WO2025153529A1

WO2025153529A1 - Adeno-associated virus vector sequence

Info

Publication number: WO2025153529A1
Application number: PCT/EP2025/050880
Authority: WO
Inventors: Jacob LUND; Karl Agger; Gert Bolt; Jonas HØJFELDT; Jazmine Pua'nani HALLINAN; Kyle Andrew HAVENS; John Christopher MOORE; Michelle Louise SCALLEY-KIM
Original assignee: Novo Nordisk AS
Current assignee: Novo Nordisk AS
Priority date: 2024-01-16
Filing date: 2025-01-15
Publication date: 2025-07-24
Anticipated expiration: 2026-07-16

Abstract

The invention relates to adeno-associated virus (AAV) vector sequences comprising a nucleotide sequence encoding a Factor VIII (FVIII) protein, compositions containing these vector sequences, and associated therapeutic uses thereof.

Description

TITLE: ADENO-ASSOCIATED VIRUS VECTOR SEQUENCE

TECHNICAL FIELD OF THE INVENTION

The present disclosure relates to adeno-associated virus (AAV) vector sequences comprising a nucleotide sequence encoding a Factor VIII (FVIII) protein. Further, the disclosure relates to AAV vector particles, gene editing compositions and pharmaceutical compositions containing these AAV vector sequences and associated therapeutic uses thereof.

BACKGROUND OF THE INVENTION

Hemophilia A is a rare genetic disease caused by mutations in the gene encoding coagulation Factor VIII (FVIII). Reduced FVIII function results in a prolonged bleeding phenotype. Today, hemophilia A is, for example, treated with replacement therapy, intravenous (iv) injections of recombinant FVIII, subcutaneous (sc) injections of FVIII mimicking bi-specific antibodies or treatment with bypassing agents. These therapies require lifelong administration and, in some instances, result in the formation of neutralizing FVIII inhibitors.

Gene therapy, in contrast, offers a potentially curative treatment through continuous production of FVIII from hepatocytes in the liver following a single treatment instance. Current FVIII gene therapy relies on AAV vectors encoding FVIII under control of a hepatocyte specific promoter. Presence of a promoter encoded in the AAV poses a potential safety concern. Moreover, durability of current forms of FVIII gene therapy appears limited to less than 5 years and it is currently presumed not feasible to re-dose due to immunogenicity of the AAV vector.

Adeno-associated virus (AAV) is a replication-defective, non-enveloped virus that infects humans and some other primate species. Several features of the AAV make this virus an attractive vehicle for delivery of genetic material for gene therapy, including, for example, that the AAV is not known to cause human disease and induces only a mild immune response, and that the AAV can infect both dividing and quiescent cells. Gene therapy using AAV vectors have been successfully used in some clinical trials, for example, for the delivery of a gene encoding human B-domain deleted FVIII to the liver for the treatment of Hemophilia A (Ozelo et al. N Engl J Med. 2022 Mar 17;386(11)).

Due to lack of durability of the current AAV-FVIII gene therapies, there is a need for novel types of gene therapies for the treatment of Hemophilia A. SUMMARY OF INVENTION

In an aspect, the invention provides novel adeno-associated virus (AAV) vector sequences. In other aspects, the invention provides AAV vector particles, gene editing compositions and pharmaceutical compositions comprising these AAV vector sequences, and therapeutic uses thereof. Further, provided herein are methods for the use of these AAV vector sequences and gene editing compositions to induce the expression, function and/or activity of coagulation Factor VIII (FVIII) in a cell by gene editing.

In certain embodiments, the present invention provides an adeno-associated virus (AAV) vector sequence comprising a nucleotide sequence which comprises a splice acceptor, a transgene encoding a linker sequence and a B-domain variant of Factor VIII (FVIII-BDV), wherein the portion of the sequence encoding the linker sequence comprises a nucleotide sequence encoding one of the following amino acid sequences:

• MFSMRIVCLVLSVVGTAWT (SEQ ID NO:1), or

• MQIELSTCFFLCLLRFCFS (SEQ ID NO:2); and wherein the portion of the nucleotide sequence encoding the B-domain of FVIII-BDV comprises a nucleotide sequence encoding one of the following amino acid sequences:

• SFSQNSRHPSQNPPVLKRHQR (SEQ ID NO:3), or SFSQNSRHPSNHTANHTANHTANHTANHTANHTSQNPPVLKRHQR (SEQ ID NO:4).

In an aspect, the present invention provides an AAV vector particle comprising, an AAV vector sequence, according to the invention, encapsidated by AAV capsid proteins.

In a particular embodiment the AAV vector sequence comprises the nucleotide sequence set forth in SEQ ID NO: 102.

In one aspect, the present invention provides a gene editing kit comprising (a) an AAV vector sequence, according to the invention, or an AAV vector particle according to the invention; and (b) an mRNA encoding a site-specific nuclease comprised in a lipid nanoparticle.

In one aspect, the present invention provides a method of editing a gene comprising providing the following to a cell: (a) an AAV vector sequence, according to the invention; and (b) a nucleotide sequence encoding a site-specific nuclease.

In one aspect, the present invention provides a pharmaceutical composition comprising an AAV vector sequence, according to the invention, or an AAV vector particle according to the invention in a pharmaceutically-acceptable solution.

In one aspect, the present invention provides a pharmaceutical kit comprising (a) an AAV vector sequence, according to the invention, or an AAV vector particle according to the invention in a pharmaceutically-acceptable solution and; (b) an mRNA encoding a sitespecific nuclease comprised in a lipid nanoparticle in a pharmaceutically-acceptable solution.

In one aspect, the present invention provides a method of treating hemophilia A in a patient comprising administering to the patient an effective amount of an AAV vector sequence according to the invention, an AAV vector particle according to the invention, a pharmaceutical composition according to the invention, or the components (a) and (b) of the pharmaceutical kit according to the invention.

In an aspect, the invention provides an AAV vector sequence that is co-delivered with a site-specific nuclease to mediate targeted integration of the FVIII transgene inside an endogenous gene in a process known as gene editing.

In an aspect, the invention provides an AAV vector sequence comprising a nucleotide sequence encoding a FVIII-BDV and necessary elements for high expression and activity of functional FVIII.

In another aspect, the invention provides a FVIII AAV vector sequence (delivered in an AAV vector particle) comprised in a gene editing composition together with a site-specific nuclease, delivered in a lipid nanoparticle as mRNA, which enables integration of a FVIII transgene for endogenous promoter driven expression in hepatocytes.

In another aspect the invention aims at providing a curative treatment of hemophilia A.

These and other aspects of the invention will be readily apparent from the following detailed description of the invention.

DETAILED DESCRIPTION OF INVENTION

The present disclosure relates to novel adeno-associated virus (AAV) vector sequences. Further, the present disclosure relates to AAV vector particles, gene editing compositions, pharmaceutical compositions and kits, and the use thereof. The present disclosure provides AAV vector sequences, and compositions, kits and methods for use thereof for gene editing to induce the expression, function and/or activity of a blood-clotting protein such as Factor VIII (FVIII) in a cell by gene editing.

In an aspect, the invention provides an AAV vector sequence, comprising a nucleotide sequence encoding a B-domain variant FVIII (FVIII-BDV), which together with a site-specific nuclease (e.g. delivered as mRNA encapsulated in a lipid nanoparticle), enables genomic integration of the nucleotide sequence encoding FVIII-BDV into the human albumin (ALB) gene for endogenous promoter driven expression in hepatocytes. AAVs and AAV vector sequences

Adeno-associated virus (AAV) is a single-stranded DNA Parvovirus with a genomic size of approximately 4.7 kilobases (kb). The AAV genome comprises the rep gene and the cap gene flanked by two inverted terminal repeats (ITR). The rep gene encodes factors required for AAV genome replication and virion assembly and the cap gene encodes the capsid proteins which determines the serotype of the AAV. Only the ITRs which play a role in vector production are necessary for recombinant AAV (rAAV) propagation and almost 96% of the genome can be removed to enable the engineering of the AAV genome for gene therapy, e.g. replacing the rep and cap genes with a nucleotide sequence comprising a therapeutic transgene, such as a sequence encoding FVIII-BDV.

As used herein, “AAV” refers to all serotypes, subtypes, and naturally-occurring AAV as well as recombinant AAV. “AAV” may be used to refer to the virus itself or a derivative thereof. The genomic sequences of various serotypes of AAV, as well as the sequences of the native inverted terminal repeats (ITRs), Rep proteins, and capsid subunits are known in the art. Such sequences may be found in the literature or in public databases such as GenBank.

An “AAV vector sequence” as used herein refers to an AAV vector sequence comprising a heterologous sequence not of AAV origin (i.e., a nucleic acid sequence heterologous to AAV), typically comprising a sequence encoding a heterologous polypeptide of interest, i.e. a transgene.

AAV vector sequence elements

In an aspect, the AAV vector sequence, which is a nucleotide sequence, either single-stranded or double-stranded, comprises a splice acceptor, a therapeutic transgene (such as a nucleotide sequence encoding a linker sequence and a FVIII-BDV), and a polyadenylation signal, flanked by inverted terminal repeat (ITR) sequences. In one aspect, the elements of the AAV vector sequence have the following order from the 5' end to the 3’ end: 5' ITR - splice acceptor - transgene - polyadenylation signal - ITR 3'.

The term "inverted terminal repeat (ITR)", as used herein, refers to the regions found at the 5' and 3' termini of the AAV genome, and the AAV vector sequence, which function as origins of DNA replication and as packaging signals for the AAV genome, and the AAV vector sequence. Methods of designing suitable ITR sequences are well known in the art.

In some embodiments, the AAV vector sequences disclosed herein comprise one, two, or three ITRs. In some embodiments, the constructs disclosed herein comprise no more than two ITRs. In one particular embodiment of the invention, the ITR sequence comprises the following sequence:

• CTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCAAAGCCCGGGCGTC GGGCGACCTTTGGTCGCCCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGA GGGAGTGGCCAACTCCATCACTAGGGGTTCCT (SEQ ID NO:5).

The term "splice acceptor" as used herein refers to a sequence that is necessary for the splicing process where a precursor mRNA containing both introns and exons is transformed into a mature mRNA through the removal of the introns and splicing back together exons. The process is catalysed by the spliceosome that recognize splice donor sequences and splice acceptor sequences in the precursor mRNA. The general function of the splicing process in mammalian cells is well known in the art.

The splice acceptor is positioned in a way that ensures that the part of the precursor mRNA encoded by the transgene, after splicing has occurred, will be fused to the precursor mRNA encoded by a gene at an endogenous target locus to generate a mature mRNA encoding both transgene and target gene and that this mRNA when translated by the ribosome will result in a functional FVIII protein.

In some embodiments, the splice acceptor is the splice acceptor from exon 2 of albumin, e.g., the splice acceptor used in the splicing together of exons 1 and 2 of albumin. In some embodiments, the splice acceptor is derived from the human albumin gene. In some embodiments, the splice acceptor is derived from the mouse albumin gene.

In particular embodiments of the invention, the splice acceptor sequence comprises one of the following sequences:

• TTAACTGTCTTTCTCATTTATCTAG (SEQ ID NO:6),

• TTAACAATCCTTTTTTTTCTTCCCTTGCCCAG (SEQ ID NO:7),

• CTGAGTAATCCTGGTTCCTCTTGAG (SEQ ID NO:56),

• CTAAAGCTTTCTTTCACACTCAAAG (SEQ ID NO:57),

• CTAACGCTTTCTTTCACACTCAAAG (SEQ ID NO:58),

• ATCACACAGAGCCATGCCTATTTAG (SEQ ID NO:59),

• TTAACGCCTCCACCATCTGACTTAG (SEQ ID NQ:60),

• TCAATCAGTCCTCTTGGCGTCTTAG (SEQ ID NO:61),

• TTCACACATCTCGGTTTTTCCTCAG (SEQ ID NO:62),

• AAAACCTACTATTATTCGTTTCTAG (SEQ ID NO:63),

• TTTATTTCTACTTCTCTGGTTTAAG (SEQ ID NO:64), TGAACAACTACTCTCTTTTCTCTAG (SEQ ID NO:65), or TCTACGCCTCCTTACGCACCCCTAG (SEQ ID NO:66).

The term "linker sequence” as used herein refers to a sequence located between the splice acceptor sequence and the FVIII-coding sequence of the transgene. The linker sequence starts with two nucleotides to keep the corrected reading frame when translating the mature mRNA. The mature mRNA comprises ALB exon 1 (Ex1), followed by a sequence that encodes a signal peptide, and a protease cleavage site that ensures that the transgene encoded FVIII-BDV, is correctly processed and that the mature protein is secreted to the bloodstream.

In particular embodiments of the invention, the linker sequence allows for translation, maturation and secretion into the bloodstream of the transgene encoded FVIII- BDV after genomic integration into the human ALB locus.

In some embodiments, the first two nucleotides of the linker sequence, at the 5’ end, are adenine (A) followed by cytosine (C).

In particular embodiments of the invention, the linker sequence allows for translation, maturation and secretion into the bloodstream of the transgene encoded FVIII- BDV protein after genomic integration into the human ALB locus.

In certain embodiments of the invention, the portion of the AAV vector sequence encoding the linker sequence comprises a nucleotide sequence encoding one of the following amino acid sequences:

• MFSMRIVCLVLSVVGTAWT (SEQ ID NO:1),

• MQIELSTCFFLCLLRFCFS (SEQ ID NO:2),

• LSTCFFLCLLRFCFS (SEQ ID NO:44), or

• an amino acid sequence having at least 80, 90%, 95% or 98% identity to SEQ ID NO:1, 2, or 44.

In particular embodiments of the invention, the nucleotide sequence of the linker sequence comprises one of the following sequences:

• ATGTTTTCCATGAGGATCGTCTGCCTGGTCCTAAGTGTGGTGGGCACA GCATGGACC (SEQ ID NQ:107),

• ATGCAAATAGAGCTCTCCACCTGCTTCTTTCTGTGCCTTTTGCGATTCTG CTTTAGT (SEQ ID NO: 108), • ATTAGCTGAATAATAGAGAAGAATTAACCTTTTGCTTCTCCAGTTGAACA TTTGTAGCAATAAACCATGCAAATAGAGCTCTCCACCTGCTTCTTTCTGT GCCTTTTGCGATTCTGCTTTAGT (SEQ ID NO:8), or

• a nucleotide sequence comprising an adenine (A) follow by a cytosine (C) at the 5’ end of the sequence, followed by a nucleotide sequence as set forth in SEQ ID 107, 108 or 8.

In a particular embodiment of the invention, the portion of the AAV vector sequence encoding the linker sequence comprises a nucleotide sequence encoding one of the following amino acid sequences:

• MFSMRIVCLVLSVVGTAWT (SEQ ID NO:1), or

• an amino acid sequence having at least 90%, 95% or 98% identity to SEQ ID NO:1.

The terms "transgene" and “therapeutic transgene” as used herein, refer to a nucleotide sequence encoding a non-natural or naturally occurring protein, for example, a human protein such as FVIII, in particular a FVI I l-BDV, which is designed to be inserted, or has been inserted, into an animal's or cell’s genome in such a way as to alter the genome of the cell into which it is inserted. A transgene, as used herein, can include AAV vector sequence elements, such as a linker sequence, which comprises nucleotide sequences useful for exogenous protein production in human cells.

The term "a polyadenylation signal" as used herein refers to a nucleotide sequence that signals the termination of transcription of an integrated nucleotide sequence, such as the transgene, and provides a signal for the cell to add a poly-A tail which improves the stability of the mRNA within the cell.

In some embodiments, the polyadenylation signal sequence is encoded at the 3’ end of AAV vector sequence. Methods of designing a polyadenylation signal sequence are well known in the art.

In particular embodiments of the invention, the polyadenylation signal sequence comprises the following sequence:

• AATAAAAGATCTTTATTTTCATTAGATCTGTGTGTTGGTTTTTTGTGTG (SEQ ID NO:9).

In a particular embodiment, the AAV vector sequence comprises a nucleotide sequence which comprises a splice acceptor, a transgene encoding a linker sequence and a B-domain variant Factor VIII (FVIII-BDV), wherein the nucleotide sequence of the splice acceptor is TTAACTGTCTTTCTCATTTATCTAG (SEQ ID NO:6), wherein the portion of the nucleotide sequence encoding the linker sequence encodes the amino acid sequence, MFSMRIVCLVLSVVGTAWT (SEQ ID NO:1) and wherein the portion of the nucleotide sequence encoding the B-domain of FVIII-BDV comprises a nucleotide sequence encoding one of the following amino acid sequences

• SFSQNSRHPSQNPPVLKRHQR (SEQ ID NO:3), or

• SFSQNSRHPSNHTANHTANHTANHTANHTANHTSQNPPVLKRHQR (SEQ ID NO:4).

In a particular embodiment, the AAV vector sequence comprises a nucleotide sequence set forth in SEQ ID NO: 102.

In a particular embodiment, the AAV vector sequence comprises a nucleotide sequence set forth in SEQ ID NQ:90.

AAV vector

In an aspect, the invention also provides an AAV vector particle comprising an AAV vector sequences of the invention.

An "AAV vector particle" or "AAV particle", herein, refers to a viral particle comprising AAV capsid proteins on the surface of the AAV vector particle and an encapsidated AAV vector sequence, such as an AAV vector sequence comprising a FVIII- BDV transgene.

AAV serotypes

The serotype of the AAV is determined by the capsid proteins on the surface of the AAV vector particle and decides the tissue tropism of the AAV vector particle. Numerous different serotypes of AAVs have been isolated from human or non-human primates (NHP) and are well characterized and several of these have been used in clinical trials in patients with various diseases, including AAV serotype 1 (AAV1), AAV serotype 2 (AAV2), AAV serotype 5 (AAV5), AAV serotype 6 (AAV6), AAV serotype 8 (AAV8) and AAV serotype 9 (AAV9) (Issa SS, et al. Cells. 2023 Mar 1 ; 12(5:785; and Samulski et al. Nature rev gen, 2020 April 256(21)).

For example, liver-specific delivery of an AAV encoded transgene, such as a sequence encoding FVIII, may advantageously be mediated by transduction of hepatocytes in the liver. Several AAV serotypes are liver tropic and can transduce hepatocytes, including, but not limited to, AAV1 , AAV5, AAV8 and AAV9. In a particular embodiment of the invention, the serotype of the AAV capsid protein is serotype AAV5. In a particular embodiment of the invention, the serotype of the AAV capsid protein is serotype AAV8.

Genomic integration

The AAV vector particles of the invention represent a tool for the in vivo delivery of a nucleotide sequence, such as a nucleotide sequence encoding FVIII-BDV of the invention, by engineering the nucleotide sequence within the AAV vector sequence and encapsidating it in either AAV5 or AAV8 capsid proteins to form AAV vector particles of either serotype 5 or serotype 8, so that it efficiently transduces hepatocytes.

The AAV vector sequences of the invention, are useful in delivery of a heterologous gene, such as FVIII, to a target cell, such as hepatocytes.

In an aspect of the invention, the aim is to integrate a FVIII encoding gene at a specific location in the genome of the hepatocytes which is referred to as "targeted integration" or “site-specific genomic integration”. In some embodiments, targeted integration is enabled by using a site-specific nuclease, to generate a double stranded break in a specific site in the genomic DNA, such as within the human ALB gene.

Upon genomic integration, the transcription of the FVIII-BDV encoding sequence is coupled to the target gene, such as the ALB gene. The splice acceptor placed in front of the FVIII-BDV sequence in the AAV vector sequence, ensures splicing to the target gene, for example the ALB gene. Depending on the architecture of the target locus and the integration site, a linker sequence may be necessary to allow for secretion and maturation of the the FVIII-BDV protein.

In a particular embodiment of the invention, the FVIII-BDV-encoding sequence is inserted within intron 1 of the human albumin (ALB) gene.

