of the genetic modification in the offspring is determined. In a still further embodiment, the determining is by direct sequencing. In one embodiment, the organism is livestock, for example, a pig, a cow, etc. In another embodiment, the organism is a pet, for example, a dog, a cat, a fish, etc.
In certain situations, for example, when modifying the genome of a target cell by the addition of a nucleic-acid sequence, it can be advantageous to insert the nucleic-acid sequence into a safe-harbor location, in part to reduce the risks associated with random insertion. Certain embodiments are therefore directed to a method for inserting a nucleic-acid sequence into a safe-harbor location. In one embodiment, the cell is a human cell and the safe-harbor location is the AAVS1 locus. In another embodiment, the cell is a rodent cell and the safe-harbor location is the Rosa26 locus. In one embodiment, the cell is further contacted with one or more nucleic acids encoding one or more repair templates. Other embodiments are directed to a kit for altering the DNA sequence of a cell. In one embodiment, the cell is a human cell, and the target DNA molecule comprises a nucleotide sequence that encodes the AAVS1 locus. In another embodiment, the cell is a rodent cell, and the target DNA molecule comprises a nucleotide sequence that encodes the Rosa26 locus. Other embodiments are directed to a method for generating a reporter cell by contacting the cell with one or more nucleic acids encoding one or more gene-editing proteins and one or more nucleic acids encoding one or more repair templates. In one embodiment, the one or more repair templates comprise DNA. In another embodiment, the one or more repair templates encode one or more fluorescent proteins. In a further embodiment, the one or more repair templates encode at least part of the promoter region of a gene.
In certain situations, for example, when generating a library of gene-edited cells, it can be beneficial to increase the efficiency of gene editing, in part to reduce the cost of cell characterization. It has now been discovered that gene-editing efficiency can be increased by repeatedly contacting a cell with synthetic RNA encoding one or more gene-editing proteins. Certain embodiments are therefore directed to a method for gene editing a cell by repeatedly contacting the cell with one or more nucleic acids encoding one or more gene-editing proteins. In one embodiment, the cell is contacted at least twice during five consecutive days. In another embodiment, the cell is contacted twice at an interval of between about 24 hours and about 48 hours.
In cancer, the survival and proliferation of malignant cells can be due in part to the presence of specific genetic abnormalities that are not generally present in the patient. It has now been discovered that gene-editing proteins can be used to target survival and proliferation-associated pathways, and that when used in this manner, gene-editing proteins and nucleic acids encoding gene-editing proteins can constitute potent anti-cancer therapeutics. Certain embodiments are therefore directed to an anti-cancer therapeutic. In one embodiment, the therapeutic is a therapeutic composition that inhibits the survival and/or prevents, slows or otherwise limits the proliferation of a cell. In another embodiment, the cell is a cancer cell. In a further embodiment, the therapeutic comprises one or more gene-editing proteins or a nucleic acid that encodes one or more gene-editing proteins. In a still further embodiment, the one or more gene-editing proteins target one or more sequences that promote survival and/or proliferation of the cell. Such sequences include, but are not limited to: apoptosis-related genes, including genes of the inhibitor of apoptosis (IAP) family (See, e.g., Table 2 and Table 2 of U.S. Provisional Application No. 61/721,302, the contents of which are hereby incorporated by reference), such as BIRC5, sequences associated with telomere maintenance, such as the gene telomerase reverse transcriptase (TERT) and the telomerase RNA component (TERC), sequences affecting angiogenesis, such as the gene VEGF, and other cancer-associated genes, including: BRAF, BRCA1, BRCA2, CDKN2A, CTNNB1, EGFR, the MYC family, the RAS family, PIK3CA, PIK3R1, PKN3, TP53, PTEN, RET, SMAD4, KIT, MET, APC, RB1, the VEGF family, TNF, and genes of the ribonucleotide reductase family. Example gene-editing protein target sequences for BIRC5 are set forth in Table 3 and in Table 3 of U.S. Provisional Application No. 61/721,302, the contents of which are hereby incorporated by reference, and are given by way of example, and not by way of limitation. In one embodiment, at least one of the one or more sequences is present in both malignant and non-malignant cells. In another embodiment, at least one of the one or more sequences is enriched in malignant cells. In a further embodiment, at least one of the one or more sequences is enriched in non-malignant cells. In one embodiment, the therapeutic composition further comprises a nucleic acid encoding one or more repair templates. In another embodiment, the one or more gene-editing proteins induce the cells to express an inactive or dominant-negative form of a protein. In a further embodiment, the protein is a member of the IAP family. In a still further embodiment, the protein is survivin.







TABLE 2







Exemplary Inhibitor of Apoptosis (IAP) Genes













BIR
CARD
RING


Name
Length/aa
Domains
Domain
Domain














BIRC1 (neuronal apoptosis-
1,403
3
N
N


inhibitory protein)


BIRC2 (c-IAP1 protein)
604
3
Y
Y


BIRC3 (c-IAP2 protein)
618
3
Y
Y


BIRC4 (X-linked IAP)
497
3
N
Y


BIRC5 (survivin protein)
142
1
N
N


BIRC6 (BRUCE/apollon
4845
1
N
N


protein)


BIRC7 (livin protein)
298
1
N
Y


ILP2 (tissue-specific
236
1
N
Y


homolog of BIRC4)
















TABLE 3







Exemplary Gene Editing-Protein Target Sequences for BIRC5













SEQ

SEQ




ID

ID


Target
Left
NO.
Right
NO.





UTR
TAAGAGGGCGTGCGCTCCCG
83
TCAAATCTGGCGGTTAATGG
84


 


Start Codon
TTGGCAGAGGTGGCGGCGGC
85
TGCCAGGCAGGGGGCAACGT
86


 


Exon 1
TTGCCCCCTGCCTGGCAGCC
16
TTCTTGAATGTAGAGATGCG
17


 


Exon 2
TCCACTGCCCCACTGAGAAC
87
TCCTTGAAGCAGAAGAAACA
88


 


Exon 4
TAAAAAGCATTCGTCCGGTT
89
TTCTTCAAACTGCTTCTTGA
90


 


Exon 5
TTGAGGAAACTGCGGAGAAA
91
TCCATGGCAGCCAGCTGCTC
92









Other embodiments are directed to a method for treating cancer comprising administering to a patient a therapeutically effective amount of a gene-editing protein or a nucleic acid encoding one or more gene-editing proteins. In one embodiment, the treatment results in the growth of cancer cells in the patient being reduced or halted. In another embodiment, the treatment results in delayed progression or remission of the cancer. In one embodiment, the target DNA molecule comprises the BIRC5 gene. In another embodiment, the target DNA molecule comprises a sequence selected from: SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, and SEQ ID NO: 15. In a further embodiment, a plurality of adjacent binding sites are at least about 50% or at least about 60% or at least about 70% or at least about 80% or at least about 90% or at least about 95% or at least about 98%, or at least about 99% homologous to one or more sequences listed in Table 3, Table 4, Table 3 of U.S. Provisional Application No. 61/721,302, the contents of which are hereby incorporated by reference, Table 1 of U.S. Provisional Application No. 61/785,404, the contents of which are hereby incorporated by reference or Table 1 of U.S. Provisional Application No. 61/842,874, the contents of which are hereby incorporated by reference. In certain situations, a gene-editing protein with a truncated N-terminal domain can be used to eliminate the first-base-T restriction on the binding-site sequence. In some embodiments, the cancer is glioma. In one embodiment, the patient has previously undergone surgery and/or radiation therapy and/or concurrently undergoes surgery and/or radiation therapy. In another embodiment, the administering is by one or more of: intrathecal injection, intracranial injection, intravenous injection, perfusion, subcutaneous injection, intraperitoneal injection, intraportal injection, and topical delivery.







TABLE 4







Exemplary BIRC5 Binding Sites















SEQ

SEQ





ID

ID













Gene
#
Left
NO
Right
NO
Spacing





BIRC5
 1
TGGGTGCCCCGACGT
 18
TGCGGTGGTCCTTGA
 19
14




TGCCC

GAAAG




 


BIRC5
 2
TGGGTGCCCCGACGT
 93
TAGAGATGCGGTGGT
 94
20




TGCCC

CCTTG




 


BIRC5
 3
TGCCCCGACGTTGCCC
 95
TAGAGATGCGGTGGT
 96
16




CCTG

CCTTG




 


BIRC5
 4
TGCCCCGACGTTGCCC
 97
TGTAGAGATGCGGTG
 98
18




CCTG

GTCCT




 


BIRC5
 5
TCAAGGACCACCGCA
 20
TGCAGGCGCAGCCCT
 21
20




TCTCT

CCAAG




 


BIRC5
 6
TCTCTACATTCAAGAA
 99
TCACCCGCTCCGGGG
100
20




CTGG

TGCAG




 


BIRC5
 7
TCTACATTCAAGAACT
101
TCACCCGCTCCGGGG
102
18




GGCC

TGCAG




 


BIRC5
 8
TCTACATTCAAGAACT
103
TCTCACCCGCTCCGG
104
20




GGCC

GGTGC




 


BIRC5
 9
TACATTCAAGAACTG
105
TCACCCGCTCCGGGG
106
16




GCCCT

TGCAG




 


BIRC5
10
TACATTCAAGAACTG
107
TCTCACCCGCTCCGG
108
18




GCCCT

GGTGC




 


BIRC5
11
TTCAAGAACTGGCCC
109
TCTCACCCGCTCCGG
110
14




TTCTT

GGTGC




 


BIRC5
 1
TCCCTTGCAGATGGCC
111
TGGCTCGTTCTCAGT
112
15




GAGG

GGGGC




 


BIRC5
 2
TCCCTTGCAGATGGCC
113
TCTGGCTCGTTCTCA
114
17




GAGG

GTGGG




 


BIRC5
 3
TGGCCGAGGCTGGCT
 22
TGGGCCAAGTCTGGC
 23
15




TCATC

TCGTT




 


BIRC5
 4
TCCACTGCCCCACTGA
115
TCCTTGAAGCAGAAG
116
18




GAAC

AAACA




 


BIRC5
 5
TGCCCCACTGAGAAC
117
TCCAGCTCCTTGAAG
118
19




GAGCC

CAGAA




 


BIRC5
 6
TGCCCCACTGAGAAC
119
TTCCAGCTCCTTGAA
120
20




GAGCC

GCAGA




 


BIRC5
 7
TTGGCCCAGTGTTTCT
 24
TCGTCATCTGGCTCC
 25
16




TCTG

CAGCC




 


BIRC5
 8
TGGCCCAGTGTTTCTT
121
TCGTCATCTGGCTCC
122
15




CTGC

CAGCC




 


BIRC5
 9
TGGCCCAGTGTTTCTT
123
TGGGGTCGTCATCTG
124
20




CTGC

GCTCC




 


BIRC5
10
TGTTTCTTCTGCTTCA
125
TACATGGGGTCGTCA
126
16




AGGA

TCTGG




 


BIRC5
11
TGTTTCTTCTGCTTCA
127
TTACATGGGGTCGTC
128
17




AGGA

ATCTG




 


BIRC5
12
TTTCTTCTGCTTCAAG
129
TACATGGGGTCGTCA
130
14




GAGC

TCTGG




 


BIRC5
13
TTTCTTCTGCTTCAAG
131
TTACATGGGGTCGTC
132
15




GAGC

ATCTG




 


BIRC5
14
TTCTTCTGCTTCAAGG
133
TTACATGGGGTCGTC
134
14




AGCT

ATCTG




 


BIRC5
 1
TTTTCTAGAGAGGAA
135
TGACAGAAAGGAAA
136
15


 

CATAA

GCGCAA




 


BIRC5
 2
TTTTCTAGAGAGGAA
137
TTGACAGAAAGGAA
138
16




CATAA

AGCGCA




 


BIRC5
 3
TTTTCTAGAGAGGAA
139
TCTTGACAGAAAGGA
140
18




CATAA

AAGCG




 


BIRC5
 4
TAGAGAGGAACATAA
141
TGCTTCTTGACAGAA
142
17




AAAGC

AGGAA




 


BIRC5
 5
TAAAAAGCATTCGTC
143
TCTTCAAACTGCTTC
144
14




CGGTT

TTGAC




 


BIRC5
 6
TAAAAAGCATTCGTC
145
TTCTTCAAACTGCTT
146
15




CGGTT

CTTGA




 


BIRC5
 7
TAAAAAGCATTCGTC
147
TAATTCTTCAAACTG
148
18




CGGTT

CTTCT




 


BIRC5
 8
TAAAAAGCATTCGTC
149
TTAATTCTTCAAACT
150
19




CGGTT

GCTTC




 


BIRC5
 9
TTCGTCCGGTTGCGCT
151
TCACCAAGGGTTAAT
152
20




TTCC

TCTTC




 


BIRC5
10
TCGTCCGGTTGCGCTT
153
TCACCAAGGGTTAAT
154
19




TCCT

TCTTC




 


BIRC5
11
TCGTCCGGTTGCGCTT
155
TTCACCAAGGGTTAA
156
20




TCCT

TTCTT




 


BIRC5
12
TCCGGTTGCGCTTTCC
157
TCACCAAGGGTTAAT
158
16




TTTC

TCTTC




 


BIRC5
13
TCCGGTTGCGCTTTCC
159
TTCACCAAGGGTTAA
160
17




TTTC

TTCTT




 


BIRC5
14
TTGCGCTTTCCTTTCT
161
TCAAAAATTCACCAA
162
19




GTCA

GGGTT




 


BIRC5
15
TTGCGCTTTCCTTTCT
163
TTCAAAAATTCACCA
164
20




GTCA

AGGGT




 


BIRC5
16
TGCGCTTTCCTTTCTG
 26
TCAAAAATTCACCAA
 27
18




TCAA

GGGTT




 


BIRC5
17
TGCGCTTTCCTTTCTG
165
TTCAAAAATTCACCA
166
19




TCAA

AGGGT




 


BIRC5
18
TGCGCTTTCCTTTCTG
167
TTTCAAAAATTCACC
168
20




TCAA

AAGGG




 


BIRC5
19
TTTCCTTTCTGTCAAG
169
TTCAAAAATTCACCA
170
14




AAGC

AGGGT




 


BIRC5
20
TTTCCTTTCTGTCAAG
171
TTTCAAAAATTCACC
172
15




AAGC

AAGGG




 


BIRC5
21
TTTCCTTTCTGTCAAG
173
TCCAGTTTCAAAAAT
174
20




AAGC

TCACC




 


BIRC5
22
TTCCTTTCTGTCAAGA
175
TTTCAAAAATTCACC
176
14




AGCA

AAGGG




 


BIRC5
23
TTCCTTTCTGTCAAGA
177
TCCAGTTTCAAAAAT
178
19




AGCA

TCACC




 


BIRC5
24
TCCTTTCTGTCAAGAA
179
TCCAGTTTCAAAAAT
180
18




GCAG

TCACC




 


BIRC5
25
TCCTTTCTGTCAAGAA
181
TGTCCAGTTTCAAAA
182
20




GCAG

ATTCA




 


BIRC5
26
TTTCTGTCAAGAAGC
183
TCCAGTTTCAAAAAT
184
15




AGTTT

TCACC




 


BIRC5
27
TTTCTGTCAAGAAGC
185
TGTCCAGTTTCAAAA
186
17




AGTTT

ATTCA




 


BIRC5
28
TTTCTGTCAAGAAGC
187
TCTGTCCAGTTTCAA
188
19




AGTTT

AAATT




 


BIRC5
29
TTCTGTCAAGAAGCA
189
TCCAGTTTCAAAAAT
190
14




GTTTG

TCACC




 


BIRC5
30
TTCTGTCAAGAAGCA
191
TGTCCAGTTTCAAAA
192
16




GTTTG

ATTCA




 


BIRC5
31
TTCTGTCAAGAAGCA
193
TCTGTCCAGTTTCAA
194
18




GTTTG

AAATT




 


BIRC5
32
TTCTGTCAAGAAGCA
195
TCTCTGTCCAGTTTC
196
20




GTTTG

AAAAA




 


BIRC5
33
TCTGTCAAGAAGCAG
197
TGTCCAGTTTCAAAA
198
15




TTTGA

ATTCA




 


BIRC5
34
TCTGTCAAGAAGCAG
199
TCTGTCCAGTTTCAA
200
17




TTTGA

AAATT




 


BIRC5
35
TCTGTCAAGAAGCAG
201
TCTCTGTCCAGTTTC
202
19




TTTGA

AAAAA




 


BIRC5
36
TCTGTCAAGAAGCAG
203
TTCTCTGTCCAGTTTC
204
20




TTTGA

AAAA




 


BIRC5
37
TGTCAAGAAGCAGTT
205
TCTGTCCAGTTTCAA
206
15




TGAAG

AAATT




 


BIRC5
38
TGTCAAGAAGCAGTT
207
TCTCTGTCCAGTTTC
208
17




TGAAG

AAAAA




 


BIRC5
39
TGTCAAGAAGCAGTT
209
TTCTCTGTCCAGTTTC
210
18




TGAAG

AAAA




 


BIRC5
40
TGTCAAGAAGCAGTT
211
TTTCTCTGTCCAGTTT
212
19




TGAAG

CAAA




 


BIRC5
41
TCAAGAAGCAGTTTG
213
TCTCTGTCCAGTTTC
214
15




AAGAA

AAAAA




 


BIRC5
42
TCAAGAAGCAGTTTG
215
TTCTCTGTCCAGTTTC
216
16




AAGAA

AAAA




 


BIRC5
43
TCAAGAAGCAGTTTG
217
TTTCTCTGTCCAGTTT
218
17




AAGAA

CAAA




 


BIRC5
44
TTTGAAGAATTAACC
219
TCTTGGCTCTTTCTCT
220
15




CTTGG

GTCC




 


BIRC5
45
TTGAAGAATTAACCC
221
TCTTGGCTCTTTCTCT
222
14




TTGGT

GTCC




 


BIRC5
46
TTGAAGAATTAACCC
223
TTCTTGGCTCTTTCTC
224
15




TTGGT

TGTC




 


BIRC5
47
TGAAGAATTAACCCT
225
TTCTTGGCTCTTTCTC
226
14




TGGTG

TGTC




 


BIRC5
48
TGAAGAATTAACCCT
227
TGTTCTTGGCTCTTTC
228
16




TGGTG

TCTG




 


BIRC5
49
TTAACCCTTGGTGAAT
229
TACAATTTTGTTCTT
230
17




TTTT

GGCTC




 


