JP2024511851A - Modified plasma coagulation factor VIII and its use - Google Patents

Abstract

Modified human Factor VIII polypeptides are described that have enhanced Factor VIII activity. In some embodiments, the modified human Factor VIII polypeptide is at positions A20, T21, F57, L69, I80, L178, R199, H212, I215, R269, I310, L318, S332, R378, I610 and/or I661. contains one or more amino acid substitutions. Such polypeptides and viral vectors encoding such polypeptides can be used for the treatment of FVIII deficiency diseases such as hemophilia A.

Description

FIELD OF THE INVENTION This application relates generally to medical treatments, and specifically to modified plasma coagulation factor VIII polypeptides and their use in the treatment of hemophilia A.

Hemophilia A is an X-linked recessive disorder caused by a deficiency of functional plasma coagulation factor VIII (hFVIII). In patients with hemophilia A, the blood does not clot properly, resulting in excessive bleeding when injured. The bleeding phenotype is generally related to residual factor activity: people with severe disease (factor activity <1% of normal) bleed frequently and suddenly; people with moderate disease (factor activity <1% of normal); people with mild disease (factor activity 5% to 40% of normal) bleed with minor trauma, but rarely spontaneously; and people with mild disease (factor activity 5% to 40% of normal) bleed after minor trauma; or bleed during trauma.

Current treatments for severe hemophilia A (<1% factor VIII activity) require regular intravenous infusions of recombinant factor VIII (rFVIII) or plasma-enriched factor VIII. Individuals with moderate and mild hemophilia A can be treated as needed without a regular prophylactic schedule. Injection procedures are expensive and introduce the risk of infectious disease. rFVIII therapy has proven costly due to production, purification and formulation costs. rFVIII therapy still requires intravenous access for delivery due to limited bioavailability from other delivery routes. The cost and limited availability of rFVIII has prevented widespread implementation of this treatment strategy.

Gene therapy offers an alternative to injection procedures. However, current gene therapy requires high viral vector doses, increasing treatment-related costs. However, difficulties in implementing gene therapy techniques include vector toxicity and insufficient expression levels of factor VIII.

Therefore, bioengineered FVIII for improved clotting activity, reflecting increased secretion, increased specific activity, or both, would significantly improve rFVIII production in cell culture manufacturing or transgenic animals. as well as increase the likelihood of success of gene therapy strategies for hemophilia A. Therefore, there is a need for improved vectors and constructs that can efficiently express sufficient amounts of hFVIII protein to increase FVIII production or reduce the required dose of viral vector to an acceptable level. It is.

One aspect of the present application relates to modified hFVIII polypeptides (mhFVIII) that contain one or more mutations compared to a wild-type hFVIII polypeptide or a reference polypeptide.

In some embodiments, mhFVIII has one or more amino acid substitutions at positions A20, T21, F57, L69, I80, L178, R199, H212, I215, R269, I310, L318, S332, R378, I610 and/or I661. including.

In some embodiments, mhFVIII comprises one or more amino acid substitutions selected from the group consisting of the amino acid substitutions set forth in Table 1.

In some embodiments, mhFVIII is selected from the group consisting of A20K, T21I, T21V, F57L, L69V, I80V, L178F, R199K, H212Q, I215V, R269K, I310V, L318F, S332P, R378S, I610M, and I661V. Contains one or more amino acid substitutions at a position. In some embodiments, mhFVIII contains a single amino acid substitution.

In some embodiments, mhFVIII comprises amino acid substitutions at each of amino acids A20K and T21I.

In some embodiments, mhFVIII includes amino acid substitutions A20K and T21V.

In some embodiments, mhFVIII includes amino acid substitutions T21I, L69V, and I80V.

In some embodiments, mhFVIII includes amino acid substitutions T21I, L69V, I80, and L178F.

In some embodiments, mhFVIII includes amino acid substitutions T21I, L69V, I80V, and I661V.

In some embodiments, mhFVIII includes amino acid substitutions T21I, L69V, I80, L178F, and I661V.

In some embodiments, mhFVIII includes amino acid substitutions R199K, H212Q, I215V, R269K, I310V, L318F, and S332P.

In some embodiments, mhFVIII includes amino acid substitutions T21I, L69V, I80V, L178F, H212Q, I215V, R269K, L318F, and I661V.

In some embodiments, mhFVIII includes amino acid substitutions A20K, T21V, L69V, I80V, L178F, H212Q, I215V, R269K, L318F, and I661V.

In some embodiments, mhFVIII comprises amino acid substitutions T21I, L69V, I80V, L178F, R199K, H212Q, I215V, R269K, I310V, L318F, S332P and I661V.

In some embodiments, mhFVIII comprises amino acid substitutions A20K, T21V, L69V, I80V, L178F, R199K, H212Q, I215V, R269K, I310V, L318F, S332P and I661V.

In some embodiments, mhFVIII consists of a single polypeptide comprising the A1, A2, A3, C1 and C2 domains of hFVIII.

In some embodiments, mhFVIII consists of a single polypeptide comprising (1) the A1, A2, A3, C1 and C2 domains of hFVIII; and (2) the truncated B domain of hFVIII.

In some embodiments, mhFVIII consists of a heavy chain polypeptide comprising the A1 and A2 domains of hFVIII and a light chain polypeptide comprising the A3, C1 and C2 domains of hFVIII. In some embodiments, the heavy chain polypeptide further comprises a truncated B domain of hFVIII and the light chain polypeptide comprises the A3, C1 and C2 domains of hFVIII.

In some embodiments, the mhFVIII comprises a human FVIII heavy chain and a FVIII light chain from a different species, eg, a canine FVIII light chain.

Another aspect of the present application relates to isolated polynucleotides encoding mhFVIII of the present application.

Another aspect of the present application relates to an expression cassette comprising a polynucleotide of the present application; and a regulatory sequence operably linked to the polynucleotide.

Another aspect of the present application relates to expression vectors comprising the polynucleotides of the present application. In some embodiments, the expression vector is a plasmid. In some embodiments, the expression vector is a viral vector. In some embodiments, the expression vector is an AAV vector.

Another aspect of the present application relates to host cells containing the expression vectors of the present application.

Another aspect of the present application relates to a pharmaceutical composition comprising the mhFVIII of the present application and a pharmaceutically acceptable carrier.