Factor VIII

In an aspect of the invention, the AAV vector sequence comprises a nucleotide sequence encoding a functional FVIII protein, such as a FVIII-BDV protein.

Factor VIII

Coagulation Factor VIII (FVIII) is a large, complex glycoprotein that is primarily produced by liver sinusoidal cells and is encoded by the F8 gene. The native (wild type) human FVIII protein consists of 2351 amino acids, including a signal peptide, and contains several distinct domains. The FVIII protein circulates in plasma as two chains, a heavy chain (HC) and a light chain (LC). The chains are connected by bivalent metal ion-bindings.

Endogenous FVIII molecules circulate in vivo as a pool of molecules with B-domains of various sizes. These FVIII molecules with B-domains of different length all have full procoagulant activity. Upon activation by thrombin, the activated FVIII molecule is termed FVIIIa. The activation allows interaction of FVIIIa with phospholipid surfaces like activated platelets, and activated factor IX (FIXa), i.e. the tenase complex is formed, allowing efficient activation of factor X (FX).

The terms “Factor VI 11 (a)” and “FVIII(a)” include both FVIII and FVIIIa. “Factor VIII” or “FVIII” as used herein refers to a human plasma glycoprotein that is a member of the intrinsic coagulation pathway and is essential to blood coagulation. “Native FVIII” is the human FVIII molecule derived from the full-length sequence as shown in SEQ ID NO:10 (amino acid 1-2332).

FVIII activity

In an aspect of the invention, FVIII protein, encoded in the AAV vector sequence, is capable of functioning in the coagulation cascade in a manner that is functionally similar, or equivalent, to native FVIII, inducing the formation of FXa via interaction with FIXa on an activated platelet and supporting the formation of a blood clot. FVIII activity can be assessed in vitro using techniques well known in the art. Clot analyses, FX activation assays (often termed chromogenic assays), thrombin generation assays and whole blood thromboelastography are examples of such in vitro techniques.

As used herein, a "functional FVIII" is a FVIII protein that has the functionality of a native FVIII protein in vitro, when expressed in cultured cells, or in vivo, when expressed in cells or body tissues.

In some embodiments, the FVIII protein, encoded in the AAV vector sequence, has FVIII activity that is at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, 100% or even more than 100% as compared to the activity of endogenous FVIII in healthy subjects.

Previously known nucleotide sequences encoding FVIII transgenes may have had problems with expression of functional FVIII protein. This is thought to be due to inefficient expression of mRNA, protein misfolding with subsequent intracellular degradation, and inefficient transport of the primary translation product from the endoplasmic reticulum to the Golgi apparatus. The inventors of the current invention have found that the nucleotide sequence encoding the FVIII-BDV provided by the invention causes surprisingly high levels of expression of functional FVIII protein both in vitro and in vivo. This means that the FVIII- BDV nucleotide sequence, of the invention, could be used in gene therapy to treat hemophilia A. Further, the FVIII-BDV nucleotide sequence, due to its smaller size, can more effectively be comprised into an AAV vector sequence and AAV vector particles.

FVIII-BDV

B-domain variant FVIII

The B-domain in FVIII spans amino acids 741-1648 of SEQ ID NO:10. The B- domain is subject to limited proteolysis at several different sites, generating large heterogeneity in circulating plasma FVIII molecules. The exact function of the heavily glycosylated B-domain is unknown. What is known is that the B-domain is dispensable for FVIII activity in the coagulation cascade. Recombinant FVIII is thus frequently produced in the form of B-domain-modified variants, including B-domain-deleted and B-domain truncated variants.

In particular embodiments of the invention, the nucleotide sequence of the AAV vector sequence encoding the transgene comprises a novel nucleotide sequence encoding a B-domain variant FVIII (FVIII-BDV) protein.

A "B-domain variant FVIII" or "FVIII-BDV", herein, refers to a FVIII protein comprising a B-domain with a modified amino acid sequence compared to the native sequence (741-1648 of SEQ ID NO: 10).

In certain embodiments the FVIII-BDV comprises a shortened B-domain sequence, fully or partially based on sequences from the full-length B-domain (residue 741-1648 of SEQ ID NQ:10).

In certain embodiments, the FVIII protein, encoded in the AAV vector sequence, is a FVIII-BDV wherein the remaining domains of FVIII correspond closely to the sequences as set forth in amino acid numbers 1-740 and 1649-2332 of SEQ ID NQ:10.

In certain aspects, having a shortened B-domain sequence is an advantage for expression of the FVIII protein, encoded in the AAV vector sequence.

In certain embodiments of the invention, FVIII gene sequence is a FVIII-BDV gene sequence.

In particular embodiments of the invention, the portion of the AAV vector sequence encoding the B-domain of FVIII-BDV comprises a nucleotide sequence encoding one of the following amino acid sequences: • SFSQNSRHPSQNPPVLKRHQR (SEQ ID NO:3),

• SFSQNSRHPSNHTANHTANHTANHTANHTANHTSQNPPVLKRHQR (SEQ ID NO:4),

• SFSQNATNVSNNSNTSKRHQR (SEQ ID NO:11),

• SFSQNSRHPSNHTNHTNHTNHTNHTNHTSQNPPVLKRHQR (SEQ ID NO:12),

• SFSQNSRHPSNTTNATNNTNQTNSTNHTSQNPPVLKRHQR (SEQ ID NO:13),

• SFSQNSRHPSNTTANATANNTANQTANSTANHTSQNPPVLKRHQR (SEQ ID NO:14),

• SFSQNSRHPSNHTANHTANHTANHTSQNPPVLKRHQR (SEQ ID NO:15), or

• SFSQNSRHPSGGNGTGGNGTGGNGTGGNGTSQNPPVLKRHQR (SEQ ID NO:16).

In particular embodiments, the portion of the nucleotide sequence encoding the B- domain of the FVIII protein encodes an amino acid sequence comprising a sequence having at least 80%, 90%, 95%, 98% or 99% identity to SEQ ID NO: 3, 4, 11 , 12, 13, 14, 15 or 16.

In some embodiments, the portion of the nucleotide sequence encoding the B- domain of the FVIII protein encodes an amino acid sequence comprising the sequence of SEQ ID NO: 3, 4, 11, 12, 13, 14, 15 or 16 with up to two amino acid substitutions in the amino acid residues. In one embodiment, there is up to one substitution in the amino acid residues, with the proviso that no asparagine residues are being substituted.

In a particular embodiment of the invention, the FVIII-BDV gene sequence comprises a nucleotide sequence encoding one of the following amino acid sequences:

• SFSQNSRHPSNHTANHTANHTANHTANHTANHTSQNPPVLKRHQR (SEQ ID NO:4),

• an amino acid sequence having up to two substitutions compared to SEQ ID NO:4, in the amino acid residues which are not asparagine, or

• an amino acid sequence having at least 90%, 95% or 98% identity to SEQ ID NO:4.

In a particular embodiment of the invention, the B-domain is a B-domain variant, having the following nucleotide sequence:

• AGCTTCTCCCAGAATTCAAGACACCCTTCTAACCACACGGCCAACCACACG GCGAACCACACTGCTAACCATACCGCAAACCATACTGCTAATCACACTTCT CAAAACCCACCAGTCTTGAAACGCCATCAACGG (SEQ ID NO: 109). In some embodiments, the nucleic acid sequence encoding the FVIII protein, such as FVIII-BDV, is codon optimized.

Compositions and kits for gene editing

In an aspect, the invention provides compositions and kits for gene editing.

As used herein, the terms “gene editing” and ’’genome editing” refer to the substitution, deletion, and/or introduction of genetic material at a target site in the genome of the cell, which restores, corrects, disrupts, and/or modifies expression of a gene or gene product. The process of integrating non-native nucleic acid into genomic DNA in a predefined genomic location is an example of gene editing.

In an aspect of the invention, the gene editing composition comprises an AAV composition. As used herein, the term an “AAV composition” refers to a composition comprising an AAV vector sequence, preferably comprised in AAV vector particle.

In certain embodiments, the AAV composition comprises an AAV vector sequence comprised in an AAV vector particle where the AAV vector sequence comprises a nucleotide sequence encoding a therapeutic transgene, such as FVIII-BDV.

In certain embodiments, the composition for gene editing further comprises an mRNA encoding a site-specific nuclease.

In an aspect, the invention provides kits for gene editing. In an aspect of the invention, the gene editing kit comprises (a) an AAV composition and (b) a composition comprising a nucleotide sequence encoding a site-specific nuclease preferably comprised in a lipid nanoparticle.

In certain embodiments, the sequence encoding a site-specific nuclease is an mRNA encoding a megaTAL.

The gene editing compositions and kits contemplated are useful for editing a target site in the genome of a cell or a population of cells. In some embodiments, the cells are human hepatocytes and in some embodiments the target site in the genome is the human ALB gene (ALB gene).

The compositions and kits contemplated, in particular embodiments, comprise an AAV vector sequence, comprising a nucleotide sequence encoding a FVIII-BDV, comprised in AAV vector particles; and site-specific nuclease, such as a megaTAL, encoded by a mRNA nucleotide sequence formulated in lipid nanoparticle. These compositions and kits can be used in methods for editing a genome, such as the site-specific genomic integration of a FVIII gene in hepatocytes, and for treating a subject, e.g. a patient with hemophilia A. Gene

Several gene editing technologies are available and enable direct targeting and modification of genomic sequences in eukaryotic cells and mammals. Such technologies include, but are not limited to, transcription activator-like effector nucleases (TALENs), zinc- finger nucleases (ZFNs), clustered regularly interspaced short palindromic repeat (CRISPR)- Cas-associated nucleases, and homing endonucleases (also referred herein to as meganucleases). “Homing endonuclease” and “meganuclease” are used interchangeably. Common to all of these editing techniques is that they create a double strand break in the DNA at the target nucleotide sequence, while the natural cellular repair mechanisms are left to re-ligate the nucleotide sequence. These editing technologies make use of site-specific nucleases.

In some embodiments, the site-specific nuclease is a CRISPR-Cas-associated nuclease. In a particular embodiment the site-specific nuclease is a CRISPR-Cas9- associated nuclease.

Site-specific nucleases

In some embodiments, a site-specific nuclease is a gene-editing fusion polypeptide comprising a DNA-binding domain, a homing endonuclease, and optionally an endprocessing enzyme (e.g., exonuclease). The term “end-processing enzyme” refers to an enzyme that modifies the exposed ends of a polynucleotide chain.

In particular embodiments, the genome editing fusion polypeptide comprises a DNA- binding domain and a homing endonuclease variant that binds a target site in the genome and generates a double strand break in the DNA. Homing endonuclease variants contemplated in particular embodiments can be designed to bind to any suitable target sequence (such as in the human ALB gene). Homing endonuclease variants do not exist in nature and can be obtained by recombinant DNA technology or by random mutagenesis. Homing endonuclease variants may be obtained by making one or more amino acid alterations, e.g., mutating, substituting, adding, or deleting one or more amino acids, in a naturally occurring homing endonuclease or homing endonuclease variant.

In various embodiments, a homing endonuclease or meganuclease is reprogrammed to introduce double-strand breaks (DSBs) at a target site within a target gene.

In some embodiments, the nucleic acid encoding the site-specific nuclease, such as a DNA endonuclease, is a mRNA sequence. Hybrid nucleases, as the megaTAL technology, use a fusion of TALE DNA binding domains and the cleaving ability of homing endonucleases (meganuclease), taking advantage of both the tunable DNA binding and specificity of the TALEs, as well as the cleavage sequence specificity of the homing endonucleases.

In particular embodiments of the invention, the site-specific nuclease is a fusion polypeptide comprising a megaTAL.

A “megaTAL” herein refers to a polypeptide comprising a TALE DNA-binding domain and a homing endonuclease variant that binds and cleaves a DNA target sequence in a target gene, and optionally could further comprise one or more linkers and/or additional functional domains, e.g., an end-processing enzymatic domain of an end-processing enzyme that exhibits 5'-3' exonuclease, 5'-3' alkaline exonuclease, 3'-5' exonuclease (e.g., Trex2, Exol or ExoX), 5' flap endonuclease, helicase or template-independent DNA polymerase activity.

In particular embodiments, the site-specific nucleaseis a megaTAL linked by a linker domain (e.g., a polypeptide linker) to an exonuclease (e.g., an ExoX exonuclease), or biologically active fragment thereof.

A “TALE DNA-binding domain” is the DNA-binding portion of transcription activatorlike effectors (TALE or TAL-effectors), which mimics plant transcriptional activators to manipulate the plant transcriptome (see e.g., Kay et al., 2007. Science 318:648-651). Illustrative examples of TALE proteins for deriving and designing DNA-binding domains are disclosed in U.S. Patent No. 9,017,967, and references cited therein, all of which are incorporated herein by reference in their entireties.

Various site-specific nucleases are contemplated herein, including, but not limited to, homing endonuclease variants, such as megaTALs.

Lipid nanoparticles

In one aspect, the present invention provides lipid nanoparticles (LNPs) used to deliver one or more site-specific nucleases, to a target cell type in a patient.

The terms “lipid nanoparticle”, “LNP particle”, and “lipid particle” herein, refer to a lipid formulation that can be used to deliver a nucleotide sequence (e.g. mRNA encoding a site-specific nuclease) to a target cell type of interest (such as hepatocytes).

In some embodiments, the lipid nanoparticle comprises a cationic lipid, a noncationic lipid, and a mRNA sequence that is encapsulated in the lipid nanoparticle. Lipid nanoparticles of this type have been shown to efficiently deliver mRNA to the hepatocytes of the liver of rodents, primates and humans. The encapsulated mRNA undergoes a process of endosomal escape mediated by the ionizable nature of the cationic lipid. This delivers the mRNA into the cytoplasm where mRNA can be translated into the encoded protein.

In certain embodiments, the lipid nanoparticle is a nucleic acid comprising-lipid nanoparticle (i.e. the nucleic acid is encapsulated in the other components of the lipid nanoparticle), comprising: (a) a cationic lipid, a non-cationic lipid (e.g., a phospholipid), a cholesterol, and a conjugated lipid that prevents aggregation of the particle (e.g., a PEG- lipid); and (b) an mRNA molecule.

In particular embodiments, the lipid nanoparticle comprises (i) a cationic lipid, (ii) a non-cationic lipid, (iii) a conjugated lipid that prevents aggregation of the particle (e.g., a PEG-lipid), (iv) a cholesterol, and (v) an mRNA sequence encoding a site-specific nuclease, such as a megaTAL (being encapsulated in the other components of the lipid nanoparticle).

Encapsulation of the site-specific nucleases, such as a mRNA, within lipid nanoparticles confers one or more advantages, such as protecting the mRNA from nuclease degradation in the bloodstream and providing a means of mRNA entry into the cellular cytoplasm where the mRNA can express the encoded gene editing protein.

In one aspect, the present invention provides nucleic acid comprising-lipid nanoparticles, i.e. lipid nanoparticles comprising one or more mRNA molecules encapsulated in lipid nanoparticles.

In particular embodiments, the lipid nanoparticles comprise: (a) mRNA encoding a site-specific nuclease, such as a megaTAL; (b) one or more cationic lipids; (c) one or more non-cationic lipids; and (d) one or more conjugated lipids that inhibit aggregation of particles.

In one aspect, the present invention provides lipid nanoparticles comprising a mRNA sequence encoding a site-specific nuclease such as an megaTAL.

Methods of gene editing

Provided herein is a method of gene editing inserting a FVIII gene, such as a nucleotide sequence encoding FVIII-BDV, into a pre-defined location in the genome of a cell. This method can be used to treat a subject, e.g. a patient of hemophilia A. The genomic DNA of the cell is edited using the materials and methods described herein.

In an aspect, provided herein, the method of editing a genome in a cell comprises providing the following to the cell: (a) an AAV vector sequence, comprising a nucleotide sequence encoding a FVIII protein within an AAV vector particle; and (b) a nucleotide sequence encoding a site-specific nuclease within a lipid nanoparticle. In some embodiments, a knock-in strategy involves knocking-in a transgene consisting of a sequence encoding FVIII, such as FVIII-BDV, into a genomic site.

Human ALB gene

In some embodiments, the genomic site where the FVIII encoding nucleotide sequence, such as a FVIII-BDV encoding nucleotide sequence, is inserted at, or within, the human ALB gene. In some embodiments, the targeted integration of a FVIII encoding nucleotide sequence occurs in an intron of the ALB gene that is highly expressed in the cell type of interest, e.g. human hepatocyte.

In some embodiments, the target site is within one of the introns of the ALB gene locus. In some embodiments, the target site within one of the exons of the ALB gene locus. In some embodiments, the target site is at one of the junctions between an intron and exon (or vice versa) of the ALB gene locus. In some embodiments, the target site is in the first intron (or intron 1) of the ALB gene.

In a particular embodiment, the targeted integration of a FVIII-BDV encoding nucleotide sequence occurs within intron 1 of the ALB gene.

The transcription of the FVIII-encoding nucleotide sequence from the ALB promoter can result in a pre-mRNA that contains exon 1 of ALB, part of intron 1 and the integrated FVIII-encoding nucleotide sequence. When this pre-mRNA undergoes the natural splicing process to remove the introns, the splicing machinery can join the splice donor at the 3' side of ALB exon 1 to the next available splice acceptor which will be the splice acceptor at the 5' end of the FVIII-encoding sequence of the inserted AAV vector sequence. This can result in a mature mRNA containing ALB exon 1 fused to the nucleotide sequence encoding FVIII.

In some embodiments, according to any of the methods of editing a genome in a cell described herein, the nucleotide sequence encoding a FVIII protein is expressed under the control of the endogenous ALB promoter.

In an aspect, provided herein is a method of genome editing integrating a FVIII encoding nucleotide sequence, such as a FVIII-BDV encoding nucleotide sequence, in a safe harbour locus relying on an endogenous promoter. This precludes the need to include an exogenous promoter and has the advantage of mitigating the potential safety concerns related to exogenous promoter activity. In addition, the smaller AAV genome increases the ratio of capsid containing complete genomes.

In particular embodiments, provided herein is a method of inserting a FVIII encoding nucleotide sequence into the ALB gene of the genome of a human hepatocyte, by introducing into the cell an AAV vector sequence, consisting of a nucleotide sequence encoding a FVIII-BDV protein, comprised in an AAV vector particle, and a mRNA sequence encoding a megaTAL, and encapsulated in a lipid nanoparticle, wherein the megaTAL is capable of cleaving the target sequence in the human ALB gene.

Administration regime

In some embodiments, according to any of the methods of editing a genome in a cell described herein, the lipid nanoparticles comprising the nucleotide sequence of the sitespecific nuclease are introduced into the cell a sufficient time following introduction of the AAV vector particles, comprising the nucleotide sequence encoding the transgene, to allow time for the AAV vector sequence to enter the cell nucleus.

In an embodiment of the method of editing a genome in a cell, lipid nanoparticles comprising the nucleotide sequence of the site-specific nuclease, such as megaTAL mRNA, is introduced into the cell 1 day following introduction of the AAV vector particles, comprising the nucleotide sequence encoding the transgene, such as a FVIII-BDV encoding nucleotide sequence.

In an embodiment of the method of editing a genome in a cell, lipid nanoparticles comprising the nucleotide sequence of the site-specific nuclease, such as megaTAL mRNA, is introduced into the cell 2 days following introduction of the AAV vector particles, comprising the nucleotide sequence encoding the transgene, such as a FVIII-BDV encoding nucleotide sequence.

In an embodiment of the method of editing a genome in a cell, lipid nanoparticles comprising the nucleotide sequence of the site-specific nuclease, such as megaTAL mRNA, is introduced into the cell 3 days following introduction of the AAV vector particles, comprising the nucleotide sequence encoding the transgene, such as a FVIII-BDV encoding nucleotide sequence.

