CA2951882A1

CA2951882A1 - Factor viii mutation repair and tolerance induction and related cdnas, compositions, methods and systems

Info

Publication number: CA2951882A1
Application number: CA2951882A
Authority: CA
Inventors: Tom E. HOWARD
Original assignee: Individual
Current assignee: Individual
Priority date: 2014-06-11
Filing date: 2015-06-11
Publication date: 2015-12-17
Also published as: EP3155098A1; EP3155098A4; WO2015191899A1; WO2015191899A9

Abstract

The present disclosure relates to methods, systems, and compositions to repair one or more mutations in a Factor VIII gene sequence of a subject by introducing into a cell of the subject one or more polynucleotides encoding a DNA scission enzyme (DNA-SE) and one or more repair vehicles (RVs) containing at least a cDNA-repair sequence (RS) such that insertion of the cDNA-RS through homologous recombination with the F8 gene of the subject (sF8) provides a repaired F8 gene (rF8), the repaired F8 gene (rF8) upon expression forming a functional FVIII conferring improved coagulation functionality to the FVIII protein encoded by the sF8. The present disclosure also relates to cells derived using the methods, systems and compositions described.

Description

FACTOR VIII MUTATION REPAIR AND TOLERANCE INDUCTION AND
RELATED cDNAs, COMPOSITIONS, METHODS AND SYSTEMS
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority to U.S. Provisional Application 62/011,019, entitled "Factor VIII mutation repair and tolerance induction" and filed on June 11, 2014, and is also a continuation-in-part application of U.S. Non-Provisional Application No.
14/649,910, filed on June 4, 2015, which, in turn, is a U.S. national stage entry of International Patent Application No. PCT/U52013/073751, filed on December 6, 2013, which, in turn, claims priority from U.S. Provisional Application No. 61/734,678, filed on December 7, 2012, and U.S. Provisional Application No. 61/888,424, filed on October 8, 2013. All such applications are incorporated herein by reference in their entirety.
STATEMENT OF GOVERNMENT GRANT

[0002] The U.S. government has certain rights in the inventions pursuant to Grant Nos grant # 1R41MD008156-01A1 and 1R41MD008808-01 awarded by the National Institutes of Health (NIH).
FIELD

[0003] The present disclosure relates to gene mutation repairs and related materials, methods and systems, and in particular relates to Factor VIII mutation repair and tolerance induction and related cDNAs compositions, methods and systems.
BACKGROUND

[0004] Factor VIII (FVIII) is a blood-clotting protein, also known as anti-hemophilic factor (AHF), encoded by a Factor VIII gene (F8 gene or F8).

[0005] Certain mutations in the F8 gene (F8) result in production of a dysfunctional version of the Factor VIII protein (qualitative deficiency), and/or in production of Factor VIII in insufficient amounts (quantitative deficiency) which cause hemophilia in subjects having the mutations.

[0006] Despite developments of various options to manage hemophilia, prophylaxis and treatment of hemophilia in subjects remains challenging.
SUMMARY

[0007] Provided herein are methods and systems and related cDNA, polynucleotides, vehicles and compositions which allow in several embodiments to selectively target and repair one or more mutations in the sequence of Factor VIII gene of a subject, and in particular the one or more mutations of the Factor VIII gene resulting in hemophilia.

[0008] According to a first aspect, a method for repairing one or more mutations in a Factor VIII gene (F8 gene) sequence of a subject is described. The method comprises introducing into a cell of the subject one or more polynucleotides encoding a DNA scission enzyme (DNA-SE) such as a nuclease or nickase and one or more repair vehicles (RVs) containing at least a cDNA-repair sequence (RS) comprising a repaired version of the F8 gene sequence of the subject comprising the one or more mutations within a cDNA sequence encoding for a truncated Factor VIII.
The DNA-SE is selected to be capable of targeting a portion of the F8 gene of the subject and to create a first break in one strand of the F8 gene and a second break in the other strand of the F8 gene for subsequent repair by the cDNA-RS. The cDNA-RS is comprised in each of the one or more repair vehicles (RVs) flanked by an upstream flanking sequence (uFS) and a downstream flanking sequence (dFS) to form a DNA donor within the RVs. The upstream flanking sequence (uFS) is homologous to a nucleic acid sequence upstream of the first break in the one strand of the F8 gene and the downstream flanking sequence (dFS) homologous to a nucleic acid sequences downstream of the second break in the other strand of the F8 gene.
In the method, introducing into a cell of the subject one or more polynucleotides encoding a DNA scission enzyme (DNA-SE) and one or more repair vehicles (cDNA-RS) is performed to allow insertion of the cDNA-RS through homologous recombination of the upstream flanking sequence (uFS) and the downstream flanking sequence (dFS) with the subject's F8 gene (sF8) to provide a repaired F8 gene (rF8). In the method, the repaired F8 gene (rF8) upon expression forms functional FVIII that confers improved coagulation functionality to the FVIII protein encoded by the sF8 without the repair.

[0009] According to a second aspect, a system for repairing one or more mutations in a Factor VIII gene (F8 gene) sequence of a subject is described. The system comprises one or more polynucleotides encoding a DNA scission enzyme (DNA-SE) herein described and one or more repair vehicles (RVs) herein described.
In the system, the DNA scission enzyme (DNA-SE), and the and one or more repair vehicles (RVs) are selected and configured so that upon insertion of the cDNA-RS
through homologous recombination of the upstream flanking sequence (uFS) and the downstream flanking sequence (dFS) of the DNA donor sequence with the subject's F8 gene (sF8) a repaired F8 gene (rF8) is provided. In the system, the repaired F8 gene (rF8) upon expression forms functional FVIII that confers improved coagulation functionality to the FVIII protein encoded by the sF8 without the repair.

[0010] According to a third aspect, a cDNA is described configured to be used as a cDNA-RS in methods and systems of the disclosure for repairing one or more mutations in a Factor VIII gene (F8 gene) sequence of a subject. The cDNA encodes a truncated Factor VIII
polypeptide consisting essentially of the amino acid sequence encoded by each of exons 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26 of a F8 gene or an in frame combination thereof In some embodiments, the each of the exons has a sequence of a corresponding exon in the F8 gene of the subject.

[0011] According to a fourth aspect a repair vehicle (RV) is described configured to be used in methods and systems of the disclosure for repairing one or more mutations in a Factor VIII
gene (F8 gene) sequence of a subject. The repair vehicle is a polynucleotide configured for use in combination with a DNA scission enzyme (DNA-SE) selected to target a portion of the F8 gene of the subject and to create a first break in one strand of the F8 gene and a second break in the other strand of the F8 gene. The repair vehicle comprises a cDNA-repair sequence (RS) comprising a repaired version of the F8 gene sequence of the subject comprising the one or more mutations within a cDNA sequence encoding for a truncated Factor VIII. In the repair vehicle (RV), the cDNA-RS is flanked by an upstream flanking sequence (uFS) and a downstream flanking sequence (dFS) to form a DNA donor within the RV. The upstream flanking sequence (uFS) is homologous to a nucleic acid sequence upstream of the first break in the one strand of the F8 gene and the downstream flanking sequence (dFS) homologous to a nucleic acid sequences downstream of the second break in the other strand of the F8 gene.

[0012] According to a fifth aspect a polynucleotide encoding a DNA scission enzyme (DNA-SE) is described configured for use in methods and systems of the disclosure for repairing one or more mutations in a Factor VIII gene (F8 gene) sequence of a subject. The DNA scission enzyme is selected to be capable of targeting a portion of the F8 gene of the subject and to create a first break in one strand of the F8 gene and a second break in the other strand of the F8 gene for subsequent repair by the cDNA-RS.

[0013] According to a sixth aspect, a cell is described comprising one or more repair vehicles (RVs) herein described and one or more polynucleotide encoding a DNA scission enzyme (DNA-SE) herein described.

[0014] According to a seventh aspect, a composition for repairing one or more mutations in a Factor VIII gene (F8 gene) sequence of a subject is described. The composition comprises one or more polynucleotides encoding a DNA scission enzyme (DNA-SE) herein described and one or more repair vehicles (RVs) herein described together with a suitable excipient. In some embodiments, the composition is a pharmaceutical composition for treatment of hemophilia and/or promotion of immune tolerance to a Factor VIII replacement protein in a subject and the suitable excipient is a pharmaceutically acceptable excipient.

[0015] Methods and systems and related cDNA, polynucleotides, vehicles and compositions are expected in several embodiments to provide a repaired F8 gene and corresponding functional Factor VIII in a subject with hemophilia in a form and amount remedying the qualitative and/or quantitative deficiencies of the Factor VIII of the subject, thus allowing treatment of the hemophilia in the subject.

[0016] Methods and systems and related cDNA, polynucleotides, vehicles and compositions are expected in several embodiments to provide a repaired F8 and corresponding functional Factor VIII formed by sequences of the subject thus minimizing production of Factor VIII
inhibitor in the subject.

[0017] Methods and systems and related cDNA, polynucleotides, vehicles and compositions are expected in several embodiments to provide a repaired F8 gene expressing a functional FVIII which allows inducing immune tolerance to a FVIII replacement product ((r)FVIII) in a subject having a FVIII deficiency and who will be administered, is being administered, or has been administered a (r)FVIII product.

[0018] The methods and systems and related cDNA, polynucleotides, vehicles and compositions herein described, can be used in connection with applications wherein repair of mutations in Factor VIII gene of a subject is desired, in particular in connection with treatment and/or prophylaxis of various forms of hemophilia and in particular hemophilia A, in subjects. Exemplary applications comprise medical applications, biological analysis, research and diagnostics including but not limited to clinical, therapeutic and pharmaceutical applications, and additional applications identifiable by a skilled person.

[0019] The details of one or more embodiments of the disclosure are set forth in the accompanying drawings and the description below. Other features and objects will be apparent from the description and drawings, and from the appended claims.
BRIEF DESCRIPTION OF THE DRAWINGS

[0020] The accompanying drawings, which are incorporated into and constitute a part of this specification, illustrate one or more embodiments of the present disclosure and, together with the description of example embodiments, serve to explain the principles and implementations of the disclosure.

[0021] FIG. 1 is a schematic illustration of the wild-type and intron-22-inverted FVIII loci (F8 & F81221) and their expressed protein products (FVIIIFL & FVIIIB for F8 and FVIIII22I
& FVIIIB for F8I 221).

[0022] FIG. 2 is a schematic illustration of a TALEN-mediated genomic editing that can be used to repair the human intron-22 (I22)-inverted F8 locus, F81221.

[0023] FIG. 3 shows a functional heterodimeric TALEN, comprised of its left and right monomer subunits (TALEN-L and TALEN-R), targeting the human F8 gene.

[0024] FIG. 4 shows a functional heterodimeric TALEN, comprised of its left and right monomer subunits (TALEN-L and TALEN-R) targeting the canine F8 gene

[0025] FIG. 5 illustrates the TALEN approach linking Exon 22 of the F8 gene to a nucleic acid encoding a truncated FVIII polypeptide encoding exons 23-26.

[0026] FIG. 6 illustrates the TALEN approach linking Intron 22 to a F8 3' splice acceptor site operably linked to a nucleic acid encoding a truncated FVIII polypeptide.

[0027] FIG. 7 shows a comparison of expected genomic DNA, spliced RNA and proteins pre and post repair.

[0028] FIG. 8 shows PCR primer design to confirm correct integration of exons 23-26 to repair the human intron-22 0221-inverted F8 locus, F81221.

[0029] FIG. 9 illustrates the donor plasmid targeting the F8 Exon22/Intron22 junction using a TALEN, ZFN, CRISPR/Cas, CRISPR-PN, and CRISPR-RFN approach.

[0030] FIG. 10 illustrates the donor plasmid targeting the F8 Exonl/Intronl junction using a TALEN, ZFN, CRISPR/Cas, CRISPR-PN, and CRISPR-RFN approach.

[0031] FIG. 11 illustrates the donor plasmid targeting the F8 Intron 22 region using a TALEN, ZFN, CRISPR/Cas, CRISPR-PN, and CRISPR-RFN approach.

[0032] FIG. 12 illustrates the donor plasmid targeting the F8 Intron 1 region using a TALEN, ZFN, CRISPR/Cas, CRISPR-PN, and CRISPR-RFN approach.

[0033] FIG. 13 illustrates the CRISPR/Cas9-mediated F8 repair strategy targeting intron 1.

[0034] FIG. 14 illustrates examples of severe HA-causing F8 mutations that can be cured with the exon-2 I targeted CasPN therapeutics of our personalized 3' gene repair system.

[0035] FIG. 15 is a schematic diagram of exon-21 targeted, CasPN mediated personalized repair of the intron-22 inversion mutation (F8I221).

[0036] FIG. 16 is a schematic diagram of the repair vehicle, donor sequence used in the repair of FIG. 15.

[0037] FIG. 17 shows a series of graphs displaying results obtained from flow cytometry using CRISPR/Cas9 plasmids pH0007, pH0009 as well as a repair plasmid (labeled as "Donor").

[0038] FIG. 18 is an image of an agarose gel electrophoresis assay displaying results from a T7E1 assay done on cells transfected with CRISPR/Cas9 plasmids pH0007, pH0009, pH0011 and pH0013.

[0039] FIG. 19 is a bar graph showing estimated NHEJ rates for CRISPR
constructs pH0007, pH0009, pH0011 and pH0013.

[0040] FIG. 20 is an image of an agarose gel electrophoresis assay displaying results from a RFLP assay done on cells transfected with CRISPR/Cas9 plasmids pH0007, pH0009 as well as a repair plasmid (labeled as "Donor").

[0041] FIG. 21 is a bar graph showing the percentage of homologous recombination in cells following Intron 22-targeted CRISPR treatment.
DETAILED DESCRIPTION

[0042] Provided herein are methods and systems and related cDNA, polynucleotides, vehicles and compositions which allow in several embodiments to selectively target and repair one or more mutations in the sequence of Factor VIII gene of a subject.

[0043] The term "Factor VIII" or "FVIII" as used herein indicates an essential cofactor in the blood coagulation pathway provided by a large plasma glycoprotein that functions in the blood coagulation cascade as a cofactor for the factor IXa-dependent activation of factor X.
Factor VIII is tightly associated in the blood with von Willebrand factor (VWF), which serves as a protective carrier protein for factor VIII. In particular Factor VIII circulates in the bloodstream in an inactive form, bound to von Willebrand factor (VWF). Upon injury, FVIII
is activated. The activated protein (FVIIIa) interacts with coagulation factor IX, leading to clotting as will be understood by a skilled person.

[0044] FVIII is encoded in a subject by a F8 gene containing 26 exons and spanning 186 kb (Gitschier, et al. Nature 314: 738-740, 1985). In human the F8 gene is located in the X
chromosome. In some subjects (e.g. humans, monkeys, rats) the sequences F8 gene also contains an F8A gene and an F8B gene within intron 22. The F8A gene is intron-less, is contained entirely in intron 22 of the F8 gene in reverse orientation to the F8 gene, and is therefore transcribed in the opposite direction to F8. The F8B gene is also located in intron 22 and is transcribed in opposite direction from F8A gene; its first exon lies within intron 22 and is spliced to exons 23-26.

[0045] The term "orientation" with reference to a gene indicates the direction of the 5' ¨> 3' DNA strand which provides the sense strand in the double stranded polynucleotide comprising the gene. Accordingly, 5'->3' DNA strand is designated, for a given gene, as 'sense', 'plus' or 'coding' strand when its sequence is identical to the sequence of the premessenger (premRNA), except for uracil (U) in RNA, instead of thymine (T) in DNA. An antisense strand is instead the 3'->5' strand complementary to the sense strand in a double stranded polynucleotide coding for the gene. The antisense transcribed by the RNA
polymerase and is also designated as "template" DNA. Accordingly two genes or sequences thereof within the F8 genomic locus encoded by a same polynucleotide are in a same orientation when their respective sense strands are located on a same strand of the polynucleotide and are in in reverse or opposite orientation when respective sense strands are located on different strand of the polynucleotide. Accordingly two genes or coding sequences within the F8 genomic locus encoded by a same polynucleotide are in a same orientation when their respective sense strands are located on a same strand of the polynucleotide. Two genes or coding sequences within the F8 genomic locus are in reverse or opposite orientation when their respective sense strands are located on the opposing strand of the polynucleotide.

[0046] FVIII is synthesized primarily in the liver of s subject and the primary translation product of 2332 amino acids undergoes extensive post-translational modification, including N- and 0-linked glycosylation, sulfation, and proteolytic cleavage. The latter event divides the initial multi-domain protein (A 1 -A2-B-A3-C1-C2) into a heavy chain (A 1 -A2-B) and a light chain (A3-C1-C2) and the protein is secreted as a two-chain molecule associated through a metal ion bridge (Lenting et al., The life cycle of coagulation FVIII in view of its structure and function. Blood 1998; 92: 3983-96).

[0047] Mutations in the F8 gene can result in production of a dysfunctional version of the Factor VIII protein (qualitative deficiency), and/or in production of Factor VIII in insufficient amounts (quantitative deficiency) causing hemophilia in subjects having the mutations.

[0048] Accordingly, a Factor VIII is indicated as functional when it is produced in a form and an amount allowing a coagulation functionality comparable with the coagulation functionality of the wild type FVIII protein in a healthy subject. FVIII
function is evaluated by routine clinical laboratory methods that are well established in the art and apparent to one of ordinary skill in the art (Barrowcliffe TW, Raut S, Sands D, Hubbard AR:
Coagulation and chromogenic assays of factor VIII activity: general aspects, standardization, and recommendations. Semin Thromb Hemost 2002 Jun;28(3):247-256).

[0049] A non-functional Factor VIII instead indicates an FVIII protein functioning aberrantly or FVIII proteins present in circulating blood in a reduced or absent amount, leading to the reduction of or absence of the ability to clot in response to injury by the subject. FVIII
function is evaluated by routine clinical laboratory methods that are well established in the art and apparent to one of ordinary skill in the art (Banowcliffe TW, Raut S, Sands D, Hubbard AR: Coagulation and chromogenic assays of factor VIII activity: general aspects, standardization, and recommendations. Semin Thromb Hemost 2002 Jun;28(3):247-256).

[0050] Over 2100 different hemophilia A (HA)-causing mutations have thus far been identified in the F8 loci of unrelated patients which result in the expression of a non-functional and/or deficient FVIII protein. In particular, defects within the F8 affect about one in 5000 newborn males (Jones et al., Identification and removal of promiscuous CD4+ T cell epitope from the Cl domain of factor VIII. J. Throm. Haemost. 2005; 3: 991-1000).

[0051] Mutations of the F8 gene resulting in a non-functional Factor VIII
include point mutations, deletions, insertion and inversion as will be understood by a skilled person. For example, of the 2100 unique mutations identified in human F8 gene, over 980 of them being missense mutations, i.e., a point mutation wherein a single nucleotide is changed, resulting in a codon that codes for a different amino acid than its wild-type counterpart (see HAMSTeRS
Database: at the http :// web page: hadb.org.uk/WebP ages/PublicF
iles/Mutation Summary.htm). One of the most common mutations resulting in a non-functional and/or deficient FVIII protein includes inversion of intron 22, which leads to a severe type of HA.

[0052] Accordingly, a mutation in an F8 gene of a subject resulting in a non-functional Factor VIII results in an F8 gene comprising at least one Factor VIII
functional coding sequence and at least one Factor VIII non-functional coding sequence.

[0053] The wording "functional coding sequence" of Factor VIII refers to an F8 gene sequence that is configured to be transcribed and contains one or more exons of the F8 gene with an open reading frame resulting in a functional Factor VIII or in a portion thereof Exemplary functional coding sequences comprise the sequence of E1-E22 and E23-E26 of the wild type F8 genomic locus in FIG. 1, the sequence of E1-E22 of the Intron-22 inverted F8 locus of FIG. 1, the sequence of human F8 cDNA of FIG. 2, the sequence of Exons 1-22 and Ex 23-26 of the normal F8 gene in FIG. 7, the sequence of Ex 1-22 of the Intron 22 inversion of the F8 gene in FIG. 7, the sequence of Ex 1-22 and Ex 23-26 of the repaired F8 gene of FIG. 7, the cDNA sequence of Exons 23-26 of the repair vehicle of FIG.
9, the cDNA

sequence of Exons 2-26 of the repair vehicle of FIG. 10, the cDNA sequence of Exons 23-26 of the repair vehicle of FIG. 11, the cDNA sequence of Exons 2-26 of the repair vehicle of FIG. 12, the cDNA of exons 23-26 of the repair vehicle of Table 51, the cDNA
sequence of exons 23-26 of the repair vehicle of Table 52, the cDNA sequence of exons 2-26 or 2-13 of the repair vehicle of Tables 53 and 54, respectively.

[0054] Functional coding sequences can include introns or be formed by exons only or a portion thereof Exemplary functional coding sequences comprise the sequence of and E23-E26 of the wild type F8 genomic locus in FIG. 1, the sequence of E1-E22 of the Intron-22 inverted F8 locus of FIG. 1, Exons 1-22 and respective intervening introns of the Intron-22 inversion human F8 locus of FIG. 2, the sequence of Exons 1-22 and Exons 23-26 of the normal F8 gene in FIG. 7, the sequence of Exons 1-22 of the Intron 22 inversion of the F8 gene in FIG. 7, the sequence of Exons 1-22 and Exons 23-26 of the repaired F8 gene of FIG. 7.

[0055] Functional coding sequences can be included in the same orientation as the wild type F8 gene or in an opposite orientation as the wild type F8 gene. Exemplary functional coding sequences in a same orientation as the wild type F8 gene comprise the sequence of E1-E22 and E23-E26 of the wild type F8 genomic locus in FIG. 1, the sequence of Exons 1-22 and Exons 23-26 of the normal F8 gene in FIG. 7, the cDNA sequence of Exons 2-26 of the repair vehicle of FIG. 10, the cDNA sequence of Exons 2-26 of the repair vehicle of FIG. 12, the cDNA of exons 23-26 of the repair vehicle of Table 51, the cDNA sequence of exons 23-26 of the repair vehicle of Table 52, the cDNA sequence of exons 2-26 or 2-13 of the repair vehicle of Tables 53 and 54, respectively. Exemplary functional coding sequences in an opposite orientation as compared to wild type F8 gene comprise the sequence of E1-E22 of the Intron-22 inverted F8 locus of FIG. 1, the sequence of human F8 cDNA of FIG. 2, the sequence of Ex 1-22 of the Intron 22 inversion of the F8 gene in FIG. 7, the sequence of Ex 1-22 and Ex 23-26 of the repaired F8 gene of FIG. 7, the cDNA sequence of Exons 23-26 of the repair vehicle of FIG. 9, the cDNA sequence of Exons 2-26 of the repair vehicle of FIG.
10, the cDNA sequence of Exons 23-26 of the repair vehicle of FIG. 11, the cDNA sequence of Exons 2-26 of the repair vehicle of FIG. 12.

[0056] The wording "non-functional coding sequence" of the F8 gene refers to an F8 gene sequence that is not configured to be transcribed and/or contains one or more exons of the F8 gene with an open reading frame resulting in a non-functional Factor VIII or in a portion thereof. In particular, coding sequences can be non-functional, and therefore result in a non-functional Factor VIII, due to point mutations resulting in a sequence coding for an amino acid, in an insertion or deletion of coding sequences resulting in frame shift or a different open reading frame, with respect to an open reading frame (such as the open reading frame of the wild type F8 gene), which results in a functional Factor VIII.

[0057] Exemplary non-functional coding sequences resulting from F8 gene mutations comprise the sequence of E24 in the case of a F8 c.6761 T>A nonsense mutation that results in a stop codon at codon 2178 in place of the leucine (Leu)-encoding codon that is present at codon 2178 in the non-mutated form of the F8 gene as seen in FIG. 14, the sequence of E25 in the case of a F8 c.6917 T>G missense mutation that results in a codon encoding arginine (Arg) at codon 2230 in place of the leucine (Leu)-encoding codon that is present at that codon 2230 in the non-mutated form of the F8 gene as seen in FIG. 14, the sequence of sequence of E24, E25 and E26 in the case of a F8 IVS-23 +1 G>A splice site mutation that results in a non-functional pre-mRNA splice site immediately downstream of exon 23 of the F8 gene as seen in FIG. 14, sequence of E26 in the case of a F8 Exon 26 del.[A] small deletion and frameshift mutation that results in a frameshift of the gene-encoding sequence which changes the downstream sequence by a single base-pair deletion frameshift and introduction of a novel terminating stop codon in the gene-encoding sequence as seen in FIG. 14.

[0058] Non-functional coding sequences can be included in the same orientation as the wild type F8 gene or in an opposite orientation of the wild type F8 gene. Exemplary non-functional coding sequences in a same orientation of the wild type F8 gene comprise the sequence of El B and the sequence of E23-E26 of the Intron-22 inverted F8 genomic locus of FIG. 1, the sequence of exons 23c and 24c of the Intron-22 inverted human locus of FIG. 2A, the sequence of Exons 23-26 of the Intron 22 Inversion of the F8 gene in FIG.
7, the sequence of E24 in the case of a F8 c.6761 T>A nonsense mutation that results in a stop codon at codon 2178 in place of the leucine (Leu)-encoding codon that is present at codon 2178 in the non-mutated form of the F8 gene as seen in FIG. 14, the sequence of E25 in the case of a F8 c.6917 T>G missense mutation that results in a codon encoding arginine (Arg) at codon 2230 in place of the leucine (Leu)-encoding codon that is present at that codon 2230 in the non-mutated form of the F8 gene as seen in FIG. 14, the sequence of sequence of E24, E25 and E26 in the case of a F8 IVS-23 +1 G>A splice site mutation that results in a non-functional pre-mRNA splice site immediately downstream of exon 23 of the F8 gene as seen in FIG. 14, sequence of E26 in the case of a F8 Exon 26 del.[A] small deletion and frameshift mutation that results in a frameshift of the gene-encoding sequence which changes the downstream sequence by a single base-pair deletion frameshift and introduction of a novel terminating stop codon in the gene-encoding sequence as seen in FIG. 14. Exemplary non-functional coding sequences comprise in opposite orientation of the wild type F8 gene comprise the sequence of exons E23C and E24C of the Intron-22 inverted F8 genomic locus of FIG. 1.

[0059] In embodiments, herein described non-functional coding sequences are replaced by a cDNA-repair sequence (RS).

[0060] The term cDNA or complementary DNA indicates double-stranded DNA that can be synthesized from a messenger RNA (mRNA) template in a reaction catalysed by the enzyme reverse transcriptase. Accordingly cDNA can be synthesized from mature (fully spliced) mRNA using the enzyme reverse transcriptase or be synthesized synthetically based on the mRNA sequence as will be understood by a skilled person.

[0061] The terms "polynucleotide", "oligonucleotide" and "nucleic acid," are used interchangeably and refer to an organic polymer composed of two or more monomers including nucleotides, nucleosides or analogs thereof. The term "nucleotide"
refers to any of several compounds that consist of a ribose or deoxyribose sugar joined to a purine or pyrimidine base and to a phosphate group and that is the basic structural unit of nucleic acids.
The term "nucleoside" refers to a compound (such as guanosine or adenosine) that consists of a purine or pyrimidine base combined with deoxyribose or ribose and is found especially in nucleic acids. The term "nucleotide analog" or "nucleoside analog" refers respectively to a nucleotide or nucleoside in which one or more individual atoms have been replaced with a different atom or a with a different functional group. Exemplary functional groups that can be comprised in an analog include methyl groups and hydroxyl groups and additional groups identifiable by a skilled person. In general, an analogue of a particular nucleotide has the same base-pairing specificity; i.e., an analogue of A will base-pair with T.

[0062] Exemplary monomers of a polynucleotide comprise deoxyribonucleotide, and ribonucleotides. The term "deoxyribonucleotide" refers to the monomer, or single unit, of DNA, or deoxyribonucleic acid. Each deoxyribonucleotide comprises three parts:
a nitrogenous base, a deoxyribose sugar, and one or more phosphate groups. The nitrogenous base is typically bonded to the l' carbon of the deoxyribose, which is distinguished from ribose by the presence of a proton on the 2' carbon rather than an -OH group.
The phosphate group is typically bound to the 5' carbon of the sugar. The term "ribonucleotide" refers to the monomer, or single unit, of RNA, or ribonucleic acid. Ribonucleotides have one, two, or three phosphate groups attached to the ribose sugar.

[0063] Accordingly, the term "polynucleotide", "oligonucleotide includes nucleic acids of any length, and in particular DNA, RNA, analogs thereof, and fragments thereof Polynucleotides can typically be provided in single-stranded form or double-stranded form (herein also duplex form, or duplex).

[0064] A "single-stranded polynucleotide" refers to an individual string of monomers linked together through an alternating sugar phosphate backbone. In particular, the sugar of one nucleotide is bond to the phosphate of the next adjacent nucleotide by a phosphodiester bond. Depending on the sequence of the nucleotides, a single-stranded polynucleotide can have various secondary structures, such as the stem-loop or hairpin structure, through intramolecular self-base-paring. A hairpin loop or stem loop structure occurs when two regions of the same strand, usually complementary in nucleotide sequence when read in opposite directions, base-pairs to form a double helix that ends in an unpaired loop. The resulting lollipop-shaped structure is a key building block of many RNA
secondary structures. The term "small hairpin RNA" or "short hairpin RNA" or "shRNA" as used herein indicate a sequence of RNA that makes a tight hairpin turn and can be used to silence gene expression via RNAi.

[0065] A "double-stranded polynucleotide", "duplex polynucleotide" refers to two single-stranded polynucleotides bound to each other through complementarily binding.
The duplex typically has a helical structure, such as double-stranded DNA (dsDNA) molecule or double stranded RNA, is maintained largely by non-covalent bonding of base pairs between the strands, and by base stacking interactions.

[0066] In embodiments, herein described a cDNA-repair sequence (RS) is a double stranded polynucleotide comprising a repaired version of the entire F8 gene non-functional coding sequence of the subject or of a portion thereof In particular in methods and compositions herein described the cDNA-RS comprise at least a repaired version the portion of the non-functional sequence of the Factor VIII of the subject comprising the one or more mutations in the Factor VII of the subject. In some embodiments, cDNA-RS described herein further comprises introns and/or exons located upstream and/or downstream to the non-functional coding sequence. In embodiments described herein, the cDNA-RS is designed so that once recombined into the desired region in the F8 genomic locus it remains in-frame with functional coding upstream and downstream functional coding sequences.

[0067] Accordingly in methods systems and related cDNA vehicles and compositions herein described a cDNA-RS are designed based on the one or more mutations within the subject's F8 gene targeted for replacement and repair. For example, when repairing a point mutation, the cDNA-RS includes only a small number of replacement nucleotide sequences compared with, for example, a cDNA-RS derived for repairing an inversion such as an intron 22 inversion. Therefore, a cDNA-RS can be of any length, for example between 2 and 10,000 nucleotides in length (or any integer value there between or there above), e.g. between about 100 and 1,000 nucleotides in length (or any integer there between), between about 200 and 500 nucleotides in length (or any integer there between). Exemplary cDNA-RS
herein described comprise the sequence of human F8 cDNA of FIG. 2, the cDNA sequence of Exons 23-26 of the repair vehicle of FIG. 9, the cDNA sequence of Exons 2-26 of the repair vehicle of FIG. 10, the cDNA sequence of Exons 23-26 of the repair vehicle of FIG. 11, the cDNA sequence of Exons 2-26 of the repair vehicle of FIG. 12, the cDNA
sequence of exons 23-26 of the repair vehicle of Table 51, the cDNA sequence of exons 23-26 of the repair vehicle of Table 52, the cDNA sequence of exons 2-26 or 2-13 of the repair vehicle of Tables 53 and 54, respectively.

[0068] In an embodiment, the gene mutation targeted for repair is a point mutation, and the cDNA-RS includes a nucleic acid sequence that replaces the point mutation with a functional sequence for Factor VIII that does not include the point mutation, for example, the wild-type F8 sequence. In one embodiment, the gene mutation targeted for repair is a deletion and the cDNA-RS includes a nucleic acid sequence that replaces the deletion with a functional Factor VIII sequence that does not include the deletion, for example, a corresponding F8 sequence of the wild-type F8 sequence.

[0069] In one embodiment, the gene mutation targeted for repair is an inversion, and the cDNA-RS includes a nucleic acid sequence that encodes a truncated FVIII
polypeptide that, upon insertion into the F8 genome, repairs the inversion and provides for the production of a functional FVIII protein. In one embodiment, the gene mutation targeted for repair is an inversion of intron 1. In one embodiment, the gene mutation targeted for repair is an inversion of intron 22, and the donor sequence includes a nucleic acid that encodes all of exons 23-25 and the coding sequence of exon-26 to be inserted in frame with the inverted exons 1-22 in opposite orientation with the F8 gene.

[0070] In the methods and compositions described herein, the cDNA-RS can contain sequences that are homologous, but not identical (for example, contain nucleic acid sequence encoding wild-type amino acids or differing ns-SNP amino acids), to subject's genomic sequences in the region of interest, thereby stimulating homologous recombination to insert a non-identical sequence in the region of interest.

[0071] The term "homologous" and "homology" when referred to protein or polynucleotide sequences is defined in terms of sequence similarities and percent identity between sequences. Accordingly homologous sequences indicate sequences having a percent identify of at least 80% versus sequences with a percentage identify lower than 80%, which are instead indicated as non-homologous. The terms "percent homology" and "sequence similarity" are often used interchangeably. Sequence regions that are homologous are also called conserved.

[0072] Thus, in certain embodiments, portions of the cDNA-RS that are homologous to sequences in the region of interest exhibit between about 80 to about 99%
sequence identity to the subject's genomic sequence that is replaced. In other embodiments, the homology between the cDNA-RS and the subject's genomic sequence is higher than 99%, for example if only 1 nucleotide differs as between the cDNA-RS and the subject's genomic sequences of over 100 contiguous base pairs. In certain cases, a non-homologous portion of the cDNA-RS
contains sequences not present in the region of interest, such that new sequences are introduced into the region of interest. In these instances, the non-homologous sequence is generally flanked by sequences of 50-1,000 base pairs, or any number of base pairs greater than 1,000, that are homologous or identical to the subject's sequences in the region of interest. In other embodiments, the cDNA-RS containing non-homologous sequence is inserted into the subject's genome by homologous recombination mechanisms.

[0073] Accordingly, cDNA-RS herein described can be comprised within a cDNA
sequence encoding for a truncated Factor VIII. The term "truncated FVIII polypeptide"
refers to a polypeptide that contains less than the full length of FVIII protein. The truncated FVIII
polypeptide is encoded in a portion of the full length F8 gene such as a partial F8 cDNA
replacement sequence (cDNA-RS). For example, for FVIII polypeptide that is truncated from the corresponding 5' end of the oligonucleotide sequence, a variable amount of the oligonucleotide sequence can be missing from the 5' end of the gene. In one embodiment, the truncated FVIII polypeptide is encoded by exons 23-26. In one embodiment, the truncated FVIII polypeptide is encoded by exons 2-26. In one embodiment, the truncated FVIII polypeptide is encoded by exons 15-26.

[0074] In embodiments herein described the cDNA-RS are designed in combination with the selection of DNA scission Enzyme (DNA-SE) and the related target site.

[0075] A DNA scission enzyme indicates an enzyme that catalyzes the hydrolytic cleavage of phosphodiester linkages in the DNA backbone in a specific target site. DNA
scission refers to the breaking of the chemical bonds between adjacent nucleotides on a nucleotide strand or sequence. DNA scission enzymes comprise nucleases and nickases. "Nucleases" or "Deoxyribonucleases" are enzymes capable of hydrolyzing phosphodiester bonds that link nucleotides. A wide variety of deoxyribonucleases are known, which differ in their substrate specificities, chemical mechanisms, and biological functions. DNA-SEs described herein break the genomic DNA at a target site on the F8 gene upstream from a region to be replaced by a repair vehicle comprising a cDNA-RS. The target site is preferentially located about 50-100 base pairs upstream of the desired region to be replaced on the F8 genomic locus so as to optimize recombination by the repair vehicle, donor plasmid, or editing cassette comprising the cDNA-RS. In studies, it was seen that when a target site is located about 50-100 base pairs upstream of the desired region to be replaced on the F8 genomic locus, optimal recombination was observed by the repair vehicle, donor plasmid, or editing cassette comprising the cDNA-RS. Following recombination of the repair vehicle, donor plasmid, or editing cassette into the target site, expression of the repaired F8 gene segment results in expression of a repaired and functional FVIII protein. DNA-SEs described herein comprise nucleases or nickases coupled to nucleotide sequences that specifically guide the nuclease or nickase to the target site. DNA-SEs described herein include heterodimeric nucleases that bind to specific regions of the F8 gene, nucleases or nickases guided to specific sites of the F8 gene by short RNA sequences or combinations thereof. Exemplary nucleases include transcription activator¨like effector nuclease (TALEN), a zinc finger nuclease (ZFN), a CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)-associated (Cas) nuclease, Paired CRISPR, or CRISPR with ZFN. "Nickases" are enzyme that causes nicks (breaks in one strand) of double stranded nucleic acid, allowing it to unwind.
An exemplary nickase is Cas9n (the DlOA mutant nickase version of Cas9).