Another aspect of the present application relates to a pharmaceutical composition comprising an expression vector of the present application and a pharmaceutically acceptable carrier.

Another aspect of the present application relates to a method of treating a subject with Factor VIII deficiency. The method includes administering to a subject an effective amount of mhFVIII, an expression vector, or a host cell of the present application.

Another aspect of the present application is a recombinant AAV vector comprising a nucleotide encoding mhFVIII, wherein mhFVIII is A20K, T21I, T21V, F57L, L69V, I80V, L178F, R199K, H212Q, I215V, R269K, I310V, A recombinant AAV vector comprising one or more amino acid substitutions at a position selected from the group consisting of L318F, S332P, R378S, I610M and I661V, wherein the AAV vector is capable of expressing mhFVIII in a host cell. In some embodiments, mhFVIII comprises a truncated B domain of hFVIII.

Another aspect of the present application relates to methods of expressing mhFVIII. The method includes (a) a polynucleotide comprising a nucleotide sequence encoding a signal peptide and a nucleotide sequence encoding mhFVIII, wherein mhFVIII is A20K, T21I, T21V, F57L, L69V, I80V, L178F, R199K, H212Q, a polynucleotide comprising one or more amino acid substitutions at a position selected from the group consisting of I215V, R269K, I310V, L318F, S332P, R378S, I610M and I661V; and an expression comprising a regulatory sequence operably linked to the polynucleotide (b) growing the host cells under conditions suitable for expression and secretion of mhFVIII; (c) harvesting the culture medium from the host cells; and (d) harvesting the culture medium from the host cells. It includes the step of purifying mhFVIII from the culture medium.

FIG. 2 shows an expression plasmid (pANG-CAG-hBDDF8) encoding wild-type human FVIII (hBDDF8) with a deletion in the B domain. The hBDDF8 coding sequence (including heavy and light chains) was utilized to assay the functional activity of various modified hBDDF8 proteins (also referred to as "mutant hFVIII" or "hFVIII variants") of the present application. It is under the control of the CAG promoter. Figure 2 shows an alignment between hBDDF8 heavy chain (hBDDF8-HC) and a modified hBDDF8 heavy chain containing 17 substitution mutations at 16 amino acid positions (qwBDDF8-HC) for analysis of hFVIII mutant activity. be. Additional constructs containing their various single, double and multiple substitutions are further described in FIG. 3 and Example 1. Further analysis of the relative functional activities of such mutants is shown in Figures 4-9, as described further below. Exemplary substitution mutants in hBDDF8-HC for analysis of hFVIII functional activity are summarized. Figure 3 shows the relative functional activity of single amino acid substitution hFVIII variants (ie, modified hBDDF8 protein) in HuH7 cells compared to the functional activity of hFVIII (ie, unmodified hBDDF8 protein). Figure 2 shows the relative functional activity of a single amino acid substitution hFVIII variant (ie, modified hBDDF8 protein) in HEK 293T cells compared to that of hFVIII (ie, unmodified hBDDF8 protein). Relative of a specific single amino acid substitution mutation at amino acid 21 of the hFVIII heavy chain (i.e., hBDDF8 protein modified at amino acid position 21) in HEK 293T cells compared to the functional activity of hFVIII (i.e., unmodified hBDDF8 protein). Shows functional activity. Native hBDDF8 protein and single amino acid substitution mutant T21I as well as T21I in combination with various substitutions at amino acid 20 of the hFVIII heavy chain (i.e., modification at amino acid position 21) in HRK 293 cells compared to each other. Represents the relative functional activity of double amino acid substitutions. Figure 2 shows the functional activity of various hFVIII-HC mutants with single or multiple mutations (as indicated) in HuH7 cells compared to hBDDF8 cDNA in pANG-CAG-hBDDF8 at 24 or 48 hours post-transfection. . Comparison of functional activity of hFVIII (i.e., unmodified hBDDF8 protein) at 24, 48 and 72 hours post-transfection in HEK 293T cells, including those with single and multiple mutations (as shown). The functional activity of hFVIII-HC mutants is shown. Various hFVIII-HCs, including those with single or multiple mutations (as indicated), in CHO cells compared to the functional activity of hFVIII (i.e., unmodified hBDDF8 protein) at 24 or 48 hours post-transfection. Showing the functional activity of the mutants. Figure 2 is a graph showing the structural domains of selected hFVIII mutants (and wild type) and human/canine hybrid FVIII mutants. HEK 293T of hFVIII mutant without B domain (and wild type) and human/dog hybrid FVIII mutant in FIG. It is a graph showing functional activity in cells. FIG. 2 shows an expression plasmid (pANG-TTR-hBDDF8) similar to the expression plasmid in FIG. 1, except that the CAG promoter has been replaced by a TTR promoter. Figure 13 is a graph showing the functional activity of hBDDF8 and various mhFVIII constructs expressed in HuH7 cells from rAAV2 vectors 72 hours after transfection.

I. DEFINITIONS Various terms relating to the biomolecules of this application are used herein above and throughout the specification and claims.

The phrase "FVIII with enhanced activity (actFVIII or actF8)" means that the encoded protein has at least 10%, 20%, 30%, 40%, 50% of the activity when compared to unmodified wild type hFVIII. %, 60%, 70%, 80%, 90% or 100%. The nucleotide sequence for unmodified wild type hFVIII is shown in SEQ ID NO: 1, which contains a nucleotide sequence encoding a 19 amino acid signal peptide (MQIELSTCFLCLLRFCFS (SEQ ID NO: 2)). The amino acid sequence for unmodified wild type hFVIII, including the signal peptide, is shown in SEQ ID NO:3. The amino acid sequence for unmodified wild type hFVIII without signal peptide is shown in SEQ ID NO:4.

The term "hBDDF8 protein", "hBDDF8 polypeptide" or "hBDDF8" refers to a wild type hFVIII protein that has a deletion in the B domain. In some embodiments, the deletion encompasses most of the B domain and includes sequences that are responsive to multiple cleavages within the wild-type B domain. Exemplary hBDDF8 polypeptides have the amino acid sequence shown in SEQ ID NO: 5 (with signal peptide), or SEQ ID NO: 6 (without signal peptide), which includes the hFVIII heavy chain (SEQ ID NO: 7), a truncated Contains the B domain (SEQ ID NO: 8) and hFVIII light chain (SEQ ID NO: 9).