BIRC5
50
TAACCCTTGGTGAATT
231
TACAATTTTGTTCTT
232
16




TTTG

GGCTC




 


BIRC5
51
TAACCCTTGGTGAATT
233
TACATACAATTTTGT
234
20




TTTG

TCTTG




 


BIRC5
52
TTGGTGAATTTTTGAA
235
TACATACAATTTTGT
236
14




ACTG

TCTTG




 


BIRC5
 1
TTATTTCCAGGCAAA
237
TCCGCAGTTTCCTCA
238
17




GGAAA

AATTC




 


BIRC5
 2
TTATTTCCAGGCAAA
239
TCTCCGCAGTTTCCT
240
19




GGAAA

CAAAT




 


BIRC5
 3
TTATTTCCAGGCAAA
241
TTCTCCGCAGTTTCC
242
20




GGAAA

TCAAA




 


BIRC5
 4
TATTTCCAGGCAAAG
243
TCCGCAGTTTCCTCA
244
16




GAAAC

AATTC




 


BIRC5
 5
TATTTCCAGGCAAAG
245
TCTCCGCAGTTTCCT
246
18




GAAAC

CAAAT




 


BIRC5
 6
TATTTCCAGGCAAAG
247
TTCTCCGCAGTTTCC
248
19




GAAAC

TCAAA




 


BIRC5
 7
TATTTCCAGGCAAAG
249
TTTCTCCGCAGTTTC
250
20




GAAAC

CTCAA




 


BIRC5
 8
TCCAGGCAAAGGAAA
251
TCTCCGCAGTTTCCT
252
14




CCAAC

CAAAT




 


BIRC5
 9
TCCAGGCAAAGGAAA
253
TTCTCCGCAGTTTCC
254
15




CCAAC

TCAAA




 


BIRC5
10
TCCAGGCAAAGGAAA
255
TTTCTCCGCAGTTTC
256
16




CCAAC

CTCAA




 


BIRC5
11
TTTGAGGAAACTGCG
257
TCCATGGCAGCCAGC
258
16




GAGAA

TGCTC




 


BIRC5
12
TTTGAGGAAACTGCG
259
TCAATCCATGGCAGC
260
20




GAGAA

CAGCT




 


BIRC5
13
TTGAGGAAACTGCGG
261
TCCATGGCAGCCAGC
262
15




AGAAA

TGCTC




 


BIRC5
14
TTGAGGAAACTGCGG
263
TCAATCCATGGCAGC
264
19




AGAAA

CAGCT




 


BIRC5
15
TGAGGAAACTGCGGA
265
TCCATGGCAGCCAGC
266
14




GAAAG

TGCTC




 


BIRC5
16
TGAGGAAACTGCGGA
267
TCAATCCATGGCAGC
268
18




GAAAG

CAGCT









Certain embodiments are directed to a method for treating cancer comprising: a. removing a biopsy containing one or more cancerous cells from a patient, b. determining the sequence of a cancer-associated genetic marker in the one or more cancerous cells, and c. administering to the patient a therapeutically effective amount of a gene-editing protein or a nucleic acid encoding a gene-editing protein, wherein the sequence of the target DNA molecule is at least about 50% or about 60% or about 70% or about 80% or about 90% or about 95% or about 98%, or about 99% homologous to the sequence of the cancer-associated genetic marker. In one embodiment, the method further comprises comparing the sequence of one or more cancer-associated genetic markers in the one or more cancerous cells to the sequence of the same cancer-associated genetic markers in one or more non-cancerous cells, selecting a cancer-associated genetic marker having a sequence that is different in the one or more cancerous cells and the one or more non-cancerous cells, and wherein the sequence of the target DNA molecule or binding site is at least about 50% or about 60% or about 70% or about 80% or about 90% or about 95% or about 98% or about 99% homologous to the sequence of the selected cancer-associated genetic marker.
Many cancer cells express survivin, a member of the inhibitor of apoptosis (IAP) protein family that, in humans, is encoded by the BIRC5 gene. Using RNA interference to reduce expression of certain mRNA molecules, including survivin mRNA, can transiently inhibit the growth of certain cancer cells. However, previous methods of using RNA interference to reduce expression of survivin mRNA yield temporary effects, and result in only a short increase in mean time-to-death (TTD) in animal models. It has now been discovered that inducing a cell to express one or more gene-editing proteins that target the BIRC5 gene can result in disruption of the BIRC5 gene, can induce the cell to express and/or secrete a non-functional variant of survivin protein, can induce the cell to express and/or secrete a dominant-negative variant of survivin protein, can trigger activation of one or more apoptosis pathways in the cell and nearby cells, can slow or halt the growth of the cell and nearby cells, can result in the death of the cell and nearby cells, can inhibit the progression of cancer, and can result in remission in a cancer patient. Certain embodiments are therefore directed to a gene-editing protein that targets the BIRC5 gene. In one embodiment, the gene-editing protein binds to one or more regions in the BIRC5 gene. In another embodiment, the gene-editing protein binds to one or more regions of a sequence selected from: SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, and SEQ ID NO: 15. In a further embodiment, the gene-editing protein binds to one or more sequences selected from: SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, and SEQ ID NO: 27. In a still further embodiment, the gene-editing protein binds to one or more nucleic-acid sequences that encode SEQ ID NO: 34 or a biologically active fragment, variant or analogue thereof. In a still further embodiment, the gene-editing protein binds to one or more sequences selected from Table 3, Table 4, Table 3 of U.S. Provisional Application No. 61/721,302, the contents of which are hereby incorporated by reference, Table 1 of U.S. Provisional Application No. 61/785,404, the contents of which are hereby incorporated by reference or Table 1 of U.S. Provisional Application No. 61/842,874, the contents of which are hereby incorporated by reference or to one or more sequences that is at least about 50% or at least about 60% or at least about 70% or at least about 80% or at least about 90% or at least about 95% or at least about 98%, or about 99% homologous to one or more sequences selected from Table 3, Table 4, Table 3 of U.S. Provisional Application No. 61/721,302, the contents of which are hereby incorporated by reference, Table 1 of U.S. Provisional Application No. 61/785,404, the contents of which are hereby incorporated by reference or Table 1 of U.S. Provisional Application No. 61/842,874, the contents of which are hereby incorporated by reference. In one embodiment, the gene-editing protein creates one or more nicks or double-strand breaks in the DNA of the cell. In another embodiment, the one or more nicks or double-strand breaks is created in the BIRC5 gene. In a further embodiment, the one or more nicks or double-strand breaks is created in one or more exons of the BIRC5 gene. In a still further embodiment, the one or more nicks or double-strand breaks is created in a sequence selected from: SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, and SEQ ID NO: 15. In a still further embodiment, the one or more nicks or double-strand breaks is created within a sequence that encodes an inhibitor of apoptosis domain (aka. “IAP”, “IAP domain”, “IAP repeat”, “baculovirus inhibitor of apoptosis protein repeat”, “BIR”, etc.). In a still further embodiment, the gene-editing protein binds to one or more sequences selected from Table 5, Table 2 of U.S. Provisional Application No. 61/785,404, the contents of which are hereby incorporated by reference or Table 2 of U.S. Provisional Application No. 61/842,874, the contents of which are hereby incorporated by reference or to one or more sequences that is at least about 50% or at least about 60% or at least about 70% or at least about 80% or at least about 90% or at least about 95% or at least about 98% homologous to one or more sequences selected from Table 5, Table 2 of U.S. Provisional Application No. 61/785,404, the contents of which are hereby incorporated by reference or Table 2 of U.S. Provisional Application No. 61/842,874, the contents of which are hereby incorporated by reference. In yet another embodiment, the gene editing protein binds to a sequence that encodes one or more genes selected from Table 2, Table 5, Table 6, Table 7, Table 4 of U.S. Provisional Application No. 61/721,302, the contents of which are hereby incorporated by reference, Table 2 of U.S. Provisional Application No. 61/785,404, the contents of which are hereby incorporated by reference or Table 2 of U.S. Provisional Application No. 61/842,874, the contents of which are hereby incorporated by reference.







TABLE 5







Exemplary Cancer-Associated Gene Binding Sites















SEQ

SEQ





ID

ID













Gene
#
Left
NO
Right
NO
Spacing





CDK1
1
TTTAGGATCTACCATAC
269
TCTCTATTTTGGTAT
270
15




CCA

AATCT




 


CDK1
2
TTTAGGATCTACCATAC
271
TTCTCTATTTTGGTA
272
16




CCA

TAATC




 


CDK1
3
TTTAGGATCTACCATAC
273
TTTCTCTATTTTGGT
274
17




CCA

ATAAT




 


CDK1
4
TTAGGATCTACCATACC
275
TCTCTATTTTGGTAT
276
14




CAT

AATCT




 


CDK1
5
TTAGGATCTACCATACC
277
TTCTCTATTTTGGTA
278
15




CAT

TAATC




 


CDK1
1
TCACACAGCATATTATT
279
TACCCTTATACACA
280
17




TAC

ACTCCA




 


CDK1
2
TCACACAGCATATTATT
281
TCTACCCTTATACAC
282
19




TAC

AACTC




 


CDK1
3
TACTTTGTTTCAGGTAC
283
TGTAGTTTTGTGTCT
284
14




CTA

ACCCT




 


CDK1
4
TACTTTGTTTCAGGTAC
285
TGACCTGTAGTTTTG
286
19




CTA

TGTCT




 


CDK1
5
TTTGTTTCAGGTACCTA
287
TGACCTGTAGTTTTG
288
16




TGG

TGTCT




 


CDK2
1
TGACCCGACTCGCTGGC
289
TCCGATCTTTTCCAC
290
15




GCT

CTTTT




 


CDK2
2
TGACCCGACTCGCTGGC
291
TCTCCGATCTTTTCC
292
17




GCT

ACCTT




 


CDK2
3
TCGCTGGCGCTTCATGG
293
TACGTGCCCTCTCCG
294
17




AGA

ATCTT




 


CDK2
4
TTCATGGAGAACTTCCA
295
TACACAACTCCGTA
296
19




AAA

CGTGCC




 


CDK2
5
TCATGGAGAACTTCCAA
297
TACACAACTCCGTA
298
18




AAG

CGTGCC




 


CDK2
1
TTTCCCAACCTCTCCAA
299
TCTCGGATGGCAGT
300
14




GTG

ACTGGG




 


CDK2
2
TTCCCAACCTCTCCAAG
301
TCTCTCGGATGGCA
302
15




TGA

GTACTG




 


CDK2
3
TCCCAACCTCTCCAAGT
303
TCTCTCGGATGGCA
304
14




GAG

GTACTG




 


CDK2
4
TCTCCAAGTGAGACTGA
305
TAAGCAGAGAGATC
306
18




GGG

TCTCGG




 


CDK2
5
TCTCCAAGTGAGACTGA
307
TTAAGCAGAGAGAT
308
19




GGG

CTCTCG




 


CDK3
1
TGTTTCCCAGGCAGCTC
309
TCTCCGATCTTCTCT
310
19




TGT

ACCTT




 


CDK3
2
TTTCCCAGGCAGCTCTG
311
TCTCCGATCTTCTCT
312
17




TGG

ACCTT




 


CDK3
3
TTCCCAGGCAGCTCTGT
313
TCTCCGATCTTCTCT
314
16




GGC

ACCTT




 


CDK3
4
TCCCAGGCAGCTCTGTG
315
TCTCCGATCTTCTCT
316
15




GCC

ACCTT




 


CDK3
5
TGGATATGTTCCAGAAG
317
TACACCACCCCATA
318
15




GTA

GGTGCC




 


CDK3
1
TGCCCACGGCTGTGCCC
319
TGGCAGTGCTTGGG
320
19




TTG

ACCCCC




 


CDK3
2
TGTGCCCTTGTTTCTTG
321
TCCCTGATGGCAGT
322
16




CAG

GCTTGG




 


CDK3
3
TTTCTTGCAGGGAGATG
323
TGAGCAGCGAGATC
324
20




GAG

TCCCTG




 


CDK3
4
TTCTTGCAGGGAGATGG
325
TGAGCAGCGAGATC
326
19




AGG

TCCCTG




 


CDK3
5
TTCTTGCAGGGAGATGG
327
TTGAGCAGCGAGAT
328
20




AGG

CTCCCT




 


CDK4
1
TGTGATTGTAGGGTCTC
329
TGGCTCATATCGAG
330
14




CCT

AGGTAG




 


CDK4
2
TGATTGTAGGGTCTCCC
331
TCAGCCACTGGCTC
332
20




TTG

ATATCG




 


CDK4
3
TTGTAGGGTCTCCCTTG
333
TCAGCCACTGGCTC
334
17




ATC

ATATCG




 


CDK4
4
TGTAGGGTCTCCCTTGA
335
TCAGCCACTGGCTC
336
16




TCT

ATATCG




 


CDK4
5
TAGGGTCTCCCTTGATC
337
TCAGCCACTGGCTC
338
14




TGA

ATATCG




 


CDK4
1
TTGAAAAGTGAGCATTT
339
TCGGGATGTGGCAC
340
16




ACT

AGACGT




 


CDK4
2
TTGAAAAGTGAGCATTT
341
TTCGGGATGTGGCA
342
17




ACT

CAGACG




 


CDK4
3
TGAAAAGTGAGCATTTA
343
TCGGGATGTGGCAC
344
15




CTC

AGACGT




 


CDK4
4
TGAAAAGTGAGCATTTA
345
TTCGGGATGTGGCA
346
16




CTC

CAGACG




 


CDK4
5
TGAAAAGTGAGCATTTA
347
TCAGTTCGGGATGT
348
20




CTC

GGCACA




 


CDK5
1
TACGAGAAACTGGAAA
349
TGCAGGAACATCTC
350
15




AGAT

GAGATT




 


CDK5
2
TACGAGAAACTGGAAA
351
TTGCAGGAACATCT
352
16




AGAT

CGAGAT




 


CDK5
3
TACGAGAAACTGGAAA
353
TCTTGCAGGAACAT
354
18




AGAT

CTCGAG




 


CDK5
1
TCCTTCCCCTAGGCACC
355
TGAGTCTCCCGGTTT
356
15




TAC

TTGGC




 


CDK5
2
TCCTTCCCCTAGGCACC
357
TCATGAGTCTCCCG
358
18




TAC

GTTTTT




 


CDK5
3
TCCTTCCCCTAGGCACC
359
TCTCATGAGTCTCCC
360
20




TAC

GGTTT




 


CDK5
4
TTCCCCTAGGCACCTAC
361
TCATGAGTCTCCCG
362
15




GGA

GTTTTT




 


CDK5
5
TTCCCCTAGGCACCTAC
363
TCTCATGAGTCTCCC
364
17




GGA

GGTTT




 


CDK6
1
TGTGCCGCGCTGACCAG
365
TAGGCGCCCTCCCC
366
15




CAG

GATCTC




 


CDK6
2
TGTGCCGCGCTGACCAG
367
TCCCATAGGCGCCC
368
20




CAG

TCCCCG




 


CDK6
3
TGCCGCGCTGACCAGCA
369
TCCCATAGGCGCCC
370
18




GTA

TCCCCG




 


CDK6
4
TGCCGCGCTGACCAGCA
371
TTCCCATAGGCGCC
372
19




GTA

CTCCCC




 


CDK6
5
TGACCAGCAGTACGAA
373
TGAACACCTTCCCAT
374
19




TGCG

AGGCG




 


CDK6
1
TCTAGGTTGTTTGATGT
375
TAGTTTGGTTTCTCT
376
14




GTG

GTCTG




 


CDK6
2
TCTAGGTTGTTTGATGT
377
TAAAGTTAGTTTGGT
378
20




GTG

TTCTC




 


CDK6
3
TAGGTTGTTTGATGTGT
379
TAAAGTTAGTTTGGT
380
18




GCA

TTCTC




 


CDK6
4
TTGTTTGATGTGTGCAC
381
TAAAGTTAGTTTGGT
382
14




AGT

TTCTC




 


CDK6
5
TTGATGTGTGCACAGTG
383
TCAAACACTAAAGT
384
18




TCA

TAGTTT




 


EGFR
1
TCCGGGACGGCCGGGG
385
TCGCCGGGCAGAGC
386
15




CAGC

GCAGCC




 


EGFR
1
TCTTCCAGTTTGCCAAG
387
TCAAAAGTGCCCAA
388
14




GCA

CTGCGT




 


EGFR
2
TCTTCCAGTTTGCCAAG
389
TGATCTTCAAAAGT
390
20




GCA

GCCCAA




 


EGFR
3
TTCCAGTTTGCCAAGGC
391
TGATCTTCAAAAGT
392
18




ACG

GCCCAA




 


EGFR
4
TCCAGTTTGCCAAGGCA
393
TGATCTTCAAAAGT
394
17




CGA

GCCCAA




 


EGFR
5
TCACGCAGTTGGGCACT
395
TGAACATCCTCTGG
396
14




TTT

AGGCTG




 


HIF1A
1
TGAAGACATCGCGGGG
397
TGTCGTTCGCGCCGC
398
15




ACCG

CGGCG




 


HIF1A
2
TGAAGACATCGCGGGG
399
TTGTCGTTCGCGCCG
400
16




ACCG

CCGGC




 


HIF1A
3
TGAAGACATCGCGGGG
401
TCTTGTCGTTCGCGC
402
18




ACCG

CGCCG




 


HIF1A
4
TGAAGACATCGCGGGG
403
TTCTTGTCGTTCGCG
404
19




ACCG

CCGCC




 


HIF1A
5
TGAAGACATCGCGGGG
405
TTTCTTGTCGTTCGC
406
20




ACCG

GCCGC




 


HIF1A
1
TCTCGTGTTTTTCTTGTT
407
TCTTTTCGACGTTCA
408
14




GT

GAACT




 


HIF1A
2
TCTCGTGTTTTTCTTGTT
409
TTCTTTTCGACGTTC
410
15




GT

AGAAC




 


HIF1A
3
TCTCGTGTTTTTCTTGTT
411
TTTCTTTTCGACGTT
412
16




GT

CAGAA




 


HIF1A
4
TCTCGTGTTTTTCTTGTT
413
TTTTCTTTTCGACGT
414
17




GT

TCAGA




 


HIF1A
5
TTCTTGTTGTTGTTAAG
415
TCGAGACTTTTCTTT
416
14




TAG

TCGAC




 


HSPA4
1
TGGTGGGCATAGACCTG
417
TGCCGCCGGCGCGG
418
20




GGC

GCCACA




 