[0076] In embodiments described herein, DNA-SEs are designed to comprise multiple elements to efficiently target a specific target site within the F8 gene and function as heterodimers or heterodimeric nucleases; Such DNA-SEs are referenced in FIG.
2, FIG. 3, FIG. 4, FIG. 5 and FIG. 6 as TALENL and TALENR. Such heterodimeric nucleases comprise two monomers (a left monomer and a right monomer) that each comprise a nuclear localization signal, a monomer subunit for binding to a specific region of the F8 gene and a Fokl nuclease domain. Further, the monomer subunit for binding of the left monomer binds upstream (5') of the target site, while the monomer subunit of the right monomer binds to a region downstream (3') of the target site, as depicted in FIG. 3 by TALENL and TALENR. In such embodiments, a double-stranded break in the DNA of the target region is mediated by dimerization of the Fok-1 nucleases. The monomer binding subunits are designed such that off-target binding non-specific DNA breaks are minimized and such that the location of the target site is optimally placed upstream from a region to be replaced by a repair vehicle comprising a cDNA-RS.

[0077] In embodiments described herein, DNA-SEs are designed to efficiently target a specific target site within the F8 gene by using a short RNA to guide a nuclease to the desired target site; such a DNA-SE is referenced in FIG. 13 as the CRISPR-Associated Gene Editing system. Such DNA-SEs comprise at least a complementary single strand RNA
(CRISPR
RNA, labeled as CRISPR g-RNA in FIG. 13, for example) that localizes a Cas9 nuclease to a target site on F8 gene. The CRISPR RNA binds to a region upstream of a desired target site, allowing the Cas9 nuclease to cause a double-strand break. The CRISPR RNA is designed such that off-target binding non-specific DNA breaks are minimized and such that the location of the target site is optimally placed upstream from a region to be replaced by a repair vehicle comprising a cDNA-RS. In embodiments described herein, such a DNA-SE is modified to further minimize off-target DNA scission events by modifying the CRISPR-Associated Gene editing system DNA-SE described above to carry a mutated Cas9 that functions as a nickase (Cas9-nickase); such a DNA-SE is referenced in FIG. 14 and in FIG.
15. In such embodiments, CRISPR RNA (labeled as CRISPR gRNAi in FIG. 15) that is longer in length than the CRISPR RNA of the DNA-SE referenced in FIG. 13 is used to guide a first Cas9-nickase to a target site. The Cas9-nickase then makes a single strand break in the DNA at the target site. A second Cas9-nickase is guided to a second target on the complementary DNA strand site by a second CRISPR RNA (labeled as CRISPR g-RNA2 in FIG. 15) and the second Cas9-nickase makes a single strand break in the complementary DNA strand. The two nicking target sites can be separated by 0-30 nucleotides.

[0078] In the methods and compositions set forth herein, the DNA-SEs that targets a mutation in F8 for repair are, for example, a transcription activator¨like effector nuclease (TALEN), a zinc finger nuclease (ZFN), a CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)-associated (Cas) nuclease, Paired CRISPR, or CRISPR with ZFN, as described in detail below.

[0079] In the methods and systems and related compositions set forth herein, the DNA-SEs is selected for the DNA-SE ability to target a mutation in the F8 gene for repair cleaving the F8 gene sequence for subsequent repair by the cDNA-RS. In particular in methods and systems and related compositions herein described a DNA-SE is for the capability of creating a first break in one strand of the F8 gene and a second break in the other strand of the F8 gene defining a target site located in a position of the F8 gene configured to allow replacement of the F8 gene non-functional coding sequence by a cDNA-RS.

[0080] In methods and systems herein described, the DNA-SE has a target site upstream of the F8 gene nonfunctional coding sequence.

[0081] The wording "upstream" as used herein refers to a position in a polynucleotide relative to a 5' end of the reference point in the polynucleotide. Therefore a sequence or series of nucleotide residues that is "upstream" relative to a site, region or sequence indicates a sequence or series of nucleotides before the 5' end site, region or sequence of the polynucleotide in a 5'to 3' direction. Accordingly, making reference to the exemplary illustration of FIG. 7, Exons 1-22 are located upstream of Exons 23-26 at the normal genomic DNA (gDNA). Additionally, making reference to FIG. 3, TALEN-L binds to a nucleotide sequence upstream of the target site.

[0082] The wording "downstream" as used herein refers to a position in a polynucleotide relative to a 3' end of the reference point in the polynucleotide. Therefore a sequence or series of nucleotide residues that is "downstream" relative to a site, region or sequence indicates a sequence or series of nucleotides after the 3' end site, region or sequence of the polynucleotide in a 5' to 3' direction. Accordingly, making reference to the exemplary illustration of FIG. 7, Exons 23-26 are located downstream of Exons 1-22 at the genomic DNA (gDNA). Additionally, making reference to FIG. 13, the Protospacer Adjacent Motif (PAM) is downstream of the target site.

[0083] In methods and systems herein described, the cDNA-RS is designed to provide a repaired version of the F8 gene nonfunctional coding sequence or a portion thereof encompassing the one or more mutations to be repaired in frame with the F8 gene functional coding sequence upstream of the DNA-SE target site.

[0084] A sequence or series of nucleotide residues that is "in-frame" or "in frame" with a F8 gene functional sequence refers to a sequence or series of nucleotide residues that does not cause a shift in the open reading frame of the F8 functional sequence. An open reading frame (ORF) is the part of a reading frame of a coding sequence that encodes for a protein or peptide according to the standard genetic code, in this case a functional Factor VIII. An ORF
is a continuous stretch of DNA beginning with a start codon, usually methionine (ATG), and ending with a stop codon (TAA, TAG or TGA in most genomes) as will be understood by a skilled person. Accordingly, sequence or series of nucleotide residues is "out of frame" or "out-of-frame" with an F8 functional sequence when to the sequence or series of nucleotide residues causes a shift in the open reading frame of the F8 functional sequence thus resulting in a sequence coding for a non-functional Factor VIII.

[0085] For example in some embodiments, the cDNA-RS provides a repaired version of the F8 nonfunctional sequence in a same orientation with the wild type F8 gene. In some embodiments, the cDNA-RS provides a repaired version of the F8 nonfunctional sequence in opposite orientation with the wild type F8 gene in frame with the functional sequence of the F8 gene following the inversion. In particular in some embodiments the cDNA-RS
for the inversion of intron 22 provides repaired version of the F8 non-functional sequence downstream the inverted exons 1-22 encompassing sequences for exons 23-26 in opposite orientation to the F8 gene.

[0086] In embodiments, herein described selection of a suitable DNA-SE is performed by selecting a target site among candidate target sites on the F8 gene based on the one or more mutations of the F8 gene to be repaired and based on the features of the cDNA-RS to be used on the repair and/or the related donor sequence comprising the cDNA-RS flanked by flanking sequence is homologous to nucleic acid sequences of the F8 gene.

[0087] The wording "flanked" as used herein refers to a position relative to ends of a reference item. More specifically, in referring to a polynucleotide sequences, "flanked" refers to having a sequences upstream and downstream the end of the polynucleotide sequences. In particular, a flanked referenced polynucleotide has a first sequence or series of nucleotide residues positioned adjacent to the 5' end of the referenced polynucleotide and a second sequence or series of nucleotide residues positioned adjacent to the 3' end of the referenced polynucleotide. For example, in Figure 2B, the human F8 cDNA is flanked by a left homology arm (homologY0 and a right homology arm (homologyr).

[0088] In some embodiments, selection based on the one or more mutations of the F8 gene to be repaired can be performed with algorithms or other means directed to minimize off-target effects associated with the DNA-SEs. For example, in some embodiments a program such as PROGNOS can be used to identify the target site. The PROGNOS algorithm locates for example potential TALEN off-target sites by searching through the genome for sequences similar to the intended TALEN design. It ranks these similar sequences according to various features of TALEN-DNA interactions, including RVD base preferences, polarity of TALEN
specificity (5' end is more specific), context dependent compensation of strong RVDs (such as NN and HD), and a model of dimeric TALEN interactions. The PROGNOS model has been shown to accurately predict the majority of all known TALEN off-target sites as discussed in Fine et al. Nucleic Acids Research 2013, incorporated herein by reference. As another example, an algorithm employed for ranking potential CRISPR off-target sites disclosed in Hsu et al. Nature Biotech 2013, incorporate herein by reference, uses a position-weight-matrix (PWM) to determine the importance of different types of mismatches at each position in the target sequence (both the DNA bases targeted by the guide strand as well as the protospacer adjacent motif sequence). This PWM was derived by experimentally observing the drop in nuclease activity at a target site of artificial guide strands (relative to a perfectly matched guide strand) containing different types of mismatches. This PWM is then used to screen potential sites in the genome with homology to the intended target and assign them a score indicating their likelihood of off-target activity.

[0089] In embodiments herein described a target site is selected based on the features of a cDNA-RS used for repair. Factors influencing the location of the target site include the desired length and sequence of cDNA-RS, proximity of the target site to upstream and downstream functional coding sequences, proximity of the target site to upstream and downstream non-functional coding sequences, likelihood of off-target or non-specific DNA
scission, likelihood of off-target or non-specific homologous recombination of the cDNA-RS, homology to off-target genomic sites and nature of the DNA scission enzyme used.

[0090] In particular in some embodiments the target site is selected to have a location relative to the desired region of replacement on the F8 genomic locus that optimizes the recombination rate of the cDNA-RS. For instance, in some embodiments, the target site is selected to be from 50-100 nucleotides upstream of the desired region of replacement on the F8 genomic locus so as to optimize the recombination of the cDNA-RS following scission of the genomic DNA. Location of the target site within about 50-100 base pairs upstream of the desired region to be replaced on the F8 genomic locus results in optimal recombination by the repair vehicle, donor plasmid, or editing cassette comprising the cDNA-RS.
Optimal recombination is an important aspect as it results in an increase in the likelihood that the cDNA-RS will be incorporated at the targeted site within an individual cell and/or population of cells following exposure to the cDNA ¨RS. Also, following recombination of the repair vehicle, donor plasmid, or editing cassette into the target site, expression of the repaired F8 gene segment results in expression of a repaired and functional FVIII protein.
Thus, conditions promoting optimal recombination greatly contribute towards achieving optimal expression of a repaired and functional protein for treatment and/or induction of immune tolerance.

[0091] In embodiments herein described a target site is also be selected based on the features of the donor DNA comprising the cDNA-RS flanked by an upstream flanking sequence (uFS) and a downstream flanking sequence (dFS).

[0092] In particular, in embodiments herein described in a donor sequence, the cDNA-RS is flanked on each side by regions of nucleic acids which are homologous to the subject's F8 gene that are called flanking sequences. Each of the flanking sequence can include about 20, 50, 75, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000 or more nucleotides homologous to regions within the subject's F8 gene. In particular, the upstream flanking sequence (uFS) is homologous to a nucleic acid sequence upstream of the first break in the one strand of the F8 gene by a selected DNA-SE and the downstream flanking sequence (dFS) homologous to a nucleic acid sequences downstream of the second break in the other strand of the F8 gene by the selected DNA-SE.

[0093] In some embodiments, each of the homologous regions flanking the donor sequence is between about 200 to about 1,200 nucleotides, e.g. between 400 and about 1000, between about 600 and about 900, or between about 800 and about 900 nucleotides. Thus, each donor sequence includes a cDNA-RS replacing an endogenous mutation in the subject's F8 gene, and 5' and 3' flanking sequences which are homologous to the F8 gene. In preferred embodiments the length of the homologous regions flanking the donor sequence are between 700 ¨ 800 nucleotides in length. Exemplary homologous regions or arms are the left and right homology arms shown in FIG. 9, FIG. 10, FIG. 11 and FIG. 12.

[0094] In some embodiments, the cDNA-RS is comprised within an editing cassette together with one or more transcriptional elements and the upstream flanking sequence (uFS) and downstream flanking sequence (dFS) are located adjacent at the 5' end and at 3' end of the editing cassette, respectively.

[0095] The wording "adjacent" as used herein refers to a location and/or position nearest in space or position; immediately adjoining without intervening space. More specifically, when referring to a sequence or series of nucleotide residues that is "adjacent" to a site or sequence, "adjacent" refers to a location and/or position next to or proximate to the reference site or position without intervening nucleotide residues. An example is seen in FIG. 9 where the left homology arm (700 bp) is located adjacent to Exons 23-26 (cDNA sequence).

[0096] In some embodiments, where the cDNA-RS codes for the 3' terminal sequence of the F8 gene the cDNA-RS is within an editing cassette also comprising a sequence for a polyA
site at the 3' end of the cDNA-RS sequence. In some embodiments where the target site is on a portion of the F8 gene having downstream intron sequences, the 3' terminal sequence of the F8 gene the cDNA-RS is within an editing cassette also comprising a splice acceptor at the 5' end of the cDNA-RS sequence. In particular in some embodiment the editing cassette comprise (i) a nucleic acid encoding a truncated FVIII polypeptide or (ii) a native F8 3' splice acceptor site operably linked to a nucleic acid encoding a truncated FVIII
polypeptide that contains a non-mutated portion of the FVIII protein.

[0097] As used throughout, "operably linked" is defined as a functional linkage between two or more elements. In particular, the term "operably linked" or "operably connected" indicates an operating interconnection between two elements finalized to the expression and translation of a sequence. Functional linkages between elements in the sense of the present disclosure are identifiable by a skilled person. For example, an operable linkage between a polynucleotide of interest and a regulatory sequence (i.e., a promoter) comprise a functional link that allows for expression of the polynucleotide of interest. Another example of operable linkage is provided by a control sequence ligated to a coding sequence in such a way that expression of the coding sequence is achieved under conditions compatible with the control sequences.
Operably linked elements are contiguous or non-contiguous and comprise polynucleotides in a same or different reading frame. In an embodiment, each of the operably linked polynucleotide is comprised within the editing cassette. The cassette additionally contains at least one additional gene to be co-transformed into the organism (e.g. a selectable marker gene). One or more additional genes can also be provided on multiple expression cassettes that can further comprise a plurality of restriction sites and/or recombination sites for insertion of other polynucleotides.

[0098] In embodiments herein described, editing cassettes refers to a mobile genetic element that contains a gene and a sequence used to repair an F8 non-functional coding sequence.
Editing cassettes carry at least a cDNA-repair sequence (RS) flanked by an upstream flanking sequence (uFS) and a downstream flanking sequence (dFS) to form a DNA donor.
The cDNA-RS is a repaired version of the F8 non-functional F8 gene sequence. The upstream flanking sequence (uFS) is homologous to a nucleic acid sequence upstream of a target site on the F8 gene and the downstream flanking sequence (dFS) is homologous to a nucleic acid sequences downstream of a target site on the F8 gene. In embodiments described herein, the cDNA-RS of the editing cassette is designed and oriented such that when recombined into the desired region on the F8 gene, it is in-frame with upstream and downstream functional coding sequences. Exemplary editing cassettes include the sequence comprising the left homology arm, cDNA of Exons 23-26, the human growth hormone polyadenylation signal sequence and the right homology arm of the plasmid in FIG. 9, the sequence comprising the left homology arm, cDNA of Exons 2-26, the human growth hormone polyadenylation signal sequence and the right homology arm of the plasmid in FIG. 10, the sequence comprising the left homology arm, cDNA of Exons 23-26, the human growth hormone polyadenylation signal sequence and

99 the right homology arm of the plasmid in FIG. 11, the sequence comprising the left homology arm, cDNA of Exons 2-26, the human growth hormone polyadenylation signal sequence and the right homology arm of the plasmid in FIG. 12.
[0099] In embodiments herein described, following identification of a target site a DNA-SE
is configured for binding to the F8 gene at the selected target site. The DNA-SE is modified to target a target site that is preferentially located about 50-100 base pairs upstream of the desired region to be replaced on the F8 genomic locus so as to optimize recombination by the repair vehicle, donor plasmid, editing cassette comprising the cDNA-RS.
Location of the target site within about 50-100 base pairs upstream of the desired region to be replaced on the F8 genomic locus results in optimal recombination by the repair vehicle, donor plasmid, or editing cassette comprising the cDNA-RS. Optimal recombination is an important aspect as it results in an increase in the likelihood that the cDNA-RS will be incorporated at the targeted site within an individual cell and/or population of cells following exposure to the cDNA ¨RS.
Also, following recombination of the repair vehicle, donor plasmid, or editing cassette into the target site, expression of the repaired F8 gene segment results in expression of a repaired and functional FVIII protein. Thus, conditions promoting optimal recombination greatly contribute towards achieving optimal expression of a repaired and functional protein for treatment and/or induction of immune tolerance. DNA-SEs described herein are modified to comprise nucleases or nickases coupled to nucleotide sequences that specifically guide the nuclease or nickase to the target site. DNA-SEs described herein include heterodimeric nucleases that bind to specific regions of the F8 gene, nucleases or nickases guided to specific sites of the F8 gene by short RNA sequences or combinations thereof A DNA-SE
can be designed and assembled using molecular techniques commonly known and available to one of ordinary skill in the art and as described in Ran, F. A. et al. Genome engineering using the CRISPR-Cas9 system. Nat Protoc 8, 2281-2308 (2013).

[0100] In embodiments described herein, polynucleotides and vectors comprising the DNA-SE and the DNA donor are provided for introduction into a cell of a subject having a mutated F8 gene. In particular the DNA-SE comprises nucleases or nickases coupled to nucleotide sequences that specifically guide the nuclease or nickase to the target site.
DNA-SEs described herein include heterodimeric nucleases that bind to specific regions of the F8 gene, nucleases or nickases guided to specific sites of the F8 gene by short RNA
sequences or combinations thereof The polynucleotides and vectors comprising the DNA-SE and DNA

donor vary in design and function as a function of the type of gene editing system that is utilized. For instance, different polynucleotides and vectors are used for TALENs, CRISPR/Cas9 nuclease, CRISPR/Cas9n nickase, and CRISPR/Cas9 RFN.

[0101] In embodiments herein described, a "donor plasmid" refers to a mobile genetic element in the form of a plasmid, vector, sequence or strand that is be used as a means to deliver or donate a polynucleotide sequence to a specific genomic site. The donor plasmid contains DNA and/or cDNA. Embodiments of donor plasmids described herein consist of at least the following elements: a cDNA-RS for repair of a non-functional F8 coding sequence flanked by an upstream flanking sequence (uFS) and a downstream flanking sequence (dFS).
The upstream flanking sequence (uFS) is homologous to a nucleic acid sequence upstream of the first break in the one strand of the F8 gene and the downstream flanking sequence (dFS) homologous to a nucleic acid sequences downstream of the second break in the other strand of the F8 gene. Donor plasmids are designed and configured to optimally integrate by homologous recombination at a target site following DNA scission by a DNA-SE.
The cDNA-RS of donor plasmid designed and oriented such that when recombined into the desired region on the F8 gene, it is in-frame with upstream and downstream functional coding sequences. Exemplary donor plasmids include the plasmids referenced in FIG. 9, FIG. 10, FIG. 11 and FIG. 12.

[0102] In embodiments herein described the DNA donor is comprised within a repair vehicle (RV). The RV can be a sequence of DNA in the form of a circular plasmid. The RV can be a linear sequence of DNA. The RV provides the template, through which by homologous recombination, a targeted DNA sequence can be introduced into the genomic DNA
of the subject at the site of a targeted double strand break. In addition to a cDNA-RS, optionally an editing cassette and flanking sequences of the DNA donor, a RV can also contain sequences important for the preparation of the DNA sequence in bacteria, such as an antibiotic resistance gene for ampicillin, an antibiotic resistance gene for kanamycin, and/or other antibiotic resistance genes. The RV can also contain intervening DNA sequences important for the integrity of the plasmid or linear sequence of DNA, such as sequences that are located between antibiotic-resistance gene-encoding sequences and cDNA-RS, and which intervening DNA sequences can contain gene-encoding sequences or alternatively can contain sequences that do not encode for a gene.

[0103] In methods and systems herein described polynucleotides coding for a DNA-SE and one or more repair vehicles are introduced into a cell of a subject having a mutated F8 for a time and under condition allowing homologous recombination of the upstream flanking sequence (uFS) and the downstream flanking sequence (dFS) of the donor DNA to corresponding sequences of the F8 gene.

[0104] In particular, in some embodiments herein described, the targeting and repair of a mutated F8 gene in a subject, by introducing into a subject's cell one or more plasmids encoding a DNA-SE that specifically targets the F8 mutation of the subject.
Each subject's mutation for targeting and repair can be determined using techniques known in the art. The identified mutation in the subject is then directly targeted by DNA-SE for correction according e.g. by selecting a DNA-SE target site at the 5' of the mutated non-functional F8 gene sequence. Alternatively, the subject's F8 gene mutations can be corrected by targeting a region of the F8 gene upstream (or 5') from the non-functional coding sequence (e.g. where the mutation occurred), and adding back the corresponding downstream coding regions of the F8 gene. For example, intron 14 could be targeted by the DNA-SE. This allows for gene repair of downstream mutations (i.e. missense mutations in exon 15 to exon 26) and inversions (such as the intron 22 inversion), due to the replacement of exons 15 to 26 with the cDNA-RS discussed above. In other embodiments, the F8 gene can be targeted at additional regions upstream, in order to capture an increasing proportion of F8 gene mutations. Thus, the DNA-SE can be engineered to specifically target a subject's F8 mutation, or alternatively, can target regions upstream of a subject's F8 mutation, in order to correct the mutation in combination with a donor sequence which provides cDNA-RS, which is a partial F8 gene during homologous recombination that replaces, and thus repairs, the mutated portion of the subject's F8 gene and possibly includes functional coding sequences upstream of the non-functional coding sequence of the mutated F8 gene.

[0105] In particular in some embodiments of methods and systems herein described the repairing is performed introducing into a cell of the subject one or more nucleic acids encoding a DNA scission enzyme (DNA-SE) having a DNA-SE target site located upstream from a 5' end of at least one Factor VIII non-functional coding sequence to be repaired, the DNA-SE target site located about 50 bp to about 100 bp upstream from a 5' end of the Factor VIII non-functional coding sequence to be repaired; and introducing into the cell of the subject a cDNA repair editing cassette comprising a cDNA repair sequence (cDNA-RS) coding for a repaired version of the Factor VIII non-functional coding sequence, the cDNA
repair sequence in frame with the Factor VIII functional coding sequence. In those embodiments, location of the target site within about 50-100 base pairs upstream of the desired region to be replaced on the F8 genomic locus results in optimal recombination by the repair vehicle, donor plasmid, or editing cassette comprising the cDNA-RS.
Optimal recombination is an important aspect as it results in an increase in the likelihood that the cDNA-RS will be incorporated at the targeted site within an individual cell and/or population of cells following exposure to the cDNA ¨RS. Also, following recombination of the repair vehicle, donor plasmid, or editing cassette into the target site, expression of the repaired F8 gene segment results in expression of a repaired and functional FVIII protein.
Thus, conditions promoting optimal recombination greatly contribute towards achieving optimal expression of a repaired and functional protein for treatment and/or induction of immune tolerance.

[0106] Also in those embodiments the cDNA repair editing cassette within a DNA
donor where the cDNA repair editing cassette is flanked by an upstream flanking sequence (uFS) homologous to a genomic nucleic acid sequence of at least 200 bp from the DNA-SE target site and a downstream flanking sequence (dFS) homologous to a genomic nucleic acid sequences of at least 200 bp downstream of the DNA-SE target site. In those embodiments introducing one more nucleic acids encoding a DNA scission enzyme (DNA-SE) and introducing a cDNA repair editing cassette is performed to allow homologous recombination of the upstream flanking sequence (uFS) and the downstream flanking sequence (dFS) with corresponding genomic sequences of the Factor VIII gene of the subject.

[0107] In some embodiments, the DNA-SE target site is adjacent to a 3' end of the Factor VIII functional coding sequence, and in particular the 3' end of the functional coding sequence can be a 3' end of a Factor VIII exon.

[0108] In some embodiments, the upstream flanking sequence (uFS) is homologous to a genomic nucleic acid sequence of at least about 400 bp from the DNA-SE target site and the downstream flanking sequence (dFS) is homologous to a genomic nucleic acid sequences of at least about 400 bp downstream of the DNA-SE target site.

[0109] In some embodiments, the upstream flanking sequence (uFS) is homologous to a genomic nucleic acid sequence of at least about 400-800 bp from the DNA-SE
target site and the downstream flanking sequence (dFS) is homologous to a genomic nucleic acid sequences of at least about 400-800 bp downstream of the DNA-SE target site.

[0110] In some embodiments, the uFS is homologous to a genomic nucleic acid sequence of at least about 800-3000 bp from the DNA-SE target site and the dFS is homologous to a genomic nucleic acid sequences of at least about 800-3000 bp downstream of the DNA-SE
target site.

[0111] In some embodiments, the cDNA repair sequence (cDNA-RS) encodes for one or more repaired Factor VIII non-functional sequence consisting essentially of the amino acid sequence encoded by exons 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 26, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, or an in frame portion or combination thereof

[0112] In some embodiments, the methods and compositions set forth herein, the DNA-SEs that targets a mutation in F8 for repair are, for example, a transcription activator¨like effector nuclease (TALEN), a zinc finger nuclease (ZFN), a CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)-associated (Cas) nuclease (CasN), a pair of wild-type CasN each containing its own CRISPR-single-guide-RNA (CRISPR-sgRNA) targeting a deep intronic sequence of a F8 intron flanking the two sides of a large F8 exonic duplication (to repair a HA-causing F8 mutation comprised of a large duplication of one or more F8 exons by introducing a double-stranded DNA (dsDNA) break on each side of large exonic duplication such that intervening genomic DNA sequence comprising the duplication can be deleted, thereby restoring the transcriptional and post-transcriptional functionality to the repair F8 sequence), a pair of missense mutant Cas nickases -- each capable of introducing only a single-stranded DNA (ssDNA) break -- using paired CRISPR guide RNAs, or CRISPR
with RFN, as described in detail below.

[0113] To minimize off-target effects associated with the DNA-SEs, a program such as PROGNOS is used. The PROGNOS algorithm locates for example potential TALEN off-target sites by searching through the genome for sequences similar to the intended TALEN
design. It ranks these similar sequences according to various features of TALEN-DNA
interactions, including RVD base preferences, polarity of TALEN specificity (5' end is more specific), context dependent compensation of strong RVDs (such as NN and HD), and a model of dimeric TALEN interactions. The PROGNOS model has been shown to accurately predict the majority of all known TALEN off-target sites as discussed in Fine et al. Nucleic Acids Research 2013, incorporated herein by reference in their entirety.

[0114] The algorithm employed for ranking potential CRISPR off-target sites described in Hsu et al. Nature Biotech 2013, incorporate herein by reference, uses a position-weight-matrix (PWM) to determine the importance of different types of mismatches at each position in the target sequence (both the DNA bases targeted by the guide strand as well as the protospacer adjacent motif sequence). This PWM was derived by experimentally observing the drop in nuclease activity at a target site of artificial guide strands (relative to a perfectly matched guide strand) containing different types of mismatches. This PWM is then used to screen potential sites in the genome with homology to the intended target and assign them a score indicating their likelihood of off-target activity.

[0115] In some embodiments the DNA-SE is Transcription Activator-Like Effector Nucleases (TALENs) which provides an alternative to zinc finger nucleases (ZFNs) for certain types of genome editing. The C-terminus of the TALEN component carries nuclear localization signals (NLSs), allowing import of the protein to the nucleus.
Downstream of the NLSs, an acidic activation domain (AD) is also present, which is probably involved in the recruitment of the host transcriptional machinery. The central region harbors a series of nearly identical 34/35 amino acids modules repeated in tandem. Residues in positions 12 and 13 are highly variable and are referred to as repeat-variable di-residues (RVDs). Studies of TALENs such as AvrBs3 from X axonopodis pv. vesicatoria and the genomic regions (e.g., promoters) they bind, led two teams to "crack the TALE code" by recognizing that each RVD
in a repeat of a particular TALE determines the interaction with a single nucleotide. Most of the variation between TALEs relies on the number (ranging from 5.5 to 33.5) and/or the order of the quasi-identical repeats. Estimates using design criteria derived from the features of naturally occurring TALEs suggest that, on average, a suitable TALEN target site can be found every 35 base pairs in genomic DNA. Compared with ZFNs, the cloning process of TALENs is easier, the specificity of recognized target sequences is higher, and off-target effects are lower. In one study, TALENs designed to target chemokine receptor 5 (CCR5) were shown to have very little activity at the highly homologous chemokine receptor 2 (CCR2) locus, as compared with CCR5-specific ZFNs that had similar activity at the two sites.

[0116] FIG. 2 and FIG. 3 provide exemplary illustrations outlining the use of a repair vehicle encoding a TALEN nuclease that is used to repair the F8 gene in, for example, a human with an intron-22 (I22)-inverted F8 locus, F8122I. As illustrated in Figure 2(A), the major transcription unit of the F8122I locus consists of 24 exons, which are designated exons 1-22 (a functional coding sequence) and exons 23C & 24C (a non-functional coding sequence).
The first 22 are the same as exons 1-22 of the wild-type FVIII structural locus (F8) but the last two (exon-23C & exon-24C) are cryptic and non-functional in non-hemophilic individuals as well as in patients whose HA is caused by F8 gene abnormalities other than the 1221-mutation. As illustrated in Figure 2(B) the strategy to repair the 1221-mutation consists of introducing in the cell of the subject a repair vehicle encoding a functional TALEN --which is a heterodimeric nuclease comprised of a monomer subunit that binds 5' of the desired genome editing site (TALEN-L) and one that binds 3' of it (TALEN-R) --that is specific for a DNA sequence that is present in only a single copy per haploid human genome, which is approximately 1 kb downstream of the 3 '-end of exon-22. Upon expression, once both monomers are bound to this specific sequence, their individual Fokl nuclease domains dimerize to form the active enzyme that catalyzes a double-stranded (ds) break in the DNA
between their binding sites. If a ds-DNA break occurs in the presence of a second nucleic acid, for example a cDNA-RS (a functional coding sequence) comprising a native FVIII 3' splice acceptor site operably linked to a nucleic acid encoding a truncated FVIII polypeptide encoding exons 23-26 (i.e., a "donor plasmid (DP)" or donor sequence), which is flanked by a stretch of DNA with a left homology (HL) arm and right homology (HL) arm that have identical DNA sequences to that in the native chromosomal DNA 5' and 3' of the region flanking the break-point, homologous recombination (HR) occurs very efficiently. Following HR, the cDNA-RS segment between the left and right homology arms (which as shown in Figure 2 contains a partial human F8 cDNA that contains, in-frame, all of exons 23-25 and the coding sequence of exon-26, with a functional 3'-splice site at its 5'-end) becomes permanently ligated/inserted into the chromosome. Since the cDNA-RS fused at its 5'-end to a functional 3'-splice site, this TALEN catalyzes repair and converts F8122I
into wild-type F8-like locus and restore its ability to drive synthesis of a full-length fully functional wild-type FVIII protein. Figure 3 shows the details of a functional heterodimeric TALEN, comprised of left and right monomer subunits (TALEN-L and TALEN-R), bound to its target "editing" sequence in intron-22 (122) of the human FVIII structural locus (F8), ¨1 kb downstream of the 3 '-end of exon-22 (Figure 3).

[0117] Likewise, FIG. 4 shows a functional heterodimeric TALEN targeting a F8 mutation in canine, comprised of its left and right monomer subunits (TALEN-L and TALEN-R), bound to its target "editing" sequence in the 122 of the canine F8 structural locus (cF8), ¨0.25 kb downstream of the 3'-end of exon-22. Because the target binding sequence of each monomer is the same in both a wild-type canine F8 (cF8) and an I22-inverted F8 gene (cF8-I221), this TALEN edits each locus equally well. Following binding of this TALEN's monomeric subunits to their target I22-sequences in the cF8-122I locus of a dog with severe HA caused by the 1221-mutation, their individual Fokl nuclease domains are able to form a homo-dimer, i.e. the active form of the enzyme, which catalyzes a double-stranded (ds) break in the DNA
between the monomer binding sites; this site is labeled as the target site. If a ds-DNA break occurs in the presence of a donor sequence or plasmid, which contains a stretch of DNA with left and right arms that have identical DNA sequences to that in the native chromosomal DNA, in the region flanking the break-point (see Figure 3 for the human F8 locus), homHR
occurs very efficiently. Following HR, the DNA segment between the left and right homology arms (which contains a partial cF8 cDNA that contains, in-frame, all of exons 23-25 and the coding sequence of exon-26, with a functional 3'-splice site at its 5'-end) becomes permanently ligated/inserted into the canine X-chromosome. Because the DNA
segment between the left and right homology arms comprises a partial cF8 cDNA (which, as shown in Figure 2 for the human F8-122I, contains, in-frame, all of canine exons 23-25 and the coding sequence of canine exon-26) fused at its 5'-end to a functional 3'-splice site, this TALEN
catalyzes repair and converts cF8-122I into a wild-type cF8-like locus that restores its ability to drive synthesis of a full-length fully functional wild-type canine FVIII.

[0118] FIG. 5 illustrates a TALEN-mediated strategies to repair the human Factor VIII
(FVIII) gene (F8) mutations in >50% of all patients with severe hemophilia-A
(HA), including the highly recurrent intron-22 (I22)-inversion (1221)-mutation.
Figure 5 highlights the TALEN approach linking Exon 22 of the F8 gene to a nucleic acid including exons 23-26 encoding a truncated FVIII polypeptide. Panel A of Figure 5 shows the specific F8 genomic DNA sequence (spanning positions 126,625 - 126,693) within which a double-stranded DNA
break (DSDBs) is introduced (designated "Endonuclease domain" and "target site" in Panel B) by this strategy's functional TALEN dimer. The left and right TALEN protein sequences for the variable DNA-binding domain are listed as Seq. ID. No. 4 and Seq. ID.
No. 6, respectively. An example of DNA sequences encoding the left and right TALEN
DNA-binding domains are listed as Seq. ID. No 5 and Seq. ID. No. 7, respectively.
Because of the degeneracy of the genetic code, there are many possible constructs that can be used to encode TALEN DNA-binding domains. In some embodiments, the codons are optimized for expression of the DNA constructs. Panel A in Figure 5 also shows the F8 genomic DNA
sequence containing (i) the recognition sites for the left (TALENL-hF8E22422) and right (TALENR-hF8E22n22) TALEN monomers comprising F8-TALEN-5 and (ii) the intervening spacer region within which the F8-TALEN-5's endonuclease activity creates the double-stranded DNA breaks (DSDBs) required for inducing the physiologic cellular machinery that mediates the homology-dependent DNA repair pathway. Panel A in Figure 5 also shows important orienting landmarks, including the following: (i) Nucleotide coordinates of this region (based on the February, 2009, human genome assembly [UCSC Genome Browser:
http://genome.ucsc.edu/]) are numbered with respect to the wild-type F8 transcription unit, where the initial (5'-most) base of the F8 pre-mRNA (5'-base of exon-1 [El]) is designated +1 or 1 (note that this base corresponds to X-chromosome position 154,250,998) and includes the appropriate intronic sequence bases in calculating the genomic base positioning; (ii) Relative location of the X-chromosome's centromere (X-Cen) and its long-arm telomere (Xq-Tel), as transcription of the wild-type F8 locus and all of its mutant alleles causing HA
¨with the exception of its two recurrent intronic inversions, the intron-1 (I1)-inversion (In)-and the 1221-mutations¨ is oriented towards X-Cen. Transcription of the Ii-and I22-inverted F8 loci, in contrast, are oriented towards Xq-Tel. This strategy repairs (i) the highly recurrent 1221-mutation ¨ also designated F81221 ¨ which causes ¨45% of all unrelated patients with severe hemophilia-A (HA) and (ii) mutant F8 loci in ¨20% of all other patients with severe HA, who are either known or found to have any one of the >200 distinct mutations that have been found (according to the HAMSTeRS database of HA-causing F8 mutations) thus far to reside down-stream (i.e., 3') of exon-22 (E22). The last codon of exon 22 encodes methionine (Met [M]) as translated residue 2,143 (2,124 in the mature FVIII
protein secreted into plasma). Most mutations repaired are "previously known"
(literature and/or HAMSTeRS or other databases), some have never been identified previously; the F8 abnormalities in this latter category are "private" (found only in this particular) to the patient/family.