The phrase "one or more" after a list of elements or species is intended to encompass any permutation of the elements or species in the list. Thus, for example, the phrase "one or more substitution mutations selected from the group consisting of A, B, C, D, E and F" refers to substitutions containing A, B, C, D, E and/or F. Any combination of mutations may be included.

As used herein, ranges may represent from one particular integer value to another particular integer value. When such a range is expressed, each and every integer value within the range defines a separate embodiment of this application, and the complete scope of the embodiments included within the range is the same as the integer value within the initial range. It is to be understood that it further includes any and all subranges between any pair of numerical values.

With respect to nucleic acids of this application, the term "isolated nucleic acid" when applied to DNA refers to immediately adjacent (in the 5' and 3' directions) in the naturally occurring genome of the organism from which it originates. refers to a DNA molecule that has been separated from the sequence that contains it. For example, an "isolated nucleic acid" may include a DNA or cDNA molecule inserted into a vector, such as a plasmid or a viral vector, or integrated into prokaryotic or eukaryotic DNA. Nucleic acid codons can be optimized for enhanced expression in mammalian cells.

With respect to RNA molecules in the present application, the term "isolated nucleic acid" primarily refers to RNA molecules encoded by isolated DNA molecules as defined above. Alternatively, the term refers to an RNA molecule that is sufficiently separated from the RNA molecule with which it would be associated in its natural state (i.e., in a cell or tissue) such that it exists in "substantially pure" form. (The term "substantially pure" is defined below).

With respect to proteins, the term "isolated protein" or "isolated and purified protein" is used herein in connection with a protein produced by expression of an isolated nucleic acid molecule of the present application. be done. Alternatively, the term may refer to a protein that is sufficiently separated from other proteins with which it is naturally associated such that it exists in "substantially pure" form.

The term "hFVIII polypeptide" refers to full-length human FVIII protein, fragments of human FVIII protein, domains and combinations of domains of human FVIII protein that substantially maintain the biological function of hFVIII.

The term "mutant hFVIII polypeptide" or "modified hFVIII polypeptide" refers to a polypeptide that differs from a reference hFVIII polypeptide by one or more amino acids (eg, one or more amino acid substitutions). Reference hFVIII polypeptides include wild-type hFVIII proteins with or without a signal peptide, wild-type hFVIII proteins with modifications, such as wild-type hFVIII proteins with deletions in the B domain (e.g., hBDDF8) or hFVIII without the B domain. , a fragment of hFVIII, a domain or a combination of domains of hFVIII with or without further modification. In some embodiments, a "reference hFVIII polypeptide" of a "modified hFVIII polypeptide" refers to the hFVIII polypeptide before modification. In some embodiments, the term "mutant hFVIII polypeptide" or "modified hFVIII polypeptide" refers to a hybrid FVIII comprising a human FVIII heavy chain and a FVIII light chain from a different species, such as a light chain from canine FVIII. Refers to polypeptide.

The term "hFVIII variant" as used herein refers to a "mutant hFVIII protein" or "modified hFVIII protein" that substantially maintains the biological function of hFVIII.

A "conservative amino acid substitution" is a replacement of an amino acid residue by a functionally similar residue. Examples of conservative substitutions include replacing a nonpolar (hydrophobic) residue such as isoleucine, valine, leucine or methionine with another; between arginine and lysine, between glutamine and asparagine, or between threonine and Substitution of a charged or polar (hydrophilic) residue with another, such as between serine; Substitution of basic residues, such as lysine or arginine, with another; Displacement of acidic residues, such as aspartic acid or glutamic acid. substitution of an aromatic residue such as phenylalanine, tyrosine or tryptophan with another; or substitution of alanine or glycine. The variant FVIII proteins of the present application may contain one or more conservatively substituted amino acids relative to the reference protein and retain some or all of the activity of the reference protein, as described herein. .

The term "expression cassette" as used herein refers to a nucleic acid construct that contains sufficient nucleic acid elements for the expression of a polynucleotide of interest. Typically, an expression cassette includes a polynucleotide of interest operably linked to regulatory sequences such as a promoter and enhancer. In some embodiments, the expression cassette includes additional elements, such as introns, polyadenylation sites, woodchuck hepatitis virus post-transcriptional response elements (WPREs), secretory signal sequences, and/or that affect the expression level of coding sequences. Other elements known to have such effects may also be included.

The term "regulatory sequence" refers to the transcriptional control sequences of a gene, which may be found 3' or 5' to or within the coding region, or within an intron. Examples of regulatory sequences include, but are not limited to, promoters and enhancers.

The term "promoter" as used herein refers to a nucleotide sequence capable of controlling the expression of a coding sequence or functional RNA. Generally, the polynucleotide of interest is located 3' to the promoter sequence. In some embodiments, the promoter is derived in its entirety from the native gene. In some embodiments, the promoter is composed of different elements derived from different naturally occurring promoters. In some embodiments, the promoter comprises a synthetic nucleotide sequence. It will be understood by those skilled in the art that different promoters direct the expression of genes in different tissues or cell types, or at different stages of development, or in response to different environmental conditions or the presence or absence of drugs or transcriptional cofactors. will be done. Ubiquitous promoters, cell type-specific promoters, tissue-specific promoters, developmental stage-specific promoters, and conditional promoters, such as drug-responsive promoters (eg, tetracycline-responsive promoters), are well known to those skilled in the art. Examples of promoters include, but are not limited to, the phosphoglycerate kinase (PKG) promoter, CAG, NSE (neuron-specific enolase), synapsin or NeuN promoter, SV40 early promoter, mouse mammary tumor virus LTR. Promoter; adenovirus major late promoter (Ad MLP); herpes simplex virus (HSV) promoter, cytomegalovirus (CMV) promoter, e.g., CMV immediate early promoter region (CMVIE), SFFV promoter, Rous sarcoma virus (RSV) promoter, Examples include synthetic promoters and hybrid promoters. Promoters can be of human origin or of other species, including murine origin. In addition, sequences derived from non-viral genes such as the mouse metallothionein gene promoter are also used herein. In some embodiments, the promoter is a heterologous promoter. In some embodiments, the promoter sequence consists of proximal and more distal upstream elements and can include enhancer elements.

The term "heterologous promoter" as used herein refers to a promoter that is not found operably linked to a given coding sequence in nature.

