WO2020104480A1

WO2020104480A1 - Adeno-associated virus vectors for expressing fviii mimetics and uses thereof

Info

Publication number: WO2020104480A1
Application number: PCT/EP2019/081846
Authority: WO
Inventors: Ying Poi LIU; Sander Jan Hendrik Van Deventer; Erhard Seifried; Joachim SCHWÄBLE; Karin Nien HUBER
Original assignee: Uniqure Biopharma B.V.; DRK-Blutspendedienst Baden-Württemberg-Hessen gemeinnützige GmbH
Priority date: 2018-11-19
Filing date: 2019-11-19
Publication date: 2020-05-28

Abstract

The present inventors now sought to provide for a gene therapy approach for the treatment of hemophilia A that is both safe and effective for human use and that is not affected by the presence of FVIII neutralizing antibodies. The current inventors now provide for means, methods and uses of nucleic acid sequences encoding a variant human FIX protein that mimic FVIII activity, that can be applied in a gene therapy approach suitable and effective for humans.

Description

Adeno-associated virus vectors for expressing FVIII mimetics and uses thereof Background

Clotting disorders such as haemophilia, wherein patients lack or have a defect in a component of the clotting cascade, typically result in prolonged and excessive bleeding, which may occur spontaneously or following an injury or certain medical procedures. Clotting disorders are also referred to as coagulopathies and apart from prolonged and excessive bleedings, coagulopathies are also characterized by chronic hemophilic arthropathy, due to chronic injury of endothelial cells lining the bloodvessels. Hemophilia A is such a clotting disorder and is characterized by a defect in or lack of FVIII (Furie and Furie, Cell, 1988). Hemophilia A can be hereditary or can be an acquired disease. Current treatment involves protein replacement therapy, i.e. wherein the patient receives infusions of FVIII clotting factor, or e.g. variants thereof with an extended half-life. Some haemophilia A patients cannot tolerate infusions of FVIII because these patients have developed an immune response towards FVIII and produce antibodies targeting FVIII thereby neutralizing FVIII and thus inhibiting its action. Hence, these patients need to resort to alternative protein replacement therapies. Such alternative treatments can involve activated FVII or activated prothrombin complex concentrate (aPCC) (Antunes et a , Haemophilia, 2014; Konkle et a , J Thromb Haemost 2007; Leissinger et a , NEJM, 2011). Although very successful, these therapies are used on- demand due to their short half-life and therefore cannot prevent chronic hemophilic arthropathy. Although rare, thrombotic events have been reported with the usage of these therapies (Guillet et a , 2002; Peerlinck and Vermylen, 1999). An alternative treatment with non-factor molecules recently approved, involves the use of a bispecific antibody, emicizumab (Hemlibra), which binds both FIX and FX, and by bridging activated FIX and FX, replaces the function of the missing activated FVIII. Another alternative treatment with non-factor molecules which is under development involves an RNAi approach targeting antithrombin (fitusiran) (Pasi et a , N Engl J Med 2017). However, using such agents in clinical trials, unexpected thrombotic events were reported warranting an assessment of the safety profiles of such new drugs (Oldenburg et al. N Engl J Med 2017, Pasi et a , N Engl J Med 2017).

In addition to protein replacement therapies, currently gene therapy approaches, including AAV-mediated gene therapies, for coagulopathies are being tested in clinical trials, including gene therapies for hemophilia A (Rangarajan et a , N Engl J Med 2017). However, in such AAV-based gene therapies aimed to get high expression levels of FVIII, safety concerns have been raised as elevations of the liver enzyme ALT were observed in patients treated with high doses of gene therapy vector suggesting potential hepatocyte toxicity and patients needed to be treated with steroids for immunosuppression (see i.a. ClinicalTrials.gov registry numbers by the U.S. National Library of Medicine: NCT01687608, NCT02484092, NCT02618915). Treated patients have also shown a decline in FVIII expression levels over time (Biomarin, 2018 IR call, May 22, 2018, World Federation of Hemophilia Congress). Furthermore, such FVIII based gene therapy approaches currently are not an option for hemophilia A patients which have inhibitors against FVIII, because FVIII protein induced by the therapeutic transgene would be neutralized by those inhibitors and consequently these patients are excluded from clinical trials. Hence, in spite of protein replacement therapies having some success, improving the lives of haemophilia A patients, and that the current gene therapy approaches for haemophilia A achieved some success in the clinic, concerns with regards to safety and/or activity remain. In addition, there is still a need for new and improved therapies that are suitable for use in patients suffering from hemophilia A with and without inhibitors.

Summary of the invention

The present inventors now sought to provide for a gene therapy approach for the treatment of hemophilia A that is both safe and effective for human use and that is not affected by the presence of FVIII neutralizing antibodies. Previously variants of FIX that can activate FX in the absence of FVIII and showed FVIII mimetic activity have been proposed for the treatment of haemophilia in e.g. a gene therapy approach (Milanov et al., Blood, 2012;

Quade-Lyssy et al., J Thromb Hemost, 2014) (see figure 4). The highest levels of FIX protein expression that are currently obtained and as reported in hemophilia B patients with gene therapy for wild-type FIX are at most 12% FIX (Doshi and Arruda, Ther. Adv. Hematology, 2017), which, in view of an only modest dose response represents an upper level of FIX achieved in hemophilia B patients (Miesbach et al., Blood, 2018). Furthermore, increasing dosages of AAV may result in liver toxicity. Hence, gene therapy approaches in hemophilia B patients have resorted to utilizing hyperactive variants having 8-9 fold more activity to increase clotting activity in human patients (George et al., N Engl J M, 2017). Based on results of AAV mediated FIX transgene expression in human patients, assuming similar expression levels could be obtained, FIX data can be extrapolated to reported FVIII mimetic activities of said variants in the literature, which is expected to result in clotting activity which is too low to be considered meaningful. This is because about at least 10% FVIII activity is considered meaningful for haemophilia A patients (den Uijl, Haemophilia, 2011), and with reported FVIII mimetic activity of FIX variants of at most about 15%, in the current clinical setting this would surmount to, in the most optimistic scenario, in 1.8 % clotting activity in hemophilia A patients (i.e. 12% x 15% = 1.8%). This is not expected to surmount to any meaningful therapeutic effect in the human hemophilia A population.

The scenario described above is based on several assumptions, and one has to realize that results obtained with FIX variants were only obtained in mice utilizing hydrodynamic injections of minicircle DNA which is far removed from any acceptable clinical application suitable for humans. Furthermore, expression of FIX variants may not necessarily be the same as observed for their wild-type counterpart and can be reduced. Hence, even the scenario outlined above may be regarded to be based on too optimistic assumptions.

As said, FIX variants can activate coagulation in the absence of FVIII. Emicizumab (Hemlibra) may be regarded to mimic FVIII activity. However, as indicated above, with Emicizumab and fitusiran, adverse events related to unexpected thrombotic events were reported. Hence, because of these adverse events observed in humans, the use of FIX variants mimicking FVIII activity may raise potential concerns from a safety perspective, in particular when used in a gene therapy approach which is irreversible. Such safety perspective is currently lacking for the FIX variants.

Easily, as a large numbers of FIX variants have been described in the art, the optimal candidate from the viewpoint of FVIII mimetic activity, safety and expression levels for use in humans with hemophilia A, remains unknown.

To conclude, the treatment of hemophilia A patients utilizing FIX variants having FVIII mimetic activity has neither been demonstrated to be feasible nor has clinical application in humans been supported in previous work.

The current inventors now have addressed the issues and now provide for means, methods and uses of nucleic acid sequences encoding a variant human FIX protein having at least amino acid substitutions corresponding to VI 8 II, K265A and 1383 V, which were found to be useful in a gene therapy approach suitable for humans. Said variant human FIX protein further comprising either F6F or E185D. Most preferably, the variant human FIX protein has amino acid substitutions F6F, V181I, K265A and I383V. In particular, means, methods and uses are provided utilizing AAV vectors encoding said FIX variants that allow for a treatment suitable and effective for human use.

These variant human FIX proteins can activate the coagulation cascade in the absence of FVIII and therefore FVIII inhibitors and/or potential immune responses against FVIII in hemophilia A patients would not impact therapy. Furthermore, the inventors found that gene therapy mediated expression of such variant FIX protein was safe, as in vitro it could e.g. be combined with FVIIa or aPCC without an unexpected or exaggerated increase in thrombin generation, and when combined with FVIII, there is a potentiation of the thrombin potential, that showed to have a linear correlation, which argues for a lower dose of FVIII when combined with the expressed variant FIX proteins for human use.

Variant FIX proteins will ideally have similar activation and regulation properties as wild-type FIX, in addition to their FVIII mimetic properties and analyses are ongoing.

Immunological screening in silico and in vitro furthermore indicated that the amino acid substitutions present in the variant FIX proteins expressed upon gene therapy, are predicted not to present a high immunogenicity risk such as has been observed for native FVIII, or variants of FVIII (therapeutic proteins based on FVIII that have been modified, e.g. to extend half-life).

Lastly, and importantly, the amount of variant FIX protein expression levels that needs to be achieved in humans for therapeutic action could be obtained in primates upon AAV mediated expression of FIX variants, showing that the gene therapy can be applied and is feasible in primates, and is expected to be effective also in humans. Combined, the inventors provide for a gene therapy approach, utilizing variant human FIX protein having amino acid substitutions corresponding to VI 8 II, K265A and I383V, preferably combined with L6F, that is both safe and effective for human use. Such a gene therapy preferably utilizes an AAV vector, most preferably of the AAV5 serotype. Said FIX variants being preferably expressed from a highly efficient liver specific promoter, such as Q1 or a variant thereof. The human use of this gene therapy approach includes administration to hemophilia A patients having inhibitors against FVIII. Hemophilia A patients that do not have inhibitors against FVIII will benefit from such a gene therapy treatment as well.

Definitions

As used in the description of the invention, clauses and clauses appended claims, the singular forms“a”,“an” and“the” are used interchangeably and intended to include the plural forms as well and fall within each meaning, unless the context clearly indicates otherwise. Also, as used herein,“and/or” refers to and encompasses any and all possible combinations of one or more of the listed items, as well as the lack of combinations when interpreted in the alternative (“or”).

As used herein, the term“about” will be understood by persons of ordinary skill in the art and will vary to some extent depending upon the context in which it is used. If there are uses of the term which are not clear to persons of ordinary skill in the art given the context in which it is used,“about” will mean up to plus or minus 10% of the particular term.

“Sequence identity” and“sequence similarity” can be determined by alignment of two peptides or two nucleotide sequences using global or local alignment algorithms, depending on the length of the two sequences. Sequences of similar lengths are preferably aligned using a global alignment algorithm (e.g. Needleman Wunsch) which aligns the sequences optimally over the entire length. A global alignment is suitably used to determine sequence identity when the two sequences have similar lengths. Generally, the GAP default parameters are used, with a gap creation penalty = 50 (nucleotides) / 8 (proteins) and gap extension penalty = 3 (nucleotides) / 2 (proteins). For nucleotides the default scoring matrix used is nwsgapdna and for proteins the default scoring matrix is Blosum62 (Henikoff & Henikoff, 1992, PNAS 89, 915-919). Sequence alignments and scores for percentage sequence identity may be determined using computer programs, such as the GCG Wisconsin Package, Version 10.3, available from Accelrys Inc., 9685 Scranton Road, San Diego, CA 92121-3752 USA, or using open source software, such as the program“needle” (using the global Needleman Wunsch algorithm) or“water” (using the local Smith Waterman algorithm) in Emboss WIN version 2.10.0, using the same parameters as for GAP above, or using the default settings (both for‘needle’ and for‘water’ and both for protein and for DNA alignments, the default Gap opening penalty is 10.0 and the default gap extension penalty is 0.5; default scoring matrices are Blossum62 for proteins and DNAFull for DNA). Alternatively, percentage similarity or identity may be determined by searching against public databases, using algorithms such as FASTA, BEAST, etc.

As used herein, "gene therapy" is the delivery of nucleic acid sequences into individual's cells and/or tissues to treat a disease. The nucleic acid can be a functional mutant allele that replaces or supplements a defective one. (e.g., a nucleic acid or expression cassettes encoding a FIX variant as defined herein). Such transgenes may be exogenous. An exogenous molecule or sequence is understood to be molecule or sequence not normally occurring in the cell, tissue and/or individual to be treated. Both acquired and congenital diseases are amenable to gene therapy.

A "gene therapy vector" in accordance with the invention refers to a vehicle for delivery of nucleic acids into an individual's cells and/or tissues to treat a disease. Examples of gene therapy vectors include non- viral gene therapy vectors, i.e. a nucleic acid which is comprised in e.g. a liposome, lipoplex or conjugated with a ligand, such that the nucleic acid is delivered to cells and/or tissues of an individual to treat a disease. Examples of gene therapy vectors also include viral gene therapy vectors. Viral gene therapy vectors may also be referred to as "viral vectors" herein. Suitable viral gene therapy vectors may be lentiviral vectors, retrotransposon based vector systems, or AAV vectors. A viral gene therapy vector is distinguished from a wild-type virus since at least part of the viral genome has been replaced with a transgene, i.e. an inserted nucleic acid, which is a non-native nucleic acid with respect to the wild-type viral nucleic acid sequence. The viral vector comprises similar constituents as a wild- type virus. A viral vector comprises a viral vector genome, with its inserted nucleic acid, which viral vector is comprised in a virus envelop/capsid, or a derivative thereof.

An "AAV vector" refers to a gene therapy vector which is based on a recombinant adeno-associated virus (AAV) vector which is derived from wild- type AAV. An AAV5 gene therapy vector thus refers to an AAV5 vector for use in gene therapy. An AAV vector is distinguished from a wild-type AAV since at least part of the viral genome has been replaced with a transgene, which is a non-native nucleic acid with respect to the wild-type AAV nucleic acid sequence. Hence, an AAV vector may consist of similar constituents as a wild- type virus, such as a capsid comprising a vector genome, wherein the vector genome comprises a transgene (e.g. an expression cassette). The vector genome most preferably does not express any viral proteins (such as VP1, VP2, VP3 and/or Rep40/52, Rep60/78), and typically may have flanking inverted terminal repeats (ITRs) as found e.g. in a wild-type AAV. The vector genome as comprised in an AAV vector may comprise ITRs flanking an expression cassette and further non- AAV derived sequences e.g. stuffer sequences.

A "serotype" is traditionally defined on the basis of a lack of cross-reactivity between antibodies to one virus as compared to another virus. Such cross-reactivity differences are usually due to differences in capsid protein sequences/antigenic determinants (e.g., due to VP1, VP2, and/or VP3 capsid sequence differences of AAV serotypes). Under the traditional definition, a serotype means that the virus of interest has been tested against serum specific for all existing and characterized serotypes for neutralizing activity and no antibodies have been found that neutralize the virus of interest. As more naturally occurring virus isolates are discovered and capsid mutants generated, there may or may not be serological differences with any of the currently existing serotypes. For the sake of convenience, AAV5 serotypes include AAV with capsid sequence modifications that have not been characterized as being a distinct serotype, which may also constitute a subgroup or variant of the AAV5 serotype.

Such variants in general having substantial sequence identity and having similar tropism with e.g. AAV5 vectors having capsids such as described by Urabe et a , J Virol. 2006 and As used herein, the term“saturating agent” refers to an agent that is capable of saturating the scavenger function of the reticuloendothelial system (RES). The RES may include, but are not limited to, Kupffer cells, sinusoidal endothelial cells (SEC), and hepatic stellate cells (HSC). For the purposes of the present disclosure, precise compound or composition is not crucial, so long as the administration of the compound or composition can occupy the scavenging function of the RES such that the RES is unable to scavenge AAV gene therapy vectors that are concurrently or subsequently administered.

As used herein, the phrase“scavenger function of the RES” refers to the phagocytic activity of the RES. Kupffer cells and liver sinusoidal endothelial cells (SEC) are responsible for approximately 90% of the total phagocytic capacity of the RES. In some embodiments, the saturating agent is an agent that is capable of saturating the phagocytic capacity of the RES. In some embodiments, the saturating agent is an agent that is capable of saturating the phagocytic capacity of the Kupffer cells.

As used herein, the phrase“saturating the phagocytic capacity” refers to the phagocytic uptake of an agent (e.g., the saturating agent) that prevents the phagocytic cell from taking up another agent (e.g., the AAV gene therapy vector). Thus, in some

embodiments, a saturating agent is an agent that is capable of saturating the phagocytic activity of the RES, such that the phagocytic cells of the RES take up the saturating agent instead of another agent, such as the AAV gene therapy vector and/or therapeutic agent. In some embodiments, a saturating agent is an agent that is capable of saturating the phagocytic activity of Kupffer cells, such that the Kupffer cells take up the saturating agent instead of another agent, such as the AAV gene therapy vector and/or therapeutic agent.

Detailed description

The present invention provides for a variant of factor IX (also referred to as FIX), wherein the variant of Factor IX is characterized in that it has clotting activity in the absence of factor VIII or activated factor VIII, said variant being encoded by a nucleic acid. Said nucleic acids being most preferably comprised in AAV vectors suitable for use in methods of treatments of humans having hemophilia A. Hence, the current invention relates to a nucleic acid encoding a variant of human factor IX, said variant factor IX comprising at least amino acid substitutions corresponding to VI 8 II, K265A and I383V of a polypeptide having an amino acid sequence as listed in SEQ ID NO. 1. Preferably, said variant factor IX comprises further an amino acid substitution selected from the group consisting of L6F and E185D, most preferably, the amino acid substitution is L6F. A variant factor IX comprising L6F, V181I, K265A and I383V may be referred to herein as factor IX FIAV, FIX-FIAV, or FIAV (See Figure 1 and SEQ ID NO.l). A variant factor IX comprising V181I, E185D, K265A and I383V may be referred herein as factor IX IDAV, FIX-ID AV or IDAV.

Human factor IX, and a nucleic acid encoding human factor IX, within this patent application can refer to human factor IX cDNA which encodes human factor IX protein such as listed respectively in Refseq. NM_000133.3 and Refseq. NP_000124.1. The human factor IX amino acid sequence represents a precursor molecule or propeptide. The first 46 amino acid residues contain the hydrophobic signal and the hydrophilic propeptide sequence that is cleaved upon secretion of the mature protein of 415 amino acids, factor IX, is secreted from the cell in which it is expressed (see i.a. Figure 1). FIX consists of a calcium-binding Gla domain, two EGF (epidermal Growth factor)-like domains (EGF1 and EGF2), an activation peptide and the protease domain. Activation of FIX results in the formation of two chains: light chain (Gla-EGF1-EGF2) and the heavy chain (protease domain) and the release of the activation peptide (Figure 1) (Zogg T and Brandstetter Prog Mol Biol Transl Sci 2011). Hence, a factor IX protein, or a variant thereof in accordance to the invention, may correspond with or comprises the 415 amino acids of the mature protein. The numbering of amino acid substitutions in e.g. FIAV and IDAV corresponding with the positions of the mature protein as depicted in figure 1 and of SEQ ID NO.l and amino acid positions corresponding therewith in the propeptide SEQ ID NO.2 and mature FIX are indicated in figure 2. Preferably, the variant of the factor IX protein in accordance with the invention comprises a factor IX propeptide corresponding with SEQ ID NO. 2, i.e. having a sequence in accordance with SEQ ID NO. 2 having e.g. the FIAV or IDAV mutations. It is understood that SEQ ID NO.l and SEQ ID NO.2 represents examples of wild-type human FIX protein sequences. The human FIX protein sequences allow for sequence variation in humans and it is understood that variants of FIX in accordance with the invention may be based on such natural variation, from which mutations associated with clotting disorders such as hemophilia B are excluded. Hence, a human FIX protein based upon which FIX variants in accordance with the invention can be made, may have an amino acid sequence as found in humans, and which has a human FIX clotting activity similar to a human FIX as listed e.g. in SEQ ID NO.l, such clotting activity being determined e.g. as shown in the examples.

In one embodiment, a nucleic acid is provided encoding the variant of human factor IX in accordance with the invention, wherein the FIX variant has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity with the amino acid sequences of SEQ ID NO 1 or SEQ ID NO.2, wherein it is understood that the amino acid substitutions at positions corresponding with VI 8 II, K265A, 1383V and selected from L6F and E185D, are not taken into account when determining sequence identity. A mature and propeptide FIX-FIAV that are provided in accordance with the invention have a sequence as listed in SEQ ID NO. 5 and 6, respectively. In a further embodiment, a nucleic acid in accordance with the invention is provided encoding a variant of human factor IX, wherein said nucleic acid has at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with SEQ ID NO.3 or SEQ ID NO.4,

representing respectively an open reading frame encoding FIX either codon optimized or as can be found in humans. SEQ ID NO. 3 and SEQ ID NO.4 encode wild-type propeptide FIX. It is understood that the codons encoding the amino acid substitutions at positions corresponding with V181I, K265A, I383V and selected from L6F and E185D of the mature FIX variant are not taken into account when determining sequence identity. It is also understood that a nucleic acid encoding a protein sequence may comprise an intron. A nucleic acid encoding a FIX variant in accordance with the invention may thus be

interspersed by an intron. It also understood that the coding sequence may be interspersed by more than one intron. Hence, in determining sequence identity, such intronic sequences are not to be taken into account. In determining sequence identity, the open reading frame sequence that is translated into a FIX variant (i.e. a sequence without intronic sequences, which may be an RNA or DNA sequence), or a sequence that corresponds with an open reading frame sequence (e.g. a reverse complementary sequence) are thus aligned with SEQ ID NO.3 or SEQ ID NO.4 (or to the reverse complement of SEQ ID NO.3 or SEQ ID NO.4). A nucleic acid encoding FIX-FIAV in accordance with the invention may have a sequence as listed in SEQ ID NO. 7.

The nucleic acid according to the invention may further be operably linked to a promoter sequence for expression of the variant FIX. Hence, the nucleic acid encoding the variant FIX in accordance with the invention may be comprised in an expression cassette for expression of the variant FIX. Preferably said promoter sequence is highly active in the liver, preferably a human liver, and more preferably the promoter sequence is liver- specific. Fiver- specific means that high expression can be obtained in human liver cells, whereas in other cell types, e.g. lung or muscle, expression is low. This is as opposed to promoters such as CMV or the like, which in general provide for high expression levels in many cell types. Hence, preferably the promoter is not a ubiquitous promoter and is active in the liver.