[0119] Panel B in Figure 5 shows the functional aspects of the TALENs including the overall DNA-binding domain (DBD) and the DBD-subunit repeats of the left and right monomers (TALENL-hF8E22422 and TALENR-hF8E22/122). Also shown are the (i) specific DNA
sequences recognized by each TALEN monomer (shown in bold font immediately below each DBD-subunit); (ii) the spacer region between the DNA recognition sequences of the TALEN monomers contains the sequence within which the dimerized Fokl catalytic domains, which form a functional endonuclease, introduce a double-stranded DNA
break (DSDB) ; this site is indicated as the target site. As shown in the lower left portion of Figure 5, the introduction of a DSDB in the presence of homologous repair vehicle no.
5 (HRV5), the nucleotide sequence of which is provided below as Seq. ID. No. 12, results in the in-frame integration, immediately 3' to exon 22, of the partial human F8 cDNA
comprising exons 23, 24 and 25 and the protein coding sequence, or CDS, of exon 26 (designated hF8[E23-E25/E26cDs]). In one embodiment, the TALEN constructs depicted in Figure 5 can be used to repair all 1221 inversion mutations (See #1 pathway). In another embodiment, the same constructs can be used to repair non-I221 F8 mutations that occur 3' (i.e. downstream) of the exon-22/intron-22 junction (See #2 pathway).

[0120] FIG. 6 illustrates a TALEN-mediated strategy to repair the human F8 mutations in >50% of all patients with severe HA, including the highly recurrent 1221-mutation. Figure 6 highlights the TALEN approach linking intron-22 of the F8 to a nucleic acid encoding a truncated FVIII polypeptide encoding exons 23-26. Panel A shows the specific F8 genomic DNA sequence within which a DSDB is introduced (designated "Endonuclease domain" in Panel B and "target site") by this strategy's functional TALEN dimer. The left and right TALEN protein sequences for the variable DNA-binding domain are listed as Seq.
ID. No. 8 and Seq. ID. No. 10, respectively. Examples of DNA sequences encoding the left and right TALEN DNA-binding domains are listed as Seq. ID. No. 9 and Seq. ID. No. 11, respectively.
Because of the degeneracy of the genetic code, there are many possible constructs that can be used to encode TALEN DNA-binding domains. In some embodiments, the codons are optimized for expression of the DNA constructs. Panel A in Figure 6 also shows important orienting landmarks, including the: (i) nucleotide coordinates of this region (based on the February, 2009, human genome assembly available at the UCSC Genome Browser:
http://genome.ucsc.edu/) are numbered with respect to the wild-type F8 transcription unit, where the initial (5'-most) base of the F8 pre-mRNA (5' most base of exon-1 [El]) is designated +1 or 1 (note that this base corresponds to X-chromosome position 154,250,998) and includes the appropriate intronic sequence bases in calculating the genomic base positioning; (ii) relative location of the X-chromosome's centromere (X-Cen) and its long-arm telomere (Xq-Tel), as transcription of the wild-type F8 locus and all of its mutant alleles causing HA ¨with the exception of its two recurrent intronic inversions, Ill-and the 1221-mutations ___________________________________________________________ is oriented towards X-Cen; Transcription of the Ii- and I22-inverted F8 loci, in contrast, is oriented towards Xq-Tel. This strategy repairs (i) the highly recurrent 1221-mutation ¨ also designated F81221 ¨ which causes ¨45% of all unrelated patients with severe HA and (ii) mutant F8 loci in ¨20% of all other patients with severe HA, who are either known or found to have any one of the >200 distinct mutations that have been found (according to the HAMSTeRS database of HA-causing F8 mutations) thus far to reside down-stream (i.e., 3') of exon-22 (E22). The last codon of E22 entirely encodes methionine (Met [M]) as translated residue 2,143 (2,124 in the mature FVIII secreted into plasma). Most mutations repaired are "previously known" (literature and/or HAMSTeRS or other databases), but some have never been identified previously. The F8 abnormalities in this latter category are "private" (found only in this particular) to the patient/family.

[0121] Panel B in Figure 6 shows the functional aspects of the TALENs including the overall DBD and the DBD-subunit repeats of the left and right monomers (TALENL-hF822 and TALENR-hF8I22). Also shown are the (i) specific DNA sequences recognized by each TALEN monomer (shown in bold font immediately below each DBD-subunit); (ii) the spacer region between the DNA recognition sequences of the TALEN monomers contains the sequence within which the dimerized Fokl catalytic domains, which form a functional endonuclease, introduce a DSDB; this site is indicated as the target site.. As shown in the lower left portion of Figure 6, the introduction of a DSDB in the presence of a homologous repair vehicle, the nucleotide sequence of which is listed as Seq. ID. No. 13, results in the integration into intron-22 of a native F8 3' splice acceptor site operably linked to a nucleic acid encoding F8 exons-23, 24 and 25 and the protein coding sequence, or CDS, of exon-26 (designated hF8[E23-E25/E26cDs]). In one embodiment, the TALEN constructs depicted in Figure 6 can be used to repair all 1221 inversion mutations (See #1 pathway).
In another embodiment, the same constructs are used to repair non-I221 F8 mutations that occur 3' (i.e.
downstream) of the exon-22/intron-22 junction (See #2 pathway).

[0122] FIG. 7 shows a comparison of expected genomic DNA, spliced RNA and proteins pre and post repair. Several examples of functional and non-functional coding sequences are depicted in the gDNA panel of FIG. 7. Example functional coding sequences include exons 1-22 and exons 22-23 of the wild-type F8 genomic DNA (Normal), exons 1-22 of the 1221 mutant F8 genomic DNA (1221), and exons 1-22 of the 1221 mutant F8 genomic DNA
and exons 23-26 of the wild-type F8 cDNA (Repaired). Example non-functional coding sequences include exons 23-26 of the 1221 mutant F8 genomic DNA (1221) and exons 23-26 of the 1221 mutant F8 genomic DNA (right, Repaired).

[0123] In some embodiments, nucleic acids encoding nucleases specifically target intron-1, intron-14, or intron-22. In some embodiments, nucleic acids encoding nucleases specifically target the exon-1/intron-1 junction; exon-14/intron-14 junction; or the exon-22/intron-22 junction.

[0124] Figure 9 illustrates an example of a donor plasmid that can be used to repair the F8 at the exon-22/intron-22 junction using a TALEN, ZFN, CRISPR/Cas, CRISPR-PN, and CRISPR-RFN approach. The donor plasmid contains the cDNA sequence for exons 23-26 of the F8 (labeled as functional coding sequence) and a polyadenylation signal sequence flanked by two regions of homology to the F8. The left homology region contains a DNA
sequence (approximately 700 base pairs) that is homologous to part of intron-21 and exon-22 of the F8.
The right homology region contains a DNA sequence (approximately 700 base pairs) that is homologous to part of intron-22 of the F8. Upon successful homologous recombination into the F8 locus, the integrated construct expresses the resulting mRNA encoding the wild-type (corrected) version of the FVIII. The sequence of the plasmid depicted in Figure 9 is listed as Seq. ID. No. 12. The annotation of Seq. ID. No. 12 is provided in Table 1 below.
Table 1: Repair vehicle targeted to the Exon 22 ¨ Intron 22 junction of F8 LOCUS RepairVehicle 7753 bp DNA linear FEATURES Location/Qualifiers misc_feature 21..327 /note="fl origin (-)"
misc_feature 6765..7625 /note="<= Ampicillin"
misc_feature 471..614 /label=<= lacZ A
misc_feature 626..644 /note="T7 promoter =>"
misc_feature 5564..5583 /note="T3 promoter =>"
misc_feature 6765..7625 /note="<= Orfl"
misc_feature 7667..7695 /note="<= AmpR promoter"
misc_feature 658..740 /note="MCS"

Table 1: Repair vehicle targeted to the Exon 22 ¨ Intron 22 junction of F8 misc_feature 1446..2072 /note="Exons 23-26 (cDNA seq)"
misc_feature 1730..1737 /note="Create NotI site"
misc_feature 2082..2707 /note="hGH polyA"
misc_feature 1785..1787 /note="ns-SNP: A6940G (M2238V)"
misc_feature 3408..4160 /note="HSV-TK promoter "
misc_feature 4161..5546 /note="HSV-TK gene and TK pA Terminator "
misc_feature 741..745 /note="Create site for cloning"
misc_feature 5547..5551 /note="Create site for cloning"
misc_feature 746..1445 /note="Left homolgy arm (700 bp)"
misc_feature 1290..1445 /note="Exon 22"
misc_feature 1433..1445 /note="Partial Left TALEN recognition site"
misc_feature 2708..3407 /note="Right homology arm (700 bp)"
misc_feature 2708..2716 /note="Partial Right TALEN recognition site"
misc_feature 2708..3407 /note="Partial Intron 22"
misc_feature 746..1289 /note="Partial Intron 21"
source 1..7753 /dnas_title="RepairVehicle E22-I22 pBluescript"

[0125] FIG. 10 illustrates an example of a donor plasmid that can be used to repair the F8 using a TALEN, ZFN, CRISPR/Cas, CRISPR-PN, and CRISPR-RFN approach. The donor plasmid contains the cDNA sequence for exons2-26 of the F8 (labeled as functional coding sequence) flanked by two regions of homology to the F8. The left homology region contains a DNA sequence that is homologous to part of the F8 promoter and part of exon-1. The right homology region contains a DNA sequence that is homologous to part of intron-1. Upon successful homologous recombination into the F8, the integrated construct expresses the resulting mRNA encoding the wild-type (corrected) version of the FVIII. The donor sequence is cloned into plasmid (p)BlueScript-II KS-minus (pBS-II-KS[). The donor plasmid is used with a TALEN, ZFN, CRISPR/Cas, CRISPR-PN, and CRISPR-RFN genomic editing strategy. The sequence of the plasmid depicted in Figure 10 is listed as Seq.
ID. No. 13. The annotation of Seq. ID. No. 13 is provided in Table 2 below.
Table 2: Repair vehicle targeted to the Exon 1 ¨ Intron 1 junction of F8 LOCUS RepairVehicle 11418 bp DNA linear Table 2: Repair vehicle targeted to the Exon 1 ¨ Intron 1 junction of F8 FEATURES Location/Qualifiers misc_feature 21..327 /note="fl origin (-)"
misc_feature 10430..11290 /note="<= Ampicillin"
misc_feature 471..614 /label=<= lacZ A
misc_feature 626..644 /note="T7 promoter =>"
misc_feature 9229..9248 /note="<= T3 promoter"
misc_feature 10430..11290 /note="<= Orfl"
misc_feature 11332..11360 /note="<= AmpR promoter"
misc_feature 658..740 /note="MCS"
misc_feature 5780..6405 /note="hGH polyA"
misc_feature 7073..7825 /note="HSV-TK promoter "
misc_feature 7826..9211 /note="HSV-TK gene and TK pA Terminator "
misc_feature 740..745 /note="Create site for cloning"
misc_feature 1540..5770 /note="Exons 2-26 BDD (cDNA seq)"
misc_feature 2664..2669 /note="Create ClaI site"
misc_feature 2903..2905 /note="ns-SNP: G1679A (R484H)"
misc_feature 3680..3685 /note="BDD (5er743 - G1n1638)"
misc_feature 5428..5435 /note="Create NotI site"
misc_feature 5768..5768 /dnas_title="Stop"
/vntifkey="21"
/label=Stop misc_feature 5483..5485 /note="ns-SNP: A6940G (M2238V)"
insertion_seq 3934..5770 /dnas_title="Tg"
/vntifkey="14"
/label=Tg misc_feature 9212..9217 /note="Create site for cloning"
misc_feature 9212..9212 /note="MCS"
misc_feature 746..1539 /note="Left homolgy arm (794bp)"
misc_feature 746..1237 /note="Partial F8 promoter"
misc_feature 1238..1539 /note="Partial Exon 1"
misc_feature 6406..7072 /note="Right homology arm (667 bp)"
misc_feature 6406..7072 /note="Partial intron 1"

Table 2: Repair vehicle targeted to the Exon 1 ¨ Intron 1 junction of F8 source 1..11418 /dnas_title="RepairVehicle El-I1 pBluescript"

[0126] Figure 11 illustrates an example of a donor plasmid that is used to repair the F8 in intron-22 using a TALEN, ZFN, CRISPR/Cas, CRISPR-PN, and CRISPR-RFN approach.
The donor plasmid contains a 3' splice site, the cDNA sequence for exons 23-26 of the F8 (labeled as functional coding sequence), and a polyadenylation signal sequence flanked by two regions of homology to the F8. The left homology region contains a DNA
sequence (approximately 700 base pairs) that is homologous to part of intron-22 of the F8. The right homology region contains a DNA sequence (approximately 700 base pairs) that is homologous to part of intron-22 of the F8. Upon successful homologous recombination into the F8 locus, the integrated construct expresses the resulting mRNA encoding the wild-type (corrected) version of the FVIII. The sequence of the plasmid depicted in Figure 11 is listed as Seq. ID. No. 14. The annotation of Seq. ID. No. 14 is provided in Table 3 below.
Table 3: Repair vehicle targeted to Intron 22 of F8 LOCUS RepairVehicle 7755 bp DNA linear FEATURES Location/Qualifiers misc_feature 21..327 /note="fl origin (-)"
misc_feature 6767..7627 /note="<= Ampicillin"
misc_feature 471..614 /label=<= lacZ A
misc_feature 626..644 /note="T7 promoter =>"
misc_feature 5566..5585 /note="T3 promoter =>"
misc_feature 6767..7627 /note="<= Orfl"
misc_feature 7669..7697 /note="<= AmpR promoter"
misc_feature 658..740 /note="MCS"
misc_feature 1448..2074 /note="Exons 23-26 (cDNA seq)"
misc_feature 1732..1739 /note="Create NotI site"
misc_feature 2084..2709 /note="hGH polyA"
misc_feature 1787..1789 /note="ns-SNP: A6940G (M2238V)"
misc_feature 3410..4162 /note="HSV-TK promoter "
misc_feature 4163..5548 /note="HSV-TK gene and TK pA Terminator "

Table 3: Repair vehicle targeted to Intron 22 of F8 misc_feature 741..745 /note="Create site for cloning"
misc_feature 5549..5553 /note="Create site for cloning"
misc_feature 746..1445 /note="Left homology arm (700 bp)"
misc_feature 1437..1445 /note="Partial Left TALEN recognition site"
misc_feature 2710..3409 /note="Right homolgy arm (700 bp)"
misc_feature 2710..2719 /note="Partial Right TALEN recognition site"
misc_feature 746..1445 /note="Partial Intron 22"
misc_feature 2710..3409 /note="Partial Intron 22"
misc_feature 1446..1447 /note="3' spice site"
source 1..7755 /dnas title="RepairVehicle 122 pEluescript"

[0127] Figure 12 illustrates an example of a donor plasmid that is used to repair the F8 in intron-1 using a TALEN, ZFN, CRISPR/Cas, CRISPR-PN, and CRISPR-RFN approach.
The donor plasmid contains a 3' splice site, the cDNA sequence of the F8 for exons 2-26 lacking the B-domain (B-domain deleted (BDD) version of the F8) (labeled as functional coding sequence), and a polyadenylation signal sequence flanked by two regions of homology to the F8. The left homology region contains a DNA sequence (approximately 700 base pairs) that is homologous to part of exon-1 and intron-1 of the F8 gene. The right homology region contains a DNA sequence (approximately 700 base pairs) that is homologous to part of intron-1 of the F8. Upon successful homologous recombination into the F8 locus, the integrated construct expresses the resulting mRNA encoding the wild-type (corrected) version of the FVIII. The sequence of the plasmid depicted in Figure 12 is listed as Seq. ID.
No. 15. The annotation of Seq. ID. No. 15 is provided in Table 4 below.
Table 4: Repair vehicle targeted to Intron 1 of F8 LOCUS RepairVehicle 11359 bp DNA linear FEATURES Location/Qualifiers misc_feature 21..327 /note="fl origin (-)"
misc_feature 10371..11231 /note="<= Ampicillin"
misc_feature 471..614 /label=<= lacZ A
misc_feature 626..644 /note="T7 promoter =>"

Table 4: Repair vehicle targeted to Intron 1 of F8 misc_feature 9170..9189 /note="<= T3 promoter"
misc_feature 10371..11231 /note="<= Orfl"
misc_feature 11273..11301 /note="<= AmpR promoter"
misc_feature 658..740 /note="MCS"
misc_feature 874..1187 /note="Exon 1"
misc_feature 1436..1445 /note="Partial Left TALEN recognition site"
misc_feature 5688..6313 /note="hGH polyA"
misc_feature 6314..7013 /note="Right homology arm (700 bp)"
misc_feature 6314..6322 /note="Partial Right TALEN recognition site"
misc_feature 7014..7766 /note="HSV-TK promoter "
misc_feature 7767..9152 /note="HSV-TK gene and TK pA Terminator "
misc_feature 746..1445 /note="Left homolgy arm (700 bp)"
misc_feature 746..873 /note="Partial F8 promoter"
misc_feature 740..745 /note="Create site for cloning"
misc_feature 6314..7013 /note="Partial Intron 1"
misc_feature 1448..5678 /note="Exons 2-26 BDD (cDNA seq)"
misc_feature 1446..1447 /note="3' spice site"
misc_feature 2572..2577 /note="Create ClaI site"
misc_feature 2811..2813 /note="ns-SNP: G1679A (R484H)"
misc_feature 3588..3593 /note="BDD (Ser743 - G1n1638)"
misc_feature 5336..5343 /note="Create NotI site"
misc_feature 5676..5676 /dnas_title="Stop"
/vntifkey="21"
/label=Stop misc_feature 5391..5393 /note="ns-SNP: A6940G (M2238V)"
insertion_seq 3842..5678 /dnas_title="Tg"
/vntifkey="14"
/label=Tg misc_feature 9153..9158 /note="Create site for cloning"
misc_feature 9153..9153 /note="MCS"
source 1..11359 /dnas_title="RepairVehicle Ii pBluescript"

[0128] In one embodiment, the integration matrix component for each of the distinct homologous donor plasmid is either a cDNA that is linked to the immediately upstream exon or a cDNA that has a functional 3'-intron-splice-junction so that the cDNA
sequence is linked through the RNA intermediate following removal of the intron. In one embodiment, the donor plasmid is personalized, on an individual basis, so that each patient's gene that is repaired expresses the form of the FVIII that they are maximally tolerant of

[0129] In some embodiments the DNA-SE used for F8 targeting is a ZFN. ZFNs are hybrid proteins containing the zinc-finger DNA-binding domain present in transcription factors and the non-specific cleavage domain of the endonuclease Fokl. (Li et al., In vivo genome editing restores hemostasis in a mouse model of hemophilia, Nature 2011 Jun 26;
475(7355):217-21).

[0130] The same sequences targeted by the TALEN approach, discussed above, can also be targeted by the ZFN approach for genome editing. ZFNs are a class of engineered DNA-binding proteins that facilitate targeted editing of the genome by creating DSDB at user-specified locations. Each ZFN consists of two functional domains: 1) a DBD
comprised of a chain of two-finger modules, each recognizing a unique hexamer (6 bp) sequence of DNA, wherein two-finger modules are stitched together to form a ZFN, each with specificity of?
24 bp, and 2) a DNA-cleaving domain comprised of the nuclease domain of Fok 1.
The DNA-binding and DNA-cleaving domains are fused together and recognize the targeted genomic sequences, allowing the Fokl domains to form a heterodimeric enzyme that cleaves the DNA by creating double stranded breaks.

[0131] ZFNs can be readily made by using techniques known in the art (Wright DA, et al.
Standardized reagents and protocols for engineering zinc finger nucleases by modular assembly. Nat Protoc. 2006;1(3):1637-52). Engineered ZFNs can stimulate gene targeting at specific genomic loci in animal and human cells. The construction of artificial zinc finger arrays using modular assembly has been described. The archive of plasmids encoding more than 140 well-characterized zinc finger modules together with complementary web-based software for identifying potential zinc finger target sites in a gene of interest has also been described. These reagents enable easy mixing-and-matching of modules and transfer of assembled arrays to expression vectors without the need for specialized knowledge of zinc finger sequences or complicated oligonucleotide design (Wright DA, et al.
Standardized reagents and protocols for engineering zinc finger nucleases by modular assembly. Nat Protoc. 2006;1(3):1637-52). Any gene in any organism can be targeted with a properly designed pair of ZFNs. Zinc-finger recognition depends only on a match to the target DNA
sequence (Carroll, D. Genome engineering with zinc-finger nucleases. Genetics Society of America, 2011, 188(4), pp 773-782).

[0132] In some embodiments the DNA-SE used for F8 gene targeting comprises Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR Associated (Cas) Nucleases based on CRISPR technology. (Mali P, Yang L, Esvelt KM, Aach J, Guell M, DiCarlo JE, Norville JE, Church GM. RNA-guided human genome engineering via Cas9.
Science. 2013 Feb 15;339(6121):823-6; Gasiunas G, Barrangou R, Horvath P, Siksnys V.
Cas9-crRNA ribonucleoprotein complex mediates specific DNA cleavage for adaptive immunity in bacteria. Proc Natl Acad Sci U S A. 2012 Sep 25;109(39):E2579-86.
Epub 2012 Sep 4).

[0133] The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR Associated (Cas) system was discovered in bacteria and functions as a defense against foreign DNA, either viral or plasmid. In bacteria, the endogenous CRISPR/Cas system targets foreign DNA with a short, complementary single-stranded RNA
(CRISPR
RNA or crRNA) that localizes the Cas9 nuclease to the target DNA sequence. The DNA
target sequence can be on a plasmid or integrated into the bacterial genome.
The crRNA can bind on either strand of DNA and the Cas9 cleaves both strands (double strand break, DSB).
A recent in vitro reconstitution of the Streptococcus pyogenes type II CRISPR
system demonstrated that crRNA fused to a normally trans-encoded tracrRNA is sufficient to direct Cas9 protein to sequence-specifically cleave target DNA sequences matching the crRNA.
The fully defined nature of this two-component system allows it to function in the cells of eukaryotic organisms such as yeast, plants, and even mammals. By cleaving genomic sequences targeted by RNA sequences, such a system greatly enhances the ease of genome engineering.

[0134] The crRNA targeting sequences are transcribed from DNA sequences known as protospacers. Protospacers are clustered in the bacterial genome in a group called a CRISPR
array. The protospacers are short sequences (-20bp) of known foreign DNA
separated by a short palindromic repeat and kept like a record against future encounters. To create the CRISPR targeting RNA (crRNA), the array is transcribed and the RNA is processed to separate the individual recognition sequences between the repeats. In the Type II system, the processing of the CRISPR array transcript (pre-crRNA) into individual crRNAs is dependent on the presence of a trans-activating crRNA (tracrRNA) that has sequence complementary to the palindromic repeat. When the tracrRNA hybridizes to the short palindromic repeat, it triggers processing by the bacterial double-stranded RNA-specific ribonuclease, RNase III.
Any crRNA and the tracrRNA can then both bind to the Cas9 nuclease, which then becomes activated and specific to the DNA sequence complimentary to the crRNA. (Mali P, Yang L, Esvelt KM, Aach J, Guell M, DiCarlo JE, Norville JE, Church GM. RNA-guided human genome engineering via Cas9. Science. 2013 Feb 15;339(6121):823-6; Gasiunas G, Barrangou R, Horvath P, Siksnys V. Cas9-crRNA ribonucleoprotein complex mediates specific DNA cleavage for adaptive immunity in bacteria. Proc Natl Acad Sci U
S A. 2012 Sep 25;109(39):E2579-86. Epub 2012 Sep 4).

[0135] The DSDB induced by the TALEN approach overlaps with the 6 distinct sites of DSDB induced by Cas9, via targeting by 6 distinct CRISPR-guide RNAs [F8-CRISPR/Cas9-1 (F8-Exl/Int1), F8-CRISPR/Cas9-2 (F8-Intl), F8-CRISPR/Cas9-3 (F8-Ex14/Intl 4), F8-CRISPR/Cas9-4 (F8-Int14), F8-CRISPR/Cas9-5 (F8-Ex22/Int22), F8-CRISPR/Cas9-6 (F8-Int22)]. This allows use of the same 6 distinct homologous donor sequences with all three genome editing approaches, including the TALEN nuclease, ZFN, and the Cas nuclease.

[0136] Figure 13 illustrates a CRISPR/Cas9-mediated strategy to repair the human Factor VIII (FVIII) gene (F8) mutations in ¨95% of all patients with severe hemophilia-A (HA), including the highly recurrent intron-1 (I1)-inversion (M)-mutation as well as the intron-22 (I22)-inversion (122I)-mutation. Figure 13 shows the specific F8 genomic DNA
sequence (spanning genic base positions 172 ¨ 354 at intron 1) within which a double-stranded (ds)-DNA break is introduced (designated "Endonuclease target" or "target site" in this panel) by this strategy's wild-type (wt) CRISPR/Cas9 ds-DNase in which both of its endonuclease domains are catalytically functional ("hF8-CRISPR/Cas9wt-1"). This panel also shows important orienting landmarks, including the following: (i) Nucleotide coordinates of this region (based on the February, 2009, human genome assembly [UCSC Genome Browser:
http://genome.ucsc.edu/]) are numbered with respect to the wild-type F8 transcription unit, where the initial (5'-most) base of the F8 pre-mRNA (5'-base of exon-1 [El]) is designated +1 or 1 (note that this base corresponds to X-chromosome position 154,250,998) and include the appropriate intronic sequence bases in calculating the genomic base positioning; (ii) Relative location of the X-chromosome's centromere (X-Cen) and its long-arm telomere (Xq-Tel), as transcription of the wild-type F8 locus and all of its mutant alleles causing HA
¨with the exception of its two recurrent intronic inversions, the In- and the mutations ___________________________________________________________ is oriented towards X-Cen. Transcription of the Ii- and I22-inverted F8 loci, in contrast, are oriented towards Xq-Tel. This strategy repairs (i) the highly recurrent 1221-mutation ¨ also designated F81221 ¨ which causes ¨45% of all unrelated patients with severe hemophilia-A (HA) and (ii) mutant F8 loci in ¨90-95% of all other patients with severe HA, who are either known or found to have any one of the >1,500 distinct mutations that have been found (according to the HAMSTeRS database of HA-causing F8 mutations) thus far to reside down-stream (i.e., 3') of exon-1 (El). The last codon of El partially encodes the translated residue 48 (29 in the mature FVIII protein secreted into plasma). Most mutations repaired are "previously known" (literature and/or HAMSTeRS or other databases). Some have never been identified previously. These F8 abnormalities in this latter category are "private" (found only in this particular) to the patient/family.
Finally, Figure 13 shows the functional aspects of hF8-CRISPR/Cas9wt-1 including the overall DNA-binding domain of the CRISPR-associated guide (g)RNA as well as the (i) Protospacer adjacent motif (PAM), which is the site at which the DNase function of Cas9 introduces the ds-DNA break (DSDB); and (ii) The Transactivating Crispr-RNA (TrCr-RNA), which is covalently attached the gRNA as is what brings the Cas9 endonuclease to the genomic DNA target for digestion.
The introduction of a DSDB in the presence of a homologous repair vehicle, results in the in-frame integration, immediately 3' to El, of one of either two partial human F8 cDNAs comprising either (i) exons 2-25 and the protein coding sequence, or CDS, of exon 26 (designated hF8[E2-E25/E26cDs]), which effects repair of the F8 gene such that it now encodes a full-length wild-type FVIII protein; or (ii) Exons 2-13 entirely linked next to the very 5'-most end of exon-14 (E14), which in turn is linked covalently to the very 3'-most end of E14 (i.e., a B-domain-deleted [BDD]-F8 cDNA), which is then covalently linked to Exons 15-25 entirely, and then the protein coding sequence, or CDS, of exon 26 (designated hF8[E2-E13/E14-BDD/E15-E25/E26cDs]), which effects repair of the F8 gene such that it now encodes a BDD-engineered FVIII protein, which is fully functional in FVIII:C activity.
The homologous repair vehicle is selected to have a F8 cDNA with the appropriate alleles at all ns-SNP sites so that the patient can receive a "matched" gene repair or at least a least mismatched repair.

[0137] The left homology arm of the homologous repair vehicle for Homologous Repair Vehicle No. 1 (HRV1) for hF8-CRISP/Cas9wt-1 is listed as Seq. ID. No. 17 and comprises the first 1114 bases of the human F8 genomic DNA (which is shown here as single-stranded and representing the sense strand) and contains 800 bp of the immediately 5'-promoter region of the human F8 gene and all 314 bp of the F8 exon-1 (El), including its 171 bp 5'-UTR and its 143 bp of protein (en)coding sequence (CDS). The actual left homologous arm (LHA) of the homologous repair vehicle (HRV1), which is used for this CRISPR/Cas9-mediated F8 gene repair (that occurs at the El/intron-1 [Ii] junction of a given patient's endogenous mutant F8), contains at least 500 bp of this genomic DNA sequence (i.e., from it's very 3'-end, which corresponds to the second base of the codon for translated residue 48 of the wild-type FVIII protein and residue 29 of the mature FVIII protein found in the circulation) and could include it all, if, for example, we find that full-length F8 gene repair can be effected efficiently in the future. In this instance, the integration matrix would then follow the LHA
of this HRV1, and be covalently attached to it, and this integration matrix contains (in-frame with each other and with the 3'-end of the patient's native exon-1, which is utilized in situ, along with his native F8 promoter, to regulate expression of the repaired F8 gene), all of F8 exons 2-25, and the protein CDS of exon-26, followed by the functional mRNA 3'-end forming signals of the human growth hormone gene (hGH-pA). The F8 cDNA from exons 2-25 and the CDS of exon-26 to be used in the homologous repair vehicle is listed as Seq. ID.
No. 18 and follows the left homology arm, and in this example represents the haplotype (H)3 encoding wild-type variant of F8, which can be used to cure, for example, patients with the In-mutation and the 1221-mutation, that arose on an H3-background haplotype.
This following protein encoding cDNA sequence contains 6,909 bp of the entire 7,053 bp of F8 protein encoding sequence (i.e., the first 144 bp of protein CDS from FVIII, from its initiator methionine, is not shown, as this is contained in exon-1, which is provided by the patient's own endogenous exon-1, providing it is not mutant and thus precluding the repair event).
The right homology arm of the homologous repair vehicle for the cas nuclease approach is listed as Seq. ID. No. 19 and includes 1109 bases of human F8 genomic DNA
(which is shown here as single-stranded and representing the sense strand) from the F8 gene intron 1.

[0138] In some embodiments, the DNA-SE is a CRISPR Paired Nickase. A single CRISPR
nuclease targets a total of 22 bp of DNA sequence, which is much less than what is targeted by dimeric TALENs (30-40 bp) or ZFNs (30-36 bp); as a result, some CRISPR
nucleases can have substantial off-target activity throughout the rest of the genome. The Cas9 protein has two nuclease domains (an HNH domain and a RuvC domain) which each cleave one of the strands of the DNA helix in order to cause a double-strand break. By inactivating one of the nuclease domains in Cas9 (through the amino acid mutation D1 OA or H840A), the Cas9 molecule becomes a `nickase' which can only cause a break in one strand of DNA
thereby creating a nick rather than a double-strand break. However, by targeting to Cas9-nickase molecules to nearby regions of DNA, offset nicks can in effect cause a double-strand break with DNA overhangs similar to how the two FokI dimers in ZFNs and TALENs come together to create a double-strand DNA break with overhanging bases.
Guidelines for how to orient the paired target sites for Cas9-nickases were developed by Ran FA, Hsu PD et al. Cell 2013, incorporated herein by reference, and it was shown that similar on-target activity was able to be achieved by correctly oriented paired Cas9-nickases as by a single Cas9-nuclease. Importantly, it was also shown that at sites previously identified as having off-target activity when using a certain guide strand with the Cas9 nuclease that when using the Cas9-nickase the off-target activity was reduced ¨1400 fold. The hypothesis for the reduction in off-target activity is that although at the previously identified off-target site there was homology to one of the guide strands (which allowed off-target activity using the Cas9-nuclease), in that region of the genome there was not also homology to the other guide strand in the pair; binding of a single Cas9-nickase does not induce DNA mutations, it is only when both guide strands bind in proper orientation that nicks are made in both DNA
strands to create a double strand break which can lead to mutations through the NHEJ
pathway. By creating the requirement that both guide strands bring the two nickases to the same region of the genome, the effective targeting length of the paired Cas9-nickase system is 44 bp, compared to 22 bp of the Cas9-nuclease system, greatly enhancing specificity in large genomes such as the human genome.

[0139] Example of repair at the exon21/intron-21 junction (the 3'-end of exon-21), using paired nickase are described below. Repair of the F8 at exon-21/intron-21 junction, i.e. the 3'-end of exon-21 would correct HA in patients with mutations in exons 22, 23, 24, 25, or 26, as well as the common 1221 mutation. Examples of known patient mutations in exons 22-26 are detailed in Figure 14, including, but not limited to (i) the F8 c.6761 T>A
nonsense mutation that results in a stop codon at codon 2178 in place of the leucine (Leu)-encoding codon that is present at codon 2178 in the non-mutated form of the F8; (ii) the F8 c.6917 T>G missense mutation that results in a codon encoding arginine (Arg) at codon 2230 in place of the leucine (Leu)-encoding codon that is present at that codon 2230 in the non-mutated form of the F8; (iii) the F8-122I mutation that is detailed above;
(iv) the F8 IVS-23 +1 G>A splice site mutation that results in a non-functional pre-mRNA splice site immediately downstream of exon-23 of the F8; (v) the F8 del exons 24-26 multi-exonic deletion mutation that results in deletion of exons 24-26 of the F8; and (vi) the F8 exon-26 del.[A] small deletion and frameshift mutation that results in a frameshift of the gene-encoding sequence which changes the downstream sequence by a single base-pair deletion frameshift and introduction of a novel terminating stop codon in the gene-encoding sequence.
Creating the double-strand break at exon-21/intron-21 junction can be accomplished by using DNA-SE including such as TALENs, Cas9-nuclease, paired Cas9-nickases, or RNA-guided Fold Nucleases disclosed herein. An example of how to create such a break in F8 with paired Cas9-nickases is illustrated in Figure 15. Specifically, Cas9-nickases are shown binding near the exon-21/intron-21 junction of F8. The Cas9-nickases create nicks on both strands of F8 DNA, thereby generating a double-strand break that will trigger homology directed repair;
the site of the break is indicated as the "target site." An engineered homologous repair vehicle (HRV) disclosed herein is then introduced to the cells along with the DNA-SE in order to be used as a template in the homology directed repair pathway. An example of a RV
to be used at the exon-21/intron-21 junction is shown here Figure 16.
Regardless of the mechanism used to create the DNA-break at the exon-21/intron-21 junction the same RV can be used to alter the gene sequence. This RV has a LHA corresponding to the sequence 5' of the DNA break labeled as "target break" (exon-21 and a portion of intron-20), the cDNA
sequence encoding the downstream exons of the F8 (exons 22-26), a polyadenylation signal (such as the signal from the hGH gene labeled as "target break," hGH-pA), and aRHA
corresponding to the sequence 3' of the DNA break (intron-21). After homology directed repair takes place, the gDNA sequence now contains a healthy copy of exons 22-26 fused to exon-21, allowing expression of the full-length F8. The RV can also contain SNPs in order to haplotypically match a certain patient; an example SNP (6940 A>G) is shown here.

[0140] In some embodiments the DNA-SE comprises CRISPR-RNA-guided Fokl nucleases (CRISPR-RFN). Although the paired Cas9-nickases dramatically increased the specificity of CRISPR systems, low levels of off-target activity were still observed at some sites (Ran FA
and Hsu PD et al. Cell 2013), presumably due to the occasional repair of DNA
nicks through the error-prone NHEJ pathway rather than the error free base-excision-repair pathway. In contrast to a Cas9-nickase, which will cut one strand of DNA even in the absence of its corresponding pair, the FokI nuclease requires dimerization in order to cleave DNA; the presence of a single Fold monomer will not make any modification to the DNA.
The Cas9 molecule can have all of its DNA cleavage activity removed by mutating both DNA cleavage domains (using the amino acid substitutions D 1 OA and H840A) which is known as "dead"
Cas9 or dCas9. When the FokI domain is fused to dCas9, two properly oriented guide strands can bring the two Fold domains in close proximity where they can dimerize and create a double-strand break, in a similar manner to ZFNs and TALENs. Tsai SQ
et al (Nature Biotech 2014), incorporated herein by reference, determined that with correct orientation of guide strands and fusing Fold to the N-terminus of dCas9, double-strand breaks can be made efficiently by these RNA-guided Fold Nucleases, termed "RFNs".
Tsai et al further characterized the off-target activity of these RFNs and found that they had even lower levels of off-target activity than the paired Cas9-nickases targeted to the same locations; in almost all cases the off-target activity of the RFNs was below the detection limit of the deep-sequencing-based assay employed. A further method in which RFNs reduce off-target activity is that they are more limited in what orientations they can efficiently cleave DNA
compared to paired Cas9-nickases. This reduces the possibility for off-target sites, but also limits the types of sequences which can be targeted by RFNs; several 3' ends of the exons in the F8 gene did not contain the required sequence motifs to be able to be effectively targeted by RFNs. Overall, RFNs have benefits and drawbacks compared to the paired Cas9-nickases, but nonetheless represent another addition to the toolkit of nucleases available to create double-strand breaks in order to trigger homology-directed repair.