The term "enhancer" refers to a nucleotide sequence that is capable of stimulating promoter activity and can be a native element of the promoter or a heterologous element inserted to enhance the level or tissue specificity of the promoter.

The terms "operably linked" or "operably linked" refer to the association of two or more nucleic acid fragments on a single nucleic acid fragment such that its function is affected by the other. Point. For example, a promoter is operably linked to a coding sequence if it can affect the expression of that coding sequence (eg, the coding sequence is under the transcriptional control of the promoter). Coding sequences can be operably linked to regulatory sequences in sense or antisense orientation.

As used herein, the terms "secretion signal sequence", "signal peptide", or variations thereof, refer to the levels of secretion of an operably linked polypeptide from a cell compared to the level of secretion seen with the native polypeptide. It is intended to refer to an amino acid sequence that functions (as defined above) to enhance secretion. As defined above, "enhanced" secretion means that the relative proportion of polypeptides synthesized by the cell that is secreted from the cell is increased, and the absolute amount of secreted protein is also increased. There's no need. In some embodiments, essentially all (ie, at least 95%, 97%, 98%, 99% or more) of the polypeptide is secreted. However, it is not necessary that essentially all, or even most, of the polypeptide be secreted, so long as the level of secretion is enhanced compared to the native polypeptide. Generally, secretory signal sequences are cleaved within the endoplasmic reticulum, and in some embodiments, secretory signal sequences are cleaved prior to secretion. However, the secretion signal sequence need not be cleaved as long as secretion of the polypeptide from the cell is enhanced and the polypeptide is functional. Thus, in some embodiments, the secretory signal sequence is partially or completely retained. The secretory signal sequence may be derived in whole or in part from the secretion signal of the secreted polypeptide (ie, from a precursor) and/or may be wholly or partially synthetic. The length of the secretory signal sequence is not critical, and generally known secretory signal sequences are about 10-15 to 50-60 amino acids long. Additionally, known secretion signals from secreted polypeptides can be altered or modified so long as the resulting secretion signal sequence functions to enhance secretion of the operably linked polypeptide (e.g., (by amino acid substitution, deletion, truncation or insertion). The secretion signal sequences of the invention may comprise, consist essentially of, or consist of naturally occurring secretion signal sequences or modifications thereof (as described above). A large number of secreted proteins and sequences that direct their secretion from cells are known in the art. Secretion signal sequences of the invention may further be wholly or partially synthetic or artificial. Synthetic or artificial secretory signal peptides are known in the art and are described, for example, in Barash et al., “Human secretory signal peptide description by hidden Markov model and generation,” which is incorporated herein in its entirety. f a strong artificial "Signal peptide for secreted protein expression", Biochem. Biophys. Res. Comm 294:835-42 (2002). The term "operably linked" means that the regulatory sequences necessary for expression of the coding sequence are placed in a DNA molecule in appropriate positions relative to the coding sequence so as to effect expression of the coding sequence. It means there is. This same definition may apply to sequences of coding sequences and transcriptional control elements (eg, promoters, enhancers, and termination elements) in expression vectors. This definition may also apply to the arrangement of the nucleic acid sequences of the first and second nucleic acid molecules from which a hybrid nucleic acid molecule is created.

The term "substantially pure" refers to a preparation containing at least 50-60% by weight of the compound of interest (eg, nucleic acid, oligonucleotide, protein, etc.). More preferably, the preparation contains at least 75% by weight, most preferably 90-99% by weight of the compound of interest. Purity is determined for the compound of interest by an appropriate method (eg, chromatographic methods, agarose or polyacrylamide gel electrophoresis, HPLC analysis, etc.).

The phrase "consisting essentially of" when referring to a particular nucleotide or amino acid sequence means a sequence having the properties of the given SEQ ID NO. For example, when used in reference to an amino acid sequence, the phrase includes molecular modifications that would not affect the sequence itself, as well as the basic and novel characteristics of the sequence.

The term "oligonucleotide" refers herein to the primers and probes of the present application and is defined as a nucleic acid molecule composed of two or more ribo- or deoxyribonucleotides, preferably more than three ribo- or deoxyribonucleotides. Ru. The exact size of an oligonucleotide depends on a variety of factors and the particular application for which the oligonucleotide is used. The term "probe" as used herein refers to a nucleic acid that is naturally occurring in a purified restriction enzyme digest or synthetically capable of annealing or specifically hybridizing to a sequence complementary to the probe. Refers to oligonucleotides, polynucleotides or nucleic acids, whether produced as RA or DNA. Probes can be either single-stranded or double-stranded. The exact length of the probe depends on many factors including temperature, origin of the probe and method of use. For example, for diagnostic applications, depending on the complexity of the target sequence, oligonucleotide probes typically contain 15 to 25 or more nucleotides, which may contain fewer nucleotides. You can leave it there.

The term "percent identity" is used herein with respect to comparisons between nucleic acid or amino acid sequences. Nucleic acid and amino acid sequences are often compared using computer programs, such as in the National Library of Medicine's BLAST alignment program.

Any "corresponding" nucleic acid or amino acid or sequence as used herein has the same structure and/or function as a site in a FVIII molecule of another species, but may not have the same number of nucleic acids or amino acids. FVIII or a mutant FVIII molecule, or a fragment thereof. A sequence "corresponding to" another FVIII sequence substantially corresponds to such sequence and hybridizes under stringent conditions to the human FVIII DNA sequence designated SEQ ID NO: 1. A sequence “corresponding to” another FVIII sequence also provides for the expression of FVIII or a claimed procoagulant hybrid FVIII or a fragment thereof, and apart from redundancy in the genetic code, a nucleic acid molecule comprising SEQ ID NO: 1 containing sequences that will hybridize to.

A "unique" amino acid residue or sequence herein refers to an amino acid sequence or residue in a FVIII molecule of one species that is different from the homologous residue or sequence in a FVIII molecule of another species.

"Specific activity" as used herein refers to an activity that corrects the coagulation defect of human factor VIII deficiency plasma. Specific activity is measured in units of clotting activity per milligram of total FVIII protein in a standard assay that compares the clotting time of human FVIII-deficient plasma with that of normal human plasma. One unit of FVIII activity is the activity present in one milliliter of normal human plasma. In this assay, the shorter the time for clot formation, the higher the activity of FVIII assayed. Hybrid human/pig FVIII has clotting activity in the human FVIII assay. This activity, as well as the activity of other hybrid FVIII molecules or hybrid equivalent FVIII molecules, or fragments thereof, may be lower than or equal to the activity of either plasma-derived or recombinant human FVIII; It can be higher than that.