A suitable promoter sequence that may be selected is the FP1 promoter as listed in SEQ ID NO.8. Preferably, a promoter sequence that may be selected is SEQ ID NO.16-18. The latter promoter sequences being synthetic liver specific promoter sequences that provide for higher and more selective expression in human liver cells as the LP1 promoter. SEQ ID NO. 18 is referred to as Q1 herein, and SEQ ID NO. 16 and 17 are variants of Ql. Variants of the Ql promoter suitable for use in accordance with the invention can comprise one or more, preferably at least 4, most preferably all of HNF1/HNF3 (SEQ ID NO: 9), HNF3/HNF3 (SEQ ID NO: 10), c/EBP/HNF4 (SEQ ID NO: 11), HS_CRM2/HNF3 (SEQ ID NO: 12), and CRM8 (SEQ ID NO.13), transcription factor binding elements, and, optionally and preferably, a minimal promoter sequence G6PC (SEQ ID NO: 15). It is understood that instead of SEQ ID NO. 13 transcription factor binding element, a variant thereof may be selected, e.g. a sequence selected from the group consisting of SEQ ID NO. 14 and SEQ ID NOs. 30 - 43. The CRM8 sequence (SEQ ID NO. 13) and variant SEQ ID NO. 14 are comprised in the Ql promoter and Ql variant 1 (and LP1 as well). The variant of CRM8 represented by SEQ ID NO. 30 is comprised in Ql variant 2. Variants of SEQ ID NO. 13 may be considered exchangeable for the CRM8 sequence as comprised e.g. in the Ql promoter as these have very similar promoter strength in human liver cells when comprised in a Ql promoter. It is understood that of one or more of the transcription factor-binding elements the reverse complementary sequence may be selected. This means that for a transcription factor binding element the reverse complement sequence of the said sequence as listed in one of SEQ ID NO.9- 13 may be selected and be operably linked to the other elements. Hence, variants of Ql in accordance with the invention may comprise one or more, preferably at least 4, most preferably all of HNF1/HNF3 (SEQ ID NO: 9), HNF3/HNF3 (SEQ ID NO: 10), c/EBP/HNF4 (SEQ ID NO: 11), HS_CRM2/HNF3 (SEQ ID NO: 12) and CRM8 (SEQ ID NO: 13) transcription factor binding elements, or a reverse complementary sequence of one or more thereof, and, optionally and preferably, a minimal promoter sequence G6PC (SEQ ID NO: 15). It is understood that instead of the CRM8 sequence represented by SEQ ID NO. 13, one may select instead a variant selected from the group consisting of SEQ ID NO. 14, and 30-43, or the reverse complement thereof. The expression cassette comprising the nucleic acid sequence operably linked to the promoter sequence may further comprise and be operably linked to elements for expression of the variant factor IX, such as 5' and 3' untranslated terminal regions (UTR), intronic sequences, poly A sequence, and post-transcriptionally regulatory elements such as PRE, WPRE, CTE, and the like. An intronic sequence that may be preferred is an SV40 intronic sequence. Examples of suitable expression cassettes are listed in SEQ ID NOs. 19-22. In another embodiment, a gene therapy vector is provided comprising a nucleic acid encoding the human variant factor IX in accordance with the invention. In a further embodiment, said gene therapy vector comprises an expression cassette comprising the nucleic acid encoding the human variant FIX in accordance with the invention. Such an expression cassette preferably being an expression cassette such as described above.

Preferably, gene therapy vectors are used that can stably transfer the nucleic acid and/or expression cassette to cells in a human patient such that expression of the FIX variants can be achieved. Suitable vectors may be lentiviral vectors, retrotransposon based vector systems, or AAV vectors. It is understood that as e.g. lentiviral vectors carry an RNA genome, the RNA genome (a nucleic acid) will encode for the said expression cassette such that after transduction of a cell and reverse transcription a double stranded DNA sequence is formed comprising the nucleic acid sequence and/or said expression cassette in accordance with the invention.

Preferably the gene therapy vector that is used is an AAV vector. AAV sequences that may be used in the present invention for the production of AAV vectors, e.g. as produced in insect or mammalian cell lines, can be derived from the genome of any AAV serotype. The production of AAV vectors comprising an expression cassette of interest is described i.a. in; W02007/046703, WO2007/148971, W02009/014445, W02009/104964, WO2011/122950, W02013/036118, which are incorporated herein in its entirety. Generally, the AAV serotypes have genomic sequences of significant homology at the amino acid and the nucleic acid levels, provide an identical set of genetic functions, produce virions, and replicate and assemble by practically identical mechanisms. For the genomic sequence of the various AAV serotypes and an overview of the genomic similarities see e.g. GenBank Accession number U89790; GenBank Accession number J01901; GenBank Accession number AF043303;

GenBank Accession number AF085716; (Chlorini et al. J. Vir. 1997: 6823-33; Srivastava et al. J. Vir. 1983; Chlorini et al. J. Vir. 1999, 73:1309- 1319; Rutledge et al. J. Vir. 1998; Wu et al. J. Vir. 2000). AAV serotypes 1, 2, 3, 4 and 5 may be preferred source of AAV nucleotide sequences for use in the context of the present invention. Preferably the AAV ITR sequences for use in the context of the present invention are derived from AAV1, AAV2, and/or AAV5. Likewise, the Rep52, Rep40, Rep78 and/or Rep68 coding sequences are preferably derived from AAV1, AAV2 and AAV5. The sequences coding for the VP1, VP2, and/or VP3 capsid proteins for use in the context of the present invention may preferably be taken from AAV1, AAV2, AAV3, AAV4, AAV5, AAV7, AAV8, AAV9, AAVrhlO and AAV10, as these are serotypes that are suitable for use in transducing the liver. Also, newly developed AAV-like particles obtained by e.g. capsid shuffling techniques and AAV capsid libraries comprising mutations (insertions, deletions, substitutions), derived from AAV capsid sequences, and selected from such libraries as being suitable for liver transduction may be contemplated. Examples of such capsids are e.g. AAV3 LK03 (Lisowski et al., Nature, 2014), AAV3-ST (Li et al., Molecular Therapy, 2015), AAV-DJ (Grimm et al., Journal of Virology, 2008), and AAV-Anc80L65 (Zinn et al., Cell Reports, 2015). AAV capsids may consist of VP1, VP2 and VP3 capsid proteins, but may also consist of VP1 and VP3 capsid proteins. AAV capsids may not contain any substantial amount of VP2 capsid protein. This is because the VP2 capsid protein may not be essential for transduction.

A preferred AAV vector that may be used in accordance with the inventions is an AAV vector of serotype 5. AAV of serotype 5 (also referred to as AAV5) is in particularly useful for transducing human liver cells. AAV vectors comprising AAV5 capsids can comprise AAV5 VP1, VP2 and VP3 capsid proteins. AAV vectors comprising AAV5 capsids can also comprise AAV5 VP1 and VP3 capsid proteins, while not comprising AAV5 VP2 capsid proteins or at least not comprising any substantial amount of VP2 capsid proteins. In a wild-type derived AAV5 capsid protein, the VP1, VP2 and VP3 capsid proteins comprise identical amino acid sequences at their C-termini. The VP3 sequence is comprised in the VP2 sequence, and the VP2 sequence is comprised in the VP1 sequence. The N-terminal part of the VP1 amino acid sequence that is not contained in the VP2 and VP3 capsid proteins is positioned at the interior of the virion. This N-terminal VP1 sequence may e.g. be exchanged with an N-terminal sequence of another serotype, e.g. from serotype 2, whereas the VP2 and VP3 amino acid sequences may be entirely based on the AAV5 serotype. Such non-natural capsids comprising hybrid VP1 sequences, and such hybrid vectors are also understood to be AAV5 viral vectors in accordance with the invention. Such a hybrid vector of the AAV5 serotype is i.a. described by Urabe et al., J Virol. 2006. Furthermore, AAV5 capsid sequences may also have one or more amino acids inserted or replaced to enhance manufacturing and/or potency of a vector, such as i.a. described in WO2015137802. Such modified AAV5 capsids are also understood to be of the AAV5 serotype. Examples of suitable VP1-VP3 amino acid sequences are represented by SEQ ID NOs. 23-25, wherein SEQ ID N0.23 represents an open reading frame of wild-type AAV5, SEQ ID N0.24 describes an AAV5 capsid sequence as described in WO2015137802, and SEQ ID N0.25 of an AAV5 hybrid capsid sequence such as described by U rabe et al. J Virol. 2006.

For SEQ ID N0.23 the indicated amino acid sequence represents the full VP1 sequence of wild-type AAV5 as comprised in a capsid. The bold threonine at position 137 indicated in the sequence represent the translational start for VP2 as comprised in the AAV5 capsid. Herein the first amino acid of VP2 is a methionine as there exists a translational initiation context prior to the threonine. Similarly, the bold methionine at position 193 represents the translational start for the VP3. For SEQ ID NO.24 the indicated amino acid sequence represents an adapted AAV5 sequence as described in WO2015137802. Of note is the addition of an alanine in the second position to aid translation of the capsid subunits in optimal stoichiometry in insect cells. Similarly, the full sequence represents the VP1 sequence as comprised in a capsid while the bold threonine (position 138) and methionine (position 194) represent the translational start of VP2 and VP3 respectively. SEQ ID NO.25 represent the amino acid sequence of a hybrid AAV5 where the VP1 unique sequence is substituted by the VP1 unique sequence of AAV2 to aid manufacturability in insect cells. The VP2 and VP3 amino acids sequences are as described for SEQ ID N0.23 and 24.

In one embodiment, the viral vector in accordance with the invention comprises an expression cassette for a FIX variant in accordance with the invention, comprised in a viral vector of the AAV serotype 5. Preferably, said FIX variant being a FIX-FIAV variant. In another embodiment, the viral vector in accordance with the invention comprises an expression cassette for a FIX variant in accordance with the invention, comprised in a viral vector of the AAV serotype 5, said FIX variant being a FIX-FIAV variant having the amino acid sequence as listed in SEQ ID NO.6. In another embodiment, the viral vector in accordance with the invention comprises an expression cassette for a FIX variant in accordance with the invention, comprised in a viral vector of the AAV serotype 5, said FIX variant being a FIX-FIAV variant encoded by the nucleic acid sequence as listed in SEQ ID NO.7.

In one embodiment, the viral vector of the AAV5 serotype comprises an expression cassette for a FIX variant in accordance with the invention, said expression cassette comprising a liver specific promoter sequence, wherein said liver specific promoter sequence is selected from the group consisting of SEQ ID NO.16-18. In this embodiment, preferably said FIX variant is a FIX-FIAV variant. Preferably, said FIX variant being a FIX-FIAV variant having the amino acid sequence as listed in SEQ ID NO.6 or said FIX variant being a FIX-FIAV variant encoded by the nucleic acid sequence as listed in SEQ ID NO.7. In one embodiment, the viral vector of the AAV5 serotype comprises an expression cassette for a FIX variant in accordance with the invention, said expression cassette comprising a liver specific promoter sequence according to SEQ ID NO.16, SEQ ID NO. 17, or SEQ ID NO.18, and encoding a FIX variant having the amino acid sequence as listed in SEQ ID NO.6. In one embodiment, the viral vector of the AAV5 serotype comprises an expression cassette for a FIX variant in accordance with the invention, said expression cassette comprising a liver specific promoter sequence according to SEQ ID NO.16, SEQ ID NO. 17, or SEQ ID NO.18, and encoding FIX-FIAV as by the nucleic acid sequence as listed in SEQ ID NO.7.

In one embodiment, the viral vector of the AAV5 serotype is produced with an amino acid sequence of the VP1 capsid as shown in SEQ ID NO.25, said capsid being produced having VP2 and VP3 capsids corresponding with wild- type AAV5 sequences, said viral vector comprises an expression cassette for a FIX variant in accordance with the invention, said expression cassette comprising a liver specific promoter sequence, wherein said liver specific promoter sequence is selected from the group consisting of SEQ ID NO.16-18. In this embodiment, preferably said FIX variant is a FIX-FIAV variant. Preferably, said FIX variant being a FIX-FIAV variant having the amino acid sequence as listed in SEQ ID NO.6 or said FIX variant being a FIX-FIAV variant encoded by the nucleic acid sequence as listed in SEQ ID NO.7. In one embodiment, the viral vector of the AAV5 serotype comprises an expression cassette for a FIX variant in accordance with the invention, said expression cassette comprising a liver specific promoter sequence according to SEQ ID NO.16, SEQ ID NO. 17, or SEQ ID NO.18, and encoding a FIX variant having the amino acid sequence as listed in SEQ ID NO.6. In one embodiment, the viral vector of the AAV5 serotype comprises an expression cassette for a FIX variant in accordance with the invention, said expression cassette comprising a liver specific promoter sequence according to SEQ ID NO.16, SEQ ID NO. 17, or SEQ ID NO.18, and encoding FIAV FIX as by the nucleic acid sequence as listed in SEQ ID NO.7.

In one embodiment, the viral vector is of an AAV serotype, or a variant thereof, and comprises an expression cassette for a FIX variant in accordance with the invention, said expression cassette comprising a liver specific promoter sequence, wherein said liver specific promoter sequence is selected from the group consisting of SEQ ID NO.16-18. In this embodiment, preferably said FIX variant is a FIX-FIAV variant. Preferably, said FIX variant being a FIX-FIAV variant having the amino acid sequence as listed in SEQ ID NO.6 or said FIX variant being a FIX-FIAV variant encoded by the nucleic acid sequence as listed in SEQ ID NO.7. In one embodiment, the viral vector is of an AAV serotype, or a variant thereof, and comprises an expression cassette for a FIX variant in accordance with the invention, said expression cassette comprising a liver specific promoter sequence according to SEQ ID NO.16, SEQ ID NO. 17, or SEQ ID NO.18, and encoding a FIX variant having the amino acid sequence as listed in SEQ ID NO.6. In one embodiment, the viral vector is of an AAV serotype, or a variant thereof, and comprises an expression cassette for a FIX variant in accordance with the invention, said expression cassette comprising a liver specific promoter sequence according to SEQ ID NO.16, SEQ ID NO. 17, or SEQ ID NO.18, and encoding FIX-FIAV as by the nucleic acid sequence as listed in SEQ ID NO.7.

In another embodiment, an expression cassette is provided, comprising a liver specific promoter as listed in a sequence selected from the group consisting of SEQ ID NOs. 16-18, and comprising a nucleic acid encoding a FIX-FIAV variant having the amino acid sequence as listed in SEQ ID NO.6 or said FIX variant being a FIX-FIAV variant encoded by the nucleic acid sequence as listed in SEQ ID NO.7.

It is understood that herein throughout that the FIX-FIAV variant is preferred for the viral vectors or nucleic acids in accordance with the invention. However, as an alternative, having somewhat lesser FVIII mimetic activity as shown in the examples, the FIX-ID AV variant may be selected as well instead. Examples of useful sequences for FIX-ID AV are represented by SEQ ID NO.26 (mature protein), SEQ ID N0.27 (propeptide), and SEQ ID NO.28 (open reading frame encoding propeptide FIX-IDAV).

In one embodiment, a viral vector or a nucleic acid in accordance with the invention is provided, wherein said viral vector is for use in a medical treatment of a primate, such as a human subject. Hence, methods are provided herein for treatment of a human subject, wherein said method comprises the administration of a viral vector or a nucleic acid in accordance with the invention. As shown in the examples, in particular in non-human primates, it was shown that sufficient levels of FIX variants in accordance with the invention can be obtained, which represent a suitable model in particular for human use. Said human subject preferably suffering from a coagulopathy, such as hemophilia A. Hemophilia A is characterized by a deficiency in FVIII. Hemophilia A may be inherited or may be acquired. Acquired hemophilia A occurs due to the development of auto-antibodies against FVIII. Haemophilia A can also occur in patients due to a defect in the gene encoding FVIII on the X-chromosome. Such a defect found in patient is usually inherited but may also have occurred due to a de novo mutation. Coagulation factor concentration is expressed in international units (IU); 1 IU is defined as the concentration of coagulation factor in 1 mF of normal pooled plasma. Healthy individuals have a FVIII plasma concentration of 0.50-1.50 IU/ml, which can also be expressed as percentages of normal pooled plasma (defined as 100%), with normal levels between 50% and 150% (Fijnvandraat K et al BMJ 2012). 100% corresponds to 200 ng/ml (Pittman DD et al. Thromb Haemostas 1989, Hoyer FW et al. Hemostasis and Thrombosis 1994). Hemophilia A patients can be classified in accordance with the severity of the disease, ranging from mild, to moderate, to severe. A patient with mild haemophilia can be classified as having a percentage of factor VIII in the range of 5% - 40%, moderate haemophilia is in the range of 1% - 5% of normal, whereas severe hemophila A patients have less than 1% of normal. It is understood that the % of normal refers to the amount of FVIII in blood plasma, e.g. when expression of FVIII is reduced in a hemophilia A patient, or can refer to the FVIII clotting activity as determined in blood plasma with e.g. an activated partial thromboplastin time (APTT) or a chromogenic assay, 100% corresponding to the activity of a reference blood plasma sample having e.g. 200 ng / ml of FVIII, which can be human plasma with a defined FVIII concentration. Human plasma calibrators are provided with the commercially available assays for FVIII activity.

In one embodiment, a viral vector or a nucleic acid in accordance with the invention is provided, wherein said medical treatment reduces the severity of joint bleeds. In another embodiment, a viral vector or a nucleic acid in accordance with the invention is provided, wherein said medical treatment reduces the frequency of joint bleeds. In another embodiment, a viral vector or a nucleic acid in accordance with the invention is provided, wherein said medical treatment reduces the duration of joint bleeds. In another embodiment, a viral vector or a nucleic acid in accordance with the invention is provided, wherein said medical treatment reduces the severity of cerebral bleeds. In another embodiment, a viral vector or a nucleic acid in accordance with the invention is provided, wherein said medical treatment reduces the frequency of cerebral bleeds.

In another embodiment, a viral vector or a nucleic acid in accordance with the invention is provided, wherein said medical treatment reduces the /duration of cerebral bleeds. In still another embodiment, a viral vector or a nucleic acid in accordance with the invention is provided, wherein said medical treatment reduces the hemophilia from severe to moderate or mild hemophilia A. In another further embodiment, a viral vector or a nucleic acid in accordance with the invention is provided, wherein said medical treatment reduces the hemophilia from moderate to mild hemophilia A. Preferably, in these embodiments, the viral vector or nucleic acid in accordance with the invention encodes a factor IX FIAV or IDAV, most preferably FIAV. Most preferably, the viral vector is an AAV vector.

Hemophilia A patients that have a genetic defect in the X-chromosome usually receive protein replacement therapy, consisting of regular infusions of human FVIII, which may be isolated from blood donors, i.e. plasma derived or be made recombinantly. Human patients receiving such FVIII protein replacement therapy are suitable candidates for the medical use of the viral vector or nucleic acid in accordance with the invention. In addition, hemophilia A patients that have auto-antibodies against FVIII, or that have developed antibodies against FVIII due to FVIII protein replacement therapy, are suitable candidates for the medical use of the viral vector or nucleic acid in accordance with the invention. These antibodies against FVIII are also called inhibitory antibodies, or inhibitors, which render the standard replacement therapy ineffective in patients with inhibitors. Patients with severe haemophilia often develop inhibitors. Hence, the viral vector or the nucleic acid in

accordance with the invention is for use in a medical treatment, wherein the medical treatment is of a human subject having antibodies against Factor FVIII, such a subject being a patient suffering from hemophilia A. Preferably, patients eligible for treatment have been diagnosed with severe or moderate hemophila A.

As shown in the examples, the FIX variants in accordance with the invention, can be combined with standard treatments of hemophilia A patients, said treatments comprising recombinant activated FVII (rFVIIa; NovoSeven®, Novo Nordisk, Bagsvaerd, Denmark) or plasma derived activated prothrombin complex concentrate (aPCC; FEIBA®, Shire,

Lexington, KY, USA) or recombinant porcine FVIII (rpFVIII; Obizur, Baxter, Vienna, Austria) or the like (Ljung R et al. European J of Haematol 2018). Hence, when e.g. a hemophilia A patient has received a medical treatment, e.g. a viral vector in accordance with the invention or a nucleic acid in accordance with the invention, which improves the classification of hemophilia A in the patient from severe to mild or moderate, such a patient may still benefit from treatment with products such as FVIIa, activated prothrombin complex concentrate, recombinant porcine FVIII, or the like. This may be the case e.g. when undergoing surgery or having a traumatic injury. Such treatment may be as safe and effective and at the same dosage as used for hemophilia A patients that have not received a viral vector or a nucleic acid in accordance with the invention. Hence, in one embodiment, a viral vector for use in a medical treatment in accordance with the invention is provided, wherein the medical treatment is of a human subject, wherein subsequently said human subject is eligible for treatment with recombinant activated Factor VII, or a variant thereof. In a further embodiment, a viral vector for use in a medical treatment according to the invention is provided, wherein the medical treatment is of a human subject, wherein subsequently said human subject is eligible for treatment with an activated prothrombin complex concentrate or a variant thereof. In another further embodiment, a factor VII or Factor Vila or a variant thereof is provided, for use in a medical treatment of a human subject, wherein said human subject has been treated with a viral vector or a nucleic acid in accordance with the invention. In still another further embodiment, an activated prothrombin complex concentrate or a variant thereof is provided, for use in a medical treatment of a human subject, wherein said human subject has been treated with a viral vector or a nucleic acid in accordance with the invention. It is understood that preferably, in these embodiments, the viral vector or nucleic acid in accordance with the invention encodes a factor IX FIAV or IDAV, most preferably FIAV.