In an embodiment of the method of editing a genome in a cell, lipid nanoparticles comprising the nucleotide sequence of the site-specific nuclease, such as megaTAL mRNA, is introduced into the cell simultaneously or on the same day as introduction of the AAV vector particles, comprising the nucleotide sequence encoding the transgene, such as a FVIII-BDV encoding nucleotide sequence.

In a particular embodiment of the method of editing a genome in a cell, lipid nanoparticles comprising megaTAL mRNA are introduced into the cell 1 day following introduction of the AAV vector particles, comprising the nucleotide sequence encoding FVIII- BDV. In some embodiments, according to any of the methods of editing a gene in a cell described herein, one or more additional administrations of (a) the AAV vector particles comprising the nucleotide sequence encoding the transgene and/or (b) the lipid nanoparticles comprising the nucleotide sequence of the site-specific nuclease are provided to the cell following the first administration of (a) the AAV vector particles and/or (b) the sitespecific nuclease.

Pharmaceutical compositions and pharmaceutical kits

In an aspect, the present invention provides pharmaceutical compositions and pharmaceutical kits.

Herein, a “pharmaceutical composition” refers to a composition formulated in pharmaceutically-acceptable or physiologically-acceptable solutions for administration to an animal such as a human, either alone, or in combination with one or more other compositions or agents of therapy. There is no limit to other components that may also be included in the compositions, provided that the additional agents do not adversely affect the composition.

The phrase “pharmaceutically acceptable” is employed herein to refer to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.

The pharmaceutical composition may comprise a diluent, adjuvant, excipient, or vehicle, such as saline solutions and aqueous dextrose or sucrose and glycerol solutions, particularly for injectable solutions. Suitable pharmaceutical excipients in particular embodiments include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene glycol, water, ethanol and the like. Except insofar as any conventional agent is incompatible with the active ingredient, its use in the therapeutic compositions is contemplated. Supplementary active ingredients can also be incorporated into the compositions.

It would be understood by a skilled artisan that particular embodiments of pharmaceutical compositions contemplated herein may comprise other pharmaceutically- acceptable or physiologically-acceptable solutions, such as those that are well known in the pharmaceutical art, and are described, for example, in Remington: The Science and Practice of Pharmacy, volume I and volume II. 22^nd Edition. Edited by Loyd V. Allen Jr. Philadelphia, PA: Pharmaceutical Press; 2012, which is incorporated by reference herein, in its entirety. In some embodiments, the pharmaceutical composition is suitable for parenteral administration, e.g., intravascular (intravenous or intraarterial), intraperitoneal or intramuscular administration. In particular embodiments, the pharmaceutical composition is suitable for intravenous administration.

The term “buffer” as used herein refers to a solution or liquid whose chemical makeup neutralizes acids or bases without a significant change in pH.

In an aspect of the invention, a pharmaceutical composition comprises an AAV vector sequence of the invention, formulated in pharmaceutically-acceptable or physiologically-acceptable solutions for administration to a human.

In some embodiments of the invention, a pharmaceutical composition comprises an AAV vector sequence of the invention comprised in an AAV vector particle, formulated in pharmaceutically-acceptable or physiologically-acceptable solutions. In certain embodiments, a pharmaceutical composition comprises an AAV vector sequence comprising a nucleotide sequence encoding a therapeutic transgene, such as FVIII-BDV, comprised in an AAV vector particle.

In another aspect of the invention, a pharmaceutical composition comprises an mRNA encoding a site-specific nuclease comprised in a lipid nanoparticle (such as the mRNA is encapsulated by the other components of the lipid nanoparticle). In particular embodiments, the mRNA encoding the site-specific nuclease is an mRNA encoding a megaTAL.

The AAV vector sequence of the invention and a site-specific nuclease could either formulated in a pharmaceutical composition together or formulated separately. In certain embodiments of the invention, the AAV vector sequence of the invention and the site-specific nuclease are formulated separately in separate pharmaceutical compositions.

In another aspect, provided herein is a pharmaceutical kit comprising two components: (a) an AAV vector sequence of the invention comprised in an AAV vector particle; and (b) an mRNA encoding a site-specific nuclease comprised in a lipid nanoparticle. These components (a) and (b), of the pharmaceutical kit, can be administered together or separately to a patient.

In some embodiments the pharmaceutical kit comprises two components: (a) an AAV vector sequence comprising a nucleotide sequence encoding a therapeutic transgene, such as FVIII-BDV, comprised in an AAV vector particle; and (b) an mRNA encoding a sitespecific nuclease, such as megaTAL, comprised in a lipid nanoparticle.

In a particular embodiment the pharmaceutical kit comprises (a) an AAV vector sequence, comprising the nucleotide sequence set forth in SEQ ID NO: 102, comprised in an AAV vector particle where the AAV capsid proteins of the AAV vector particle are of serotype AAV8; and (b) an mRNA encoding a megaTAL comprised in a lipid nanoparticle, and wherein the megaTAL mRNA comprises the nucleotide sequence set forth in SEQ ID NO:113.

Methods of treatment

In an aspect, the present invention provides a method of treating hemophilia A in a subject, i.e. a patient, comprising administering to a subject an effective amount of any of the AAV vector sequences, the AAV vector particles, the pharmaceutical composition, or the components (a) and (b) of the pharmaceutical kit of the invention.

Hemophilia A (HA) is a rare congenital life-threatening bleeding disorder with variable severity caused by coagulation factor VIII deficiency predominantly affecting males. If untreated, patients with severe hemophilia A (FVIII activity <1%) often experience markedly reduced life expectancy due to intra cranial hemorrhage and noticeably increased morbidity namely due to frequent and over time potentially disabling joint and muscle bleeds leading to irreversible damage and reduced HR-QoL.

Today, hemophilia A is, for example, treated with replacement therapy, i.e. injections of recombinant FVIII. A common side effect of replacement therapy in HA is the development of neutralizing antibodies (referred to as inhibitors). This phenomenon is observed in approximately 30% of patients with severe HA after regular exposure to FVIII containing products. The presence of inhibitors renders FRT with FVIII containing products partially or fully ineffective. The course and clinical impact of inhibitors in HA is highly variable, e.g., some FVIII inhibitors are low- other high titre, some are transient while others are persistent. Inhibitors can successfully be eradicated in up to 80% of cases using immune induction therapy (ITI) typically consisting of daily FVIII infusions for a year or longer.

In some embodiments, the present invention provides a method of treating hemophilia A patients without inhibitors of all ages (including pediatric patients), and a severe bleeding phenotype.

In some embodiments, the present invention provides a method of treating hemophilia A patients with a history of FVIII inhibitors (after successful ITI) and subgroups of patients with persistent inhibitors may also be in scope for the treatment.

In an aspect, the present invention provides AAV vector sequences, the AAV vector particles, the pharmaceutical composition, or the components (a) and (b) of the pharmaceutical kit of the invention for the use in the prevention, treatment, and amelioration of a disease or disorder, or ameliorating a disease condition or symptom associated therewith. In some embodiments, AAV vector sequences, the AAV vector particles, the pharmaceutical composition, or the components (a) and (b) of the pharmaceutical kit of the invention are used in methods of treating, preventing, or inhibiting a disease for which the single cause is a defective gene, such as hemophilia A with or without inhibitors, or ameliorating a disease condition or symptom associated with a disease, such as hemophilia A.

In a further aspect, the invention provides for a use of any of the AAV vector sequences, the AAV vector particles, the pharmaceutical composition, or the components (a) and (b) of the pharmaceutical kit of the invention for preparation of a medicament for the treatment of hemophilia A.

In an aspect, the invention provides a pharmaceutical kit comprising (a) an AAV vector sequence of the invention comprised in an AAV vector particle; and (b) an mRNA encoding a site-specific nuclease comprised in a lipid nanoparticle. These components (a) and (b) of the pharmaceutical kit, can be administered together or separately to a patient.

These components (a) and (b), can be administered in combination or separately to a patient. Where the term “administered in combination” or “combined administration” means that two or more compositions are administered to a subject at the same time or within an interval such that there can be an overlap of an effect of each composition on the patient.

In some embodiments, the composition comprising a nucleotide sequence encoding a site-specific nuclease comprised in a lipid nanoparticle is administered to a subject, i.e. a patient, within 1 day to 10 days or within 1 days to 5 days or within 1 days to 3 days after administration of the AAV composition comprising an AAV vector sequence of the invention.

In some embodiments, the composition comprising a nucleotide sequence encoding a site-specific nuclease comprised in a lipid nanoparticle is administered to a subject, i.e. a patient, simultaneously or on the same day as an AAV vector sequence comprised of a nucleotide sequence encoding a transgene, such as FVIII, comprised in an AAV vector particle.

As used herein, the term “gene therapy” refers to the introduction of extra genetic material into the genetic material in a cell of a patient that restores, corrects, or modifies expression of a gene or gene product, or for the purpose of expressing a transgene (e.g., therapeutic transgene). In particular embodiments, introduction of genetic material into the genome of a cell of a subject, e.g. a patient by gene editing that restores, corrects, disrupts, or modifies expression of a gene or gene product, or for the purpose of expressing a transgene (e.g., therapeutic transgene) is considered gene therapy. Methods of preparation

AAV vector sequences, AAV vector particles, gene editing compositions, lipid nanoparticles, pharmaceutical compositions, pharmaceutical kits described herein, could be prepared according to standard methods of preparation known to a person skilled in the art. (e.g. see Issa SS, et al. Cells. 2023 Mar 1 ;12(5:785; and Samulski et al. Nature rev gen, 2020 April 256(21)). For example, the production of AAV vector particles of serotype 8 is described further in J Virol. 2007 Nov;81 (22): 12260-71.

With respect to the preparation of AAV vector sequences and AAV vector particles, and formulation and storage of the AAV vector particles, as an example of a reference, the review article by P. Grossen et al. (European Journal of Pharmaceutics and Biopharmaceutics 190 (2023) 1-23) describes and refers to methods that could be used for these purposes. Production of an AAV vector particle necessarily includes production of an AAV vector sequence, as such a vector sequence is contained within an AAV vector particle. In some embodiments, the AAV vector of the invention is prepared by triple transfection of a production cell line with a transfer plasmid containing the vector genome sequence as well as two addition plasmids encoding genes necessary to produce and package AAV vector particles.

With respect to the preparation of gene editing compositions, particularly mRNA of site-specific nucleases, as an example of a reference, Boissel S., et al, Methods Mol Biol. 2015;1239:171-96 (PMID: 25408406) describes and refer to methods that could be used for these purposes.

With respect to the preparation of lipid nanoparticles and formulation and storage of lipid nanoparticles, as an example of a reference, the review articles by C. Webb et al. (Molecular Pharmaceutics 19 (2022) 1047-1058) and M. Youssef et al. (Biomolecules 13 (2023) 1497) describe and refer to methods that could be used for these purposes.

Further, specific methods of preparation are described in the Example section below, particularly in the “General Methods of Preparation” section.

DEFINITIONS

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one skilled in the art to which the claimed subject matter belongs. It is to be understood that the detailed descriptions are exemplary and explanatory only and are not restrictive of any subject matter claimed.

In this application, the use of the singular includes the plural unless specifically stated otherwise. It must be noted that, as used in the specification, the singular forms “a”, “an” and “the” include plural referents unless the context clearly dictates otherwise. In this application, the use of “or” means “and/or” unless stated otherwise.

Although various features of the disclosures may be described in the context of a single embodiment, the features may also be provided separately or in any suitable combination.

The terms “polypeptide”, “polypeptide sequence”, “peptide”, “peptide sequence”, “protein”, “protein sequence” and “amino acid sequence” are used interchangeably herein to designate a linear series of amino acid residues connected one to the other by peptide bonds, which series may include proteins, polypeptides, oligopeptides, peptides, and fragments thereof. The terms “polypeptide”, “peptide”, and “protein” includes fusion proteins, including, but not limited to, fusion proteins with a heterologous amino acid sequence, fusions with heterologous and homologous sequences.

The term "polynucleotide”, “polynucleotide sequence”, “oligonucleotide”, “oligonucleotide sequence”, “oligomer”, “oligo”, “nucleic acid sequence” or “nucleotide sequence” used interchangeably herein, refer to a polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides. Thus, this term includes, but is not limited to, single-, double-, or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or a polymer having purine and pyrimidine bases or other natural, chemically or biochemically modified, non-natural, or derivatized nucleotide bases.

“Percentage of sequence identity is determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide or polypeptide sequence in the comparison window may have additions or deletions (i.e. gaps) as compared to the reference sequence (which does not have additions or deletions). In some cases the percentage can be calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity.

The terms “identical” or “percent identity” in the context of two or more nucleic acid or polypeptide sequences, refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same (e.g. 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% identity over a specified region, e.g., the entire polypeptide sequences or individual domains of the polypeptides), when compared and aligned for maximum correspondence over a comparison window or designated region as measured using a sequence comparison algorithm or by manual alignment and visual inspection.

As used herein, the term “codon optimized” refers to substituting codons in a polynucleotide encoding a polypeptide in order to increase the expression, stability and/or activity of the polypeptide. Factors that influence codon optimization include, but are not limited to one or more of: (i) variation of codon biases between two or more organisms or genes or synthetically constructed bias tables, (ii) variation in the degree of codon bias within an organism, gene, or set of genes, (iii) systematic variation of codons including context, (iv) variation of codons according to their decoding tRNAs, (v) variation of codons according to GC %, either overall or in one position of the triplet, (vi) variation in degree of similarity to a reference sequence for example a naturally occurring sequence, (vii) variation in the codon frequency cutoff, (viii) structural properties of mRNAs transcribed from the DNA sequence, (ix) prior knowledge about the function of the DNA sequences upon which design of the codon substitution set is to be based, (x) systematic variation of codon sets for each amino acid, and/or (xi) isolated removal of spurious translation initiation sites.

As used herein, “endogenous gene” or “endogenous sequence” in the context of nucleic acid, refers to a nucleic acid sequence or gene that is naturally present in the genome of a cell, without being introduced via any artificial means.

The invention may also solve further problems that will be apparent from the disclosure of the exemplary embodiments.

EXAMPLES

List of Abbreviations

• FVIII-BDV : B-domain variant FVI 11

• ALB-FVIII-BDV fusion proteins: Protein encoded by the nucleotide sequence of exon 1 of human ALB fused to a nucleotide sequence encoding FVIII- BDV.

General Methods of Preparation

Cloning of Plasmids for Recombinant Expression of FVIII-BDV and ALB-FVIII-BDV Fusion

Proteins

DNA encoding the FVIII-BDV proteins and ALB-FVIII-BDV fusion proteins was synthesized and inserted between the Nhel and Asci sites of the mammalian expression vector pQMCF-5 (Icosagen AS) using standard molecular biology methods. The resulting plasmid were used for transient recombinant protein expression. ALB-FVIII-BDV fusion proteins constitutes the sequence encoding the exon(s) 1, 1-2, 1-3 or 1-4 of the human ALB gene followed by the sequence encoding FVIII-BDV.

Recombinant Expression of FVIII-BDV and ALB-FVIII-BDV Fusion Proteins

Cultures of the suspension adapted Chinese hamster ovary (CHO) cell line CHOEBNALT85 (Icosagen AS) were seeded at 0.3E6 cells/mL in a 500 mL Erlenmeyer flask with 1:1 CD CHO and SFM II cell culture medium (Thermo Fisher Scientific) and 20 mg/L puromycin (Thermo Fisher Scientific) and incubated in a CO2 shaker (125 rpm orbital shaking, 36.5 °C, and 8 % CO2) for 3 days. Cells from this culture were electroporated with the individual plasmids encoding FVIII-BDV constructs or ALB-FVIII-BDV fusion protein constructs. For each plasmid, 1.0E7 cells suspended in 0.7 mL of the above cell culture medium was transferred to a 4 mm electroporation cuvette (Gene Pulser Cuvette, BioRad) along with 10 pg plasmid. Electroporation was carried out in a GenePulser Xcell (Biorad). Subsequently the cells from each cuvette were transferred to a 125 mL Erlenmeyer flask (Corning) with 30 mL of the above cell culture medium and incubated in a CO2 shaker (125 rpm orbital shaking, 37 °C, and 8 % CO2). Four days after transfection, the culture volumes were increased 20 % by addition of CHO CD Efficient Feed B (Thermo Fisher Scientific), and the incubation temperature was reduced to 30 °C. After three days of incubation at 30 °C, the culture volumes were further increased 10 % by addition of CHO CD Efficient Feed B (Thermo Fisher Scientific). After four days of incubation at 30 °C, cell culture supernatants were collected and frozen in aliquots with 20 pM imidazol pH 7.0 (Sigma) and 0.2 % Tween 80 (Merck).

Preparation of AAV Vector Particles

The AAV vector particles were produced using the triple plasmid transfection method in the Viral production cells 2.0, suspension cell line (derived from HEK293F; ThermoFisher). Transfection was performed using the AAV-MAX Helper-Free AAV Production System (Thermo Fisher Scientific) according to manufacturer’s instructions for the 3 plasmids expressing: 1) the AAV vector sequence (pAAV-040), 2) the Rep and Cap genes (pRep2Cap8) and 3) adenoviral Helper genes (pHelper). Following incubation for 66-72 hours, cells were chemically lysed using AAV-MAX Lysis Buffer, and residual genomic and plasmid DNA was enzymatically digested prior to clarification. Lysate was clarified by either by depth or tangential flow filtration and AAV particles were purified via Affinity Capture Chromatography using POROS™ CaptureSelect™ AAVX Affinity Resin (Thermo Fisher Scientific). Following purification, enrichment of full AAV particles was performed via anion exchange polish chromatography. The fraction enriched for full capsids was subject to buffer exchange and sterile filtration, before final fill and finish. Production of AAV vector particles of serotype 8 is described further in J Virol. 2007 Nov;81 (22): 12260-71.

Preparation of mRNA

The mRNA production process generally follows published methods (E. Wen et al. BioPhorum (2023) 8-13), but with optimizations for megaTAL mRNA. mRNA is produced through in vitro transcription (IVT), from a linearized DNA template containing an T7 RNA polymerase promoter. Vendors can supply either plasmid DNA or a linearized plasmid as the starting material. If starting with a circular plasmid DNA template, it must first be linearized through an overnight restriction digest. The digested plasmid is then purified using tangential flow filtration (TFF).

TFF is a process that recirculates a solution across a filter, retaining the desired material while allowing smaller undesired components, such as small proteins or nucleic acid fragments, to pass through. This method enables the concentration of plasmid or mRNA and the exchange of its buffer to water for injection (WFI). TFF allows for tight control of the nucleic acid and its background matrix prior to each step, where buffer components are reintroduced. Therefore, TFF is performed after each reaction or chromatography step.

IVT involves incubating the reagents at 37°C for 4 hours. The reagents include RNA polymerase, linearized plasmid, nucleotide triphosphates, modified pseudouridine, buffer components, and supporting enzymes. The reaction is quenched with EDTA prior to TFF. dT Deoxythymidilic acid (dT) affinity chromatography is a process that purifies full- length mRNA by binding the polyadenylated 3’-end (poly(A) tail) of the mRNA to the dT resin. Impurities are washed away with a pH 7.0 salt buffer. The mRNA is eluted under ambient conditions with a salt-free buffer. This process removes incomplete mRNA fragments, the bulk of residual plasmid, and proteins from previous processes that were retained during TFF. After chromatography, TFF is used again to prepare the mRNA for further processing. dT affinity chromatography is performed twice: once after IVT and once prior to sterile filtration to ensure full length, potent mRNA.

The 5’-cap of mRNA is essential, and the vaccinia capping system, along with 2'-O- Methyltransferase, produces Cap1 mRNA species that are efficiently translated in vivo. The capping reaction is incubated at 37°C for 2 hours. The mRNA is then treated with DNase to remove any residual plasmid. The DNase buffer and enzyme are added directly to the capping mixture and incubated at 37°C for 1 hour. A double-stranded RNA (dsRNA) depletion chromatography step further purifies the mRNA from dsRNA side products, which is followed by TFF.