"Subunits" of human or animal FVIII, as used herein, are the heavy and light chains of the protein. The heavy chain of FVIII contains three domains: A1, A2 and B. The light chain of FVIII also contains three domains: A3, CI and C2.

The terms "epitope", "antigenic site" and "antigenic determinant" are used interchangeably herein and are the portion of human, animal, hybrid or hybrid equivalent FVIII that is specifically recognized by an antibody. , or a fragment thereof. This may consist of any number of amino acid residues and may depend on the primary, secondary or tertiary structure of the protein. Hybrid FVIII, hybrid FVIII equivalents, or any fragments containing at least one epitope of the present disclosure can be used as reagents in the diagnostic assays described below. In some embodiments, the hybrid FVIII or hybrid equivalent FVIII, or fragment thereof, is not cross-reactive or has less cross-reactivity with all naturally occurring inhibitory FVIII antibodies than with human or porcine FVIII. .

The term "immunogenic site" as used herein refers to FVIII, hybrid, hybrid in humans or animals, as measured by routine protocols such as immunoassays, e.g., ELISA, or Bethesda assays, as described herein. Defined as a region of human or animal FVIII, a hybrid, or a hybrid equivalent FVIII, or a fragment thereof, that specifically elicits the production of antibodies against the equivalent, or a fragment thereof. This may consist of any number of amino acid residues and may depend on the primary, secondary or tertiary structure of the protein. In some embodiments, the hybrid or hybrid equivalent FVIII, or fragment thereof, is not or less immunogenic in animals or humans than human or porcine FVIII.

"FVIII deficiency" is defined herein as being caused by (1) defective production of FVIII; (2) insufficient or no production of FVIII; or (3) partial or total inhibition of FVIII. refers to a deficiency in coagulation activity. Hemophilia A is a type of FVIII deficiency resulting from a defect in the X-linked gene and the absence or deficiency of the FVIII protein it encodes.

II. Modified hFVIII polypeptide (mhFVIII)
One aspect of the present application relates to modified hFVIII polypeptides (mhFVIII) that contain one or more mutations compared to a wild-type hFVIII polypeptide or an unmodified reference polypeptide. In some embodiments, mhFVIII, when expressed in a host cell, increases hFVIII activity.

Human FVIII encodes a 2351 amino acid protein with a 19 amino acid signal peptide and a 2332 amino acid mature protein. It is arranged in a series of structural "domains": NH ₂ -SP-A1-al-A2-a2-B-a3-A3-Cl-C2-COOH. As used herein, a FVIII "domain" is defined, for example, by a contiguous sequence of amino acids characterized by internal amino acid sequence identity to structurally related domains and by a site of proteolytic cleavage by thrombin. Furthermore, it should be understood that the term "no domain" or "lacking a domain" means that at least 95% or 100% of the domain is deleted. Unless otherwise specified, FVIII domains are defined by the following amino acid residues arranged in hFVIII from amino-terminus to carboxy-terminus (as shown in SEQ ID NO: 3): SP, amino acid residue 1 ~19; Al domain, amino acid residues 20-354; a1 domain, amino acid residues 355-391; A2 domain, amino acid residues 392-728; a2 domain, amino acid residues 729-760; B domain, amino acid residues 761 ~l667; a3 domain, amino acid residues 1668-l708; A3 domain, amino acid residues 1709-2039; C1 domain, amino acid residues 2040-2192; and C2 domain, amino acid residues 2193-2351.

The A1-a ₁ -A2-a ₂ -B (aa 20-1667) or A1-a ₁ -A2-a ₂ (aa 1-740) sequence is commonly referred to as hFVIII heavy chain. The a ₃ -A3-C1-C2 sequence (aa 1668-2351) is commonly referred to as hFVIII light chain. FVIII is proteolytically activated by thrombin or factor Xa, which dissociates it from von Willebrand factor to form FVIIIa, which has procoagulant functions. The biological function of FVIIIa is to increase the catalytic efficiency of Factor IXa for activation of Factor X by several orders of magnitude. Thrombin-activated FVIIIa is a 160 kDa A1-a ₁ /A2-a ₂ /a ₃ -A3-C1-C2 heterotriple complex that forms a complex with factor IXa and factor X on the surface of platelets or monocytes. It is a quantity.

The cDNA sequence encoding wild-type human FVIII has the nucleotide sequence shown in SEQ ID NO:1. In SEQ ID NO: 1, the first 57 nucleotides of the FVIII open reading frame encode a signal peptide sequence (SEQ ID NO: 2) that is typically cleaved from the mature FVIII protein.

In some embodiments, the modified hFVIII polypeptides of the present application include one or more amino acid substitutions in the region corresponding to amino acid residues 20-171 of the wild-type hFVIII amino acid sequence set forth in SEQ ID NO:3. In some embodiments, the modified hFVIII polypeptides of the present application are located at positions A20, T21, F57, L69, I80, L178, R199, H212, I215, R269, I310, L318, S332, R378, I610, and I661. Contains one or more substitutions.

With respect to the variants or modifications described herein, the nomenclature of the position represented by the single letter code for the amino acid followed by a number refers to the amino acid residue and its position in wild-type hFVIII (SEQ ID NO: 3). For example, the nomenclature "A20" refers to the amino acid residue alanine (A) at position 20 of the wild-type hFVIII sequence (SEQ ID NO: 3). Similarly, the nomenclature for substitutions represented by the one-letter code for the first amino acid, followed by a numerical value, followed by the one-letter code for the second amino acid is as follows: refers to the substitution of the original amino acid residue at the numerically indicated position. For example, the nomenclature "A20K" refers to the substitution of the amino acid residue alanine (A) with the amino acid residue lysine (K) at position 20 of the wild-type hFVIII sequence (SEQ ID NO: 3). Amino acid position nomenclature and substitution nomenclature also apply to domains of hFVIII, hFVIII heavy and light chains, fragments of hFVIII, polypeptides that share consensus sequences with hFVIII, and/or from other hFVIII, such as hBDDF8. Also applies to arrays.