[0141] In methods and systems and related cDNA, vehicles and composition herein descried the gene targeting and repair approaches using the different nucleases of the disclosure can be carried out using many different target cells. For example, the transduced cells can include endothelial cells, hepatocytes, or stem cells. In one embodiment, the cells can be targeted in vivo. In one embodiment, the cells can be targeted using ex vivo approaches and reintroduced into the subject.

[0142] In one embodiment, the target cells from the subject are endothelial cells. In one embodiment, the endothelial cells are blood outgrowth endothelial cells (BOECs).
Characteristics that render BOECs attractive for gene repair and delivery include the: (i) ability to be expanded from progenitor cells isolated from blood, (ii) mature endothelial cell, stable, phenotype and normal senescence (-65 divisions), (iii) prolific expansion from a single blood sample to 1019 BOECs, (iv) resilience, which unlike other endothelial cells, permits cryopreservation and hence multiple doses for a single patient prepared from a single isolation. Methods of isolation of BOECs are known, where the culture of peripheral blood provides a rich supply of autologous, highly proliferative endothelial cells, also referred to as blood outgrowth endothelial cells (BOECs). Bodempudi V, et al., Blood outgrowth endothelial cell-based systemic delivery of antiangiogenic gene therapy for solid tumors.
Cancer Gene Ther. 2010 Dec;17(12):855-63.

[0143] Studies in animal models have revealed properties of blood outgrowth endothelial cells that indicate that they are suitable for use in ex vivo gene repair strategies. For example, a key finding concerning the behavior of canine blood outgrowth endothelial cells (cBOECs) is that cBOECs persist and expand within the canine liver after infusion.
Milbauer LC, et al.
Blood outgrowth endothelial cell migration and trapping in vivo: a window into gene therapy.
2009 Apr;153(4):179-89. Whole blood clotting time (WBCT) in the HA model was also improved after administration of engineered cBOECs. WBCT dropped from a pretreatment value of under 60 mm to below 40 min and sometimes below 30 min. Milbauer LC, et al., Blood outgrowth endothelial cell migration and trapping in vivo: a window into gene therapy.
2009 Apr;153(4):179-89.

[0144] In one embodiment, the target cells from the subject are hepatocytes.
In one embodiment, the cell is a liver sinusoidal endothelial cell (LSECs). Liver sinusoidal endothelial cells (LSEC) are specialized endothelial cells that play important roles in liver physiology and disease. Hepatocytes and liver sinusoidal endothelial cells (LSECs) are thought to contribute a substantial component of FVIII in circulation, with a variety of extra-hepatic endothelial cells supplementing the supply of FVIII.

[0145] In one embodiment, the present disclosure targets LSEC cells, as LSEC
cells likely represent the main cell source of FVIII. Shahani, T, et al., Activation of human endothelial cells from specific vascular beds induces the release of a FVIII storage pool.
Blood 2010;
115(23):4902-4909. In addition, LSECs are believed to play a role in induction of immune tolerance. Onoe, T, et al., Liver sinusoidal endothelial cells tolerize T
cells across MHC
barriers in mice. J Immunol 2005; 175(1):139-146. Methods of isolation of LSECs are known in the art. Karrar, A, et al., Human liver sinusoidal endothelial cells induce apoptosis in activated T cells: a role in tolerance induction. Gut. 2007 February;
56(2): 243-252.

[0146] In one embodiment, the transduced cells from the subject are stem cells. In one embodiment, the stem cells are induced pluripotent stem cells (iPSCs). Induced pluripotent stem cells (iPSCs) are a type of pluripotent stem cell artificially derived from a non-pluripotent cell, typically an adult somatic cell, by inducing expression of specific genes and factors important for maintaining the defining properties of embryonic stem cells. Induced pluripotent stem cells (iPSCs) have been shown in several examples to be capable of site specific gene targeting by nucleases. Ru, R. et al. Targeted genome engineering in human induced pluripotent stem cells by penetrating TALENs. Cell Regeneration. 2013, 2:5; Sun N, Zhao H. Seamless correction of the sickle cell disease mutation of the HBB
gene in human induced pluripotent stem cells using TALENs. Biotechnol Bioeng. 2013 Aug 8.
Induced pluripotent stem cells (iPSCs) can be isolated using methods known in the art.
Lorenzo, IM. Generation of Mouse and Human Induced Pluripotent Stem Cells (iPSC) from Primary Somatic Cells. Stem Cell Rev. 2013 Aug;9(4):435-50.

[0147] As discussed above, a number of different cells types can be targeted for repair.
However, in some cases, pure populations of some cell types may not promote sufficient homing and implantation upon reintroduction to provide extended and sufficient expression of the corrected F8 gene. Therefore, some cell types may be co-cultured with different cell types to help promote cell properties (i.e. ability of cells to engraft in the liver).

[0148] In one embodiment, the transduced cells are from blood outgrowth endothelial cells (BOECs) that have been co-cultured with additional cell types. In one embodiment, the transduced cells are from blood outgrowth endothelial cells (BOECs) that have been co-cultured with hepatocytes or liver sinusoidal endothelial cell (LESCs) or both. In one embodiment, the transduced cells are from blood outgrowth endothelial cells (BOECs) that have been co-cultured with induced pluripotent stem cells (iPSCs).

[0149] In embodiments of methods and systems herein described and related vehicles composition methods and systems, the polynucleotide encoding for the DNA-SE
and repair vehicles RVs comprising the DNA donor can be delivered to the cells with methods of nucleic acid delivery well known in the art. (See, e.g., WO 2012051343). In the methods provided herein, the described nuclease encoding nucleic acids can be introduced into the cell as DNA or RNA, single-stranded or double-stranded and can be introduced into a cell in linear or circular form. In one embodiment, the nucleic acids encoding the nuclease are introduced into the cell as mRNA. The donor sequence can introduced into the cell as DNA
single-stranded or double-stranded and can be introduced into a cell in linear or circular form.
If introduced in linear form, the ends of the nucleic acids can be protected (e.g., from exonucleolytic degradation) by methods known to those of skill in the art. For example, one or more dideoxynucleotide residues are added to the 3' terminus of a linear molecule and/or self-complementary oligonucleotides are ligated to one or both ends. See, for example, Chang et al. (1987) Proc. Natl. Acad. Sci. USA 84:4959- 4963; Nehls et al.
(1996) Science 272:886-889.
Additional methods for protecting exogenous polynucleotides from degradation include, but are not limited to, addition of terminal amino group(s) and the use of modified intemucleotide linkages such as, for example, phosphorothioates, phosphoramidates, and 0-methyl ribose or deoxyribose residues.

[0150] The nucleic acids can be introduced into a cell as part of a vector molecule having additional sequences such as, for example, replication origins, promoters and genes encoding antibiotic resistance. Moreover, the nucleic acids can be introduced as naked nucleic acid, as nucleic acid complexed with an agent such as a liposome or poloxamer, or can be delivered by viruses (e.g., adenovirus, AAV, herpesvirus, retrovirus, lentivirus).

[0151] The nucleic acids can be delivered in vivo or ex vivo by any suitable means. Methods of delivering nucleic acids are described, for example, in U.S. Patent Nos.
6,453,242;
6,503,717; 6,534,261; 6,599,692; 6,607,882; 6,689,558; 6,824,978; 6,933,113;
6,979,539;
7,013,219; and 7,163,824.

[0152] Any vector systems can be used including, but not limited to, plasmid vectors, retroviral vectors, lentiviral vectors, adenovirus vectors, poxvirus vectors;
herpesvirus vectors and adeno-associated virus vectors, etc. See, also, U.S. Patent Nos.
6,534,261; 6,607,882;
6,824,978; 6,933,113; 6,979,539; 7,013,219; and 7,163,824. Furthermore, any of these vectors can comprise one or more of the sequences needed for treatment. Thus, when one or more nucleic acids are introduced into the cell, the nucleases and/or donor sequence nucleic acids can be carried on the same vector or on different vectors. When multiple vectors are used, each vector can comprise a sequence encoding a nuclease, a nickase, or a donor sequence nucleic acid. Alternatively, two or more of the nucleic acids can be contained on a single vector.

[0153] Conventional viral and non- viral based gene transfer methods can be used to introduce nucleic acids encoding the nucleic acids in cells (e.g., mammalian cells) and target tissues. Non- viral vector delivery systems include DNA plasmids, naked nucleic acid, and nucleic acid complexed with a delivery vehicle such as a liposome or poloxamer. Viral vector delivery systems include DNA and RNA viruses, which have either episomal or integrated genomes after delivery to the cell. Methods of non- viral delivery of nucleic acids include electroporation, lipofection, microinjection, biolistics, virosomes, liposomes, immunoliposomes, polycation or lipid:nucleic acid conjugates, naked DNA, artificial virions, and agent- enhanced uptake of DNA. Sonoporation using, e.g., the Sonitron 2000 system (Rich- Mar) can also be used for delivery of nucleic acids.

[0154] Additional exemplary nucleic acid delivery systems include those provided by Amaxa Biosystems (Cologne, Germany), Maxcyte, Inc. (Rockville, Maryland), BTX
Molecular Delivery Systems (Holliston, MA) and Copernicus Therapeutics Inc, (see for example U56008336). Lipofection is described in e.g., U.S. Patent Nos. 5,049,386;
4,946,787; and 4,897,355) and lipofection reagents are sold commercially {e.g., TransfectamTM
and LipofectinTm). Cationic and neutral lipids that are suitable for efficient receptor-recognition lipofection of polynucleotides include those of Feigner, WO 91/17424, WO
91/16024.

[0155] The preparation of lipid:nucleic acid complexes, including targeted liposomes such as immunolipid complexes, is well known to one of skill in the art (see, e.g., Crystal, Science 270:404-410 (1995); Blaese et al, Cancer Gene Ther. 2:291-297 (1995); Behr et al, Bioconjugate Chem. 5:382-389 (1994); Remy et al, Bioconjugate Chem. 5:647-654 (1994);
Gao et al, Gene Therapy 2:710-722 (1995); Ahmad et al, Cancer Res. 52:4817-4820 (1992);
U.S. Pat. Nos. 4,186,183, 4,217,344, 4,235,871, 4,261,975, 4,485,054, 4,501,728, 4,774,085, 4,837,028, and 4,946,787).

[0156] Additional methods of delivery include the use of packaging the nucleic acids to be delivered into EnGeneIC delivery vehicles (EDVs). These EDVs are specifically delivered to target tissues using bispecific antibodies where one arm of the antibody has specificity for the target tissue and the other has specificity for the EDV. The antibody brings the EDVs to the target cell surface and then the EDV is brought into the cell by endocytosis.
Once in the cell, the contents are released (see MacDiarmid et al (2009) Nature Biotechnology 27(7):643).

[0157] The use of RNA or DNA viral based systems for the delivery of nucleic acids take advantage of highly evolved processes for targeting a virus to specific cells in the body and trafficking the viral payload to the nucleus. Viral vectors can be administered directly to patients (in vivo) or they can be used to treat cells in vitro and the modified cells are administered to patients (ex vivo). Conventional viral based systems for the delivery of nucleic acids include, but are not limited to, retroviral, lentivirus, adenoviral, adeno-associated, vaccinia and herpes simplex virus vectors for gene transfer.

[0158] The tropism of a retrovirus can be altered by incorporating foreign envelope proteins, expanding the potential target population of target cells. Lentiviral vectors are retroviral vectors that are able to transduce or infect non-dividing cells and typically produce high viral titers. Selection of a retroviral gene transfer system depends on the target tissue. Retroviral vectors are comprised of cz's-acting long terminal repeats with packaging capacity for up to 6-10 kb of foreign sequence. The minimum cz's-acting LTRs are sufficient for replication and packaging of the vectors, which are then used to integrate the therapeutic gene into the target cell to provide permanent transgene expression. Widely used retroviral vectors include those based upon murine leukemia virus (MuLV), gibbon ape leukemia virus (GaLV), Simian Immunodeficiency virus (SIV), human immunodeficiency virus (HIV), and combinations thereof (see, e.g., Buchscher et al, J. Virol. 66:2731-2739 (1992); Johann et al, J. Virol.
66:1635-1640 (1992); Sommerfelt et al., Virol. 176:58-59 (1990); Wilson et al, J. Virol.
63:2374-2378 (1989); Miller et al, J. Virol. 65:2220- 2224 (1991); PCT
U594/05700).

[0159] In applications in which transient expression is preferred, adenoviral based systems can be used. Adenoviral based vectors are capable of very high transduction efficiency in many cell types and do not require cell division. With such vectors, high titer and high levels of expression have been obtained. This vector can be produced in large quantities in a relatively simple system. Adeno-associated virus ("AAV") vectors are also used to transduce cells with target nucleic acids, e.g., in the in vitro production of nucleic acids and peptides, and for in vivo and ex vivo gene therapy procedures (see, e.g., West et al, Virology 160:38-47 (1987); U.S. Patent No. 4,797,368; WO 93/24641; Kotin, Human Gene Therapy 5:793-801 (1994); Muzyczka, J. Clin. Invest. 94:1351(1994). Construction of recombinant AAV
vectors is described in a number of publications, including U.S. Pat. No.
5,173,414; Tratschin et al, Mol Cell. Biol. 5:3251-3260 (1985); Tratschin, et al, Mol. Cell. Biol.
4:2072-2081 (1984); Hermonat & Muzyczka, PNAS 81:6466-6470 (1984); and Samulski et al, J.
Virol.

63:03822-3828 (1989).

[0160] At least six viral vector approaches are currently available for gene transfer in clinical trials, which utilize approaches that involve complementation of defective vectors by genes inserted into helper cell lines to generate the transducing agent. pLASN and MFG-S are examples of retroviral vectors that have been used in clinical trials (Dunbar et al, Blood 85:3048-305 (1995); Kohn et al, Nat. Med. 1 :1017-102 (1995); Malech et al, PNAS 94:22 12133-12138 (1997)). PA317/pLASN was the first therapeutic vector used in a gene therapy trial. (Blaese et al, Science 270:475-480 (1995)). Transduction efficiencies of 50% or greater have been observed for MFG-S packaged vectors. (Ellem et al, Immunol Immunother.
44(1):10-20 (1997); Dranoff et al, Hum. Gene Ther. 1:111-2 (1997). Recombinant adeno-associated virus vectors (rAAV) are an alternative gene delivery systems based on the defective and nonpathogenic parvovirus adeno-associated type 2 virus. All vectors are derived from a plasmid that retains only the AAV 145 bp inverted terminal repeats flanking the transgene expression cassette. Efficient gene transfer and stable transgene delivery due to integration into the genomes of the transduced cell are key features for this vector system.
(Wagner et al, Lancet 351 :9117 1702-3 (1998), Kearns et al, Gene Ther. 9:748-55 (1996)).
Other AAV serotypes, including AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9 and AAVrh.10 and any novel AAV serotype can also be used in accordance with the present disclosure. In a particular embodiment, the vector is based on a hepatotropic adeno-associated virus vector, serotype 8 (see, e.g., Nathwani et al., Adeno-associated viral vector mediated gene transfer for hemophilia B, Blood 118(21):4-5, 2011).

[0161] Replication-deficient recombinant adenoviral vectors (Ad) can be produced at high titer and readily infect a number of different cell types. Most adenovirus vectors are engineered such that a transgene replaces the Ad El a, El b, and/or E3 genes;
subsequently the replication defective vector is propagated in human 293 cells that supply deleted gene function in trans. Ad vectors can transduce multiple types of tissues in vivo, including non-dividing, differentiated cells such as those found in liver, kidney and muscle. Conventional Ad vectors have a large carrying capacity. An example of the use of an Ad vector in a clinical trial involved polynucleotide therapy for antitumor immunization with intramuscular injection (Sterman et al, Hum. Gene Ther. 7:1083-9 (1998)). Additional examples of the use of adenovirus vectors for gene transfer in clinical trials include Rosenecker et ah, Infection 24:1 5-10 (1996); Sterman et ah, Hum. Gene Ther. 9:7 1083-1089 (1998); Welsh et ah, Hum.

Gene Ther. 2:205-18 (1995); Alvarez et al, Hum. Gene Ther. 5:597-613 (1997);
Topf et al, Gene Ther. 5:507-513 (1998); Sterman et al, Hum. Gene Ther. 7:1083-1089 (1998).

[0162] Packaging cells are used to form virus particles that are capable of infecting a host cell. Such cells include 293 cells, which package adenovirus, and tv2 cells or PA317 cells, which package retrovirus. Viral vectors used in gene therapy are usually generated by a producer cell line that packages a nucleic acid vector into a viral particle.
The vectors typically contain the minimal viral sequences required for packaging and subsequent integration into a host (if applicable), other viral sequences being replaced by an expression cassette encoding the protein to be expressed. The missing viral functions are supplied in trans by the packaging cell line. For example, AAV vectors used in gene therapy typically only possess inverted terminal repeat (ITR) sequences from the AAV genome which are required for packaging and integration into the host genome. Viral DNA is packaged in a cell line, which contains a helper plasmid encoding the other AAV genes, namely rep and cap, but lacking ITR sequences. The cell line is also infected with adenovirus as a helper. The helper virus promotes replication of the AAV vector and expression of AAV genes from the helper plasmid. The helper plasmid is not packaged in significant amounts due to a lack of ITR
sequences. Contamination with adenovirus can be reduced by, e.g., heat treatment to which adenovirus is more sensitive than AAV.

[0163] In many applications, it is desirable that the g vector be delivered with a high degree of specificity to a particular tissue type. Accordingly, a viral vector can be modified to have specificity for a given cell type by expressing a ligand as a fusion protein with a viral coat protein on the outer surface of the virus. The ligand is chosen to have affinity for a receptor known to be present on the cell type of interest. For example, Han et ah, Proc. Natl. Acad.
Sci. USA 92:9747- 9751 (1995), reported that Moloney murine leukemia virus can be modified to express human heregulin fused to gp70, and the recombinant virus infects certain human breast cancer cells expressing human epidermal growth factor receptor.
This can be used with other virus-target cell pairs, in which the target cell expresses a receptor and the virus expresses a fusion protein comprising a ligand for the cell- surface receptor. For example, filamentous phage can be engineered to display antibody fragments (e.g., FAB or Fv) having specific binding affinity for virtually any chosen cellular receptor. Although the above description applies primarily to viral vectors, the same principles can be applied to non-viral vectors. Such vectors can be engineered to contain specific uptake sequences which favor uptake by specific target cells.

[0164] Vectors can be delivered in vivo by administration to an individual patient, typically by systemic administration (e.g., intravenous, intraperitoneal, intramuscular, subdermal, or intracranial infusion) or topical application, as described below.
Alternatively, vectors can be delivered to cells ex vivo, such as cells explanted from an individual patient (e.g., lymphocytes, bone marrow aspirates, tissue biopsy) or universal donor hematopoietic stem cells, followed by re-implantation of the cells into a patient, usually after selection for cells which have incorporated the vector.

[0165] Vectors (e.g., retroviruses, adenoviruses, liposomes, etc.) containing the nucleic acids described herein can also be administered directly to an organism for transduction of cells in vivo. Alternatively, naked DNA can be administered.

[0166] Administration is by any of the routes normally used for introducing a molecule into ultimate contact with blood or tissue cells including, but not limited to, injection, infusion, topical application and electroporation. Suitable methods of administering such nucleic acids are available and well known to those of skill in the art, and, although more than one route can be used to administer a particular composition, a particular route can often provide a more immediate and more effective reaction than another route.

[0167] Vectors suitable for introduction of the nucleic acids described herein include non-integrating lentivirus vectors (IDLV). See, for example, Ory et al. (1996) Proc. Natl. Acad.
Sci. USA 93:11382-11388; Dull et al. (1998) J. Virol. 72:8463-8471; Zuffery et al. (1998) J.
Virol. 72:9873-9880; Follenzi et al. (2000) Nature Genetics 25:217-222; U.S.
Patent Publication No 2009/054985.

[0168] The nucleic acids encoding the monomers of the DNA scission enzymes can be expressed either on separate expression constructs or vectors, or can be linked in one open reading frame. Expression of the nuclease can be under the control of a constitutive promoter or an inducible promoter.

[0169] Administration can be by any means in which the polynucleotides are delivered to the desired target cells. For example, both in vivo and ex vivo methods are contemplated. In one embodiment, the nucleic acids are introduced into a subject's cells that have been explanted from the subject, and reintroduced following F8 gene repair.

[0170] For in vivo administration, for example, intravenous injection of the nucleic acids to the portal vein is a method of administration. Other in vivo administration modes include, for example, direct injection into the lobes of the liver or the biliary duct and intravenous injection distal to the liver, including through the hepatic artery, direct injection into the liver parenchyma, injection via the hepatic artery, and/or retrograde injection through the biliary tree. Ex vivo modes of administration include transduction in vitro of resected hepatocytes or other cells of the liver, followed by infusion of the transduced, resected hepatocytes back into the portal yasculature, liver parenchyma or biliary tree of the human patient, see e.g., Grossman et ah, (1994) Nature Genetics, 6:335-341.

[0171] If ex vivo methods are employed, cells or tissues can be removed and maintained outside the body according to standard protocols well known in the art. The compositions can be introduced into the cells via any gene transfer mechanism as described above, such as, for example, calcium phosphate mediated gene delivery, electroporation, microinjection, proteoliposomes, or viral vector delivery. The transduced cells can then be infused (e.g., in a pharmaceutically acceptable carrier) or homotopically transplanted back into the subject per standard methods for the cell or tissue type. Standard methods are known for transplantation or infusion of various cells into a subject.

[0172] In some embodimentsõ the one or more mutations cause hemophilia in the subject and the repair results in treatment of the hemophilia in the subject. The term "treatment" as used herein indicates any activity that is part of a medical care for, or deals with, a condition, medically or surgically.

[0173] The term "subject" as used herein is meant an individual and refers to a single biological organism such animals and in particular higher animals and in particular vertebrates such as mammals and in particular human beings. . Thus, the "subject" can include domesticated animals, such as cats, dogs, etc., livestock (e.g., cattle, horses, pigs, sheep, goats, etc.), laboratory animals (e.g., mouse, rabbit, rat, guinea pig, etc.) and birds.
Thus, veterinary uses and medical formulations are contemplated herein. In some embodiments, the subject is a mammal such as a primate, for example, a human.

[0174] The term "haemophilia" indicates a group of hereditary genetic disorders that impair the body's ability to control blood clotting, which is used to stop bleeding when a blood vessel is broken.

[0175] Haemophilia A (HA) (clotting factor VIII deficiency) is the most common form of the disorder, present in about 1 in 5,000-10,000 male births and is caused by loss-of-function mutations in the X-linked Factor (F) VIII gene. Haemophilia B (HB) (factor IX
deficiency) occurs in around 1 in about 20,000-34,000 newborn male births.

[0176] The levels of functional FVIII in circulation determine the severity of the disease, with plasma levels 5-25% of normal being mild, 1-5% being moderate, and <1%
being severe (Brettler et al., Clinical aspects of and therapy for hemophilia A. Churchill Livingstone, New York, NY 1995; pp. 1648-63). As such, only a small amount of circulating protein is necessary to provide protection from spontaneous bleeding episodes.

[0177] The 1221-mutation of the F8 accounts for ¨45% of severe HA and is caused by an intra-chromosomal recombination within the gene. FIG. 1 shows a schematic illustration of the wild-type and 1221 F8 loci (F8 & F8122I). Indicated in FIG.1 are the exon-1B (E1B) and exon-1 to exon-22 (E1-E22) functional coding sequences as well as the exons-23C (E23C), -24C (E24C), and exon-23 (E23C), exon-24C (E24C) and exon-23 (E23) to exon-26 (E26) non-functional coding sequences. Transcription from the F8 promoter of both the F8 (wild-type) & F8122I loci, which is normally functioning in both forms, yields polyadenylated mRNAs. The F8 (wild-type) mRNA has 26 exons, exon-1 (El) to exon-22 (E22) and exon-23 (E23) to exon-26 (E26), all of which encode the amino acids found in the FVIII.
Conversely, the F8122I mRNA has at least 24 exons, El-E22 (they are the same in F8 and thus encode FVIII amino acid sequence), and E23C & E24C (they are cryptic and encode no FVIII amino acid sequence). The sequence of intron-22, in both F8 & F8122I, contains a bi-directional promoter that transcribes two additional mRNAs from the two genes:
F8A, which is oriented oppositely to that of F8 & F8122I and contains a single exon (box designated ElA), and F8B, which contains five exons that are oriented similarly transcriptionally to that of F8 & F8122I and contains a single non-F8 first exon within 122 (box designated ElB) followed by four additional exons, which are identical to E23-E26 of F8. The F8A mRNA
encodes the FVIIIA protein, which is now known as HAP40 (a cytoskeleton-interacting protein involved in endocytosis and thus functionally unrelated to the coagulation system) and has no FVIII amino acid sequence. The F8B mRNA encodes FVIII B, a protein with unknown function that has 8 non-FVIII amino acid residues at its N-terminus followed by 208 residues that represent FVIII residues 2125-2332.

[0178] Infusion of replacement plasma-derived (pd) or recombinant (r) FVIII is the standard of care to manage this chronic disease. Currently available rFVIII replacement products include the commercially available Kogenate0 (Bayer) and Hefixate (ZLB
Behring), Recombinate0 (Baxter) and Advate0 (Baxter), and the B-domain deleted Refacto0 (Pfizer) and Xyntha0 (Pfizer). Patients unable to be treated with FVIII experience more painful, joint bleeding and over time, a greater loss of mobility than patients whose HA is able to be managed with FVIII. Infusion of replacement FVIII, however, is not a cure for HA.
Spontaneous bleeding remains a serious problem especially for those with severe HA, defined as circulating levels of FVIII coagulant activity (FVIII: C) below 1%
of normal.
Furthermore, the formation of anti-FVIII antibodies occurs in about 20% of all patients and more often in certain subpopulations of HA patients, such as African Americans (Viel KR, Amen i A, Abshire TC, et al. Inhibitors of factor VIII in black patients with hemophilia. N
Engl J Med. 360: 1618-27, 2009). There is therefore also a critical need to identify ways to avoid FVIII inhibitor development and to abate a FVIII inhibitor response.

[0179] In some embodiments herein described, the methods and compositions described herein are directed to treating a subject with hemophilia and in particular hemophilia A
comprising selectively targeting and replacing a portion of the subject's genomic F8 gene sequence containing a mutation in the gene with a partial F8 cDNA replacement sequence (cDNA-RS). In one embodiment, the resultant repaired F8 gene containing the cDNA-RS, upon expression, produces functional FVIII that confers improved coagulation functionality to the encoded FVIII protein of the subject. The levels of functional FVIII in circulation are believed to obviate or reduce the need for infusions of replacement FVIII in the subject. In one embodiment, expression of functional FVIII reduces whole blood clotting time (WBCT).
In one embodiment, the repaired F8 gene, upon expression, provides for the immune tolerance induction (ITI) to an administered replacement FVIII protein product. In one embodiment, the subject is a human.

[0180] In one aspect, a method of treating hemophilia A in a subject is provided comprising introducing into a cell of the subject one or more repair vehicles (RV) containing at least a cDNA-RS and one or more plasmids encoding a DNA scission enzyme (DNA-SE) such as a nuclease or nickase. The DNA-SE targets a portion of the F8 gene containing a mutation that causes hemophilia A and creates a first break in one strand of the F8 gene and a second break in the other strand of the F8 gene for subsequent repair by the cDNA-RS. In some embodiments, the first break and the second break are a double-stranded DNA
break. In other embodiments, the first break and the second break are off-set paired and complementary single-stranded DNA nicks. The cDNA-RS comprises (i) a nucleic acid encoding a truncated FVIII polypeptide or (ii) a native F8 3' splice acceptor site operably linked to a nucleic acid encoding a truncated FVIII polypeptide. The RV
further comprises flanking sequences comprising an upstream flanking sequence (uFS) that is homologous to the nucleic acid sequences upstream of the first break in the DNA of the subject's F8 gene and a downstream flanking sequence (dFS) that is homologous to the nucleic acid sequences downstream of the second break in the DNA of the subject's F8 gene. The 5' end of the cDNA-RS is flanked by the uFS and the 3' end of the cDNA-RS is flanked by dFS
to form a donor sequence that is a portion of the RV. After insertion of the cDNA-RS
through homologous recombination into the subject's F8 gene (sF8), a repaired F8 gene (rF8) is formed, which upon expression forms functional FVIII that confers improved coagulation functionality to the FVIII protein encoded by the sF8 without the repair.

[0181] In one aspect, methods and systems for repairing F8 gene can be used to induce immune tolerance to a FVIII replacement product (FVIIIT) such as a recombinant FVIII
(rFVIII) or a plasma derived FVIII (pdFVIII) in a subject having a FVIII
deficiency and who will be administered, is being administered, or has been administered a replacement FVIII
product is disclosed. The method comprises introducing into cells of the subject one or more RVs encoding a cDNA-RS and one or more plasmids encoding a DNA-SE. The DNA-SE
targets a portion of the F8 gene containing a mutation that causes hemophilia A and creates a first break in one strand of the F8 gene and a second break in the other strand of the F8 gene for subsequent repair by the cDNA-RS. In some embodiments, the first break and the second break are a double-stranded DNA break. In other embodiments, the first break and the second break are off-set paired and complementary single-stranded DNA nicks.
The cDNA-RS comprises (i) a nucleic acid encoding a truncated FVIII polypeptide or (ii) a native F8 3' splice acceptor site operably linked to a nucleic acid encoding a truncated FVIII polypeptide.
The RV further comprises flanking sequences comprising an upstream flanking sequence (uFS) that is homologous to the nucleic acid sequences upstream of the first break in the DNA of the subject's F8 gene and a downstream flanking sequence (dFS) that is homologous to the nucleic acid sequences downstream of the second break in the DNA of the subject's F8 gene. The 5' end of the cDNA-RS is flanked by the uFS and the 3' end of the cNDA-RS is flanked by dFS to form a donor sequence that is a portion of the RV. After insertion of the cDNA-RS through homologous recombination into the subject's F8 gene (sF8), a repaired F8 gene (rF8) is formed, which upon expression forms functional FVIII that provides immune tolerance induction (ITI) to an administered replacement FVIII protein product. In some cases, the person administered the cells may have no anti-FVIII antibodies or have anti-FVIII
antibodies as detected by ELISA or Bethesda assays. In one embodiment, the truncated FVIII polypeptide amino acid sequence shares homology with a portion of the FVIIIrp's amino acid sequence. In one embodiment, the truncated FVIII polypeptide amino acid sequence shares homology with a similar portion of the FVIIIrp's amino acid sequence. In one embodiment, the truncated FVIII polypeptide amino acid sequence shares complete homology with a similar portion of the FVIIIrp's amino acid sequence.

[0182] In some embodiments, the repaired version of the Factor VIII non-functional coding sequence comprises Factor VIII exons of a replacement FVIII protein product and the repair results in inducing immune tolerance to the FVIII replacement product.

[0183] In some embodiments disclosed herein, the cDNA, polynucleotides repair vehicles plasmids and vehicles herein described are provided as a part of systems to repair F8 gene in a subject. The systems can be provided in the form of a kits of part. In a kit of parts, the cDNA, polynucleotides repair vehicles plasmids and vehicles herein described and other reagents to repair one or more mutations of the F8 gene can be comprised in the kit independently. The cDNA, polynucleotides repair vehicles plasmids and vehicles herein described can be included in one or more compositions, and each capture agent can be in a composition together with a suitable excipient.

[0184] In some embodiments, additional components of the system include reagents, antibodies and enzymes that can be used to verify proper integration and expression of the cDNA-RS. Proper integration can be assessed through a variety of means that would be apparent to one of ordinary skill in the art, including DNA sequencing by Sanger technique or by next-generation sequencing techniques of the desired genomic DNA site of cDNA-RS
integration to ensure proper integration of the donor sequence. Expression of a repaired FVIII
can be assessed through a variety of means that would be apparent to one of ordinary skill in the art including using ELISA assays to measure repaired FVIII expression both intracellularly expressed and secreted into the medium and commercially-available coagulation and FVIII assays for measuring coagulation activity.

[0185] In particular, in some embodiments components of the kit are provided, with suitable instructions and other necessary reagents, in order to perform the methods here described.
The kit will normally contain the compositions in separate containers.
Instructions, for example written or audio instructions, on paper or electronic support such as tapes or CD-ROMs, for carrying out the assay, will usually be included in the kit. The kit can also contain, depending on the particular method used, other packaged reagents and materials (e.g.
Chromogenix Coamatic Factor VIII kit, available from Diapharma (Itttp://www.diaphartna.comlaspiproductdetails.asp?ID=1 00080) can be used for measuring FVIII activity).

[0186] In some embodiments, the cDNA, polynucleotides repair vehicles plasmids and vehicles herein described herein described can be included in pharmaceutical compositions together with an excipient or diluent. In particular, in some embodiments, disclosed are pharmaceutical compositions which contain at least one cDNA, polynucleotides repair vehicles plasmids and vehicles herein described in combination with one or more compatible and pharmaceutically acceptable excipients, and in particular with pharmaceutically acceptable diluents or excipients. In those pharmaceutical compositions the multi-ligand capture agent can be administered as an active ingredient for treatment or prevention of a condition in an individual.

[0187] The term "excipient" as used herein indicates an inactive substance used as a carrier for the active ingredients of a medication. Suitable excipients for the pharmaceutical compositions herein described include any substance that enhances the ability of the body of an individual to absorb the multi-ligand capture agents or combinations thereof. Suitable excipients also include any substance that can be used to bulk up formulations with the peptides or combinations thereof, to allow for convenient and accurate dosage.
In addition to their use in the single-dosage quantity, excipients can be used in the manufacturing process to aid in the handling of the peptides or combinations thereof concerned.
Depending on the route of administration, and form of medication, different excipients can be used. Exemplary excipients include, but are not limited to, antiadherents, binders, coatings, disintegrants, fillers, flavors (such as sweeteners) and colors, glidants, lubricants, preservatives, sorbents.

[0188] The term "diluent" as used herein indicates a diluting agent which is issued to dilute or carry an active ingredient of a composition. Suitable diluents include any substance that can decrease the viscosity of a medicinal preparation.

[0189] Further details concerning the identification of the suitable carrier agent or auxiliary agent of the compositions, and generally manufacturing and packaging of the kit, can be identified by the person skilled in the art upon reading of the present disclosure.
EXAMPLES

[0190] The methods and system herein disclosed are further illustrated in the following examples, which are provided by way of illustration and are not intended to be limiting.

[0191] In particular, the following examples illustrate exemplary embodiments in accordance with exemplary procedures in accordance to the present disclosure. A person skilled in the art will appreciate the applicability of the features described in detail for the exemplified embodiments to different methods, different applications and different reaction conditions and reagents in accordance with the present disclosure.
Example 1: Ex vivo Gene Repair

[0192] Examples are provided of an ex vivo gene repair strategies that can be performed without the use of viral vectors. Genetic materials are delivered to restore secretion of a wild-type full-length FVIII to lymphoblastoid cells derived from a human HA patient with the F81221, using electroporation and TALENs. A similar strategy can be used as an example to repair the naturally-occurring 1221-mutation in cells from an animal model of HA (dogs of the HA canine colony located at the University of North Carolina in Chapel Hill).
Canine (adipose) tissue, which can be induced to acquire many properties of hepatocytes, can be used.

[0193] Use of autologous cells is an attractive therapy for several reasons as levels of blood clotting proteins needed to maintain hemostasis may be more readily produced by expansion of large populations of cells ex vivo and reintroduction into the patient.
Repair of the F8122I
gene residing in a B-lymphoblastoid cell-line derived from a patient with severe HA caused by the 1221-mutation is effected by using electroporation to deliver (i) two distinct mRNAs encoding a highly specific heterodimeric TALEN that targets a single human genome site located in F8 near the 5'-end of 122 and (ii) the corresponding donor plasmid that carries the "editing cassette", which is comprised of a functional 3'-intron splice site ligated immediately 5' of a partial F8 cDNA matched in sequence with the wild-type sequence of exons 23-26 in the patient's own F8122I locus, flanked by "left" and "right"
homology arms.

[0194] The use of viral-free methods to derive autologous cells of various phenotypes and to stably introduce genetic information into the genome is attractive. These methods can be effectively used to successfully "repair" the F8122I, which arises through a highly-recurrent mutational event essentially restricted to the male germ-line. This same F8 abnormality, which is widely known as the 1221-mutation, occurs naturally in dogs, and results in spontaneous bleeding. Two large colonies of HA dogs have been established, one at the University of North Carolina in Chapel Hill. Investigation of F8122I at the molecular genetic, biochemical, and cellular levels to characterize its expression products have been studied in order to determine the immune response to replacement FVIII. Extensive sequencing efforts and analyses of the F8122I and its mRNA transcripts allow for an innovative gene repair strategy that exploits nuclease technology, for example, transcription activator-like effector TALEN technology to repair the 1221-mutation.