HSPA4
2
TGGGCATAGACCTGGG
419
TGCCGCCGGCGCGG
420
17




CTTC

GCCACA




 


HSPA4
3
TAGACCTGGGCTTCCAG
421
TCGATGCCGCCGGC
422
15




AGC

GCGGGC




 


HSPA4
4
TAGACCTGGGCTTCCAG
423
TCTCGATGCCGCCG
424
17




AGC

GCGCGG




 


HSPA4
5
TAGACCTGGGCTTCCAG
425
TAGTCTCGATGCCG
426
20




AGC

CCGGCG




 


HSPA4
1
TCTTAAGTGCTTTTTTTG
427
TGAACGATTCTTAG
428
20




TC

GACCAA




 


HSPA4
2
TTAAGTGCTTTTTTTGTC
429
TGAACGATTCTTAG
430
18




TT

GACCAA




 


HSPA4
3
TTAAGTGCTTTTTTTGTC
431
TTGAACGATTCTTAG
432
19




TT

GACCA




 


HSPA4
4
TAAGTGCTTTTTTTGTCT
433
TGAACGATTCTTAG
434
17




TC

GACCAA




 


HSPA4
5
TAAGTGCTTTTTTTGTCT
435
TTGAACGATTCTTAG
436
18




TC

GACCA




 


HSP90
1
TGCCCCCGTGTTCGGGC
437
TCCCGAAGGGAGGG
438
15


AA1

GGG

CCCAGG




 


HSP90
2
TGCCCCCGTGTTCGGGC
439
TGTCCCGAAGGGAG
440
17


AA1

GGG

GGCCCA




 


HSP90
3
TCCTGGGCCCTCCCTTC
441
TCGCGCGGGTATTC
442
20


AA1

GGG

AGCACT




 


HSP90
4
TGGGCCCTCCCTTCGGG
443
TCGCGCGGGTATTC
444
17


AA1

ACA

AGCACT




 


HSP90
5
TCCCTTCGGGACAGGGA
445
TCCAGACGGTCGCG
446
19


AA1

CTG

CGGGTA




 


HSP90
1
TCCAGAAGATTGTGTTT
447
TCTTGGTACCAGTTA
448
14


AA1

ATG

ACAGG




 


HSP90
2
TGTGTTTATGTTCCCAG
449
TTGGGCCTTTTCTTG
450
14


AA1

CAG

GTACC




 


HSP90
3
TCCCAGCAGGGCACCTG
451
TGCCAGAGAAACAC
452
17


AA1

TTA

TTGGGC




 


HSP90
4
TAACTGGTACCAAGAA
453
TCCAGACACCATCA
454
15


AA1

AAGG

GATGCC




 


HSP90
5
TAACTGGTACCAAGAA
455
TGGATCCAGACACC
456
19


AA1

AAGG

ATCAGA




 


MYC
1
TCCAGCAGCCTCCCGCG
457
TAGTTCCTGTTGGTG
458
15




ACG

AAGCT




 


MYC
2
TCCAGCAGCCTCCCGCG
459
TCATAGTTCCTGTTG
460
18




ACG

GTGAA




 


MYC
3
TCCCGCGACGATGCCCC
461
TCGAGGTCATAGTT
462
14




TCA

CCTGTT




 


MYC
4
TCCCGCGACGATGCCCC
463
TAGTCGAGGTCATA
464
17




TCA

GTTCCT




 


MYC
5
TCCCGCGACGATGCCCC
465
TCGTAGTCGAGGTC
466
20




TCA

ATAGTT




 


PKN3
1
TGCAGCCTGGGCCGAG
467
TGGCCCGGCGGATC
468
20




CCAG

ACCTCC




 


PKN3
2
TGGGCCGAGCCAGTGG
469
TGGATGGCCCGGCG
470
17




CCCC

GATCAC




 


PKN3
3
TGGGCCGAGCCAGTGG
471
TCTGGATGGCCCGG
472
19




CCCC

CGGATC




 


PKN3
4
TGGGCCGAGCCAGTGG
473
TTCTGGATGGCCCG
474
20




CCCC

GCGGAT




 


PKN3
5
TGGCCCCCAGAGGATG
475
TCAGCTCTTTCTGGA
476
15




AGAA

TGGCC




 


RRM2
1
TGGGAAGGGTCGGAGG
477
TGGCTTTGGTGCCCC
478
16




CATG

GGCCC




 


RRM2
2
TGGGAAGGGTCGGAGG
479
TTGGCTTTGGTGCCC
480
17




CATG

CGGCC




 


RRM2
3
TCGGAGGCATGGCACA
481
TTCCCATTGGCTTTG
482
14




GCCA

GTGCC




 


RRM2
4
TGGCACAGCCAATGGG
483
TCCCGGCCCTTCCCA
484
14




AAGG

TTGGC




 


RRM2
5
TGCACCCTGTCCCAGCC
485
TGGAGGCGCAGCGA
486
17




GTC

AGCAGA




 


APC
1
TATGTACGCCTCCCTGG
487
TGGTACAGAAGCGG
488
15




GCT

GCAAAG




 


APC
2
TGTACGCCTCCCTGGGC
489
TGAGGGTGGTACAG
490
19




TCG

AAGCGG




 


APC
3
TACGCCTCCCTGGGCTC
491
TGAGGGTGGTACAG
492
17




GGG

AAGCGG




 


APC
4
TCGGGTCCGGTCGCCCC
493
TCCAGGACCCGAGA
494
18




TTT

ACTGAG




 


APC
5
TCCGGTCGCCCCTTTGC
495
TGCTCCAGGACCCG
496
16




CCG

AGAACT




 


APC
1
TTAAACAACTACAAGG
497
TCAATCTGTCCAGA
498
18




AAGT

AGAAGC




 


APC
2
TAAACAACTACAAGGA
499
TCAATCTGTCCAGA
500
17




AGTA

AGAAGC




 


APC
3
TACAAGGAAGTATTGA
501
TAATAAATCAATCT
502
16




AGAT

GTCCAG




 


APC
4
TATTGAAGATGAAGCTA
503
TAAGACGCTCTAAT
504
16




TGG

AAATCA




 


APC
5
TATTGAAGATGAAGCTA
505
TTAAGACGCTCTAA
506
17




TGG

TAAATC




 


BRCA
1
TGGATTTATCTGCTCTT
507
TGCATAGCATTAAT
508
15


1

CGC

GACATT




 


BRCA
2
TGGATTTATCTGCTCTT
509
TCTGCATAGCATTA
510
17


1

CGC

ATGACA




 


BRCA
3
TTATCTGCTCTTCGCGT
511
TAAGATTTTCTGCAT
512
20


1

TGA

AGCAT




 


BRCA
4
TATCTGCTCTTCGCGTT
513
TAAGATTTTCTGCAT
514
19


1

GAA

AGCAT




 


BRCA
5
TCTGCTCTTCGCGTTGA
515
TAAGATTTTCTGCAT
516
17


1

AGA

AGCAT




 


BRCA
1
TGCTAGTCTGGAGTTGA
517
TGCAAAATATGTGG
518
19


1

TCA

TCACAC




 


BRCA
2
TGCTAGTCTGGAGTTGA
519
TTGCAAAATATGTG
520
20


1

TCA

GTCACA




 


BRCA
3
TAGTCTGGAGTTGATCA
521
TGCAAAATATGTGG
522
16


1

AGG

TCACAC




 


BRCA
4
TAGTCTGGAGTTGATCA
523
TTGCAAAATATGTG
524
17


1

AGG

GTCACA




 


BRCA
5
TAGTCTGGAGTTGATCA
525
TACTTGCAAAATAT
526
20


1

AGG

GTGGTC




 


BRCA
1
TGCCTATTGGATCCAAA
527
TGCAGCGTGTCTTA
528
17


2

GAG

AAAATT




 


BRCA
2
TGCCTATTGGATCCAAA
529
TTGCAGCGTGTCTTA
530
18


2

GAG

AAAAT




 


BRCA
3
TGCCTATTGGATCCAAA
531
TGTTGCAGCGTGTCT
532
20


2

GAG

TAAAA




 


BRCA
4
TATTGGATCCAAAGAG
533
TTGCAGCGTGTCTTA
534
14


2

AGGC

AAAAT




 


BRCA
5
TATTGGATCCAAAGAG
535
TGTTGCAGCGTGTCT
536
16


2

AGGC

TAAAA




 


BRCA
1
TAGATTTAGGACCAATA
537
TGGAGCTTCTGAAG
538
16


2

AGT

AAAGTT




 


BRCA
2
TTAGGACCAATAAGTCT
539
TAGGGTGGAGCTTC
540
16


2

TAA

TGAAGA




 


BRCA
3
TTAGGACCAATAAGTCT
541
TATAGGGTGGAGCT
542
18


2

TAA

TCTGAA




 


BRCA
4
TTAGGACCAATAAGTCT
543
TTATAGGGTGGAGC
544
19


2

TAA

TTCTGA




 


BRCA
5
TAGGACCAATAAGTCTT
545
TATAGGGTGGAGCT
546
17


2

AAT

TCTGAA




 


TP53
1
TCACTGCCATGGAGGA
547
TGACTCAGAGGGGG
548
15




GCCG

CTCGAC




 


TP53
2
TCACTGCCATGGAGGA
549
TCCTGACTCAGAGG
550
18




GCCG

GGGCTC




 


TP53
3
TCACTGCCATGGAGGA
551
TTCCTGACTCAGAG
552
19




GCCG

GGGGCT




 


TP53
4
TCACTGCCATGGAGGA
553
TTTCCTGACTCAGAG
554
20




GCCG

GGGGC




 


TP53
5
TGCCATGGAGGAGCCG
555
TCCTGACTCAGAGG
556
14




CAGT

GGGCTC




 


APP
1
TTCTTTCAGGTACCCAC
557
TGGCAATCTGGGGT
558
18




TGA

TCAGCC




 


APP
2
TCTTTCAGGTACCCACT
559
TGGCAATCTGGGGT
560
17




GAT

TCAGCC




 


APP
3
TTTCAGGTACCCACTGA
561
TGGCAATCTGGGGT
562
15




TGG

TCAGCC




 


APP
4
TTCAGGTACCCACTGAT
563
TGGCAATCTGGGGT
564
14




GGT

TCAGCC




 


APP
5
TACCCACTGATGGTAAT
565
TGCCACAGAACATG
566
20




GCT

GCAATC




 


IAPP
1
TGGGCATCCTGAAGCTG
567
TGGTTCAATGCAAC
568
15




CAA

AGAGAG




 


IAPP
2
TGGGCATCCTGAAGCTG
569
TCAGATGGTTCAAT
570
20




CAA

GCAACA




 


IAPP
3
TGCAAGTATTTCTCATT
571
TGGGTGTAGCTTTCA
572
17




GTG

GATGG




 


IAPP
4
TGCTCTCTGTTGCATTG
573
TTACCAACCTTTCAA
574
14




AAC

TGGGT




 


IAPP
1
TGTTACCAGTCATCAGG
575
TGCGTTGCACATGT
576
17




TGG

GGCAGT




 


IAPP
2
TTACCAGTCATCAGGTG
577
TGCGTTGCACATGT
578
15




GAA

GGCAGT




 


IAPP
3
TACCAGTCATCAGGTGG
579
TGCGTTGCACATGT
580
14




AAA

GGCAGT




 


IAPP
4
TCATCAGGTGGAAAAG
581
TGCCAGGCGCTGCG
582
18




CGGA

TTGCAC




 


IAPP
5
TCATCAGGTGGAAAAG
583
TTGCCAGGCGCTGC
584
19




CGGA

GTTGCA




 


SNCA
1
TTTTGTAGGCTCCAAAA
585
TTACCTGTTGCCACA
586
14




CCA

CCATG




 


SNCA
2
TTTTGTAGGCTCCAAAA
587
TGGAGCTTACCTGTT
588
20




CCA

GCCAC




 


SNCA
3
TTTGTAGGCTCCAAAAC
589
TGGAGCTTACCTGTT
590
19




CAA

GCCAC




 


SNCA
4
TTGTAGGCTCCAAAACC
591
TGGAGCTTACCTGTT
592
18




AAG

GCCAC




 


SNCA
5
TGTAGGCTCCAAAACCA
593
TGGAGCTTACCTGTT
594
17




AGG

GCCAC




 


SOD1
1
TAGCGAGTTATGGCGAC
595
TGCACTGGGCCGTC
596
16




GAA

GCCCTT




 


SOD1
2
TTATGGCGACGAAGGC
597
TGCCCTGCACTGGG
598
14




CGTG

CCGTCG




 


SOD1
3
TTATGGCGACGAAGGC
599
TGATGCCCTGCACT
600
17




CGTG

GGGCCG




 


SOD1
4
TTATGGCGACGAAGGC
601
TGATGATGCCCTGC
602
20




CGTG

ACTGGG




 


SOD1
5
TATGGCGACGAAGGCC
603
TGATGCCCTGCACT
604
16




GTGT

GGGCCG




 













SOD1
1
TAATGGACCAGTGAAG
605
TGCAGGCCTTCAGT
606
14




GTGT

CAGTCC




 













SOD1
2
TAATGGACCAGTGAAG
607
TCCATGCAGGCCTTC
608
18




GTGT

AGTCA




 


SOD1
3
TGGACCAGTGAAGGTG
609
TCCATGCAGGCCTTC
610
15




TGGG

AGTCA




 


SOD1
4
TGGACCAGTGAAGGTG
611
TGGAATCCATGCAG
612
20




TGGG

GCCTTC




 