In some embodiments, the present application provides a method for applying a compound selected from the group consisting of: mhFVIII containing amino acid substitutions at the above amino acid residues is provided. mhFVIII may contain any permutation of mutations encompassing these 16 amino acid positions. An exemplary mhFVIII for use in this application is described in Figure 3, which identifies single and multiple mutations that fill in the boxes.

In some embodiments, the mhFVIII of the present application is from the group consisting of A20K, T21I, T21V, F57L, L69V, I80V, L178F, R199K, H212Q, I215V, R269K, I310V, L318F, S332P, R378S, I610M, and I661V. Contains one or more selected amino acid substitutions.

In some embodiments, the mhFVIII of the present application comprises an amino acid substitution at position T21. Preferred substitutions include T21I and T21V. In some embodiments, mhFVIII is at one or more positions selected from the group consisting of A20, F57, L69, I80, L178, R199, H212, I215, R269, I310, L318, S332, R378, I610, and I661. further comprising amino acid substitutions.

In some embodiments, mhFVIII of the present application comprises amino acid substitutions at positions A20 and T21. In some embodiments, the mhFVIII of the present application comprises amino acid substitutions A20K and T21I (2M1 variant), or amino acid substitutions A20K and T21V (2M2 variant).

In some embodiments, mhFVIII of the present application comprises amino acid substitutions at positions T21, L69 and I80. In some embodiments, the mhFVIII of the present application comprises amino acid substitutions T21I, L69V, and I80V (3M1 variant).

In some embodiments, mhFVIII of the present application comprises amino acid substitutions at positions T21, L69, I80, and L178. In some embodiments, the mhFVIII of the present application comprises the amino acid substitutions T21I, L69V, I80, and L178F (4M1 variant).

In some embodiments, mhFVIII of the present application comprises amino acid substitutions at positions T21, L69, I80 and I661. In some embodiments, mhFVIII of the present application comprises amino acid substitutions T21I, L69V, I80V and I661V (4M3 variant).

In some embodiments, mhFVIII of the present application comprises amino acid substitutions at positions T21, L69, I80, L178 and I661. In some embodiments, the mhFVIII of the present application comprises amino acid substitutions 21I, L69V, I80V, L178F and I661V (5M4 variant).

In some embodiments, mhFVIII of the present application comprises amino acid substitutions at positions R199, H212, I215, R269, I310, L318 and S332. In some embodiments, the mhFVIII of the present application comprises amino acid substitutions R199K, H212Q, I215V, R269K, I310V, L318F and S332P (7M2 variant).

In some embodiments, the mhFVIII of the present application comprises amino acid substitutions at positions T21, L69, I80, L178, H212, I215, R269, L318 and I661. In some embodiments, the mhFVIII of the present application comprises amino acid substitutions T21I, L69V, I80V, L178F, H212Q, I215V, R269K, L318F and I661V (9M1 variant).

In some embodiments, mhFVIII of the present application comprises amino acid substitutions at positions A20, T21, L69, I80, L178, H212, I215, R269, L318 and I661. In some embodiments, mhFVIII of the present application comprises amino acid substitutions A20K, T21V, L69V, I80V, L178F, H212Q, I215V, R269K, L318F and I661V (10M1 variant).

In some embodiments, mhFVIII of the present application comprises amino acid substitutions at positions T21, L69, I80, L178, R199, H212, I215, R269, I310, L318, S322 and I661. In some embodiments, the mhFVIII of the present application comprises amino acid substitutions T21I, L69V, I80V, L178F, R199K, H212Q, I215V, R269K, I310V, L318F, S322P and I661V (12M1 variant).

In some embodiments, mhFVIII of the present application comprises amino acid substitutions at positions A20, T21, L69, I80, L178, R199, H212, I215, R269, I310, L318, S332 and I661. In some embodiments, the mhFVIII of the present application comprises amino acid substitutions A20K, T21V, L69V, I80V, L178F, R199K, H212Q, I215V, R269K, I310V, L318F, S332P and I661V (13M1 variant).

In some embodiments, amino acid positions corresponding to the above amino acid substitutions may be substituted with other conservative substitutions. Table 1 provides a list of exemplary amino acid substitutions at amino acid positions A20, T21, L69, I80, L178, R199, H212, I215, R269, I310, L318, S332 and I661.

In some embodiments, the mhFVIII described above comprises a deletion of the B domain ("no B domain"). Examples of hFVIII polypeptides containing B domain deletions are described in U.S. Pat. is incorporated herein by reference.

In some embodiments, the mhFVIII of the present application comprises a single chain polypeptide. In some embodiments, the mhFVIII of the present application comprises a single chain hFVIII polypeptide with a truncated or deleted B domain. In some embodiments, the mhFVIII of the present application comprises a heterodimer of a heavy chain (HC) comprising an A1 domain and an A2 domain, and a light chain (LC) comprising an A3 domain, a C1 domain and a C2 domain. In some embodiments, the mhFVIII of the present application has a heavy chain (HC) comprising an A1 domain, an A2 domain, and a full-length or truncated B domain, and a light chain (LC) comprising an A3 domain, a C1 domain, and a C2 domain. ) contains a heterodimer of In some embodiments, the mhFVIII of the present application comprises a heterotrimer of a polypeptide comprising an A1 domain, a polypeptide comprising an A2 domain, and a polypeptide comprising an A3 domain, a C1 domain, and a C2 domain.

In some embodiments, the mhFVIII described above is derived from wild-type hBDDF8 containing a deletion of the B domain with a native hFVIII signal peptide set forth in SEQ ID NO:5. The secreted form of mhFVIII does not contain the signal peptide sequence shown in SEQ ID NO:2.

In some embodiments, the mhFVIII of the present application, when expressed in vitro or in vivo, is 5% to 100 times, 10% to 50 times, that of wild type hFVIII or the reference polypeptide from which it is derived; 50% to 25 times, 2 to 100 times, 2 to 80 times, 2 to 60 times, 2 to 40 times, 2 to 20 times, 2 to 10 times, 2 to 5 times, at least 2 times, at least 3 times, at least Provides a 4-fold, at least 5-fold, at least 10-fold, at least 20-fold, at least 30-fold, at least 40-fold, at least 50-fold, at least 70-fold, at least 80-fold or at least 100-fold increase in FVIII activity. In vitro FVIII activity can be determined by analyzing tissue culture media from cells expressing mhFVIII. In vivo FVIII activity can be determined by analyzing plasma collected from an individual receiving an injection of mhFVIII or an expression vector expressing mhFVIII.