[0195] Lymphoblastoid cells derived from HA patient with the 1221-mutation is obtained.
The left (TALEN-L) and right (TALEN-R) monomers comprising the heterodimeric TALEN
is shown in Figure 3, which was specifically designed to cleave within the human F8 I22-sequence, ¨1 kb downstream of the 3'-end of exon-22. In alternative embodiments, the TALENs target sequences throughout the FVIII gene, with replacement of the corresponding FV8 gene sequence on the donor sequence.

[0196] An example of a sequence that can be targeted includes a sequence within intron 22 (tactatgggatgagttgcagatggcaagtaagacactggggagattaaat (SEQ. ID No.
1)), where the underlined regions of sequence are recognized by the left TAL Effector DNA-binding domain and the right TAL Effector DNA-binding domain). Another example of a sequence that can be targeted includes a sequence at the junction of exon 22 with intron 22 (Iggaaccttaatggtatgtaattagtcatttaaagggaatgcctgaata (SEQ. ID No. 2)), where the underlined regions of sequence are recognized by the left TAL Effector DNA-binding domain and the right TAL Effector DNA-binding domain). Another example of a sequence that can be targeted within intron 22 is depicted in Figure 3 (ttagtattatagtttctcagattatcaccagtgatactatggga (SEQ. ID No. 3)), where the underlined regions of sequence are recognized by the left TAL

Effector DNA-binding domain and the right TAL Effector DNA-binding domain).
The two TALEN expression plasmids that target these sequences (or the mRNA) are co-transfected with the donor plasmid. The donor plasmid contains flanking homology regions to the intron 22 locus, which allows for recombination of the donor plasmid into the chromosome. The cDNA of exons 23 to 26 of the F8 gene is contained between the flanking homology regions of the donor plasmid. The donor plasmid can also contain a suicide gene (such as the thymidine kinase gene from the herpes simplex virus), which allows counter-selection to avoid random and multi-copy integration into the genome.

[0197] Electroporation (AMAXA Nucleofection system) and chemical transfection (with a commercial reagent optimized to this cell type) can be used as transfection methods for the lymphoblastoid cells. A plasmid containing the green fluorescent protein (GFP) gene is introduced into the cells using both methods. The cells are analyzed by fluorescent microscopy to obtain an estimate of transfection efficiency, and the cells are observed by ordinary light microscopy to determine the health of the transfected cells.
Any transfection method that gives a desirable balance of high transfection efficiency and preservation of cell health in the lymphoblastoid cells can be used. The TALEN mRNAs and the gene repair donor plasmid is then introduced into the lymphoblastoid cells using a transfection method.
The TALENs for the human lymphoblastoid cells and their target site are shown in Figure 3.

[0198] Repair of the F81221 in the adipose tissue-derived hepatocyte-like cells from the 1221 HA canine animal model is effected using electroporation to deliver mRNAs encoding an analogous TALEN that targets the 5'-end of 122 in canine F8 and an analogous donor plasmid carrying a "splice-able" cDNA spanning canine F8 exons 23-26.

[0199] Adipose tissue is collected from these FVIII deficient dogs by standard liposuction.
Stromal cells from the adipose tissue are reprogrammed into induced pluripotent stem cells (iPSC), as described by Sun et al. ("Feeder-free derivation of induced pluripotent stem cells from adult human adipose stem cells" Proc Natl Acad Sci USA. 106: 720-5, 2009) with two modifications: (i) mRNA of the reprogramming factors are used in place of lentiviral vectors and (ii) the reprogramming is performed under conditions of hypoxia, 5% 02, and in the presence of small molecules that have been found to increase the reprogramming efficiency.
Once produced and characterized, pluripotent canine cells are obtained.

[0200] The defective FVIII sequence in iPSC is replaced by the correct sequence using site-specific TALE nucleases (see Figure 4). The iPSC with repaired Factor VIII are differentiated into hepatocytes using well established protocols (see, for example, Hay et al.
"Direct differentiation of human embryonic stem cells to hepatocyte-like cells exhibiting functional activities" Cloning Stem Cells. 9: 51-62, 2007; Si-Tayeb et al.
"Highly efficient generation of human hepatocyte-like cells from induced pluripotent stem cells"
Hepatology.
51: 297-305, 2010; and Cayo et al. "JD induced pluripotent stem cell-derived hepatocytes faithfully recapitulate the pathophysiology of familial hypercholesterolemia"
Hepatology.
May 31, 2012). In short, small colonies of iPSC are induced to differentiate for the first 3 days into definitive endoderm by treatment with 50 ng/mL Wnt3a and 100 ng/mL
Activin A, and then into the hepatocyte lineage by 20 ng/mL BMP4. Two expression plasmids necessary to produce mRNAs encoding a functional TALEN are obtained. These are designed to cleave and yield a double-stranded DNA break at only a single site within the canine genome, located within canine F8 122, ¨0.3 kb downstream of the 3'-end of exon-22.
The left (TALEN-L) and right (TALEN-R) monomers comprising this heterodimeric TALEN
is shown above in Figure 4.
102011 A donor plasmid containing the sequence of the 3'-end of canine F8 intron-22 and all of canine F8 exon-22 as the left homologous sequence and the 5'-end of canine F8 intron-23 as the right homologous sequence to provide an adequate length of genomic DNA
for efficient homologous recombination at the target site (i.e., the TALEN cut site) is created.
The TALEN mRNAs and the gene repair donor plasmid are introduced into the pluripotent canine cells using a transfection method described herein.
[0202] Likewise, in humans, human iPSCs are electroporated with the human F8 TALENs &
donor plasmid described above, to assess candidate genome-editing tools (which were designed to be equally capable of "editing" the I22-sequence in the wild-type and I22-inverted F8 loci, F8 and F8122I, respectively) for their efficiency of site-specific gene repair.
The genomic DNA at the repaired F8 loci, as well as the mRNAs and expression products synthesized by, the cells described above are assessed before and after electroporation.
[0203] The TALEN gene repair method described above inserts F8 exons 23-26 immediately downstream (telomeric) to F8 exons 1-22 to encode a FVIII protein. Genomic DNA, spliced mRNA, and protein sequences differ among normal, repaired, and unrepaired cells (see Figure 5). Gene repair is verified in genomic DNA through the use of PCR.
Specific PCR

primers are designed to amplify across the homologous recombination target sequence in unrepaired and repaired cells. A common primer is placed toward the end of exon-22. An 1221-specific primer is placed in the sequence telomeric to exon-22 in the 1221-inverted cells.
A Repaired-specific primer is placed in the inserted exon 23-26 sequence.
Primer design is shown in Figure 8. In FIG. 8, Exons 1-22 (top schematic) and Exons 1-22 and 23-26 (left, bottom schematic) represent functional coding sequences, while Exons 23-26 (top schematic) and Exons 23-26 (right, bottom schematic) represent non-functional coding sequences.
Separate sets of primers are designed for human and canine sequences.
[0204] Characterization of the genomic DNA at the repaired F8 loci, as well as the mRNAs and expression products synthesized by, the cells described above, before and after electroporation are performed.
[0205] A quantitative RT-PCR test that specifically detects and quantifies the mRNA
transcripts from normal and 1221 cells is used. The quantitative RT-PCR test uses three separate primer sets: one set to detect exons 1-22, one set to detect exons 23-26, and one set that spans the exon-22/exon-23 junction. mRNA is purified from cells before and after transfection. The existing primer design to probe mRNA from the human cells is used.
Primers against canine sequences are designed using the same strategy and then the mRNA
from the canine cells is probed using these new primers. An increased signal from the exon-22/exon-23 junction reaction in repaired cells, relative to unrepaired cells should be observed.
[0206] Monoclonal antibody ESH8, which is specific for the C2-domain of the FVIII protein, is be used. NIH3T3 cells were transfected with expression constructs encoding full-length and 1221 F8 genes and then assayed by flow cytometry. Signal from the ESH8 antibody was high in cells transfected with the full-length construct but virtually absent in cells transfected with the 1221 construct. The ESH8 antibody is used to test transfected cells.
There should be an increased signal in repaired cells relative to unrepaired cells. Secreted FVIII levels, as measured by ELISA, are dramatically lower in 1221 cells relative to normal cells. Whole-cell lysates and supemates from transfected cells are obtained and tested for FVIII
concentration by ELISA. There should be an increase in FVIII concentration in the supernates from repaired cells relative to unrepaired cells.
[0207] In another example, canine blood outgrowth endothelial cells (cBOECs) and canine iPSCs derived from canine adipose tissue can be transfected with TALENs that target the F81221 canine gene and a plasmid repair vehicle that carries exons 23-26 of cF8. TALENs are expected to make DSBs in the F8122I DNA at the target site to allow "homologous recombination and repair" of the canine F8 1221 gene by insertion of exons 23-26 of the canine F8. The TALENS are designed to cleave and yield a DSB at only a single site within the canine genome, located within canine F8 122, (-0.3 kb) downstream of the 3'-end of exon-22. The donor plasmid contains the sequence of canine F8 exons 23-26 flanked by the 3 '-end of canine F8 intron-22 and all of canine F8 exon-22 as the left homologous sequence and the 5'-end of canine F8 intron-23 as the right homologous sequence to provide an adequate length of genomic DNA for efficient homologous recombination at the target site.
[0208] Feasibility of deriving canine iPSCs is well established. An mRNA
transcript that enables expression of the so called "Yamanaka" genes coding for transcription factors OCT4, SOX2, KLF4 and C-MYC to induce iPSCs from canine adipose derived stem cells (hADSCs). iPSCs have been transfected using Nucleofector. For transfection, Qiagen's Polyfect transfection reagents can be used with TALENs for many cell types, including BOECs. Transfection methods can be assessed using commercial reagents and transfected cells can be analyzed by fluorescent microscopy to obtain an estimate of transfection efficiency, while viability can be determined by Trypan Blue dye exclusion.
The transfection method that gives the best balance of high transfection efficiency and preservation of cell health can be used.
[0209] Prior to commencing transfection with the TALENS and repair plasmid, the cleavage activity of the TALENs against the target site can be analyzed. This can be done by monitoring TALEN induced mutagenesis (Non-Homologous End Joining Repair) via a Endonuclease assay. To assess potential risk of unintended genomic modification induced by the selected repair method, off-site activity is analyzed following transfection. In silico identification based on homologous regions within the genome can be used to identify the top 20 alternative target sites containing up to two mismatches per target half-site. PCR primers can be synthesized for the top 20 alternative sites and Surveyor Nuclease (Cel-I) assays (Transgenomics, Inc.) can be performed for each potential off-target site.
[0210] Transfection for expression and secretion of FVIII can be assessed in the various cell types before and after transfection. Genomic DNA is isolated from cells before and after transfection. Purified genomic DNA is used as template for PCR. Primers are designed for amplification from a FVIII 1221-specific primer only in unrepaired cells, and amplification from the repaired-specific primer only in repaired cells. RT-PCR can specifically detect and quantify the mRNA hF8 transcripts from normal and 1221 cells. The quantitative RT-PCR
test uses three separate primer sets: one set to detect exons 1-22, one set to detect exons 23-26, and one set that spans the exon-22/exon-23 junction. mRNA is purified from cells before and after transfection, with an increased signal from the exon-22/exon-23 junction reaction in repaired cells, relative to unrepaired cells. Flow-cytometry based assays may also be used for FVIII protein in peripheral blood mononuclear cells (PBMCs).
[0211] iPSCs derived from canine adipose tissue engineered can be conditioned to secrete FVIII to hepatocyte-like tissue. Canine iPSCs are conditioned toward hepatocyte like cells using a three step protocol as described by Chen et al. that incorporates hepatocyte growth factor (HGF) in the endodermal induction step (Chen YF, Tseng CY, Wang HW, Kuo HC, Yang VW, Lee OK. Rapid generation of mature hepatocyte-like cells from human induced pluripotent stem cells by an efficient three-step protocol. Hepatology. 2012 Apr;55(4):1193-203).
[0212] Subpopulations of cBOECs are segregated and expanded and then characterized for the expression of endothelial markers, such as Matrix Metalloproteinases (MMPs), and cell-adhesion molecules (JAM-B, JAM-C, Claudin 3, and Claudin 5) using RT-PCR.
Detailed RT-PCR methods, including primers for detecting expression of mRNA transcripts of the cell-adhesion molecules of interest and detailed immunohistochemistry methods to detect the proteins of interest, including a list of high affinity antibodies have been published by Geraud et al. (Geraud C, et al. Unique cell type-specific junctional complexes in vascular endothelium of human and rat liver sinusoids. PLoS One. 2012;7(4):e34206).
Antibodies that detect JAM-B, JAM-C, Claudin 3, and Claudin 5 may be purchased from LifeSpan Biosciences (www.lsbio.com).
[0213] One subpopulation of co-cultured cBOECs can be prepared and segregated early (before ¨4 passages of outgrowth). Later segregation of the subpopulation can occur after ¨10 passages. After 1 week of co-culture, two cBOECs subpopulations can be compared for expression and secretion of FVIII, and suitability for engraftment in the canine liver. Co-culturing of hepatocytes can be done with several cell types including human umbilical vein endothelial cells (HUVECs). cBOECs can be used as surrogates for HUVECS in this system.

Once the repaired cBOECs (with the repaired FVIII gene) are obtained, the cells can be used to induce immune tolerance in canines with high titer-antibodies to FVIII.
Example 2: Protocol for Factor VIII Gene Repair in Humans Obtaining a blood sample [0214] A protocol for gene repair of the F8 gene in blood outgrowth endothelial cells (BOECs) is described in the following example. First, a blood sample is obtained, with 50-100mL of patient blood samples obtained by venipuncture and collection into commercially-available, medical-grade collecting devices that contain anticoagulants reagents, following standard medical guidelines for phlebotomy. Anticoagulant reagents that are used include heparin, sodium citrate, and/or ethylenediaminetetraacetic acid (EDTA).
Following blood collection, all steps proceed with standard clinical practices for aseptic technique.
Isolating appropriate cell populations from blood sample [0215] Procedures for isolating and growing blood outgrowth endothelial cells (BOECs) have been described in detail by Hebbel and colleagues (Lin, Y., Weisdorf, D. J., Solovey, A. &
Hebbel, R. P. Origins of circulating endothelial cells and endothelial outgrowth from blood. J
Clin Invest 105, 71-77 (2000)). Peripheral blood mononuclear cells (PBMCs) are purified from whole blood samples by differential centrifugation using density media-based separation reagents. Examples of such separation reagents include Histopaque-1077, Ficoll-Paque, Ficoll-Hypaque, and Percoll. From these PBMCs multiple cell populations can be isolated, including BOECs. PBMCs are resuspended in EGM-2 medium without further cell subpopulation enrichment procedures and placed into 1 well of a 6-well plate coated with type I collagen. This mixture is incubated at 37 C in a humidified environment with 5%
CO2. Culture medium is changed daily. After 24 hours, unattached cells and debris are removed by washing with medium. This procedure leaves about 20 attached endothelial cells plus 100-200 other mononuclear cells. These non-endothelial mononuclear cells die within the first 2-3 weeks of culture.
Cell culture for growing target cell population [0216] BOECs cells are established in culture for 4 weeks with daily medium changes but with no passaging. The first passaging occurs at 4 weeks, after approximately a 100-fold expansion. In the next step, 0.025% trypsin is used for passaging cells and tissue culture plates coated with collagen-I as substrate. Following this initial 4-week establishment of the cells in culture, the BOECs are passaged again 4 days later (day 32) and 4 days after that (day 36), after which time the cells should number 1 million cells or more.
In vitro gene repair [0217] In order to affect gene repair in BOECs, cells are transfected with 0.1-10 micrograms per million cells of each plasmid encoding left and right TALENs and 0.1-10 micrograms per million cells of the repair vehicle plasmid. Transfection is done by electroporation, liposome-mediated transfection, polycation-mediated transfection, commercially available proprietary reagents for transfection, or other transfection methods using standard protocols.
Following transfection, BOECs are cultured as described above for three days.
Selection of gene-repaired clones [0218] Using the method of limiting serial dilution, the BOECs are dispensed into clonal subcultures, and grown as described above. Cells are examined daily to determine which subcultures contain single clones. Upon growth of the subcultures to a density of >100 cells per subculture, the cells are trypsinized, re-suspended in medium, and a 1/10 volume of the cells is used for colony PCR. The remaining 9/10 of the cells are returned to culture. Using primers that detect productively repaired F8 genes, each 1/10 volume of colonies are screened by PCR for productive gene repair. Colonies that exhibit productive gene repair are further cultured to increase cell numbers. Using the top 20 predicted potential off-site targets of the TALENs, each of the colonies selected for further culturing is screened for possible deleterious off-site mutations. The colonies exhibiting the least number of off-site mutations are chosen for further culturing.
Preparation of cells for re-introduction into patients by conditioning and/or outgrowth [0219] Prior to re-introducing the cells into patients, the BOECs are grown in culture to increase the cell numbers. In addition to continuing cell culture in the manner described above, other methods can be used to condition the cells to increase the likelihood of successful engraftment of the BOECs in the liver sinusoidal bed of the recipient patient.
These other methods include: 1) co-culturing the BOECs in direct contact with hepatocytes, wherein the hepatocytes are either autologous patient-derived cells, or cells from another donor; 2) co-culturing the BOECs in conditioned medium taken from separate cultures of hepatocytes, wherein the hepatocytes that yield this conditioned medium are either autologous patient-derived cells, or cells from another donor; or 3) culturing the BOECs as spheroids in the absence of other cell types.
[0220] Co-culturing endothelial cells with hepatocytes is described further in the primary scientific literature (e.g. Kim, Y. & Rajagopalan, P. 3D hepatic cultures simultaneously maintain primary hepatocyte and liver sinusoidal endothelial cell phenotypes.
PLoS ONE 5, e15456 (2010)). Culturing endothelial cells as spheroids is also described in the scientific literature (e.g. Korff, T. & Augustin, H. G. Tensional forces in fibrillar extracellular matrices control directional capillary sprouting. J Cell Sci 112 (Pt 19), 3249-3258 (1999)). Upon growing the colonies of cells to a total cell number of at least 1 billion cells, the number of cells needed for injection (>50 million cells) into the patient are separated from the remainder of the cells and used in the following step for injection into patients. The remainder of the cells are aliqouted and banked using standard cell banking procedures.
Injection of gene-repaired BOECs into patients [0221] BOECs that have been chosen for injection into patients are resuspended in sterile saline at a dose and concentration that is appropriate for the weight and age of the patient.
Injection of the cell sample is performed in either the portal vein or other intravenous route of the patient, using standard clinical practices for intravenous injection.
Example 3: Nuclease sites for repair at different exon-intron junctions [0222] Because mutations causing Hemophilia A occur throughout the FVIII gene, different repair strategies may be employed at different exon-intron junctions in order to allow the use of repair vehicles which correct a wider range of patient mutations. All gene repairs employ the methodology described herein of using a DNS scission enzyme (DNA-SE) such as a zinc finger nuclease, a TALEN, or a CRISPR to induce a double-strand break near the 3' end of an exon, thereby allowing homologous recombination to incorporate a therapeutic repair vehicle encoding the cDNA for the downstream exons of the gene into the genome in order to be operably linked to the 3' end of that exon.
[0223] In order to choose CRISPR target sites in exons 1-22, several considerations were taken into account. The ¨100 bp of the 3' end of each exon (hg19 human genome build) were searched for CRISPRICas9 binding sites using an online algorithm described by Hsu et al. in Nature Biotechnology 2013, incorporated herein by reference. Single guide RNAs (sgRNAs) were chosen based on low potential for off-target activity, the proximity of the cleavage site to the 3' end of the exon, and guidelines for increasing the likelihood of high on-target activity (Wang T et al., Science 2014). Paired nickases were chosen by adding the additional consideration that they be orientated to create 5' overhangs and be spaced apart within the recommended range for optimal activity (Shen B, et al., Nature Methods 2014).
[0224] In order to choose TALEN binding sites in exons 1-22, several considerations were taken into account. The ¨100 bp of the 3' end of each exon (hg19 human genome build) were searched for TALEN binding sites using the SAPTA algorithm as described by Lin Y, Fine EJ, et al. in Nucleic Acids 2014, incorporated herein by reference. Potential binding sites were then screened using the TALEN v2.0 algorithm of the PROGNOS tool as described by Fine EJ et al. in Nucleic Acids Research 2013, incorporated herein by reference to ensure that no highly scored potential off-target sites existed in the human genome.
[0225] Sequences listed in Table 5 below contain identified binding sites for CRISPRs within exons 1-22 respectively. If a homologous sequence in the canine genome (canFam3 build) exists that permits the possibility of CRISPR/Cas9 cleavage using the same guide strand as used for the human exon, it is listed with any mismatches in lowercase bold;
if no reasonable homology exists, it is listed as "N/A".
Table 5 FVIII Gene Genome Editing Genomic Target of SG/PG RNAs Target of SG/PG RNAs in Dogs (Region) (Desired Activity) (DNA Sequence) (DNA
Sequence) single nuclease 5 ' ¨AAGATACTACCTGGGTGCAGtGG 5 ' ¨AAaATACTACCTcGGTGCAGtGG
Exon 1 paired nickase (5') 5' ¨CACTAAAGCAGAATCGCAAAaGG N/A
paired nickase (3') 5' ¨AAGATACTACCTGGGTGCAGtGG N/A
Exon 2 single nuclease 5 ' ¨TTTTCAACATCGCTAAGCCAaGG N/A
paired nickase (5') 5 ' ¨AGTCTTTTTGTACACGACTGaGG N/A
paired nickase (3') 5 ' ¨TTTTCAACATCGCTAAGCCAaGG N/A
Exon 3 single nuclease 5 ' ¨ATGCTGTTGGTGTATCCTACtGG 5 ' ¨AcGCTGTTGGTGTATCCTAttGG
paired nickase (5') 5 ' ¨CAGCATGAAGACTGACAGGAtGG N/A
paired nickase (3') 5' ¨ATGCTGTTGGTGTATCCTACtGG N/A
Exon 4 single nuclease 5' ¨GACTTGAATTCAGGCCTCATtGG 5' ¨GACcTGAATTCAGGCCTCATtGG
paired nickase (5') 5 ' ¨TATGAGTAGGTAAGGCACAGtGG N/A
paired nickase (3') 5 ' ¨GACTTGAATTCAGGCCTCATtGG N/A
Exon 5 single nuclease 5 ' ¨AAGTAGTATAAATTTGTGCAaGG N/A
paired nickase (5') 5 ' ¨AAGTAGTATAAATTTGTGCAaGG N/A

SUBSTITUTE SHEET (RULE 26) Table 5 FVIII Gene Genome Editing Genomic Target of SG/PG RNAs Target of SG/PG RNAs in Dogs (Region) (Desired Activity) (DNA Sequence) (DNA
Sequence) paired nickase (3') 5 ' -CTTTTTGCTGTATTTGATGAaGG N/A
Exon 6 single nuclease 5 ' -CAGTCAATGGTTATGTAAACaGG 5 ' -CcaTCAATGGcTATGTAAACaGG
paired nickase (5') 5' -GACTGTGTGCATTTTAGGCCaGG N/A
paired nickase (3') 5' -CAGTCAATGGTTATGTAAACaGG N/A
Exon 7 single nuclease 5' -CAAACACTCTTGATGGACCTtGG N/A
paired nickase (5') 5' -GCGAGATTTCCAAGGACGCCtGG N/A
paired nickase (3') 5' -CAAACACTCTTGATGGACCTtGG N/A
Exon 8 single nuclease 5' -ACATTACATTGCTGCTGAAGaGG N/A
paired nickase (5') 5' -TCTTGGCAACTGAGCGAATTtGG N/A
paired nickase (3') 5' -ACATTACATTGCTGCTGAAGaGG N/A
Exon 9 single nuclease 5' -GAAGCTATTCAGCATGAATCaGG 5' -GAAGCTATTCAGtATGAATCaGG
paired nickase (5') 5' -AATAGCTTCACGAGTCTTAAaGG N/A
paired nickase (3') 5' -GAAGCTATTCAGCATGAATCaGG N/A
Exon 10 single nuclease 5' -GGACATCAGTGATTCCGTGAgGG N/A
paired nickase (5') 5' -GGACATCAGTGATTCCGTGAgGG N/A
paired nickase (3') 5' -ATGTCCGTCCTTTGTATTCAaGG N/A
Exon 11 single nuclease 5' -GATCTAGCTTCAGGACTCATtGG 5' -GATCTAGCTTCAGGACTCATtGG
paired nickase (5') 5' -AACGAAACTAGAGTAATAGCgGG N/A
paired nickase (3') 5' -GATCTAGCTTCAGGACTCATtGG N/A
Exon 12 single nuclease 5' -CGCTTTCTCCCCAATCCAGCtGG N/A
paired nickase (5') 5' -AGCGTTGTATATTCTCTGTGaGG N/A
paired nickase (3') 5' -CGCTTTCTCCCCAATCCAGC tGG N/A
Exon 13 single nuclease 5' -AGAAACTGTCTTCATGTCGAtGG 5' -AGAAACTGTCTTCATGTCaAtGG
paired nickase (5') 5' -ATAGACCATTTTGTGTTTGAaGG 5' -ATAGACCATTTTGTGTTTGAaGG
paired nickase (3') 5' -AGAAACTGTCTTCATGTCGAtGG 5' -AGAAACTGTCTTCATGTCaAtGG
Exon 14 single nuclease 5' -ACACTATTTTATTGCTGCAGtGG 5' -ACACTATTTcATTGCTGCAGtGG
paired nickase (5') 5' -TTTTCTTTTGAAAGCTGCGGgGG 5' -TTTTCTTTTGAAAGCTGCGGaGG
paired nickase (3') 5' -ACACTATTTTATTGCTGCAGtGG 5' -ACACTATTTcATTGCTGCAGtGG
Exon 15 single nuclease 5' -TCAACTTCTGCTCTTATATAtGG 5' -TCAACTTCTGCTCTTATATAtGG
paired nickase (5') 5' -ACGGTATAAGGGCTGAGTAAaGG N/A
paired nickase (3') 5' -AAATGAACATTTGGGACTCCtGG N/A
Exon 16 single nuclease 5' -ATGAGTTTGACTGCAAAGCCtGG 5' -ATGAGTTTGACTGCAAAGCCtGG
paired nickase (5') 5' -CAGTCAAACTCATCTTTAGTgGG 5' -CAGTCAAACTCATCTTTAGTgGG
paired nickase (3') 5' -ATGAGTTTGACTGCAAAGCCtGG 5' -ATGAGTTTGACTGCAAAGCCtGG
Exon 17 single nuclease 5' -GGCTCCCTGCAATATCCAGAtGG 5' -aGCTCCCTGCAATgTCCAGAaGG
paired nickase (5') 5' -TTCAGTGAAGTACCAGCTTTtGG N/A
paired nickase (3') 5' -GGCTCCCTGCAATATCCAGAtGG N/A
Exon 18 single nuclease 5' -GTTCACTGTACGAAAAAAAGaGG 5' -GTTCACTGTACGAAAAAAAGaGG
paired nickase (5') 5' -GTCCACTGAAATGAATAGAAtGG N/A
paired nickase (3') 5' -GTTCACTGTACGAAAAAAAGaGG N/A
Exon 19 single nuclease 5' -CAAAGCTGGAATTTGGCGGGtGG N/A
paired nickase (5') 5' -CGCCAAATTCCAGCTTTGGAtGG N/A
paired nickase (3') 5' -ATTGGCGAGCATCTACATGCtGG N/A
Exon 20 single nuclease 5'-TGTCCAGAAGCCATTCCCAGgGG N/A
paired nickase (5') 5'-TGTCCAGAAGCCATTCCCAGgGG N/A
paired nickase (3') 5'-GATTTTCAGATTACAGCTICaGG N/A
Exon 21 single nuclease 5'-AATCAATGCCTGGAGCACCAaGG 5'-AATCAATGCCTGGAGCACCAaGG
paired nickase (5') 5'-TGATCCGGAATAATGAAGTCtGG 5'-TGATCCGGAATAATGAAGTCtGG

SUBSTITUTE SHEET (RULE 26) paired nickase (3') 5'-AATCAATGCCTGGAGCACCAaGG 5'-AATCAATGCCTGGAGCACCAaGG
Exon 22 single nuclease 5'-AAGAAGTGGCAGACTTATCGaGG N/A
paired nickase (5') 5'-AGATAAACTGAGAGATGTAGaGG N/A

SUBSTITUTE SHEET (RULE 26) Table 5 FVIII Gene Genome Editing Genomic Target of SG/PG RNAs Target of SG/PG RNAs in Dogs (Region) (Desired Activity) (DNA Sequence) (DNA
Sequence) paired nickase (3') 5 ' -AAGAAGTGGCAGACTTATCGaGG N/A
[0226] Sequences contain the top 20 potential off-target sites computationally identified in the human genome for the previously mentioned CRIPSR binding sites in exons 1-22 are listed in tables 6-27, respectively below.
[0227] Top-ranked Potential Off-Target Sites for sgRNAs in Human Genome [0228] The top twenty potential off-target sites in the human genome (hgl 9 genome build) for single guide strands were located using an online tool (Hsu et al., Nature Biotechnology 2013). Mismatches to the intended binding sequence are shown in bold. The genomic region is annotated and the gene name given in parentheses.
Table 6. Targeting Exon 1 Genome Coordinates Sequence Genomic Region chrX:154250739 AGATACTACCTGGGTGCAGtGG Exon Coding Sequence (F8) chr5:65751749 AAACACAACCTGGGTGCAGgGG Intergenic chr9:17600130 AAAAAGTACCTGGGTGCAGaAG Intron (SH3GL2) chr9:100168533 AGAAACTACATGGGTGCAGaGG Intergenic chr21:45748293 GGCGACCACCTGGGTGCAGcAG Intergenic chr2:144598347 ATTTACCAACTGGGTGCAGcAG Intergenic chr3:89701232 ATTTACCATCTGGGTGCAGgGG Intergenic chr10:43493946 AGATGCTTCCTGGGTGCAGcAG Intergenic chr18:37552785 ACAAACTCCCTGGGTGCAGaGG Intergenic chr7 :63413239 ACACACTGCCTGGGTGCAGcAG Intergenic chr7:157859920 GGAGACACCCTGGGTGCAGgAG Intron (PTPRN2) chr22:48920664 AGGAACGCCCTGGGTGCAGaAG Intron (FAM19A5) chr1:153919242 GGAAGCTACCTGGGTGCAGgGG Promoter (DENND4B) chr11:71136741 AGATACCCTCTGGGTGCAGaAG Intergenic chr2:145627680 AGATACCCTCTGGGTGCAGgAG Intron (TEX41) chr2:145629372 AGATACCCTCTGGGTGCAGgAG Intron (TEX41) chr4:60481509 AGATACTGCCTGGGTCCAGaGG Intergenic chr6:35192631 AGATACTCCCTGGGTCCAGcAG Intron (SCUBE3) chr10:132278858 GGATACTAGATGGGTGCAGaGG Intergenic chr3:86928921 AGAGACTACAAGGGTGCAGtGG Intergenic chr5: 61074999 CAACACTACCTGGGTGCAAaAG Intergenic Table 7. Targeting Exon 2 Genome Coordinates Sequence Genomic Region chrX:154227766 TTTCAACATCGCTA_AGCCAaGG Exon Coding Sequence (F8) SUBSTITUTE SHEET (RULE 26) Table 27. Targeting Exon 22:
Genome Coordinates Sequence Genomic Region chX:154124374 (target) AGAAGTGGCAGACTTATCGaGG Exon Coding Sequence (F8) chr21:42038990 AGAAGCAGCAGACTTATCCaGG Intron (DSCAM) chr12:69990980 GGAAGTTGCAAACTTATCGaGG Exon Coding Sequence (CCT2) chr7:110964978 GGATGTGGCAGACTTATCTtAG Intron (IMMPL2) chr8:42174378 CTGAGTGGCAGGCTTATCGgGG Exon Coding Sequence (IKBKB) chr3:57930763 AGAACAGGCAGACTTATCTtAG
Intergenic chr1:52997435 AGAAGAGGCATACTTATCTgAG Intron (ZCCHC11) chr15:27460224 GAAACTGGCAGACTTATCTaGG Intron (GABRG3) chr2:102965996 AGAAGTGGCAGAGTTATCCtGG Intron (IL1RL1) chr20:2306018 AGGAGTGGCTGACTTATCTaAG Intron (TGM3) chr8: 92580265 AAAAATGGTAGACTTATCAaAG I
ntergenic chr13:113875149 AGAAGTCGCAGGCTTATGGgAG Intron (CUL4A) chr18:30300891 AGAAGAGGAAGACTTATGGaAG Intron (KLHL14) chr2:135308659 AGTGCTGGCAGACTTATTGcAG Intron (TMEM163) chr11:133197425 AGGAGGGGCAGATTTATCGaAG Intron (OPCML) chr12:102978261 AGAAGTAGAAAACTTATCAtAG I
ntergenic chr3:30382779 AGCAGTGGCAGACATATTGaAG I
ntergenic chr6:118027061 AGAAGTGGATGACTTATTGcAG Intron (NUS1) chr9 :117888881 GCAAGTGGCAGGCTTATCTgGG Intron (L0C101928748) chr2 :51293036 GCAAGTGGCAGACTTTTCCaAG I
ntergenic chr21:36105270 AAGAGTGGCAGACTTCTCAtGG Non-coding Exon (LINC00160) [0229] Sequences listed in Table 28 contain identified binding sites for TALENs within exons 1-22 respectively. If a similar sequence existed in the homologous exon in the canine genome (canFam3 genome build), that corresponding binding site is shown with any mismatches in lowercase red; if insufficient homology to permit a reasonable possibility of the TALENs being able to cleave the canine exon, the site is listed as "N/A".
Table 28 FVIII Gene Genome Editing Genomic Target of TALEN Target of TALEN in Dogs (Region) Position (DNA Sequence) (DNA Sequence) 5' Half-Site 5 ' -TGGAACTGTCATGGGAC N/A
Exon 1 3' Half-Site 5 ' -TCCACAGGCAGCTCACCGAG N/A
5' Half-Site 5 ' -TCTGTTTGTAGAATTCACGG N/A
Exon 2 3' Half-Site 5 ' -TGGCCTTGGCTTAGCGAT N/A
5' Half-Site 5 ' -TACACTTAAGAACATGGCT N/A
Exon 3 3' Half-Site 5 ' -TACACCAACAGCATGAAGAC N/A
5' Half-Site 5 ' -TGTGCCTTACCTACTCATATCT
N/A
Exon 4 3' Half-Site 5 ' -TGAATTCAAGTCTTTTACCAG N/A
E 5' Half-Site 5 ' -TCTGGCCAAGGAAAAGACACAGAC 5 -TCTGGCCAAGAAAgGACACAGAC
xon 5 3' Half-Site 5 ' -TTCATCAAATACAGCAAAAAGTAG 5T -TTCATCAAATACAGCAAAAAGTAG
5' Half-Site 5 ' -TGCTGCATCTGCTCGGG N/A
Exon 6 3' Half-Site 5 ' -TTTACATAACCATTGACTGTGT
N/A
5' Half-Site 5 ' -TCTCGCCAATAACTTTCC N/A
Exon 7 3' Half-Site 5 ' -TGTCCAAGGTCCATCAAGAG N/A