After the second dT affinity chromatography, the mRNA is exchanged into the final formulation buffer by TFF. The mRNA is then sterile filtered using a 0.22 pm filter. The integrity and purity of the mRNA are evaluated by capillary electrophoresis and UV-Vis spectrometry. The poly(A) tail length can be assessed using Liquid Chromatorgraphy-Mass Spectrometry or Polyacrylamid gel electrophoresis. The dsRNA content, capping efficiency and structure, as well as residual plasmid DNA (pDNA) content, are also analyzed. The potency of the mRNA is evaluated in vitro prior to encapsulation into lipid nanoparticles.

Encapsulation of mRNA in lipid nanoparticles follows published methods (for example E. Wen et al., BioPhorum (2023) 8-13) and can be carried out successfully by a skilled person.

For the encapsulation of the mRNA in lipid nanoparticles, the manufactured mRNA was buffer exchanged to an acidic buffer (pH range of 4-6). A defined lipid mixture of a cationic lipid, a non-cationic lipid, cholesterol and a PEG-conjugated lipid was prepared in >99.8% ethanol. The lipid nanoparticle formation took place at a concentration which enables a ratio of positive charges from the cationic lipid to negative charges of the mRNA between 3 and 9 which is most common and effective. The formation of lipid nanoparticles with high encapsulation efficacy of the mRNA took place in a well-defined and reproduceable process in an appropriate mixing technique; mixing parameter are dependent on the used mixer type (S.J. Shepherd et al., Biomaterials 274 (2021) 120826). The ethanol amount in the mixture was then reduced by buffer dilution and afterward buffer exchanged and concentrated by tangential flow filtration. A sterile filtration completes the manufacturing process.

Quality attributes of lipid nanoparticles was set to a size of <100 nm and a monodisperse distribution. The encapsulation efficiency is defined as the percentage of mRNA encapsulated inside the lipid nanoparticles and therefore not accessible by a fluorescent dye in the Ribogreen assay (L.J. Jones et al., Analytical Biochemistry 265 (1998) 368-374) and was set to >85%.

In vivo Genome Editing in Mice

Hemophilia A mice (F8'^/7RAG2'^/- mice, C57BL/6 background) and hALB mice (mALB^_/7hALB^+/+, C57BL/6 background) were purchased from Taconic Biosciences (Denmark) and wild-type C57BI/6 mice from Janvier Labs (France). Mice of both genders were used in experimental studies. All animal procedures were performed in accordance with the Danish Animal Experimentation Act and Novo Nordisk Animal Welfare Guidelines.

Animals were anaesthetised with isoflurane (Baxter, induction: 5% isoflurane, 0.7 L/min N2O, 0.3 L/min O2, maintenance: 2% isoflurane, 0.7 L/min N2O, 0.3 L/min O2) and a tail vein catheter inserted. The AAV vector particles and the lipid nanoparticles were then administered intravenously via the catheter before it was removed, and animals allowed to recover.

Blood samples were collected from the retro-orbital venous plexus of anaesthetised mice (Baxter, induction: 5% isoflurane, 0.7 L/min N₂O, 0.3 L/min O2, maintenance: 2% isoflurane, 0.7 L/min N2O, 0.3 L/min O2) using uncoated capillaries (Vitrex Medical, Ref: 174513) with blood transferred to tubes containing 10% v/v sodium citrate (Merck) for FVIII antigen and activity measurements, EDTA coated capillaries (Vitrex Medical, Ref: 178213) for albumin, alanine transaminase (ALT), aspartate aminotransferase (AST) measurement, and for blood cell counts EDTA coated capillaries and tubes containing cell pack (Sysmex).

Tail vein transection was performed in 12- to 18-week-old F8'^/7RAG2'^/' mice as described in Carol Illa et al. (J Vis Exp. 2021). Isoflurane anaesthetised animals (Baxter, induction: 5% isoflurane, 0.7 L/min N2O, 0.3 L/min O2, maintenance: 2% isoflurane, 0.7 L/min N2O, 0.3 L/min O2) in the prone position had their tails submerged in saline 37°C for 5 minutes. Tails were then removed from the saline and the left lateral tail vein transected using a scalpel (#11 Scalpel blade, Swann-Morton) and guide template (Novo Nordisk A/S). Tails were then returned to the saline and bleeding time monitored and recorded for 40 minutes, with blood collected in the saline tubes. The clot was challenged at 10 minutes, 20 minutes, and 30 minutes post-injury by gently wiping the cut twice with wet gauze if there was no bleeding. After 40 minutes animals were euthanized. Total bleeding time was then calculated, and blood loss quantified by amount of hemoglobin in the saline. To quantify hemoglobin, blood collection tubes were centrifuged 4000g x g for 5 minutes at room temperature to pellet erythrocytes. The supernatant was then discarded, and erythrocytes lysed in 2-14 mL RBC lysing solution (Lysebio, ABX Diagnostics) until a light coffee colour. Samples were kept refrigerated until 550 nm absorbance assessed on a ACLTOP 550CTS (Instrumentation Laboratory Co.). Absorbance was then converted to nmol hemoglobin using a standard curve prepared from human hemoglobin (HemoCue Calibrator, HemoCue) and corrected for RBC lysing solution dilution.

Liver tissue was collected from the left lateral lobe of euthanized animals. For molecular biology analysis liver tissue was snap frozen in liquid nitrogen and stored at -80°C until analysis. For histological analysis, tissue was fixed in formaldehyde (4%, VWR Chemicals) for 3-5 days, before transfer to 70% ethanol.

In vivo Genome Editing in Non-Human Primates

Purpose bred naive male cynomolgus monkeys (Macaca fascicularis) aged 1-2 years were sourced from Noveprim, Maritus. Animals were pre-screened for relevant AAV serotype neutralizing antibodies and single nucleotide polymorphisms (SNPs) in the ALB sequence for megaTAL cleavage site before inclusion in the study. Animals were socially housed in two story cages (3 or 5 animals/cage) compliant with the European Union Directive on the Protection of Animals Used for Scientific Purposes (Directive 2010/63/EU). Animals were acclimatized for minimum 6 weeks prior to dose initiation. Animals were fed Lab Diet Certified Primate Diet 5S48 pellets and ad libitum access fresh drinking water and provided environmental enrichment in the form of dietary supplements and cage enrichment devices.

Animals were temporarily held in restraining chairs during administration, and a temporary catheter inserted into the tail vein for intravenous (IV) administration of AAV vector sequences, encoding FVIII-BDV, comprised in AAV vector particles and megaTAL mRNAs comprised in a lipid nanoparticles. Administration site was shaved and cleaned with sterile wipes prior to dose administration. NHPs were intravenously dosed into the tail vein first with AAV vector particles (2.0E13 vg/kg or 4.0E13 vg/kg) as a 1-3 minutes duration IV bolus injection, followed by a 60 minute infusion of lipid nanoparticles (2.5 mg/kg mRNA), targeting human ALB gene, using a calibrated pump accepted for use if within +/-10% of target volume. Administration was completed within 6 hours of formulation preparation completion.

Blood samples were collected by venipuncture from the femoral (or other suitable) vein. Animals were not fasted before sampling. Blood was collected into K2 EDTA tubes for hematology, cytokine, complement and pharmacokinetic parameter evaluation, 3.2% (w/v) trisodium citrate tubes for assessment of coagulation, thrombin and anti-FVIll antibodies, FVIII antigen and activity and lithium heparin for clinical chemistry. Blood samples, except for hematology, were then processed as below and resultant plasma collected. Cytokine samples were mixed gently kept on crushed wet ice and centrifuged (1500 g, 10 minutes, 4°C) within 60 minutes. Complement samples were mixed gently kept on crushed wet ice and centrifuged (2000 g, 10 minutes, 4°C) within 60 minutes. Plasma was stabilized with futhan (FUT-175). Pharmacokinetic samples were mixed gently and centrifuged (2000 g, 15 minutes, 4°C). Samples for assessing coagulation, anti-FVIll antibody, FVIII antigen and FVIII activity were gently inverted 10 times and centrifuged (4000 g, 5 minutes, room temperature) within 30 minutes of collection. Blood samples were also collected for AAV8 neutralizing antibody assessment and were left at room temperature for at least 30 minutes before processing to serum by centrifugation (2000 x g, 15 minutes, room temperature) within 90 minutes. All plasma and serum samples were then frozen immediately over dry ice and stored at -80°C.

All animals were euthanized by IV injection of sodium pentobarbitone, followed by terminal body weight measurement then exsanguination. Animals were then subjected to a complete necropsy examination and organs weighed and tissue collected and either snap frozen or fixed in NBF for further processing and macroscopic, microscopic and histological evaluation.

General Methods of Detection and Characterisation

FVIII Chromogenic Activity Assay for Recombinant FVIII Proteins in Conditioned Media using Coatest SP

The FVIII activity (FVIII :C) of recombinant FVIII proteins in cell culture supernatant was measured in a chromogenic FVIII assay using Coatest SP reagents (Chromogenix) as follows: a FVIII standard e.g. recombinant FVIII (turoctocog alfa, NovoEight®) calibrated against the 7th international FVIII standard from NIBSC (2009 # 07/350), was diluted to 5, 4, 3, 2, 1, 0.5 and 0.25 mll/mL in Coatest assay buffer (50 mM Tris, 150 mM NaCI, 1 %(w/v) BSA, pH 7.3, with preservative). Cell culture supernatants were in the same buffer. At least two dilutions were analysed. Fifty pL of samples, standards, and buffer negative control were added to 96-well microtiter plates in duplicates. The factor IXa/factor X reagent, the phospholipid reagent and CaCh from the Coatest SP kit were mixed 5:1:3 (vol:vol:vol) and 75 pL of this added to the wells. After 15 min incubation at room temperature, 50 pL of the factor Xa substrate S-2765/thrombin inhibitor 1-2581 mix was added and the reactions incubated 5 min at room temperature before 25 pL 1 M citric acid, pH 3, was added. The absorbance at 405 nm was measured on a microtiter plate reader with absorbance at 620 nm used as reference wavelength. The value for the negative control was subtracted from all samples and calibrators and the activity of the samples calculated based on a calibration curve prepared by plotting the absorbance values vs. FVIII concentration of the FVIII calibrator dilutions.

FVIII Chromogenic Activity in Mouse Plasma and Recombinant FVIII in Buffer System

FVIII chromogenic was can also be assess using the chromogenix FVIII activity kit (COATEST SP4 FVIII, cat. no. 82 4086 63), according to manufacturer’s description with minor adjustments. Briefly, a calibration curve and quality controls (QC) were made by spiking FVIII (NovoEight® secondary reference material) in the provided buffer. 20 pL of FVIII sample/calibrator/QC were applied to a half area 96 well plate (Corning ¹Z> area Non-treated plates, cat. no. 3695). Then 40 pL of a mixture of provided FIX/FX, phospholipid and calcium were added, mixed shortly, and incubated for 15 minutes at room temperature allowing the FXase complex to form. Then 20 pL substrate, a FXa-chromogenic substrate (S-2765), were added and incubated for 10 minutes at room temperature. The substrate S-2765 is cleaved by FXa liberating the chromophoric group pNA (p-nitroaniline). After stopping this reaction with 20 pL 2 %(v/v) citric acid, the plates were read photometrically at 405 (A405) and 620 (A620) nm, and absorbance reported as A405 subtracted A620. The intensity of the color is proportional to the FVIII activity of the sample. Minimal required sample dilution was 80x and normally samples are run in 80x and 160x dilution. Acceptance of an analytical run was based on back-calculated calibration concentrations for the NovoEight® calibrator and quality control samples (based on max. +/-20% RE and below 15% CV on signal). The analytical range of the assay in F8'^/7RAG2'^/' plasma spans from 0.25 - 20 ll/L with an LLOQ of 80 x 0.25 = 20 U/L.

FVIII Antigen Assay for Recombinant FVIII Proteins in Conditioned Media using VisuLize™ Factor VIII Antigen Kit

The FVIII antigen concentration was determined in an ELISA (Factor VI11 antigen (FVIIkC), Affinity Biologicals) using a polyclonal anti-FVIll antibody both for catching and detection. Microtiter plates (96 well plates, NUNC Maxisorp) were coated overnight at 4°C with 100 pL capture antibody from the kit (F8C-EIA-C). After 5 times washing in PBS + 0.05 %(v/v) Tween 20 and 15 min incubation with the buffer at room temperature, the wells were blocked 30 min at room temperature with 3 %(w/v) casein. After washing, 100 pl cell culture supernatant diluted minimum 10-fold, FVIII calibrator (turoctocog alfa, Novoeight®) diluted to 100, 33, 11 , 3.70, 1.23, 0.41 and 0.14 ng/mL (corresponding to 600, 200, 67, 22, 7.4, 2.5 and 0.8 pM) in 0.1 M Hepes, 0.1 M NaCI, 10 g/L BSA and 0.1 %(v/v) Tween 20, pH 7.0, and buffer negative control were added in duplicates to the wells and incubated 1-1.5 hours at room temperature. Plates were washed, and 100 pL HRP-labelled antibody from the kit (F8C-EIA-D) added to wells and incubated 1 hours. Plates were washed, 100 pL TMB One™ Substrate (Kem-En-Tek) added, and colour development allowed to take place for approximate five min before the reaction was stopped by adding 100 pL 4 M Phosphoric acid. The absorbance at 450 nm with 620 nm as reference was measured on a microtiter plate reader. The value for the negative control was subtracted from all samples and calibrators, and FVIII antigen in the samples calculated relative to the FVIII calibrator.

FVIII LC LOCI Assay for Mouse Plasma Sample and Recombinant FVIII in Buffer System FVIII antigen was measured in plasma samples and conditioned media using Luminescence Oxygen Channelling Immunoassay (LOCI). The employed LOCI system contains a streptavidin-coated donor beads with a photosensitive dye, acceptor beads coated with a FVIII light chain specific antibody (in-house antibody, 1212-0000-0080) and a secondary biotinylated antibody (in-house, 1212-0000-0149) also specific for the FVIII light chain. Upon illumination, the generated chemiluminescent signal, resulting from complex formation of donor and acceptor beads via FVII and the anti-FVIll antibodies, was proportional to the FVIII contraction. A calibration curve and quality control standards were made by spiking recombinant FVIII (NovoEight® secondary reference material) in 1 % rat plasma. For samples with expected FVIII levels below 200 U/L, a so-called analogue calibrator curve was made by spiking NovoEight® secondary reference material in F8'^/7RAG2'^/' mouse plasma (or any other mouse plasma).

Working solutions of both acceptor beads/biotinylated antibody and donor beads were prepared in a LOCI buffer with high salt (25 mM HEPES pH 7.4, 550 mM NaCI, 10 mM EDTA, 0.5 %(w/v) BSA, 0.1 %(w/v) bovine IgG, 2 mg/mL dextran T500, 0.1 %(v/v) Tween 20, 0.1 %(v/v) proclin 300, 0.01 %(v/v) gentamycin, 0.2 mg/mL HBR1). Five pL for FVIII sample/calibrator/QC was applied per well. Then 15 pL of the working solution, containing 7.5 nM biotinylated antibody and 33.3 pg/mL antibody-coated acceptor beads, were added to each well. The plates were sealed and covered with a black lid. The assay was allowed to incubate for 1 hour at 18-22 °C. Under green light, 30 pL of 66.67 pg/mL streptavidin-coated donor beads were added per well. The plate was covered with a black lid and incubated for 30 minutes at 18-22 °C. Finally, the plate was read in Envision (PerkinElmer). Acceptance of an analytical run was based on back-calculated calibration concentrations for the NovoEight® calibrator and quality control samples (based on max. +/-20% RE and below 15% CV on signal). The analytical range of the assay in F8'^/7RAG2'^/' mouse plasma spans from 3 - 2100 U/L with a lower limit of quantification (LLOQ) of 5 U/L.

Human FVIII Catching Chromogenic Activity Assay

FVIII activity was measured by so-called catching chromogenic activity assay. Five pg/mL GMA-8024 (Green Mountain Antibodies, human specific mAb to the FVIII A2 domain) in PBS was coated overnight at 4°C (25 pL per well; half area high binding Corning ref # 3690). Plates were washed with TBS/T and blocked with 3 %(w/v) casein in TBS/T (25 pL) for 2 hours at room temperature. Following another wash step, the calibrator (0 - 2000 ll/L NovoEight® in citrated cynomolgus monkey plasma pool), QC samples (100, 400, and 1600 ll/L NovoEight®) and the FVIII protein sample (in several dilution in the citrated cyno plasma pool) (20 pL) were incubated for 1 hour at room temperature followed by a final wash step. Thereafter, an optimized chromogenic activity assay was performed, using the chromogenix FVIII activity kit (COATEST SP4 FVIII). 40 pL of a mixture of 2x more concentrated FIX/FX (48 %), PL (20 %) and calcium (32%) was added and incubated for 15 minutes at room temperature. Then 20 pL of 2x more concentration substrate, S-2765, was added and incubated for 10 minutes at room temperature, after which the reaction was stopped with 20 pL 2 %(v/v) citric acid. Plates were read at OD405/620 and the FVIII concentration were obtained by comparing the absorbance levels of tested FVIII proteins to the calibrator. An average was taken of all points that are within range of the calibrator. Acceptance of an analytical run was based on back-calculated calibration concentrations for the NovoEight® calibrator and quality control samples (based on max. +/-20% RE and below 15% CV on signal).

Human FVIII Full Length Antigen Assay

FVIII antigen was measured by an MSD assay using a heavy chain antibody GMA- 8023 (Green Mountain Antibodies, human specific mAb to the FVIII A2 domain) as capture and a light chain antibody (in-house mAb against the C2-domain, hF8-4F45, 1212-0000- 0080). Five pg/mL GMA-8023 (Green Mountain Antibodies) in PBS was coated overnight at 4°C (50 pL per well; 96 well plate from mesoscale ref # L15XA-3/L11XA-3). Plates were washed with TBS/T and blocked with 3 %(w/v) casein in TBS/T (150 pL) for 1 hour at room temperature. Following another wash step, the calibrator (0 - 2000 U/L NovoEight® in citrated cyno plasma pool), QC samples (100, 400, and 1600 U/L NovoEight® ) and the FVIII proteins (in several dilutions in the citrated cyno plasma pool) (25 pL) were incubated for 30 min at room temperature followed by an overnight incubation at 4°C. Plates were washed, 1 pg/mL sulfo-tagged hF8-4F45 (25 pL) in TBS/T + 1 %(w/v) BSA was incubated for 1 hour at room temperature and washed again. 150 pL MSD GOLD Read Buffer A (ref # R92TG-2) was added and plates were read on an MSD Sector Imager 6000. The FVIII concentration were obtained by comparing the signal counts of FVIII proteins to the calibrator. An average was taken of all points that are within range of the calibrator. The assay was qualified by a fit- for-purpose qualification. Acceptance of an analytical run was based on back-calculated calibration concentrations for the NovoEight® calibrator and quality control samples (based on max. +/-20% RE and below 15% CV on signal).

Specific Activity of Recombinant FVIII Proteins

The specific activities of the FVIII proteins were calculated by dividing the activity of the samples with the FVIII antigen concentration. FVIII activity is determined using one of the following methods: “FVIII Chromogenic Activity Assay for Recombinant FVIII Proteins in Conditioned Media using Coatest SP”, “FVIII Chromogenic Activity in Mouse Plasma and Recombinant FVIII in Buffer System” or “Human FVIII Catching Chromogenic Activity Assay”. FVIII antigen concentration is determined using one of the following methods: “FVIII Antigen Assay for Recombinant FVIII Proteins in Conditioned Media using VisuLize™ Factor VIII Antigen Kit”, “FVIII LC LOCI Assay for Mouse Plasma Sample and Recombinant FVIII in Buffer System” or “Human FVIII Full Length Antigen Assay”.