SOD1
5
TGTGGGGAAGCATTAA
613
TCATGAACATGGAA
614
15




AGGA

TCCATG









In some embodiments, the target DNA molecule comprises a gene that is overexpressed in cancer. Example genes that are overexpressed in cancer include, but are not limited to: ABL1, BIRC5, BLK, BTK, CDK family members, EGFR, ERBB2, FAS, FGR, FLT4, FRK, FYN, HCK, HIF1A, HRAS, HSP90AA1, HSP90AA1, HSPA4, KDR, KIF11, KIF11, KIF20A, KIF21A, KIF25, KIT, KRAS, LCK, LYN, MAPK1, MET, MYC, MYH1, MYO1G, NRAS, NTRK1, PDGFB, PDGFRA, PDGFRB, PKN3, PLK1, RAF1, RB1, RET, RRM1, RRM2, SRC, TNF, TPM2, TYRO3, VEGFA, VEGFB, VEGFC, YES1, and ZAP70. In some embodiments, the target DNA molecule comprises a gene selected from: ABL1, BIRC5, BLK, BTK, a CDK family member, EGFR, ERBB2, FAS, FGR, FLT4, FRK, FYN, HCK, HIF1A, HRAS, HSP90AA1, HSP90AA1, HSPA4, KDR, KIF11, KIF11, KIF20A, KIF21A, KIF25, KIT, KRAS, LCK, LYN, MAPK1, MET, MYC, MYH1, MYO1G, NRAS, NTRK1, PDGFB, PDGFRA, PDGFRB, PKN3, PLK1, RAF1, RB1, RET, RRM1, RRM2, SRC, TNF, TPM2, TYRO3, VEGFA, VEGFB, VEGFC, YES1, and ZAP70 or a fragment or variant thereof. In other embodiments, the target DNA molecule comprises a gene that is mutated in cancer. Example genes that are mutated in cancer include, but are not limited to: AIM1, APC, BRCA1, BRCA2, CDKN1B, CDKN2A, FAS, FZD family members, HNF1A, HOPX, KLF6, MEN1, MLH1, NTRK1, PTEN, RARRES1, RB1, SDHB, SDHD, SFRP1, ST family members, TNF, TP53, TP63, TP73, VBP1, VHL, WNT family members, BRAF, CTNNB1, PIK3CA, PIK3R1, SMAD4, and YPEL3. In some embodiments, the target DNA molecule comprises a gene selected from: AIM1, APC, BRCA1, BRCA2, CDKN1B, CDKN2A, FAS, a FZD family member, HNF1A, HOPX, KLF6, MEN1, MLH1, NTRK1, PTEN, RARRES1, RB1, SDHB, SDHD, SFRP1, a ST family member, TNF, TP53, TP63, TP73, VBP1, VHL, a WNT family member, BRAF, CTNNB1, PIK3CA, PIK3R1, SMAD4, and YPEL3 or a fragment or variant thereof. In one embodiment, the method further comprises administering to a patient a therapeutically effective amount of a repair template.
Mutations in certain genes can increase the likelihood of a cell becoming cancerous. In certain situations, however, it can be detrimental to inactivate a cancer-associated gene in non-cancerous cells, for example, if the non-mutated form of the cancer-associated gene is beneficial. It has now been discovered that gene-editing proteins can be used to specifically inactivate, partially or completely, mutated forms of genes. Examples of cancer-associated mutations include, but are not limited to: ALK (F1174, R1275), APC (R876, Q1378, R1450), BRAF (V600), CDKN2A (R58, R80, H83, D84, E88, D108G, W110, P114), CTNNB1 (D32, S33, G34, S37, T41, or S45), EGFR (G719, T790, L858), EZH2 (Y646), FGFR3 (S249, Y373), FLT3 (D835), GNAS (R201), HRAS (G12, G13, Q61), IDH1 (R132), JAK2 (V617), KIT (D816), KRAS (G12, G13), NRAS (G12, G13, Q61), PDGFRA (D842), PIK3CA (E542, E545, H1047), PTEN (R130), and TP53 (R175, H179, G245, R248, R249, R273, W282). Certain embodiments are therefore directed to a gene-editing protein that binds to a disease-associated mutation. In one embodiment, the gene-editing protein binds to DNA containing a specific mutation with greater affinity than DNA that does not contain the mutation. In another embodiment, the disease is cancer.
Neurodegenerative diseases, including Alzheimer's disease, Parkinson's disease, and dementia with Lewy bodies, are characterized by the progressive loss of function and/or death of cells of the central and/or peripheral nervous systems. Disease progression can be accompanied by the accumulation of protein-rich plaques that can comprise the protein α-synuclein (encoded, in humans, by the SNCA gene). As a result, researchers have sought to develop therapeutics that can break up these plaques, for example, by means of an antibody that binds to the plaque and tags it for destruction by the immune system. However, in many cases, breaking up plaques has little or no effect on patient symptoms or the progression of the disease. It has now been discovered that the failure of existing therapies that target neurodegenerative disease-associated plaques is due in part to the inability of the nervous system to repair the damage to cells that occurs during the early stages of plaque formation. It has been further discovered that inducing a cell to express one or more gene-editing proteins that target the SNCA gene can result in disruption of the SNCA gene, can induce the cell to express a plaque-resistant variant of α-synuclein protein, can slow or halt the growth of neurodegenerative disease-associated plaques, can protect the cell and nearby cells from the damaging effects of neurodegenerative disease-associated plaques, can slow and/or halt the progression of neurodegenerative diseases, including Alzheimer's disease, Parkinson's disease, and dementia with Lewy bodies, and can result in a reduction of symptoms and/or gain of function in patients with neurodegenerative diseases, including Alzheimer's disease, Parkinson's disease, and dementia with Lewy bodies. Other neurodegenerative diseases include, for example, vision loss, including blindness, hearing loss, including deafness, balance disorders, loss of taste and/or smell, and other sensory disorders. Certain embodiments are therefore directed to a gene-editing protein that targets the SNCA gene. In one embodiment, the gene-editing protein binds to one or more regions in the SNCA gene. In another embodiment, the gene-editing protein binds to one or more nucleic-acid sequences that encode SEQ ID NO: 51 or a biologically active fragment, variant or analogue thereof. Other embodiments are directed to a method for treating a neurodegenerative disease comprising administering to a patient a therapeutically effective amount of a gene-editing protein or a nucleic acid encoding a gene-editing protein, wherein the gene-editing protein is capable of binding to a nucleotide sequence that encodes a protein that forms disease-associated plaques, and resulting in a reduction of disease-associated plaques in the patient and/or delayed or halted progression of the disease. In one embodiment, the nucleotide sequence comprises the SNCA gene. In another embodiment, the nucleotide sequence encodes α-synuclein. In a further embodiment, the neurodegenerative disease is selected from: Parkinson's disease, Alzheimer's disease, and dementia.
Certain embodiments are directed to a method for identifying a disease-causing toxicant comprising transfecting a cell with a gene-editing protein or a nucleic acid encoding a gene-editing protein to alter the DNA sequence of the cell, wherein the altered DNA sequence confers susceptibility to a disease, contacting the cell with a suspected disease-causing toxicant, and assessing the degree to which the cell exhibits a phenotype associated with the disease. In one embodiment, the disease is a neurodegenerative disease, autoimmune disease, respiratory disease, reproductive disorder or cancer. Other embodiments are directed to a method for assessing the safety of a therapeutic substance comprising transfecting a cell with a gene-editing protein or a nucleic acid encoding a gene-editing protein to alter the DNA sequence of the cell, wherein the altered DNA sequence confers susceptibility to one or more toxic effects of the therapeutic substance, contacting the cell with the therapeutic substance, and measuring one or more toxic effects of the therapeutic substance on the cell. Still other embodiments are directed to a method for assessing the effectiveness of a therapeutic substance comprising transfecting a cell with a gene-editing protein or a nucleic acid encoding a gene-editing protein to alter the DNA sequence of the cell, wherein the altered DNA sequence causes the cell to exhibit one or more disease-associated phenotypes, contacting the cell with the therapeutic substance, and measuring the degree to which the one or more disease-associated phenotypes are reduced.
In some embodiments, the patient is diagnosed with a proteopathy. Example proteopathies and proteopathy-associated genes are given in Table 6, and are included by way of example, and not by way of limitation. In one embodiment, the proteopathy is selected from: AA (secondary) amyloidosis, Alexander disease, Alzheimer's disease, amyotrophic lateral sclerosis, aortic medial amyloidosis, ApoAI amyloidosis, ApoAII amyloidosis, ApoAIV amyloidosis, bibrinogen amyloidosis, cardiac atrial amyloidosis, cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy, cerebral β-amyloid angiopathy, dialysis amyloidosis, familial amyloid cardiomyopathy, familial amyloid polyneuropathy, familial amyloidosis (Finnish type), familial British dementia, familial Danish dementia, frontotemporal lobar degeneration, hereditary cerebral amyloid angiopathy, hereditary lattice corneal dystrophy, Huntington's disease, inclusion body myositis/myopathy, lysozyme amyloidosis, medullary thyroid carcinoma, odontogenic (Pindborg) tumor amyloid, Parkinson's disease, pituitary prolactinoma, prion diseases, pulmonary alveolar proteinosis, retinal ganglion cell degeneration in glaucoma, retinitis pigmentosa with rhodopsin mutations, senile systemic amyloidosis, serpinopathies, synucleinopathies, tauopathies, type II diabetes, dementia pugilistica (chronic traumatic encephalopathy), frontotemporal dementia, frontotemporal lobar degeneration, gangliocytoma, ganglioglioma, Hallervorden-Spatz disease, lead encephalopathy, lipofuscinosis, Lytico-Bodig disease, meningioangiomatosis, progressive supranuclear palsy, subacute sclerosing panencephalitis, tangle-predominant dementia, and tuberous sclerosis. In another embodiment, the target DNA molecule comprises a gene selected from: APOA1, APOA2, APOA4, APP, B2M, CALCA, CST3, FGA, FGB, FGG, FUS, GFAP, GSN, HTT, IAPP, ITM2B, LYZ, MAPT, MFGE8, NOTCH3, NPPA, ODAM, PRL, PRNP, RHO, a SAA family member, a SERPIN family member, SFTPC, SNCA, a SOD family member, TARDBP, TGFBI, and TRR or a fragment or variant thereof. In a further embodiment, the target DNA molecule encodes a gene selected from Table 6 or a fragment thereof, and the patient is diagnosed with the corresponding disease listed in Table 6.







TABLE 6







Exemplary Proteopathies and Proteopathy-Associated Genes








Gene/Family
Disease/Condition





APOA1
ApoAI amyloidosis


APOA2
ApoAII amyloidosis


APOA4
ApoAIV amyloidosis


APP
Cerebral β-amyloid angiopathy


APP
Retinal ganglion cell degeneration in glaucoma


APP
Inclusion body myositis/myopathy


APP, MAPT
Alzheimer's disease


B2M
Dialysis amyloidosis


CALCA
Medullary thyroid carcinoma


CST3
Hereditary cerebral amyloid angiopathy (Icelandic)


FGA, FGB, FGG
Fibrinogen amyloidosis


GFAP
Alexander disease


GSN
Familial amyloidosis, Finnish type


HTT
Huntington's disease


IAPP
Type II diabetes


ITM2B
Familial British dementia


ITM2B
Familial Danish dementia


LYZ
Lysozyme amyloidosis


MAPT
Tauopathies (multiple)


MFGE8
Aortic medial amyloidosis


NOTCH3
Cerebral autosomal dominant arteriopathy



with subcortical infarcts and



leukoencephalopathy (CADASIL)


NPPA
Cardiac atrial amyloidosis


ODAM
Odontogenic (Pindborg) tumor amyloid


PRL
Pituitary prolactinoma


PRNP
Prion diseases (multiple)


RHO
Retinitis pigmentosa with rhodopsin mutations


SAA family genes
AA (secondary) amyloidosis


SERPIN family
Serpinopathies (multiple)


genes


SFTPC
Pulmonary alveolar proteinosis


SNCA
Parkinson's disease and other synucleinopathies



(multiple)


SNCA
Other synucleinopathies


SOD family genes,
Amyotrophic lateral sclerosis (ALS)


TARDBP, FUS


TARDBP, FUS
Frontotemporal lobar degeneration (FTLD)


TGFBI
Hereditary lattice corneal dystrophy


LMNA
Hutchinson-Gilford Progeria Syndrome


TRR
Senile systemic amyloidosis (SSA), familial



amyloid polyneuropathy (FAP), familial amyloid



cardiomyopathy (FAC)









Example tauopathies include, but are not limited to Alzheimer's disease, Parkinson's disease, and Huntington's disease. Other example tauopathies include: dementia pugilistica (chronic traumatic encephalopathy), frontotemporal dementia, frontotemporal lobar degeneration, gangliocytoma, ganglioglioma, Hallervorden-Spatz disease, lead encephalopathy, lipofuscinosis, Lytico-Bodig disease, meningioangiomatosis, progressive supranuclear palsy, subacute sclerosing panencephalitis, tangle-predominant dementia, and tuberous sclerosis. In some embodiments, the patient is diagnosed with a tauopathy. In one embodiment, the tauopathy is selected from: Alzheimer's disease, Parkinson's disease, and Huntington's disease. In another embodiment, the tauopathy is selected from: dementia pugilistica (chronic traumatic encephalopathy), frontotemporal dementia, frontotemporal lobar degeneration, gangliocytoma, ganglioglioma, Hallervorden-Spatz disease, lead encephalopathy, lipofuscinosis, Lytico-Bodig disease, meningioangiomatosis, progressive supranuclear palsy, subacute sclerosing panencephalitis, tangle-predominant dementia, and tuberous sclerosis.
Autoimmune diseases, including but not limited to lupus, multiple sclerosis (MS), amyotrophic lateral sclerosis (ALS), and transplant rejection, are characterized by symptoms caused in part by one or more elements of the immune system attacking uninfected and non-cancerous isogenic cells and/or tissues. Certain embodiments are therefore directed to a method for treating an autoimmune disease. In one embodiment, the autoimmune disease is selected from: lupus, multiple sclerosis (MS), amyotrophic lateral sclerosis (ALS), and transplant rejection. In another embodiment, the target DNA molecule encodes a polypeptide sequence that can be recognized by the host immune system.
Infectious agents can contain nucleic acid sequences that are not present in the host organism. It has now been discovered that gene-editing proteins can be used to eliminate, reduce or otherwise alter, in whole or in part, infectious agents and/or the effects of infection, and that when used in this manner, gene-editing proteins and nucleic acids encoding gene-editing proteins, can constitute potent anti-infection therapeutics. Infectious agents that can be treated in such a manner include, but are not limited to: viruses, bacteria, fungi, yeast, and parasites. Certain embodiments are therefore directed to a method for inducing a cell to express a gene-editing protein that targets one or more infectious agent-associated sequences. In one embodiment, the cell is one of: a bacterial cell, a fungal cell, a yeast cell, and a parasite cell. In another embodiment, the cell is a mammalian cell. In a further embodiment, the cell is a human cell. Other embodiments are directed to a therapeutic composition comprising a nucleic acid that encodes one or more gene-editing proteins that targets one or more infectious agent-associated sequences. Certain embodiments are directed to a method for inducing a cell to express a gene-editing protein that targets one or more sequences associated with susceptibility or resistance to infection. Other embodiments are directed to a therapeutic composition comprising a nucleic acid that encodes one or more gene-editing proteins that targets one or more sequences associated with susceptibility or resistance to infection. In one embodiment, the cell is transfected with a nucleic acid encoding one or more gene-editing proteins and a nucleic acid encoding one or more repair templates. In another embodiment, the repair template contains a resistance gene or a biologically active fragment or variant thereof. In a further embodiment, the repair template contains an RNAi sequence. In a still further embodiment, the RNAi sequence is a shRNA. Other embodiments are directed to a method for treating an infectious disease comprising administering to a patient a therapeutically effective amount of a gene-editing protein or a nucleic acid encoding a gene-editing protein, wherein the gene-editing protein is capable of binding to one or more nucleotide sequences that are present in the infectious agent.
It has now been discovered that the ratio of non-homologous end joining events to homologous recombination events can be altered by altering the expression and/or function of one or more components of a DNA-repair pathway. Non-limiting examples of genes that encode components of a DNA-repair pathway include, but are not limited to: Artemis, BLM, CtIP, DNA-PK, DNA-PKcs, EXOl, FEN1, Ku70, Ku86, LIGIII, LIGIV, MRE11, NBS1, PARP1, RAD50, RAD54B, XLF, XRCC1, XRCC3, and XRCC4. Certain embodiments are therefore directed to a method for altering the expression and/or function of one or more components of a DNA-repair pathway. In certain embodiments, the expression and/or function is increased. In other embodiments, the expression and/or function is decreased. DNA-dependent protein kinase (DNA-PK) is a component of the non-homologous end-joining DNA-repair pathway. It has now been discovered that repair via homologous recombination can be increased by altering the expression of DNA-PK. In one embodiment, a cell is contacted with a DNA-PK inhibitor. Example DNA-PK inhibitors include, but are not limited to: Compound 401 (2-(4-Morpholinyl)-4H-pyrimido[2,1-a]isoquinolin-4-one), DMNB, IC87361, LY294002, NU7026, NU7441, OK-1035, PI 103 hydrochloride, vanillin, and wortmannin.
Genetic mutations can affect the length of a protein product, for example, by introducing a stop codon and/or disrupting an open reading frame. Certain diseases, including Duchenne muscular dystrophy, can be caused by the production of truncated and/or frameshifted proteins. It has now been discovered that gene-editing proteins can be used to treat diseases that are associated with the production of one or more truncated and/or frameshifted proteins. In one embodiment, the gene-editing protein creates a double strand break within about 1 kb or about 0.5 kb or about 0.1 kb of an exon containing a disease-contributing mutation. In another embodiment, the gene-editing protein is co-expressed with a DNA sequence comprising one or more wild-type sequences. In certain embodiments, the DNA is single-stranded. In other embodiments, the DNA is double-stranded. Diseases caused by the expression of truncated proteins can be treated by exon skipping. It has now been discovered that gene-editing proteins can be used to induce exon skipping. In one embodiment, the gene-editing protein creates a double-strand break within about 1 kb or about 0.5 kb or about 0.1 kb of the exon to be skipped. In another embodiment, the gene-editing protein creates a double-strand break within about 1 kb or about 0.5 kb or about 0.1 kb of an intron upstream of the exon to be skipped. In another embodiment, the gene-editing protein creates a double-strand break within about 1 kb or about 0.5 kb or about 0.1 kb of the splice-acceptor site of an intron upstream of the exon to be skipped.
Nucleic acids, including liposomal formulations containing nucleic acids, when delivered in vivo, can accumulate in the liver and/or spleen. It has now been discovered that nucleic acids encoding gene-editing proteins can modulate gene expression in the liver and spleen, and that nucleic acids used in this manner can constitute potent therapeutics for the treatment of liver and spleen diseases. Certain embodiments are therefore directed to a method for treating liver and/or spleen disease by delivering to a patient a nucleic acid encoding one or more gene-editing proteins. Other embodiments are directed to a therapeutic composition comprising a nucleic acid encoding one or more gene-editing proteins, for the treatment of liver and/or spleen disease. Diseases and conditions of the liver and/or spleen that can be treated include, but are not limited to: hepatitis, alcohol-induced liver disease, drug-induced liver disease, Epstein Barr virus infection, adenovirus infection, cytomegalovirus infection, toxoplasmosis, Rocky Mountain spotted fever, non-alcoholic fatty liver disease, hemochromatosis, Wilson's Disease, Gilbert's Disease, and cancer of the liver and/or spleen. Other examples of sequences (including genes, gene families, and loci) that can be targeted by gene-editing proteins using the methods of the present invention are set forth in Table 7, and are given by way of example, and not by way of limitation.







TABLE 7







Exemplary Gene Editing-Protein Targets








Disease/Condition
Gene/Family/Locus





Age-related macular
VEGF family


degeneration


Alzheimer's disease
APP, PSEN1, PSEN2, APOE, CR1,



CLU, PICALM, BIN1, MS4A4, MS4A6E,



CD2AP, CD33, EPHA1


Amyotrophic lateral
SOD1


sclerosis


Cancer
BRCA1, EGFR, MYC family, TP53,



PKN3, RAS family, BIRC5, PTEN,



RET, KIT, MET, APC, RB1, BRCA2,



VEGF family, TNF, HNPCC1,



HNPCC2, HNPCC5


Cystic fibrosis
CFTR


Diabetes
GCK, HNF1A, HNF4A, HNF1B


Duchenne muscular
DMD


dystrophy


Fanconi anemia
BRCA2, FANCA, FANCB, FANCC,



FANCD2, FANCE, FANCF, FANCG,



FANCI, FANCJ, FANCL, FANCM,



FANCN, FANCP, RAD51C


Hemochromatosis
HFE, HJV, HAMP, TFR2, SLC40A1


Hemophilia
F8, F9, F11


HIV/AIDS
CCR5, CXCR4


Huntington's disease
HTT


Marfan's syndrome
FBN1


Neurofibromatosis
NF1, NF2


Parkinson's disease
SNCA, PRKN, LRRK2, PINK1,



PARK7, ATP13A2


Safe-harbor locus
AAVS1


in humans


Safe-harbor locus
Rosa26


in mice and rats


Sickle-cell anemia
HBB


Tay-Sachs disease
HEXA


Xeroderma pigmentosum
XPA, XPB, XPC, XPD, DDB2,



ERCC4, ERCC5, ERCC6, RAD2,



POLH


Psoriasis, Rheumatoid
TNF


arthritis, Ankylosing


spondylitis, Crohn's


disease, Hidradenitis


suppurativa, Refractory


asthma


Psoriasis, Rheumatoid
JAK family


arthritis, Polycythemia


vera, Essential


thrombocythemia,


Myeloid metaplasia


with myelofibrosis









Certain embodiments are directed to a combination therapy comprising one or more of the therapeutic compositions of the present invention and one or more adjuvant therapies. Example adjuvant therapies are set forth in Table 8 and Table 5 of U.S. Provisional Application No. 61/721,302, the contents of which are hereby incorporated by reference, and are given by way of example, and not by way of limitation.