As said and as shown in the examples, the FIX variants in accordance with the invention, may also be combined with standard treatments of haemophilia A patients, such a treatment may comprise treatment with FVIII, or a FVIII variant thereof. However, as also shown in the examples, such a treatment will be with a lower dosage as compared with a dosage in a patient that has not been treated with a viral vector or nucleic acid in accordance with the invention, said viral vector or nucleic acid preferably encoding a factor IX FIAV or IDAV, most preferably FIX-FIAV. Hence, in one embodiment, a viral vector for use in a medical treatment in accordance with the invention is provided, wherein the medical treatment is of a human subject, wherein subsequently said human subject is eligible for treatment with Factor VIII, wherein said treatment with Factor VIII comprises a lower dose as compared with the human subject not being subjected to the medical treatment with the viral vector. In a further embodiment, human FVIII is provided, or a variant thereof, for use in a medical treatment of a human subject, wherein said human subject has been treated with a viral vector or a nucleic acid in accordance with the invention. It is understood that preferably, in these embodiments, the viral vector or nucleic acid in accordance with the invention encodes a factor IX FIAV or IDAV, most preferably FIAV. As said and as shown in the examples, the FIX variants in accordance with the invention, may also be combined with standard treatments of

haemophilia A patients, such a treatment may comprise treatment with emicizumab, or an equivalent thereof (e.g. another bispecific antibody binding both activated FIX and FX). However, as also shown in the examples, such a treatment will be with a lower dosage as compared with a dosage in a patient that has not been treated with a viral vector or nucleic acid in accordance with the invention, said viral vector or nucleic acid preferably encoding a factor IX FIAV or IDAV, most preferably FIX-FIAV. Hence, in one embodiment, a viral vector for use in a medical treatment in accordance with the invention is provided, wherein the medical treatment is of a human subject, wherein subsequently said human subject is eligible for treatment with emicizumab, or an equivalent thereof, wherein said treatment with emicizumab, or an equivalent thereof, comprises a lower dose as compared with the human subject not being subjected to the medical treatment with the viral vector. In a further embodiment, emicizumab, or an equivalent thereof is provided, for use in a medical treatment of a human subject, wherein said human subject has been treated with a viral vector or a nucleic acid in accordance with the invention. It is understood that preferably, in these embodiments, the viral vector or nucleic acid in accordance with the invention encodes a factor IX FIAV or IDAV, most preferably FIAV. In a further embodiment, a viral vector or nucleic acid in accordance with the invention is provided for use in a medical treatment, wherein the variant FIX is expressed in the human patient at a level of at least 1 pg/ml of plasma. For example, when the variant FIX-FIAV would be expressed in blood plasma at a level of 1 ug/ml, this results in an estimated FVIII mimetic activity of about 5% FVIII mimetic activity (1 pg/ml / 5 pg/ml x 28% FVIII mimetic activity = 5,6 %). Such an increase in clotting activity would change a severe haemophilia A patient to a mild haemophilia A patient and can change a moderate patient to a mild patient as well. Preferably, the amount of the FIX-FIAV variant as expressed in the human patient is at least 1 pg/ml, at least 2 pg/ml, at least 3pg/ml, most preferably at least 4 pg/ml. Preferably, the amount of the FIX-ID AV variant as expressed in the human patient is at least 2 pg/ml, at least 3 pg/ml, at least 4pg/ml, most preferably at least 5 pg/ml.

In a further embodiment, a viral vector or a nucleic acid for use in a medical treatment according to the invention is provided, wherein the clotting activity of the plasma of the human patient is at least 5%, at least 6%, at least 7%, at least 8%, or at least 9% of the clotting activity of a healthy human patient. More preferably, the clotting activity is at least 10%. as a clotting activity above 10% has been associated with little variation in joint bleeds in mild hemophilia patients (den Uijl et al., Haemophilia, 2011, 17:41-44). It may be preferred to have a clotting activity in a human patient of at least 11%, 12%, 13%, 14% or at least 15%. It may be preferred to have an even higher clotting activity in a human patient of at least 20%, 25%, 30%, 40% or at least 45%.

For example, for the FIAV variant, as shown in the examples, a FVIII mimetic activity of at least 5%, 6%, 7%, 8%, 9%, or more preferably at least 10%, of normal can be obtained in blood plasma of the treated patient, (i.e. normal corresponding to the clotting activity of a healthy patient, i.e. normal being 100% and corresponding to plasma from a healthy patient having approximately 200 ng / ml of FVIII, or 1 IU/ml of FVIII), or the clotting activity of plasma from a severe haemophilia A patient not having inhibitors being supplemented with 200 ng / ml of FVIII, or 1 IU/ml of FVIII). Likewise, for the IDAV variant, as shown in the examples, a FVIII mimetic activity of at least 5%, 6%, 7%, 8%, 9%, or more preferably at least 10%, of normal is obtained in blood plasma of the treated patient, (i.e. normal corresponding to the clotting activity of a healthy patient, i.e. normal being 100% and corresponding to plasma from a healthy patient having 200 ng / ml of FVIII, or 1 IU/ml of FVIII), or the clotting activity of plasma from a severe haemophilia A patient not having inhibitors being supplemented with 200 ng / ml of FVIII, or 1 IU/ml of FVIII). Hence, it is understood that in accordance with the invention, the clotting activity of a healthy individual is defined as having 200 ng / ml of FVIII, or 1 IU/ml of FVIII. Such clotting activity of a healthy person can be easily determined by using commercially available assays such as described in the examples as disclosed herein. Hence, as clotting activity relates to

concentration of FVIII in blood plasma, the percentage of clotting activity of a treated patient can be easily measured by determining clotting activity of its plasma and comparing it with the clotting activity of a reference sample (e.g. a dilution series, such as shown in the examples). Hence, in a further embodiment, a viral vector for use in a medical treatment according to the invention is provided, wherein the clotting activity of the plasma of the human patient is at least 5%, 6%, 7%, 8%, 9%, or more preferably at least 10% of the clotting activity of a healthy human person, wherein the healthy human individual’s plasma is defined as comprising 200 ng/ml of human FVIII. It is understood that preferably, in these

embodiments, the viral vector or nucleic acid in accordance with the invention encodes a factor IX FIAV or IDAV, most preferably FIAV.

In a further embodiment, a viral vector or nucleic acid for use in a medical treatment according to the invention is provided, wherein said variant is expressed for at least 2 months, preferably 3 months, more preferably 6 months, most preferably 1 year, 2 years, 3 years, or more, and clotting activities as defined herein are maintained at a sufficient level benefiting the patients for these periods as well. Preferably, said expression levels as defined herein are maintained for at least 2 months, preferably 3 months, more preferably 6 months, most preferably 1 year, 2 years, 3 years, or more.

In one embodiment, the viral vector or nucleic acid for use in a medical treatment according to the invention is provided, wherein the variant FIX does not induce antibodies against the variant FIX. As shown in the examples, the variant FIX has low immunogenicity risk when assessing the sequence divergence with a reference wild-type FIX protein. The FIX variant may thus not be recognized as being foreign and may thus not raise immunological issues, such as observed e.g. with FVIII replacement therapies in hemophilia A patients, if not completely avoided.

In a further embodiment, a viral vector or a nucleic acid for use in a medical treatment according to invention is provided, wherein said viral vector is administered via the bloodstream. Preferably, said viral vector or nucleic acid is delivered to the liver. As it is most preferred to have the FIX variants expressed by the liver, administration, such as shown in the examples with an AAV vector, e.g. AAV5, of the viral vector or nucleic acid of the invention is preferably via the bloodstream such that it is delivered to the liver. Preferably, the viral vector or nucleic acid in accordance with the invention encodes a factor IX FIAV or IDAV, most preferably FIAV. In further embodiments, these factor IX variants are expressed using an LP1 promoter or a Q1 promoter, or a variant of a Q1 promoter, such as described in the examples.

In a further embodiment, a viral vector for use in a medical treatment of a human subject according to invention is provided, wherein said viral vector is an AAV vector in accordance with the invention, wherein prior to said use in a medical treatment, the human subject has been treated with a saturating agent. Preferably, said AAV vector for use in a medical treatment according to invention, wherein prior to said medical use, the human subject has been treated with a lipid formulation which is administered via the bloodstream.

As AAV vectors are most preferred in accordance with the invention, in one embodiment, this may be combined with the use of a saturating agent, wherein the saturating agent is taken up by one or more cells of the reticuloendothelial system (RES) as found e.g. in the liver. In some embodiments, the saturating agent comprises an emulsion, and in further embodiments, the emulsion preferably is a lipid-based emulsion. A lipid-based emulsion may be selected from INTRALIPID® 10%, INTRALIPID® 20%, and INTRALIPID® 30%. In some embodiments, the lipid-based emulsion is Clinolipid. In some embodiments, the lipid- based emulsion is selected from LIPOSYN®, LIPOSYN® II, and LIPOSYN® III.

INTRALIPID® 10% contains 10% soybean oil, 1.2 % egg yolk phospholipids, 2.25% glycerin, and water. INTRALIPID® 20% contains 20% soybean oil, 1.2% egg yolk phospholipids, 2.25% glycerin, and water. INTRALIPID® 30% contains 30% soybean oil, 1.2% egg yolk phospholipids, 2.25% glycerin, and water. Clinolipid emulsions contain refined olive oil and refined soybean oil in an approximate ratio of 4:1. Lor example, clinolipid 20% contains approximately 16% of olive oil, 4% of soybean oil, 1.2% egg phospholipids, 2.25% glycerin, 0.03% sodium oleate, and water. LIPOSYN® emulsions may be formulated as a 10% or 20% emulsion. LIPOSYN® II 10% contains 5% safflower oil, 5% soybean oil, up to 1.2% egg phosphatides added as an emulsifier and 2.5% glycerin in water. LIPOSYN® II 20% contains 10% safflower oil, 10% soybean oil, 1.2% egg phosphatides and 2.5% glycerin in water. LIPOSYN® III 10% contains 10% soybean oil, up to 1.2% egg phosphatides added as an emulsifier and 2.5% glycerin in water. LIPOSYN® III 20% contains 20% soybean oil, 1.2% egg phosphatides and 2.5% glycerin in water. One of skill in the art would understand that similar lipid and/or fat-based emulsions can be prepared using other acceptable lipids and/or oils in similar percentages, and such emulsions would likewise be suitable for use in the disclosed methods.

In some embodiments, the saturating agent is taken up by one or more cells of a reticuloendothelial system (RES). In some embodiments, the saturating agent may be taken up selectively or preferentially by any one of the aforementioned RES cell types, while in some embodiments, the saturating agent may be taken up by all of these cell types. In other words, the saturating agent may be taken up by one or more, two or more, or three or more cell types of the RES. For example, the saturating agent may be taken up selectively or preferentially by Kupffer cells and SECs, or it may be taken up selectively or preferentially by Kupffer cells and HSCs or SECs and HSCs.

In one embodiment, the saturating agent is administered to the human subject prior to the administration of the AAV vector in accordance with the invention. For example, the dosage and/or route of administration as shown in the example section of an emulsion such as Intralipid, or the like, resulted in a triglyceride level as measured in the blood plasma of above 1.33 mmol/L or higher, which may improve transduction. Any dosage and/or route of administration may be selected in order to achieve a triglyceride level in the blood of at least 2 mmol/L, 3 mmol/L, 4 mmol/L, 5 mmol/L, 6 mmol/L, 7 mmol/L, 8 mmol/L, 9 mmol/L, 10 mmol/L or more. In other words, wherein a subject is treated with a food supplement, such as a lipid and/or fat based emulsion formulation (e.g. Intralipid), thereby achieving a triglyceride level in the blood of at least 2 mmol/L, 3 mmol/L, 4 mmol/L, 5 mmol/L, 6 mmol/L, 7 mmol/L, 8 mmol/L, 9 mmol/L, 10 mmol/L or more, followed by subsequent administration of an AAV gene therapy vector. The dosage of the emulsion (g) per body weight (kg) selected (g/kg) may hence be for example, 0.5g/kg, lg/kg, 1.5g/kg, 2g/kg, 2.5g/kg, 3g/kg, 3.5g/kg, 4g/kg, 4.5g/kg, 5g/kg, 5.5g/kg, 6g/kg, 6.5g/kg, 7g/kg, 7.5g/kg, 8g/kg, 8.5g/kg, 9g/kg, 9.5g/kg, or 10 g/kg or more. In some embodiments, the dosage of the emulsion may be less than 5 g/kg, 5.5g/kg, 6g/kg, 6.5g/kg, 7g/kg, 7.5g/kg, 8g/kg, 8.5g/kg, 9g/kg, 9.5g/kg, or lOg/kg. In some embodiments, the dosage of the emulsion is between 0.5g/kg and 5g/kg. In some embodiments, the dosage of the emulsion is about 2g/kg or about 4g/kg.

Hence, the use of a saturating agent, preferably a lipid emulsion as described above, is combined with the administration of an AAV vector in accordance with the invention. An adeno-associated virus (AAV) gene therapy vector is thus provided for use in a medical treatment of a human subject, wherein said human subject has been administered a saturating agent. Preferably said saturated agent comprises a lipid emulsion, preferably a lipid emulsion. Furthermore, an adeno-associated virus (AAV) gene therapy vector for use in a medical treatment of a human subject according to the invention is provided, wherein the saturating agent comprises triglycerides, and wherein the plasma concentration of triglycerides in the blood of the human subject is at least 3 mmol/L prior to administration of the AAV gene therapy vector. Furthermore, the invention comprises an adeno-associated virus (AAV) gene therapy vector for use in a medical treatment of a human subject according to the invention, wherein the saturating agent has been administered to the human subject between at least 15 minutes or more prior to administration of the AAV gene therapy vector. It is understood that in the uses of these embodiments, preferably, the AAV vector in accordance with the invention encodes a factor IX FIAV or IDAV, most preferably FIAV, and furthermore preferably is expressed from an LP1 promoter or a Q1 promoter, most preferably a Q1 promoter or a variant thereof.

In another aspect of the invention, said AAV gene therapy vector as described above is administered at a dosage based on genomic copies. Genomic copies meaning the amount of vector genomes contained in the AAV preparation. The gc titer of an AAV vector preparation can easily be determined by using a qPCR that quantifies a vector genomic sequence.

Preferably said AAV gene therapy vector is used at a dosage corresponding with at least 5 x 10¹¹ gc/kg of body weight. A dosage of at least 5 x 10¹¹ capsid/kg of body translates to about 5 x10 gc per patient or about 10 gc / L blood volume of a patient, based on an average body weight of about 85 kg and average blood volume of 5L. Hence, whatever dose range contemplated, these may easily be recalculated. The dosage selected may be at least 1 x 10 12 gc/kg of body weight, at least 2 x 10 12 gc/kg of body weight, or 4 x 1012 gc/kg of body weight. The dosage used in the example section being of about 5 x 10 12 gc/kg of body weight and about 2 x 10 gc/kg of body weight. Although there may not be an upper limit, this may be set to correspond to a dosage corresponding with at most 10¹⁵ gc/kg of body weight.

In another aspect of the invention, the AAV gene therapy vector is an AAV5 gene therapy vector as described above and is administered at a dosage based on genomic copies. Genomic copies meaning the amount of vector genomes contained in the AAV5 preparation. The gc titer of an AAV5 vector preparation can easily be determined by using a qPCR that quantifies a vector genomic sequence. Preferably said AAV5 gene therapy vector is used at a dosage corresponding with at least 3 x 10 gc/kg of body weight. A dosage of at least 3 x 10¹³ capsid/kg of body translates to about 2.5 xlO¹⁵ gc per patient or about 5 x 10¹⁴ gc / L blood volume of a patient, based on an average body weight of about 85 kg and average blood volume of 5L. Hence, whatever dose range contemplated, these may easily be recalculated based on these parameters. The dosage selected may be at least 6 x 10 13 gc/kg of body weight, or at least 9 x 10 13 gc/kg of body weight. The dosage used in the example section being 9 x 10 gc/kg of body weight. These dosages being suitable for human use and allowing for expression of FIX variants such as FIAV or IDAV that can allow for sufficient FVIII mimetic activity (of at least 5%, preferably at least 10%). Preferably said FIX variants FIAV or IDAV are expressed from an LP1 promoter or a Q1 promoter or a variant thereof, more preferably a Q1 promoter or a variant thereof, such as one of SEQ ID NO. 16, 17 or 18. Said FIX variants preferably being encoded by a nucleic acid having a sequence such as SEQ ID NO. 7 or 28.

The viral vector or nucleic acid according to the invention is preferably comprised in a pharmaceutical acceptable formulation. Said pharmaceutical formulation allows for administration of the viral vector or nucleic acid, which is preferably an AAV vector of the AAV5 serotype to the human patient. Such administration preferably comprising

administration via the bloodstream, e.g. via intravenous infusion. Hence, preferably said pharmaceutical formulation is compatible therewith. It is understood that the pharmaceutical formulation may be a liquid but may also be e.g. a lyophilized formulation. Said liquid or solid being subsequently combined with e.g. a solution for injection or infusion.

Embodiments

1. An adeno-associated viral (AAV) vector, comprising a nucleic acid encoding a variant of human factor IX, said variant factor IX comprising at least amino acid substitutions corresponding to V181I, K265A and I383V of a polypeptide having an amino acid sequence as listed in SEQ ID NO. 1.

2. An AAV vector according to embodiment 1, wherein said variant factor IX comprises further an amino acid substitution selected from the group consisting of L6F, or E185D.

3. An AAV vector in accordance with embodiment 1 or embodiment 2, wherein said polypeptide has at least 90% sequence identity with SEQ ID NO 1 or SEQ ID NO.2.

4. An AAV vector, in accordance with any one of embodiments 1 - 3, wherein said nucleic acid sequence has at least 70% sequence identity with SEQ ID NO.4. 5. An AAV vector according to any one of embodiments 1-4, wherein said nucleic acid is operably linked to a promoter sequence for expression of the variant factor IX.

6. An AAV vector according to embodiment 5, wherein said promoter is for expression of the variant factor IX in the human liver.

7. An AAV vector according to embodiment 6, wherein the promoter is selected from the group consisting of SEQ ID NOs. 8, 16, 17 or 18.

8. An AAV vector according to any one of embodiments 1-7, wherein the AAV vector is based on serotype 5.

9. An AAV vector according to embodiment 8, wherein the AAV serotype 5 comprises a capsid protein as listed in SEQ NO.23, 24 or 25.

10. An AAV vector according to any one of embodiments 1-9, wherein the nucleic acid is flanked by AAV ITR sequences.

11. An AAV vector according to any one of embodiments 1-10, comprised in a pharmaceutical acceptable formulation.

12. An AAV vector according to any one of embodiments 1-11, for use in a medical treatment of a primate.

13. An AAV vector according to any one of embodiments 1-12, for use in a medical treatment of a human subject.

14. A use in a medical treatment according to any one of embodiments 12 and 13, wherein the medical treatment is of a human subject having a coagulopathy.

15. A use in a medical treatment according to any one of embodiments 12-14, wherein the medical treatment is of a human subject having hemophilia A. 16. A use in a medical treatment according to any one of embodiments 12-15, wherein the medical treatment is of a human subject having antibodies against Factor FVIII.

17. A use in a medical treatment in accordance with any one of embodiments 12-16, wherein the medical treatment is of a human subject, wherein subsequently said human subject is eligible for treatment with Factor VII or Factor Vila or a variant thereof.

18. A use in a medical treatment according to any one of embodiments 12-17, wherein the medical treatment is of a human subject, wherein subsequently said human subject is eligible for treatment with an activated prothrombin complex concentrate or a variant thereof.

19. A use in a medical treatment in accordance with any one of embodiments 12-18, wherein the medical treatment is of a human subject, wherein subsequently said human subject is eligible for treatment with Factor VIII, wherein said treatment with Factor VIII comprises a lower dose as compared with the human subject not being subjected to the medical treatment with the AAV vector.

20. A factor VII or Factor Vila or a variant thereof, for use in a medical treatment of a human subject, wherein said human subject has been treated with an AAV vector in accordance with any one of embodiments 1-11.

21. A factor VII or Factor Vila or a variant thereof, for use in a medical treatment of a human subject, in accordance with embodiment 20, wherein said human subject has a coagulopathy, preferably said coagulopathy is a deficiency in FVIII.

22. An activated prothrombin complex concentrate or a variant thereof, for use in a medical treatment of a human subject, wherein said human subject has been treated with an AAV vector in accordance with any one of embodiments 1-11.

23. An activated prothrombin complex concentrate or a variant thereof, for use in a medical treatment of a human subject, in accordance with embodiment 22, wherein said human subject has a coagulopathy, preferably said coagulopathy is a deficiency in FVIII.

24. A factor VIII or a variant thereof, for use in a medical treatment of a human subject, wherein said human subject has been treated with an AAV vector in accordance with any one of embodiments 1-11.

25. A factor VIII or a variant thereof, for use in a medical treatment of a human subject, in accordance with embodiment 24, wherein said human subject has a coagulopathy, preferably wherein said coagulopathy is a deficiency in FVIII.

26. A use in a medical treatment according to any one of embodiments 12-22, wherein the medical treatment comprises the administration of the at least 2 x 10 genomic copies of the viral vector per kg of body weight of the human subject.

24. A use in a medical treatment according to any one of embodiments 12-23, wherein the variant FIX is expressed in the human patient at a level of at least 1 m g/m 1 of plasma.

25. A use in a medical treatment according to any one of embodiments 12-24 wherein said variant is expressed for at least 1 year.

26. A use in a medical treatment according to any one of embodiments 12-25, wherein the clotting activity of the plasma of the human patient is at least 10 % of the clotting activity of a healthy human individual.

27. A use in a medical treatment according to any one of embodiments 12-26, wherein the clotting activity of the plasma of the human patient is at least 10 % of the clotting activity of a healthy human individual, wherein the healthy human individual is defined to comprise 1 IU/ml of human FVIII.

28. A use in a medical treatment according to any one of embodiments 12-27, wherein the variant FIX does not induce antibodies against the variant FIX.

29. A use in a medical treatment according to any one of embodiments 12-28, wherein said viral vector is administered via the bloodstream.

30. A use in a medical treatment according to any one of embodiments 12-29, wherein said viral vector is delivered to the liver. 31. A use in a medical treatment according to any one of embodiments 12-30, wherein prior to the administration of said AAV vector, the human subject has been treated with a lipid formulation.

32. A use in a medical treatment according to any one of embodiments 12-31, wherein prior to the administration of said AAV vector, the human subject has been treated with a lipid formulation which is administered via the bloodstream.

33. A use in a medical treatment according to any one of embodiments 12-32, wherein said lipid formulation is Intralipid.

34. A use in a medical treatment according to any one of embodiments 12-33, wherein said medical treatment reduces the severity, frequency, and/or duration of joint bleeds.

35. A use in a medical treatment according to any one of embodiments 12-34, wherein said medical treatment reduces the severity, frequency, and/or duration of spontaneous bleeds.

36. A use in a medical treatment according to any one of embodiments 12-35, wherein said medical treatment reduces the severity, frequency, and/or duration of cerebral bleeds.

37. A use in a medical treatment according to any one of embodiments 12-35, wherein said medical treatment reduces the haemophilia from severe to moderate or mild hemophilia.

38. A use in a medical treatment according to any one of embodiments 12-35, wherein said medical treatment reduces the hemophilia from moderate to mild hemophilia.

Examples

Introduction

Studies have been carried out using vectors encoding FIX variants, and isolated FIX variants, that could activate coagulation independent from FVIII. In vitro and in vivo studies have been conducted to evaluate the mechanism of action of different variants. Several FIX variants were initially selected encoding amino acids modifications to induce coagulation in the absence of FVIII, combined with amino acid mutations that provide FIX hyperactivity. Of these variants, two FIX variants, FIAV and IDAV, were selected for further testing. Under normal conditions FIX is an enzyme that has very low enzymatic activity and only in the presence of its cofactor VIII, its substrate activity is enhanced by approximately 200,000-fold (Van Dieijen G et al. Thromb Haemost 1985). Recombinant FIX variant proteins were generated and characterized for their ability to induce clotting. Further, to assess the efficacy and mechanism of action, a proof of concept study was conducted in hemophilia A mice. A non-human primates’ study was conducted to assess the feasibility of expressing protein level that will translate to therapeutic meaningful FVIII mimetic activity. In addition,

immunological analyses have been carried out to assess the possible immunological consequences of FIX variant expression. Together detailed supportive information is provided for human use as well as mechanistic safety supportive information regarding the activity of the FIX variants in hemophilia A patients.