SUBSTITUTE SHEET (RULE 26) Table 28 FVIII Gene Genome Editing Genomic Target of TALEN Target of TALEN in Dogs (Region) Position (DNA Sequence) (DNA Sequence) 5' Half-Site 5 ' -TCAGTTGCCAAGAAGCATCCTAA 5' -TCAGTTGCCAAGAAGCATCCTAA
Exon 8 3' Half-Site 5 ' -TCCTCCTCTTCAGCAGCAATGT 5' -TCCTCCTCTCAGCAGCAATT
5' Half-Site 5 ' -TTCAGCATGAATCAGGAA N/A
Exon 9 3' Half-Site 5 ' -TCTCCAACTTCCCCATAA N/A
5' Half-Site 5 ' -TATAACATCTACCCTCACGG N/A
Exon 10 3' Half-Site 5 ' -TCTCCTTGAATACAA_AGGAC N/A
5' Half-Site 5'-TCTAGCTTCAGGACTCAT 5'-TCTAGCTTCAGGACTCAT
Exonll 3' Half-Site 5'-TCTACAGATTCTTTGTAGCAG 5'-TCTACAGATTCTTTGTAGCAG
5' Half-Site 5'-TCACAGAGAATATACAACG N/A
Exon 12 3' Half-Site 5'-TCCTCAAGCTGCACTCCAGCT N/A
E 13 5' Half-Site 5'-TGTCTTCTTCTCTGGAT 5'-TGTCTTCTTCTCTGGAT
xon 3' Half-Site 5'-TGTGTCTTCATAGACCATTTT 5'-TGTGTCTTCATAGACCATTTT
5' Half-Site 5'-TCAAAAGAAAACACGACACTATTT 5'-TCAAAAGAAAACACGACACTATTT
Exon 14 3' Half-Site 5'-TCATCCCATAATCCCAGAGCCTCT 5'-TCATCCCATAATCCCAGAGaCgCT
5' Half-Site 5'-TCAGCCCTTATACCGTGGAG 5'-TCAGCCCTTATACCGTGGAG
Exon15 3' Half-Site 5'-TATGGCCCCAGGAGTCCCAA 5'-TATGGCCCCAaGAGTCCCAA
5' Half-Site 5T-TATGGCACCCACTAAAGATGAG 5'-TATGGCACCCACTAAAGATGAG
Exon 16 3' Half-Site 5'-TCAGAGAAATAAGCCCAG 5'-TCAGA&AAATAAGCCCAG
5' Half-Site 5'-TCTTTGATGAGACCAAA N/A
Exon 17 3' Half-Site 5'-TCTTTCCATATTTTCAG N/A
5' Half-Site 5'-TCTATTCATTTCAGTGGAC N/A
Exon 18 3' Half-Site 5'-TATACTCCTCTTTTTTTCG N/A
5' Half-Site 5'-TGTTACCATCCAAAGCT N/A
Exon 19 3' Half-Site 5'-TGCTCGCCAATAAGGCATTCC N/A
5' Half-Site 5'-TCCCCTGGGAATGGCTTCTGG N/A
Exon 20 3' Half-Site 5 ' -TGTCCTGAAGCTGTA_ATCTGAA N/A
5' Half-Site 5 ' -TGGGCCCCAAAGCTGGCCAG 5' -TGGGCCCCAAAGCTGGCCAG
Exon 21 3' Half-Site 5' -TGCTCCAGGCATTGATTGAT 5' -TGCTCCAGGCATTGATTGAT
5' Half-Site 5 ' -TCTACATCTCTCAGTTTAT N/A
Exon 22 3' Half-Site 5 ' -TCTGCCACTTCTTCCCATCAAG N/A
[0230] Sequences listed in Tables 29-50 below contain the top 20 potential off-target sites computationally identified in the human genome for the previously mentioned TALEN
binding sites in exons 1-22, respectively. Off-target analysis was performed using the PROGNOS algorithm (Fine et al., Nucleic Acids Research 2013) "TALEN v2.0" on the hg19 build of the human genome. The top 20 potential off-target sites are given for each TALEN
pair. Homodimers were allowed in the search and spacing between the TALENs of 10-30 bp.
The right half-site is listed as the sequence on the same strand as the left half-site; the right half-site is therefore listed in the reverse anti-sense orientation to the sequence which is bound by the TALEN. Left and right half-sites are given as the 5' (left) and 3' (right) binding sites on the positive strand of the chromosome; the "left" and "right"
annotation may therefore differ from the annotation for TALENs designed to genes on the negative strand of SUBSTITUTE SHEET (RULE 26) chromosomes. Mismatches to the intended binding sequence are depicted in lowercase SUBSTITUTE SHEET (RULE 26) ak 02951882 2016-12-09 letters.
Table 29. Targeting Exon 1:
Genome Left Half-Site Right Half-Site Genomic Region Coordinates chrX:154250691 TCCACAGGCAGCTCACCGAG GTCCCATGACAGTTCCA Exon (F8) chr14:45095676 TGGAACTcTCATGGaAC GagCaATGACtGTTCCA Intergenic chr6:26839581 aGGAgCTGTCAgtcaAC GTCtCATGACAGTTaCA Intron (GUSBP2) chr10:45462110 TGGAACTGTCATGGtgC CTCaGaGAGtTGCCTGgttA Intron (RASSF4) chr11:101870316 TGaAACTGTCATatGAC tgCCCATGACtccTCCA Exon (KIAA1377) chr15:20414578 TGaAgCTGTCATGaaAC cTtCCATtAtAGTTttA Intergenic chr16:33444315 TaaAACTaTaATGGaAg GTttCATGACAGcTtCA Intergenic chr5:61534127 TGaAgCTGTCATGaaAC cTtCCATtAtAGTTttA Intergenic chr7:44551672 TGGAcCcagCATGGGgC GTtCCtTGACAtTTCCA Intergenic chr1:165095506 TGGAACTGTCATGtGAg GTtCCATGgCAGaTaCt Intergenic chrX:15724565 TaGgACTGTCcTGaGcC GgCtCAgGACAGTcCCA Intergenic chr7:67809648 TaGAACTaTCATGGGAa GgCttcTGAgAcTTCCA Intergenic chr6:13204828 TGGcAtTGTCATGGaAC GTCCtAgGtagGTTCCA Intron (PHACTR1) chr2:37743218 TGaAACccTCATGaGcC GTCCtATGAgAtTTCtA Intergenic chr10:78301531 TGtAAaTGTCATGGaAC GTCtCATttCAGTgtaA Intron (ClOorf11) chrX:106781486 TGGAAaTGTCATaGaAC cTCCatTGACAGaTCtt Intergenic chr12:70809983 TaGgtCTGTCtTGGGtC GctCCATGtCAGTTtCA Intron (KCNMB4) chr11:46818282 TatAACTGTCAaGaGAC GTCCaATttCAGTcCaA Intron (CKAP5) chr3:30945924 TGGAgCTGaaAaGcaAC GTCtCcTGACAGcTCCA Intergenic chr9:13642916 TaGAACTaaCATaaaAC GTgtCATtAtAGTTgCA Intergenic chr14:27743308 TaGAAaTaTCcTGGGAt aTtgCATGAtAGTTCCA Intergenic Table 30. Targeting Exon 2:
Genome Left Half-Site Right Half-Site Genomic Region Coordinates chrX:154227764 TGGCCTTGGCTTAGCGAT CCGTGAATTCTACAAACAGA Exon (F8) chr12:51122429 TGGaCTTGGCTTcGCGcT ATgGaaAAGCCAAGGagA Exon (DIP2B) chr14:83666273 TaGCCTTGGCTTAGaaAa cTgGCTAAGCaAAGataA Intergenic chr15:99285268 gGaaCTTGaCTTAGCccT cctGCTAAGCCAAGGCtA Intron (IGF1R) chr15:29750773 TGcCCTgGaCTTgGaGgT AgaGaTAAGCCAAGGtCA Intron (FAM189A1) chr20:59053322 TGGCCTTGGtTTAGaaAa AgCGaTAAGgaAAGGttA Intergenic chr1:163956121 TCTaTTTGTAGAATTactaG tTgGtTAAGCCAAttCCA Intergenic chr2:123622749 TCTtTTTGTAaAAaTgACGa ATtcCgAAGCCAAGGatA Intergenic chr12:92444873 TGtCCaTGGCcTgGgGgT ATCttgAAGCCAAGGCtA Intron (L0C256021) chr14:86193436 caGCCTTGGCTTgtgGAT tTtaCTAAGaCAAGGCCA Intergenic chr8:1184501 TGaCCTctcCTTAaCcAT ATttCTAAaCtAAGGtCA Intergenic chr4:60350711 TGGCaaTGcCTTAGaaAT ATtGCTAAGtCAAatCaA
Intergenic chr2:109270631 TttCCTTGGCTTAGtGAT ATtGCTAActCAAtcaCA Promoter (LIMS1) Promoter (LIMS3-chr2:110655405 TttCCTTGGCTTAGtGAT ATtGCTAActCAAtcaCA L0C440895) Promoter (LIMS3-chr2:111231206 TGtgaTTGagTTAGCaAT ATCaCTAAGCCAAGGaaA L0C440895) chr7:105518314 ctGCCcTGGCTgAaCcAT ATCGCTAAGCCAgtGttA Intergenic chrX:12453009 TtGCaTTtaCTcAGCcAT ATCttTtAGCCAAtGCCA
Intron (FRMPD4) chr9:133831225 TGGCCTgaGCTTtGgGgT ActGCTAAGaCAAGcCCA Intergenic chr7:27778567 TgTGcTTaTAaAATTCACtG CaGTtAtTTCTACtAcCAGA Promoter (TAX1BP1) chr8:22054601 TaGggcTGGCTTgGCGAg gTaGCTAAGtCAAGGCtA
Intron (BMP1) chr6:102761808 TGGCagTaGCTctGCcAT AattCTAAGCtAAGGCCA Intergenic Table 31. Targeting Exon 3:
Genome Left Half-Site Right Half-Site Genomic Region Coordinates SUBSTITUTE SHEET (RULE 26) Table 31. Targeting Exon 3:
Genome Left Half-Site Right Half-Site Genomic Region Coordinates chrX:154225270 TACACCAACAGCATGAAGAC AGCCATGTTCTTAAGTGTA Exon (F8) chr2:175647194 aACAaTcAgGctCATGGCa AGCCATGTTtTTAAGaGTA Intergenic chr4:164801896 TAtACTTAAaAACATaGCT AGtgATtTTtTTcAaTGaA Intron (MARCH1) chr3:1591042 TACAtTTAAaAACATGtCT AGCtATcTTaTTcAtTtTA
Intergenic chr21:39750804 TACgCTgcAGAgCtgGGCa AGaCATtTTtTTAAGTGTA Intron (ERG) chrX:46478957 TACACaTAAcAACATGGCT AGCCAgacaCTaAAaTaTA
Intron (SLC9A7) chrX:99327213 aAtcCTTAAGAACATGaCT AtCCtTGTTCTTAtGTtcA Intergenic chr8:103196820 cACACTgAAGAcCATGGCT GTCTTCATcaTGTTaGTGTc Intergenic chr9:76364644 TAgACTTAAtcAtgTaGCT gGCtATGTTCTTAAGTGTc Intergenic Intron chr8:19520723 TACACTTgtGAAgATGGaT AGgCtTGTaCTTAAtTGTA (CSGALNACT1) chr1:7465386 TACACTTAgaAAaAaaGCT GTtTgttTGCTGTTGtTGTt Intron (CAMTA1) chrX:151388800 TACACTTAtGtgttTGGCT AtCCATGTTgTTgAGTGTA Intron (GABRA3) chr8:52110351 aACACTTAAaAACAgGGCT AtCtATtTaCTaAAtTGTt Intergenic chr11:42440454 aACAaaTAAtAtCATcaCT AtCtATGTTCTTAAGTcTA Intergenic chr2:74468885 cgCACaaAAaAACATGGaT AGgCATGTTtTTAAGTGgg Intron (SLC4A5) chr6:82600824 cACAtTTgAGAACATGGCT GctTTCAgtCTGgTGGTtTA Intergenic chr2:65094538 TgCACTTAAaAAtATGaCa AGCacaGTgCTTAAGTGcA Intergenic chrX:87497023 TACACTgAAGAgaATGGag AGCaATGTTtTTAAGTGat Intergenic chr13:74882688 TtCAtTgAAGAAaAaaGCT aTtTTtATGCTGTTGGaGTA Intergenic chr21:25077810 TACAtTTAAGcAtATGGCT tGCttTagTCTTAAtTGTA Intergenic chr10:92935297 TACcCcTgtGAACATGGaa tGCttTGTTCTTAAaTGTA Intron (PCGF5) Table32.TargetingExon4:
Genome Left Half-Site Right Half-Site Genomic Region Coordinates chrX:154221245 TGAATTCAAGTCTTTTACCAG AGATATGAGTAGGTAAGGCACA Exon (F8) chr5:166223644 TGAATTCAAaTCTTTTtCCtG tTGGaAAAAtcCcTtAATaCA Intergenic Promoter chr3:48957213 TGAtTTCtAGTtTTgTgCCAa tTaGTAAAtGACcTGAATTCA (C3orf71) chr1:14460511 TGAcaTtAAGaCaTTTAaCAG CTGGgAAAAGAagTGgATTCA Intergenic chr8:26674607 gaAAggCAAGcCaTaTACtAG
CTGaTAAAtGACTTGtATTCA Intron (ADRA1A) chr15:41366843 TGcATaCAAtTCcTTTACCAa CTGaTAAAcaAtTTtAATTtA Intron (IN080) chr6:134930070 TaAAgTCActTCcTTTACgAc aTGGTtgAtGACTTGAATTCA Intergenic chr6:121097474 TGAATcCAAaaCTTTTACCtG CTGGgttAAtACaTttATTtA Intergenic chr11:49119615 gGAATTaAAGTCcTTcACata tTGGTtAcAGACTTGAAgTCA Intergenic chr1:74307557 gGAATTCAAtTCaaTaACaAG tgGGcAAAAGACcTGAATTgA Intergenic chr18:38466162 TGtATTCAAGTCcTTaAaaAG tTGGTtAAAattTTGAAcTCA Intergenic chr20:45113912 atAATTCtAGTCTTaggaCAG CTGGgAAAAGttTgGAATTtA Intergenic chr5:26641542 TGAATTCcttcCTTgTACCAt tgGaTtAAAGACTTGAATgCA Intergenic chr3:160034110 TGAAagCAAaTCTTTccCCAG CTGGTcAAtGcCTTGctTgCA Intron (IFT80) chr2:241783612 TGAcTTCAAGTCTTTaAaCAa aTcagAAAAtctTTGAATcCA Intergenic chr6:123852751 gGTcaCTaAtCTACTCtTATCT AGATATGAacAGGTAAGGCACt Intron (TRDN) chr2:89343189 TGAATTCAAcTCTTTagaCAG gTaaggAAAGctTTGAATTCA Intergenic chr2:90195655 TGAATTCAAagCTTTccttAc CTGtctAAAGAgTTGAATTCA Intergenic chr8:13349868 TGAAaTtgAaTCTgaTtCCAG
tTtGTcAAAGACTTGtATTtA Intron (DLC1) chrY:4231090 TGAATTCAAtTCTTcagCCAG
tcaGaAAAAtctTTGAATcCA Intergenic chrX:90035974 TGAATTCAAtTCTTcagCCAG tcaGaAAAAtctTTGAATcCA Intergenic Table 33. Targeting Exon 5:
Genome Left Half-Site Right Half-Site Genomic Region Coordinates chrX:154215513 TTCATCAAATACAGCAAAAAGTAG
GTCTGTGTCTTTTCCTTGGCCAGA Exon (F8) chr8:65938903 TCTaGCCAAGccAgAGgCACtGAC GgCTcTGTCTTTTCCTctGCCAcA Intergenic SUBSTITUTE SHEET (RULE 26) chr1:26774318 TTCAaCAAcaACAaCAAAAAagca cTCTGTGcCaTgTaCTTGGCCAGA Intron (DHDDS) chr10:102225665 cTCAcCAAgcAttGCAtAAAGctG
CTACTTTTaGgTGTATTTtATGAA Intron (WNT8B) SUBSTITUTE SHEET (RULE 26) ak 02951882 2016-12-09 Table 33. Targeting Exon 5:
Genome Left Half-Site Right Half-Site Genomic Region Coordinates chr7:14755743 TTCATCAAcTcCAGgAAAAAcaAc GTaTaTGTgTTTTCacTGGaCAGA Intron (DGKB) chr8:124089292 TTCATaAtATcaAGtAAtAcGTga GTtTGgGTtTTTTtCTTtGaCAGA Intron (WDR67) chr6:70049288 TCTGGCCAtGacAgAtAaACgctC
aTACTTTTTGCTGTgTTTGATtcA Exon (BAI3) Promoter chr17:37764808 TCaaaCCAAGGgAAAGACAgAGAa GTCTGTGcCTcTgCaTgGGCgtGt (NEUROD2) chr2:92285124 TCTtGCCAcaaAAAAtACACAGAa CTACgTTgTGaTGTgTTTacTcAA Intergenic chr11:80679047 TTaATaAAgTgaAaCtAAAAGTAa GTCTGTaTgTTTTatTTtGCtAGA Intergenic chr7:49746821 TCaGaCCAAGccAgAGgtgCAcAC GgCTtTGTCaTTTCCTTGGCCtGt Intergenic chr2:92283421 TCTGGCCAcaaAAActACACAGAa CTACgTTgTGaTGTgTTTacTcAA Intergenic chr6:53622618 TCcacCCAAGGAAtAGgCAgAGAg CTAaTcTTTGCTGTATTTtATtgA Intergenic chr7:64186025 gcCAaCAgcaACAGCAAcAAaaAG GTtTtTGTCTTTTttTTaGaCAGA Intergenic chr8:76622826 TCatGaaAAatAAAAGAaACAGta GTtTtTtTtTTTTCtTgGGaCAGA Intergenic chr13:27818295 TCTGtCCAAaaAAAAaAaAaAaAa gTttTgTTTcCTGaATTTGATaAA Intergenic chr18:68100701 TCaGGCCAAtaAAAAacaACAaAC tgcCTTTTTttTtTtTTTttTGAA Intergenic chr5:72817667 TCTaGCaAAGaAAAAtAaACAaAa tTaTtTtTCTTTTttTTttCCAGc Intergenic chr15:43320939 TCaaaCaAAaaAAAAaAaACAaAC
aTaTaTaTaTaTTCCTTGGCCgGA Intron (UBR1) chr4:12953588 TaCATaAAAcACAaCAAgAAaTAG tTACTTacattTGTATTTGAaGAt Intergenic chr22:49683417 TCTGGCaAAaGgAtAGcCACAGAt tTgTGTtTCTTTTtCcTGGgCAtg Intergenic Table 34. Targeting Exon 6:
Genome Left Half-Site Right Half-Site Genomic Region Coordinates chrX:154212976 TTTACATAACCATTGACTGTGT CCCGAGCAGATGCAGCA Exon (F8) chr3:140445224 TGCTGCATtaGCTCaGa CCaGAGCAGAgGCAGCt Intergenic chr8:56002214 TaCTGCATCTtCTCtGG CtgGAGtAGgcGCtGCA Intergenic chr12:49424040 gGtgGCATCTGCTCttG CCCGgGCAGAgGCAGCA Exon (MLL2) chr1:70622888 TtCTaCtTCTGCTttaG tCtGtGtAGATGCAGCA Intron (LRRC40) chr4:184357162 TtCTGCcTCTGCTCGaG ttttAcaAGATGCAGCA Intergenic chr5:172342828 TGCaGCcTCTGCTCaGa CCtGAGCtGggGttGCA Intron (ERGIC1) chr6:115061184 TGtTaCAcCTGCTCtGG gCtGAGCAtATGCAGgA Intergenic chr12:39726775 TGaTGCATCTGtTtcGa CCtGAGCAGgTGCAtCA Exon (KIF21A) chr7:88799625 TTTACcTAACCAaTGAaaGTGT CCtttGtAGATGCAGaA
Intron (ZUF804B) chr20:17949040 TGCTGCAgCaaCTCGGG CtCGAGCAGggGCcGCc Exon (SNX5) chr1:189751560 TttTcCATCaGCTCaGa CCtGAGCAGcTtCAGCA Intergenic chr21:42907464 TGCcaCATCaGCTCtGG CCaGAGCAGcaGgAGCA Intergenic chr5:2548607 TGCTGCcTCTGCcttca CatGAGCAGgTGCAGCA Intergenic chr8:19923395 TtCTaCATCTGCTCaGa tCCtgGgAagTGCAGCA Intergenic chr6:15883284 TGCTGtcTCTGCTCaGG CCtGAGCgGAaGCAGag Intergenic chr17:81092958 TGCaGCcTCTGCTCcaG tCCcAGgAGATGtAGaA Intergenic chrX:153711226 TGCTGCATCTaCTCctG CCCGgGCAGATctAttg Intergenic chr1:3370563 TGCaGCcTCTGCcCGGG tCCcAGCAGgcGgAGCA Promoter (ARHGEF16) chr17:58495805 TaCTGCATCTtCTCaGa CaaaAGCAGtTtCAaCA Intergenic chr5:169541385 TGtTGCATCaGCTCGGG CCtGAtCAGcgaCAGCc Intergenic Table35.TargetingExon7:
Genome Left Half-Site Right Half-Site Genomic Region Coordinates chrX:154197644 TGTCCAAGGTCCATCAAGAG GGAAAGTTATTGGCGAGA Exon (F8) chr2:18105031 TGTCaAAaaTCaATCAAaAa tTaTTGATtGAttTTtGACA
Intron (KCNS3) chr7:26500117 TGTCCAAaGTCCATtttGAG tTtTTcATGGACacTGGgCA
Intron 1L0C441204) chr4:27239786 TGTCacAGGTCCtTaAAGAG atAAAGTTATTGGgGtGA Intergenic chr4:27428400 TCTtaCCAATcACTTTCt GGAAAGgcAgTGGtGAGA Intergenic chrX:79810036 TGTCCAAaGTCacTtgAGAG GGAAAGTTgTTtGaGAGt Intergenic chr1:172943650 TaTCCAgacTCCATCcAcAG tTaTgGAaGGAgtTTGGACA Intergenic SUBSTITUTE SHEET (RULE 26) ak 02951882 2016-12-09 Table 35. Targeting Exon 7:
Genome Left Half-Site Right Half-Site Genomic Region Coordinates chr18:40289853 aGTCCAAcaTCCAgCAAGAa CTCTTGATtGAgCTTaGAac Intergenic chr17:53122291 TCTtttCAATAACTgTCC CTaTTGATGGACaTTaGACt Intron ISTXBP4) chr1:184048225 TCTgGCCAATAACcgTtC CTCTTaATGatCtTTGGAtA Intergenic chr19:32600353 TGaCCctGaTCCATCcAGAG GacAAGTTAgTGGCcAGA Intergenic chr3:29286452 TGcCaAAGagCCATCAAGAa ttAAAGTTATgGGaaAGA Intergenic chrX:145253799 TGTCCAAGGTCCcaCAgttG CTCTTGATGccCaTTGtAgA Intergenic chr9:85073714 TcctCAAGGgCaATCtAGAG CTCTTGATtGtCtTgGGtCA Intergenic chr22:25490404 TGTCCAAGGcCCcTCAgcAG GGgAAGTaAaaGGtGAGA
Intron (KIAA1671) chr8:61847049 TCcaGagAcTAACTTTgC CcCTTGATtGACCTaGGACA Intergenic chr4:177996308 TGTCCAgaGTCCAagAAaAa CaCTTGAaGGAtggTGGAaA Intergenic chr2:63471205 TaTCaAAGGTCtcTCAAaAc CTCTTGAattAttTTGGgCA
Intron (WDPCP) chr14:101569007 TGTCCAcatTCCcTCcAGAG CcCaTGATGGACCcaGccCA Intergenic chr2:75005696 ctTCCAAGGcCCAcagAGAG CcCcTGATtGcCtTTGGAtA Intergenic chr18:36812500 TCTCtCCAATAACTgTga tgCTTcATGtAtCTTGGcCA Intron (L00647946) Table36.TargedngExon8:
Genome Left Half-Site Right Half-Site Genomic Region Coordinates chrX:154194740 TCCTCCTCTTCAGCAGCAATGT
TTAGGATGCTTCTTGGCAACTGA Exon (F8) chr5:33245024 cCAGaTtCCAAGAgaCATCaTAA
ACATgGCaGCTGAAGAGGAtGt Intergenic chr3:159590558 TCCTCCTCaTCAGtAatAATGT
TTAGaATGtTcagTtGCAAtTGt Intron (SCHIP1) chrY:14031090 TCAtTTtCaAtGgAtCATCCTAA
ACATgGagGagGAgGAGGAGGA Intergenic chr10:83854828 TCctTTtCCtgGAAGCtTtCTcA
TTtGGATGCTTtTgGGaAcCTGA Intron (NRG3) chr12:86811646 TCAaaaGCCAAaAAaCAagCaAA
TTAttATGCTcaTTtGCAAaTGA Intron (MGAT4C) chr6:43379997 TgAGaTaCCAttAcaCATCCTAg AaAgTGCTGgTGAAGAtGtGGA Intergenic chr15:60816292 TCtgCCTCcTCccCAcCcATaT
TTAGGcTGCTTCTTGGCAcCTtc Intron (RORA) chr4:104036767 TtAaaaGCCAgGAAGCATCCTAA
ttATTGaTtaTGAAtgcGAGGA Intron (CENPE) chr2:220922430 aCAaTTcCacAGAAtCATCCaAA
aatGGATGCTcCTTGGCAtCaGA Intergenic chr6:151256031 TCAGcTaCCAAGAgaaATtCTAA
TTgGGAcatTTaTTtGCAcCTGg Intron (MTHFD1L) chr12:14116257 TCtcCCTCaTCAGCAGaAATGa gCATgaCaGCTGtAGtGGAGGg Intron (GRIN2B) chr11:41540671 TttTCaTCTTCAtCtGtgATtT
caATTGCTGCTGAAGgtGAGGA Intergenic chr10:607478 TaCTCCTCTaaAaCcaCAATGg acAGGATGgTTCTcaGCcACTGA Intron (DIP2C) chr18:64076819 TCAtTTaCCAAacAGaATtaTAA
gTAaGATGtTTCcTGatttCTGA Intergenic chr3:159590555 TCaTCCTCcTCAtCAGtAATaa TTAGaATGtTcagTtGCAAtTGt Intron (SCHIP1) chr2:25775417 TCCcCaTCaTtAGCAGCAATGc TcAGGtTtCcTtTTGcaAACaGA Intron (DTNB) chr5:60672404 aCCTCCaCTTCAGtAatAATGa TTAGaATGtgTtaTGtCAttTGA Intron (ZSWIM6) chr2:158235451 TCAaaTGaCAtaAcaCATtCTAA
tCATTatTaCTGAAGtGGAGGt Intergenic chr11:131914316 TCtGagGCCAAaAAGaAaaaTAA AtgTgtCTGtTcAAGAGGAGGA Intron (NTM) chrY:3867095 aCAGTTaCCAAaAAGCAaaaTAA
gCAagatgGCTGAAtAGGAaGA Intergenic Table 37. Targeting Exon 9:
Genome Left Half-Site Right Half-Site Genomic Region Coordinates chrX:154194255 TCTCCAACTTCCCCATAA TTCCTGATTCATGCTGAA Exon (F8) chr4:150672318 TTCAGCtTaAcaCtGGAt TTCCTGATTCcTGaTGAA Intergenic chr2:89399484 TgCAGCATagATCAGGgA TcCCTGgTTtcTGCTGAA Intergenic chr5:19372097 TTCAtCATaAAgCtaaAA TTCtTaATTaATGCTGAA Intergenic chr4:56376997 TTCAGaATGAAaCAGGAA TTCCTGAgaCAaGaTGgg Intron (CLOCK) chr14:98831622 TtTCCtcCTTCCCCATAc gTtCTGATTCATGaTGAA Intergenic chr20:6216194 TTCAGCATGAAgCAaGAA TTCCTGAaaCATcaacAA Intergenic chr3:76350178 TTCAGCtTGAATtAGGAA cTtgTGtTTaATGaTGAA Intergenic chr6:79957598 TTCAGCATaAATaAtaAA TTCtTGtTTaATtCTcAA Intergenic chr5:129714571 TTCAcCATctATCtGaAA TTtCTGAggCATGtTGAA Intergenic chr2:183992955 aTCAaCATGtAaCAGaAA TTttTGATTCATGtaGgA Intron (NUP35) SUBSTITUTE SHEET (RULE 26) Table 37. Targeting Exon 9:
Genome Left Half-Site Right Half-Site Genomic Region Coordinates chr11:100927598 TTCAatATGAtTaAGtAt TTgaTGATTtATGCTGAA Intron (PGR) chr5:118162509 TgCAGCAgtAAaCAtGAA TTtCTaATTCATGCTaAA Intergenic chr7:136796091 TgCAGCATaAATtAaGgA aTCCTGggTCATGtTGAA Intron(L0C349160) chrX:114442244 TTCcaCATaAAaaAGGAc TTCCTGtTgtAgGCTGAA Intron (LRCH2) chr17:70147587 TTaAaaATGAATCAaaAc TTtCaGATcaATGCTGAA Intergenic chr22:17414552 TgCAGCATGAATtAGGAg TcCCTGgTTtcTGCTGAt Intergenic chr1:220485886 TTCAGgAgaAATCgaGAA TTCCTGATatATGtTGAg Intergenic chr2:89292060 TgCAGCATagATCAGGAg TcCCTGgTTttTGCTGAt Intergenic chr2:89309611 TgCAGCATagATCAGGAg TcCCTGgTTttTGCTGAt Intergenic chr2:90260070 aTCAGCAaaAAcCAGGgA cTCCTGATctATGCTGcA Intergenic Table38.Targeting Exon 10:
Left Half-Site Right Half-Site Genomic Genome Coordinates Region chrX:154189360 TCTCCTTGAATACAAAGGAC CCGTGAGGGTAGATGTTATA Exon (F8) chr6:129821493 TgTCCTTaAAaACAAAGGAC CttTGAGGtTAcATGTTAgA Intron(LAMAZ
chr2:147755789 TtTCCTTGgATACAAAGaAC aaaaTTTaTATgCAAGGAGg Intergenic chr15:35542434 TATAAgATaTACCCTaAtGG tTCCTgTGTcTTCAAaGAGA Intergenic chrX:106606342 TCTCCcTGcATACAgAGatC GTtCTTTGTATaagAGGAGg Intergenic chr11:116391255 TCTCCaaaAATAaAAAaGAa GcCtaTTGTATTCcAGGAaA Intergenic chr4:174370428 TaTCtTcaAATtCAAAGGAC aTCCTTTGTAgTCAAGGAtg Intergenic chrX:48388946 TgTCCTTGcATgCAAAatAC cTCtTTTGTtTTtttGGAGA Intergenic chr1:184030566 TCTtaTTattTACAAAGagC GTCtcTTtTATTgAAGGAGA Intron(TSEN15) chr8:105838647 aCatCTTaAATACAAAGaAC GgCaTcTGTAaTCAAGtgGA Intergenic chr14:60101345 TCTCCaTaAATACAAAGGga CaGaGgGGGaAaATtTTAcA Intron(RTN1) chr6:32447046 gCTCtTTGtgaACAAAGGcC tTCCTTTGTATTtActGAGA Intergenic chr6_cibl_hap6:3707956 gCTCtTTGtgaACAAAGGcC tTCCTTTGTATTtActGAGA Intergenic chr6_apd_hap1:3761430 gCTCtTTGtgaACAAAGGcC tTCCTTTGTATTtActGAGA Intergenic chr6:153043585 TgTAAtATtTtCCCcCAaGc GTatTTTGTATTCAAtGtGA bmn(NWCT1) chrX:129578399 TCaCCaTcAgTgCAAgaGAC GgCtTTgGTATTaAAtGAGA Intergenic chr2:237165553 TCTCgTaGAAagCAAAGaAa tTttTcTGTATTtAAaGAGA Mtron(ASB18) chr14:74504800 TATcttATCTcCCCTaAtaG GTCCTTTGTATTCAttGAaA Mtron(C14off45) chr14:94651285 TCTCCTgGggaAtgAAGGtC GatacTTGTATTCAAGGAGA Mtron(PPP4R4) chr14:42051030 TtTCCTaGtATACAAAaGAt aTCtTTTGTATaCtAGGAaA Intergenic chr11:31557496 caTCCTTGgATACAgAGGgC GattTTgGTATTCAtGGAGt Mtron(ELP4) Table39.TargetingExon11:
Genome Left Half-Site Right Half-Site Genomic Region Coordinates chrX:154185248 TCTACAGATTCTTTGTAGCAG ATGAGTCCTGAAGCTAGA Exon (F8) chr8:91254790 TCTAGtTTCAGcAgTatT ATGAGTCaTGAAGCTtGA Intron(LINC00534) chr2:220340352 TCcAtCTTCAGGACTCAc AgGAGcCCTGAAGtTtGg Intron (SPEG) chr13:65583211 TtTACAGATgCTTTaTAGCAG CTGgcAatAAacATCTGTAGA Intergenic chr8:136213502 cCTACAaATcCTTTGTgGCAG ATGgGctCTGgAGCcAGA Intergenic chr4:79545446 TtcAcCTTCctGACTCAT ATGAGTtCTGggGCTAGA Intergenic chr6:105454604 TCTcaCTTCAGGACcCAg ATaAGTttTGAAGCagGA
Intron (LIN28B) chr17:50618031 TCcAaCcTCAGaACTCAT cTGAGTtCTGAgGtTgGg Intergenic chr21:40482039 TCTAaaaTCAGGACTCcT gTGAtTgtTGAAGCcAGA Intergenic chr11:132218577 TCTcaCTTaAGGACTtAc tTGAGTCCaGAAGtTtGA Intergenic chr2:27385297 TCTgtCTTCAGaAgTCcT gTGAGTtCTGAAtCTgGA Intergenic chr14:22481030 TCTAcCTTCAGcACTCtg tTttGTtCTGAAGCcAGA Intergenic chr3:31348185 TCTcGCaTCAaGACcCAT tgGAGTtCaGAtGCTAaA Intergenic chr4:87584049 aCTACAGcTaCTTgGaAGCAG tTGAGcCCaGAAGtTtGA
Intron (PTPN13) SUBSTITUTE SHEET (RULE 26) ak 02951882 2016-12-09 Table39.Targeting Exon 11:
Genome Left Half-Site Right Half-Site Genomic Region Coordinates chr4:71281490 TCaAaCTcCtGacCTCAT tTGtTtCAAAtAATtTGTAtA Intergenic chr2:108857249 TCTctCTcCAGtACTCAT ATGtGTgCTGtgGgTAGA
Intergenic chrX:47785928 TgTAGCTTCtGtACTacT ATaAGTCtTGAAGtcAGA
Intergenic chr8:79584265 TCTtGCcTgAGGACTCAT tgGgGaCtTGAAGtTAGA
Intron (ZC2HC1A) chr1:216023388 TCaAGaTcCAGaACTCAa ATaAGTaCTGAAGCTAtt Intron (USH2A) chr17:50619873 TaTAcaTaCAGaACTtAT ATGAGTtCTGAgGtTAGg Intergenic chr13:20930589 aCTAGCTTCAttAtTCAT ATtAGTCtTGAAGtatGA
Intergenic Table40.Targeting Exon 12:
Genome Left Half-Site Right Half-Site Genomic Region Coordinates chrX:154182199 TCCTCAAGCTGCACTCCAGCT CGTTGTATATTCTCTGTGA
Exon (F8) chr7:156430074 TCCaCAAGCTGgACTCCAaCT atTTGaAcAcTtTCTGTGA Intergenic chr9:43597045 TCACAaAGAATAaACAACt CtaTGTATATaaTCTtTtA
Intergenic chr10:899227 TCcCAGtGAATATAaAAat tGTTGTATATTtaaTGTGA
Intron(LARP4B) chr5:44595593 TCAaAGtGgAaATACAACa CtTTGTATATTtTCTtTtA
Intergenic chr12:13837730 TCcCAGAGAAaATACcAaG CGTTaTcTcTTtTtTGTGA Intron(GRIN2B) chr10:85585731 TCAtAGAaAATAagaAACt tGTTGTATATTCTgTGTcA Intergenic chr10:64580474 TCcCAGAGgcTATAaAcCa AaCTGttGTGaAGCTTGAGGA Intergenic chrX:38783417 TCCTCAAaCTGCtCTCCAaCa CtTccTATtTgtTCTtTGA Intergenic chr2:193570138 TtACAtAGAATtTACAAta CaTTGTAaATTCTaTGTGA Intergenic chr7:110741635 TaAtAcAGAATATACAtaG tcTTGTATATTtcCTGTGA Intron(IMMP2L) chr3:191344909 TCcCAaAGAcTgTtCtAaG gGTgtTATATTCTCTGTGA Intergenic chr9:39389206 TaAaAGAttATATACAtaG ttTTGTtTATTCTtTGTGA
Intergenic chr9:39918509 TaAaAGAttATATACAtaG ttTTGTtTATTCTtTGTGA
Intergenic chr9:40733954 TaAaAGAttATATACAtaG ttTTGTtTATTCTtTGTGA
Intergenic chr9:41293775 TCACAaAGAATAaACAAaa CtaTGTATATaaTCTtTtA
Intergenic chr9:65476200 TCACAaAGAATAaACAAaa CtaTGTATATaaTCTtTtA
Intergenic chrX:50790890 gCACAGActATAggCAgCc CaTgGTATATTCTtTGTGA
Intergenic chr5:5141262 TCCcCAAcCTttcCTCCttCT CGTTGctTATTCTCaGTGA Intron(ADAMTS16) Intron(LOC10087306 chrX:22329605 TCAaAtgGAgTAaACAACt CtTTGTAcATTtTCTGTGt 5) chr7:105616909 TCACAGAGcATATACtcCa ttTaGTATATTCaCaGTcA
Intron (CDHR3) Table41.Targeting Exon 13:
Genome Left Half-Site Right Half-Site Genomic Region Coordinates chrX:154176028 TGTGTCTTCATAGACCATTTT
ATCCAGAGAAGAAGACA Exon (F8) chr19:31555212 TaTCTTCTTCTCTGGAT cTCCAtgGAAGAAaAaA
Intergenic chr11:98185196 TaTcTCTTaATAGcCCATTTT ATaCAGAGAAGAAaACA Intergenic chr9:126179092 TGTGTCTTtATgGAaCAacTa ATtCAGAGAAtAAGACA Intron (DENND1A) chr1:197582736 aGTtcTCaTCcCTGtAT cTCCAGAGAAGAAGACA
Intron (DENND1B) chr9:25886338 TtTtTaCTTCTCaGaAT ATtCAGAGAAGcAGAtA
Intergenic chr16:65046771 TGcCTTCTTCTCTGaAT cTCtAGAccAaAAGtCA
Intron (CDH11) chr6:37769405 TGaGTCTTCATAGAaCATTTT AgCtgGAagAGAAGACc Intergenic chr4:53116406 TGgCTTCTgCTCTGtgT AgCCAGAGAtGAAGtCA
Intergenic chr10:117955396 acTaaaCTTCTCTGaAT AgCCAGAGAtGAAGACA
Intron (GFRA1) chr4:157999316 TaTaTTCTTaTaTGGAg AAggTGGTtTATGAAGACACA Intron (GLRB) chr4:172676113 TGTCaTCTTCTCTGtAT tTtaAGAGAAaAAtACt Intergenic chr7:70692951 TGcCTTCTTCcCTGGAT cgatAGAGgAGgAGACA
Intron (WBSCR17) chr1:153460499 TGTCTTCTTCTCTGtcT ATCtAGAGAAtggGAgt Intergenic chr17:55521352 gGTCaTCaTCTtTGGtT AgCCAGgGAAGAAGACA
Intron (MSI2) chr15:37159972 TGTtTTCTTCTCTGcAT tAAATaaTCTATGAtGAgAtA Intron(L0C145845) chr10:81475753 TcTCTTCTTCTCTGtAT AggCAtAGAtGAtGgCA
Intergenic chr10:88997979 TcTCTTCTTCTCTGtAT AggCAtAGAtGAtGgCA
Intergenic SUBSTITUTE SHEET (RULE 26) Table 41. Targeting Exon 13:
Genome Left Half-Site Right Half-Site Genomic Region Coordinate s ch r10 :89259535 TGcCaTCaTCTaTGccT ATaCAGAGAAGAAGAgA Intergenic chr2:12846210 ctTCTTCTTCTCTGaAT ATatAtAGAAGAAtAtA Intergenic chr13:107009889 TGTCTcCcaCTCTGctg AT a CAGAGAAGAAG gCA Intergenic Table 42. Targeting Exon 14:
Genome Left Half-Site Right Half-Site Genomic Coordinates Region chrX:154156874 TCATCCCATAATCCCAGAGCCTCT AAATAGTGTCGTGTTTTCTTTTGA Exon (F8) Intron chr6:17669261 TgAAAAaAAAAaAaaACACTATTa AAATAcctTttTtTTTTtTTTTGA (NUP153) Intron chr11:12730893 TaAAAAaAAAAaACcAgAaTAaTT ttATAGTtTtGTtTcTTtTTTTGA ITEAD1) chill: 68651384 TCAAAAaAAAcCAaaACACTtaTT
AAtTAaTtTtaTtTaTTtTTTTGA Intergenic Intron chr5:132729450 TagAAAGgAgACAaGggtCTAgTT AGAaGCTCTGtGAgTtTGGGATGA (FSTL4) chr5:102197872 TCAAAAaAAAAaAaaAaAaaAaTT AcATAtTGTCtTtTTTTtTTTTaA Intergenic Intron chr6:150020193 TCAAAAaAAAAaAaGgCACTATcT AGtaGgTtaGGGtTTcTGaaATGA (LATS1) Intron chr8:102067589 TCAgAAaAtAAtAtGACACTtTTg AAATttTGTCaTGTTTgCTTTaGA (FLJ42969) Intron chr5:96436598 aaAAAAaAAAAaAaaAgAaTATaT AAtTAGTGTtGTcTTTTCcTgTGA (LIX1) chr22:31430439 TCAAAAaAAAAaAaGcCcCTgTcc AtATAtTtTttTtTTTTtTTTTGA Intergenic Intron chr5:96436600 aaAAAAaAAAAaAaGAataTATaT AAtTAGTGTtGTcTTTTCcTgTGA (LIX1) chr8:129874245 TtAAAAGAAAcagCGACACTATTT AtAaAaTagCaTtTTcTCTTcTGA Intergenic chr8:76048195 TaAcAcagAAtCACctCACTATaT tAATAGTtTttTtTTTTtTTTTGA Intergenic Intron chr3:167630709 TtAAAAaAAAAaAaaAgcCTATTT AAATtGTGaCaTcTTTTtTTTTaA (L00646168) chr17:79330592 TCAAAAaAAAAaAaaAaAtTATTT tttTttTGTttTGTTTTgTTTTGt Intergenic Intron chr7:56511801 aaAAAAGAAAACtgGtgtCaATTT AAAaAGTGTCGgGTTTTtTTTTtt (L00650226) Intron chrX:108947147 TaAAAAaAAAAaAattCACTATgT AAATAtTGTgGgGTTTTtTTgTtg (ACSL4) chr12:123230886 TCAAtAaAAAtaAaaAtAaaATTT tAATAGTaTttTtTTTTtTTTTGA
Intergenic chr3:163374286 TaAAccaAAAACtCaACAaTcaTT AAATAtgGTtGgtTTgTtTTTTGA Intergenic Intron chr12:9357687 TCAAAAaAAAACAaaACAaagTTT gAAaAGTcTttTcTTTTtTaTTtA (PEP) chr2:188514899 TCAAAAGtAAAaAgtAaACTATTT tAATAGTGagGTaaTTTCTTTatA Intergenic Table 43. Targeting Exon 15:
Genome Left Half-Site Right Half-Site Genomic Region Coordinates chrX:154134726 TATGGCCCCAGGAGTCCCAA CTCCACGGTATAAGGGCTGA Exon (F8) chr1:43805061 TATGGCCCCAGagaTCCCAA tcCCACGGTcatAcaGCTGA Exon (MPL) chr17:48220703 TATaGCCCCcatgGTCaCcA CTtCAgGGcATAgGGGCTGA Intron (PPP1R9B) chr6:10659136 TCAatCCTTATgCCaaGGAG TctGGtCTCCTGtGGtCAcA Intergenic chr4:138564864 TATGaCCCaAaGAaaCCaAA tTCtAtGtTAaAAGtGaTGA Intergenic chr1:242357075 TgTGaCCCCAGGAGTCatAA CTtCAaGGgcTAtGGGagGA Intron (PLD5) chr20:53898975 TCAaCCCTaATtCCtTaGAG CTCtAgGGgATAAGGctTcA Intergenic chr16:10915221 TcTGaCCCtAaGAaTCaCcA TTGGGgtTCCTGGaGtCATg Intergenic chr10:134224399 TgTGGCCCCAGGgGcCCaAc agGGGACTttTGGGGgCgTA Intron (PWWP2B) chrX:17609569 TaAGCCCTTATAatGgGtAG tTCCAtGGTATttGGtaTGA Intron (NHS) chr12:4412126 TggGcCCCaAGGAGTCCCAc TTGGGAaTCtTGGaGCCtaA Exon (CCND2) c1ir22:48089574 TgTGGgCCCAGGAGTCaCgA CcCCAgGGTATcAGGGtgGc Intergenic chr17:1538247 TgTGGCCCCAGGAagCCCAg TTGGGgCTCtgGccGaCAgA Exon (SCARF1) SUBSTITUTE SHEET (RULE 26) chr19:35657806 TAccaCCCCAGcAGTCaCAA tggCAgGGaAcAAGGGCTGA Intron (FXYD5) chr1:158375793 TcTaGCtCCAtaAGTCCCtA TTGGGtCTCtTGGGatCtgA Intergenic chr14:99426061 TCAGCaCTTATcCaGTGGAc TTGGGACaCCaGaGaaCAcA Intergenic chr1:34177797 cATcaCaCCAGGAtTCCCAA TgGGGtCcCCTGGGGtCAgg Intron (CSMD2) chr13:19522623 cCAcCCCcccTACaGgGGAG TgGGcACTCCTGGGcCCATA Intergenic chr11:17783271 TcTGGCCCCAtGgaTCCCAA caGaGcCTCCTGGGGCacaA Intron (KCNC1) chr14:71921590 TCtGCCCTTtTACtGTGGAG acGGGACaCCTGatGtCAcA Intergenic chr10:132968471 TCAGCCaTTccACCGTGGAa acGGctCTCCgGGGGCCAct Intron (TCERG1L) SUBSTITUTE SHEET (RULE 26) Table 44. Targeting Exon 16:
Genome Left Half-Site Right Half-Site Genomic Coordinates Region chrX:154133096 TCAGAGAAATAAGCCCAG CTCATCTTTAGTGGGTGCCATA Exon (F8) chr7:25537263 TCtGtGcAATAAtCtCAG CTGtGCTTATTTaTtTGA Intergenic chr1:85241221 TaAaAaAAAaAAGCCCAG CTGGGCTTtcTTCTggGA Intergenic chr17:49365434 TCcaAGAAAcAAaCCCAa CaGGtgTTAcTTCTCTGA Exon (UTP18) Intron chr10:15407376 TATGaCAtCaACTAAAGATGcG agGGGCTTAaTTCcCaGA (FAM171A1) chr6:66455619 cCAGAcAgAgAAcCCCAG CTGGGtTTATTgCaCTGA Intergenic chr2:168339348 TCAaAaAAgaAAGCCaAG CTGtGCTTATaTCTCTcA Intergenic Intron chr8:3275497 TCAGtGAcATAAGCCCAG CTGtGCTTgTTaaaaTGA (CSMD1) chr1:172577364 TCAtAGtAATAAaCagAG tTGtGtTTATTTCTCTaA Intron (SUCO) chr9:131943933 gaAGgGgAATAgGCCCAa CTGGcCTTATTTCTCTGt Intergenic chr14:30487657 TCAtAGAAATAtGCCCAa CTGaGCTcATgggTtTGA Intergenic chr3:82950355 aCAtAtAAATAAGaaCAt CTtGGCTTATTTtaCTGA Intergenic Intron chr22:40341367 TCAGAGAAATgAGCCCct tcGGctTTAaTcCTCTGA (GRAP2) Intron chr20:19686090 TtgGAaAAATAAtCCCAG taGGGCTTATTTgctTGA (SLC24A3) Intron chr4:20811976 TCAGAGAcAatAtCaaAG gTGGGtTTATTTgTCTGA (KCNIP4) chrX:97284124 TCAGgGcAATcAGCCCAG CTGGGgTTtcTTgTCTGg Intergenic chr18:41220996 TCAaAtgAATAAGaCaAt tTGGttTTgTTTCTCTGA Intergenic chrY:19504648 TCAGgaAAAaAAtCCCAG CTtGttTTATTctcCTGA Intergenic chr6:11989807 TCAtAtAAATgAGCtCAt CTtGGCTTcTTTCaCTGA Intergenic Intron chr11:100111323 TaAaAttAATgAGCCCAG tTtGGCTTATTTCcaTGA (CNTN5) Intron chr13:26279732 agAGAGAAAaAgGCCgAG tTGGGtTTATTTtTCTaA (ATP8A2) Table 45. Targeting Exon 17:
Genome Left Half-Site Right Half-Site Genomic Region Coordinates chrX:154132638 TCTTTCCATATTTTCAG TTTGGTCTCATCAAAGA Exon (F8) chr11:86435291 aCTTTCCATAgTTTCAG CTGAAAATATtGAAtGA Intergenic chr17:191390 TCTTaGAgGAcACCAAA TTTGGTgTCATCtAAGA Intron (RPH3AL) chrX:16807199 TCTaTCCtTtTTTTCAG tTGAAAATATtGAAAGA Intron (TXLNG) chrX:4909433 TtTTTCCATATTTTCAG TcaGtTtTCtTCAAAGA Intergenic chr15:98192520 TCTTTCCAcATTTTCAG CTGAAAATATtaAAtaA Intergenic chr3:65632758 TCTTTGAaaAGACCAAA CTGAcAAcAgGGAAAaA Intron (MAGI1) chrX:81782933 TCaTTtaATATTTTtgG CTGAAAATgTGGAAAGA Intergenic chr20:48433923 TCTTTaATGAtACCAAA TTaGGTCTttTCAgAaA
Intron (SLC9A8) chr8:84366161 TCaTTtCATATTTTCAG CTGAAAtTgTGGAAAGt Intergenic chr1:93406669 atTTTGATaAGAtCAAA TTTGGTgTCATCtAAGA Intron (FAM69A) chr3:23702529 TaTTTGATttaAtCAAA TTTGGTtTCATgAAAGA Intergenic chr4:127360864 TCTTTCCAcATTcTCtG gTTGGTtTCATCcAAGA Intergenic chr9:10862420 TtTTaGAaGAaAaCAAA TTTGGTgTCAgCAAAGA Intergenic chr2:30136701 TCTcTCCATATTcTCca CTGAAAATAcaGAAAGA Intron (ALK) Intron chr2:8966383 TtTTTaATaAtcCCAAA TTgGGgCTCATtAAAGA (KIDINS220) chr10:106620765 TCcTgGgTGAGACCcAA TcTGGTtTCATCAAgGA Intron (SORCS3) chrX:108769761 TaTTTGATGAGACCAAc aTGAgAATATaGcAAGA Intergenic chr1:111227475 TCaTTtaATATTTTCAG CTGAAAtTATGGAAAGc Intergenic chr3:114347859 TCTTTGATGAaAaCcAA TTTGtTtTCAcaAAtGA
Intron (ZBTB20) chr6:24241996 TCTTTCCATATTTTaAt taGAAtATATGaAtAGA Intron (DCDC2) SUBSTITUTE SHEET (RULE 26) Table 46. Targeting Exon 18:
Genome Left Half-Site Right Half-Site Genomic Region Coordinates chrX:154132208 TATACTCCTCTTTTTTTCG GTCCACTGAAATGAATAGA Exon (F8) chr3:89963270 TCTATTCATTaCtGTttAC GTCCAtTGAAtTGcATAaA Intergenic SUBSTITUTE SHEET (RULE 26) ak 02951882 2016-12-09 Table 46. Targeting Exon 18:
Genome Left Half-Site Right Half-Site Genomic Region Coordinates chr13:71330234 TtTATTCATTTCAtTGaAa GTCtAtTtAAATaAAgAGA Intergenic chr7:52504835 TCTATaCATTTCAGaacAC GcaCACTaAAAaGAAcAGA Intergenic chr7:93233952 aATACTCCTCcTTcTTTtt aTaCACTGAAATGgATAGA Intergenic chr20:8957392 TATAaaCgTtTaTTTTTCt GTtaACTGAAATGAcTAGA Intergenic Intron chr2:55547229 TATACTtCTCTTTTgTTCa tGAAAAAAtGtGtAcTAgA (CCDC88A) chr6:55916123 cATACTCCTCTTaTTTTCa tgCCACTGAAATGAcTttt Intergenic chr8:93952422 TCTATcCATgTCAaaGaAC GTCttCTcAAATGtAcAGA Intron (TRIQK) chr14:61101496 TCTATcCATTTCtGTGtAC tGcAAAtAAaAGtAGTATt Intergenic chr11:33381162 TATACTtCTaTTTTTTTat aGAAAAAgAGAGtAGTAcA Intergenic chr6:84078984 TCTATTacTgaCAcTGaAC GTCtACTGAAgTGAActGA Intron (ME1) chr11:123025415 aATcCcCCTCaTTTTTctG tTCCACTGAAATGAtTAtA Intron (CLMP) chr1:58698828 TAatCaCCTCTTTTTcTCc GTatAtTGAAATGtAgAGA Intron (DAB1) chr13:90438048 TCTATTaATaTCAGTaaAC GgCCAaTGAAAcaAATgGc Intergenic chr3:20841157 TCTtccCATTTCtGTGaAa GTtaAaTGgAATGAATAGA Intergenic chr5:22000977 TCTATTaAaaTCAaTaGAC GTttACTtAcATtAtTAGA Intron (CDH12) chr5:69306485 TCTATTaAaaTCAaTaGAC GTttACTtAcATtAtTAGA Intergenic chr5:70181567 TCTATTaAaaTCAaTaGAC GTttACTtAcATtAtTAGA Intergenic chr3:62322281 aCTATaCATTTCAaTaGtC tTCCACTGtAATtAgTAtA Intergenic chr1:239837471 TtaAaTtATTTCcGTGGAa GTCCACaGAtATGAATAtA Intron (CHRM3) Table 47. Targeting Exon 19:
Genome Left Half-Site Right Half-Site Genomic Region Coordinates chrX:154130370 TGCTCGCCAATAAGGCATTCC AGCTTTGGATGGTAACA Exon (F8) chr4:53352906 TGTgACCATCCAAgGCT AGCaTTGGAgGGgAACA Intergenic chr21:36529769 TGTTcCCAcCCAAAtCT AGaTTTGGgTGGggACA Intergenic chr9:76182583 aaTTACaAaCaAAAGCc tGCTTTtGATGGTAAtA Intergenic chr3:81470457 TGTTACttTgCAAAtgc AatTTTGGATGGTAACA Intergenic chr1:203239036 TGTTACCAgCCAAAcCT AGggaTGGAgGGTtgCA Intergenic chr3:65643349 TGTTtCCtTtaAAAtCT AGCTTTGtcTGGTAACA
Intron (MAGI1) chr2:52456162 TaTTgCCtTCatcAGCT AGCTTTGGAaGGTAtCA Intergenic chr4:150055809 TtTcACCATCCAAAtCT AttgTTGGgTGGTAAgA Intergenic Intron chr11:43851516 TacTACCATaCAAAGCT tGgaTTGGATGtTcACA (HSD17B12) chr7:114250318 TaTTACtgTCtAtAtCT AGCTTTGaATGGTAAaA
Intron (FOXP2) chr3:167657104 TGTgAaCATCCAAgGCT AGCTcTtGATGGTcACt Intergenic chrX:149844333 TGgTgCCtaCCAcAcCT AGCTTTGGATGGTcAgA Intergenic chr9:29156612 TGaTAaCtTCCAAgaCT gtCTTTGGAaGGTAACA
Intron (LING02) chr4:70236889 TaTTACCATCaAAAtCa AGCTTTtGtaGGTAAtg Intergenic chr3:151160745 aaTTcCaAcCCAAAGgT AGCcTTGGATGGTAACc Exon (IGSF10) chr13:35431619 TtTTACCcTCCAAAcCc AGCTTTGGAaaaTAACA Intergenic chr4:29377428 TGTTAaaATCCtAAtCc AcCTTTGGATGGTAAtt Intergenic chr13:62451673 TGTTcCCAcCCAAAtCT AGagTTGGAgGGaAgtA Intergenic chr12:95616056 TtTTcCCATttAgAtCT AttTTTGtATGGTAACA Intron (VEET) chr18:28761651 TagaACCATCCAAAaCT AGaTTTGcATGtTtAaA Intergenic Table 48. Targeting Exon 20:
Genome Left Half-Site Right Half-Site Genomic Region Coordinates chrX:154129651 TGTCCTGAAGCTGTAATCTGAA CCAGAAGCCATTCCCAGGGGA Exon (F8) chr8:31295960 TCCCCTaGGAcTGaCTTCaGa CCAGActCtATTgCCAtGtGg Intergenic chr2:165151202 aaTCCaGAAGCaGTAAcCaGtA CgtGAAtCCtTTCCCAGGGGA Intergenic chr15:66216735 TCCCCaGGGAATGGgaTCTGG aCAGggGtCtcTCCCAGtGGt Intron (MEGF11) chr14:97246034 TgCCaTGGGAtTtGCTTCTGc CCAGAAGCagTcttCAGGGGA Intergenic chr1:17425225 TCCaCTGaaAtgacCTTCTGG CCtGtAGtCATgCCCAtGGGA
Intron (PADI2) SUBSTITUTE SHEET (RULE 26) ak 02951882 2016-12-09 Table 48. Targeting Exon 20:
Genome Left Half-Site Right Half-Site Genomic Region Coordinates chr19:11752845 TCCCCTGGGAcactCagCTtt CCAGAttCCATTCCttGGGGA Intergenic chr6:165113924 TCCCtTGGcAATtGCTTCTct CCccAttCCATTCaCAGGGGA Intergenic chr3:18310932 TtCCCTGattATaGCTTtctG CCAGAAGaCATTtCaAGGaGA Intergenic chr16:54478454 TCtCCaGaGAgaGGCTTCTaG CCtGAtGtCcTTCCtttGGGA Intergenic chr2:100885233 TCCtCaGtcAATGGCTTCTGG atgGAAaCCAgTCCaAGGGaA Intergenic chr6:160576093 TgCtCTtGGgATGtCTTCTGG taAGAAtCCATTCCtAGGatA Intron (SLC22A1) chr1:888254 TaCCCTGGccATGGCcTCaGG agAGAgGCCcTcCCCtGGGGA Intron (NOC2L) chr11:24688064 TCCatTGaaAATaGCTcCTGa gCAGgAGCtATTCtCAGacGA Intron (LUZP2) chr3:188747522 TCCCtTGtGAATGGCTTggtG aCcGtAGtCATTCCCAtGaGA Intergenic chr10:74502577 TcTCCTGAAGaTGTAATtaGAg CCtGAgGtgATTtCtAGGGGg Intron (MCU) Intron chrX:28644076 TCCaCaGaGAATaGtTTaTGc CttGtAcCCATTCCatGGGGA (IL1RAPL1) chr2:167140954 cGTCCTtAcGCTGTcATCaGAA gCAGAAGCtgTcCattGGGGA Intron (SCN9A) chr10:3095266 gCaCCTtGaAATGGgcaCTGG CCgGAAGCCATTCCaAatGGA Intergenic chr5:73250307 TCCCCTGGGAActGCTgaTGG CCAGAAGggATggtaAaGGGA Intergenic chr1:145822030 TCaCCTGGGAATaGtaTCTaG CaAGAAGaaAacaCtAGaGGA Intron (GPR89A) Table 49. Targeting Exon 21:
Genome Left Half-Site Right Half-Site Genomic Region Coordinates chrX:154128167 TGCTCCAGGCATTGATTGAT
CTGGCCAGCTTTGGGGCCCA Exon (F8) chr10:123955374 TGGtCCCacAgGCTGGCCAG CTGGaCAGCTcTGGGcCCCA Intron (TACC2) chr6:73606839 TaCTCCAGGCATaGAagGAg tTGGaCcaCTTTGGGGCCCA Intron (KCNQ5) chr15:87990891 aGaGCCCCAtAtCTccCaAG ATCAgTCAtTGtCTGGAGCA Intergenic chr13:104866433 TGCTtCAGaCAcTGATTGAg aTtGCCAcaTTTGGGGCCCA Intergenic Intron chr21:44889451 TGGtCCCCAAAcCTGGCCAa CTGGaCAGaTgccaGGgCCA (LINC00313) chr15:72922848 gGaGgCCCAAAcgTGGCCtt CTaGCCAGCTcTGGGGCCCA Intergenic chr8:20252698 TGCTCattGCAcTGgTgGAT CTGGCaAGCTTTGGGGtCtg Intergenic chr18:32975516 TGtGgCCCAtAGCTGGCCAG CTGGCCAGCTaTGGGttttc Intergenic chr16:989379 TGcGCCaCAAAGCTGGCCAc AgCAATaAAaaCCaGGAaCA Intron (LMF1) chr20:44515651 TGGGCCCCAggcCTGGgCAG
CTGctCAGCTTTctGGCtCA Exon (SPATA25) chr2:240861687 TaGGCaCCtcAGCTGGCCAa CTGGgCAGCcTgGGaGCCCt Intergenic chr9:132364724 TGaGCCaCtgAGCTGGCCAG cTtAtTCctTGtCTGGAGaA Intergenic Intron chr1:151341446 TGGtCtaCtgAGCTGGCaAG tTGtgCAGCTTTGGGGCCCg (SELENBP1) Intron chr12:1996302 TGGaCCCCcAAGaTGGCCAt CaGaaCAGCTTTGGaGCtag (CACNA2D4) chr16:68354549 TGCTgCAGagATTtgTTtAT
tTGGCCAGaTTTGGGGgCCt Intron (PRMT7) Intron chr3:64099060 TGGGgCCCcAgcCTGGCCAc tTGGgtAcCTTgGGGGCCCA (PRICKLE2) chr12:133199141 TGGtCCCCAcAGCcaGCCAG CTGcCCAGgcTgGGaGtgCA Intergenic chr12:53741716 TaaGaaCCAAAGCTaatCAG tTcttCAGtTTTGtGGCCCA Intergenic chr16:3006381 TGGGgCCCAAAtgaaGCCAG CctGCCAGCcTTGGGGtCCt Intergenic chr5:53389184 aGcaCCCCAAAcCTGGCCtG
tTGGgCAGCaTTtGGcCCCA Intron (ARL15) Table 50. Targeting Exon 22:
Genome Left Half-Site Right Half-Site Genomic Region Coordinates chrX:154124384 TCTGCCACTTCTTCCCATCAAG ATAAACTGAGAGATGTAGA Exon (F8) chr17:55200444 TCaACATCTgTCAGacgAT ATAAAaTGAGAGtTGTAGc Intergenic Intron chr7:149959793 TCTACATCTaaCAtTTTAT ATAAAtgGAaAacTGgAGA
(ACTR3C) chr3:182164176 TgcACATCTCTCAcTTTAa AaAAgCTGAGAGAgGTtGA
Intergenic chr8:85206496 TgTgCtTaTCTaAGTacAT gcAAAtTGAGAGATGTAGA
Intron (RALYL) SUBSTITUTE SHEET (RULE 26) Table 50. Targeting Exon 22:
Genome Left Half-Site Right Half-Site Genomic Region Coordinates chr1:107949372 TtTACATCTaTCAGTTTAT AaAAACTGAGctAcagAGg Intron (NTNG1) Promoter chr3:150421949 TCTtCgTCTCTCAGcTTAT CTTGggtGGAgGAAGTGGCttc (FAM194A) chr8:22075977 gCTcCATCTCaaAaaTaAT ATAAAaTGAtAGATGcAGA Intergenic Intron chr5:56152387 TaTACATtTCTCAtTTTAT tTtAgtcGtGAGATGgAGA (MAP3K1) chrX:147805582 TtgGCCACTTCTTCCCATCccG tTAAcCTGAaAcATGgAGA
Intron (AFF2) SUBSTITUTE SHEET (RULE 26) Table 50. Targeting Exon 22:
Genome Left Half-Site Right Half-Site Genomic Region Coordinates chr3:59243225 aCgAtATCaCTatGTTTAc ATAAtCTGAGAGtTGTAtA Intergenic chr15:88546432 TCTAgATCTaaCtGacaAT ATAAACTGgGAGgcGTAGA Intron (NTRK3) chr3:101738660 TCTAgATCTCTCAGgTTAa caActCTGtGAGATGaAGA Intergenic Intron chr15:64473144 TCTAgtTCTCTCAGTTTAT ATAgACTtAGtGcTGatGt (CSNK1G1) chr15:96928325 agTACATCTtTtAaTTTAT CcTGATGGGAAGAAtTaGaAGA Intergenic chr11:85386305 cCatCcTCaCTaAGTTTAa tTAAAgTGAGAGATGTAtA Intergenic chr5:117743942 TCTcCATCTggCAaTTgAg cTAAACTGgaAGATGTAGA Intergenic chr1:5052686 TaTACATtTCTCAGTTgAT CTTGtTctGAcGAtGctGCAGA Intergenic chr6:9920117 caTACATCTCTCAcTTTAT tTAAACTtAGtGAgGaAGg Intergenic chr1:159052090 TCTcCATgTCTCAGTTTgT ATAgACTaAGtGActTAtA Intergenic chr20:25560526 TCTACAaaTgTaAaaTTcT AaAAACTGAGAGATtTtGA Intron (NINL) [0231] In all exons 1-22, favorable sites were able to be located for TALENs, Cas9-nuclease, Cas9 paired-nickase, and dCas9 RNA-guided FokI Nucleases (RFNs). These sites met guidelines established for predicting high on-target activity (using the SAPTA
algorithm for TALENs and avoiding stretches of pyrimidines in the PAM-proximal region of the target).
These sites also met guidelines established for being relatively unique throughout the genome and having no high-scoring predicted off-target sites. Analysis of TALEN sites using PROGNOS yielded no sites generating warnings as scoring substantially similar to the designated target site. Analysis of Cas9-nuclease off-target sites found in almost all cases that no sites existed with fewer than two mismatches to the target sequence;
furthermore, sites with few mismatches typically had mismatches in disruptive regions such as the PAM, or the 12 bp PAM-proximal 'seed region'. Cas9-nickases and RFNs have been shown to have very low off-target activity approaching the detection limit of deep-sequencing assays (Ran & Hsu et al. Cell 2013, Tsai SQ et al. Nature Biotech 2014).
[0232] Taken together, this example identified the sequences to repair the F8 gene at the 3' end of any exon 1-22 for TALENs, Cas9-nucleases, Cas9-nickases, or RFNs; by using the above-mentioned selected target sites. High on-target activity allows efficacious clinical repair of HA and low off-target activity ensures the safety of the proposed therapy.
Example 4: Homologous Repair Vehicles for repair at different exon-intron junctions [0233] Repair at different exon-intron junctions throughout the FVIII gene employ methodology similar to example 3 described above, the repair vehicles used however are different for each junction. This example describes various repair vehicles.