TABLE 8







Exemplary Adjuvant Therapies









Therapy Class
Disease/Condition
Example Therapy





Acetylcholinesterase inhibitors
Myasthenia gravis, Glaucoma, Alzheimer's
Edrophonium



disease, Lewy body dementia, Postural



tachycardia syndrome


Angiotensin-converting-enzyme
Hypertension, Congestive heart failure
Perindopril


inhibitor


Alkylating agents
Cancer
Cisplatin


Angiogenesis inhibitors
Cancer, Macular degeneration
Bevacizumab


Angiotensin II receptor
Hypertension, Diabetic nephropathy,
Valsartan


antagonists
Congestive heart failure


Antibiotics
Bacterial infection
Amoxicillin


Antidiabetic drugs
Diabetes
Metformin


Antimetabolites
Cancer, Infection
5-fluorouracil




(5FU)


Antisense oligonucleotides
Cancer, Diabetes, Amyotrophic lateral
Mipomersen



sclerosis (ALS), Hypercholesterolemia


Cytotoxic antibiotics
Cancer
Doxorubicin


Deep-brain stimulation
Chronic pain, Parkinson's disease, Tremor,
N/A



Dystonia


Dopamine agonists
Parkinson's disease, Type II diabetes,
Bromocriptine



Pituitary tumors


Entry/Fusion inhibitors
HIV/AIDS
Maraviroc


Glucagon-like peptide-1 agonists
Diabetes
Exenatide


Glucocorticoids
Asthma, Adrenal insufficiency,
Dexamethasone



Inflammatory diseases, Immune diseases,



Bacterial meningitis


Immunosuppressive drugs
Organ transplantation, Inflammatory
Azathioprine



diseases, Immune diseases


Insulin/Insulin analogs
Diabetes
NPH insulin


Integrase inhibitors
HIV/AIDS
Raltegravir


MAO-B inhibitors
Parkinson's disease, Depression, Dementia
Selegiline


Maturation inhibitors
HIV/AIDS
Bevirimat


Nucleoside analog reverse-
HIV/AIDS, Hepatitis B
Lamivudine


transcriptase inhibitors


Nucleotide analog reverse-
HIV/AIDS, Hepatitis B
Tenofovir


transcriptase inhibitors


Non-nucleoside reverse-
HIV/AIDS
Rilpivirine


transcriptase inhibitors


Pegylated interferon
Hepatitis B/C, Multiple sclerosis
Interferon beta-1a


Plant alkaloids/terpenoids
Cancer
Paclitaxel


Protease inhibitors
HIV/AIDS, Hepatitis C, Other viral
Telaprevir



infections


Radiotherapy
Cancer
Brachytherapy


Renin inhibitors
Hypertension
Aliskiren


Statins
Hypercholesterolemia
Atorvastatin


Topoisomerase inhibitors
Cancer
Topotecan


Vasopressin receptor antagonist
Hyponatremia, Kidney disease
Tolvaptan









Pharmaceutical preparations may additionally comprise delivery reagents (a.k.a. “transfection reagents”) and/or excipients. Pharmaceutically acceptable delivery reagents, excipients, and methods of preparation and use thereof, including methods for preparing and administering pharmaceutical preparations to patients (a.k.a. “subjects”) are well known in the art, and are set forth in numerous publications, including, for example, in US Patent Appl. Pub. No. US 2008/0213377, the entirety of which is hereby incorporated by reference.
For example, the present compositions can be in the form pharmaceutically acceptable salts. Such salts include those listed in, for example, J. Pharma. Sci. 66, 2-19 (1977) and The Handbook of Pharmaceutical Salts; Properties, Selection, and Use. P. H. Stahl and C. G. Wermuth (eds.), Verlag, Zurich (Switzerland) 2002, which are hereby incorporated by reference in their entirety. Non-limiting examples of pharmaceutically acceptable salts include: sulfate, citrate, acetate, oxalate, chloride, bromide, iodide, nitrate, bisulfate, phosphate, acid phosphate, isonicotinate, lactate, salicylate, acid citrate, tartrate, oleate, tannate, pantothenate, bitartrate, ascorbate, succinate, maleate, gentisinate, fumarate, gluconate, glucaronate, saccharate, formate, benzoate, glutamate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate, camphorsulfonate, pamoate, phenylacetate, trifluoroacetate, acrylate, chlorobenzoate, dinitrobenzoate, hydroxybenzoate, methoxybenzoate, methylbenzoate, o-acetoxybenzoate, naphthalene-2-benzoate, isobutyrate, phenylbutyrate, α-hydroxybutyrate, butyne-1,4-dicarboxylate, hexyne-1,4-dicarboxylate, caprate, caprylate, cinnamate, glycollate, heptanoate, hippurate, malate, hydroxymaleate, malonate, mandelate, mesylate, nicotinate, phthalate, teraphthalate, propiolate, propionate, phenylpropionate, sebacate, suberate, p-bromobenzenesulfonate, chlorobenzenesulfonate, ethylsulfonate, 2-hydroxyethylsulfonate, methylsulfonate, naphthalene-1-sulfonate, naphthalene-2-sulfonate, naphthalene-1,5-sulfonate, xylenesulfonate, tartarate salts, hydroxides of alkali metals such as sodium, potassium, and lithium; hydroxides of alkaline earth metal such as calcium and magnesium; hydroxides of other metals, such as aluminum and zinc; ammonia, and organic amines, such as unsubstituted or hydroxy-substituted mono-, di-, or tri-alkylamines, dicyclohexylamine; tributyl amine; pyridine; N-methyl, N-ethylamine; diethylamine; triethylamine; mono-, bis-, or tris-(2-OH-lower alkylamines), such as mono-; bis-, or tris-(2-hydroxyethyl)amine, 2-hydroxy-tert-butylamine, or tris-(hydroxymethyl)methylamine, N,N-di-lower alkyl-N-(hydroxyl-lower alkyl)-amines, such as N,N-dimethyl-N-(2-hydroxyethyl)amine or tri-(2-hydroxyethyl)amine; N-methyl-D-glucamine; and amino acids such as arginine, lysine, and the like.
The present pharmaceutical compositions can comprises excipients, including liquids such as water and oils, including those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. The pharmaceutical excipients can be, for example, saline, gum acacia, gelatin, starch paste, talc, keratin, colloidal silica, urea and the like. In addition, auxiliary, stabilizing, thickening, lubricating, and coloring agents can be used. In one embodiment, the pharmaceutically acceptable excipients are sterile when administered to a subject. Suitable pharmaceutical excipients also include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like. Any agent described herein, if desired, can also comprise minor amounts of wetting or emulsifying agents, or pH buffering agents.
In various embodiments, the compositions described herein can administered in an effective dose of, for example, from about 1 mg/kg to about 100 mg/kg, about 2.5 mg/kg to about 50 mg/kg, or about 5 mg/kg to about 25 mg/kg. The precise determination of what would be considered an effective dose may be based on factors individual to each patient, including their size, age, and type of disease. Dosages can be readily ascertained by those of ordinary skill in the art from this disclosure and the knowledge in the art. For example, doses may be determined with reference Physicians' Desk Reference, 66th Edition, PDR Network; 2012 Edition (Dec. 27, 2011), the contents of which are incorporated by reference in its entirety.
The active compositions of the present invention may include classic pharmaceutical preparations. Administration of these compositions according to the present invention may be via any common route so long as the target tissue is available via that route. This includes oral, nasal, or buccal. Alternatively, administration may be by intradermal, subcutaneous, intramuscular, intraperitoneal or intravenous injection, or by direct injection into cancer tissue. The agents disclosed herein may also be administered by catheter systems. Such compositions would normally be administered as pharmaceutically acceptable compositions as described herein.
Upon formulation, solutions may be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective. The formulations may easily be administered in a variety of dosage forms such as injectable solutions, drug release capsules and the like. For parenteral administration in an aqueous solution, for example, the solution generally is suitably buffered and the liquid diluent first rendered isotonic with, for example, sufficient saline or glucose. Such aqueous solutions may be used, for example, for intravenous, intramuscular, subcutaneous and intraperitoneal administration. Preferably, sterile aqueous media are employed as is known to those of skill in the art, particularly in light of the present disclosure.
Exemplary subjects or patients refers to any vertebrate including, without limitation, humans and other primates (e.g., chimpanzees and other apes and monkey species), farm animals (e.g., cattle, sheep, pigs, goats, and horses), domestic mammals (e.g., dogs and cats), laboratory animals (e.g., rodents such as mice, rats, and guinea pigs), and birds (e.g., domestic, wild and game birds such as chickens, turkeys and other gallinaceous birds, ducks, geese, and the like). In some embodiments, the subject is a mammal. In some embodiments, the subject is a human.
This invention is further illustrated by the following non-limiting examples.
EXAMPLES
Example 1 RNA Synthesis
RNA encoding the human proteins Oct4, Sox2, Klf4, c-Myc-2 (T58A), and Lin28 or TALENs targeting the human genes XPA, CCR5, TERT, MYC, and BIRC5, and comprising various combinations of canonical and non-canonical nucleotides, was synthesized from DNA templates using the T7 High Yield RNA Synthesis Kit and the Vaccinia Capping System kit with mRNA Cap 2′-O-Methyltransferase (all from New England Biolabs, Inc.), according to the manufacturer's instructions and the present inventors' previously disclosed inventions (U.S. application Ser. No. 13/465,490 (now U.S. Pat. No. 8,497,124), U.S. Provisional Application No. 61/637,570, U.S. Provisional Application No. 61/664,494, International Application No. PCT/US12/67966, U.S. Provisional Application No. 61/785,404, U.S. application Ser. No. 13/931,251, and U.S. Provisional Application No. 61/842,874, the contents of all of which are hereby incorporated by reference in their entirety) (Table 9, FIG. 1A, FIG. 1B, and FIG. 15). The RNA was then diluted with nuclease-free water to between 100 ng/μL and 200 ng/μL. For certain experiments, an RNase inhibitor (Superase-In, Life Technologies Corporation) was added at a concentration of 1 μL/100 μg of RNA. RNA solutions were stored at 4° C. For reprogramming experiments, RNA encoding Oct4, Sox2, Klf4, c-Myc-2 (T58A), and Lin28 was mixed at a molar ratio of 3:1:1:1:1.