In some embodiments, the mhFVIII of the present application described above has reduced antigenicity, increased stability, increased circulating half-life through binding to serum binding proteins, increased protein secretion, factor IXa and/or or increased affinity for factor X, decreased affinity for von Willebrand factor, increased glycosylation, altered inactivation cleavage, altered at least one calcium binding site, and/or increased shelf life. It may be further modified to additionally include, delete, or modify other FVIII sequences to confer other desired properties such as.

For example, in some embodiments, the mhFVIII of the present application is R503A, R503G, P504A, L505S, Y506L, Y506A, S507A, S507L, R508A, R508S, R509G, L510S, P511L, P511A, K512A, G513S, V514A, K5 15M , H516L, L517S, K518M, D519A, F520A, P521L, I522M, L523M, P524A, G525A, E526G, I527M, I528A, M2218I, F2219L, V2242A, K2246E, L2271F or any combination thereof. Patent No. 5,859 , 204, U.S. Patent No. 6,770,744, and U.S. Patent Application Publication No. 2003/0166536, adding amino acid substitutions that contribute to the immunogenicity and/or antigenicity of human FVIII. It may be modified to include

In other embodiments, the mhFVIII of the present application described above is in accordance with US patent application Ser. It may also be modified to include additional amino acid substitutions that provide increased secretion, as described.

In other embodiments, the mhFVIII of the present application described above may be modified to additionally contain amino acid substitutions that confer greater stability of activated FVIII by virtue of the fused A2 and A3 domains. good. In particular, a variant in which FVIII is modified by substituting cysteine residues at positions 683 and 1845 (i.e., Y683C, T1845C) to form a C683-Cl845 disulfide bond that covalently links the A2 and A3 domains. FVIII.

In other embodiments, the mhFVIII of the present application described above may be further modified to include additional amino acid substitutions that confer altered inactivating cleavage sites. For example, A355 or A581 are substituted and used to reduce the susceptibility of mutant FVIII to cleavage enzymes that normally inactivate wild-type FVIII.

In other embodiments, the mhFVIII of the present application described above may be further modified to include additional amino acid substitutions that confer enhanced affinity for Factor IXa.

In other embodiments, the mhFVIII of the present application described above may be further modified to include additional amino acid substitutions that confer increased circulating half-life. This can be accomplished through a variety of approaches including, but not limited to, by reducing interaction with heparan sulfate.

In other embodiments, the mhFVIII of the present application described above may be further modified to include additional amino acid substitutions that confer recognition sequences for glycosylation at asparagine residues. Such modifications may be useful for evading detection by existing inhibitory antibodies (low antigenic FVIII) and by reducing the likelihood of developing inhibitory antibodies (low immunogenic FVIII). In one exemplary embodiment, the modified FVIII is mutated to incorporate a consensus amino acid sequence for N-linked glycosylation, such as NX--S/T.

In other embodiments, the mhFVIII of the present application described above (i) deletes the von Willebrand factor binding site, (ii) adds an A759 mutation, and/or (iii) It may be further modified to include additional mutations that add an amino acid sequence spacer in between, where the amino acid spacer is of sufficient length such that upon activation, the procoagulant-active FVII protein becomes a heterodimer. It belongs to

In some embodiments, the mhFVIII of the present application is a hybrid FVIII comprising a human FVIII heavy chain and a FVIII light chain from a different species, such as a light chain from canine FVIII. In some embodiments, the human FVIII heavy chain further comprises one or more amino acid substitutions described in this application. In some embodiments, the hybrid FVIII comprises a truncated B domain. In some embodiments, a hybrid FVIII comprises (1) a wild-type human FVIII heavy chain sequence or a modified human FVIII heavy chain sequence, and (B) a FVIII light chain sequence from a different species, e.g., a light chain from canine FVIII. Consists of a single polypeptide containing. In some embodiments, the modified human FVIII heavy chain sequence comprises one or more of the amino acid substitutions described in this application. In some embodiments, the modified human FVIII heavy chain sequence comprises a hBDDF8 sequence or a modified hBDDF8 sequence having one or more of the amino acid substitutions described in this application.

III. Polynucleotides, expression cassettes and expression vectors encoding mhFVIII Another aspect of the present application is that the polynucleotides of the present application include all possible nucleic acids encoding the breadth of substitutions and/or other mutations described herein. An isolated polynucleotide encoding mhFVIII. Isolated nucleic acids can be RNA or DNA.

In certain embodiments, the polynucleotide encodes an mhFVIII polypeptide that is codon-optimized for expression in various human, primate, or mammalian cells, such as HuH7, HEK293T or CHO cells. Polynucleotides encoding mhFVIII of the present application may be codon-optimized to improve activity, stability, or expression in a host cell without altering the encoded amino acid sequence.

A codon consists of a set of three nucleotides and either codes for a specific amino acid or causes the termination of translation (ie, a stop codon). The genetic code is redundant in that multiple codons specify the same amino acids, ie, there are a total of 61 codons that code for 20 amino acids. Codon optimization replaces codons present in a polynucleotide sequence with preferred codons that encode the same amino acid, eg, preferred codons for mammalian expression. Therefore, the amino acid sequence is not changed during the process. Codon optimization can be performed using gene optimization software. The codon-optimized nucleotide sequence is translated and aligned with the original protein sequence to confirm that no changes have been made to the amino acid sequence. Methods of codon optimization are known in the art and are described, for example, in US Application Publication No. 2008/0194511 and US Patent No. 6,114,148.

In some embodiments, the mhFVIII protein is expressed in the form of a single-chain B domain-less mhFVIII. In some embodiments, the mhFVIII protein is expressed from one or more nucleic acids in the form of a double-stranded (DC) protein that includes a heavy chain (HC) and a light chain (LC) of hFVIII. In some embodiments, the mhFVIII protein is derived from one or more nucleic acids in the form of a heterotrimer of a polypeptide comprising an A1 domain, a polypeptide comprising an A2 domain, and a polypeptide comprising A3, C1, and C2 domains. expressed.