Estimation of Targeted Integration

Integration frequency of AAV donor at the ALB locus was determined using a QX200 Droplet Digital PCR (ddPCR) System (BIO-RAD). Briefly, purification of total RNA and genomic DNA (gDNA) from tissues were done using the All Prep DNA/RNA Mini Kit (Qiagen). Frozen liver pieces were dissolved in RLT-buffer containing 40mM DTT and homogenized using a Tissuelyzer (Qiagen, frequency 30 1/s, time: 0.15 min/sec). RNA was eluted is 50 pL and gDNA in 100 pL TE and the concentration was determined using a Lunatic UV/Vis absorbance spectrometer (Unchained labs). Integration frequency of AAV donor at the ALB locus was determined by ddPCR using 100-200ng gDNA as input.

For in vivo studies in mice, the ddPCR assay was designed to detect the borders between the integrated AAV and the genome using the following primers and probes: SEQ ID NO:17-19 were used for detection of targeted integration in the 5’ end, whereas SEQ ID NO 20, 21 and 22 were used for detection of targeted integration in the 3’ end. Here, SEQ ID NO 19 and 22 were modified with 5’ 56-FAM fluorophore as well as internal ZEN and 3’ 3IABkFQ quenchers. Primers detecting an unlinked (constant) region of the genome was used as reference: SEQ ID NO: 23, 24 and 25, where SEQ ID NO:25 is modified with 5’ HEX fluorophore as well as internal ZEN and 3’ 3IABkFQ quenchers.

For in vivo studies in non-human primates, primers and probes were designed to detect the borders between the integrated AAV and the genome using the following primers and probes: SEQ ID NO 18, 26 and 27 were used for detection of targeted integration in the 5’ end, whereas SEQ ID NO 20, 28 and 29 were used for detection of targeted integration in the 3’ end. Here, SEQ ID NO 27 and 29 were modified with 5’ 56-FAM fluorophore as well as internal ZEN and 3’ 3IABkFQ quenchers. Primers detecting an unlinked (constant) region of the genome was used as reference: SEQ ID NO 30, 31 and 32, where SEQ ID NO:32 is modified with 5’ HEX fluorophore as well as internal ZEN and 3’ 3IABkFQ quenchers.

The reference amplicon was multiplexed with the integration site amplicon each with a different color probe (integration: FAM and reference: HEX). Droplet generation was done using an automated droplet generator (BIO-RAD). The PCR was performed on C1000 touch thermal cycler (BIO-RAD) with deep block attachment using the thermocycler program described in the following: (1) One initiation cycle at 95 °C for 10 minutes, (2) Thirty-eight amplification cycles consisting of 94 °C for 30 seconds followed by 59 °C for 105 seconds and 72 °C for 150 seconds and (3) one termination cycle of 98 °C for 10 minutes followed by 10 °C for indefinitely. The droplets were read on a QX200 droplet reader system (BIO-RAD) and the frequency of integration site amplicons relative to the reference amplicon was determined using QuantaSoft Version 1.7.4 (BIO-RAD).

The average genomic integration levels in hemophilia A (F8'^/7RAG2'^/') mice were observed to range from 0.1 to 4% two weeks after administration of 2.0E13 Vg/kg AAV vector particles and 3 mg/mL lipid nanoparticles.

Example 1: Mimicking insertion of FVIII-BDV encoding DNA in intron 1 , 2, 3, or 4 of the human ALB genomic locus

Selection of an appropriate integration locus is key to ensure mRNA splicing and maturation leading the FVIII-BDV expression. To assess the ideal integration site for the AAV vector sequence, DNA constructs simulating targeted integration of the AAV vector sequence encoding FVIII-BDV in each of the first four introns of the human ALB gene were synthesized and transient FVIII-BDV expression was assessed in CHO cells.

Generation of the plasmids encoding FVIII-BDV or ALB fusion proteins hereof were generated as described in the “Cloning of Plasmids for Recombinant Expression of FVIII- BDV and ALB-FVIII-BDV Fusion Proteins” method under the “General Method of Preparation” section resulting in the pGB1014 (SEQ ID NO:33) encoding the FVIII-BDV protein alone and the plasmids pGB1099 (SEQ ID NO:34), pGB1100 (SEQ ID NO:35), PGB1101 (SEQ ID NO:36), and pGB1102 (SEQ ID NO:37), all encoding various ALB-FVIII- BDV fusion proteins. The proteins were expressed recombinantly according to the “Recombinant Expression of FVIII-BDV and ALB-FVIII-BDV Fusion Proteins” method described under the “General Method of Preparation” section. Cell culture supernatant was subsequently analysed by chromogenic FVIII assay (“FVIII chromogenic activity assay for recombinant FVIII proteins in conditioned media using Coatest SP”) and by FVIII ELISA (“FVI II antigen assay for recombinant FVIII proteins in conditioned media using VisuLize™ Factor VIII Antigen Kit”) as described in General Methods of Detection and Characterisation, giving rise to the results shown in Table 1.

The plasmids pGB1099, pGB1100, pGB1101, and pGB1102 encodes hybrid proteins containing as linker sequence, a complete FVIII signal peptide (Q2E amino acid substitution) (SEQ ID NO:38) and N-terminal extension consisting of the amino acids encoded by human ALB gene exon 1 (pGB1099), exon 1 and 2 (pGB1100), exon 1, 2, and 3 (pGB1101), and exon 1, 2, 3, and 4 (pGB1102). As the ALB exons sometimes terminates within a codon, the relevant nucleotides are added between the ALB exons and the FVI 11- BDV encoding sequence to complete the codon. pGB1099 induced FVIII-BDV expression with similar antigen and activity levels as that secreted from cells transfected with a standard FVIII-BDV encoding plasmid without ALB sequences (pGB1014). In contrast pGB1100, pGB1101, and pGB1102 induced no or very little FVIII-BDV secretion. This demonstrates that secretion of functional FVIII-BDV can be obtained from insertion of FVIII-BDV encoding DNA in intron 1 of the human ALB gene, but also surprisingly demonstrates that intron 2, 3, or 4 from the same gene are much less favourable sites for insertion of FVIII-BDV encoding DNA.

As shown in the present example, functional FVIII-BDV can be secreted from cells expressing a precursor protein consisting of from the N-terminus the human ALB exon 1 (SEQ ID NO:39, containing the albumin signal peptide [1-18], the albumin pro-peptide [19-24] and amino acid 1-2 of mature albumin [25-26]), followed by a histidine residue to complete the partial codon 27 encoded in human ALB exonl, a linker sequence encoding the complete FVIII signal peptide (Q2E amino acid substitution, SEQ ID NO:38) and the FVIII-BDV protein. The present example further demonstrates a limit to the allowed length of the ALB sequence in front of the FVIII signal peptide.

Table 1 : FVIII-BDV antigen and activity estimates for simulation of AAV vector integration into the human ALB gene intron 1 , 2, 3 and 4

Example 2: Identification of the N-terminal sequences required for secretion of functional FVIII-BDV expressed from DNA inserted in intron 1 of the human ALB gene

Following targeted integration of the AAV vector sequence, the mRNA encoding FVIII-BDV will be spliced to the mRNA encoded by the upstream ALB gene fragment during transcription, resulting in mature mRNA encoding an ALB-FVIII-BDV fusion protein. In turn this fusion protein needs to be processed to allow efficient secretion of FVIII-BDV without residual amino acids from the ALB gene. The present example describes the simulation of expressing FVIII-BDV proteins from DNA inserted in intron 1 of the human ALB gene by transfecting CHO cells with plasmids directing the transcription of mRNA encoding the same amino acid sequence as the FVIII-BDV encoding mRNA that will be transcribed from the engineered human ALB gene.

Generation of the plasmids encoding FVIII-BDV or ALB-FVIII-BDV fusion proteins were generated as described in the “Cloning of Plasmids for Recombinant Expression of FVIII-BDV and ALB-FVIII-BDV Fusion Proteins” method under the “General Method of Preparation” section resulting in the plasmids pGB1015 (SEQ ID NO:40), pGB1016 (SEQ ID NO:41), pGB1017 (SEQ ID NO:42), and pGB1018 (SEQ ID NO:43), containing sequence encoding full-length or truncated forms of the native FVIII signal peptides. The proteins were expressed recombinantly according to the “Recombinant Expression of FVIII-BDV and ALB- FVIII-BDV Fusion Proteins” method described under the “General Method of Preparation” section. Cell culture supernatant was subsequently analysed by chromogenic FVIII assay (“FVIII chromogenic activity assay for recombinant FVIII proteins in conditioned media using Coatest SP”) and by FVIII ELISA (“FVIII antigen assay for recombinant FVIII proteins in conditioned media using VisuLize™ Factor VIII Antigen Kit”) as described in General Methods of Detection and Characterisation, giving rise to the results shown in Table 2.

From the chromogenic FVIII activity assay and the FVIII ELISA it was evident that active FVIII-BDV was secreted equally well from cells transfected with pGB1015 as from cells transfected with a standard FVIII-BDV encoding plasmid without ALB sequences (pGB1014). This surprisingly demonstrated that that the amino acids encoded by ALB exon 1 do not appear to interfere with FVIII-BDV secretion, if a FVIII signal peptide is inserted between the ALB sequence and the mature FVIII-BDV sequence. Interestingly, active FVIII- BDV was secreted equally well from cells transfected with pGB1016 as from cells transfected with pGB1014 or pGB1015, whereas no or very little FVIII-BDV was secreted from cells transfected with pGB1017 or pGB1018. Thus, the FVIII signal peptide can be truncated from the N-terminus but only to a limited extent. The specific activity of the FVIII-BDV secreted from cells transfected with pGB1014, pGB1015, or pGB1016 were in the normal range of FVIII-BDV, suggesting that the pGB1015- and pGB1016-encoded FVIII-BDV precursors synthesized with a N-terminus encoded by ALB exon 1 followed by the FVIII signal peptide were processed into fully active FVIII-BDV.

Thus, in conclusion it appears from the present data that functional a FVIII-BDV can be produced from a nucleotide sequence inserted in exon 1 of the human ALB gene, if a functional signal peptide is present immediately at the N-terminus of the mature FVIII-DBV protein.

Table 2: Optimization of linker sequence for increase FVIII expression

Example 3: In vitro screening of Linker sequences

Following correct integration of the AAV vector nucleotide sequence into intron 1 of the human ALB gene, the FVIII-BDV encoding nucleotide sequence will be spliced to exon 1 of the ALB gene, and a nucleotide sequence is inserted to enhance the expression of mature FVIII without residual peptide sequence derived from human ALB attached. This nucleotide sequence is here termed “linker sequence” and is located between the splice acceptor and the FVIII-BDV encoding sequence in the AAV vector sequence. To enable screening of different linker sequences allowing for efficient maturation of the FVIII-BDV protein and to optimize the FVIII-BDV expression after integration into ALB intron 1 , several synthetic DNA constructs mimicking the FVIII-BDV encoding sequence inserted into intron 1 of the human ALB gene and with different linker sequences, were generated

In the present example, the following linker sequences were investigated for the effect on functional FVIII-BDV expression when inserted between the human ALB exon 1 and the sequence encoding FVIII-BDV: L1015 (SEQ ID NO:38, encompassing a complete FVIII signal peptide sequence with Q2E amino acid substitution), L1016 (SEQ ID NO:44, encompassing the FVIII signal peptide with a 4 residue N-terminal truncation), L1018 (SEQ ID NO:45, encompassing the FVIII signal peptide with 12 residue N-terminal truncation), L1040 (SEQ ID NO:1, encompassing the full-length Fibrinogen alpha signal peptide) and L1042 (SEQ ID NO:46, encoding a canonical Furin recognition site). In another approach, the linker sequence L1021 (SEQ ID NO:8, encompassing stop codons in all reading frames, followed by nucleotide sequence containing 5’-UTR sequence from native F8 gene, a Kozak sequence and the full-length FVIII signal peptide) was designed to terminate the ALB exon 1 open reading frame and to initiate a new open reading frame starting with a full-length FVIII signal peptide sequence with Q2E amino acid substitution (SEQ ID NO:38). For comparison, a construct containing the full-length FVIII signal peptide (L1014) followed by the sequence encoding FVIII-BDV (i.e. without human ALB exon 1 sequence upstream) was included (SEQ ID NO:33).

To screen various linker sequences for optimal output of secreted and functional FVIII-BDV from cells, expression constructs were stably integrated into cell lines and FVIII-BDV expression was analysed by chromogenic FVIII assay (“FVIII chromogenic activity assay for recombinant FVIII proteins in conditioned media using Coatest SP”) and by FVIII ELISA (“FVIII antigen assay for recombinant FVIII proteins in conditioned media using VisuLize™ Factor VIII Antigen Kit”) as described in General Methods of Detection and Characterisation. Specific FVIII activity was calculated as described in the “Specific Activity of Recombinant FVIII Proteins” method. Different linker designs were cloned in between human ALB exonl sequence and the encoding sequence of FVIII-BDV. mRNA levels in cell pellets were measured by RNA isolation with “RNeasy Kit” (Qiagen), cDNA synthesis with “High-Capacity RNA-to-cDNA Kit” (ThermoFisher) and qPCR with “SYBR Green Master Mix” (ThermoFisher) using primers specific for FVIII-BDV (SEQ ID NO:47-48) and for GAPDH (SEQ ID NQ:49-50).

Constructs encoding ALB-FVIII-BDV fusion proteins with different linkers were generated as described in the “Cloning of Plasmids for Recombinant Expression of FVIII- BDV and ALB-FVIII-BDV Fusion Proteins” method under the “General Method of Preparation” section. This generates ALB-FVIII-BDV fusion proteins with linkers listed in Table 3. From these constructs, a Notl/Nhel fragment was subcloned into multiple cloning site (Notl/Xbal digested) in expression vector “PB513B-1 - pB-CMV-MCS-EF1-CopGFP” (Sanbio B.V.). This generates vectors where expression is driven by a CMV promoter and where the expression vector also includes sites for transposase mediated insertion into cells. Co-transfection of the vector and transposase into HepG2 cells resulted in cells with stable expression of construct. To normalize for vector copy number and potential insertion site influence on expression level, the expression constructs have a separate promoter driving GFP expression. All cells were sorted by GFP expression level to generate comparable pools for FVIII-BDV expression analysis. In the sorted pool of cells, FVIII-BDV encoding mRNA is measured in cell pellets by qPCR and compared to GAPDH, and FVIII-BDV protein and activity was measured in the cell supernatant after 48 hours.

The results in Table 3 demonstrate that both FVIII-BDV expression level and specific activity was affected by the different linker sequences inserted between the splice acceptor and FVIII-BDV encoding sequence. Using the full-length FVIII signal peptide (L1015) or 4 amino acid truncated FVIII signal peptide (L1016) as linker resulted in expression of FVIII-BDV protein with high specific activity, comparable to control without human ALB exon 1 sequence upstream (L1014). Truncating the signal peptide further (L1018) resulted in reduced specific activity confirming finding in example 2. Surprisingly, terminating the ALB open reding frame and initiating a new using a Kozak sequence (L1021) resulted in comparable expression and specific activity as L1015. However, using the Fibrinogen alpha signal peptide (L1040) increased both the expression level and specific activity, whereas cleaving off albumin residual residues with a Furin recognition site (L1042) yield poor specific activity.

Table 3: In vitro test of different linker sequences

Example 4: Production and characterization of FVIII-BDV expressed from DNA encoding FVIII-BDV precursor proteins with the same amino acid sequence as precursor proteins that can be expressed from DNA inserted in intron 1 of the human ALB gene.

Efficient post-translational processing of the ALB-FVIII-BDV fusion protein formed from the splicing of the exon 1 sequence of the human ALB gene to the FVIII-BDV encoding nucleotide sequence is required to secure sufficient expression of functional FVIII-BDV protein. The present example describes the recombinant production and characterization of ALB-FVIII-BDV fusion proteins expressed in a manner that simulates expressing FVIII-BDV fusion proteins from an AAV vector sequence inserted in intron 1 of the human ALB gene. This was carried out by transfecting HEK293 cells with plasmids directing the transcription of mRNA encoding the same amino acid sequence as the FVIII-BDV encoding mRNA that will be transcribed from the engineered human ALB gene. The secreted protein was analysed for N-terminal processing, intact mass and Y1680 sulphatation.

DNA encoding the FVIII-BDV proteins was inserted in the mammalian expression vector pTT5 (Durocher et al. 2002, Nucleic Acids Res 30: E9) giving rise to the plasmids pGB1300 (SEQ ID NO:51) and pGB1301 (SEQ ID NO:52), respectively. A similar plasmid encoding FVIII-BDV without 5’ human ALB exon 1 sequence was used as reference. In these plasmids, the proteins of interest are under the transcriptional control of the CMV promoter. Suspension adapted human embryonal kidney 293F (HEK293F) cells (ThermoFisher Scientific) in Freestyle 293 Expression Medium (ThermoFisher Scientific) were seeded at a density of 1.0E6 cells/mL in 3000 mL Erlenmeyer shaker flasks (Corning Inc.). For each 1000 mL culture, 1000 pg plasmid was diluted in Freestyle 293 Expression Medium to a volume of 33.3 mL. Furthermore, for each 1000 mL culture, 1333 pL 293fectin reagent was diluted in 32 mL Freestyle 293 Expression Medium and incubated for 5 minutes at room temperature. Subsequently, each plasmid solution was gently mixed with a 293fectin reagent solution. The DNA complexation reactions were incubated for 20 minutes at room temperature and then gently transferred to the HEK293F. The cell cultures were incubated in a CO2 shaker (85 rpm orbital shaking, 36.5 °C, and 8 % CO2). Five days after transfection, cell culture supernatants were collected and utilized for purification of FVIII-BDV protein. For all following purification steps, buffers contained CaCh, Tween80, and glycerol for stabilization of the FVIII-BDV protein. First, protein was captured directly from supernatant by affinity chromatography on an anti-FVIll column (CNBr activated Sepharose coupled with anti-FVIll mAb F25). Protein was eluted from the affinity column in a buffer also containing 50% ethylene glycol. Subsequently, and after dilution, the protein was polished by anion exchange chromatography on a POROS 50HQ column. The protein was eluted in the following final buffer: Imidazole (20 mmol/kg), NaCI (approx. 500 mmol/kg), CaCh (10 mmol/kg), Tween80 (0.02% v/v), glycerol (approx. 1 mol/kg), pH 7.3.

The plasmids pGB1300 and pGB1301 both comprises DNA encoding the amino acids encoded by the human ALB exon 1 (SEQ ID NO:39), a complete or partial FVIII signal peptide, and mature FVIII-BDV. Thus, the difference between the two constructs lies in the length of the encoded FVIII signal peptide. pGB1300 and pGB1301 encodes FVIII signal peptides of 19 (complete) or 15 amino acids, respectively. The purified FVIII-BDV proteins produced by cells transfected with plasmid pGB1300, pGB1301 or reference plasmid were primarily characterised by LC-MS analysis and N-terminal sequencing. PNGaseF treatment removing N-glycans was performed prior to LC-MS analysis in order to obtain useful signals. LC-MS analysis was performed on a Waters Synapt G2 instrument. Furthermore N-terminal sequencing performed on a Shimadzu PPSQ-51A sequencer. Sulfation at Y1680 was examined using trypsin-based peptide-mapping using LC-MS on a Thermo Orbitrap Fusion instrument.