TABLE 9







RNA Synthesis












Reaction
ivT


Template
Nucleotides
Volume/μL
Yield/μg













Oct4
A, G, U, C
10
64.9


Oct4
A, G, 0.25 4sU, C
10
64.3


Oct4
A, G, 0.5 4sU, C
10
62.8


Oct4
A, G, 0.75 4sU, C
10
51.9


Oct4
A, G, 4sU, C
10
0


Oct4
A, 0.5 7dG, 0.75 4sU, 0.25 piC
20
70.1


Sox2
A, 0.5 7dG, 0.75 4sU, 0.25 piC
10
29.6


Klf4
A, 0.5 7dG, 0.75 4sU, 0.25 piC
10
29.5


c-Myc-2 (T58A)
A, 0.5 7dG, 0.75 4sU, 0.25 piC
10
25.9


Lin28
A, 0.5 7dG, 0.75 4sU, 0.25 piC
10
36.7


Oct4
A, 0.5 7dG, 0.75 4sU, 0.5 piC
20
51.7


Sox2
A, 0.5 7dG, 0.75 4sU, 0.5 piC
10
23.0


Klf4
A, 0.5 7dG, 0.75 4sU, 0.5 piC
10
22.3


c-Myc-2 (T58A)
A, 0.5 7dG, 0.75 4sU, 0.5 piC
10
21.4


Lin28
A, 0.5 7dG, 0.75 4sU, 0.5 piC
10
23.3


Oct4
A, 0.5 7dG, 0.8 4sU, 0.2 5mU, 0.5 piC
20
50.8


Oct4
A, 0.5 7dG, 0.7 4sU, 0.3 5mU, 0.5 piC
20
58.3


Oct4
A, 0.5 7dG, 0.6 4sU, 0.4 5mU, 0.5 piC
20
58.3


Oct4
A, 0.5 7dG, 0.5 4sU, 0.5 5mU, 0.5 piC
20
68.2


Oct4
A, 0.5 7dG, 0.4 4sU, 0.6 5mU, 0.5 piC
20
78.7


Oct4
A, G, psU, 5mC
10
110.4


Oct4
A, G, psU, 0.5 piC
10
85.0


Oct4
A, 0.5 7dG, psU, 0.5 piC
10
58.3


Oct4
A, 0.5 7dG, psU, 5mC
10
27.0


Oct4
A, 0.5 7dG, 0.5 5mU, 0.5 piC
20
109.0


Oct4
A, 0.5 7dG, 0.6 5mU, 0.5 piC
20
114.8


Oct4
A, 0.5 7dG, 0.7 5mU, 0.5 piC
20
107.2


Oct4
A, 0.5 7dG, 0.8 5mU, 0.5 piC
20
110.9


Oct4
A, 0.5 7dG, 0.9 5mU, 0.5 piC
20
103.4


Oct4
A, 0.5 7dG, 5mU, 0.5 piC
20
97.8


Oct4
A, 0.5 7dG, psU, 0.5 piC
20
124.5


Sox2
A, 0.5 7dG, psU, 0.5 piC
20
109.0


Klf4
A, 0.5 7dG, psU, 0.5 piC
20
112.8


c-Myc-2 (T58A)
A, 0.5 7dG, psU, 0.5 piC
20
112.8


Lin28
A, 0.5 7dG, psU, 0.5 piC
20
126.5


Oct4
A, G, psU, 5mC
20
213.4


Sox2
A, G, psU, 5mC
10
107.2


Klf4
A, G, psU, 5mC
10
106.1


c-Myc-2 (T58A)
A, G, psU, 5mC
10
97.8


Lin28
A, G, psU, 5mC
10
95.9


Oct4
A, 0.5 7dG, psU, 0.5 piC
20
124.2


Sox2
A, 0.5 7dG, psU, 0.5 piC
10
57.3


Klf4
A, 0.5 7dG, psU, 0.5 piC
10
59.6


c-Myc-2 (T58A)
A, 0.5 7dG, psU, 0.5 piC
10
66.7


Lin28
A, 0.5 7dG, psU, 0.5 piC
10
65.2


Oct4
A, 0.5 7dG, psU, 0.3 piC
10
60.5


Sox2
A, 0.5 7dG, psU, 0.3 piC
10
58.8


Klf4
A, 0.5 7dG, psU, 0.3 piC
10
57.9


c-Myc-2 (T58A)
A, 0.5 7dG, psU, 0.3 piC
10
62.0


Lin28
A, 0.5 7dG, psU, 0.3 piC
10
64.3


Oct4
A, 0.5 7dG, 0.5 5mU, 5mC
10
64.7


Sox2
A, 0.5 7dG, 0.5 5mU, 5mC
10
62.4


Klf4
A, 0.5 7dG, 0.5 5mU, 5mC
10
75.6


c-Myc-2 (T58A)
A, 0.5 7dG, 0.5 5mU, 5mC
10
69.4


Lin28
A, 0.5 7dG, 0.5 5mU, 5mC
10
60.7


Oct4
A, 0.5 7dG, 0.5 4sU, 0.5 5mU, 5mC
10
48.3


Sox2
A, 0.5 7dG, 0.5 4sU, 0.5 5mU, 5mC
10
54.0


Klf4
A, 0.5 7dG, 0.5 4sU, 0.5 5mU, 5mC
10
58.7


c-Myc-2 (T58A)
A, 0.5 7dG, 0.5 4sU, 0.5 5mU, 5mC
10
54.7


Lin28
A, 0.5 7dG, 0.5 4sU, 0.5 5mU, 5mC
10
54.1


Oct4
A, 0.5 7dG, 0.3 5mU, 5mC
10
69.6


Sox2
A, 0.5 7dG, 0.3 5mU, 5mC
10
69.6


Klf4
A, 0.5 7dG, 0.3 5mU, 5mC
10
87.4


c-Myc-2 (T58A)
A, 0.5 7dG, 0.3 5mU, 5mC
10
68.1


Lin28
A, 0.5 7dG, 0.3 5mU, 5mC
10
74.3


Oct4
A, 0.5 7dG, 0.4 5mU, 5mC
10
71.3


Sox2
A, 0.5 7dG, 0.4 5mU, 5mC
10
69.7


Klf4
A, 0.5 7dG, 0.4 5mU, 5mC
10
74.8


c-Myc-2 (T58A)
A, 0.5 7dG, 0.4 5mU, 5mC
10
83.7


Lin28
A, 0.5 7dG, 0.4 5mU, 5mC
10
69.9


XPA-L1
A, G, psU, 5mC
20
120.0


XPA-L2
A, G, psU, 5mC
20
114.0


XPA-R1
A, G, psU, 5mC
20
159.6


CCR5-L1
A, G, psU, 5mC
20
170.4


CCR5-L2
A, G, psU, 5mC
20
142.8


CCR5-R1
A, G, psU, 5mC
20
132.0


CCR5-R2
A, G, psU, 5mC
20
154.8


CCR5-L1
A, G, psU, 5mC
10
56.6


CCR5-L2
A, G, psU, 5mC
10
58.5


CCR5-R1
A, G, psU, 5mC
10
56.8


CCR5-R2
A, G, psU, 5mC
10
58.7


TERT-L
A, G, U, C
10
49.4


TERT-R
A, G, U, C
10
37.6


MYC-L
A, G, U, C
10
39.6


MYC-R
A, G, U, C
10
33.7


BIRC5-L
A, G, U, C
10
63.0


BIRC5-R
A, G, U, C
10
44.5


TERT-L
A, 0.5 7dG, 0.4 5mU, 5mC
10
50.8


TERT-R
A, 0.5 7dG, 0.4 5mU, 5mC
10
58.3


MYC-L
A, 0.5 7dG, 0.4 5mU, 5mC
10
40.8


MYC-R
A, 0.5 7dG, 0.4 5mU, 5mC
10
41.4


BIRC5-L
A, 0.5 7dG, 0.4 5mU, 5mC
10
35.8


BIRC5-R
A, 0.5 7dG, 0.4 5mU, 5mC
10
41.5


Oct4 (SEQ ID NO: 8)
A, 0.5 7dG, 0.4 5mU, 5mC
300
2752.0


Sox2 (SEQ ID NO: 9)
A, 0.5 7dG, 0.4 5mU, 5mC
100
965.0


Klf4 (SEQ ID NO: 10)
A, 0.5 7dG, 0.4 5mU, 5mC
100
1093.8


c-Myc-2 (T58A)
A, 0.5 7dG, 0.4 5mU, 5mC
100
1265.6


Lin28
A, 0.5 7dG, 0.4 5mU, 5mC
100
1197.8


Oct4
A, 0.5 7dG, 0.35 5mU, 5mC
30
155.7


Sox2
A, 0.5 7dG, 0.35 5mU, 5mC
15
79.8


Klf4
A, 0.5 7dG, 0.35 5mU, 5mC
15
90.0


c-Myc-2 (T58A)
A, 0.5 7dG, 0.35 5mU, 5mC
15
83.2


Lin28
A, 0.5 7dG, 0.35 5mU, 5mC
15
74.0


APP UTR_L (Rat)
A, 0.5 7dG, 0.4 5mU, 5mC
20
37.9


APP UTR_R (Rat)
A, 0.5 7dG, 0.4 5mU, 5mC
20
40.0


APP Exon2L (Rat)
A, 0.5 7dG, 0.4 5mU, 5mC
20
38.6


APP Exon2R (Rat)
A, 0.5 7dG, 0.4 5mU, 5mC
20
37.9


APP 6L (Human)
A, 0.5 7dG, 0.4 5mU, 5mC
20
43.1


APP 6R (Human)
A, 0.5 7dG, 0.4 5mU, 5mC
20
43.7


APP 7L (Human)
A, 0.5 7dG, 0.4 5mU, 5mC
20
42.1


APP 7R (Human)
A, 0.5 7dG, 0.4 5mU, 5mC
20
36.2


APP 670L (Rat)
A, 0.5 7dG, 0.4 5mU, 5mC
20
27.0


APP 670R (Rat)
A, 0.5 7dG, 0.4 5mU, 5mC
20
28.3


APP 678L (Rat)
A, 0.5 7dG, 0.4 5mU, 5mC
20
30.1


APP 678R (Rat)
A, 0.5 7dG, 0.4 5mU, 5mC
20
26.2


APP 680L (Rat)
A, 0.5 7dG, 0.4 5mU, 5mC
20
8.1


APP 680R (Rat)
A, 0.5 7dG, 0.4 5mU, 5mC
20
25.4


APP 6L (Human)
A, 0.5 7dG, 0.4 5mU, 5mC
40
48.6


APP 6R (Human)
A, 0.5 7dG, 0.4 5mU, 5mC
40
48.6


APP 6L (Human)
A, G, U, C
10
54.0


APP 6R (Human)
A, G, U, C
10
61.0


APP 6L (Human)
A, 0.5 7dG, 0.4 5mU, 5mC
10
35.4


APP 6R (Human)
A, 0.5 7dG, 0.4 5mU, 5mC
10
48.0









Example 2 Transfection of Cells with Synthetic RNA
For transfection in 6-well plates, 2 μg RNA and 6 μL transfection reagent (Lipofectamine RNAiMAX, Life Technologies Corporation) were first diluted separately in complexation medium (Opti-MEM, Life Technologies Corporation or DMEM/F12+10 μg/mL insulin+5.5 μg/mL transferrin+6.7 ng/mL sodium selenite+2 μg/mL ethanolamine) to a total volume of 60 μL each. Diluted RNA and transfection reagent were then mixed and incubated for 15 min at room temperature, according to the transfection reagent-manufacturer's instructions. Complexes were then added to cells in culture. Between 30 μL and 240 μL of complexes were added to each well of a 6-well plate, which already contained 2 mL of transfection medium per well. Plates were shaken gently to distribute the complexes throughout the well. Cells were incubated with complexes for 4 hours to overnight, before replacing the medium with fresh transfection medium (2 mL/well). Volumes were scaled for transfection in 24-well and 96-well plates. Alternatively, between 0.5 μg and 5 μg of RNA and between 2-3 μL of transfection reagent (Lipofectamine 2000, Life Technologies Corporation) per μg of RNA were first diluted separately in complexation medium (Opti-MEM, Life Technologies Corporation or DMEM/F12+10 μg/mL insulin+5.5 μg/mL transferrin+6.7 ng/mL sodium selenite+2 μg/mL ethanolamine) to a total volume of between 5 μL and 100 μL each. Diluted RNA and transfection reagent were then mixed and incubated for 10 min at room temperature. Complexes were then added to cells in culture. Between 10 μL and 200 μL of complexes were added to each well of a 6-well plate, which already contained 2 mL of transfection medium per well. In certain experiments, DMEM+10% FBS or DMEM+50% FBS was used in place of transfection medium. Plates were shaken gently to distribute the complexes throughout the well. Cells were incubated with complexes for 4 hours to overnight. In certain experiments, the medium was replaced with fresh transfection medium (2 mL/well) 4 h or 24 h after transfection.
Example 3 Toxicity of and Protein Translation from Synthetic RNA Containing Non-Canonical Nucleotides
Primary human fibroblasts were transfected according to Example 2, using RNA synthesized according to Example 1. Cells were fixed and stained 20-24 h after transfection using an antibody against Oct4. The relative toxicity of the RNA was determined by assessing cell density at the time of fixation.
Example 4 Transfection Medium Formulation
A cell-culture medium was developed to support efficient transfection of cells with nucleic acids and efficient reprogramming (“transfection medium”):
DMEM/F12+15 mM HEPES+2 mM L-alanyl-L-glutamine+10 μg/mL insulin+5.5 μg/mL transferrin+6.7 ng/mL sodium selenite+2 μg/mL ethanolamine+50 μg/mL L-ascorbic acid 2-phosphate sesquimagnesium salt hydrate+4 μg/mL cholesterol+1 μM hydrocortisone+25 μg/mL polyoxyethylenesorbitan monooleate+2 μg/mL D-alpha-tocopherol acetate+20 ng/mL bFGF+5 mg/mL treated human serum albumin.
A variant of this medium was developed to support robust, long-term culture of a variety of cell types, including pluripotent stem cells (“maintenance medium”):
DMEM/F12+2 mM L-alanyl-L-glutamine+10 μg/mL insulin+5.5 μg/mL transferrin+6.7 ng/mL sodium selenite+2 μg/mL ethanolamine+50 μg/mL L-ascorbic acid 2-phosphate sesquimagnesium salt hydrate+20 ng/mL bFGF+2 ng/mL TGF-β1.
Transfection medium, in which the treated human serum albumin was treated by addition of 32 mM sodium octanoate, followed by heating at 60° C. for 4 h, followed by treatment with ion-exchange resin (AG501-X8(D), Bio-Rad Laboratories, Inc.) for 6 h at room temperature, followed by treatment with dextran-coated activated charcoal (C6241, Sigma-Aldrich Co. LLC.) overnight at room temperature, followed by centrifugation, filtering, adjustment to a 10% solution with nuclease-free water, followed by addition to the other components of the medium, was used as the transfection medium in all Examples described herein, unless otherwise noted. For reprogramming experiments, cells were plated either on uncoated plates in DMEM+10%-20% serum or on fibronectin and vitronectin-coated plates in transfection medium, unless otherwise noted. The transfection medium was not conditioned, unless otherwise noted. It is recognized that the formulation of the transfection medium can be adjusted to meet the needs of the specific cell types being cultured. It is further recognized that treated human serum albumin can be replaced with other treated albumin, for example, treated bovine serum albumin, without negatively affecting the performance of the medium. It is further recognized that other glutamine sources can be used instead of or in addition to L-alanyl-L-glutamine, for example, L-glutamine, that other buffering systems can be used instead of or in addition to HEPES, for example, phosphate, bicarbonate, etc., that selenium can be provided in other forms instead of or in addition to sodium selenite, for example, selenous acid, that other antioxidants can be used instead of or in addition to L-ascorbic acid 2-phosphate sesquimagnesium salt hydrate and/or D-alpha-tocopherol acetate, for example, L-ascorbic acid, that other surfactants can be used instead of or in addition to polyoxyethylenesorbitan monooleate, for example, Pluronic F-68 and/or Pluronic F-127, that other basal media can be used instead of or in addition to DMEM/F12, for example, MEM, DMEM, etc., and that the components of the culture medium can be varied with time, for example, by using a medium without TGF-β from day 0 to day 5, and then using a medium containing 2 ng/mL TGF-β after day 5, without negatively affecting the performance of the medium. It is further recognized that other ingredients can be added, for example, fatty acids, lysophosphatidic acid, lysosphingomyelin, sphingosine-1-phosphate, other sphingolipids, ROCK inhibitors, including Y-27632 and thiazovivin, members of the TGF-β/NODAL family of proteins, IL-6, members of the Wnt family of proteins, etc., at appropriate concentrations, without negatively affecting the performance of the medium, and that ingredients that are known to promote or inhibit the growth of specific cell types and/or agonists and/or antagonists of proteins or other molecules that are known to promote or inhibit the growth of specific cell types can be added to the medium at appropriate concentrations when it is used with those cell types without negatively affecting the performance of the medium, for example, sphingosine-1-phosphate and pluripotent stem cells. The present invention relates equally to ingredients that are added as purified compounds, to ingredients that are added as parts of well-defined mixtures, to ingredients that are added as parts of complex or undefined mixtures, for example, animal or plant oils, and to ingredients that are added by biological processes, for example, conditioning. The concentrations of the components can be varied from the listed values within ranges that will be obvious to persons skilled in the art without negatively affecting the performance of the medium. An animal component-free version of the medium was produced by using recombinant versions of all protein ingredients, and non-animal-derived versions of all other components, including semi-synthetic plant-derived cholesterol (Avanti Polar Lipids, Inc.).
Example 5 Reprogramming Human Fibroblasts Using Synthetic RNA Containing Non-Canonical Nucleotides
Primary human neonatal fibroblasts were plated in 6-well plates coated with recombinant human fibronectin and recombinant human vitronectin (each diluted in DMEM/F12 to a concentration of 1 μg/mL, 1 mL/well, and incubated at room temperature for 1 h) at a density of 10,000 cells/well in transfection medium. The following day, the cells were transfected as in Example 2, using RNA containing A, 0.5 7 dG, 0.5 5 mU, and 5 mC, and an RNA dose of 0.5 μg/well on day 1, 0.5 μg/well on day 2, 2 μg/well on day 3, 2 μg/well on day 4, and 4 μg/well on day 5. Small colonies of cells exhibiting morphology consistent with reprogramming became visible as early as day 5. The medium was replaced with maintenance medium on day 6. Cells were stained using an antibody against Oct4. Oct4-positive colonies of cells exhibiting a morphology consistent with reprogramming were visible throughout the well (FIG. 2).
Example 6 Feeder-Free, Passage-Free, Immunosuppressant-Free, Conditioning-Free Reprogramming of Primary Adult Human Fibroblasts Using Synthetic RNA
Wells of a 6-well plate were coated with a mixture of recombinant human fibronectin and recombinant human vitronectin (1 μg/mL in DMEM/F12, 1 mL/well) for 1 h at room temperature.
Primary adult human fibroblasts were plated in the coated wells in transfection medium at a density of 10,000 cells/well. Cells were maintained at 37° C., 5% CO2, and 5% O2. Beginning the following day, cells were transfected according to Example 2 daily for 5 days with RNA synthesized according to Example 1. The total amount of RNA transfected on each of the 5 days was 0.5 μg, 0.5 μg, 2 μg, 2 μg, and 4 μg, respectively. Beginning with the fourth transfection, the medium was replaced twice a day. On the day following the final transfection, the medium was replaced with transfection medium, supplemented with 10 μM Y-27632. Compact colonies of cells with a reprogrammed morphology were visible in each transfected well by day 4 (FIG. 8).
Example 7 Efficient, Rapid Derivation and Reprogramming of Cells from Adult Human Skin Biopsy Tissue
A full-thickness dermal punch biopsy was performed on a healthy, 31 year-old volunteer, according to an approved protocol. Briefly, an area of skin on the left, upper arm was anesthetized by topical application of 2.5% lidocaine. The field was disinfected with 70% isopropanol, and a full-thickness dermal biopsy was performed using a 1.5 mm-diameter punch. The tissue was rinsed in phosphate-buffered saline (PBS), was placed in a 1.5 mL tube containing 250 μL of TrypLE Select CTS (Life Technologies Corporation), and was incubated at 37° C. for 30 min. The tissue was then transferred to a 1.5 mL tube containing 250 μL of DMEM/F12-CTS (Life Technologies Corporation)+5 mg/mL collagenase, and was incubated at 37° C. for 2 h. The epidermis was removed using forceps, and the tissue was mechanically dissociated. Cells were rinsed twice in DMEM/F12-CTS. Phlebotomy was also performed on the same volunteer, and venous blood was collected in Vacutainer SST tubes (Becton, Dickinson and Company). Serum was isolated according to the manufacturer's instructions. Isogenic plating medium was prepared by mixing DMEM/F12-CTS+2 mM L-alanyl-L-glutamine (Sigma-Aldrich Co. LLC.)+20% human serum. Cells from the dermal tissue sample were plated in a fibronectin-coated well of a 6-well plate in isogenic plating medium. Many cells with a fibroblast morphology attached and began to spread by day 2 (FIG. 3A). Cells were expanded and frozen in Synth-a-Freeze (Life Technologies Corporation).
Cells were passaged into 6-well plates at a density of 5,000 cells/well. The following day, the medium was replaced with transfection medium, and the cells were transfected as in Example 2, using RNA containing A, 0.5 7 dG, 0.4 5 mU, and 5 mC, and an RNA dose of 0.5 μg/well on day 1, 0.5 μg/well on day 2, 2 μg/well on day 3, 2 μg/well on day 4, and 2 μg/well on day 5. Certain wells received additional 2 μg/well transfections on day 6 and day 7. In addition, certain wells received 2 ng/mL TGF-β1 from day 4 onward. The medium was replaced with maintenance medium on day 6. Colonies of cells exhibiting morphology consistent with reprogramming became visible between day 5 and day 10 (FIG. 3B). Colonies grew rapidly, and many exhibited a morphology similar to that of embryonic stem-cell colonies (FIG. 3C). Colonies were picked and plated in wells coated with recombinant human fibronectin and recombinant human vitronectin (each diluted in DMEM/F12 to a concentration of 1 μg/mL, 1 mL/well, incubated at room temperature for 1 h). Cells grew rapidly, and were passaged to establish lines.
Example 8 Synthesis of RiboSlice Targeting CCR5
RiboSlice pairs targeting the following sequences: L1: TCATTTTCCATACAGTCAGT (SEQ ID NO: 615), L2: TTTTCCATACAGTCAGTATC (SEQ ID NO: 616), R1: TGACTATCTTTAATGTCTGG (SEQ ID NO: 617), and R2: TATCTTTAATGTCTGGAAAT (SEQ ID NO: 618) were synthesized according to Example 1 (FIG. 4A and FIG. 4B). These pairs target 20-bp sites within the human CCR5 gene on the sense (L1 and L2) or antisense strand (R1 and R2). The following pairs were prepared: L1&R1, L1&R2, L2&R1, and L2&R2.
Example 9 Measurement of CCR5 Gene-Editing Efficiency Using a Mismatch-Detecting Nuclease
Primary human fibroblasts were plated in 6-well plates coated with recombinant human fibronectin and recombinant human vitronectin (each diluted in DMEM/F12 to a concentration of 1 μg/mL, 1 mL/well, and incubated at room temperature for 1 h) at a density of 10,000 cells/well in transfection medium. The following day, the cells were transfected as in Example 2 with RNA synthesized according to Example 8. Two days after the transfection, genomic DNA was isolated and purified. A region within the CCR5 gene was amplified by PCR using the primers F: AGCTAGCAGCAAACCTTCCCTTCA (SEQ ID NO: 619) and R: AAGGACAATGTTGTAGGGAGCCCA (SEQ ID NO: 620). 150 ng of the amplified PCR product was hybridized with 150 ng of reference DNA in 10 mM Tris-Cl+50 mM KCl+1.5 mM MgCl2. The hybridized DNA was treated with a mismatch-detecting endonuclease (SURVEYOR nuclease, Transgenomic, Inc.) and the resulting products were analyzed by agarose gel electrophoresis (FIG. 4C and FIG. 4D).
Example 10 High-Efficiency Gene Editing by Repeated Transfection with RiboSlice
Primary human fibroblasts were plated as in Example 9. The following day, the cells were transfected as in Example 2 with RNA synthesized according to Example 8. The following day cells in one of the wells were transfected a second time. Two days after the second transfection, the efficiency of gene editing was measured as in Example 9 (FIG. 4E).
Example 11 Gene-Editing of CCR5 Using RiboSlice and DNA-Free, Feeder-Free, Immunosuppressant-Free, Conditioning-Free Reprogramming of Human Fibroblasts
Primary human fibroblasts were plated as in Example 9. The following day, the cells were transfected as in Example 2 with RNA synthesized according to Example 8. Approximately 48 h later, the cells were reprogrammed according to Example 5, using RNA synthesized according to Example 1. Large colonies of cells with a morphology characteristic of reprogramming became visible as in Example 5 (FIG. 4F). Colonies were picked to establish lines. Cell lines were subjected to direct sequencing to confirm successful gene editing (FIG. 4G).
Example 12 Personalized Cell-Replacement Therapy for HIV/AIDS Comprising Gene-Edited Reprogrammed Cells
Patient skin cells are gene-edited and reprogrammed to hematopoietic cells according to the present inventors' previously disclosed inventions (U.S. application Ser. No. 13/465,490, U.S. Provisional Application No. 61/637,570, and U.S. Provisional Application No. 61/664,494) and/or Example 11. Cells are then enzymatically released from the culture vessel, and CD34+/CD90+/Lin− or CD34+/CD49f+/Lin− cells are isolated. Between about 1×103 and about 1×105 cells are infused into a main vein of the patient. Hematopoietic cells home to the bone marrow cavity and engraft.
Example 13 Production of APP-Inactivated Rat Embryonic Stem Cells
Rat embryonic stem cells are plated in 6-well plates at a density of 10,000 cells/well in rat stem cell medium. The following day, the cells are transfected as in Example 2 with 0.5 μg/well of RiboSlice synthesized according to Example 1 targeting the following sequences: L: TTCTGTGGTAAACTCAACAT (SEQ ID NO: 621) and R: TCTGACTCCCATTTTCCATT (SEQ ID NO: 622) (0.25 μg L and 0.25 μg R).
Example 14 Production of APP-Knockout Rats Using APP-Inactivated Rat Embryonic Stem Cells
Rat embryonic stem cells are gene-editing according to Example 13 and microinjected into rat blastocysts. The microinjected blastocysts are then transferred to a pseudopregnant female rat.
Example 15 Production of APP-Inactivated Embryos for the Generation of Knockout Rats
A RiboSlice pair targeting the following sequences: L: TTCTGTGGTAAACTCAACAT (SEQ ID NO: 623) and R: TCTGACTCCCATTTTCCATT (SEQ ID NO: 624) is synthesized according to Example 1. RiboSlice at a concentration of 5 μg/μL is injected into the pronucleus or cytoplasm of a 1-cell-stage rat embryo. The embryo is then transferred to a pseudopregnant female rat.
Example 16 Transfection of Cells with Synthetic RNA Containing Non-Canonical Nucleotides and DNA Encoding a Repair Template
For transfection in 6-well plates, 1 μg RNA encoding gene-editing proteins targeting exon 16 of the human APP gene, 1 μg single-stranded repair template DNA containing a PstI restriction site that was not present in the target cells, and 6 μL transfection reagent (Lipofectamine RNAiMAX, Life Technologies Corporation) were first diluted separately in complexation medium (Opti-MEM, Life Technologies Corporation) to a total volume of 120 μL. Diluted RNA, repair template, and transfection reagent were then mixed and incubated for 15 min at room temperature, according to the transfection reagent-manufacturer's instructions. Complexes were added to cells in culture. Approximately 120 μL of complexes were added to each well of a 6-well plate, which already contained 2 mL of transfection medium per well. Plates were shaken gently to distribute the complexes throughout the well. Cells were incubated with complexes for 4 hours to overnight, before replacing the medium with fresh transfection medium (2 mL/well). The next day, the medium was changed to DMEM+10% FBS. Two days after transfection, genomic DNA was isolated and purified. A region within the APP gene was amplified by PCR, and the amplified product was digested with PstI and analyzed by gel electrophoresis (FIG. 16).
Example 17 Insertion of a Transgene into Rat Embryonic Stem Cells at a Safe Harbor Location
Rat embryonic stem cells are plated in 6-well plates at a density of 10,000 cells/well in rat stem cell medium. The following day, the cells are transfected as in Example 13 with RiboSlice targeting the following sequences: L: TATCTTCCAGAAAGACTCCA (SEQ ID NO: 625) and R: TTCCCTTCCCCCTTCTTCCC (SEQ ID NO: 626), synthesized according to Example 1, and a repair template containing a transgene flanked by two regions each containing approximately 400 bases of homology to the region surrounding the rat Rosa26 locus.
Example 18 Humanized LRRK2 Rat
Rat embryonic stem cells are plated and transfected as in Example 13 with RiboSlice targeting the following sequences: L: TTGAAGGCAAAAATGTCCAC (SEQ ID NO: 627) and R: TCTCATGTAGGAGTCCAGGA (SEQ ID NO: 628), synthesized according to Example 1. Two days after transfection, the cells are transfected according Example 17, wherein the transgene contains the human LRRK2 gene, and, optionally, part or all of the human LRRK2 promoter region.
Example 19 Insertion of a Transgene into Human Fibroblasts at a Safe Harbor Location
Primary human fibroblasts are plated as in Example 9. The following day, the cells are transfected as in Example 2 with RiboSlice targeting the following sequences: L: TTATCTGTCCCCTCCACCCC (SEQ ID NO: 629) and R: TTTTCTGTCACCAATCCTGT (SEQ ID NO: 630), synthesized according to Example 1, and a repair template containing a transgene flanked by two regions each containing approximately 400 bases of homology to the region surrounding the human AAVS1 locus.
Example 20 Inserting an RNAi Sequence into a Safe Harbor Location
Primary human fibroblasts are plated and transfected according to Example 19, wherein the transgene contains a sequence encoding an shRNA, preceded by the PolIII promoter.
Example 21 Gene Editing of Myc Using RiboSlice
Primary human fibroblasts were plated in 6-well plates at a density of 50,000 cells/well in DMEM+10% FBS. Two days later, the medium was changed to transfection medium. Four hours later, the cells were transfected as in Example 2 with 1 μg/well of RiboSlice targeting the following sequences: L: TCGGCCGCCGCCAAGCTCGT (SEQ ID NO: 631) and R: TGCGCGCAGCCTGGTAGGAG (SEQ ID NO: 632), synthesized according to Example 1. The following day gene-editing efficiency was measured as in Example 9 using the following primers: F: TAACTCAAGACTGCCTCCCGCTTT (SEQ ID NO: 633) and R: AGCCCAAGGTTTCAGAGGTGATGA (SEQ ID NO: 634) (FIG. 5).
Example 22 Cancer Therapy Comprising RiboSlice Targeting Myc
HeLa cervical carcinoma cells were plated in 6-well plates at a density of 50,000 cells/well in folate-free DMEM+2 mM L-alanyl-L-glutamine+10% FBS. The following day, the medium was changed to transfection medium. The following day, the cells were transfected as in Example 21.
Example 23 Gene Editing of BIRC5 Using RiboSlice
Primary human fibroblasts were plated in 6-well plates at a density of 50,000 cells/well in DMEM+10% FBS. Two days later, the medium was changed to transfection medium. Four hours later, the cells were transfected as in Example 2 with 1 μg/well of RiboSlice targeting the following sequences: L: TTGCCCCCTGCCTGGCAGCC (SEQ ID NO: 16) and R: TTCTTGAATGTAGAGATGCG (SEQ ID NO: 17), synthesized according to Example 1. The following day gene-editing efficiency was measured as in Example 9 using the following primers: F: GCGCCATTAACCGCCAGATTTGAA (SEQ ID NO: 635) and R: TGGGAGTTCACAACAACAGGGTCT (SEQ ID NO: 636) (FIG. 6).
Example 24 Cancer Therapy Comprising RiboSlice Targeting BIRC5
HeLa cervical carcinoma cells were plated in 6-well plates at a density of 50,000 cells/well in folate-free DMEM+2 mM L-alanyl-L-glutamine+10% FBS. The following day, the medium was changed to transfection medium. The following day, the cells were transfected as in Example 23 (FIG. 7A and FIG. 7B).
Example 25 Culture of Cancer-Cell Lines
The cancer cell lines HeLa (cervical carcinoma), MDA-MB-231 (breast), HCT 116 (colon), U87 MG (glioma), and U-251 (glioma) were propagated in culture. Cells were cultured in DMEM+10% FBS or DMEM+50% FBS and maintained at 37° C., 5% CO2, and either ambient 02 or 5% O2. Cells grew rapidly under all conditions, and were routinely passaged every 2-5 days using a solution of trypsin in HBSS.
Example 26 RiboSlice Gene-Editing RNA Design Process and Algorithm
The annotated DNA sequence of the BIRC5 gene was retrieved from NCBI using the eFetch utility and a python script. The same python script was used to identify the DNA sequences encoding the protein within each of the four exons of the BIRC5 gene. The script then searched these sequences, and the 40 bases flanking each side, for sequence elements satisfying the following conditions: (i) one element exists on the primary strand, the other on the complementary strand, (ii) each element begins with a T, and (iii) the elements are separated by no fewer than 12 bases and no more than 20 bases. Each element was then assigned a score representing its likelihood of binding to other elements within the human genome using Qblast (NCBI). This score was computed as the sum of the inverse of the nine lowest E-values, excluding the match to the target sequence. Pair scores were computed by adding the scores for the individual elements.
Example 27 Synthesis of RNA Encoding Gene-Editing Proteins (RiboSlice)
RNA encoding gene-editing proteins was designed according to Example 26, and synthesized according to Example 1 (Table 10, FIG. 9). The RNA was diluted with nuclease-free water to between 200 ng/μL and 500 ng/μL, and was stored at 4° C.