Materials and methods

Animal studies:

To study the expression of the different FIX variants in C57B16 mice upon injection of AAV5 vectors, 6-8 weeks old male wt C57/BL6JRj mice (n=5) received 5x10 gc/kg of different AAV5-hFIX vectors (5 in total: FIX-wt, FIX-ID AV, FIX-FIAV, FIX-ID A V+KLW and FIX-FIAV-KLW in their tail vein. At week 1, 2, 4, 6 and 8 weeks post-treatment, blood samples were taken to determine the FIX protein and activity levels in the plasma. At sacrifice, livers were taken from the mice to extract DNA and RNA for quantification.

Plasma samples of week 8 post-treatment were also analyzed for TAT and D-dimer levels. Hemophilic male mice (8 to 12 weeks old) that are FVIII-/- deficient with disruption in exon 16 of the FVIII gene were injected with AAV-hFIX vectors via a tail vein injection at a vector dose of 5x10 gc/kg. At week 1, 2, 5 and 8 weeks post-treatment, blood samples were taken to determine the FIX protein and FVIII mimetic activity in the plasma. At sacrifice, livers were taken from the mice to extract DNA and RNA for quantification.

Cynomolgus macaques (Macaca fascicularis) (n=2) received 9x10 gc/kg of AAV5 vectors; AAV5-LP1-FIAV, AAV5-Q1-IDAV and AAV5-Q1-FIAV intravenously after pretreatment with intralipid. One animal received the vehicle after pretreatment with intralipid. Sequential venous bleeds were taken 1 and 2 weeks, and 1 day before administration of the test materials and throughout the study, on day 4, and weeks 2, 4, 6, 8, 10, 13, 14, 15 and 16. The schematic outline of the study is depicted in Figure 24. At sacrifice, several tissues were snap frozen for subsequent Q-PCR and RT-QPCR analyses to quantify vector DNA and mRNA.

Recombinant FIX protein expression and purification

HEK 293T cells were transfected into HEK293 cells using Lipofectamine2000 per the manufacturer’s instructions and as described previously (Verhoef D et al. Nature

Communications 2017). Single cell-derived clones were analysed for their FIX protein expression. High FIX-expressing HEK293 clones were propagated and expanded in a cell factory (10-chambers, 2528 cm2) that was pretreated with Poly-D Lysin Hydrobromide (5 mg in 1 L PBS; one hour at 37°C). Once the cells were confluent after ~3-4 days propagation, the media was switched to FIX-expression media (DMEM-F12 media without phenol red, without FBS and supplemented with 6 pg/ml vitamin K (Konakion) and 10 pg/ml insulin- transferrin- selenite (ITS)). FIX-containing conditioned media was collected for 10 days and replaced for new FIX-expression media every 24hrs. The conditioned media was filtrated using an 0.45 pm membrane, 10 mM of the serine protease inhibitor benzamidine was added. For each recombinant FIX variant 20 F of conditioned media was collected. The conditioned media was applied to a size 6 A ultrafiltration hollow fiber cartridge with a molecular weight cut-off of 10 kDa using an Akta flux 6 instrument (GE Healthcare), 10-fold concentrated, 10- fold diafiltrated to a final volume of 450 mH in 20 mM Hepes, 0.15 M NaCl, pH 7.4 (HBS). For each recombinant FIX variant, -500 mF of HBS concentrate was prepared. The overall FIX recovery after concentration and diafiltration varied from 50% to >100%; the wide range in % recovery is likely due to some variation in the FIX-specific clotting assay, as the samples of the final concentrate were measured. To purify the recombinant FIX proteins the concentrated medium was subjected to anion exchange chromatography by applying to a 4.8 x 4 cm Q-Sepharose Fast Flow (Q-FF) column (GE Healthcare) equilibrated in 20 mM Tris, 0.15 M NaCl, pH 7.4 at ambient temperatures. Upon washing with the same buffer, bound protein was eluted with a linear 0.15-0.75 M NaCl gradient (7.5x column volumes (cV)). Total protein (Abs280corrected) and FIX-clotting activity were assessed for each fraction (8 mF/fraction); factor IX-containing fractions were stored at -80 °C until further use. Further purification was performed by hydrophobic affinity chromatography to separate fully carboxylated FIX from non carboxylated FIX. The FIX-containing Q-FF fractions were pooled, and two times dialyzed at 4°C for 2hrs to 1 mM Na2HP04/NaH2P04, pH 6.8 (4 F), followed by overnight dialysis to 5 mM Na2HP04/NaH2P04, pH 6.8 (4 F). The dialysate was centrifuged at 10,000# at 4°C for 20 min. and subsequently applied to a Bio-Scale CHT20-I hydroxyapatite column (Bio-Rad; cV 20 ml) equilibrated in 5 mM

Na2HP04/NaH2P04, pH 6.8 at ambient temperatures. Following washing with the same buffer, bound protein was eluted with a linear 5-400 mM Na2HP04/NaH2P04 gradient (20x cV) at a flow rate of 3 ml/min. Total protein (Abs280corrected) and FIX-clotting activity were assessed for each fraction (8 ml/fraction). Factor IX-containing fractions were analyzed employing SDS-PAGE analysis, stored at -80 °C until further use. The elution profiles of each recombinant FIX variant were assessed by SDS-PAGE analysis.

FIX ELISA

The amount of hFIX protein in the murine plasma was determined with an Enzyme-Linked Immunosorbent Assay (ELISA) using the VisuLize FIX Antigen kit (Product # FIX-AG, Affinity Biologicals) according to the manufacturer’s instructions. The plasma samples will be diluted in provided dilution buffer to obtain an OD value which fits in the reference standard curve and each plasma sample was measured in duplicate. Reference curve will be generated by preparing a serial dilution of a standard plasma present in the ELISA kit according to the supplier’s protocol.

Human FIX antigen was detected with an ELISA assay using a mouse monoclonal antibody specific to hFIX. Briefly, flat-bottomed 96- well plates were coated with the monoclonal antibody; AHIX-5041 (Haematologic Technologies Inc, Essex Junction, VT, EISA) at a dilution of 1:3000 at 4°C overnight. Next day, wells were emptied and washed three times using PBS/0,05% Tween-20 (wash buffer) and blocked for 1 hour at room temperature using PBS/0,05% Tween containing 6% of Bovine Serum Albumin (BSA). Serial dilutions of human plasma in PBS/0,05% Tween containing 2% BSA served as standard. NHP plasma samples were diluted 100-fold in wash buffer loaded into the wells and incubated for 1 hour at room temperature. After washing, horseradish peroxidase-conjugated polyclonal goat IgG against human FIX (Cedarlane laboratories, Burlington, ON, Canada) was added and incubated for 1 hour at room temperature. After the final wash, plates were developed using o-Phenylenediamine (Thermo Fisher Scientific, Waltham, MA, ETSA) and stopped after 10 minutes using 2M sulfuric acid (Merck, Darmstadt, Germany). Optical density was assessed spectrophotometrically at 490 nm using the Versamax Molecular Devices (San Jose, CA, EISA). The relation between hFIX levels and OD is determined by a 4-parameter nonlinear regression of the calibrator curve. FIX activity measured by chromogenic assay

Factor IX activity in the non-human primate plasma samples was determined using the Biophen Factor IXa kit (ACT. FIX, Ref. 221812, Hyphen Biomed) according to the manufacturer’s protocol. The plasma samples were diluted in provided dilution buffer to obtain an OD value which fits in the reference standard curve and each plasma sample was measured in duplicate. BIOPHEN normal control plasma that is calibrated against an

International standard for factor IX (Aniara) was used to make a reference standard curve.

APTT clotting assay to determine FIX and FVIII-like clotting activity

Factor IX expression was monitored employing FIX-specific clotting activity in a modified one- step APTT assay by mixing conditioned media with FIX-depleted human plasma (Stago) in a 1:1 ratio. A reference curve of normal pooled plasma serially diluted in Owren-Koller diluent (Stago) mixed in a 1 : 1 ratio with FIX-depleted human plasma was used to calculate the equivalent FIX Units per ml sample. One ml of human plasma comprises 1 Unit of FIX activity.

FVIII-like clotting activity was determined using a modified FVIII- specific APTT-based clotting assay. FIX samples were serially diluted to in Owren-Koller diluent (Diagnostica Stago, Paris, France). FVIII-depleted plasma (25 mΐ; Diagnostica Stago) was mixed with sample (25 mΐ) and APTT reagent (50 mΐ; Triniclot automated activated partial thromboplastin time reagent, Diagnostica Stago), followed by a 180 s incubation period at 37 °C.

Coagulation was initiated after the addition of 50 mΐ of 25mM CaC12, upon which the coagulation time was monitored. Reference curves consisted of serial dilutions of NPP or NovoEight.

Thrombin generation assay

Thrombin generation was adapted from protocols earlier described (Hemker et al. Pathophysiol Haemost Thromb 2003). Thrombin generation curves were obtained by supplementing FVIII-depleted plasma (Diagnostica Stago) or hemophilia A patient plasma (George-King Biomedical) with TF (0.5 or lpM final), PCPS (20 mM), and 5 pg/mL FIX variant (equals 100% normal FIX plasma concentration) or varying amounts of NovoEight (0-0.2 pg/mL; equals 0-100% normal FVIII plasma concentration). Thrombin formation was initiated by adding substrate buffer (FluCa; Diagnostica Stago) to the plasma. The final reaction volume was 120 mΐ, of which 80 mΐ was plasma. Thrombin formation was determined every 20 s for 30-60 min and corrected for the calibrator using Thrombino scope software. The lag time, mean endogenous thrombin potential (ETP, the area under the thrombin generation curve), time to peak, peak thrombin generation, and velocity index were calculated from at least three individual experiments.

TAT and D-dimer assay

TAT complexes were detected using a sandwich enzyme immunoassay, Enzygnost TAT micro (Siemens Healthcare GmbH, Erlangen, Germany) according to the manufacturer’s protocol.

D-dimer levels prior and post-treatment were measured using the ASSERACHROM®D-DI ELISA kit (Diagnostica Stago, Parsippany, NJ, USA) according to the manufacturer’s instructions. Two dilutions of plasma samples were measured in duplicate.

Transfection assays and cells

The constructs encoding the FIX mutants were transfected into cells using lipofectamine 3000 reagent. A renilla luciferase plasmid was co-transfected to correct for transfection efficiency. Production of FIX in vitamin K supplemented medium was detected by harvesting the supernatant 2 days post-transfection and measuring the antigen and activity levels by ELISA (Affinity Biologicals) and chromogenic assay (Hyphen Biomed). FVIII mimetic activity was determined using the one stage APTT clotting assay in FVIII-deficient plasma. Huh7 cell were transduced with AAV5 vectors encoding the different FIX variants. Two days post-transduction, FIX protein production in conditioned medium was determined by harvesting the supernatant to analyse the FIX protein level by ELISA and FVIII mimetic activity by one-stage APTT clotting assay.

AAV 5 vectors

Recombinant AAV5 (SEQ ID NO.23 through 25) harbouring the expression cassettes were produced by infecting SF+ insect cells (Protein Sciences Corporation, Meriden, Connecticut, USA) as described (Lubelski et al. Bioprocessing Journal, 2015). Following standard protein purification procedures on a fast protein liquid chromatography system (AKTA Explorer, GE Healthcare, Chicago, Illinois, USA) using AVB sepharose (GE Healthcare, Chicago, Illinois, USA) the titer of the purified AAV was determined using qPCR. The AAV5 viral vector produced from SEQ ID NO.23 results in wild-type AAV5 capsid amino acid sequence while SEQ ID N0.24 result in a capsid amino acid sequence with one additional alanine inserted in between the first and the second position (relative to the wild- type sequence). Capsids produced with SEQ ID NO.25 result in a hybrid capsid where the VP1 unique sequence from AAV2 substitutes the VP1 unique sequence from AAV5, whereas VP2 and VP3 sequences are, like with SEQ ID NO. 23 and 24, wild-type. These modified capsids aid in

manufacturing. AAV5 viral vectors with AAV5 capsids manufactured in insect cells based on SEQ ID NO.25 were used in the studies herein. The expression cassettes were incorporated in an AAV viral vector genome. The expression cassettes comprising a promoter sequence driving the expression of FIX encoding sequences. Expression cassettes used in the examples comprise e.g. promoter sequences such as listed in SEQ ID NO.8, SEQ ID NO.17, or SEQ ID NO. 18, representing LP1, Q1 variant 1, and Q1 respectively, combined with FIX encoding sequences such as listed e.g. in SEQ ID NO. 4 (wt), SEQ ID NO. 7 (FIAV), or SEQ ID NO.28 (IDAV). Exemplary expression cassettes as used in the studies being listed in in SEQ ID NO. 19 (LP1 - FIAV), SEQ ID NO. 22 (Q1 FIAV). An example of a representative viral vector genome is listed in SEQ ID NO. 29, which comprises the Q1 FIAV expression cassette.

Vector DNA and mRNA quantification in NHP tissues

Sections of tissues obtained from NHP at sacrifice, including liver lobes, were snap -frozen for Q-PCR and RT-QPCR analyses. DNA and RNA were extracted. Q-PCR and RT-QPCR assays were performed on the extracted liver, adrenals, bone marrow, brain, heart, kidneys, lungs, pancreas, spleen and testes to measure vector DNA copies and FIX transcript levels of the FIX-WT, FIX-ID AV and FIX-FIAV using the same primers.

Fluorescent in situ hybridization and images analysis

Fluorescent in situ hybridization (FISH) was used to visualize AAV vector DNA and transgene mRNA in frozen liver sections with a fluorescent probe recognizing hFIX AAV vector DNA and transgene mRNA (ACD-Bio, Newark, California, USA). Tissue samples from the left median liver lobe from one animal per dose group were analyzed. Multiple images were acquired with a confocal microscope (Leica, SP8 confocal laser scanning microscope) and analyzed with the HALO image analysis software (Indicalabs, Corrales, New Mexico, USA). On average, images of 4000 cells were analyzed. To assess the quality of the tissue and procedure, liver positive (Ubiquitin C) and negative (DapB) control probes were used. The percentage of cells positive for hFIX AAV vector DNA and transgene mRNA was analyzed. Activation kinetics of factor IX by tissue-factor Vila (TF-FVIIa)

The activation kinetics (Km and Kcat) were determined for the substrate FIX in its conversion to FIXa by incubating TF-PCPS, i.e. TF incorporated into vesicles containing phosphatidylcholine/phosphatidylserine (1-5 nM TF in 10-25 mM PCPS) and FIX (-10-2500 nM) for 5 min at 25 °C in 20mM Hepes, 0.15M NaCl, 5mM CaC12, 0.1% (w/v) PEG8000, pH 7.5, upon which the reaction will be initiated by the addition of FVIIa (50 pM). Samples (10m1) were withdrawn at various time points (0-4 min) and quenched by mixing with 90 mΐ of 20 mM HEPES, 0.15M NaCl, 50mM EDTA, 0.1% (w/v) PEG8000, pH 7.5. Quenched samples will be further diluted in the same buffer, and initial velocities of substrate hydrolysis will be determined in a SpectraMax M2e kinetic plate reader. Measured rates will be related to the concentration of plasma derived FIXa (pd-FIXa) from the linear dependence of the initial velocity on known concentrations of pd-FIXa determined in each experiment.

Results

Selection of candidate FIX variant proteins

HEK293T cells were transfected using plasmid DNA encoding the FIX variants. The FIX variants tested were FIX-wt, FIX-ID AV, FIX-FIAV, a hyperactive variant; KFW (VI OK, R338F and S377W) and IDAV and FIAV combined with hyperactive mutation Padua (R338F) or the hyperactive variant KFW (IDAV + Padua, FIAV + Padua, IDAV + KFW and FIAV + KFW). Figure 5 shows the location of the amino acids’ modifications of the variants. Two days post-transfection FIX activity was measured by a chromogenic assay and the levels are shown in Figure 5B for the FVIII mimetic variants; FIX-ID AV and FIX-FIAV. The FVIII mimetic activity in the supernatant of the transfected cells was determined using the one stage APTT clotting assay in FVIITdeficient plasma using a standard curve of recombinant FVIII (Turoctocog alpha, NovoEight, Novo Nordisk) (Figure 5C). Results show that the FIX hyperactive mutations, Padua and KFW, did not appear to contribute to FVIII mimetic activity of FIAV and IDAV, in contrast, the Padua mutations appeared to reduce FVIII mimetic activity of FIAV and IDAV. The FIAV variant showed most FVIII mimetic activity. Both IDAV and FIAV variants were selected for further development of a gene therapy for hemophilia A patients. Due to the low FVIII mimetic activities detected in vitro using plasmid transfections, AAV expressing all variants were also tested in hemophilic mice. The data in hemophilic mice also confirmed that addition of the Padua or KFW variant did not contribute to FVIII mimetic activity of IDAV and FIAV (data not shown).

Characterization of FIX variant proteins

The FIX-FIAV and variants, as well as wild-type FIX, recombinant proteins were generated by stably expressing the FIX variants in human embryonic kidney cells (HEK293) cells. Recombinant protein was produced and purified. The FVIII mimetic activity of the recombinant FIX proteins was determined using the one stage APTT clotting assay in FVIII- deficient plasma using a standard curve of recombinant FVIII (Turoctocog alpha, NovoEight, Novo Nordisk). Characterization of the FIX variants, IDAV and FIAV, revealed a FVIII mimetic activity of approximately 14 and 28% per 100% of protein or 17 and 32% when using a serial dilution of normal human plasma as standard or a serial dilution of recombinant FVIII as a standard. The wildtype FIX protein showed a FVIII mimetic activity which was below the lower limit of detection in this assay. Table 1 provides a summary of the data for the different variants; FIX-wt, FIX-ID AV and FIX-FIAV per 100% FIX protein (i.e. 5 pg/ml of FIX = 100%).

Table 1. Overview of FVIII mimetic activities with selected FIX variants.

To further characterize the FIX variants, thrombin generation assays were performed in different plasmas including FVIII-depleted pooled plasma, hemophilia A patient plasma and plasma from hemophilia A patient with inhibitors. Thrombin generation assay is a global coagulation test that reflects the whole process of activation of the coagulation system in the presence of anticoagulant factors. Hemophilia A patients display a reduced thrombin generation capacity that is linearly related to their plasma FVIII activity levels. Thrombin generation was initiated using 0.5 or 1.0 pM Tissue Factor and 20 mM of phospholipids in FVIII-depleted plasma. At these low TF concentrations thrombin formation depends, at least partially, on FVIII and FIX (Lawson et al., 1994; Mann et al., 2003), thereby reflecting the hemophilic phenotype, with the 0.5 pM TF trigger most closely resembling the severe hemophilic phenotype (Figure 6). Thrombin generation capacity of 100% of IDAV and FIAV proteins, which corresponds to 5 pg/ml was assessed and compared to both normal pooled plasma and hemophilia A plasma reconstituted with NovoEight. Figure 6 shows the thrombin generation curves initiated by 0.5 or 1.0 pM of Tissue Factor (TF). The thrombin peak was used to calculate the FVIII mimetic activity relative to the FVIII standard, resulting in 28% for IDAV and 29% for FIAV following a TF trigger of 0.5 pM, and in the presence of 1.0 pM TF, IDAV showed FVIII mimetic activity of 21% and FIAV showed 17% of FVIII mimetic activity in the thrombin generation assay in FVIITdepleted plasma (Figure 7).

The thrombin generation potential of IDAV and FIAV were further assessed in plasma of hemophilia A patient with inhibitors (80 Bethesda Units). Figure 8 shows the thrombin generation curves. In addition, FVIII mimetic activity was determined for the IDAV and FIAV using the thrombin peak levels relative to a serial dilution of recombinant FVIII using a TF trigger of 0.5 or 1.0 pM (Figure 9) in Hemophilia A patient plasma with inhibitors (80 Bethesda Units). The FVIII mimetic activities are indicated above the bars as 18% and 13% for IDAV and 17 and 12% for FIAV in the presence of 0.5 or 1.0 pM TF.

Expression studies in vitro

In vitro, efficacy studies by plasmid transfections in HEK293T cells were carried out by using FIX constructs expressing FIX-ID AV and FIX-FIAV and compared to FIX-wt. As the Hemophilia A gene therapy will be a liver targeted approach, and FIX is a liver generated protein, transfection of different FIX variants was also performed in liver cells to generate hFIX protein variants that can mimic FVIII activity. However, it is known that liver cell transfection does not result in very high transfection efficiency. This may hamper the feasibility of expressing sufficient protein to result in detectable FVIII mimetic activity in vitro. The constructs were expressed from the strong constitutive ubiquitous CMV promoter, which was selected because high FIX levels are needed to detect FVIII mimetic activity as was previously 15% of FVIII mimetic activity was reported per 100% FIX protein expression (Quade-Fyssy et a , J Thromb Haemost, 2014).

The levels of protein expression in HEK293T and Huh-7, a human liver cell line, was tested after transfection of plasmids encoding the different FIX variants. FIX protein levels in the supernatant of the transfected cells is shown below (Figure 10). FIX protein expression in the supernatant of the transfected cells was higher in HEK293T cells than in Huh-7 cells.

As the HEK293T cells showed higher protein expression and high expression levels are needed, this cell line was selected for further testing. To evaluate the FVIII mimetic activity of the FIX variants, HEK293T cells were transfected with constructs expressing FIX- wt, FIX-ID AV and FIX-FIAV variants. FIX protein and FVIII mimetic activity was determined in unconcentrated and concentrated supernatant of the transfected cells (Figure 11). Transfection of the plasmids resulted in expression of similar FIX protein levels for the different variants (Figure 11 A). Figure 1 IB shows the FVIII mimetic activity measured for both the unconcentrated (black bars) and concentrated samples (grey bars). FIX-wt shows a background level, whereas the FVIII mimetic activity of IDAV and FIAV were higher with FIAV having more FVIII mimetic potential compared to IDAV. In summary, when expressed and also concentrated under highly similar conditions, FVIII mimetic activity could be detected above the background levels of the assay. However, in vitro expression results in relatively low FIX protein levels and consequently FVIII mimetic activity. The low levels of FVIII mimetic activity may not be very accurate as the levels are close to the lower limit of detection of the assay. However, it can be clearly observed from Figure 1 IB that the FIAV variant showed more potent FVIII mimetic property compared to the IDAV variant, which is consistent with the recombinant protein characterization data.