The comparative analysis showed that similar heavy and light chain masses were obtained from FVIII-BDV proteins produced by cells transfected with pGB1300 and pGB1301 as from the FVIII-BDV protein produced by cells transfected with a reference plasmid without human ALB sequences. Furthermore, the proteins produced and secreted by cells transfected with pGB1300 and pGB1301 were demonstrated to have the same N-termini as FVIII-BDV protein produced by cells transfected with a reference FVIII-BDV encoding plasmid without human ALB sequences. These are the same N-termini as those found on the FVIII heavy and light chain of plasma-derived or recombinant FVIII-BDV proteins. Thus, the ALB-FVIII-BDV fusion proteins synthesized from the mRNA sequences that will be transcribed upon insertion of FVIII-BDV encoding DNA in intron 1 of the human ALB gene at the position simulated by the DNA constructs pGB1300 and pGB1301 appeared to be correctly and fully processed into proteins with the correct N-termini. All amino acids derived from the human ALB gene seemed to have been removed from the FVIII-BDV protein during the intracellular processing of the hybrid ALB-FVIII-BDV precursor proteins. Finally, Y1680 was sulphated essentially to the same extent (97-98 %) in the FVIII-BDV proteins produced by cells transfected with pGB1300 and pGB1301 as FVIII-BDV protein produced by cells transfected with a reference FVIII-BDV encoding plasmid without ALB sequences. Sulphation of Y1680 is required for high-affinity binding of FVIII to von Willebrand factor, and this interaction is important for the in vivo half-life of FVIII. The results are summarized in Table 4.

In conclusion, the present data strongly suggest that FVIII-BDV in the same quality as plasma-derived and recombinant FVIII can be produced from DNA inserted in exon 1 of the human ALB gene.

Table 4: Characterization of FVIII proteins purified from supernatants of transfected HEK293F cells.

Example 5: Identification of splice acceptor sequences

In order to secure an efficient splicing of inserted AAV vector nucleotide sequence to preceding exon of target locus, a synthetic library of was screened in vitro to identify the most efficient splice acceptor sequences. Moreover, this enabled selection of a short splice acceptor to mitigate the limited DNA packaging capacity of AAV vector particles.

Briefly, a library of potential splice acceptors was generated by synthesizing a 25- nucleotide pool of oligos with a nucleotide mix used at each position that approximates the frequencies of individual bases found in human splice acceptor sites. Synthesis of such 25- mer was specified by the following Formula, Formula 1:

XZNANNXXXXXXXXXXXXXXXXAG (Formula 1), wherein N denotes equal mix of each DNA base (A, C, G and T),

X defines a nucleotide mix of 10% A, 40% C, 10% G, 40%T, and

Z defines a nucleotide mix of 10% A, 10% C, 10% G, 70%T.

The library oligo further carried 5’ (SEQ ID NO:54) and 3’ (SEQ ID NO:55) sequence with overlap to target plasmid for cloning purposes. The splice acceptor library was cloned into a vector coding for mCherry fluorescent protein but lacking mammalian promoter sequence. In the target vector, reading frame is adjusted to match the exon to which splicing is tested. Cloning was performed using 1 pmol single-strand oligo library and 0.06 pmol linearized plasmid vector in a 10 pL reaction with NEBuilder HiFi cloning system, per manufactures instructions. The mCherry reporter construct carrying individual splice acceptors or splice acceptor library are inserted into genome of human HepG2 cell line using Cas9 nuclease to target a specific genomic site for insertion. The donor vector also includes homology sequence to match target locus in genome for more efficient insertion. Knock-in was performed by transfection with Lipofectamine 3000 or Nucleofection with Amaxa 4D- nucleofector. Knock-in was detected based on mCherry fluorescence in FACS analysis, and positive cells were sorted. The fluorescence intensity of mCherry-positive cells reflects the splicing efficiency for a given splice acceptor. Screening of splice acceptor library was performed by sorting the cells with the highest mCherry signal and determine which splice acceptor sequences were enriched in this group compared to their presence in the unsorted population using Illumina sequencing.

Only functional splice acceptors are sequenced, as only mCherry positive cells are analyzed. The strength of the splice acceptors varies, and individual sequences were found enriched in different populations based on mCherry intensity. A fraction (< 5%) of splice acceptors in the library outperformed the rest of the library and was found to be enriched (>2 fold) in the cells sorted for top 10% mCherry expression relative to their abundance in unsorted, but mCherry positive cells.

From Table 5 it is evident that the splicing efficiency between the ALB exon 1 and the nucleotide sequence encoding FVIII-BDV depend on the exact splice acceptor sequence. SA291, SA292, SA293, SA294, SA295, SA296, SA297, SA298, SA299, SA300, SA301 and SA302 all were enriched more than two-fold, demonstrating efficient mRNA splicing, whereas SA303, SA304, SA305, SA306, SA307, SA308, SA309, SA310 all were functional but less efficient splice acceptor sequences. This method allowed for selection of efficient and short splice acceptor sequences.

Table 5: Fold enrichment of splice acceptor sequences

Example 6: FVIII-BDV proteins with novel B-domains comprising N-glycosylation sites Modification of the B-domain sequence with addition N-linked glycosylation sites may benefit protein production, folding and/or secretion from the hepatocytes in the liver. The present example describes FVIII-BDV proteins with B-domains comprising several potential N-glycosylation sites, utilization of the novel N-glycosylation sites and the effect on expression level.

Wild-type FVIII is synthesized with a B-domain of 908 amino acids and comprises several N-glycosylation sites. The wild-type B-domain undergoes endo-proteolysis at several different sites, generating large heterogeneity in circulating plasma FVIII molecules. In the B- domain truncated FVIII compound, turoctocog alpha, the wild-type B-domain is replaced by a 21 amino acid sequence without N-glycosylation sites (D1, SEQ ID NO:3). The amino acid sequences of new B-domains with several potential N-glycosylation sites evaluated in the following examples are D3 (SEQ ID NO:11), D4 (SEQ ID NO:12), D5 (SEQ ID NO:13), D6 (SEQ ID NO:4), D7 (SEQ ID NO:14), D12 (SEQ ID NO:75) and D13 (SEQ ID NO:76). Expression of these FVIII-BDV containing several potential N-glycosylation sites was performed in CHO cells and the degree of N-glycosylation of the B-domains were evaluation.

DNA encoding human FVIII-BDV with the B-domains shown in Table 6 was synthesized and inserted between the Nhel and Asci sites of the mammalian expression vector pQMCF-5 (Icosagen AS) giving rise to the plasmids pGB1680 (SEQ ID NO:77, encoding FVIII-BDV protein containing the D3 B-domain sequence), pGB1681 (SEQ ID NO:78, encoding FVIII-BDV protein containing the D4 B-domain sequence), pGB1682 (SEQ ID NO:79, encoding FVIII-BDV protein containing the D5 B-domain sequence), pGB1683 (SEQ ID NO:80, encoding FVIII-BDV protein containing the D6 B-domain sequence), pGB1684 (SEQ ID NO:81, encoding FVIII-BDV protein containing the D7 B-domain sequence), pGB1685 (SEQ ID NO:82, encoding FVIII-BDV protein containing the D12 B- domain sequence), and pGB1686 (SEQ ID NO:83, encoding FVIII-BDV protein containing the D13 B-domain sequence), respectively.

Cultures of the suspension adapted Chinese hamster ovary cell line CHOEBNALT85 (Icosagen AS) were seeded at 0.3E6 cells/mL in a 500 mL Erlenmeyer flask with 1:1 CD CHO and 293 SFM II cell culture medium (Thermo Fisher Scientific) supplemented with HT supplement (Thermo Fisher Scientific), GlutaMAX (Thermo Fisher Scientific), and 20 mg/L puromycin (Thermo Fisher Scientific). The flask was incubated in a CO2 shaker (125 rpm orbital shaking, 36.5 °C, and 8 % CO2) for 3 days. Cells from this culture were electroporated with the plasmids pQMCF-5 (empty vector), pGB1680, pGB1681, pGB1682, pGB1683, pGB1684, pGB1685, and pGB1686. For each plasmid, 1.0E7 cells/mL suspended in 0.7 mL of the above cell culture medium was transferred to a 4 mm electroporation cuvette (Gene Pulser Cuvette, BioRad) along with 10 microgram plasmid. Electroporation was carried out in a GenePulser Xcell (Biorad) set to exponential protocol (voltage = 300V, Capacitance = 900 pF, and Resistance = ⁰⁰ Q). Subsequently the cells from each cuvette were transferred to a 125 mL Erlenmeyer flask (Corning) with 30 mL of the above cell culture medium and incubated in a CO2 shaker (125 rpm orbital shaking, 37 °C, and 8 % CO2). Four days after transfection, the culture volumes were increased 20 % by addition of CHO CD Efficient Feed B (Thermo Fisher Scientific), and the incubation temperature was reduced to 30 °C. After three days of incubation at 30 °C, the culture volumes were further increased 10 % by addition of CHO CD Efficient Feed B (Thermo Fisher Scientific). After four days of incubation at 30 °C, cell culture supernatants were collected and frozen in aliquots with 20 pM imidazol pH 7.0 (Sigma) and 0.2 % Tween 80 (Merck).

The cell culture supernatants were analyzed by Western blotting, as described in the following. A vial with supernatant from each of the transfected cultures were thawn and 7.5 pL of each supernatant were transferred to individual tubes along with 2.5 pL 4 X NuPage LDS sample buffer (Thermo Fisher Scientific) and 1.1 pL 10 X NuPAGE sample Reducing Agent (Thermo Fisher Scientific). The tubes were incubated for 10 minutes at 70° C on a block heater and then centrifuged 20.000 X g for 2 minutes. The content of each tube was loaded in the wells of a NuPAGE 4-12 % Bis-Tris gel with 15 wells (Thermo Fisher Scientific). The samples were electrophorized in the gel for 2 hours at 200 volt in MOPS running buffer (Thermo Fisher Scientific) with NuPAGE Antioxidant (Thermo Fisher Scientific) added to the running buffer in the inner electrophoresis chamber. After electrophoresis, the gel was blotted for 7 minutes in an i Blot module (Thermo Fisher Scientific) to a nitrocellulose membrane using the iBIot Gel Transfer Stacks Nitrocellulose Mini kit (Thermo Fisher Scientific). After blotting, the membrane was incubated for 1 hour at room temperature with gentle agitation in TBS with 2 % Tween 20. Subsequently, the membrane was washed 4 times for 3 minutes in TBS with 0.1 % Tween 20 as described above. Then, the membrane was incubated with gentle agitation overnight in a cold store with polyclonal sheep anti-FVIll antibody (Cedarlane Laboratories Limited) diluted 1:500 in TBS with 0.1 % Tween 20. Subsequently, the membrane was washed 4 times for 3 minutes at room temperature with gentle agitation in TBS with 0.1 % Tween 20. Then, the membranes were incubated for 45 minutes as described above but protected against light with donkey anti-sheep Alexa fluor 680 conjugate (Molecular Probes) diluted 1:10000. Subsequently, the membranes were washed as described above 4 times for 3 minutes in TBS with 0.1 % Tween 20. Finally, the membranes were scanned in an Odyssey reader at 680 nM. The Western Blot image was analyzed by densitometry using a dedicated python script previously described (Bonde et al. Febs let. Jun; 596(12) 1567). The molecular weight (MW) of the FVIII heavy chain (HC) and light chain (LC) was estimated based on a molecular size marker and the total gel band intensities (area under the, AUC) were evaluated for each FVIII-BDV variant.

In Table 6, the estimated MW of the FVIII-BDV LC were consistently 80-83 kDa as expected and in agreement with previous published results (Thim et al. Haemophilia 2010 Mar;16(2):349). The HC of FVIII-BDV with unglycosylated D1 B-domain had an electrophoretic mobility consistent with 93 kDa MW in good agreement with Example 4 (Table 4). The electrophoretic mobility of the HC of all the present FVIII-BDV variants with potential N-glycosylation sites in the B-domain linker was substantially reduced compared to FVIII-BDV without a N-glycosylation site in the B-domain linker (D1). The electrophoretic mobility of the HC also varied substantially among the different FVIII-BDV variants with potential N-glycosylation sites in the B-domain linker, even among variants with the same number (6) of potential B-domain N-glycosylation sites suggesting different glycosylation efficiencies of the B-domain sequences. Here, FVIII-BDV with the D6 and D7 sequences resulted in estimated HC sized of 128 and 126 kDa, respectively, suggesting efficient N- glycosylation, whereas FVIII-BDV with the D12 sequence only had estimated HC size of 104 kDa suggesting poor N-glycosylation efficiency. FVIII-BDV with D4, D5 and D13 sequences resulted in intermediate HC sizes suggesting partial N-glycosylation. FVIII-BDV with the D3 sequence containing only 4 N-glycosylation sites appears fully glycosylated. Further, more than two-fold differences in expression levels as measured by the AUC was observed between the FVIII-BDV variants with different B-domain sequences, where FVIII-BDV sequences with D4, D6, D7 and D13 all expressed equal to or higher than the D1 reference.

Thus, N glycosylation sites in the present B-domain linkers were utilized, and the amino acid sequence of the B-domain linker influenced the extent of N-glycosylation site utilization.

Table 6: Gel densitometric analyses of anti-FVIHI western blot to estimate MW for FVIII-BDV

LC and HC and expression level.

Example 7: Production and characterization of FVIII-BDV proteins with N-qlycosylated B- domains

The addition of N-glycosylation sites of the B-domain of the FVIII-BDV may affect both expression level and biological activity, and thus the present example describes the production and characterization of FVIII-BDV proteins with B-domains comprising several potential N-glycosylation sites.

FVIII-BDV with B-domain sequences containing multiple potential N-lined glycosylation sites were expressed recombinantly according to the “Recombinant Expression of FVIII-BDV and ALB-FVIII-BDV Fusion Proteins” method described under the “General Method of Preparation” section using the pGB1681 (SEQ ID NO:78), pGB1682 (SEQ ID NO:79), and pGB1683 (SEQ ID NO:80) and pGB1686 (SEQ ID NO:83). Cell culture supernatant was subsequently analysed by chromogenic FVIII assay (“FVIII chromogenic activity assay for recombinant FVIII proteins in conditioned media using Coatest SP”) as described in General Methods of Detection and Characterisation, giving rise to the results shown in Table 7. The supernatants were collected and utilized for purification of FVIII-BDV protein. For all purification steps, buffers contained CalCh, Tween80, and glycerol for stabilization of the FVIII-BDV protein. First, protein was captured directly from supernatant by affinity chromatography on an anti-FVIll column (CNBr activated Sepharose coupled with anti-FVIll mAb F25). Protein was eluted from the affinity column in a buffer also containing 50% ethylene glycol. Subsequently, and after dilution, the protein was polished by anion exchange chromatography on a POROS 50HQ column. The protein was eluted in the following final buffer: Imidazole (20 mmol/kg), NaCI (approx. 500 mmol/kg), CaCh (10 mmol/kg), Tween80 (0.02% v/v), glycerol (approx. 1 mol/kg), pH 7.3. The concentrations of the purified FVIII-BDV proteins were estimated by RP-HPLC on a C18 column (Daiso DMBDMS 2.1 x 250 mm 300A 5 pm beads, buffers: water/acetonitrile with 0.1% trifluoroacetic acid). The FVIII-BDV proteins separate into subunits, and the total concentration was estimated by the sum of A280nm responses for the heavy and light chain subunits. The results of the concentration determinations are shown in Table 7.

The specific activities of the four purified FVIII-BDV proteins were calculated according to the “Specific Activity of Recombinant FVIII Proteins” method described in the “General Methods of Detection and Characterization” section. The results are shown in Table 7. The specific activities of 9-13 mll/ng measured for the three FVIII-BDV proteins with B-domains expressed from pGB1681 , pGB1682, and pGB1683 fall within the range of fully functional FVIII-BDV. In contrast, the specific activity measured for the FVIII-BDV protein expressed from pGB1686 strongly indicate that the functionality of this FVIII-BDV variant is reduced compared to plasma-derived or recombinant FVIII-BDV proteins.

In conclusion, four FVIII-BDV proteins with B-domains comprising several N-glycans were expressed in cell cultures, purified from cell culture supernatant, quantified, and assayed for chromogenic activity. Based on the calculated specific activities, SEQ ID NO:78 (pGB 1681 encoding FVIII-BDV with B-domain variant D4), SEQ ID NO:80 (pGB 1683 encoding FVIII-BDV with-B-domain variant D6), and SEQ ID NO:81 (pGB 1684 encoding FVIII-BDV with B-domain variant D7) are fully functional FVIII-BDV proteins, whereas SEQ ID NO:83 (pGB 1686 encoding FVIII-BDV with B-domain variant D13) has reduced functionality. Table 7: Analysis of purified FVIII with different B-domain sequences

Example 8: Genome editing in hemophilia A mouse model

In the previous examples, sequence elements of the AAV vector sequence necessary for inducing expression of FVIII-BDV following targeted integration of the AAV vector sequence have been evaluated in vitro in various systems. To evaluate the treatment efficacy of the genome editing treatment in vivo, hemophilia A (F8'^/7RAG2'^/ mice were treated with (a) mRNA encoding a surrogate megaTAL nuclease targeting the murine ALB gene within intron 1 encapsulated in lipid nanoparticles and (b) various AAV vector particles encompassing different AAV vector sequence designs (Table 8). In the given Example AAV8 serotype was used. The mice were dosed simultaneous with 2.0E13 Vg/Kg AAV vector particles and 3 mg/kg megaTAL mRNA encapsulated in lipid nanoparticles according to the “In vivo Genome Editing in Mice” described in “General Method of Preparation”. Table 8 provides an overview of the various AAV vector sequences tested in the hemophilia A mouse model. The vector sequences have a general structure starting from the 5’-end with a (1) splice acceptor, (2) a linker sequence connecting the residual albumin amino acid residues to the FVIII-BDV, (3) the FVIII-BDV HC domain (SEQ ID NO:10, residue 1-740), (4) a B- domain, (5) the FVIII-BDV LC domain (SEQ ID NO:10, residue 1649-2332) and (6) a polyA signal sequence (SEQ ID NO:9). The vector sequences are flanked by ITR2 sequences (SEQ ID NO:5) on each site and may contain other features or changes to the general structure (Table 8). In some AAV vector sequences, the previous tested L1015 (FVIII SP Q2E) linker sequence (SEQ ID NO:38) is swapped for the related FVIII SP (SEQ ID NO:2). Beyond the N-glycosylated B-domain sequences tested in Example 6, two additional sequences, D10 (SEQ ID NO: 15) and D15 (SEQ ID NO: 16) were included in the in vivo test. Further, in one instance the murine albumin exon2 splice acceptor (SEQ ID NO:84, mAlb168) was used. Table 8: AAV vector sequences used for genome editing in hemophilia A mouse model

FVIII-BDV expression level and chromogenic activity were assessed one- and two- weeks post treatment according to the “FVI 11 LC LOCI Assay for Mouse Plasma Sample and Recombinant FVI 11 in Buffer System” method and the “FVI 11 chromogenic activity in mouse plasma and recombinant FVI 11 in buffer system” method, respectively, both described under “General Method Detection and Characterization”. After two weeks, the bleeding phenotype was assessed using the tail vein transection according the “In vivo Genome Editing in Mice” method described in “General Method of Preparation” and the level of genomic integration was assessed according to the “Estimation of Targeted Integration” method described under “General Method Detection and Characterization”.

Table 9 shows the average FVIII-BDV antigen measurements one- and two- weeks post genome editing treatment to induce FVIII-BDV expression. The level of genomic integration is included to aid comparison of the AAV vector sequences. The FVIII-BDV expression level is observed to vary significant between AAV vector sequences. The L1040 linker sequence (SEQ ID NO:1) was observed to increase FVIII-BDV expression level more than the remaining linkers. The L1021 linker sequence (SEQ ID NO:8) enabling integration at other target sites regardless of upstream sequence is shown to be functional. The N- glycosylated B-domains D6 and D13 are demonstrated to increase FVIII-BDV further, whereas short flanking homology arms are demonstrated to increase genomic integration. Untreated mice had FVIII-BDV levels and genomic integration levels below detection limit.

Table 9: Average FVIII-BDV antigen levels in plasma one- and two- weeks post genome editing treatment.