TABLE 10







RiboSlice Synthesis










Template

Reaction
ivT


(SEQ ID of Binding Site)
Nucleotides
Volume/μL
Yield/μg













BIRC5-1.1L
A, 0.5 7dG,
20
124.1


(SEQ ID NO: 16)
0.4 5mU, 5mC


BIRC5-1.1R
A, 0.5 7dG,
20
115.6


(SEQ ID NO: 17)
0.4 5mU, 5mC


BIRC5-1.2L
A, 0.5 7dG,
20
120.3


(SEQ ID NO: 18)
0.4 5mU, 5mC


BIRC5-1.2R
A, 0.5 7dG,
20
121.3


(SEQ ID NO: 19)
0.4 5mU, 5mC


BIRC5-1.3L
A, 0.5 7dG,
20
120.3


(SEQ ID NO: 20)
0.4 5mU, 5mC


BIRC5-1.3R
A, 0.5 7dG,
20
113.7


(SEQ ID NO: 21)
0.4 5mU, 5mC


BIRC5-2.1L
A, 0.5 7dG,
20
105.3


(SEQ ID NO: 22)
0.4 5mU, 5mC


BIRC5-2.1R
A, 0.5 7dG,
20
120.3


(SEQ ID NO: 23)
0.4 5mU, 5mC


BIRC5-2.2L
A, 0.5 7dG,
20
101.5


(SEQ ID NO: 24)
0.4 5mU, 5mC


BIRC5-2.2R
A, 0.5 7dG,
20
111.9


(SEQ ID NO: 25)
0.4 5mU, 5mC


BIRC5-3.1L
A, 0.5 7dG,
20
107.2


(SEQ ID NO: 26)
0.4 5mU, 5mC


BIRC5-3.1R
A, 0.5 7dG,
20
113.7


(SEQ ID NO: 27)
0.4 5mU, 5mC


BIRC5-2.1L
A, 0.5 7dG,
300
577.9


(SEQ ID NO: 22)
0.35 5mU, 5mC


BIRC5-2.1R
A, 0.5 7dG,
300
653.6


(SEQ ID NO: 23)
0.35 5mU, 5mC









Example 28 Activity Analysis of RiboSlice Targeting BIRC5
Primary adult human fibroblasts were transfected according to Example 2 with 6 RiboSlice pairs targeting BIRC5, designed according to Example 26, and synthesized according to Example 27. Two days after transfection, genomic DNA was isolated and purified. To measure gene-editing efficiency, 150 ng of the amplified PCR product was hybridized with 150 ng of reference DNA in 10 mM Tris-Cl+50 mM KCl+1.5 mM MgCl2. The hybridized DNA was treated with the SURVEYOR mismatch-specific endonuclease (Transgenomic, Inc.), and the resulting products were analyzed by agarose gel electrophoresis (FIG. 10A). All six of the tested RiboSlice pairs efficiently edited the BIRC5 gene, as demonstrated by the appearance of bands of the expected sizes (asterisks in FIG. 10A).
Example 29 Mitosis-Inhibition Analysis of RiboSlice Targeting BIRC5
Primary adult human fibroblasts were gene edited according to Example 28, and were then propagated in culture. After 11 days, genomic DNA was isolated and purified, and gene-editing efficiency was measured as in Example 28 (FIG. 10B). None of the tested RiboSlice pairs inhibited the proliferation of the fibroblasts, as shown by the appearance of bands of the expected sizes (asterisks in FIG. 10B) in genomic DNA isolated from the proliferating cells, demonstrating the low toxicity to normal fibroblasts of these RiboSlice pairs.
Example 30 Anti-Cancer-Activity Analysis of RiboSlice Targeting BIRC5
Primary adult human fibroblasts and HeLa cervical carcinoma cells, cultured according to Example 25 were transfected with RiboSlice pairs according to Example 28. Proliferation of the fibroblasts slowed briefly due to transfection reagent-associated toxicity, but recovered within 2 days of transfection. In contrast, proliferation of HeLa cells slowed markedly, and many enlarged cells with fragmented nuclei were observed in transfected wells. After 2-3 days, many cells exhibited morphology indicative of apoptosis, demonstrating the potent anti-cancer activity of RiboSlice targeting BIRC5.
Example 31 In Vivo RiboSlice Safety Study
40 female NCr nu/nu mice were injected subcutaneously with 5×106 MDA-MB-231 tumor cells in 50% Matrigel (BD Biosciences). Cell injection volume was 0.2 mL/mouse. The age of the mice at the start of the study was 8 to 12 weeks. A pair match was conducted, and animals were divided into 4 groups of 10 animals each when the tumors reached an average size of 100-150 mm3, and treatment was begun. Body weight was measured every day for the first 5 days, and then biweekly to the end of the study. Treatment consisted of RiboSlice BIRC5-1.2 complexed with a vehicle (Lipofectamine 2000, Life Technologies Corporation). To prepare the dosing solution for each group, 308 μL of complexation buffer (Opti-MEM, Life Technologies Corporation) was pipetted into each of two sterile, RNase-free 1.5 mL tubes. 22 μL of RiboSlice BIRC5-1.2 (500 ng/μL) was added to one of the two tubes, and the contents of the tube were mixed by pipetting. 22 μL of vehicle was added to the second tube. The contents of the second tube were mixed, and then transferred to the first tube, and mixed with the contents of the first tube by pipetting to form complexes. Complexes were incubated at room temperature for 10 min. During the incubation, syringes were loaded. Animals were injected either intravenously or intratumorally with a total dose of 1 μg RNA/animal in 60 μL total volume/animal. A total of 5 treatments were given, with injections performed every other day. Doses were not adjusted for body weight. Animals were followed for 17 days. No significant reduction in mean body weight was observed (FIG. 11; RiboSlice BIRC5-1.2 is labeled “ZK1”), demonstrating the in vivo safety of RiboSlice gene-editing RNA.
Example 32 Anti-Cancer-Activity Analysis of RiboSlice Targeting BIRC5 in a Glioma Model
The U-251 glioma cell line, cultured according to Example 25, was transfected with RiboSlice pairs according to Example 28. Glioma cells responded to treatment similarly to HeLa cells: proliferation slowed markedly, and many enlarged cells with fragmented nuclei were observed in transfected wells. After 2-3 days, many cells exhibited morphology indicative of apoptosis, demonstrating the potent anti-cancer activity of RiboSlice targeting BIRC5 in a glioma model.
Example 33 Screening of Reagents for Delivery of Nucleic Acids to Cells
Delivery reagents including polyethyleneimine (PEI), various commercial lipid-based transfection reagents, a peptide-based transfection reagent (N-TER, Sigma-Aldrich Co. LLC.), and several lipid-based and sterol-based delivery reagents were screened for transfection efficiency and toxicity in vitro. Delivery reagents were complexed with RiboSlice BIRC5-1.2, and complexes were delivered to HeLa cells, cultured according to Example 25. Toxicity was assessed by analyzing cell density 24 h after transfection. Transfection efficiency was assessed by analyzing morphological changes, as described in Example 30. The tested reagents exhibited a wide range of toxicities and transfection efficiencies. Reagents containing a higher proportion of ester bonds exhibited lower toxicities than reagents containing a lower proportion of ester bonds or no ester bonds.
Example 34 High-Concentration Liposomal RiboSlice
High-Concentration Liposomal RiboSlice was prepared by mixing 1 μg RNA at 500 ng/μL with 3 μL of complexation medium (Opti-MEM, Life Technologies Corporation), and 2.5 μL of transfection reagent (Lipofectamine 2000, Life Technologies Corporation) per μg of RNA with 2.5 μL of complexation medium. Diluted RNA and transfection reagent were then mixed and incubated for 10 min at room temperature to form High-Concentration Liposomal RiboSlice. Alternatively, a transfection reagent containing DOSPA or DOSPER is used.
Example 35 In Vivo RiboSlice Efficacy Study—Subcutaneous Glioma Model
40 female NCr nu/nu mice were injected subcutaneously with 1×107 U-251 tumor cells. Cell injection volume was 0.2 mL/mouse. The age of the mice at the start of the study was 8 to 12 weeks. A pair match was conducted, and animals were divided into 4 groups of 10 animals each when the tumors reached an average size of 35-50 mm3, and treatment was begun. Body weight was measured every day for the first 5 days, and then biweekly to the end of the study. Caliper measurements were made biweekly, and tumor size was calculated. Treatment consisted of RiboSlice BIRC5-2.1 complexed with a vehicle (Lipofectamine 2000, Life Technologies Corporation). To prepare the dosing solution, 294 μL of complexation buffer (Opti-MEM, Life Technologies Corporation) was pipetted into a tube containing 196 μL of RiboSlice BIRC5-1.2 (500 ng/μL), and the contents of the tube were mixed by pipetting. 245 μL of complexation buffer was pipetted into a tube containing 245 μL of vehicle. The contents of the second tube were mixed, and then transferred to the first tube, and mixed with the contents of the first tube by pipetting to form complexes. Complexes were incubated at room temperature for 10 min. During the incubation, syringes were loaded. Animals were injected intratumorally with a total dose of either 2 μg or 5 μg RNA/animal in either 20 μL or 50 μL total volume/animal. A total of 5 treatments were given, with injections performed every other day. Doses were not adjusted for body weight. Animals were followed for 25 days.
Example 36 Synthesis of High-Activity/High-Fidelity RiboSlice In Vitro-Transcription Template
An in vitro-transcription template encoding a T7 bacteriophage RNA-polymerase promoter, 5′-untranslated region, strong Kozak sequence, TALE N-terminal domain, 18 repeat sequences designed according to Example 26, TALE C-terminal domain, and nuclease domain comprising the StsI sequence (SEQ ID NO: 1), StsI-HA sequence (SEQ ID NO: 2), StsI-HA2 sequence (SEQ ID NO: 3), StsI-UHA sequence (SEQ ID NO: 4), StsI-UHA2 sequence (SEQ ID NO: 5), StsI-HF sequence (SEQ ID NO: 6) or StsI-HF2 sequence (SEQ ID NO: 7) is synthesized using standard cloning and molecular biology techniques, or alternatively, is synthesized by direct chemical synthesis, for example using a gene fragment assembly technique (e.g., gBlocks, Integrated DNA Technologies, Inc.).
Example 37 Synthesis of High-Activity/High-Fidelity RiboSlice Gene-Editing RNA
High-Activity RiboSlice and High-Fidelity RiboSlice are synthesized according to Example 27, using in vitro-transcription templates synthesized according to Example 36.
Example 38 Generation of RiboSlice-Encoding Replication-Incompetent Virus for Treatment of Proteopathy
A nucleotide sequence comprising RiboSlice targeting a DNA sequence that encodes a plaque-forming protein sequence is incorporated into a mammalian expression vector comprising a replication-incompetent viral genome, and transfected into a packaging cell line to produce replication-incompetent virus. The culture supernatant is collected, and filtered using a 0.45 μm filter to remove debris.
Example 39 Generation of RiboSlice-Encoding Replication-Competent Oncolytic Virus for Treatment of Cancer
A nucleotide sequence comprising RiboSlice targeting the BIRC5 gene, is incorporated into a mammalian expression vector comprising a replication-competent viral genome, and transfected into a packaging cell line to produce replication-competent virus. The culture supernatant is collected and filtered, according to Example 38.
Example 40 In Vivo RiboSlice Efficacy Study Orthotopic Glioma Model, Intrathecal Route of Administration
40 female NCr nu/nu mice are injected intracranially with 1×105 U-251 tumor cells. Cell injection volume is 0.02 mL/mouse. The age of the mice at the start of the study is 8 to 12 weeks. After 10 days, animals are divided into 4 groups of 10 animals each, and treatment is begun. Body weight is measured every day for the first 5 days, and then biweekly to the end of the study. Treatment consists of RiboSlice BIRC5-2.1 complexed with a vehicle (Lipofectamine 2000, Life Technologies Corporation). To prepare the dosing solution, 294 μL of complexation buffer (Opti-MEM, Life Technologies Corporation) is pipetted into a tube containing 196 μL of RiboSlice BIRC5-1.2 (500 ng/μL), and the contents of the tube are mixed by pipetting. 245 μL of complexation buffer is pipetted into a tube containing 245 μL of vehicle. The contents of the second tube are mixed, and then transferred to the first tube, and mixed with the contents of the first tube by pipetting to form complexes. Complexes are incubated at room temperature for 10 min. During the incubation, syringes are loaded. Animals are injected intrathecally with a total dose of 1-2 μg RNA/animal in 10-20 μL total volume/animal. A total of 5 treatments are given, with injections performed every other day. Doses are not adjusted for body weight. Animals are followed for 60 days.
Example 41 Treatment of Glioma with RiboSlice—IV Perfusion
A patient with a diagnosis of glioma is administered 1 mg of High-Concentration Liposomal RiboSlice BIRC5-2.1, prepared according to Example 34 by IV infusion over the course of 1 h, 3 times a week for 4 weeks. For an initial tumor volume of greater than 500 mm3, the tumor is debulked surgically and optionally by radiation therapy and/or chemotherapy before RiboSlice treatment is begun. The patient is optionally administered TNF-α and/or 5-FU using a standard dosing regimen as a combination therapy.
Example 42 Treatment of Glioma with RiboSlice—Replication-Competent Oncolytic Virus
A patient is administered 1 mL of replicating virus particles (1000 CFU/mL), prepared according to Example 39, by intrathecal or intracranial injection.
Example 43 Treatment of Parkinson's Disease with RiboSlice Targeting SNCA
A patient with a diagnosis of Parkinson's disease is administered 50 μg of RiboSlice targeting the SNCA gene by intrathecal or intracranial injection.
Example 44 Treatment of Alzheimer's Disease with RiboSlice Targeting APP
A patient with a diagnosis of Alzheimer's disease is administered 50 μg of RiboSlice targeting the APP gene by intrathecal or intracranial injection.
Example 45 Treatment of Type II Diabetes with RiboSlice Targeting IAPP
A patient with a diagnosis of type II diabetes is administered 5 mg of RiboSlice targeting the IAPP gene by intravenous, intraperitoneal or intraportal injection.
Example 46 iRiboSlice Personalized Cancer Therapy
A biopsy is taken from a patient with a diagnosis of cancer. Genomic DNA is isolated and purified from the biopsy, and the sequence of the DNA (either the whole-genome sequence, exome sequence or the sequence of one or more cancer-associated genes) is determined. A RiboSlice pair targeting the patient's individual cancer sequence (iRiboSlice) is designed according to Example 26 and synthesized according to Example 27. The patient is administered the personalized iRiboSlice using a route of administration appropriate for the location and type of cancer.
Example 47 RiboSlice Mixtures for Genetically Diverse/Treatment-Resistant Cancer
A patient with a diagnosis of genetically diverse and/or treatment-resistant cancer is administered a mixture of RiboSlice pairs targeting multiple cancer-associated genes and/or multiple sequences in one or more cancer-associated genes.
Example 48 Mito-RiboSlice for Mitochondrial Disease
A patient with a diagnosis of a mitochondrial disease is administered 2 mg of RiboSlice targeting the disease-associated sequence and containing a mitochondrial localization sequence by intramuscular injection.
Example 49 Treatment of Eye Disease with RiboSlice Eye Drops
A patient with a diagnosis of a corneal or conjunctival disease is administered RiboSlice formulated as a 0.5% isotonic solution.
Example 50 Treatment of Skin Disease with RiboSlice Topical Formulation
A patient with a diagnosis of a skin disease is administered RiboSlice formulated as a 1% topical cream/ointment containing one or more stabilizers that prevent degradation of the RNA.
Example 51 Treatment of Lung or Respiratory Disease with RiboSlice Aerosol Formulation
A patient with a diagnosis of a lung or respiratory disease is administered RiboSlice formulated as a 0.5% aerosol spray.
Example 52 Treatment of Infectious Disease with RiboSlice Targeting a DNA Sequence Present in the Infectious Agent
A patient with a diagnosis of an infectious disease is administered RiboSlice targeting a sequence present in the specific infectious agent with which the patient is infected using a route of administration appropriate to the location and type of infection, and a dose appropriate for the route of administration and severity of the infection.
Example 53 Gene-Edited Human Zygotes for In Vitro Fertilization
A human germ cell, zygote or early-stage blastocyst is transfected with RiboSlice targeting a gene that encodes a disease-associated mutation or mutation associated with an undesired trait. The genome is characterized, and the cell is prepared for in vitro fertilization.
Example 54 Cleavage-Domain Screen for Activity, Fidelity Enhancement of Gene-Editing Proteins
A panel of RiboSlice pairs, each comprising a different cleavage domain, are designed according to Example 26 and synthesized according to Example 27. The activity of the RiboSlice pairs is determined as in Example 28.
Example 55 Gene-Edited Cells for Screening Parkinson's Disease-Causing Toxicants
Primary human adult fibroblasts are gene edited according to Example 28 using RiboSlice targeting SNCA (Table 11) and repair templates to generate cells with the SNCA A30P, E46K, and A53T mutations. Cells are reprogrammed and differentiated to dopaminergic neurons. The neurons are used in a high-throughput α-synuclein-aggregation toxicant-screening assay to identify toxicants that can contribute to Parkinson's disease.