Testing of different promoters, AAV vector testing in vitro and in vivo

To select an optimal liver specific promoter for the FIX variants, liver specific promoters were tested in their ability to induce FIX protein expression by transfection of plasmids encoding FIX-wt driven from the promoters into liver cells. Figure 12A demonstrates that expression of the FIX-wt protein was 2.8-fold higher when expressed from an exemplary newly developed synthetic liver specific promoter (SEQ ID NO. 18), termed Q1 compared to the LP1 promoter (SEQ ID NO.8), suggesting that an optimal path-forward in the human liver would be the use of the newly developed synthetic liver specific promoters. Variants of the newly synthetic liver specific promoters, comprising essentially the same promoter elements of SEQ ID NO.18 (i.e. SEQ ID NO. 9-15) were also tested for expression in primary liver cells as well as liver cell lines and all showing similarly high levels of expression as compared with SEQ ID NO. 18 (data not shown). As an example, a dose response curve is shown for expression of FIX, and FIAV from SEQ ID NO. 17 (Q1 variant 1) and 18 (Ql), which both comprise the promoter elements SEQ ID NO. 9-15 (Figure 12B). The expression cassettes as used in these experiments for expression are listed amongst SEQ ID NO. 19-22. These results show that the newly developed liver specific promoter represents a good candidate for expression of FIX variants, as high levels of liver expression are warranted.

In order to evaluate the promoter performance in vivo , AAV5 vectors were generated encoding FIX-wt protein driven from the LP1 or Q1 promoter and C57B16 mice were injected using a low dose of 5 x 10 gc/kg and a high dose of 2.5 x 10 gc/kg (n= 7). FIX protein and FIX activity levels were measured in the plasma of the animals on week 1, 2, 4, 6 and 8 following administration of AAV5 vectors expressing the different FIX transgenes. Figure 13 (left) shows the protein and FIX activity levels in the low dose groups. The mice that received the AAV encoding the Q1 -FIX-wt vectors expressed approximately 2.4-fold lower FIX protein and 1.8-fold lower FIX activity (weeks 4-8) in the plasma compared to the group that received AAV5-LP1 -FIX-wt. The difference in the high dose groups was more pronounced and was approximately 7.1 -fold and 6.0-fold for the FIX protein and FIX activity levels, respectively (measured from weeks 4-8).

The potency of the liver promoters in mice is strikingly the reverse from which is observed in vitro. The Q1 promoter, and variants thereof, were developed for expression in human liver cells, i.e. human liver cell lines and primary hepatocytes. This may indicate that the Q1 promoter, or variant thereof, is more suitable for human liver cells and less compatible with mice. This indicates that results obtained in mice when it concerns expression levels in the blood must be assessed with high caution when aiming to translate results obtained in mice to human. As similar vector genome copies were detected in the livers, the lower FIX protein expression and consequent FIX activity observed in Ql-hFIX could not be explained by other experimental variation (data not shown). The FIX mRNA copy numbers in the Q1 low and high dose groups were 4-fold reduced as compared to the LP1 groups. Hence, it appears that differences in transcription levels are the cause of the differences observed between FIX protein and activity levels in the mice groups. Based on these data, for studies in mice, the LP1 promoter was selected for the studies in mice aimed at reaching high amounts of protein levels and to evaluate efficacy in a mouse model.

AAV5 vectors encoding FIX-wt, FIX-ID A V and FIX-FIAV driven from the LP1 promoter were generated and tested by transducing Huh-7 liver cells using a multiplicity of infection (MOI) of 1 x 10⁵, 1 x 10⁶ and 1 x 10⁷ gc/cell. FIX protein levels in the supernatant of the transduced Huh-7 cells were determined by ELISA. Protein in the supernatant increased with vector dose and did not show significant differences between the different FIX variants (Figure 14, upper panel). The FVIII mimetic activities in the supernatant of the transduced cells were determined by one-stage activated partial thrombin time (APTT) clotting assay (Figure 14 lower panel). Both FIX-ID AY and FIX-FIAV show FVIII mimetic activity above the background activity levels of the FIX-wt. There is a dose-dependent increase in FVIII mimetic activity in the supernatant of the FIX-ID AV and FIX-FIAV transduced Huh-7 cells (Figure 14, lower panel).

To evaluate the expression of the FIX-ID AV and FIX-FIAV variants mediated by AAV delivery in comparison with FIX-wt expression in vivo, C57B16 mice were injected with 5 x 10 gc/kg of AAV5 vectors (under the control of the LP1 promoter) via a tail vein injection (Figure 15). FIX protein and activity levels were measured at different time points until week 8 post-treatment. At week 8, the animals were sacrificed, and the vector genome copies and mRNA expression levels were determined from the livers.

Supraphysiological FIX protein expression levels were reached in all AAV-injected mice. FIX protein expression was the highest for the group that received AAV-FIX-wt with levels up to 1200% of normal. FIX-ID AV and FIX-FIAV showed approximately 1.8-fold reduced expression compared to FIX-wt (Figure 16, upper panel). FIX activity followed the same trend as that of the expressed protein (Figure 16, lower panel) with the exception that the FIX-FIAV grouped showed greater FIX activity. Similar results have been observed when characterizing the recombinant proteins, which reflects hyperactive properties of FIAV (see figure 5B).

To address possible hypercoagulability due to expression of supraphysiological levels of the FIX variants, biomarkers for coagulation activation (TAT and D-dimers) were also measured (Figure 17). Elevated thrombin/antithrombin III complexes (TAT) are found in persons predisposed to thrombosis and disseminated intravascular coagulation (DIC) (Pelzer et a , Thromb Haemost 1988). The D-dimer is the ultimate degradation product of fibrin and is an indirect marker of coagulation activation followed by thrombolysis (Adam et a , Blood 2009). D-dimer levels are being evaluated in the clinic for diagnosing and monitoring DIC and as an aid in the identification of medical patients at high risk for venous

thromboembolism (VTE) (Tripodi, Clin Chem 2011). TAT and D-dimer levels in the mice plasma was measured at week 8 post-treatment. As a positive control (+ ctr), mouse plasma sample that was activated in vitro and thus had expected high TAT and D-dimer levels was included in both tests. Figure 17 shows that both TAT and D-dimer levels were similar in all AAV-injected mice, whereas the positive control do show an elevation in D-dimer levels.

The control sample for the TAT assay is a human plasma sample with a known TAT level (10 ± 2 pg/L).

To conclude, these results show that supraphysiological levels of the FIX variants do no result in elevated TAT and D-dimer levels, indicating that the FIX variants FIAV and IDAV do not predispose possible treated patients to thrombosis and disseminated

intravascular coagulation or increased coagulation activation.

Proof of concept study of AAV -FIX variants in hemophilia A mice

Vectors expressing the hFIX variants under the control of the LP1 promoter were

administered intravenously to transgenic mice lacking the gene encoding for FVIII

(disruption in exon 16; E16-FVIII-/- knock-out (KO)) (Figure 18). These experiments were performed to demonstrate that the AAV vectors encoding the hFIX variants had FVIII- mimetic activity. Animals were injected intravenously with 5 x 10 gc/kg and blood samples were taken on weeks 1, 2, 5, 8 and 12 (terminal) to assess FIX protein, FVIII mimetic activity was tested using an APTT assay using FVIII-deficient human plasma. These studies were performed using the liver promoter, or LP1, as we found that the LP1 promoter was optimal in mice. Prothrombin time, TAT and D-dimer levels were also assessed. Following termination (week 12) the number of DNA copies in the liver was assessed along with the vector distribution and expression.

Vector DNA distribution and hFIX mRNA in the liver where FIX is synthesized, approximately l-2/10⁶gc FIX DNA was observed, translating to approximately 5 x 10⁶ - 1.5 x 10 mRNA copies/gc of RNA, demonstrating appreciable transduction in the target organ (Figure 19). In addition, vector DNA and hFIX mRNA levels were determined in various organs; lung, adrenal gland, spleen, kidney heart and muscle which showed lower levels than the levels observed in the liver. mRNA levels in other organs was at or below the level of the limit of detection (10⁴ mRNA copies/gc). One limitation of FVIII KO mice is compromised venous access, which resulted in missed or partial injections, manifesting as reduced liver DNA copies and expression. Therefore, data from mice with reduced DNA expression (<3xl0⁵ gc/pg DNA) were excluded from further analyses.

For each of the transgenes tested a plateau in liver hFIX expression was observed around at week 5 (Figure 20) in the plasma of the mice. Consistent with recombinant protein data obtained in vitro, FVIII mimetic activity was observed. Absolute FIX-mimetic activity was plotted against FIX protein levels (Figure 21) for each of the constructs that were tested. The data indicated that FIX variants induced physiological relevant FVIITmimetic activity, which resulted in specific activity of 24% and 10%, FVIII mimetic activity per 100% FIX protein, for FIX-ID AV and FIX-FIAV, respectively, at 5-week post-treatment. This was 13% and 8% of FVIII mimetic activity per 100% protein of FIX-ID AV and FIX-FIAV, respectively, at week 8 post-treatment. In summary, the FIAV variant showed greater FVIII mimetic effects than the IDAV variant.

From a safety perspective, there was no elevation in either TAT or D-Dimer levels, compared to untreated KO mice or wildtype C57B16 mice with normal blood homeostasis, suggesting no issues with hypercoagulation. D-Dimer positive controls, as provided in the assay kit, demonstrated D-Dimer levels in excess of 500 ng/mL FEU would be suggestive of pathological changes, whereas in the treated mice, 50-fold less was observed (about D-Dimer 10 ng/ml (FEU)). Under normal circumstances prothrombin time (PT) is not affected in hemophilia, accordingly it was normal in the studies in hemophilia A mice and was not impacted in the different treatment groups tested on weeks 2, 5, 8 and 12 (Table 2).

Table 2. Data for each of the variants, and the respect C57B16 mouse background on the prothrombin time (seconds ± S.D.)

To summarize, administration of FVIII KO mice with AAV5 vectors expressing FIX variants resulted in the expression of these human FIX variants. This resulted in at least approximately appreciable FVIII mimetic activity, per 100% of FIX protein, consistent with recombinant protein data (see Figure 11). Such FVIII-mimetic (relative levels of >10 % are expected to have FVIII-mimetic capabilities, thereby reducing bleeding (den Uijl et al., Haemophilia 2011). Importantly, there appeared no increased risk for hypercoagulation, or any changes in the prothrombin time.

Risk assessment: FIX-FIAV and IDAV variants combined with standard therapy

In vivo, possible hypercoagulability by expression of the FIX variants was assessed by detecting the coagulation activation markers, TAT and D-dimer in the animal studies. The levels of these coagulation activation markers were similar in all the groups including the vehicle treated animals, indicating that there is no risk of hypercoagulability even at very high protein. As shown, the FIX variants can be safe and effective, provided that sufficient expression levels can be obtained, we next sought to determine the effects of current standard of care on different plasmas e.g. FVIII-depleted plasma, hemophilia A patient’s plasma and hemophilia A inhibitor patient’s plasma supplemented with FIAV and IDAV variants. A key question to address in the clinic is the safety of the FIX variants in combination with currently used FVIII bypassing products; such as activated prothrombin complex concentrate (also referred to as FEIBA (Factor Eight Inhibitor Bypassing Activity) and recombinant FVIIa (NovoSeven). To address this question, thrombin generation assays were performed in FVIII-depleted plasma, hemophilia A patient plasma (<1% FVIII) and hemophilia A inhibitor patient plasma of the FIX-FIAV recombinant protein in the presence of bypassing agents or recombinant FVIII at clinically relevant levels. The thrombin potential of 5 pg/ml (100% of normal) FIX-FIAV was assessed in the presence of 0.5 or 1.0 U/mF (60 or 125 U/kg) FEIBA, 1.75 pg/mF (120 pg/kg) NovoSeven or 1 U/ml (100% of normal) NovoEight (Novo8) using a TF trigger of 0.5 pM. Figure 22 shows the thrombin peak level in the different plasmas. Equal thrombin peak levels were reached when normal pooled plasma (NPP) or NovoEight (Novo8) was added to the plasmas. Addition of the FIAV variant resulted in elevated thrombin peak levels and the increase differed per plasma. There was a dose-dependent increase in peak thrombin levels after addition with FEIBA and it can be observed that the thrombin generation potential in the presence of FEIBA alone was similar. Similar results were observed for the NovoSeven. The results show that there was no potentiation of the effect when FIX-FIAV was combined with either FEIBA or NovoSeven.

Figure 23 shows the thrombin generation potential of IDAV or FIAV with or without addition of Novo8. Addition of Novo8 results in elevated thrombin peak levels that are almost 4-fold higher compared to the normal pooled plasma or the FVIII-depleted plasma with Novo8. Addition of Novo8 in the hemophilia A inhibitor patient plasma does not show any effect due to the FVIII inhibitors, while the FVIII mimetic activity of IDAV and FIAV remains. Preliminary results show that there is a linear correlation between the FVIII dose added and the thrombin generation potential in the presence of the FIX variant, indicating that the effect of FVIII provided to patients may be well-controlled and allows for safe usage of FVIII in FIX variant gene therapy treated patients, albeit at a lower dosage as compared with untreated patients.

To summarize, no synergistic thrombin generation was observed with FIX-ID AV and FIAV variants when combined with FEIBA or Novoseven. When combined with FVIII, a potentiating effect was observed. This means that the FIX variants can be used in

combination with currently used FVIII bypass products, FEIBA and FVIIa, without posing a potential hypercoagulability risk. Thrombin generation potential of the FIX variants is potentiated by the presence of FVIII, which shows a linear correlation with the amount of FVIII added (data not shown). A potentiating effect was also observed when FIX-FIAV was combined with Hemlibra in two different doses. A further dose response curve will be made to demonstrate whether this effect is linear or not. This indicates that the standard line of care in hemophilia A patients that have been subjected to a gene therapy with e.g. FIX-FIAV, may be treated, e.g. if patients have sustained injury and/or have e.g. a mild or moderate phenotype after gene therapy treatment, patients may be subjected to standard use of

Novoseven and/or FEIBA. In case FVIII is contemplated, a reduced dose thereof may be recommended as compared with the standard use prescribed, which may well be the case for Hemlibra as well. However, lower doses of Hemlibra have not yet been tested and thus it is unknown whether it also potentiates thrombin generation in a linearly fashion as FVIII.

In silico and in vitro assessment of immunogenicity potential of FIX variants.

To determine the risk of potential immunogenicity related to the FIAV and IDAV variants, an in silico analysis was performed. The full-length wild type human FIX sequence as well as the FIAV and IDAV proteins were evaluated for their immunogenic potential by use of an in silico platform for epitope identification and prediction for Class II (antigen presenting cells) and Class I (non-antigen presenting cells, such as hepatocytes) HLA. The analysis for Class II was performed with a combination of iTope™, MHC Class II binding prediction, a proprietary in silico molecular modelling technology, and TCED™, T cell epitope database (ABZENA pic, Cambridge, UK). For Class I, the IEDB peptides library (www.iedb.org,

(Vita et al., 2015)) was used.

No non-germline promiscuous major histocompatibility complex (MHC) class II binding peptides were identified in the FIAV and IDAV sequences, the mutations do not result in change in hits restricted by Class II human leukocyte antigen (HLA). Altogether, there was no significant difference between the wild type FIX and the FIAV and IDAV mutated proteins. Four non-germline promiscuous MHC class I binding peptides were found in the FIAV sequence and three in the IDAV sequence. Those MHC class I binding peptides have been determined as moderate affinity peptides and therefore poses a relatively low immunogenicity risk as compared to wildtype hFIX.

AAV-FIX variants testing in non-human primates: feasibility and risk evaluation study To understand whether the approach is clinically feasible, studies more relevant for human use were conducted in non-human primates (NHP). The main question was whether or not sufficient expression levels of the FIX variants can be achieved in NHP to allow for sufficient FVIII-mimetic activity representing a benefit that can be obtained in human patients as well, while having, minimal side-effects. In mice, in a direct comparison between FIX-wt and FIAV and IDAV variants, reduced expression levels were observed. Also, the LP1 promoter outperformed the Q1 promoter. Combined, when the mice data would translate to the NHPs then the FIX protein levels of the FIX variants in the plasma would be too low for achieving sufficient FVIII mimetic activity to potentially control bleeds. We have therefore further investigated the AAV5 vectors encoding for the different hFIX variants FIAV and IDAV in cynomolgus macaques (Macaca fascicularis).

The different promoters Q1 and LP1 were tested in the NHP, as the NHP model most closely resembles the human situation. To facilitate broader hepatic cell targeting, some NHPs received 20% Intralipid (2 g/kg in 10 mL/kg) that was administered intravenously one hour prior to administration of the AAV vectors (Kwikkers et al.,2018 manuscript in preparation) over 15 minutes. Sequential venous bleeds were taken 1 and 2 weeks, and 1 day before administration of the test materials and throughout the study, on day 4, and weeks 2, 4, 6, 8, 10, 13, 14, 15 and 16 post-treatment (Figure 24). While it was not possible to measure FVIII mimetic activity, since the experimental subjects possess normal NHP FVIII levels, hFIX protein level and function were assessed as outlined in figure 24, below, i.e. AAV5- LP1-FIAV, AAV5-Q1-FIAV and AAV5-Q1-IDAV. Again, to assess potential

hypercoagulation markers, TAT and D-dimer levels are assessed. Additionally, at the end of the study animals will be challenged with Factor Eight Inhibitor Bypass Activity (FEIBA; aPCC, FEIBA NF®, Shire, Lexington, MA, USA)) and recombinant FVIIa (recombinant FVIIa; rFVIIa, eptacog alfa, NovoSeven®, Novo Nordisk Inc, NJ, USA), existing treatments for hemophilia A, to explore thrombogenic risk of the test articles in the presence of concomitant treatments. At the end of the study, vector DNA and mRNA levels in the livers will be determined as well as biodistribution and histopathology in organs including adrenals, bone marrow, brain, heart, livers, lungs, pancreas, spleen and testes.

To further understand the safety aspects associated with the vectors limited (non- GLP) toxicological parameters were investigated, i.e. general health condition, body weight, hematology, blood chemistry including liver enzymes, off-target effects and histopathology. An additional goal was to assess safety-related parameters during the NHP pharmacology study. Parameters were recorded prior to and after administration of the respective

administration of the viral vectors containing the transgene encoding for different FIX variants. General animal health condition appeared normal. Body weight gain appeared stable before and after treatment. Following treatment behavior appeared unaffected.

Hematological parameters remained in the normal range through the course of the experiment, indicating no treatment-related anomalies. Notable in the present context platelets, PT, APTT and fibrinogen levels were unaltered up to 4 weeks after treatment, consistent with mouse studies (above). Blood chemistry results demonstrated transient changes in the liver markers AST and ALT, which returned to baseline by week 4. Therefore, the treatments were well-tolerated without impacting the general health condition of the animals.

hFIX protein and activity levels up to week 8 is provided in figure 25. Highly surprising, the data shows that the amount of FIX protein expressed by the FIAV variant in the blood plasma of NHP was about 8 -fold higher when using the Q1 promoter compared to the LP1 promoter (Figure 25, upper panel). A similar increase was seen for FIX activity (Figure 25, lower panel), with the highest activity observed for FIAV which can be associated with the hyperactive FIX properties of FIAV, as protein levels were similar. Though animal numbers are limited, it can be seen that administration of AAV5 containing the transgene encoding for the hFIX-FIAV and IDAV variants engendered pronounced protein expression and hFIX function. Surprisingly, the expression as observed in NHP shows the complete reverse of what was observed in mice (see Figure 13), with the Q1 promoter clearly significantly outperforming the LP1 promoter which indicates that the Q1 promoter, or a variant thereof, may be a preferred candidate for expression of either FIAV or IDAV in human patients.

To summarize, no serious clinical observations, or adverse effects on body weight or clinical chemistry parameters were observed in this study in which animals were followed for up to 8 weeks after administration, whereas high levels of hFIX could be obtained which, taking into account the observed 14% or 28% FVIII mimetic activity, of recombinant protein as described above, of respectively IDAV and FIAV, at 100% hFIX protein levels, allowing for obtaining a % FVIII mimetic activity in human hemophilia A patients in a gene therapy approach (see table 3), that puts a patient in the range of mild (5-40%) and even normal range is within reach (above 40%).

Table 3. Estimated FVIII mimetic activity in NHPs

AAV-FIX variants testing in non-human primates: compatibility of FIX-FIAV with aPCC and FVIIa

To study the compatibility of FIX-FIAV with activated prothrombin complex concentrate (aPCC, FEIBA, Shire) and FVIIa, NHPs received an intravenous injection of aPCC at week 13 followed by a week of wash-out and followed by administration of FVIIa (NovoSeven, Novo Nordisk) at week 15. The aPCC was administered at a dose of 50 U/kg; the standard clinical dose during bleeds is 50-100 U/kg every 6-12 hours. aPCC contains non-activated factors II, IX and X and activated factor VII. In addition, FEIBA contains factor VIII antigen (FVIII C:Ag). FVIIa was administered once at a dose of 90 pg/kg and the standard clinical regimen during an acute bleed is 90-120 pg/kg every 2-3 hours until cessation. The presence of factor IX in aPCC resulted in an increase in FIX protein as well as FIX activity after the challenge (Figure 26 A and B). Administration of FVIIa at week 15 did not result in elevations of FIX protein and activity level (Figure 26A and B).

Vector DNA and mRNA levels in the livers were determined as well as biodistribution and histopathology in organs including adrenals, bone marrow, brain, heart, livers, lungs, pancreas, spleen and testes. Vector DNA and mRNA levels were equal throughout all collected liver lobes (Figure 27). Figure 28 shows the vector DNA in the following organs: adrenals, bone marrow, brain, heart, kidneys, lungs, pancreas, spleen and testes. Vector DNA copies can be found in all organs, however the vector DNA levels varied per organ. mRNA copies could only be detected in the adrenals, bone marrow and the heart. However, the mRNA levels detected were very close to the LLOQ of the assay (4e3 copies).

Vector DNA and FIX mRNA following AAV -transduction in the liver

A liver tissue sample from the liver from one animal per treatment group was analysed for the presence of vector DNA and hFIX transgene mRNA by fluorescent in situ hybridization. The assay was performed with a probe that hybridizes to wildype FIX and FIX-FIAV. The percentage of positive liver cells for hFIX AAV vector DNA and transgene mRNA was determined and summarized in the table in Figure 29. The vector DNA/mRNA positive cells are divided into +1 till +4 categories that range from low to strongly positive. It was concluded that the use of Q1 as promoter to drive expression of FIX-FIAV in NHP resulted in a higher percentage of +4 positive cells as compared to the use of LP1 as promoter to drive expression of FIX-FIAV in NHP. In line with this, a lower percentage of +1 positive cells was observed when Q1 was used versus LP1. These results confirm the potency of Q1 as a promoter to drive strong liver- specific transgene expression in NHP.