SUBSTITUTE SHEET (RULE 26) [0234] All repair vehicles contain the same basic components: a left homology arm corresponding to the genomic sequence 5' of the relevant nuclease cut site, a cDNA sequence SUBSTITUTE SHEET (RULE 26) incorporated herein by reference. Final consideration was given to choosing individual sgRNAs which each had low potential for off-target activity throughout the human genome, as assessed by the online computational tool described by Hsu et al in Nature Biotechnology 2013, incorporated herein by reference.
[0240] Sequences listed in Table 55 below contain identified binding sites for paired CRISPR
nickases within exons 1-22 respectively.
Table 55 FVIII Genomic Target of SG/PG RNAs Genome Editing Gene (Desired Activity) (Region) (DNA Sequence) paired nickase (5') Exon 1 5'-CACTAAAGCAGAATCGCAAAaGG
paired nickase (3') 5'-AAGATACTACCTGGGTGCAGtGG
Exon 2 paired nickase (5') 5'-AGTCTTTTTGTACACGACTGaGG
paired nickase (3') 5'-TTTTCAACATCGCTAAGCCAaGG
Exon 3 paired nickase (5') 5'-CAGCATGAAGACTGACAGGAtGG
paired nickase (3') 5'-ATGCTGTTGGTGTATCCTACtGG
Exon 4 paired nickase (5') 5T-TATGAGTAGGTAAGGCACAGtGG
paired nickase (3') 5'-GACTTGAATTCAGGCCTCATtGG
Exon 5 paired nickase (5') 5'-AAGTAGTATAAATTTGTGCAaGG
paired nickase (3') 5'-CTTTTTGCTGTATTTGATGAaGG
Exon6 paired nickase (5') 5'-GACTCTGIGCATITTAGGCCaGG
paired nickase (3') 5'-CAGTCAATGGTTATGTAAACaGG
Exon 7 paired nickase (5') 5'-GCGACATITCCAAGGACGCCaGG
paired nickase (3') 5'-CAAACACTCTTGATGGACCTGG
Exon 8 paired nickase (5') 5'-ICTICGCAACTGAGCGAATTaGG
paired nickase (3') 5'-ACATTACATTGCTGCTGAAGaGG
Exon9 paired nickase (5') 5'-AATACCTTCACGAGTCTTAAaGG
paired nickase (3') 5T-GAASCIATTCAGCATGAATCaGG
Exon 10 paired nickase (5') 5'-GGACATCAGTGATTCCGTGAgGG
paired nickase (3') s'-ATGICCGICCITIGTATTCAaGG

SUBSTITUTE SHEET (RULE 26) Exon 11 paired nickase (5') 5'-AACSPAACTAGAGTAATAGCgGG

SUBSTITUTE SHEET (RULE 26) Table 55 FVIII Genomic Target of SG/PG RNAs Genome Editing Gene (Desired Activity) (Region) (DNA Sequence) paired nickase (3') ' -GAICTAGCTICAGGPTI7CATTTGG
Exon 12 paired nickase (5') 5 ' -AGCST IGTATATICICIGTGaGG
paired nickase (3') 5 ' -CGCTT ICICCCCAAT:CAGC-TGG
Exon 13 paired nickase (5') 5 -ATACA CCAT TT TGIGTITGAaGG
paired nickase (3') 5 ' -AGAAPCTGICT TCATGICGATTGG
Exon 14 paired nickase (5') 5 ' -IIIICITIIGAGCICCGGgGG
paired nickase (3') 5 ' -ACATTIAT T TAT TGCTGCAG-TGG
Exon 15 paired nickase (5') 5 ' -ACGSTATAAGGGCTGAGIAAaGG
paired nickase (3') 5 ' -AAATCAACATT TGGGACTCCT_GG
Exon 16 paired nickase (5') 5'-CAGTCAAACTCATCTTTAGTgGG
paired nickase (3') 5 ' -ATGACITTGACTGCAAAGCCcGG
Exon 17 paired nickase (5') 5'-ITCACTGAAGTACCAGCTIT-TGG
paired nickase (3') 5 ' - GGCTCCCIGCAATAT :CAGATTGG
Exon 18 paired nickase (5') 5 ' -GTCCPCTGAAATGAATAGAATTGG
paired nickase (3') 5'-GTTCPCTGTACGAAAAAAAGaGG
Exon 19 paired nickase (5') 5 ' -CGCCPAATICCACCITIGGA-GG
paired nickase (3') 5 ' -AT TSC CGACCATCTACATGCcGG
Exon 20 paired nickase (5') 5 ' -IGIC,CAGAACCCATTT,CCAGgGG
paired nickase (3') 5'-GATTITCAGATTACAGCTICaGG
Exon 21 paired nickase (5') 5 ' -TGATCCGGAATAATGAAGTC-TGG
paired nickase (3') 5'-AAT2PATGCCIGGAGCACCAaGG
Exon 22 paired nickase (5') 5'-A ,A5AL,AC'AC,,,C
paired nickase (3') 5 -AAGAAGTGCCAGACITATCGaGG
10241] The spacing requirements between the sgRNAs differ between paired CRISPR
nickases and RFNs, but the other considerations regarding on-target and off-target activity remain the same and were taken into account when searching for RFN target sites in exons 1-22.