Table 10 shows the average FVIII-BDV chromogenic activity measurements one- and two- weeks post genome editing treatment to induce FVIII-BDV expression. The level of genomic integration is included to aid comparison of the AAV vector sequences. The FVIII- BDV activity level is also observed to vary significant between AAV vector sequences in agreement with the FVI 11 l-BDV level (Table 9). Again, the L1040 linker observed to increase FVIII-BDV chromogenic activity level more than the remaining linkers and the L1021 is again shown to be functional. The N-glycosylated B-domains D6 and D13 are demonstrated to increase FVIII-BDV further, whereas short flanking homology arms are demonstrated to increase genomic integration. Untreated mice had FVIII-BDV levels and genomic integration levels below detection limit.

Table 10: Average FVIII-BDV chromogenic activity levels in plasma one- and two- weeks post genome editing treatment. Table 11 show the acute bleeding phenotype of the mice treatment with various AAV vector particles and 3 mg/kg lipid nanoparticle encapsulated megaTAL mRNA according to the “In vivo Genome Editing in Mice” method described under “General Method of Preparation”. Previous data with i.v. administration of recombinant FVIII have demonstrated average reference values in this model of 953.8 nmol blood loss and 4.3 minutes bleed time in hemophilia A mice treated with 10 ll/kg recombinant FVIII (mimicking the bleeding phenotype in wild-type animals) whereas average reference values of 5771 nmol blood loss and 21.5 minutes bleeding time was observed in vehicle treated mice (0 ll/kg recombinant FVIII). From Table 11, genomic integration of AAV vector sequences and subsequence expression of FVIII-BDV is demonstrated to reduce the bleeding phenotype in hemophilia A mice relative to the measured FVIII-BDV antigen and activity level demonstrating that the FVIII-BDV is pharmacologically active. Approximately 100 lll/L FVIII- BDV appear to be required for normalization of the bleeding phenotype and in most cases, the genomic integration of the various AAV vector sequences normalized the bleeding phenotype by reducing the bleeding to the level observed in wild-type mice. The concept is thus demonstrated effective on mitigating acute bleeding.

Table 11: Effect of gene editing treatment of hemophilia A mice with AAV vector sequences on average blood loss and bleeding time following tail vein transection.

Example 9: Effect of administration time of lipid nanoparticle encapsulated megaTAL mRNA in hemophilia A mice

Genomic integration of AAV vector sequences, as described herein the Example section, requires administration of two separate components, a mRNA encoding a megaTAL site-specific nuclease comprised in a lipid nanoparticle and a AAV vector sequence, comprising a FVIII-BDV encoding nucleotide sequence, comprised in an AAV vector particle, the relative timing of the administration of each component could potentially affect integration and subsequent FVIII-BDV expression.

Genome editing was performed in mice according to the “In vivo Genome Editing in Mice” method described in “General Method for Preparations”, but with the exception, that the administration of the lipid nanoparticles comprising megaTAL mRNA was varied in time relative to the administration the AAV vector sequence comprised in AAV vector particles which always was on day 0. In the given Example AAV8 serotype was used. Surprisingly, the level of genomic integration and FVIII-BDV expression was shown to increase when the lipid nanoparticle administration was delay relative to the AAV vector particles and appear to peak at day 1-2 (Table 12). Moreover, delaying administration of megaTAL mRNA comprised lipid nanoparticles further, resulted in a decrease in genomic integration and FVIII-BDV expression levels.

The relative time of the administration of (a) lipid nanoparticles encapsulating mRNA encoding the megaTAL nuclease specific for murine ALB gene and (b) AAV vector particles comprising the FVIII-BDV encoding AAV vector sequence is demonstrated to be important for the successful integration of the AAV vector sequence.

Table 12: Effect of varying lipid nanoparticle administration time relative to AAV administration in hemophilia A mice

Example 10: Durability assessment in hemophilia A mouse model

Durability of FVIII expression in hemophilia A (F8'^/7RAG2'^/') mice were demonstrated following treatment with (a) mRNA encoding a surrogate megaTAL nuclease targeting the murine ALB gene within intron 1 encapsulated in lipid nanoparticles and (b) AAV vector particles encompassing AAV vector sequence SEQ ID NO:90. In the given Example AAV8 serotype was used. The mice were dosed simultaneously with 2.0E13 Vg/Kg AAV vector particles and 3 mg/kg megaTAL mRNA encapsulated in lipid nanoparticles according to the “In vivo Genome Editing in Mice” method described in “General Method of Preparation”. Across the group, the average genomic integration level was 0.9% (of ALB alleles) and the average FVIII-BDV plasma antigen level and activity levels is given in Table 13.

From Table 13, the FVIII-BDV chromogenic activity and plasma antigen levels are observed to be stable for 51 weeks post F8-GE treatment of hemophilia A mice. Table 13: Durability of FVIII-BDV expression in (F8-/-/RAG2-/-) mice

Example 11 : Genome editing in hALB mouse model

In addition to the in vivo testing in the hemophilia A model, F8-GE treatment was tested in a hALB mouse model (mALB^_/7hALB^+/+), where the murine ALB gene has been replaced with the human ALB gene. The hALB mouse model enables F8-GE treatment with (a) mRNA encoding a megaTAL nuclease targeting the human ALB gene within intron 1 encapsulated in lipid nanoparticles and (b) AAV vector particles comprising AAV vector sequence SEQ ID NO:90 or SEQ ID NO: 102. FVIII-BDV expression level and chromogenic activity was assessed one- and two-weeks post treatment according to the “FVI 11 LC LOCI Assay for Mouse Plasma Sample and Recombinant FVI 11 in Buffer System” method and the “FVI 11 chromogenic activity in mouse plasma and recombinant FVI 11 in buffer system” method, respectively, both described under “General Method Detection and Characterization”. After two weeks, the level of genomic integration was assessed according to the “Estimation of Targeted Integration” method described under “General Method Detection and Characterization”.

Table 14 shows the average FVIII-BDV antigen levels (with the number of animals in parenthesis) one week and two weeks following F8-GE treatment with 2E13 Vg/kg AAV007 and 3 mg/kg megaTAL mRNA encapsulated in lipid nanoparticles dosed the same day or two days apart. Furthermore, Table 14 shows the average FVIII-BDV antigen one and two weeks following F8-GE treatment with AAV040 spanning 1 E12 Vg/kg to 2E13 Vg/kg and megaTAL mRNA encapsulated in lipid nanoparticles from 0.5 mg/kg to 3 mg/kg dosed the same day. The level of genomic integration is included to aid comparison of the AAV vector sequences.

From Table 14 the FVIII-BDV plasma antigen levels are observed to increase with increasing lipid nanoparticle and AAV particle doses. The FVIII-BDV antigen levels reach the normal human level (defined as 500-1500 I U/L).

Table 14: Average FVIII-BDV antigen level in hALB mouse model

Example 12: Durability assessment in hALB mouse model

Durability of FVIII expression in hALB (mALB^_/7hALB^+/+) mice were demonstrated following treatment with (a) mRNA encoding a megaTAL nuclease (399B05, SEQ ID NO:112) targeting the human ALB gene within intron 1 encapsulated in lipid nanoparticles and (b) AAV vector particles comprising AAV vector sequence SEQ ID NO: 102. AAV vector nucleotide sequence SEQ ID NO: 102 encodes the amino acid sequence as set forth in SEQ ID NO:114. In the given Example AAV8 serotype was used. The mice were dosed simultaneously with 2.0E13 Vg/Kg AAV vector particles and 3 mg/kg megaTAL mRNA encapsulated in lipid nanoparticles according to the “In vivo Genome Editing in Mice” described in “General Method of Preparation”. The average FVIII-BDV plasma antigen level and activity levels is given in Table 15.

From Table 15, the FVIII-BDV plasma antigen level remains stable for 26 weeks post F8-GE treatment of hALB mice. One animal was excluded from the analysis following confirmation of anti-human FVIII immunity.

Table 15: Average FVIII-BDV antigen level in hALB mouse model

Example 13: Evaluation of in vivo genome editing efficacy in non-human primates

To demonstrate the feasibility of targeted genomic integration of the nucleotide sequence encoding FVIII-BDV in non-human primates and to evaluate the efficacy of the AAV vector sequences, in vivo genome editing was performed in cynomolgus monkeys (Macaca fascicularis). The cynomolgus monkeys were treated with AAV vector particles encapsidating AAV vector sequence SEQ ID NO:90 or SEQ ID NO: 102 and lipid nanoparticles encapsulating mRNA encoding a human specific megaTAL nuclease cross reacting to nonhuman primates according to the “In Vivo Genome Editing in Non-Human Primates” method described in “General Methods of Preparation”. The animals were first administered AAV vector particles and subsequently with lipid nanoparticles either on the same day or 2 days apart. Five and/or eight days post treatment with AAV and lipid nanoparticles, FVIII-BDV antigen was measured in the cynomolgus monkey plasma using the “Human FVIII full length antigen assay” method, FVIII-BDV chromogenic activity was measured in the cynomolgus monkey plasma using the “Human FVIII catching chromogenic activity assay” method and genomic integration was measured using the “Estimation of Targeted Integration” method. All methods are described in “General Methods of Detection and Characterization”. The nonhuman primate studies are summarized in Table 16.

As the non-human primates were normocoagulant (i.e. have a normal functioning coagulation system), the FVIII-BDV antigen and chromogenic activity assays were demonstrated to be specific for human FVIII and FVIII-BDV and did not measure endogenous cynomolgus FVIII.

Table 16: Overview of studies conducted in cynomolgus monkey

Table 17 shows the average FVIII-BDV activity level (lll/L) and FVIII-BDV plasma antigen level with the %CV for the determination in parenthesis. The level of genomic integration, measured at the end of the study, is included to aid comparison of the animals. From Table 17, administration non-human primates with 4.0E13 Vg/kg AAV vector particles comprising AAV vector sequence SEQ ID NO:90 in AAV8 serotype or 2.0E13 Vg/kg AAV vector particles comprising AAV vector sequence SEQ ID NO:102 in AAV8 or AAV5 serotypes and 2.5 mg/kg human ALB-specific megaTAL mRNA encapsulated in lipid nanoparticles was shown to be both feasible and highly efficacious. Five and/or eight days post treatment, high levels of FVIII-BDV antigen and chromogenic activity was measured in the plasma samples. Moreover, a good agreement with FVIII-BDV antigen and chromogenic activity was observed, and an expected correlation between the level of genomic integration and FVIII-BDV antigen/activity level. The treated monkeys generally obtained FVIII activity levels within the normal range observed in humans (defined as 500-1500 I U/L) with few animals above and below the normal range. Thus, targeted genomic integration in intron 1 of the human ALB gene of the nucleotide sequence encoding a FVIII-BDV with this frequency would be expected to abrogate the bleeding phenotype of a hemophilia A patient.

Table 17: FVIII antigen and chromogenic activity level in cynomolgus monkey 5 and 8 days following F8-GE treatment as well as genomic integration and end of the study

To evaluate durability of FVIII-BDV effect in cynomolgus monkey and limit risks of the animals developing an immune response toward the human FVIII-BDV transgene product, cynomolgus surrogate AAV vector particles comprising AAV vector sequence SEQ ID NO: 110 were administrated to four cynomolgus monkeys (Table 16, animals E1002, E2002,

E3001 and E3002) together with lipid nanoparticles which comprised an mRNA sequence encoding a human specific megaTAL nuclease and followed for 12 weeks. The data is summarized in Table 18 where the FVIII-BDV activity level is given as the average measured level with %CV for the determination in parenthesis. Genomic integration level was measured after 84 days and found to be 6.87% of alleles (E1002), 4.14% of alleles (E2002), 3.73% of alleles (E3001) and 1.14% of alleles (E3002). FVIII chromogenic activity level was low or absent seven days before F8-GE and stable between five to eighty-four days post F8- GE treatment. Three out of four treated animals obtained stable FVIII activity levels within the normal range observed in humans (defined as 500-1500 lll/L). Table 18: Assessment of FVIII durability of effect using FVIII chromogenic activity assay following F8-GE treatment of cynomolgus monkeys

While certain features of the invention have been illustrated and described herein, many modifications, substitutions, changes, and equivalents will now occur to those of ordinary skill in the art. It is, therefore, to be understood that the appended claims are intended to cover all such modifications and changes as fall within the true spirit of the invention.

SEQUENCE LISTING

LIST OF EMBODIMENTS

The invention is further described by the following non-limiting embodiments.

Embodiment 1 : An adeno-associated virus (AAV) vector sequence comprising a nucleotide sequence which comprises a splice acceptor, and a transgene encoding a linker sequence, and a B-domain variant Factor VIII protein (FVIII-BDV), wherein the portion of the sequence encoding the linker sequence comprises a nucleotide sequence encoding one of the following amino acid sequences:

• MFSMRIVCLVLSVVGTAWT (SEQ ID NO:1),

• MQIELSTCFFLCLLRFCFS (SEQ ID NO:2),

• LSTCFFLCLLRFCFS (SEQ ID NO:44), or

• an amino acid sequence having at least 80, 90%, 95% or 98% identity to SEQ ID NO:1, 2, or 44; or wherein the nucleotide sequence of the linker sequence is any one of the following sequences:

• ATGTTTTCCATGAGGATCGTCTGCCTGGTCCTAAGTGTGGTGGGCACA GCATGGACC (SEQ ID NO: 107),

• ATGCAAATAGAGCTCTCCACCTGCTTCTTTCTGTGCCTTTTGCGATTCTG CTTTAGT (SEQ ID NO: 108), or

• ATTAGCTGAATAATAGAGAAGAATTAACCTTTTGCTTCTCCAGTTGAACA TTTGTAGCAATAAACCATGCAAATAGAGCTCTCCACCTGCTTCTTTCTGT GCCTTTTGCGATTCTGCTTTAGT (SEQ ID NO:8).

Embodiment 2: The AAV vector sequence comprising a nucleotide sequence which comprises a splice acceptor, and a transgene encoding a linker sequence, and a FVIII-BDV, wherein the portion of the sequence encoding the B-domain of FVIII-BDV comprises a nucleotide sequence encoding any one of the following amino acid sequences:

• SFSQNSRHPSQNPPVLKRHQR (SEQ ID NO:3),

• SFSQNSRHPSNHTANHTANHTANHTANHTANHTSQNPPVLKRHQR (SEQ ID NO:4),

• SFSQNATNVSNNSNTSKRHQR (SEQ ID NO:11),

• SFSQNSRHPSNHTNHTNHTNHTNHTNHTSQNPPVLKRHQR (SEQ ID NO:12), • SFSQNSRHPSNTTNATNNTNQTNSTNHTSQNPPVLKRHQR (SEQ ID NO:13),

• SFSQNSRHPSNTTANATANNTANQTANSTANHTSQNPPVLKRHQR (SEQ ID NO:14),

• SFSQNSRHPSNHTANHTANHTANHTSQNPPVLKRHQR (SEQ ID NO:15),

• SFSQNSRHPSGGNGTGGNGTGGNGTGGNGTSQNPPVLKRHQR (SEQ ID NO:16), or

• a sequence having at least 80%, 90, 95% or 98% identity to SEQ ID NO:3, 4, 11 , 12, 13, 14, 15 or 16; or wherein the sequence encoding the B-domain of FVIII-BDV comprises the following nucleotide sequence:

• AGCTTCTCCCAGAATTCAAGACACCCTTCTAACCACACGGCCAACCACA CGGCGAACCACACTGCTAACCATACCGCAAACCATACTGCTAATCACAC TTCTCAAAACCCACCAGTCTTGAAACGCCATCAACGG (SEQ ID NO: 109).

Embodiment 3: An AAV vector sequence comprising a nucleotide sequence which comprises a splice acceptor, and a transgene encoding a linker sequence, and a FVIII-BDV, wherein the portion of the sequence encoding the linker sequence comprises a nucleotide sequence encoding one of the following amino acid:

• MFSMRIVCLVLSVVGTAWT (SEQ ID NO:1),

• MQIELSTCFFLCLLRFCFS (SEQ ID NO:2),

• LSTCFFLCLLRFCFS (SEQ ID NO:44), and

• a sequence having at least 80%, 90%, 95% or 98% identity to SEQ ID NO:1 , 2, or 44; and wherein the portion of the sequence encoding the B-domain of FVIII-BDV comprises a nucleotide sequence encoding any one of the following amino acid sequences:

• SFSQNSRHPSQNPPVLKRHQR (SEQ ID NO:3),

• SFSQNSRHPSNHTANHTANHTANHTANHTANHTSQNPPVLKRHQR (SEQ ID NO:4),

• SFSQNATNVSNNSNTSKRHQR (SEQ ID NO:11),

• SFSQNSRHPSNHTNHTNHTNHTNHTNHTSQNPPVLKRHQR (SEQ ID NO:12),

• SFSQNSRHPSNTTNATNNTNQTNSTNHTSQNPPVLKRHQR (SEQ ID NO:13), • SFSQNSRHPSNTTANATANNTANQTANSTANHTSQNPPVLKRHQR (SEQ ID NO:14),

• SFSQNSRHPSNHTANHTANHTANHTSQNPPVLKRHQR (SEQ ID NO:15),

• SFSQNSRHPSGGNGTGGNGTGGNGTGGNGTSQNPPVLKRHQR (SEQ ID NO:16), and a sequence having at least 80%, 90%, 95%, 98% or 99% identity to SEQ ID NO: 3, 4, 11 , 12, 13, 14, 15 or 16.

Embodiment 4a: An AAV vector sequence comprising a nucleotide sequence which comprises a splice acceptor, and a transgene encoding a linker sequence, and a FVIII-BDV, wherein the nucleotide sequence of the linker sequence is any one of the following sequences:

• ATGCAAATAGAGCTCTCCACCTGCTTCTTTCTGTGCCTTTTGCGATTCTG CTTTAGT (SEQ ID NO: 108),

• ATTAGCTGAATAATAGAGAAGAATTAACCTTTTGCTTCTCCAGTTGAACA TTTGTAGCAATAAACCATGCAAATAGAGCTCTCCACCTGCTTCTTTCTGT GCCTTTTGCGATTCTGCTTTAGT (SEQ ID NO:8), or

• a nucleotide sequence comprising an adenine (A) follow by a cytosine (C) at the 5’ end of the sequence, followed by a nucleotide sequence as set forth in SEQ ID 107, 108 or 8; and wherein the sequence encoding the B-domain of FVIII-BDV comprises the following nucleotide sequence:

Embodiment 4b: An AAV vector sequence comprising a nucleotide sequence which comprises a splice acceptor, and a transgene encoding a linker sequence, and a FVIII-BDV, wherein the nucleotide sequence of the linker sequence is any one of the following sequences:

ATGTTTTCCATGAGGATCGTCTGCCTGGTCCTAAGTGTGGTGGGCACA

GCATGGACC (SEQ ID NO: 107), or • a nucleotide sequence comprising an adenine (A) follow by a cytosine (C) at the 5’ end of the sequence, followed by a nucleotide sequence as set forth in SEQ ID 107; and wherein the sequence encoding the B-domain of FVIII-BDV comprises the following nucleotide sequence:

Embodiment 5: The AAV vector sequence according to any one of embodiments 1-

3, wherein the portion of the sequence encoding the linker sequence comprises a nucleotide sequence encoding the following amino acid sequence:

• MFSMRIVCLVLSVVGTAWT (SEQ ID NO:1).

Embodiment 6: The AAV vector sequence according to any one of embodiments 1-

3, and 5, wherein the nucleotide sequence encoding the B-domain of FVIII-BDV is encoding one of the following amino acid sequences:

• SFSQNSRHPSQNPPVLKRHQR (SEQ ID NO:3), and

• SFSQNSRHPSNHTANHTANHTANHTANHTANHTSQNPPVLKRHQR (SEQ ID NO:4).