No elevation of prothrombotic markers due to FIX-FIAV expression

The risk of a possible prothrombotic phenotype due to administration of AAV encoding the FIX variants was assessed by determining the levels of plasma Thrombin-Antithrombin (TAT) complex and D-dimers. Expression of FIX-IDAV and FIX-FIAV is not associated with an elevation of TAT and D-dimer. However, the injection with aPCC at week 13 did result in an increase of both TAT and D-dimers, which is observed in both the vehicle and AAV treated animals (Figure 30). Thus, the use of aPCC in the absence of FIX-IDAV or FIX-FIAV expression is also associated with elevation of TAT and D-dimers. The animals that had high FIX expression did show higher D-dimer and TAT levels upon aPCC challenge compared to the vehicle. However, the temporary prothrombotic state of the animals did not result in any adverse effects.

Activation kinetics of FIX-FIAV and FIX-WT The activation kinetics (Km and Kcat) were determined for the substrate FIX in its conversion to FIXa by TF-FVIIa. Progress curves of FIX activation for FIX-WT and FIX- FIAV in a serial dilution are depicted in Figure 31. The Kcat and Km are depicted in the table below the figure and similar for FIX-WT and FIX-FIAV.

Figures

Figure 1. Schematic representation of mature wildtype FIX with modification sites indicated for FIAV and IDAV.

Figure 2. Amino acid sequence of mature wildtype FIX, with positions modified for FIAV and IDAV in bold and underlined.

Figure 3. Amino acid sequence of propeptide FIX for FIAV and IDAV

Figure 4. Schematics showing the mode of action of FVIIIa as a cofactor for FIXa for activation of FX and the mode of action of a FIX variant (FIAV) that does not require FVIIIa to activate FX.

Figure 5. In vitro testing of FIX variants by plasmid transfections into HEK293T cells. IDAV and FIAV variants were combined with KLW and Padua mutations (schematically depicted in A). The FIAV mimetic variant showed an increase in FIX activity as compared with IDAV and wild-type FIX (Figure 5B, upper panel), when combined with Padua or KLW a further increase in FIX activity can be observed (Figure 5B lower panel). C) FVIII mimetic activity was tested, indicating that FIAV has higher FVIII mimetic activity as compared to IDAV, and KLW and Padua mutations do not significantly contribute to the LVIII mimetic activity.

Ligure 6. Thrombin Generation Curves of the LIX variants in LVIITdepleted plasma using 0.5 or 1.0 pM Tissue Lactor as an inducer. The grey area indicates the levels of thrombin peak generation in 95% of the normal pooled plasma (NPP). (Data show mean ± S.D.).

Ligure 7. LVIII mimetic activity of the LIX variants as determined by the thrombin peak levels from the thrombin generation curves in the presence of 0.5 or 1.0 pM Tissue Lactor in LVIITdepleted plasma. NPP; normal pooled plasma, Novo8; recombinant LVIII, NovoEight. (Data show mean ± S.D.).

Figure 8. Thrombin generation curves of FIX-ID A V and FIAV proteins in hemophilia A patient plasma and hemophilia A inhibitor patient plasma in the presence of 0.5 or 1.0 pM Tissue Factor. The grey area indicates the levels of thrombin peak generation in 95% of the normal pooled plasma.

Figure 9. FVIII mimetic activity of the FIX variants as determined by the thrombin peak levels from the thrombin generation curves in the presence of 0.5 or 1.0 pM Tissue Factor in hemophilia A inhibitor patient plasma. (Data show mean ± S.D.).

Figure 10. In vitro, FIX protein expression in transfected HEK293T and Huh-7 cells

FIX protein expression in the supernatant was measured using an enzyme-linked

immunosorbent assay (ELISA) upon transfection of the different FIX constructs in HEK293T (black bars) and Huh-7 cells (grey bars). Data show mean ± S.D.

Figure 11. FIX protein expression and FVIII mimetic activity in vitro upon transfection of the FIX variants in HEK293T cells. A) FIX protein levels in unconcentrated and concentrated supernatant of transfected HEK293T cells were determined by ELISA. B) FVIII mimetic activity in unconcentrated and concentrated supernatant of transfected HEK293T cells were determined by APTT clotting assay. All data show mean ± S.D.

Figure 12. Higher in vitro FIX expression driven by the newly developed synthetic liver specific promoter in human liver cells. (A) FIX protein levels driven from the LP1 and the newly developed promoter (SEQ ID NO.18) was determined in the supernatant of transfected cells by ELISA. Data show mean ± S.D. (B) A dose response of FIX expression was determined after transfection of representative newly developed promoters (SEQ ID NO.17 and 18), corresponding with expression cassettes as listed in SEQ ID NO. 19 and 20.

Figure 13. Data showing that the LP1 promoter appears more potent in driving FIX-wt protein expression in vivo in mice. FIX protein (upper left) and activity levels (upper right) in the plasma of mice injected with a dose of 5 x 10 gc/kg of AAV encoding either Q1 -FIX-wt or LP1 -FIX-wt or at a high dose of 2.5 x 10¹³ gc/kg (FIX protein, lower left; FIX activity, lower right). Figure 14. FIX-ID AV and FIX-FIAV protein expression by transducing Huh-7 cells with AAV vectors resulted in a dose-dependent increase in FIX (upper panel) and consequent FVIII mimetic activity (lower panel). FIX protein levels (upper graph) and FVIII mimetic activity (lower graph) in the supernatant of transduced Huh-7 cells at an MOI of 1 x 10⁵, 1 x 10⁶ and 1 x 10⁷ gc/cell.

Figure 15. Schematic overview of an experiment to assess expression of different FIX variants in C57B16 mice upon AAV vector delivery.

Figure 16. FIX protein and activity levels in AAV-injected C57B16 mice

FIX protein (upper graph) and activity levels (lower graph) in the plasma of mice that received AAV encoding FIX-wt, FIX-ID AV and FIX-FIAV expressed via the LP1 promoter up to week 8 post-injection. A vehicle group was included as control.

Figure 17. No elevation of coagulation activation markers: D-Dimer (upper graph) and TAT (lower graph) at supraphysiological FIX expression levels in AAV-injected mice. D-Dimer and TAT levels in the plasma of mice injected with AAV vectors encoding FIX variants in plasma samples of week 8 post-treatment.

Figure 18. Schematic overview of proof of concept studies in FVIII knockout mice.

Figure 19. FIX DNA (left) and FIX mRNA (right) measured in AAV-injected hemophilia A mouse livers. DNA and mRNA were both measured as (gc/pg) wet weight. LLOQ; lower limit of quantification.

Figure 20. FIX protein levels measured from the plasma of AAV-injected hemophilia A mice expressing wild type FIX (left upper), FIX-ID AV (right upper) and FIX-FIAV (lower panel). (The legend to the right of each of the figures refers to individual mouse numbers.)

Figure 21. Data showing FVIII- mimetic activity as a function of FIX protein level, 1 week (upper left), 2 weeks (upper right), 5 weeks (lower left) and 8 weeks (lower right) after AAV administration to hemophilia A mice. Figure 22. Assessment of hypercoagulability by thrombin generation assays in the presence of FIX-FIAV with marketed clotting factor products. Thrombin peak levels were measured for FIX-FIAV recombinant proteins in the absence or presence of FEIBA or NovoSeven in different plasmas as well as the thrombin generation potential of FEIBA or NovoSeven alone. NPP; normal pooled plasma, FVIIId; FVIII-depleted plasma, HA; hemophilia A patient plasma, HA/I; hemophilia A patient plasma with inhibitors, Novo8; recombinant FVIII, NovoEight.

Figure 23. Assessment of hypercoagulability by thrombin generation assays in the presence of FIX-FIAV with recombinant FVIII and Hemlibra (lower graph). Thrombin generation potential was determined for the FIX-ID AV and FIX-FIAV proteins in the absence and presence of Novo8.

Figure 24. Schematic representing the design of the NHP primate study.

Figure 25. Data demonstrating hFIX protein levels (upper panel) and hFIX activity (lower panels) determined by chromogenic assay (B) and one stage APTT clotting assay (C) from the plasma of NHP.

Figure 26. Data demonstrating hFIX protein (A), FIX activity determined by chromogenic assay (B) and one stage APTT (C) from the NHP plasma throughout the study.

Figure 27. Data of the vector DNA (A) and mRNA (B) levels in the 8 liver lobes of the NHPs.

Figure 28. Data of the vector DNA (A) and mRNA (B) levels in different organs of the NHPs.

Figure 29. Table summarizing the FIX DNA/mRNA positive liver cells based on in situ hybridization.

Figure 30. Assessment of possibly hypercoagulability by measuring Thrombin-Antithrombin complexes and D-dimers. Figure 31. Assessment of the activation kinetics of FIX-WT and FIX-WT in its conversion to FIXa.

SEQUENCE LISTING

SEQ ID NO 1 Mature AA sequence of FIX-wt of 415 aa - mature FIX

YNSGKLEEFV QGNLERECMEEKCSFEEAREVFENTERTTEFWKQY VDGDQCESNPC

LNGGSCKDDINSYECWCPFGFEGKNCELDVTCNIKNGRCEQFCKNSADNKVVCSCT

EGYRLAENQKSCEPAVPFPCGRVSVSQTSKLTRAEAVFPDVDYVNSTEAETILDNITQ

STQSFNDFTRVVGGEDAKPGQFPWQVVLNGKVDAFCGGSIVNEKWIVTAAHCVETG

VKITVVAGEHNIEETEHTEQKRNVIRIIPHHNYNAAINKYNHDIALLELDEPLVLNSYV

TPICIADKEYTNIFLKFGSGYVSGWGRVFHKGRSALVLQYLRVPLVDRATCLRSTKFT

IYNNMFCAGFHEGGRDSCQGDSGGPHVTEVEGTSFLTGIISWGEECAMKGKYGIYTK

VSRYVNWIKEKTKLT

SEQ ID NO 2 AA sequence of FIX-wt of 461 aa - propeptide

MQRVNMIMAESPGLITICLLGYLLSAECTVFLDHENANKILNRPKRYNSGKLEEFVQ

GNLERECMEEKCSFEEAREVFENTERTTEFWKQYVDGDQCESNPCLNGGSCKDDIN

S YECWCPFGFEGKNCELD VTCNIKN GRCEQFCKNS ADNKV VCSCTEGYRLAEN QKS

CEPAVPFPCGRVSVSQTSKLTRAEAVFPDVDYVNSTEAETILDNITQSTQSFNDFTRV

V GGED AKPGQFPW Q V VLN GKVD AFCGGS IVNEKWIVTA AHC VETG VKITV V AGEH

NIEETEHTEQKRNVIRIIPHHNYNAAINKYNHDIALLELDEPLVLNSYVTPICIADKEYT

NIFLKFGSGYVSGWGRVFHKGRSALVLQYLRVPLVDRATCLRSTKFTIYNNMFCAGF

HEGGRDSCQGDSGGPHVTEVEGTSFLTGIISWGEECAMKGKYGIYTKVSRYVNWIK

EKTKLT

SEQ ID NO 3 NA sequence of FIX-wt - propeptide

ATGCAGCGCGTGAACATGATCATGGCAGAATCACCAGGCCTCATCACCATCTGC

CTTTTAGGATATCTACTCAGTGCTGAATGTACAGTTTTTCTTGATCATGAAAACGC

CAACAAAATTCTGAATCGGCCAAAGAGGTATAATTCAGGTAAATTGGAAGAGTT

TGTTCAAGGGAACCTTGAGAGAGAATGTATGGAAGAAAAGTGTAGTTTTGAAGA

AGCACGAGAAGTTTTTGAAAACACTGAAAGAACAACTGAATTTTGGAAGCAGTA

TGTTGATGGAGATCAGTGTGAGTCCAATCCATGTTTAAATGGCGGCAGTTGCAAG

GATGACATTAATTCCTATGAATGTTGGTGTCCCTTTGGATTTGAAGGAAAGAACT GTGAATTAGATGTAACATGTAACATTAAGAATGGCAGATGCGAGCAGTTTTGTA

AAAATAGTGCTGATAACAAGGTGGTTTGCTCCTGTACTGAGGGATATCGACTTGC

AGAAAACCAGAAGTCCTGTGAACCAGCAGTGCCATTTCCATGTGGAAGAGTTTC

TGTTTCACAAACTTCTAAGCTCACCCGTGCTGAGACTGTTTTTCCTGATGTGGACT

ATGTAAATTCTACTGAAGCTGAAACCATTTTGGATAACATCACTCAAAGCACCCA

ATCATTTAATGACTTCACTCGGGTTGTTGGTGGAGAAGATGCCAAACCAGGTCAA

TTCCCTTGGCAGGTTGTTTTGAATGGTAAAGTTGATGCATTCTGTGGAGGCTCTAT

CGTTAATGAAAAATGGATTGTAACTGCTGCCCACTGTGTTGAAACTGGTGTTAAA

ATTACAGTTGTCGCAGGTGAACATAATATTGAGGAGACAGAACATACAGAGCAA

AAGCGAAATGTGATTCGAATTATTCCTCACCACAACTACAATGCAGCTATTAATA

AGTACAACCATGACATTGCCCTTCTGGAACTGGACGAACCCTTAGTGCTAAACAG

CTACGTTACACCTATTTGCATTGCTGACAAGGAATACACGAACATCTTCCTCAAA

TTTGGATCTGGCTATGTAAGTGGCTGGGGAAGAGTCTTCCACAAAGGGAGATCA

GCTTTAGTTCTTCAGTACCTTAGAGTTCCACTTGTTGACCGAGCCACATGTCTTCG

ATCTACAAAGTTCACCATCTATAACAACATGTTCTGTGCTGGCTTCCATGAAGGA

GGTAGAGATTCATGTCAAGGAGATAGTGGGGGACCCCATGTTACTGAAGTGGAA

GGGACCAGTTTCTTAACTGGAATTATTAGCTGGGGTGAAGAGTGTGCAATGAAA

GGCAAATATGGAATATATACCAAGGTATCCCGGTATGTCAACTGGATTAAGGAA

AAAACAAAGCTCACT

SEQ ID NO 4 NA sequence of codon optimized sequence of FIX-wt - propeptide

ATGCAGAGGGTGAACATGATCATGGCTGAGAGCCCTGGCCTGATCACCATCTGC

CTGCTGGGCTACCTGCTGTCTGCTGAGTGCACTGTGTTCCTGGACCATGAGAATG

CCAACAAGATCCTGAACAGGCCCAAGAGATACAACTCTGGCAAGCTGGAGGAGT

TTGTGCAGGGCAACCTGGAGAGGGAGTGCATGGAGGAGAAGTGCAGCTTTGAGG

AGGCCAGGGAGGTGTTTGAGAACACTGAGAGGACCACTGAGTTCTGGAAGCAGT

ATGTGGATGGGGACCAGTGTGAGAGCAACCCCTGCCTGAATGGGGGCAGCTGCA

AGGATGACATCAACAGCTATGAGTGCTGGTGCCCCTTTGGCTTTGAGGGCAAGA

ACTGTGAGCTGGATGTGACCTGCAACATCAAGAATGGCAGATGTGAGCAGTTCT

GCAAGAACTCTGCTGACAACAAGGTGGTGTGCAGCTGCACTGAGGGCTACAGGC

TGGCTGAGAACCAGAAGAGCTGTGAGCCTGCTGTGCCATTCCCATGTGGCAGAG

TGTCTGTGAGCCAGACCAGCAAGCTGACCAGGGCTGAGGCTGTGTTCCCTGATGT

GGACTATGTGAACAGCACTGAGGCTGAAACCATCCTGGACAACATCACCCAGAG

CACCCAGAGCTTCAATGACTTCACCAGGGTGGTGGGGGGGGAGGATGCCAAGCC TGGCCAGTTCCCCTGGCAAGTGGTGCTGAATGGCAAGGTGGATGCCTTCTGTGGG

GGCAGCATTGTGAATGAGAAGTGGATTGTGACTGCTGCCCACTGTGTGGAGACT

GGGGTGAAGATCACTGTGGTGGCTGGGGAGCACAACATTGAGGAGACTGAGCAC

ACTGAGCAGAAGAGGAATGTGATCAGGATCATCCCCCACCACAACTACAATGCT

GCCATCAACAAGTACAACCATGACATTGCCCTGCTGGAGCTGGATGAGCCCCTG

GTGCTGAACAGCTATGTGACCCCCATCTGCATTGCTGACAAGGAGTACACCAAC

ATCTTCCTGAAGTTTGGCTCTGGCTATGTGTCTGGCTGGGGCAGGGTGTTCCACA

AGGGCAGGTCTGCCCTGGTGCTGCAGTACCTGAGGGTGCCCCTGGTGGACAGGG

CCACCTGCCTGAGGAGCACCAAGTTCACCATCTACAACAACATGTTCTGTGCTGG

CTTCCATGAGGGGGGCAGGGACAGCTGCCAGGGGGACTCTGGGGGCCCCCATGT

GACTGAGGTGGAGGGCACCAGCTTCCTGACTGGCATCATCAGCTGGGGGGAGGA

GTGTGCCATGAAGGGCAAGTATGGCATCTACACCAAAGTCTCCAGATATGTGAA

CTGGATCAAGGAGAAGACCAAGCTGACC

SEQ ID NO 5 mature FIX-FIAV AA sequence of 415

YNSGKFEEFV QGNFERECMEEKCSFEEAREVFENTERTTEFWKQY VDGDQCESNPC LNGGSCKDDINSYECWCPFGFEGKNCELDVTCNIKNGRCEQFCKNSADNKVVCSCT EGYRLAENQKSCEPAVPFPCGRVSVSQTSKLTRAEAVFPDVDYVNSTEAETILDNITQ STQSFNDFTRIV GGED AKPGQFPW Q V VLN GKVD AFCGGS IVNEKWIVTA AHC VETG VKITVVAGEHNIEETEHTEQKRNVIRIIPHHNYNAAINAYNHDIALLELDEPLVLNSYV TPICIADKEYTNIFLKFGSGYVSGWGRVFHKGRSALVLQYLRVPLVDRATCLRSTKFT IYNNMFCAGFHEGGRDSCQGDSGGPHVTEVEGTSFLTGIVSWGEECAMKGKYGIYT KVSRY VNWIKEKTKLT