TABLE 11







RiboSlice Pairs for Generation of SNCA A30P, E46K, and A53T.














Target








Amino
Left RiboSlice
SEQ
Right RiboSlice
SEQ



Exon
Acid
Binding Site
ID NO
Binding Site
ID NO
Spacing





1
A30
TGAGAAAACCAAA
637
TAGAGAACACCCTCT
638
20




CAGGGTG

TTTGT




 


2
E46
TGTTTTTGTAGGCT
639
TACCTGTTGCCACAC
640
16




CCAAAA

CATGC




 


2
A53
TCCAAAACCAAGG
641
TAAGCACAATGGAG
642
19




AGGGAGT

CTTACC









Example 56 Gene-Edited Cells for Screening Cancer-Causing Toxicants
Primary human adult fibroblasts are gene edited according to Example 28 using RiboSlice targeting TP53 (Table 12) and repair templates to generate cells with the TP53 P47S, R72P, and V217M mutations. Cells are reprogrammed and differentiated to hepatocytes. The hepatocytes are used in a high-throughput in vitro-transformation toxicant-screening assay to identify toxicants that can contribute to cancer.







TABLE 12







RiboSlice Pairs for Generation of TP53 P475, R72P, and V217M














Target








Amino
Left Ribo Slice
SEQ
Right Ribo Slice
SEQ



Exon
Acid
Binding Site
ID NO
Binding Site
ID NO
Spacing





4
P47
TCCCAAGCAATG
643
TGAACCATTGTTCA
644
15




GATGATTT

ATATCG




 


4
R72
TGAAGCTCCCAG
645
TAGGAGCTGCTGGT
646
19




AATGCCAG

GCAGGG




 


6
V217
TGGATGACAGAA
647
TCAGGCGGCTCATA
648
15




ACACTTTT

GGGCAC









Example 57 Design and Synthesis of RNA Encoding Engineered Gene-Editing Proteins (RiboSlice)
RNA encoding gene-editing proteins designed according to Example 26 was synthesized according to Example 27 (Table 13). Each gene-editing protein comprised a DNA-binding domain comprising a transcription activator-like (TAL) effector repeat domain comprising 35-36 amino acid-long repeat sequences, as indicated in Table 13. Sequence ID numbers are given for the 36 amino acid-long repeat sequences. The label “18” in the template name indicates that the 18th repeat sequence was 36 amino acids long. The label “EO” in the template name indicates that every other repeat sequence was 36 amino acids long. The amino acids following the label “18” or “EO” indicate the amino acids at the C-terminus of the 36 amino acid-long repeat sequence(s). The label “StsI” indicates that the nuclease domain contained the StsI cleavage domain. Templates without the “StsI” label contained the FokI cleavage domain.







TABLE 13







RiboSlice Encoding Engineered Gene-Editing Proteins.










Template

Reaction
ivT


(SEQ ID of Repeat Sequence)
Nucleotides
Volume/μL
Yield/μg













BIRC5-2.1L-18-AHGGG
A, 0.5 7dG, 0.4 5mU, 5mC
20
11.9


(SEQ ID NO: 54)


BIRC5-2.1R-18-AHGGG
A, 0.5 7dG, 0.4 5mU, 5mC
20
11.9


(SEQ ID NO: 54)


BIRC5-2.1L-18-AGHGG
A, 0.5 7dG, 0.4 5mU, 5mC
20
10.7


(SEQ ID NO: 55)


BIRC5-2.1R-18-AGHGG
A, 0.5 7dG, 0.4 5mU, 5mC
20
10.9


(SEQ ID NO: 55)


BIRC5-2.1L-18-AHGSG
A, 0.5 7dG, 0.4 5mU, 5mC
20
11.9


(SEQ ID NO: 56)


BIRC5-2.1R-18-AHGSG
A, 0.5 7dG, 0.4 5mU, 5mC
20
12.7


(SEQ ID NO: 56)


BIRC5-2.1L-18-AHGGG
A, 0.5 7dG, 0.4 5mU, 5mC
20
34.5


(SEQ ID NO: 54)


BIRC5-2.1R-18-AHGGG
A, 0.5 7dG, 0.4 5mU, 5mC
20
34.8


(SEQ ID NO: 54)


BIRC5-2.1L-18-AGHGG
A, 0.5 7dG, 0.4 5mU, 5mC
20
32.7


(SEQ ID NO: 55)


BIRC5-2.1R-18-AGHGG
A, 0.5 7dG, 0.4 5mU, 5mC
20
37.4


(SEQ ID NO: 55)


BIRC5-2.1L-18-AHGSG
A, 0.5 7dG, 0.4 5mU, 5mC
20
31.5


(SEQ ID NO: 56)


BIRC5-2.1R-18-AHGSG
A, 0.5 7dG, 0.4 5mU, 5mC
20
34.1


(SEQ ID NO: 56)


BIRC5-2.1L
A, 0.5 7dG, 0.4 5mU, 5mC
20
34.9


BIRC5-2.1R
A, 0.5 7dG, 0.4 5mU, 5mC
20
25.9


BIRC5-2.1L
A, 0.5 7dG, 0.4 5mU, 5mC
20
41.5


BIRC5-2.1R
A, 0.5 7dG, 0.4 5mU, 5mC
20
38.8


BIRC5-2.1L-StsI
A, 0.5 7dG, 0.4 5mU, 5mC
20
22.2


BIRC5-2.1R-StsI
A, 0.5 7dG, 0.4 5mU, 5mC
20
18.4


BIRC5-2.1L-EO-AGHGG
A, 0.5 7dG, 0.4 5mU, 5mC
20
21.6


(SEQ ID NO: 55)


BIRC5-2.1L
A, 0.5 7dG, 0.4 5mU, 5mC
20
17.3


BIRC5-2.1L-StsI
A, G, U, C
10
71.3


BIRC5-2.1R-StsI
A, G, U, C
10
75.1


BIRC5-2.1L-EO-AGHGG
A, G, U, C
10
66.4


(SEQ ID NO: 55)


BIRC5-2.1R-EO-AGHGG
A, G, U, C
10
52.4


(SEQ ID NO: 55)









Example 58 Activity Analysis of RiboSlice Targeting BIRC5
The activity of RiboSlice molecules synthesized according to Example 57 was analyzed according to Example 28 (FIG. 12A, FIG. 12B, and FIG. 14). High-efficiency gene editing was observed in cells expressing gene-editing proteins containing one or more 36 amino acid-long repeat sequences. Gene-editing efficiency was highest in cells expressing gene-editing proteins containing one or more repeat sequences containing the amino-acid sequence: GHGG (SEQ ID NO: 675).
Example 59 In Vivo RiboSlice AAV Safety and Efficacy Study Subcutaneous Glioma Model, Intratumoral Route of Delivery
Animals were set up with tumors comprising U-251 human glioma cells according to Example 35. AAV serotype 2 encoding GFP, BIRC5-2.1L RiboSlice, and BIRC5-2.1R RiboSlice was prepared according to standard techniques (AAV-2 Helper Free Expression System, Cell Biolabs, Inc.). Viral stocks were stored at 4° C. (short term) or −80° C. (long term). Animals received intratumoral injections of either 160 μL GFP AAV on day 1 or 80 μL BIRC5-2.1L RiboSlice AAV+80 μL BIRC5-2.1R RiboSlice AAV on day 1 and day 15. Animals were followed for 25 days. No significant reduction in mean body weight was observed (FIG. 13A), demonstrating the in vivo safety of RiboSlice AAV. Tumor growth was inhibited in the RiboSlice AAV group (FIG. 13B), demonstrating the in vivo efficacy of RiboSlice AAV.
Example 60 Treatment of Cancer with RiboSlice AAV
A patient is administered 1 mL of RiboSlice AAV virus particles, prepared according to Example 59, by intrathecal or intracranial injection. Dosing is repeated as necessary. For a patient with an initial tumor volume of greater than 500 mm3, the tumor is debulked surgically and optionally by radiation therapy and/or chemotherapy before RiboSlice AAV treatment is begun. The patient is optionally administered TNF-α and/or 5-FU using a standard dosing regimen as a combination therapy.
Example 61 iRiboSlice AAV Personalized Cancer Therapy
A biopsy is taken from a patient with a diagnosis of cancer. Genomic DNA is isolated and purified from the biopsy, and the sequence of the DNA (either the whole-genome sequence, exome sequence or sequence of one or more cancer-associated genes) is determined. A RiboSlice pair targeting the patient's individual cancer sequence (iRiboSlice) is designed according to Example 26 and synthesized according to Example 59. The patient is administered the personalized iRiboSlice AAV using a route of administration appropriate for the location and type of cancer.
Example 62 Liposome Formulation and Nucleic-Acid Encapsulation
Liposomes are prepared using the following formulation: 3.2 mg/mL N-(carbonyl-ethoxypolyethylene glycol 2000)-1,2-distearoyl-sn-glycero-3-phosphoethanolamine (MPEG2000-DSPE), 9.6 mg/mL fully hydrogenated phosphatidylcholine, 3.2 mg/mL cholesterol, 2 mg/mL ammonium sulfate, and histidine as a buffer. pH is controlled using sodium hydroxide and isotonicity is maintained using sucrose. To form liposomes, lipids are mixed in an organic solvent, dried, hydrated with agitation, and sized by extrusion through a polycarbonate filter with a mean pore size of 800 nm. Nucleic acids are encapsulated by combining 10 μg of the liposome formulation per 1 μg of nucleic acid and incubating at room temperature for 5 minutes.
Example 63 Folate-Targeted Liposome Formulation
Liposomes are prepared according to Example 62, except that 0.27 mg/mL 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[folate(polyethylene glycol)-5000] (FA-MPEG5000-DSPE) is added to the lipid mixture
Example 64 Cancer Therapy Comprising Liposomal RiboSlice Targeting BIRC5
Liposomes encapsulating RiboSlice pairs synthesized according to Example 23 are prepared according to Example 62 or Example 63. The liposomes are administered by injection or intravenous infusion, and tumor response and interferon plasma levels are monitored daily.
Example 65 Cancer Therapy Comprising Liposomal RiboSlice Targeting a Cancer-Associated Gene
Liposomes encapsulating RiboSlice targeting a cancer-associated gene, synthesized according to Example 1, are prepared according to Example 62 or Example 63. The liposomes are administered by injection or intravenous infusion, and tumor response and interferon plasma levels are monitored daily.
Example 66 Therapy Comprising Liposomal Protein-Encoding RNA
Liposomes encapsulating synthetic RNA encoding a therapeutic protein, synthesized according to Example 1, are prepared according to Example 62 or Example 63. The liposomes are administered by injection or intravenous infusion.
Example 67 Combination Cancer Therapy Comprising RiboSlice Targeting BIRC5 and TNF-α
Patients are administered isolated limb perfusion (ILP) with tumor necrosis factor alpha (TNF-α) and liposomes encapsulating RiboSlice targeting BIRC5 (see Example 64). Following warming of the limb, liposomes are injected into the arterial line of the extracorporeal ILP circuit over approximately 5 minutes, and perfusion proceeds for another 85 minutes. After 1-2 days, ILP is repeated with TNF-α injected into the arterial line of the extracorporeal ILP circuit over 3-5 minutes and perfusion continues for an additional 60 minutes. Tumor response and interferon plasma levels are monitored daily.
Example 68 Combination Cancer Therapy Comprising RiboSlice Targeting BIRC5 and Fluorouracil (5-FU)
On day 1 patients receive a 60-minute intravenous infusion of liposomes encapsulating RiboSlice targeting BIRC5 (see Example 64), followed by a 46-hour intravenous infusion of 5-FU on days 2 and 3. Tumor response and interferon plasma levels are monitored daily.
EQUIVALENTS
Those skilled in the art will recognize, or be able to ascertain, using no more than routine experimentation, numerous equivalents to the specific embodiments described specifically herein. Such equivalents are intended to be encompassed in the scope of the following claims.
INCORPORATION BY REFERENCE
All patents and publications referenced herein are hereby incorporated by reference in their entireties.

## Claims

1. A composition comprising a nucleic acid encoding a gene-editing protein, the gene-editing protein comprising: (a) a DNA-binding domain and (b) a nuclease domain, wherein:
(a) the DNA-binding domain comprises a plurality of repeat sequences and at least one of the repeat sequences comprises the amino acid sequence: LTPvQVVAIAwxyzGHGG (SEQ ID NO: 75) and is between 36 and 39 amino acids long, wherein:
“v” is Q, D or E,
“w” is S or N,
“x” is N,
“y” is D, A, H, N, K, or G, and
“z” is GGKQALETVQRLLPVLCQD (SEQ ID NO: 670) or
GGKQALETVQRLLPVLCQA (SEQ ID NO: 671); and

(b) the nuclease domain comprises a catalytic domain of a nuclease.

2. The composition of claim 1, wherein the nuclease domain is capable of forming a dimer with another nuclease domain.

3. The composition of claim 1, wherein the gene-editing protein is capable of generating a nick or double-strand break in a target DNA molecule.

4. The composition of claim 1, wherein the nucleic acid is a synthetic RNA molecule.

5. The composition of claim 4, wherein the synthetic RNA molecule comprises one or more non-canonical nucleotides.

6. The composition of claim 5, wherein the non-canonical nucleotide is 5-methylcytidine.

7. The composition of claim 1, wherein the nuclease domain comprises the catalytic domain of a protein comprising the amino acid sequence of SEQ ID NO: 53.

8. The composition of claim 1, wherein the nuclease is selected from the group consisting of FokI and StsI.

9. The composition of claim 1, wherein at least one of the repeat sequences contains a region capable of binding to a binding site in a target DNA molecule, the binding site containing a defined sequence of between 1 and 5 bases in length.