SUBSTITUTE SHEET (RULE 26) 10242] The ¨140 bp of the 3' end of each exon (hg19 human genome build) was searched for RFN binding sites matching the spacing distances using the ZiFiT targeter disclosed in Tsai SQ et al. Nature Biotech 2014, incorporated herein by reference. For some exons, there was no targetable sequence matching the PAM orientation and spacing requirements of the RFN
system. Sequences in table 56 below contain identified binding sites for RFNs within exons 1-22 respectively.
Table 56 FVIII Genome Genomic Target of RFN
Gene Editing (DNA Sequence) (Region) Position 5' Half-Site 5'-GCACCCAGGTAGTATCTTCtGG
Exon 1 3' Half-Site 5'-ACTATATGCAAAGTGATCTcGG
5' Half-Site No Compatible Sites Exon 2 3' Half-Site No Compatible Sites 5' Half-Site No Compatible Sites Exon 3 3' Half-Site No Compatible Sites 5' Half-Site 5'-ACATGAGAAAGATATGAGTaGG
Exon 4 3' Half-Site 5'-ACTTGAATTCAGGCCTCATtGG
5' Half-Site 5'-AAGGTCTGTGTCTTTTCCTtGG
Exon 5 3' Half-Site 5'-TTTTTGCTGTATTTGATGAaGG
5' Half-Site 5'-TTTTCCCTGATGAGAGAGAaGG
Exon 6 3' Half-Site 5'-ACAAAGAACTCCTTGATGCaGG
5' Half-Site 5'-GTTATTGGCGAGATTTCCAaGG
Exon 7 3' Half-Site 5'-AAACACTCTTGATGGACCTtGG
5' Half-Site No Compatible Sites Exon 8 3' Half-Site No Compatible Sites 5' Half-Site 5'-ATAGCTTCACGAGTCTTAAaGG
Exon 9 3' Half-Site 5'-TCTTGGGACCTTTACTTTAtGG
5' Half-Site No Compatible Sites Exon 10 3' Half-Site No Compatible Sites 5' Half-Site 5'-ACGAAACTAGAGTAATAGCgGG
Exon 11 3' Half-Site 5'-ATCTAGCTTCAGGACTCATtGG
5' Half-Site No Compatible Sites Exon 12 3' Half-Site No Compatible Sites 5' Half-Site No Compatible Sites Exon 13 3' Half-Site No Compatible Sites 5' Half-Site 5'-TGTTTTCTTTTGAAAGCTGoGG
Exon 14 3' Half-Site 5'-GCTGCAGTGGAGAGGCTCTgGG
5' Half-Site No Compatible Sites Exon 15 3' Half-Site No Compatible Sites 5' Half-Site 5'-AGTCAAACTCATCTTTAGTgGG
Exon 16 3' Half-Site 5'-TATTTCTCTGATGTTGACCtGG
5' Half-Site 5'-CTTTTGGTCTCATCAAAGAtGG
Exon 17 3' Half-Site 5'-AATATGGAAAGAAACTGCAgGG
5' Half-Site No Compatible Sites Exon 18 3' Half-Site No Compatible Sites 5' Half-Site 5'-GCCAAATTCCAGCTTTGGAtGG
Exon 19 3' Half-Site 5'-TTGGCGAGCATCTACATGCtGG
5' Half-Site 5'-TGTCCAGAAGCCATTCCCAgGG
Exon 20 3' Half-Site 5'-TTACAGCTTCAGGACAATAtGG
5' Half-Site 5'-GATCCGGAATAATGAAGTCtGG
Exon 21 3' Half-Site 5'-CACCAAGGAGCCCTTTTCTtGG

SUBSTITUTE SHEET (RULE 26) Table 56 FVIII Genome Genomic Target of RFN
Gene Editing (DNA Sequence) (Region) Position 5' Half-Site 5'-AGGCTGGAGAACTTCTGACgGG
Exon 22 3' Half-Site 5'-TCATCATGTATAGTCTTGAtGG
Example 6: Additional Methods and Examples for FVIII Gene Repair in Cells Purifying CRISPR/Cas9 plasmids and repair plasmids (DNA-RS) [0243] A protocol for preparing CRISPR/Cas9 plasmids (DNA-SE) and repair plasmids (DNA-RS) using endotoxin-free methods is described in the following example. For this protocol, a Qiagen EndoFree Plasmid Maxi Kit is used. The Qiagen EndoFree Plasmid Maxi Kit and its contents are stored at room temperature. Once RNAse and LyseBlue are added to Buffer P1 from the kit, this buffer is stored at 4 C. The kit also requires 100%
ethanol and isopropanol (2-propanol).
[0244] According to this protocol, at Day 1, a lmL seed culture of Escherichia coil (E. coli) in Luria Broth (LB) and appropriate antibiotic is prepared and placed on a shaker at 37 C. Whether an antibiotic is appropriate is dependent on the antibiotic resistance gene that is present in the plasmid that is being prepared and purified. For example, such an antibiotic may be ampicillin, kanamycin, or other antibiotics. Approximately 5 hours from when the seed culture is prepared, the seed culture is then used to inoculate a 100 mL
LB culture and the suspension is left shaking overnight (or for at least about 8 hours) at 37 C.
[0245] At day 2, the 100 mL culture is transferred into 2x50 mL conical tubes and spun for min at 4000g; the supernatant is dumped out. The resulting cell pellet can be stored at -C for an indefinite period of time. During the spin, Buffer P3 is placed on ice. Following the spin and removal of the supernatant, 10 mL of Buffer P1 are added to the first 50 mL tube of each prep. This solution is then vortexed to resuspend the pelleted cells.
The resuspended mixture is poured a second tube and vortexed to resuspend. Next, 10 mL of Buffer P2 are added and the suspension is inverted 6X to mix (until mixture is homogenously blue). This suspension is incubated for 3 min at room temperature. Next, 10 mL of Buffer P3 is added to each tube, and each tube is inverted ¨10X.
[0246] Next, the suspensions are centrifuged for 5 minutes at 4000g. During the spin, a fresh SUBSTITUTE SHEET (RULE 26) 50 mL tube is labeled for each abovementioned prep. A cap is screwed onto a filter cartridge and placed in the fresh 50 mL tube. After the spin, a p1000 pipette tip is used to hold back debris while pouring the liquid from the spun suspension into the cartridge.
The suspension is then incubated for 10 minutes at room temperature in the cartridge. Next, the cartridge is uncapped and a plunger is used to push the liquid into the 50 mL tube; the cartridge/plunger is trashed following this step. Next, 2.5 mL of Buffer ER is added to each tube, and each tube is inverted 10X until the liquid becomes cloudy. The suspension is incubated on ice for 30 minutes. During the incubation, Qiagen-Tip-500 tubes are labeled and placed in a clamp draining into a 1000 mL beaker. 10 mL of Buffer QBT is added to Qiagen-Tips to equilibrate the system. After the 30 minute incubation, the prep mixture is poured into the respectively labeled Qiagen-tips. Buffer QC is used to wash the tips.
[0247] Next, the Qiagen-Tip-Tubes are placed into 50 mL tubes capable of withstanding spins @ 15000g. 15 mL of Buffer QN is added to the Qiagen-Tip-Tubes and centrifuged at 4 C to allow the DNA to elute from the Qiagen-Tip-Tubes as the buffer QN
drains through.
The eluted DNA can be stored at 4 C overnight.
[0248] Next, 10.5 mL of Isopropanol is added and the suspension is inverted 10X to mix. The samples are then centrifuged at 15000g for 10 min at 4 C; The DNA will be present as a pellet. After the supernatant is dumped out, 5 mL of 70% Ethanol (Et0H) is added to the pelleted DNA. The samples are centrifuged at 15000g for 10 min at 4 C. Then, the supernatant is decanted using a p1000 pipette. The tube is then left to air-dry for 10 min.
Next, 150 uL of Tris EDTA buffer (TE) is added. Isolated plasmid concentration is then determined.
[0249] In the example described, four CRISPR plasmids were prepared using these methods, each in triplicate, in addition to the preparation of a pGFP plasmid in duplicate. These procedures yielded the results shown in Table 57:
Table 57: Concentration of isolated CRISPR and pGFP plasmid preps Sample # [DNA] Unit A260 A280 260/280 260/230 pH0007-1 273.7 ng/u1 5.475 2.881 1.9 2.28 Table 57: Concentration of isolated CRISPR and pGFP plasmid preps Sample # [DNA] Unit A260 A280 260/280 260/230 pH0007-2 262.8 ng/n1 5.257 2.771 1.9 2.26 pH0007-3 350 ng/n1 7 3.688 1.9 2.27 pH0009-1 328.1 ng/n1 6.561 3.462 1.9 2.26 pH0009-2 345 ng/n1 6.901 3.637 1.9 2.27 pH0009-3 274.9 ng/n1 5.499 2.909 1.89 2.19 pH0011-1 320.4 ng/n1 6.408 3.378 1.9 2.26 pH0011-2 295.2 ng/n1 5.905 3.122 1.89 2.25 pH0011-3 328 ng/n1 6.559 3.469 1.89 2.27 pH0013-1 323.3 ng/n1 6.466 3.388 1.91 2.27 pH0013-2 311 ng/n1 6.22 3.274 1.9 2.22 pH0013-3 306.7 ng/n1 6.135 3.23 1.9 2.28 pGFP-1 273.8 ng/n1 5.477 2.877 1.9 2.28 pGFP-2 341.9 ng/n1 6.838 3.623 1.89 2.2 Nucleofection Conditions and Methods [0250] A protocol for nucleofection is described in the following example. The protocol described uses 20uL Nucleovette Strips (Lonza). The number of cells recommended for this technique is 200,000 cells per condition or sample. The maximum mass of DNA
used in this technique is -1000ng. It is recommended that a significantly greater amount of repair plasmid be used compared to the CRISPR/Cas9 plasmid as this minimizes the likelihood of off-target effects while maximizing the likelihood of homologous recombination. Typically a ratio of 4:1 repair plasmid:CRISPR/Cas9 plasmid is used.
[0251] To facilitate all of the analyses involved with these methods, the following reaction conditions are recommended. First, for the "experimental" condition, 200ng of CRISPR/Cas9 plasmid (DNA-SE), 800ng of repair plasmid (DNA-RS), and 4Ong of MaxGFP plasmid are used for transfection. Second, for the "no repair plasmid" control condition (also suitable for T7 Endonuclease (T7E1) analysis), 200ng of CRISPR/Cas9 plasmid (DNA-SE), 800ng of stiffer plasmid (pUC19), and 40ng of MaxGFP plasmid are used for transfection.
Third, for the "no CRISPR plasmid" condition, 200ng of stuffer plasmid (pUC19), 800ng of repair plasmid (DNA-RS), and 40ng of MaxGFP plasmid are used for transfection.
Fourth, for the "GFP alone" condition, 1000ng of stuffer plasmid (pUC19) and 40ng of MaxGFP
plasmid are used for transfection.
[0252] For the method, first, 500u1 of media is added to the required number of wells in a 24 well plate. This is pre-warmed in an incubator set to 37 C, 5% CO2. Next, lng of total DNA
in minimum of 2 1 is used. Next, the DNA is setup into a new strip tubes.
[0253] Next, the cells are prepared for nucleofection. 200,000 cells per nucleofection reaction are preferred. 1.2X of master mix of cells is prepared to account for cell loss during media aspiration and pipetting errors. Next, the cells are pelleted by centrifugation at 300 xg for 5 minutes. Next, if the Nucleocuvette strip kit is used, a nucleofection solution provided with kit is used. All of the supplement is added to Nucleofector solution; 20n1 of the combined buffer is required per nucleofection.
[0254] Next, during the spin a plate is labeled. The media is then aspirated from the cells and the cells are resuspended in 1.1X Nucleofector buffer (22u1 per nucleofection -352 uL / 16 nucleofections, 374 uL / 17 reactions). Next, 20u1 of cell suspension (approx.. 200,000 cells) is aliquoted to DNA solutions. Next, the Nucleocuvette strip is placed in the 4D Nucleofector X-module and the corresponding program is selected. Next, the cuvette is allowed to incubate for 10 minute following shocking of the cells. Next, 50u1 of media from 24 well plate is added to the Nucleocuvette. All of the cell/media mix from the cuvette is then added to the 24 well plate and incubated at 37 C for 72 hours.
Protocol for QuickExtract Method for gDNA Extraction [0255] A protocol for gDNA extraction is described in the following example.
This method allows for the extraction of genomic DNA (gDNA) from live cell samples using QuickExtractTM DNA Extraction Solution (Epicentre). First, about 100,000 cells are pelleted by centrifugation. Then 80 lut of the QuickExtract solution is added to the cells and the suspension is transferred to a thermocycler tube. The suspension is then vortexed. The suspension is then run in a thermocycler for 15 min at 65 C and 8 min at 98 C;
The solution can then be stored at -20 C and freeze/thawed for at least 40 times. Next, ¨1 lut of this solution is used as the genomic DNA template per 50 [EL of PCR reaction.
Protocol for T7E1 Assay [0256] A protocol for a T7E1 assay is described in the following example.
According to the protocol, 35 cycles of PCR is used on isolated gDNA to amplify a target locus at the exon22/intron22 boundary using T7E1 primers that flank this boundary. The forward primer has a sequence of 5'-GGTAATGATGGACACACCTGTAGC-3' and the reverse primer has a sequence of 5'-GGTTTTGCCCCCTAAACTTGTC-3' and PCR with these primers results in amplicons of 623 nucleotides in length. The PCR amplicons are then purified using Wizard SV Gel and PCR Clean-up System (Promega) according to manufacturer's instructions.
[0257] Next, 200ng of purified PCR product is placed in lx NEBuffer 2 (New England Biolabs, Buffer 2, a component of the T7 Endonuclease 1 kit that is available from New England Biolabs) in a total volume of 18uL. Next, the suspension is vortexed and centrifuged. Next, the samples are placed in a thermocycler programmed with the following protocol: A) 95 C for 5 mm; B) 95-25 C in ¨1 C/s steps; C) hold at 4 C.
[0258] 10 units of T7 Endonuclease 1 is are added to the hybridized PCR
products in a 2uL
volume of lx NEBuffer 2 (for a final reaction volume of 20uL). Note that for each sample, a side-by-side negative control (no T7E1 enzyme control) is prepared, wherein 2uL volume of lx NEBuffer is used in the absence of the enzyme. Next, the suspensions are vortexed and centrifuged. The suspensions are then incubated at 37 C for 30 minutes.
Following incubation, the samples are placed on ice and stop solution is added to them.
The stop solution is prepared by adding 2.45uL 0.5M EDTA to 4.49 uL 6X loading dye for each reaction (6.94uL volume per reaction, resulting in a final concentration of 45mM EDTA and lx loading dye).
[0259] Next, the samples by agarose gel electrophoresis. The gel image can be quantified with ImageJ using the following procedure: 1) the image is inverted; 2) the background is subtracted (set to 30 pixels, check light background box); 3) rectangles are drawn about the middle of a gel lane, avoid the "smiling" on the end of the gel lanes; 4) in the analyze gel lane, "select first lane" option is selected; 5) subsequent lanes are selected; 6) Quantitative analysis is performed (fraction cleaved= area cleaved/ area of all); 7) Calculate % gene modification with the following equation:

% gene modification = 100 x (1 ¨ (1 ¨ fraction cleaved)1/2) Protocol for Restriction Fragment Length Polymorphism (RFLP) Assay [0260] A protocol for a RFLP assay is described in the following example.
According to the protocol, 35 cycles of PCR is used on gDNA to amplify a target locus at the exon22/intron22 boundary using RFLP primers that flank this boundary. The forward primer has a sequence of 5'- GTTAGGTGACTCAAATGGGTTCAC-3' and the reverse primer has a sequence of 5'-GAACAAGAAGCAGGGTAGAGAAGC-3' and PCR with these primers results in amplicons of 1667 nucleotides in length. The PCR amplicons are purified using Wizard SV
Gel and PCR Clean-up System (Promega) according to manufacturer's instructions.
[0261] Next, a mixture with 20 [it reaction with 0.5[EL (5U) of restriction enzyme, 2uL
reaction buffer (provided in the enzyme kit), and then 17.5 [it of the cleaned PCR reaction is prepared. This mixture is then incubated at 37 C for 1 hour. Next, the samples are analyzed the samples by agarose gel electrophoresis. The gel image is then quantified with ImageJ
using the following procedure: 1) the image is inverted; 2) the background is subtracted (set to 30 pixels, check light background box); 3) rectangles are drawn about the middle of a gel lane, avoid the "smiling" on the end of the gel lanes; 4) in the analyze gel lane, "select first lane" option is selected; 5) subsequent lanes are selected; 6) Quantitative analysis is performed (fraction cleaved= area cleaved/ area of all); 7) Calculation of %
homologous recombination with the following equation:
%HR = (cut band) / (cut band + uncut band) Protocol for PCR Amplification at Gene Repair Site [0262] A protocol for PCR amplification at a gene repair site is described in the following example. According to the protocol, as a first qualitative approach, PCR with RFLP primers is performed to examine the presence of a band distinct from the main band.
The primers and procedures in this method are the same as those described above in the section entitled "Protocol for Restriction Fragment Length Polymorphism (RFLP) Assay." The main (uncut) band is expected to be about 1.7kb in size, wherease the cut band is expected to be about 1.0kb in size.
[0263] In a second qualitative approach according to this protocol, a reverse RFLP primer (with sequence 5'- GAACAAGAAGCAGGGTAGAGAAGC-3') that anneals within exon 22 is paired with a primer that anneals within the gene repair site (with sequence 5'-AAGATGGCCATCAGTGGACTCTC-3') is used. This PCR will only form a product of about 1.3kb in size if there is successful gene correction.
[0264] Following analysis of the results from the PCR analyses described above, clonal colonies are grown out. This is done either through limiting dilution of the cells or by FACS
sorting of single cells into a 96-well plate. With either method, initially plate 1 cell into ¨50 uL of media. Then after 1 week add ¨150 uL of new media to the wells. After about a second week, or when there are >10,000 cells, use the QuickExtract protocol to isolate gDNA.
Proceed to perform the same two PCRs described above---the 2nd PCR method will demonstrate if there is at least monoallelic gene correction, the first PCR
(with the RFLP
primers) will demonstrate if there is biallelic correction (because all of the PCR product will be at a different band size) and also serve as a positive control to determine that the QuickExtract for that sample is a viable PCR template.
Protocol for gene repair in FVIII
[0265] A protocol for gene repair in FVIII is described in the following example. According to the protocol, seed cell cultures were prepared 2 days before transfection, with a final target density of 800,000 cells/mL on the day of transfection. Next, CRISPR/Cas9 plasmids (DNA-SE) and repair plasmids (DNA-RS) were prepared as indicated above in the protocol for endotoxin-free plasmid maxiprep. Next, the transfection setup details for nucleofection, such as plasmid concentrations and volumes, cell concentrations and volumes were determined as discussed above in the protocol for nucleofection conditions and methods.
Next, nucleofection was performed, followed by culturing the cells for 72 hours as discussed above in the protocol for nucleofection conditions and methods.
[0266] Flow cytometry analysis was used to determine % viability and % GFP+
cells in each sample on one quarter of the cells collected from the nucleofection step.
Results using the CRISPR/Cas9 plasmids pH0007 and pH0009 as well as a repair plasmid (labeled "donor") are shown in Figure 17. In Figure 17 the left-most graph for each sample displays the FSC/SSC characteristics of the population and allows for gating on non-debris in the sample;
the center graph for each sample displays in histogram format the distribution of live cells in the sample as evidenced by inclusion of propidium iodide which enters only dead cells and yields a red fluorescence; and the right-most graph for each sample displays in histogram format the distribution of cells that have been successfully transfected as evidenced by green fluorescence that is due to the presence of GFP. As can be seen from the results, the percentages for each parameter are similar across all samples, with a range for each parameter of 46.8-51.8% (non-debris), 74.9-85.0% (Live), and 22.6-26.8% (GFP+). Thus the rates of successful transfection do not differ substantially as a function of the plasmid used.
[0267] In this example, gDNA from one quarter of the cells from the nucleofection event was isolated following the protocol for gDNA extraction described above. The gDNA
was then analyzed using the following protocols described above: 1) protocol for T7 El assay; 2) protocol for RFLP assay; and 3) protocol for PCR amplification at gene repair site.
[0268] Results from the analysis following the T7E1 assay are shown in Figure 18 and in Figure 19. Figure 18 and Figure 19 show results from using_CRISPR/Cas9 plasmids pH0007, pH0009, pH0011, and pH0013. Figure 18 shows an image from an agarose gel electrophoresis assay. In Figure 18 the samples names are abbreviated such that the three pH0007 are listed as 7-1, 7-2, and 7-3, and this pattern is continued for pH0009, pH0011, and pH0013. A negative control (No DNA) and positive control (+ ctrl) in the analysis. For each sample there are two lanes: one labeled at the top of the lane with a "+"
which sample contained the T7E1 enzyme, and a second labeled with a "¨" which sample contained no T7E1 enzyme. In the absence of T7E1, no nuclease activity is present and there is a single band present in the lane. In the presence of T7E1, some cleavage occurs resulting in a second smaller band that appears. This qualitative data demonstrates that pH0007 and pH0009 yield the better result than pH0011 and pH0013 as there is a greater relative abundance of the smaller band in those samples. This is quantified in Figure 19. Figure 19 shows the calculated values for percent gene modification by NHEJ (non-homologous end joining), demonstrating that pH0007 and pH0009 cause indel formation at the target site at a rate of 66% and 72%
respectively, and that both of these yield statistically significantly superior rates of indel formation compared to pH0011 and pH0013. This statistical significance is evidenced by the error bars which display the standard error of the mean for each sample.
[0269] Results from the analysis following the RFLP assay are shown in Figure 20 and Figure 21. Figure 20 and Figure 21 show results from using CRISPR/Cas9 plasmids pH0007, pH0009, as well as a repair plasmid (labeled "Donor"). Figure 20 shows an image from an agarose gel electrophoresis assay. In Figure 20 displays the results of a simple and standard RFLP assay demonstrating that only in those samples that contain the donor plasmid along with either pH0007 or pH0009 is there a smaller band which indicates restriction digestion, the presence of the restriction site and thus successful recombination in those samples. In the other control samples, no such smaller band is seen. Figure 21 shows the calculated values for percent gene modification by following Intron 22-targeted CRISPR
treatment. As can be seen from the data, homologous recombination occurs only in those samples that were transfected with the donor plasmid and pH0007 or pH0009 at a rate of 22% and 16%
respectively. The control samples that were transfected with only donor plasmid, only pH0007, only pH0009, or none of the three show a rate of homologous recombination of 0%
for each sample.
[0270] Next, cells were cloned out either by limiting serial dilution or single-cell FACS.
Clones were cultured until the clonal colonies reach cell numbers of >20,000.
gDNA from >10,000 cells of each clonal culture using was then extracted. PCR was used to amplify across the repair site, using as template each of the extracted gDNA samples from the clonal cultures. Next, sanger sequencing methods were used to sequence the repair-site PCR
amplicons. Next, the DNA sequence immediately upstream (about 25 bases), immediately downstream (about 25 bases), and across the repair was analyzed.
[0271] Clones not displaying the desired or expected integration events were eliminated.
Next, it was determined if any DNA sequence modifications have been made at sites in the genome that have been predicted by algorithm to be the top 20 potential off-target sites in the genome. Clonal cultures for which DNA sequence modifications have been made at off-target sites in the genome we eliminated.
[0272] Remaining clones were cultured out until clonal colonies reach cell numbers of >1x106. mRNA was extracted from >100,000 cells of each clonal culture; mRNA
was also extracted from >100,000 cells of the parent culture (in which no gene repair has been performed).
[0273] Quantitative reverse-transcription PCR (qRT-PCR) primers were designed for the detection of: a) Transcription of the F8 gene, targeting an exonic site 5' of the gene repair site; b) Transcription of the F8 gene, targeting an exonic site 3' of the gene repair site; c) Transcription of the F8 gene, targeting a sequence that is unique to the gene repair site itself, that furthermore overlaps the junction of (i) the gene repair site and (ii) an endogenous, non-repaired exonic site 5' of the gene repair site. This amplified product should only be detected in cells that have been correctly repaired; and d) Transcription of house-keeping genes that can be used for normalization of F8 gene transcription, including at least the genes for beta-actin (ACTB), gamma-tubulin (TUBG1), and RNA polymerase II (POLR2A).
[0274] Using qRT-PCR methods, transcription of the F8 gene using the mRNA
extracted from each clonal culture and the parent culture was analyzed; yielded a quantitative value for each sample analyzed (ACt value).
[0275] The transcription of the F8 gene across all samples was compared.
Clonal cultures that exhibit the highest AC t values for transcription of F8 when measured using qRT-PCR
primers targeting the gene repair site itself were further isolated. These cells were cultured until the clonal colonies reach cell numbers of >5x107 [0276] Next, >5x107 cells from each culture were removed and pelleted. Cell lysate from the cell pellets was collected. A modified enzyme-linked immunosorbent assay (mELISA) was then used to detect the presence of FVIII protein in both the culture medium and the whole cell lysates from each culture. This yielded a quantitative value for each sample analyzed in units of nanograms of FVIII protein per cell number (ng/5x107 cells). FVIII
protein secretion across all samples was compared. The culture yielding the highest secretion of FVIII protein was chosen to proceed for therapeutic purposes.
[0277] The examples set forth above are provided to give those of ordinary skill in the art a complete disclosure and description of how to make and use the embodiments of the materials, compositions, systems and methods of the disclosure, and are not intended to limit the scope of what the inventors regard as their disclosure.
[0278] All patents and publications mentioned in the specification are indicative of the levels of skill of those skilled in the art to which the disclosure pertains.
[0279] The entire disclosure of each document cited (including patents, patent applications, journal articles, abstracts, laboratory manuals, books, or other disclosures) in the Background, Summary, Detailed Description, and Examples is hereby incorporated herein by reference.
All references cited in this disclosure are incorporated by reference to the same extent as if each reference had been incorporated by reference in its entirety individually. However, if any inconsistency arises between a cited reference and the present disclosure, the present disclosure takes precedence.

[0280] The terms and expressions which have been employed herein are used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the disclosure claimed. Thus, it should be understood that although the disclosure has been specifically disclosed by embodiments, exemplary embodiments and optional features, modification and variation of the concepts herein disclosed can be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this disclosure as defined by the appended claims.
[0281] It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting. As used in this specification and the appended claims, the singular forms "a," "an," and "the"
include plural referents unless the content clearly dictates otherwise. The term "plurality"
includes two or more referents unless the content 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 the disclosure pertains.
[0282] When a Markush group or other grouping is used herein, all individual members of the group and all combinations and possible subcombinations of the group are intended to be individually included in the disclosure. Every combination of components or materials described or exemplified herein can be used to practice the disclosure, unless otherwise stated. One of ordinary skill in the art will appreciate that methods, device elements, and materials other than those specifically exemplified may be employed in the practice of the disclosure without resort to undue experimentation. All art-known functional equivalents, of any such methods, device elements, and materials are intended to be included in this disclosure. Whenever a range is given in the specification, for example, a temperature range, a frequency range, a time range, or a composition range, all intermediate ranges and all subranges, as well as, all individual values included in the ranges given are intended to be included in the disclosure. Any one or more individual members of a range or group disclosed herein may be excluded from a claim of this disclosure. The disclosure illustratively described herein suitably can be practiced in the absence of any element or elements, limitation or limitations which is not specifically disclosed herein.

[0283] A number of embodiments of the disclosure have been described. The specific embodiments provided herein are examples of useful embodiments of the invention and it will be apparent to one skilled in the art that the disclosure can be carried out using a large number of variations of the devices, device components, methods steps set forth in the present description. As will be obvious to one of skill in the art, methods and devices useful for the present methods can include a large number of optional composition and processing elements and steps.
[0284] In particular, it will be understood that various modifications can be made without departing from the spirit and scope of the present disclosure. Accordingly, other embodiments are within the scope of the following claims.

Claims

1. A method for repairing one or more mutations in a Factor VIII gene (F8 gene) sequence of a subject, the method comprising introducing into a cell of the subject one or more polynucleotides encoding a DNA
scission enzyme (DNA-SE) and one or more repair vehicles (RVs) containing at least a cDNA-repair sequence (RS) flanked by an upstream flanking sequence (uFS) and a downstream flanking sequence (dFS) to form a DNA donor within each of the one or more repair vehicles (RVs), wherein the DNA-SE is selected to be capable of targeting a portion of the F8 gene of the subject and to create a first break in one strand of the F8 gene and a second break in the other strand of the F8 gene for subsequent repair by the cDNA-RS, the cDNA-RS comprises a repaired version of the F8 gene sequence of the subject comprising the one or more mutations within a cDNA sequence encoding for a truncated Factor VIII, and the upstream flanking sequence (uFS) is homologous to a nucleic acid sequence upstream of the first break in the one strand of the F8 gene and the downstream flanking sequence (dFS) homologous to a nucleic acid sequences downstream of the second break in the other strand of the F8 gene, and wherein introducing into a cell of the subject one or more polynucleotides encoding a DNA
scission enzyme (DNA-SE) and one or more repair vehicles (RVs) is performed to allow insertion of the cDNA-RS through homologous recombination of the upstream flanking sequence (uFS) and the downstream flanking sequence (dFS) with the F8 gene of the subject (sF8) to provide a repaired F8 gene (rF8), the repaired F8 gene (rF8) upon expression forming a functional FVIII conferring improved coagulation functionality to the FVIII
protein encoded by the sF8.

2. The method of claim 1, wherein the one or more mutations of Factor VIII
gene of the subject result in a mutated Factor VIII gene comprise at least one Factor VIII
functional coding sequence upstream to at least one Factor VIII non-functional coding sequence, the first break and the second break define a DNA-SE target site located upstream of a non-functional coding sequence to be repaired and the cDNA-RS is configured in the one or more repair vehicles to be in frame with the Factor VIII functional coding sequence upstream the DNA-SE target site.

3. The method of claim 2, wherein the DNA-SE target site is located about 50 bp to about 100 bp upstream from a 5' end of the Factor VIII non-functional coding sequence to be repaired.

4. The method of claim 2 or 3, wherein the upstream flanking sequence (uFS) is homologous to a genomic nucleic acid sequence of at least 200 bp from the DNA-SE target site and the downstream flanking sequence (dFS) is homologous to a genomic nucleic acid sequences of at least 200 bp downstream of the DNA-SE target site.

5. The method of any one of claims 2-4, wherein the DNA-SE target site is adjacent to a 3' end of the Factor VIII functional coding sequence.

6. The method of any one of claims 2-5, wherein the 3' end of the functional coding sequence is a 3' end of a Factor VIII exon.

7. The method of any one of claims 2-6, wherein the one or more mutations comprise a replacement of one or more wild type nucleotide residues within an exon of the Factor VIII
gene with one or more mutated nucleotide residues, the Factor VIII non-functional sequence is formed by the one or more mutated residues and the repaired version of the Factor VIII
non-functional coding sequence is formed by the one or more mutated residues replaced by the one or more wild type nucleotide residues.

8. The method of any one of claims 2-6, wherein the one or more mutations comprise an insertion of one or more nucleotide residues within an exon of the Factor VIII
gene, the Factor VIII non-functional sequence is formed by the one or more inserted nucleotide residues and the repaired version of the Factor VIII non-functional coding sequence is formed by at least two nucleotide residues adjacent to a 5' and 3' end of the one or more inserted nucleotide residues.

9. The method of any one of claims 2-6, wherein the one or more mutations comprise a deletion of one or more wild type nucleotide residues of at least one exon of the Factor VIII
gene, the Factor VIII non-functional sequence is formed by one or more nucleotide residues downstream the one or more nucleotide residue deleted from the at least one exons, and the repaired version of the Factor VIII non-functional coding sequence comprises the one or more wild type nucleotide residues deleted from the at least one exon of Factor VIII.

10. The method of any one of claims 2-6, wherein the one or more mutations comprise an intron 22 inversion, the Factor VIII functional coding sequence comprises exons 1 to 22 of the Factor VIII gene, the non-functional coding sequence comprises exons 23 to 24 of the Factor VIII gene and a repaired version of the Factor VIII non-functional coding sequence comprises exons 23 to 26 of the Factor VIII gene.

11. The method of any one of claims 2-10, wherein the upstream flanking sequence (uFS) is homologous to a genomic nucleic acid sequence of at least about 400 bp from the DNA-SE
target site and the downstream flanking sequence (dFS) is homologous to a genomic nucleic acid sequences of at least about 400 bp downstream of the DNA-SE target site.

12. The method of any one of claims 2-10, wherein the upstream flanking sequence (uFS) is homologous to a genomic nucleic acid sequence of at least about 400-800 bp from the DNA-SE target site and the downstream flanking sequence (dFS) is homologous to a genomic nucleic acid sequences of at least about 400-800 bp downstream of the DNA-SE
target site.

13. The method of any one of claims 2-10, wherein the uFS is homologous to a genomic nucleic acid sequence of at least about 800-3000 bp from the DNA-SE target site and the dFS
is homologous to a genomic nucleic acid sequences of at least about 800-3000 bp downstream of the DNA-SE target site.

14. The method of any one of claims 2-13, wherein the cDNA repair sequence (cDNA-RS) encodes for one or more repaired Factor VIII non-functional sequences consisting essentially of the amino acid sequence encoded by exons 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, or an in frame portion or combination thereof.

15. The method of any one of claims 1-14, wherein the cDNA repair sequence (cDNA-RS) is in an editing cassette further comprising a polyadenylation site located at a 3' end of the cDNA repair sequence (cDNA-RS)., the editing cassette flanked by the upstream flanking sequence (uFS) and the downstream flanking sequence (dFS).

16. The method of claim 15, wherein the editing cassette further comprises a splice acceptor operatively linked to the cDNA repair sequence (cDNA-RS).

17. The method of any one of claims 1-16, wherein the one or more mutations cause hemophilia A in the subject and the repair results in treatment of the hemophilia A in the subject.

18. The method of any one of claims 1-16, wherein the repaired version of the Factor VIII
non-functional coding sequence comprises Factor VIII exons of a replacement FVIII protein product and the repair results in inducing immune tolerance to the FVIII
replacement product.

19. A system for repairing one or more mutations in a Factor VIII gene (F8 gene) sequence of a subject, the system comprising one or more polynucleotides encoding a DNA scission enzyme (DNA-SE) and one or more repair vehicles (RVs) containing at least a cDNA-repair sequence (RS) flanked by an upstream flanking sequence (uFS) and a downstream flanking sequence (dFS) to form a DNA
donor within each of the one or more repair vehicles (RVs), wherein the DNA-SE is selected to be capable of targeting a portion of the F8 gene of the subject and to create a first break in one strand of the F8 gene and a second break in the other strand of the F8 gene for subsequent repair by the cDNA-RS, the cDNA-RS comprises a repaired version of the F8 gene sequence of the subject comprising the one or more mutations within a cDNA sequence encoding for a truncated Factor VIII, and the upstream flanking sequence (uFS) is homologous to a nucleic acid sequence upstream of the first break in the one strand of the F8 gene and the downstream flanking sequence (dFS) homologous to a nucleic acid sequences downstream of the second break in the other strand of the F8 gene, and wherein, the DNA scission enzyme (DNA-SE), and the DNA donor are selected and configured so that upon insertion of the cDNA-RS through homologous recombination of the upstream flanking sequence (uFS) and the downstream flanking sequence (dFS) of the DNA
donor sequence with the subject's F8 gene (sF8) a repaired F8 gene (rF8) is provided, the repaired F8 gene (rF8) upon expression forms functional FVIII that confers improved coagulation functionality to the FVIII protein encoded by the sF8 without the repair.

20. The system of claim 19, wherein the one or more nucleic acids encoding a DNA scission enzyme (DNA-SE) encode for a DNA-SE selected from the group consisting of zinc finder nuclease (ZFN), transcription activator-like effector nuclease (TALEN), cluster regularly interspaced short palindromic repeats (CRISPR)-associated (Cas) nuclease, CRISPR-Paired Nickase (CRISPR-PN), and CRISPR-RNA-guided Fok1 nucleases (CRISPR-RFN).

21. The system of claims 19 or 20, wherein the cDNA-RS encodes a truncated Factor VIII
polypeptide consisting essentially of the amino acid sequence encoded by each of exons 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26 of a F8 gene or an in frame combination thereof.

22. A cDNA configured to be used as a cDNA-repair sequence (RS) for repairing one or more mutations in a Factor VIII gene (F8 gene) sequence of a subject, wherein the cDNA
encodes a truncated Factor VIII polypeptide consisting essentially of the amino acid sequence encoded by each of exons 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26 of a F8 gene or an in frame combination thereof.

23. The cDNA of claim 22 wherein the each of the exons has a sequence of a corresponding exon in the F8 gene of the subject.

24. A repair vehicle (RV) configured to be used for repairing one or more mutations in a Factor VIII gene (F8 gene) sequence of a subject in combination with a DNA
scission enzyme (DNA-SE) selected to target a portion of the F8 gene of the subject and to create a first break in one strand of the F8 gene and a second break in the other strand of the F8 gene, the repair vehicle comprising a cDNA-repair sequence (RS) comprising a repaired version of the F8 gene sequence of the subject comprising the one or more mutations within a cDNA
sequence encoding for a truncated Factor VIII.
wherein the cDNA-RS is flanked by an upstream flanking sequence (uFS) and a downstream flanking sequence (dFS) to form a DNA donor within the RV. The upstream flanking sequence (uFS) is homologous to a nucleic acid sequence upstream of the first break in the one strand of the F8 gene and the downstream flanking sequence (dFS) homologous to a nucleic acid sequences downstream of the second break in the other strand of the F8 gene.

25. A polynucleotide encoding a DNA scission enzyme (DNA-SE) configured for repairing one or more mutations in a Factor VIII gene (F8 gene) sequence of a subject, the DNA
scission enzyme selected to be capable of targeting a portion of the F8 gene of the subject and to create a first break in one strand of the F8 gene and a second break in the other strand of the F8 gene for subsequent repair by a cDNA-RS flanked by an upstream flanking sequence (uFS) and a downstream flanking sequence (dFS) to form a DNA donor within each of the one or more repair vehicles (RVs), the cDNA-RS comprising a repaired version of the F8 gene sequence of the subject comprising the one or more mutations within a cDNA sequence encoding for a truncated Factor VIII, and the upstream flanking sequence (uFS) being homologous to a nucleic acid sequence upstream of the first break in the one strand of the F8 gene and the downstream flanking sequence (dFS) homologous to a nucleic acid sequences downstream of the second break in the other strand of the F8 gene.

26. A cell comprising the one or more repair vehicles (RVs) of claim 24 and one or more polynucleotide encoding the DNA scission enzyme (DNA-SE).

27. A composition for repairing one or more mutations in a Factor VIII gene (F8 gene) sequence of a subject, the composition comprising one or more repair vehicles (RVs) according to claim 24 and one or more polynucleotides encoding the DNA
scission enzyme (DNA-SE), together with a suitable excipient.

28. A pharmaceutical composition for treatment of hemophilia in a subject, the composition comprising the one or more repair vehicles (RVs) accordiong to claim 24 and one or more polynucleotides encoding the DNA scission enzyme (DNA-SE), together with a pharmaceutically acceptable excipient.