SEQ ID NO 6 FIX-FIAV AA sequence of 461

MQRVNMIMAESPGLITICLLGYLLSAECTVFLDHENANKILNRPKRYNSGKFEEFVQ

GNLERECMEEKCSFEEAREVFENTERTTEFWKQYVDGDQCESNPCLNGGSCKDDIN

S YECWCPFGFEGKNCELD VTCNIKN GRCEQFCKNS ADNKV VCSCTEGYRLAEN QKS

CEPAVPFPCGRVSVSQTSKLTRAEAVFPDVDYVNSTEAETILDNITQSTQSFNDFTRIV

GGEDAKPGQFPWQVVLNGKVDAFCGGSIVNEKWIVTAAHCVETGVKITVVAGEHNI

EETEHTEQKRNVIRIIPHHNYNAAINAYNHDIALLELDEPLVLNSYVTPICIADKEYTNI

FLKFGSGYVSGWGRVFHKGRSALVLQYLRVPLVDRATCLRSTKFTIYNNMFCAGFH

EGGRDSCQGDSGGPHVTEVEGTSFLTGIVSWGEECAMKGKYGIYTKVSRYVNWIKE

KTKLT SEQ ID NO 7 FIX-FIAV NA sequence

ATGCAGAGGGTGAACATGATCATGGCTGAGAGCCCTGGCCTGATCACCATCTGC

CTGCTGGGCTACCTGCTGTCTGCTGAGTGCACTGTGTTCCTGGACCATGAGAATG

CCAACAAGATCCTGAACAGGCCCAAGAGATACAACTCTGGCAAGTTCGAGGAGT

TTGTGCAGGGCAACCTGGAGAGGGAGTGCATGGAGGAGAAGTGCAGCTTTGAGG

AGGCCAGGGAGGTGTTTGAGAACACTGAGAGGACCACTGAGTTCTGGAAGCAGT

ATGTGGATGGGGACCAGTGTGAGAGCAACCCCTGCCTGAATGGGGGCAGCTGCA

AGGATGACATCAACAGCTATGAGTGCTGGTGCCCCTTTGGCTTTGAGGGCAAGA

ACTGTGAGCTGGATGTGACCTGCAACATCAAGAATGGCAGATGTGAGCAGTTCT

GCAAGAACTCTGCTGACAACAAGGTGGTGTGCAGCTGCACTGAGGGCTACAGGC

TGGCTGAGAACCAGAAGAGCTGTGAGCCTGCTGTGCCATTCCCATGTGGCAGAG

TGTCTGTGAGCCAGACCAGCAAGCTGACCAGGGCTGAGGCTGTGTTCCCTGATGT

GGACTATGTGAACAGCACTGAGGCTGAAACCATCCTGGACAACATCACCCAGAG

CACCCAGAGCTTCAATGACTTCACCAGGATCGTGGGGGGGGAGGATGCCAAGCC

TGGCCAGTTCCCCTGGCAAGTGGTGCTGAATGGCAAGGTGGATGCCTTCTGTGGG

GGCAGCATTGTGAATGAGAAGTGGATTGTGACTGCTGCCCACTGTGTGGAGACT

GGGGTGAAGATCACTGTGGTGGCTGGGGAGCACAACATTGAGGAGACTGAGCAC

ACTGAGCAGAAGAGGAATGTGATCAGGATCATCCCCCACCACAACTACAATGCT

GCCATCAACGCCTACAACCATGACATTGCCCTGCTGGAGCTGGATGAGCCCCTGG

TGCTGAACAGCTATGTGACCCCCATCTGCATTGCTGACAAGGAGTACACCAACAT

CTTCCTGAAGTTTGGCTCTGGCTATGTGTCTGGCTGGGGCAGGGTGTTCCACAAG

GGCAGGTCTGCCCTGGTGCTGCAGTACCTGAGGGTGCCCCTGGTGGACAGGGCC

ACCTGCCTGAGGAGCACCAAGTTCACCATCTACAACAACATGTTCTGTGCTGGCT

TCCATGAGGGGGGCAGGGACAGCTGCCAGGGGGACTCTGGGGGCCCCCATGTGA

CTGAGGTGGAGGGCACCAGCTTCCTGACTGGCATCGTGAGCTGGGGGGAGGAGT

GTGCCATGAAGGGCAAGTATGGCATCTACACCAAAGTCTCCAGATATGTGAACT

GGATCAAGGAGAAGACCAAGCTGACC

SEQ ID NO.8 NA LP1 promoter

CCCTAAAATGGGCAAACATTGCAAGCAGCAAACAGCAAACACACAGCCCTCCCT

GCCTGCTGACCTTGGAGCTGGGGCAGAGGTCAGAGACCTCTCTGGGCCCATGCC

ACCTCCAACATCCACTCGACCCCTTGGAATTTCGGTGGAGAGGAGCAGAGGTTGT

CCTGGCGTGGTTTAGGTAGTGTGAGAGGGGAATGACTCCTTTCGGTAAGTGCAGT GGAAGCTGTACACTGCCCAGGCAAAGCGTCCGGGCAGCGTAGGCGGGCGACTCA

GATCCCAGCCAGTGGACTTAGCCCCTGTTTGCTCCTCCGATAACTGGGGTGACCT

TGGTTAATATTCACCAGCAGCCTCCCCCGTTGCCCCTCTGGATCCACTGCTTAAAT

ACGGACGAGGACAGGGCCCTGTCTCCTCAGCTTCAGGCACCACCACTGACCTGG

GACAGTGAATCCGGACTCTAAGGTAAATATAAAATTTTTAAGTGTATAATGTGTT

AAACTACTGATTCTAATTGTTTCTCTCTTTTAGATTCCAACCTTTGGAACTGAATT

CTAGACCACC

SEQ ID NO.9 NA HNF1/HNF3

AAGCAAATATTTGTGGTTATGGATTAACTCGAA

SEQ ID NO.10 NA HNF3/HNF3

CTGTTTGCCCACTCTATTTGCCC

SEQ ID NO.11 NA c/EBP/HNF4

GGCGCCCTTTGGACCTTTTGCAATCCTGG

SEQ ID NO.12 NA HS_CRM2/HNF3

AGCAAACAGCAAACAC

SEQ ID NO.13 N A CRM 8

GGACTTAGCCCCTGTTTGCTCCTCCGATAACTGGGGTGACCTTGGTTAATATTCA

CCAGCAGCCTC

SEQ ID NO.14 CRM8 long

GACTCAGATCCCAGCCAGTGGACTTAGCCCCTGTTTGCTCCTCCGATAACTGGGG

TGACCTTGGTTAATATTCACCAGCAGCCTCCCCCGTTGCCCCTCTG

SEQ ID NO.15 NA G6PC

GGGCATATAAAACAGGGGCAAGGCACAGACTCATAGCAGAGCAATCACCACCA

AGCCTGGAATAACTGCAGCCACC

SEQ ID NO.16 Q1 variant 2

AAGCAAATATTTGTGGTTATGGATTAACTCGAACTGTTTGCCCACTCTATTTGCCC GGCGCCCTTTGGACCTTTTGCAATCCTGGAGCAAACAGCAAACACTCACTTTGCC

CCTATTTACTCCTCCGATGACTCAGGTGACTTTGGTTAATATTCACCAGCAGCTA

GGGGCATATAAAACAGGGGCAAGGCACAGACTCATAGCAGAGCAATCACCACC

AAGCCTGGAATAACTGCAGCCACC

SEQ ID NO.17 Q1 variant 1

AAGCAAATATTTGTGGTTATGGATTAACTCGAACTGTTTGCCCACTCTATTTGCCC

GGCGCCCTTTGGACCTTTTGCAATCCTGGAGCAAACAGCAAACACGACTCAGATC

CCAGCCAGTGGACTTAGCCCCTGTTTGCTCCTCCGATAACTGGGGTGACCTTGGT

TAATATTCACCAGCAGCCTCCCCCGTTGCCCCTCTGGGGCATATAAAACAGGGGC

AAGGCACAGACTCATAGCAGAGCAATCACCACCAAGCCTGGAATAACTGCAGCC

ACC

SEQ ID NO.18 Q1

AAGCAAATATTTGTGGTTATGGATTAACTCGAACTGTTTGCCCACTCTATTTGCCC

GGCGCCCTTTGGACCTTTTGCAATCCTGGAGCAAACAGCAAACACGGACTTAGCC

CCTGTTTGCTCCTCCGATAACTGGGGTGACCTTGGTTAATATTCACCAGCAGCCT

CGGGCATATAAAACAGGGGCAAGGCACAGACTCATAGCAGAGCAATCACCACC

AAGCCTGGAATAACTGCAGCCACC

SEQ ID NO.19 LP1 FIX cassette FIAV

CCCCTAAAATGGGCAAACATTGCAAGCAGCAAACAGCAAACACACAGCCCTCCC

TGCCTGCTGACCTTGGAGCTGGGGCAGAGGTCAGAGACCTCTCTGGGCCCATGCC

ACCTCCAACATCCACTCGACCCCTTGGAATTTCGGTGGAGAGGAGCAGAGGTTGT

CCTGGCGTGGTTTAGGTAGTGTGAGAGGGGAATGACTCCTTTCGGTAAGTGCAGT

GG A AGCTGT AC ACT GCCC AGGC A A AGCGTCCGGGC AGCGT AGGCGGGCG ACTC A

GATCCCAGCCAGTGGACTTAGCCCCTGTTTGCTCCTCCGATAACTGGGGTGACCT

TGGTTAATATTCACCAGCAGCCTCCCCCGTTGCCCCTCTGGATCCACTGCTTAAAT

ACGGACGAGGACAGGGCCCTGTCTCCTCAGCTTCAGGCACCACCACTGACCTGG

GACAGTGAATCCGGACTCTAAGGTAAATATAAAATTTTTAAGTGTATAATGTGTT

AAACTACTGATTCTAATTGTTTCTCTCTTTTAGATTCCAACCTTTGGAACTGAACT

AGTATTCTAGACCACCGCTAGCATGCAGAGGGTGAACATGATCATGGCTGAGAG

CCCTGGCCTGATCACCATCTGCCTGCTGGGCTACCTGCTGTCTGCTGAGTGCACT

GTGTTCCTGGACCATGAGAATGCCAACAAGATCCTGAACAGGCCCAAGAGATAC AACTCTGGCAAGTTCGAGGAGTTTGTGCAGGGCAACCTGGAGAGGGAGTGCATG

GAGGAGAAGTGCAGCTTTGAGGAGGCCAGGGAGGTGTTTGAGAACACTGAGAG

GACCACTGAGTTCTGGAAGCAGTATGTGGATGGGGACCAGTGTGAGAGCAACCC

CTGCCTGAATGGGGGCAGCTGCAAGGATGACATCAACAGCTATGAGTGCTGGTG

CCCCTTTGGCTTTGAGGGCAAGAACTGTGAGCTGGATGTGACCTGCAACATCAAG

AATGGCAGATGTGAGCAGTTCTGCAAGAACTCTGCTGACAACAAGGTGGTGTGC

AGCTGCACTGAGGGCTACAGGCTGGCTGAGAACCAGAAGAGCTGTGAGCCTGCT

GTGCCATTCCCATGTGGCAGAGTGTCTGTGAGCCAGACCAGCAAGCTGACCAGG

GCTGAGGCTGTGTTCCCTGATGTGGACTATGTGAACAGCACTGAGGCTGAAACC

ATCCTGGACAACATCACCCAGAGCACCCAGAGCTTCAATGACTTCACCAGGATC

GTGGGGGGGGAGGATGCCAAGCCTGGCCAGTTCCCCTGGCAAGTGGTGCTGAAT

GGCAAGGTGGATGCCTTCTGTGGGGGCAGCATTGTGAATGAGAAGTGGATTGTG

ACTGCTGCCCACTGTGTGGAGACTGGGGTGAAGATCACTGTGGTGGCTGGGGAG

CACAACATTGAGGAGACTGAGCACACTGAGCAGAAGAGGAATGTGATCAGGATC

ATCCCCCACCACAACTACAATGCTGCCATCAACGCCTACAACCATGACATTGCCC

TGCTGGAGCTGGATGAGCCCCTGGTGCTGAACAGCTATGTGACCCCCATCTGCAT

TGCTGACAAGGAGTACACCAACATCTTCCTGAAGTTTGGCTCTGGCTATGTGTCT

GGCTGGGGCAGGGTGTTCCACAAGGGCAGGTCTGCCCTGGTGCTGCAGTACCTG

AGGGTGCCCCTGGTGGACAGGGCCACCTGCCTGAGGAGCACCAAGTTCACCATC

TACAACAACATGTTCTGTGCTGGCTTCCATGAGGGGGGCAGGGACAGCTGCCAG

GGGGACTCTGGGGGCCCCCATGTGACTGAGGTGGAGGGCACCAGCTTCCTGACT

GGCATCGTGAGCTGGGGGGAGGAGTGTGCCATGAAGGGCAAGTATGGCATCTAC

ACCAAAGTCTCCAGATATGTGAACTGGATCAAGGAGAAGACCAAGCTGACCTGA

GCGGCCGCCTCGAGCGATCGCTGCTTTATTTGTGAAATTTGTGATGCTATTGCTTT

ATTTGTAACCATTATAAGCTGCAATAAACAAGTTAACAACAACAATTGCATTCAT

TTTATGTTTCAGGTTCAGGGGGAGGTGTGGGAGGTTTTTTAAA

SEQ ID NO.20 Q1 variant 2 - FIAV - cassette (underlined sequences may either be absent or present)

AAGCAAATATTTGTGGTTATGGATTAACTCGAACTGTTTGCCCACTCTATTTGCCC

GGCGCCCTTTGGACCTTTTGCAATCCTGGAGCAAACAGCAAACACTCACTTTGCC

CCTATTTACTCCTCCGATGACTCAGGTGACTTTGGTTAATATTCACCAGCAGCTA

GGGGCATATAAAACAGGGGCAAGGCACAGACTCATAGCAGAGCAATCACCACC

AAGCCTGGAATAACTGCAGCCACCATGCAGAGGGTGAACATGATCATGGCTGAG AGCCCTGGCCTGATCACCATCTGCCTGCTGGGCTACCTGCTGTCTGCTGAGTGCA

CTGTGTTCCTGGACCATGAGAATGCCAACAAGATCCTGAACAGGCCCAAGAGAT

ACAACTCTGGCAAGTTCGAGGAGTTTGTGCAGGGCAACCTGGAGAGGGAGTGCA

TGGAGGAGAAGTGCAGCTTTGAGGAGGCCAGGGAGGTGTTTGAGAACACTGAGA

GGACCACTGAGTTCTGGAAGCAGTATGTGGATGGGGACCAGTGTGAGAGCAACC

CCTGCCTGAATGGGGGCAGCTGCAAGGATGACATCAACAGCTATGAGTGCTGGT

GCCCCTTTGGCTTTGAGGGCAAGAACTGTGAGCTGGATGTGACCTGCAACATCAA

GAATGGCAGATGTGAGCAGTTCTGCAAGAACTCTGCTGACAACAAGGTGGTGTG

CAGCTGCACTGAGGGCTACAGGCTGGCTGAGAACCAGAAGAGCTGTGAGCCTGC

TGTGCCATTCCCATGTGGCAGAGTGTCTGTGAGCCAGACCAGCAAGCTGACCAG

GGCTGAGGCTGTGTTCCCTGATGTGGACTATGTGAACAGCACTGAGGCTGAAAC

CATCCTGGACAACATCACCCAGAGCACCCAGAGCTTCAATGACTTCACCAGGAT

CGTGGGGGGGGAGGATGCCAAGCCTGGCCAGTTCCCCTGGCAAGTGGTGCTGAA

TGGCAAGGTGGATGCCTTCTGTGGGGGCAGCATTGTGAATGAGAAGTGGATTGT

GACTGCTGCCCACTGTGTGGAGACTGGGGTGAAGATCACTGTGGTGGCTGGGGA

GCACAACATTGAGGAGACTGAGCACACTGAGCAGAAGAGGAATGTGATCAGGA

TCATCCCCCACCACAACTACAATGCTGCCATCAACGCCTACAACCATGACATTGC

CCTGCTGGAGCTGGATGAGCCCCTGGTGCTGAACAGCTATGTGACCCCCATCTGC

ATTGCTGACAAGGAGTACACCAACATCTTCCTGAAGTTTGGCTCTGGCTATGTGT

CTGGCTGGGGCAGGGTGTTCCACAAGGGCAGGTCTGCCCTGGTGCTGCAGTACCT

GAGGGTGCCCCTGGTGGACAGGGCCACCTGCCTGAGGAGCACCAAGTTCACCAT

CTACAACAACATGTTCTGTGCTGGCTTCCATGAGGGGGGCAGGGACAGCTGCCA

GGGGGACTCTGGGGGCCCCCATGTGACTGAGGTGGAGGGCACCAGCTTCCTGAC

TGGCATCGTGAGCTGGGGGGAGGAGTGTGCCATGAAGGGCAAGTATGGCATCTA

CACCAAAGTCTCCAGATATGTGAACTGGATCAAGGAGAAGACCAAGCTGACCTG

AGCGGCCGCCTCGAGCGATCGCTGCTTTATTTGTGAAATTTGTGATGCTATTGCTT

TATTTGTAACCATTATAAGCTGCAATAAACAAGTTAACAACAACAATTGCATTCA

TTTTATGTTTCAGGTTCAGGGGGAGGTGTGGGAGGTTTTTTAAA

SEQ ID N0.21 - Q1 variant 1- FIAV - cassette (underlined sequences may either be absent or present)

AAGCAAATATTTGTGGTTATGGATTAACTCGAACTGTTTGCCCACTCTATTTGCCC

GGCGCCCTTTGGACCTTTTGCAATCCTGGAGCAAACAGCAAACACGACTCAGATC

CCAGCCAGTGGACTTAGCCCCTGTTTGCTCCTCCGATAACTGGGGTGACCTTGGT TAATATTCACCAGCAGCCTCCCCCGTTGCCCCTCTGGGGCATATAAAACAGGGGC

AAGGCACAGACTCATAGCAGAGCAATCACCACCAAGCCTGGAATAACTGCAGCC

ACCATGCAGAGGGTGAACATGATCATGGCTGAGAGCCCTGGCCTGATCACCATC

TGCCTGCTGGGCTACCTGCTGTCTGCTGAGTGCACTGTGTTCCTGGACCATGAGA

ATGCCAACAAGATCCTGAACAGGCCCAAGAGATACAACTCTGGCAAGTTCGAGG

AGTTTGTGCAGGGCAACCTGGAGAGGGAGTGCATGGAGGAGAAGTGCAGCTTTG

AGGAGGCCAGGGAGGTGTTTGAGAACACTGAGAGGACCACTGAGTTCTGGAAGC

AGTATGTGGATGGGGACCAGTGTGAGAGCAACCCCTGCCTGAATGGGGGCAGCT

GCAAGGATGACATCAACAGCTATGAGTGCTGGTGCCCCTTTGGCTTTGAGGGCA

AGAACTGTGAGCTGGATGTGACCTGCAACATCAAGAATGGCAGATGTGAGCAGT

TCTGCAAGAACTCTGCTGACAACAAGGTGGTGTGCAGCTGCACTGAGGGCTACA

GGCTGGCTGAGAACCAGAAGAGCTGTGAGCCTGCTGTGCCATTCCCATGTGGCA

GAGTGTCTGTGAGCCAGACCAGCAAGCTGACCAGGGCTGAGGCTGTGTTCCCTG

ATGTGGACTATGTGAACAGCACTGAGGCTGAAACCATCCTGGACAACATCACCC

AGAGCACCCAGAGCTTCAATGACTTCACCAGGATCGTGGGGGGGGAGGATGCCA

AGCCTGGCCAGTTCCCCTGGCAAGTGGTGCTGAATGGCAAGGTGGATGCCTTCTG

TGGGGGCAGCATTGTGAATGAGAAGTGGATTGTGACTGCTGCCCACTGTGTGGA

GACTGGGGTGAAGATCACTGTGGTGGCTGGGGAGCACAACATTGAGGAGACTGA

GCACACTGAGCAGAAGAGGAATGTGATCAGGATCATCCCCCACCACAACTACAA

TGCTGCCATCAACGCCTACAACCATGACATTGCCCTGCTGGAGCTGGATGAGCCC

CTGGTGCTGAACAGCTATGTGACCCCCATCTGCATTGCTGACAAGGAGTACACCA

ACATCTTCCTGAAGTTTGGCTCTGGCTATGTGTCTGGCTGGGGCAGGGTGTTCCA

CAAGGGCAGGTCTGCCCTGGTGCTGCAGTACCTGAGGGTGCCCCTGGTGGACAG

GGCCACCTGCCTGAGGAGCACCAAGTTCACCATCTACAACAACATGTTCTGTGCT

GGCTTCCATGAGGGGGGCAGGGACAGCTGCCAGGGGGACTCTGGGGGCCCCCAT

GTGACTGAGGTGGAGGGCACCAGCTTCCTGACTGGCATCGTGAGCTGGGGGGAG

GAGTGTGCCATGAAGGGCAAGTATGGCATCTACACCAAAGTCTCCAGATATGTG

AACTGGATCAAGGAGAAGACCAAGCTGACCTGAGCGGCCGCCTCGAGCGATCGC

TGCTTTATTTGTGAAATTTGTGATGCTATTGCTTTATTTGTAACCATTATAAGCTG

CAATAAACAAGTTAACAACAACAATTGCATTCATTTTATGTTTCAGGTTCAGGGG

GAGGTGTGGGAGGTTTTTTAAA

SEQ ID N0.22 Q1 cassette as used in NHP

AAGCAAATATTTGTGGTTATGGATTAACTCGAACTGTTTGCCCACTCTATTTGCCC GGCGCCCTTTGGACCTTTTGCAATCCTGGAGCAAACAGCAAACACGGACTTAGCC

CCTGTTTGCTCCTCCGATAACTGGGGTGACCTTGGTTAATATTCACCAGCAGCCT

CGGGCATATAAAACAGGGGCAAGGCACAGACTCATAGCAGAGCAATCACCACC

AAGCCTGGAATAACTGCAGCCACCATGCAGAGGGTGAACATGATCATGGCTGAG

AGCCCTGGCCTGATCACCATCTGCCTGCTGGGCTACCTGCTGTCTGCTGAGTGCA

CTGTGTTCCTGGACCATGAGAATGCCAACAAGATCCTGAACAGGCCCAAGAGAT

ACAACTCTGGCAAGTTCGAGGAGTTTGTGCAGGGCAACCTGGAGAGGGAGTGCA

TGGAGGAGAAGTGCAGCTTTGAGGAGGCCAGGGAGGTGTTTGAGAACACTGAGA

GGACCACTGAGTTCTGGAAGCAGTATGTGGATGGGGACCAGTGTGAGAGCAACC

CCTGCCTGAATGGGGGCAGCTGCAAGGATGACATCAACAGCTATGAGTGCTGGT

GCCCCTTTGGCTTTGAGGGCAAGAACTGTGAGCTGGATGTGACCTGCAACATCAA

GAATGGCAGATGTGAGCAGTTCTGCAAGAACTCTGCTGACAACAAGGTGGTGTG

CAGCTGCACTGAGGGCTACAGGCTGGCTGAGAACCAGAAGAGCTGTGAGCCTGC

TGTGCCATTCCCATGTGGCAGAGTGTCTGTGAGCCAGACCAGCAAGCTGACCAG

GGCTGAGGCTGTGTTCCCTGATGTGGACTATGTGAACAGCACTGAGGCTGAAAC

CATCCTGGACAACATCACCCAGAGCACCCAGAGCTTCAATGACTTCACCAGGAT

CGTGGGGGGGGAGGATGCCAAGCCTGGCCAGTTCCCCTGGCAAGTGGTGCTGAA

TGGCAAGGTGGATGCCTTCTGTGGGGGCAGCATTGTGAATGAGAAGTGGATTGT

GACTGCTGCCCACTGTGTGGAGACTGGGGTGAAGATCACTGTGGTGGCTGGGGA

GCACAACATTGAGGAGACTGAGCACACTGAGCAGAAGAGGAATGTGATCAGGA

TCATCCCCCACCACAACTACAATGCTGCCATCAACGCCTACAACCATGACATTGC

CCTGCTGGAGCTGGATGAGCCCCTGGTGCTGAACAGCTATGTGACCCCCATCTGC

ATTGCTGACAAGGAGTACACCAACATCTTCCTGAAGTTTGGCTCTGGCTATGTGT

CTGGCTGGGGCAGGGTGTTCCACAAGGGCAGGTCTGCCCTGGTGCTGCAGTACCT

GAGGGTGCCCCTGGTGGACAGGGCCACCTGCCTGAGGAGCACCAAGTTCACCAT

CTACAACAACATGTTCTGTGCTGGCTTCCATGAGGGGGGCAGGGACAGCTGCCA

GGGGGACTCTGGGGGCCCCCATGTGACTGAGGTGGAGGGCACCAGCTTCCTGAC

TGGCATCGTGAGCTGGGGGGAGGAGTGTGCCATGAAGGGCAAGTATGGCATCTA

CACCAAAGTCTCCAGATATGTGAACTGGATCAAGGAGAAGACCAAGCTGACCTG

ACTCGATGCTTTATTTGTGAAATTTGTGATGCTATTGCTTTATTTGTAACCATTAT

AAGCTGCAATAAACAAGTTAACAACAACAATTGCATTCATTTTATGTTTCAGGTT

CAGGGGGAGGTGTGGGAGGTTTTTTAAA

SEQ ID N0.23 AA VP1-VP3 AAV5 MSFVDHPPDWLEEVGEGLREFLGLEAGPPKPKPNQQHQDQARGLVLPGYNYLGPGN

GLDRGEPVNRADEVAREHDISYNEQLEAGDNPYLKYNHADAEFQEKLADDTSFGGN

LGKAVFQAKKRVLEPFGLVEEGAKTAPTGKRIDDHFPKRKKARTEEDSKPSTSSDAE

AGPSGSQQLQIPAQPASSLGADTMSAGGGGPLGDNNQGADGVGNASGDWHCDSTW

MGDRVVTKSTRTWVLPSYNNHQYREIKSGSVDGSNANAYFGYSTPWGYFDFNRFH

SHWSPRDWQRLINNYWGFRPRSLRVKIFNIQVKEVTVQDSTTTIANNLTSTVQVFTD

DDYQLPYVVGNGTEGCLPAFPPQVFTLPQYGYATLNRDNTENPTERSSFFCLEYFPS

KMLRTGNNFEFTYNFEEVPFHSSFAPSQNLFKLANPLVDQYLYRFVSTNNTGGVQFN

KNLAGRY ANTYKNWFPGPMGRTQGWNLGSGVNRAS V S AFATTNRMELEGAS Y Q V

PPQPNGMTNNLQGSNTYALENTMIFNSQPANPGTTATYLEGNMLITSESETQPVNRV

AYNVGGQMATNNQSSTTAPATGTYNLQEIVPGSVWMERDVYLQGPIWAKIPETGA

HFHPSPAMGGFGLKHPPPMMLIKNTPVPGNITSFSDVPVSSFITQYSTGQVTVEMEWE

LKKENSKRWNPEIQYTNNYNDPQFVDFAPDSTGEYRTTRPIGTRYLTRPL

SEQ ID NO. 24 AA VP1-VP3 AAV5 (inserted alanine (bold and underlined)