Embodiment 7: The AAV vector sequence according to any one of embodiments 1- 6, wherein the nucleotide sequence of the splice acceptor comprises one of the following sequences:

• TTAACTGTCTTTCTCATTTATCTAG (SEQ ID NO:6),

• TTAACAATCCTTTTTTTTCTTCCCTTGCCCAG (SEQ ID NO:7),

• CTGAGTAATCCTGGTTCCTCTTGAG (SEQ ID NO:56),

• CTAAAGCTTTCTTTCACACTCAAAG (SEQ ID NO:57),

• CTAACGCTTTCTTTCACACTCAAAG (SEQ ID NO:58),

• ATCACACAGAGCCATGCCTATTTAG (SEQ ID NO:59),

• TTAACGCCTCCACCATCTGACTTAG (SEQ ID NQ:60),

• TCAATCAGTCCTCTTGGCGTCTTAG (SEQ ID NO:61),

• TTCACACATCTCGGTTTTTCCTCAG (SEQ ID NO:62),

• AAAACCTACTATTATTCGTTTCTAG (SEQ ID NO:63), TTTATTTCTACTTCTCTGGTTTAAG (SEQ ID NO:64), TGAACAACTACTCTCTTTTCTCTAG (SEQ ID NO:65), and TCTACGCCTCCTTACGCACCCCTAG (SEQ ID NO:66).

Embodiment 8: The AAV vector sequence according to any one of embodiments 1- 7, wherein the nucleotide sequence of the splice acceptor comprises one of the following sequences:

• TTAACTGTCTTTCTCATTTATCTAG (SEQ ID NO:6), and

• TTAACAATCCTTTTTTTTCTTCCCTTGCCCAG (SEQ ID NO:7).

Embodiment 9: An AAV vector sequence comprising a nucleotide sequence which comprises a splice acceptor, a transgene encoding a linker sequence and a B-domain variant Factor VIII (FVI I l-BDV), wherein the nucleotide sequence of the splice acceptor is TTAACTGTCTTTCTCATTTATCTAG (SEQ ID NO:6), wherein the portion of the nucleotide sequence encoding the linker sequence encodes the following amino acid sequence, MFSMRIVCLVLSVVGTAWT (SEQ ID NO:1) and wherein the portion of the nucleotide sequence encoding the B-domain of FVIII-BDV comprises a nucleotide sequence encoding one of the following amino acid sequences

• SFSQNSRHPSQNPPVLKRHQR (SEQ ID NO:3), and

• SFSQNSRHPSNHTANHTANHTANHTANHTANHTSQNPPVLKRHQR (SEQ ID NO:4).

Embodiment 10: The AAV vector sequence according to any one of embodiments 1- 9, wherein the AAV vector sequence comprises ITR sequences on the 5’ end and the 3’ end of said sequence.

Embodiment 11 : The AAV vector sequence according to embodiment 10, wherein the ITR sequence is the following sequence:

• CTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCAAAGCCCGG GCGTCGGGCGACCTTTGGTCGCCCGGCCTCAGTGAGCGAGCGAGC GCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCT (SEQ ID NO:5). Embodiment 12: The AAV vector sequence according to any one of embodiments 1- 11 comprising a nucleotide sequence according to any one of nucleotide sequences SEQ ID NO:90 and 102.

Embodiment 13: An AAV vector sequence comprising a nucleotide sequence according to any one of nucleotide sequences SEQ ID NO:90 and 102.

Embodiment 14: An AAV vector sequence comprising the nucleotide sequence set forth in SEQ ID NO: 102.

Embodiment 15: An AAV vector sequence comprising a nucleotide sequence encoding a transgene amino acid sequence as forth in SEQ ID NO:114.

Embodiment 16: An AAV vector particle comprising, an AAV vector sequence, according to any one of embodiments 1-15, encapsidated by AAV capsid proteins.

Embodiment 17: The AAV vector particle according to embodiment 16, wherein the AAV capsid proteins are of serotype AAV5 or of serotype AAV8.

Embodiment 18: The AAV vector particle according to embodiment 16, wherein the AAV capsid proteins are of serotype AAV8.

Embodiment 19: A gene editing kit comprising (a) an AAV vector sequence, according to any one of embodiments 1-15, or an AAV vector particle according to any one of embodiments 16-18; and (b) an mRNA encoding a site-specific nuclease.

Embodiment 20: The gene editing kit according to embodiment 19, wherein the mRNA encoding a site-specific nuclease is comprised in a lipid nanoparticle.

Embodiment 21 : The gene editing kit according to embodiment 20, wherein the sitespecific nuclease is selected from the group of: transcription activator-like effector nucleases, zinc-finger nucleases, clustered regularly interspaced short palindromic repeat -Cas- associated nucleases, homing endonucleases and megaTAL. Embodiment 22: The gene editing kit according to embodiment 21 , wherein the sitespecific nuclease comprises a homing endonuclease or a megaTAL.

Embodiment 23: The gene editing kit according to any one of embodiments 19-22, wherein the lipid nanoparticle comprises (i) a cationic lipid, (ii) a non-cationic lipid, (iii) a conjugated lipid that prevents aggregation of the particle (e.g., a PEG-lipid), (iv) a cholesterol, and (v) an mRNA sequence encoding a site-specific nuclease, such as a megaTAL.

Embodiment 24: The gene editing kit according to any one of embodiments 19-23, wherein the AAV vector particle comprises: (i) AAV capsid proteins and (ii) an AAV vector nucleotide sequence according to any one of embodiments 1-15, and wherein the serotype of the AAV capsid proteins is selected from serotype AAV5 and serotype AAV8.

Embodiment 25: The gene editing kit according to embodiment 24, wherein the serotype of the AAV capsid proteins is AAV8.

Embodiment 26: A gene editing kit comprising (a) an AAV vector sequence, which comprises any one of nucleotide sequences SEQ ID NO:90 and 102, comprised in an AAV vector particle wherein the AAV capsid proteins of the AAV vector particle are of serotype AAV5 or of serotype AAV8; and (b) an mRNA encoding a megaTAL comprised in a lipid nanoparticle.

Embodiment 27: A gene editing kit comprising (a) an AAV vector sequence, which comprises the nucleotide sequence set forth in SEQ ID NO: 102, comprised in an AAV vector particle wherein the AAV capsid proteins of the AAV vector particle is of serotype AAV8; and (b) an mRNA encoding a megaTAL comprised in a lipid nanoparticle.

Embodiment 28: A pharmaceutical composition comprising an AAV vector sequence, according to any one of embodiments 1-15, or an AAV vector particle according to any one of embodiments 16-18 in a pharmaceutically-acceptable solution.

Embodiment 29: A pharmaceutical composition comprising an AAV vector sequence, according to any one of embodiments 1-15, or an AAV vector particle according to any one of embodiments 16-18 in a pharmaceutically-acceptable solution for administration to an animal, such as a human.

Embodiment 30: A pharmaceutical kit comprising (a) an AAV vector sequence, according to any one of embodiments 1-15, or an AAV vector particle according to any one of embodiments 16-18 in a pharmaceutically-acceptable solution and; (b) an mRNA encoding a site-specific nuclease comprised in a lipid nanoparticle in a pharmaceutically-acceptable solution. Embodiment 31: The pharmaceutical kit according to embodiment 30, wherein the lipid nanoparticle comprises (i) a cationic lipid, (ii) a non-cationic lipid, (iii) a conjugated lipid that prevents aggregation of the particle (e.g., a PEG-lipid), (iv) a cholesterol and (v) an mRNA sequence encoding a site-specific nuclease.

Embodiment 32: The pharmaceutical kit according to any one of embodiments SO-

31 , wherein the site-specific nuclease is a megaTAL.

Embodiment 33: The pharmaceutical kit according to any one of embodiments 30 -

32, wherein the site-specific nuclease is a megaTAL, where said megaTAL comprises a nucleotide sequence encoding an amino acid sequence set forth in SEQ ID NO:111 , SEQ ID NO: 112, or where the megaTAL comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in any one of SEQ ID NOs:111-112.

Embodiment 34: The pharmaceutical kit according to any one of embodiments 30 - 31 , wherein the site-specific nuclease is a megaTAL, where said megaTAL comprises a nucleotide sequence encoding an amino acid sequence set forth in SEQ ID NO:111 or SEQ ID NO:112.

Embodiment 35: The pharmaceutical kit according to embodiment 32, wherein the mRNA comprises the polynucleotide sequence set forth in SEQ ID NO: 113.

Embodiment 36: A pharmaceutical kit comprising (a) an AAV vector sequence comprising a nucleotide sequence set forth in SEQ ID: 102 comprised in a AAV vector particle; and (b) an mRNA sequence encoding a megaTAL comprising a nucleotide sequence set forth in SEQ ID NO: 113, comprised in a lipid nanoparticle.

Embodiment 37: The pharmaceutical kit according to embodiment 30, wherein comprises (a) an AAV vector sequence comprising a nucleotide sequence set forth in SEQ ID: 102 comprised in a AAV vector particle; and (b) an mRNA sequence encoding a megaTAL comprising a nucleotide sequence set forth in SEQ ID NO:113, comprised in a lipid nanoparticle, and wherein AAV capsid proteins of the AAV vector particle are of serotype AAV8.

Embodiment 38: A method of editing a genome in a cell comprising providing the following to a cell: (a) AAV vector sequence, according to any one of embodiments 1-15, or an AAV vector particle according to any one of embodiments 16-18; and (b) an mRNA encoding a site-specific nuclease such as a megaTAL. Embodiment 39: A method of editing a genome in a cell comprising providing the following to a cell: (a) an AAV vector particle comprising an AAV vector nucleotide sequence according to any one of embodiments 1-15; and (b) a lipid nanoparticle comprising(i) a cationic lipid, (ii) a non-cationic lipid (e.g. cholesterol), (iii) a conjugated lipid that prevents aggregation of the particle (e.g., a PEG-lipid), and (iv); an mRNA sequence encoding a megaTAL.

Embodiment 40: A method of editing a genome in a cell by providing the following to the cell: (a) an AAV vector particle comprising an AAV vector nucleotide sequence according to any one of embodiments 1-15; and (b) a lipid nanoparticle comprising a nucleotide sequence encoding a mRNA sequence encoding a megaTAL site-specific nuclease, wherein the AAV vector nucleotide sequence comprises a FVIII-BDV encoding nucleotide sequence and wherein the target site for mRNA sequence encoded megaTAL site-specific nuclease is within intron 1 of the human albumin (ALB) gene.

Embodiment 41: The method of editing a genome according to any one of embodiments 38 -40, wherein the site-specific nuclease is a megaTAL, where said megaTAL comprises a nucleotide sequence encoding an amino acid sequence set forth in SEQ ID NO:111 , SEQ ID NO:112, or where the megaTAL comprises a nucleotide sequence encoding an amino acid that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in any one of SEQ ID NOs:111-112.

Embodiment 42: The method of editing a genome according to any one of embodiments 38 -40, wherein the site-specific nuclease is a megaTAL, where said megaTAL comprises a nucleotide sequence encoding an amino acid sequence set forth in SEQ ID NO:111 or SEQ ID NO:112.

Embodiment 43: The method of editing a genome according to embodiment 42, wherein the site-specific nuclease comprises an amino acid sequence set forth in SEQ ID NO:111.

Embodiment 44: The method of editing a genome according to embodiment 42, wherein the site-specific nuclease comprises an amino acid sequence set forth in SEQ ID NO:112. Embodiment 45: The method of editing a genome according to embodiment 42, wherein the mRNA comprises the polynucleotide sequence set forth in SEQ ID NO:113.

Embodiment 46: A method of treating hemophilia A in a patient comprising administering to the patient an effective amount of the AAV vector sequence according to any one of embodiments 1-15, the AAV vector particle according to embodiments 16-18, the composition of embodiments 28-29, or the components (a) and (b) of the pharmaceutical kit according to any one of embodiments 30-37.

Embodiment 47: A method of treating hemophilia A in a patient comprising administering to the patient an effective amount of (a) an AAV vector sequence comprising a nucleotide sequence according to any one of nucleotide sequences SEQ ID NO:90 and 102, and (b) an mRNA sequence encoding site-specific nuclease, such as a megaTAL, comprised in a lipid nanoparticle comprising (i) a cationic lipid, (ii) a non-cationic lipid (e.g. cholesterol), (iii) a conjugated lipid that prevents aggregation of the particle (e.g., a PEG-lipid), and (iv).

Embodiment 48: A method of treating hemophilia A in a patient comprising administering to the patient an effective amount of (a) an AAV vector sequence comprising a nucleotide sequence set forth in SEQ ID: 102 and (b) an mRNA sequence encoding a megaTAL, comprised in a lipid nanoparticle comprising (i) a cationic lipid, (ii) a non-cationic lipid (e.g. cholesterol), (iii) a conjugated lipid that prevents aggregation of the particle (e.g., a PEG-lipid), and (iv). wherein the mRNA comprises the polynucleotide sequence set forth in SEQ ID NO:113.

Embodiment 49: A method of treating hemophilia A in a patient comprising administering to the patient an effective amount of (a) an AAV vector sequence comprising a nucleotide sequence set forth in SEQ ID: 102 and (b) an mRNA sequence encoding a megaTAL comprising a nucleotide sequence set forth in SEQ ID NO:113, comprised in a lipid nanoparticle.

Embodiment 50: The method of treating hemophilia A according any one of embodiments 46-49 wherein the site-specific nuclease (component (b)) is administered one, two or three days after administration of the AAV vector sequence (component (a)). Embodiment 51: A method of treating hemophilia A in a patient, comprising (i) administering to the patient an effective amount of an AAV vector sequence, according to any one of embodiments 1-15, comprised in a AAV vector particle; and (ii) one day after step (i), administering to the patient an effective amount of an mRNA encoding a site-specific nuclease comprised in a lipid nanoparticle.

Embodiment 52: A method of treating hemophilia A in a patient, comprising (i) administering to the patient an effective amount of an AAV vector sequence, according to any one of embodiments 1-15, comprised in a AAV vector particle; and (ii) two or three days after step (i), administering to the patient an effective amount of an mRNA encoding a sitespecific nuclease comprised in a lipid nanoparticle.

Embodiment 53: Use of the AAV vector sequence according to any one of embodiments 1-15, the AAV vector particle according to embodiments 16-18, the composition of claims 28-29, or the components (a) and (b) of the pharmaceutical kit according to claims 30-37, as a medicament for treatment of hemophilia A.

Embodiment 54: Use of the AAV vector sequence according to any one of embodiments 1-15, the AAV vector particle according to embodiments 16-18, the composition of claims 28-29, or the components (a) and (b) of the pharmaceutical kit according to claims 30-37, to prepare a medicament for treatment of hemophilia A.

Embodiment 55: Use of an (a) an AAV vector sequence comprising a nucleotide sequence according to any one of nucleotide sequences SEQ ID NO:90 and 102 and (b) an mRNA sequence encoding a site-specific nuclease, such as a megaTAL, comprised in a lipid nanoparticle comprising(i) a cationic lipid, (ii) a non-cationic lipid (e.g. cholesterol), and (iii) a conjugated lipid that prevents aggregation of the particle (e.g., a PEG-lipid), as a medicament for treatment of hemophilia A.

Embodiment 56: Use of an (a) an AAV vector sequence comprising a nucleotide sequence set forth in SEQ ID NO:102 and (b) an mRNA sequence encoding a site-specific nuclease, such as a megaTAL comprising a nucleotide sequence set forth in SEQ ID NO:113, comprised in a lipid nanoparticle, as a medicament for treatment of hemophilia A.

Claims

1. An adeno-associated virus (AAV) vector sequence comprising a nucleotide sequence which comprises a splice acceptor, a transgene encoding a linker sequence and a B-domain variant Factor VIII (FVIII-BDV), wherein the portion of the sequence encoding the linker sequence comprises a nucleotide sequence encoding one of the following amino acid sequences:

• MFSMRIVCLVLSVVGTAWT (SEQ ID NO:1), or

• SFSQNSRHPSQNPPVLKRHQR (SEQ ID NO:3), or

• SFSQNSRHPSNHTANHTANHTANHTANHTANHTSQNPPVLKRHQR (SEQ ID NO:4).

2. The AAV vector sequence according to claim 1, wherein the splice acceptor comprises one of the following sequences:

• TTAACTGTCTTTCTCATTTATCTAG (SEQ ID NO:6), or

• TTAACAATCCTTTTTTTTCTTCCCTTGCCCAG (SEQ ID NO:7).

3. The AAV vector sequence according any one of claims 1-2, wherein the portion of the nucleotide sequence encoding the linker sequence comprises a nucleotide sequence encoding the following amino acid sequence:

• MFSMRIVCLVLSVVGTAWT (SEQ ID NO:1).

4. An AAV vector sequence comprising a nucleotide sequence which comprises a splice acceptor, a transgene encoding a linker sequence and a B-domain variant Factor VIII (FVIII-BDV), wherein the nucleotide sequence of the splice acceptor is TTAACTGTCTTTCTCATTTATCTAG (SEQ ID NO:6), wherein the portion of the nucleotide sequence encoding the linker sequence encodes the amino acid sequence, MFSMRIVCLVLSVVGTAWT (SEQ ID NO:1) and wherein the portion of the nucleotide sequence encoding the B-domain of FVIII-BDV comprises a nucleotide sequence encoding one of the following amino acid sequences

5. The AAV vector sequence according to any one of claims 1-4, comprising the nucleotide sequence set forth in SEQ ID NO: 102.

6. An AAV vector particle comprising, an AAV vector sequence, according to any one of claims 1-5, encapsidated by AAV capsid proteins.

7. The AAV vector particle according to claim 6, wherein the AAV capsid proteins are of serotype AAV8.

8. A gene editing kit comprising (a) an AAV vector sequence, according to any one of claims 1-5, or an AAV vector particle according to any one of claims 6-7; and (b) an mRNA encoding a site-specific nuclease comprised in a lipid nanoparticle.

9. A pharmaceutical composition comprising an AAV vector sequence, according to any one of claims 1-5, or an AAV vector particle according to any one of claims 6-7 in a pharmaceutically-acceptable solution.

10. A pharmaceutical kit comprising (a) an AAV vector sequence, according to any one of claims 1-5, or an AAV vector particle according to any one of claims 6-7 in a pharmaceutically-acceptable solution; and (b) an mRNA encoding a site-specific nuclease comprised in a lipid nanoparticle in a pharmaceutically-acceptable solution.

11. The pharmaceutical kit according to claim 10, wherein the lipid nanoparticle comprises (i) a cationic lipid, (ii) a non-cationic lipid, (iii) a conjugated lipid that prevents aggregation of the particle (e.g., a PEG-lipid), (iv) a cholesterol and (v) an mRNA sequence encoding a site-specific nuclease, such as a megaTAL.

12. The pharmaceutical kit according to any one of claims 10 -11, wherein the sitespecific nuclease is a megaTAL, and wherein said megaTAL comprises a nucleotide sequence encoding an amino acid sequence set forth in SEQ ID NO:111 or SEQ ID NO:112; or comprises a nucleotide sequence set forth in SEQ ID NO:113.

13. A pharmaceutical kit comprising (a) an AAV vector sequence, comprising the nucleotide sequence set forth in SEQ ID NO:102, comprised in an AAV vector particle where the AAV capsid proteins of the AAV vector particle are of serotype AAV8; and (b) an mRNA encoding a megaTAL comprised in a lipid nanoparticle, and wherein the megaTAL mRNA comprises the nucleotide sequence set forth in SEQ ID NO:113.

14. A method of editing a genome in a cell comprising providing the following to a cell: (a) AAV vector sequence, according to any one of claims 1-5, or an AAV vector particle according to any one of claims 6-7; and (b) an mRNA encoding a site-specific nuclease.

15. A method of treating hemophilia A in a patient comprising administering to the patient an effective amount of the AAV vector sequence according to any one of claims 1-5, the AAV vector particle according to claims 6-7, the composition of claim 9, or the components (a) and (b) of the pharmaceutical kit according to claims 10-13.

16. Use of the AAV vector sequence according to any one of claims 1-5, the AAV vector particle according to claims 6-7, the composition of claim 9, or the components (a) and (b) of the pharmaceutical kit according to claims 10-13 in the treatment of hemophilia A.