MASFVDHPPDWLEEVGEGLREFLGLEAGPPKPKPNQQHQDQARGLVLPGYNYLGPG

NGLDRGEPVNRADEVAREHDISYNEQLEAGDNPYLKYNHADAEFQEKLADDTSFGG

NLGKAVFQAKKRVLEPFGLVEEGAKTAPTGKRIDDHFPKRKKARTEEDSKPSTSSDA

EAGPSGSQQLQIPAQPASSLGADTMSAGGGGPLGDNNQGADGVGNASGDWHCDST

WMGDRVVTKSTRTWVLPSYNNHQYREIKSGSVDGSNANAYFGYSTPWGYFDFNRF

HSHWSPRDWQRLINNYWGFRPRSLRVKIFNIQVKEVTVQDSTTTIANNLTSTVQVFT

DDDY QLPYVVGNGTEGCLPAFPPQVFTLPQY GY ATLNRDNTENPTERSSFFCLEYFP

SKMLRTGNNFEFTYNFEEVPFHSSFAPSQNLFKLANPLVDQYLYRFVSTNNTGGVQF

NKNLAGRYANTYKNWFPGPMGRTQGWNLGSGVNRASVSAFATTNRMELEGASYQ

VPPQPNGMTNNLQGSNTYALENTMIFNSQPANPGTTATYLEGNMLITSESETQPVNR

VAYNVGGQMATNNQSSTTAPATGTYNLQEIVPGSVWMERDVYLQGPIWAKIPETG

AHFHPSPAMGGFGLKHPPPMMLIKNTPVPGNITSFSDVPVSSFITQYSTGQVTVEME

WELKKENSKRWNPEIQYTNNYNDPQFVDFAPDSTGEYRTTRPIGTRYLTRPL

SEQ ID NO. 25 AA VP1-VP3 AAV5 construct (hybrid VP1)

MAADGYLPDWLEDTLSEGIRQWWKLKPGPPPPKPAERHKDDSRGLVLPGYKYLGPF NGLDKGEPVNEADAAALEHDKAYDRQLDSGDNPYLKYNHADAEFQERLKEDTSFG GNLGRA VF Q AKKRVLEPLGLVEEP VKT APTGKRIDDHFPKRKKARTEEDS KPSTS SD AEAGPSGSQQLQIPAQPASSLGADTMSAGGGGPLGDNNQGADGVGNASGDWHCDS TWMGDRVVTKSTRTWVLPSYNNHQYREIKSGSVDGSNANAYFGYSTPWGYFDFNR

FHSHWSPRDWQRLINNYWGFRPRSLRVKIFNIQVKEVTVQDSTTTIANNLTSTVQVF

TDDD Y QLPYVV GN GTEGCLPAFPPQVFTLPQY GY ATLNRDNTENPTERSSFFCLEYF

PSKMLRTGNNFEFTYNFEEVPFHSSFAPSQNLFKLANPLVDQYLYRFVSTNNTGGVQ

FNKNLAGRYANTYKNWFPGPMGRTQGWNLGSGVNRASVSAFATTNRMELEGASY

QVPPQPNGMTNNLQGSNTYALENTMIFNSQPANPGTTATYLEGNMLITSESETQPVN

RVAYNVGGQMATNNQSSTTAPATGTYNLQEIVPGSVWMERDVYLQGPIWAKIPETG

AHFHPSPAMGGFGLKHPPPMMLIKNTPVPGNITSFSDVPVSSFITQYSTGQVTVEME

WELKKENSKRWNPEIQYTNNYNDPQFVDFAPDSTGEYRTTRPIGTRYLTRPL

SEQ ID NO. 26 FIX mature protein IDAV

YNSGKLEEFV QGNLERECMEEKCSFEEAREVFENTERTTEFWKQYVDGDQCESNPC LNGGSCKDDINSYECWCPFGFEGKNCELDVTCNIKNGRCEQFCKNSADNKVVCSCT EGYRLAENQKSCEPAVPFPCGRVSVSQTSKLTRAEAVFPDVDYVNSTEAETILDNITQ STQSFNDFTRIVGGDDAKPGQFPWQVVLNGKVDAFCGGSIVNEKWIVTAAHCVETG VKITV V AGEHNIEETEHTEQKRN VIRIIPHHN YN A AIN A YNHDIALLELDEPLVLNS Y V TPICIADKEYTNIFLKFGSGYVSGWGRVFHKGRSALVLQYLRVPLVDRATCLRSTKFT IYNNMFCAGFHEGGRDSCQGDSGGPHVTEVEGTSFLTGIVSWGEECAMKGKYGIYT KVSRY VNWIKEKTKLT

SEQ ID NO. 27 FIX propeptide protein IDAV

M OR VNMTM A ESPGT JTTGT ,T GYT J .SAEOTVFI DHENANKTT NR PKRYNSGKl FEE VO

GNLERECMEEKCSFEEAREVFENTERTTEFWKQYVDGDQCESNPCLNGGSCKDDIN

S YECWCPFGFEGKNCELD VTCNIKN GRCEQFCKNS ADNKV VCSCTEGYRLAEN QKS

CEPAVPFPCGRVSVSQTSKLTRAEAVFPDVDYVNSTEAETILDNITQSTQSFNDFTRIV

GGDD AKPGQFPW Q V VLN GKVD AFCGGS IVNEKWIVT A AHC VETG VKIT V V AGEHN

IEETEHTEQKRNVIRIIPHHNYNAAINAYNHDIALLELDEPLVLNSYVTPICIADKEYTN

IFLKFGSGYVSGWGRVFHKGRSALVLQYLRVPLVDRATCLRSTKFTIYNNMFCAGFH

EGGRDSCQGDSGGPHVTEVEGTSFLTGIVSWGEECAMKGKYGIYTKVSRYVNWIKE

KTKLT

SEQ ID NO. 28 full nucleic acid sequence FIX-ID AV

ATGCAGAGGGTGAACATGATCATGGCTGAGAGCCCTGGCCTGATCACCATCTGC

CTGCTGGGCTACCTGCTGTCTGCTGAGTGCACTGTGTTCCTGGACCATGAGAATG CCAACAAGATCCTGAACAGGCCCAAGAGATACAACTCTGGCAAGCTGGAGGAGT

TTGTGCAGGGCAACCTGGAGAGGGAGTGCATGGAGGAGAAGTGCAGCTTTGAGG

AGGCCAGGGAGGTGTTTGAGAACACTGAGAGGACCACTGAGTTCTGGAAGCAGT

ATGTGGATGGGGACCAGTGTGAGAGCAACCCCTGCCTGAATGGGGGCAGCTGCA

AGGATGACATCAACAGCTATGAGTGCTGGTGCCCCTTTGGCTTTGAGGGCAAGA

ACTGTGAGCTGGATGTGACCTGCAACATCAAGAATGGCAGATGTGAGCAGTTCT

GCAAGAACTCTGCTGACAACAAGGTGGTGTGCAGCTGCACTGAGGGCTACAGGC

TGGCTGAGAACCAGAAGAGCTGTGAGCCTGCTGTGCCATTCCCATGTGGCAGAG

TGTCTGTGAGCCAGACCAGCAAGCTGACCAGGGCTGAGGCTGTGTTCCCTGATGT

GGACTATGTGAACAGCACTGAGGCTGAAACCATCCTGGACAACATCACCCAGAG

CACCCAGAGCTTCAATGACTTCACCAGGATCGTGGGGGGGGACGATGCCAAGCC

TGGCCAGTTCCCCTGGCAAGTGGTGCTGAATGGCAAGGTGGATGCCTTCTGTGGG

GGCAGCATTGTGAATGAGAAGTGGATTGTGACTGCTGCCCACTGTGTGGAGACT

GGGGTGAAGATCACTGTGGTGGCTGGGGAGCACAACATTGAGGAGACTGAGCAC

ACTGAGCAGAAGAGGAATGTGATCAGGATCATCCCCCACCACAACTACAATGCT

GCCATCAACGCCTACAACCATGACATTGCCCTGCTGGAGCTGGATGAGCCCCTGG

TGCTGAACAGCTATGTGACCCCCATCTGCATTGCTGACAAGGAGTACACCAACAT

CTTCCTGAAGTTTGGCTCTGGCTATGTGTCTGGCTGGGGCAGGGTGTTCCACAAG

GGCAGGTCTGCCCTGGTGCTGCAGTACCTGAGGGTGCCCCTGGTGGACAGGGCC

ACCTGCCTGAGGAGCACCAAGTTCACCATCTACAACAACATGTTCTGTGCTGGCT

TCCATGAGGGGGGCAGGGACAGCTGCCAGGGGGACTCTGGGGGCCCCCATGTGA

CTGAGGTGGAGGGCACCAGCTTCCTGACTGGCATCGTGAGCTGGGGGGAGGAGT

GTGCCATGAAGGGCAAGTATGGCATCTACACCAAAGTCTCCAGATATGTGAACT

GGATCAAGGAGAAGACCAAGCTGACC

SEQ ID NO. 29 complete vector genome sequence

GATCCTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACC

AAAGGTCGCCCGACGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAGCGAGCGAGC

GCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCTGGAGGGGTGGAGT

CGTGACGTGAATTACGTCATAGGGTTAGGGAGGTCAGATCTAAGCAAATATTTGT

GGTTATGGATTAACTCGAACTGTTTGCCCACTCTATTTGCCCGGCGCCCTTTGGAC

CTTTTGCAATCCTGGAGCAAACAGCAAACACGGACTTAGCCCCTGTTTGCTCCTC

CGATAACTGGGGTGACCTTGGTTAATATTCACCAGCAGCCTCGGGCATATAAAAC AGGGGCAAGGCACAGACTCATAGCAGAGCAATCACCACCAAGCCTGGAATAACT

GCAGCCACCATGCAGAGGGTGAACATGATCATGGCTGAGAGCCCTGGCCTGATC

ACCATCTGCCTGCTGGGCTACCTGCTGTCTGCTGAGTGCACTGTGTTCCTGGACC

ATGAGAATGCCAACAAGATCCTGAACAGGCCCAAGAGATACAACTCTGGCAAGT

TCGAGGAGTTTGTGCAGGGCAACCTGGAGAGGGAGTGCATGGAGGAGAAGTGC

AGCTTTGAGGAGGCCAGGGAGGTGTTTGAGAACACTGAGAGGACCACTGAGTTC

TGGAAGCAGTATGTGGATGGGGACCAGTGTGAGAGCAACCCCTGCCTGAATGGG

GGCAGCTGCAAGGATGACATCAACAGCTATGAGTGCTGGTGCCCCTTTGGCTTTG

AGGGCAAGAACTGTGAGCTGGATGTGACCTGCAACATCAAGAATGGCAGATGTG

AGCAGTTCTGCAAGAACTCTGCTGACAACAAGGTGGTGTGCAGCTGCACTGAGG

GCTACAGGCTGGCTGAGAACCAGAAGAGCTGTGAGCCTGCTGTGCCATTCCCAT

GTGGCAGAGTGTCTGTGAGCCAGACCAGCAAGCTGACCAGGGCTGAGGCTGTGT

TCCCTGATGTGGACTATGTGAACAGCACTGAGGCTGAAACCATCCTGGACAACA

TCACCCAGAGCACCCAGAGCTTCAATGACTTCACCAGGATCGTGGGGGGGGAGG

ATGCCAAGCCTGGCCAGTTCCCCTGGCAAGTGGTGCTGAATGGCAAGGTGGATG

CCTTCTGTGGGGGCAGCATTGTGAATGAGAAGTGGATTGTGACTGCTGCCCACTG

TGTGGAGACTGGGGTGAAGATCACTGTGGTGGCTGGGGAGCACAACATTGAGGA

GACTGAGCACACTGAGCAGAAGAGGAATGTGATCAGGATCATCCCCCACCACAA

CTACAATGCTGCCATCAACGCCTACAACCATGACATTGCCCTGCTGGAGCTGGAT

GAGCCCCTGGTGCTGAACAGCTATGTGACCCCCATCTGCATTGCTGACAAGGAGT

ACACCAACATCTTCCTGAAGTTTGGCTCTGGCTATGTGTCTGGCTGGGGCAGGGT

GTTCCACAAGGGCAGGTCTGCCCTGGTGCTGCAGTACCTGAGGGTGCCCCTGGTG

GACAGGGCCACCTGCCTGAGGAGCACCAAGTTCACCATCTACAACAACATGTTC

TGTGCTGGCTTCCATGAGGGGGGCAGGGACAGCTGCCAGGGGGACTCTGGGGGC

CCCCATGTGACTGAGGTGGAGGGCACCAGCTTCCTGACTGGCATCGTGAGCTGG

GGGGAGGAGTGTGCCATGAAGGGCAAGTATGGCATCTACACCAAAGTCTCCAGA

TATGTGAACTGGATCAAGGAGAAGACCAAGCTGACCTGACTCGATGCTTTATTTG

TGAAATTTGTGATGCTATTGCTTTATTTGTAACCATTATAAGCTGCAATAAACAA

GTTAACAACAACAATTGCATTCATTTTATGTTTCAGGTTCAGGGGGAGGTGTGGG

AGGTTTTTTAAAAGATCTGTAGATAAGTAGCATGGCGGGTTAATCATTAACTACA

AGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCAC

TGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGGCTTTGCCCGGGCGGCCTC

AGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAA SEQ ID NO. 30 variant used in SEQ ID NO.16

TCACTTTGCCCCTATTTACTCCTCCGATGACTCAGGTGACTTTGGTTAATATTCAC

CAGCAGCTAG

SEQ ID NO. 31 CRM8 short variant a

GCCCCTGTTTGCTCCTCCGATAACTGGGGTGACCTTGGTTAATATTCACCA

SEQ ID NO. 32 variant b

TAACTTAGCCCCTGTTTGCTCCTCCGATCCCCATGGTGACCTTGGTTAATATTCAC

CAGCAGCCTC

SEQ ID NO. 33 Variant c

AGACTTAGCCCCTGTTTGCTCCTCCGATGGCTAAGGTGACCTTGGTTAATATTCA

CCAGCAGCTAG

SEQ ID NO. 34 Variant d

TAACTTAGCCCCTGTTTGCTCCTTAGATCCCCATGGTGACCTTGGTTAATATTCAC

CAGCAATCTC

SEQ ID NO. 35 Variant e

GG ACTT AGCCCCT ATTT ACTCCTCCG ATG ACTC AGGTG ACTTTGGTT A ATATTC AC CAGCAGCCTC

SEQ ID NO. 36 Variant e

GGACTTAGCCCCTGTTTGCTCCTCCGATAGACGGTGTGACCTTGGTTAATATTCA CC AT AG AGCTC

SEQ ID NO. 37 Variant f

TAACTTAGCCCCTATTTACTCCTTAGATCCCCATGGTGACTTTGGTTAATATTCAC

CAGCAATCTC

SEQ ID NO. 38 Variant g

GCGGAGGAGCAAACAGGGGCTAAGTCATAACTGGGGTGACCTTGGTTAATATTC

ACCAGCAGCCTC SEQ ID NO. 39 Variant h

GGACTTAGCCCCTGTTTGCTCCTCCGATAACTGGGGGCTGCTGGTGAATATTAAC

CAAGGTCACTC

SEQ ID NO. 40 Variant i

GGACTTAGCGGAGCAAACAGGGTCCGATAACTGGGGTGACCTTGGTTAATATTC

ACCAGCAGCCTC SEQ ID NO. 41 Variant j

GGACTTAGCCCCTGTTTGCTCCTCCGATAACTGGGGTGACCTGCTGGTGAATATT

AACCAAGCCTC

SEQ ID NO. 42 Variant k

GGACTTAGCCCCTGTTTGCTCCTCCGCCCCAGTTATTGACCTTGGTTAATATTCAC CAGCAGCCTC

SEQ ID NO. 43 Variant 1

GGACTTAGCCCCTGTTTGCTCCTCCGTGACCTTGGTTAATATTCACCAGCAG CCTC

Claims

2. An AAV vector according to claim 1, wherein said variant factor IX comprises further an amino acid substitution selected from the group consisting of L6F, or E185D.

3. An AAV vector in accordance with claim 1 or claim 2, wherein said polypeptide has at least 90% sequence identity with SEQ ID NO 1 or SEQ ID NO.2.

4. An AAV vector, in accordance with any one of claims 1 - 3, wherein said nucleic acid sequence has at least 70% sequence identity with SEQ ID NO.4.

5. An AAV vector according to any one of claims 1-4, wherein said nucleic acid is operably linked to a promoter sequence for expression of the variant factor IX.

6. An AAV vector according to claim 5, wherein said promoter is for expression of the variant factor IX in the human liver.

7. An AAV vector according to claim 6, wherein the promoter is selected from the group consisting of SEQ ID NOs. 8, 16, 17 or 18.

8. An AAV vector according to any one of claims 1-7, wherein the AAV vector is based on serotype 5.

9. An AAV vector according to claim 8, wherein the AAV serotype 5 comprises a capsid protein as listed in SEQ NO.23, 24 or 25.

10. An AAV vector according to any one of claims 1-9, wherein the nucleic acid is flanked by AAV ITR sequences.

11. An AAV vector according to any one of claims 1-10, comprised in a pharmaceutical acceptable formulation.

12. An AAV vector according to any one of claims 1-11, for use in a medical treatment of a primate.

13. An AAV vector according to any one of claims 1-12, for use in a medical treatment of a human subject.

14. An AAV vector for use in a medical treatment according to any one of claims 12 and 13, wherein the medical treatment is of a human subject having a coagulopathy.

15. An AAV vector for use in a medical treatment according to any one of claims 12-14, wherein the medical treatment is of a human subject having hemophilia A.

16. An AAV vector for use in a medical treatment according to any one of claims 12-15, wherein the medical treatment is of a human subject having antibodies against Factor FVIII.

17. An AAV vector for use in a medical treatment in accordance with any one of claims 12-16, wherein the medical treatment is of a human subject, wherein subsequently said human subject is eligible for treatment with Factor VII or Factor Vila or a variant thereof.

18. An AAV vector for use in a medical treatment according to any one of claims 12-17, wherein the medical treatment is of a human subject, wherein subsequently said human subject is eligible for treatment with an activated prothrombin complex concentrate or a variant thereof.

19. An AAV vector for use in a medical treatment in accordance with any one of claims 12-18, wherein the medical treatment is of a human subject, wherein subsequently said human subject is eligible for treatment with Factor VIII, wherein said treatment with Factor VIII comprises a lower dose as compared with the human subject not being subjected to the medical treatment with the AAV vector.

20. An AAV vector for use in a medical treatment according to any one of claims 12-19,, wherein the medical treatment comprises the administration of the at least 2 x 10 13 genomic copies of the viral vector per kg of body weight of the human subject.

21. An AAV vector for use in a medical treatment according to any one of claims 12-20, wherein the variant FIX is expressed in the human patient at a level of at least 1 Lig/ml of plasma.

22. An AAV vector for use in a medical treatment according to any one of claims 12-21 wherein said variant is expressed for at least 1 year.

23. An AAV vector for use in a medical treatment according to any one of claims 12-22, wherein the clotting activity of the plasma of the human patient is at least 10 % of the clotting activity of a healthy human individual.

24. An AAV vector for use in a medical treatment according to any one of claims 12-23, wherein the clotting activity of the plasma of the human patient is at least 10 % of the clotting activity of a healthy human individual, wherein the healthy human individual is defined to comprise 1 IU/ml of human FVIII.

25. An AAV vector for use in a medical treatment according to any one of claims 12-24, wherein the variant FIX does not induce antibodies against the variant FIX.

26. An AAV vector for use in a medical treatment according to any one of claims 12-25, wherein said viral vector is administered via the bloodstream.

27. An AAV vector for use in a medical treatment according to any one of claims 12-26, wherein said viral vector is delivered to the liver.

28. An AAV vector for use in a medical treatment according to any one of claims 12-27, wherein prior to the administration of said AAV vector, the human subject has been treated with a lipid formulation.

29. An AAV vector for use in a medical treatment according to any one of claims 12-28, wherein prior to the administration of said AAV vector, the human subject has been treated with a lipid formulation which is administered via the bloodstream.

30. An AAV vector for use in a medical treatment according to any one of claims 12-29, wherein said lipid formulation is Intralipid.

31. An AAV vector for use in a medical treatment according to any one of claims 12-30, wherein said medical treatment reduces the severity, frequency, and/or duration of joint bleeds.

32. An AAV vector for use in a medical treatment according to any one of claims 12-31, wherein said medical treatment reduces the severity, frequency, and/or duration of spontaneous bleeds.

33. An AAV vector for use in a medical treatment according to any one of claims 12-32, wherein said medical treatment reduces the severity, frequency, and/or duration of cerebral bleeds.

34. An AAV vector for use in a medical treatment according to any one of claims 12-33, wherein said medical treatment reduces the haemophilia from severe to moderate or mild hemophilia.

35. An AAV vector for use in a medical treatment according to any one of claims 12-34, wherein said medical treatment reduces the hemophilia from moderate to mild hemophilia.

36. A factor VII or Factor Vila or a variant thereof, for use in a medical treatment of a human subject, wherein said human subject has a coagulopathy or is at risk of bleeding, wherein said human subject has been treated with an AAV vector in accordance with any one of claims 1-11.

37. An activated prothrombin complex concentrate or a variant thereof, for use in a medical treatment of a human subject, wherein said human subject has a coagulopathy or is at risk of bleeding, wherein said human subject has been treated with an AAV vector in accordance with any one of claims 1-11.

38. A factor VIII or a variant thereof, for use in a medical treatment of a human subject, wherein said human subject has a coagulopathy or is at risk of bleeding, wherein said human subject has been treated with an AAV vector in accordance with any one of claims 1-11.

39. A factor VII or Factor Vila or a variant thereof, for use in a medical treatment of a human subject, wherein said human subject has a coagulopathy or is at risk of bleeding, wherein said human subject has been treated with an AAV vector in accordance with any one of claims 12-35.

40. An activated prothrombin complex concentrate or a variant thereof, for use in a medical treatment of a human subject, wherein said human subject has a coagulopathy or is at risk of bleeding, wherein said human subject has been treated with an AAV vector in accordance with any one of claims 12-35.

41. A factor VIII or a variant thereof, for use in a medical treatment of a human subject, wherein said human subject has a coagulopathy or is at risk of bleeding, wherein said human subject has been treated with an AAV vector in accordance with any one of claims 12-35.