In some embodiments, the mhFVIII-encoding polynucleotides of the present application include a coding sequence for expressing the wild-type hFVIII amino-terminal signal peptide (SEQ ID NO: 2), which is removed from the mature protein. In some embodiments, the mhFVIII of the present application is derived from the hBDDF8 protein having the amino acid sequence of SEQ ID NO: 5 (with signal peptide) or SEQ ID NO: 6 (without signal peptide). Since signal peptide sequences can influence the level of expression, polynucleotides encoding mhFVIII can be engineered to express mhFVIII with any of a variety of heterologous N-terminal signal peptides known in the art. It's okay.

In some embodiments, polynucleotides encoding mhFVIII of the present application described above have reduced antigenicity, increased stability, and circulation through binding to serum binding proteins, as further described above. increased half-life, increased protein secretion, increased affinity for factor IXa and/or factor X, decreased affinity for von Willebrand factor, increased glycosylation, altered inactivation cleavage, one or modified to additionally include, delete, or modify other FVIII sequences that confer multiple calcium binding site changes and/or other desired properties such as increased shelf life. You can leave it there.

Further aspects of the present application relate to expression cassettes for expressing mhFVIII as described herein. In some embodiments, the expression cassette comprises a nucleotide sequence encoding mhFVIII of the present application and a regulatory sequence operably linked to the nucleotide sequence. In some embodiments, the regulatory sequence includes a promoter.

A further aspect of the present application relates to expression vectors capable of expressing mhFVIII of the present application in vitro and/or in vivo. In some embodiments, the expression vector is a non-viral vector, such as a plasmid. In some embodiments, the expression vector is a viral vector, such as an AAV vector or a lentiviral vector.

Expression vectors for expressing mhFVIII of the present application typically include one or more regulatory sequences operably linked to the polynucleotide sequence to be expressed. It will be appreciated by those skilled in the art that the design of the expression vector may depend on factors such as the choice of host cell to be transformed, the level of expression of the desired protein, and the like. The expression vectors of the present application can be introduced into host cells to thereby produce mhFVIII as described herein.

Suitable expression vectors for directing expression in mammalian cells generally include a promoter and other transcriptional and translational control sequences known in the art. In certain embodiments, mammalian expression vectors are capable of directing the expression of polynucleotides preferentially in particular cell types (e.g., tissue-specific regulatory elements are used to express polynucleotides). ). Tissue-specific regulatory elements are known in the art and can include, for example, liver cell-specific promoters and/or enhancers (eg, albumin promoter, a-1 antitrypsin promoter, apoE enhancer). Alternatively, constitutive promoters active in virtually any cell type (eg, HCMV) may be used.

In certain preferred embodiments, the expression vector is a viral vector. The viral vector typically has one or more viral genes removed and inserted into the viral genome insertion site for insertion of an exogenous transgene, including the mutant FVIII gene described herein. Contains a gene/promoter cassette. The necessary functions of the removed gene can be supplied by a cell line that has been engineered to express the gene product of the early gene in trans. Exemplary viral vectors include, but are not limited to, adeno-associated virus (AAV) vectors, retroviral vectors such as lentiviral vectors, adenoviral vectors, herpesvirus vectors, and alphavirus vectors. Other viral vectors include astrovirus, coronavirus, orthomyxovirus, papovavirus, paramyxovirus, parvovirus, picornavirus, poxvirus, and togavirus viral vectors. Viral vectors may contain any suitable nucleic acid construct, eg, a DNA or RNA construct, and may be single-stranded, double-stranded, or duplex.

Once the DNA construct of the present application has been prepared, it is ready to be integrated into a host cell. Accordingly, another aspect of the present application relates to methods of producing recombinant cells containing mhFVIII nucleic acids. Essentially, this involves introducing a DNA construct into cells via transformation, transduction, electroporation, calcium phosphate precipitation, liposomes, etc., and selecting for cells in which the DNA has been episomally introduced or integrated into the host genome. accompanied by something. In some embodiments, cells expressing mhFVIII are transplanted into a subject for the treatment of hemophilia.

In some embodiments, mhFVIII protein is expressed from a viral vector for administration to patients with hemophilia. In some embodiments, the viral vector is an AAV vector. In some embodiments, the viral vector is a lentiviral vector. In some embodiments, the viral vector is an adenovirus vector. In some embodiments, the viral vector is a retroviral vector. In some embodiments, the viral vector is a herpesvirus vector. In some embodiments, methods of administering one or more AAV vectors encoding mhFVIII are provided.

Recombinant AAV and lentiviral vectors have found wide utility for a variety of gene therapy applications. Their utility for such applications is largely due to the high efficiency of in vivo gene transfer achieved in the context of various organs. AAV and lentiviral particles can be used to advantage as vehicles for effective gene delivery. Such virions have several desirable properties for such applications, including a tropism for dividing and non-dividing cells. Initial clinical experiments using these vectors also demonstrated no sustained toxicity, and immune responses were minimal or undetectable. AAV is known to infect a wide variety of cell types in vitro and in vivo, either by receptor-mediated endocytosis or by transcytosis. These vector systems have been tested in humans to target retinal epithelium, liver, skeletal muscle, airways, brain, joints, and hematopoietic stem cells. Plasmid DNA or minicircle-based non-viral vectors may also be suitable gene transfer vectors for large genes such as those encoding FVIII.

In some embodiments, the mhFVIII coding sequence is provided as a component of a viral vector packaged into a capsid. In some embodiments, AAV vectors are used for in vivo delivery of mhFVIII of the present application. In this case, the AAV vector comprises at least one mhFVIII and associated expression control sequences for controlling the expression of the mhFVIII sequence. Exemplary AAV vectors for expressing mhFVIII sequences include promoter-enhancer regulatory regions for FVIII expression, as well as promoter replication and packaging of mhFVIII nucleic acids into AAV capsids and integration of mhFVIII nucleic acids into the genome of target cells. Cis-acting ITRs may be mentioned that function to enable integration. Preferably, the AAV vector has its rep and cap genes and its associated expression control sequences deleted and replaced by mhFVIII sequences. The mhFVIII sequence is typically inserted adjacent to (ie, flanking) one or two AAV TRs or TR elements appropriate for viral replication. Most preferably, only the essential parts of the vector are included, eg, only the ITR and LTR elements, respectively. In some embodiments, more than one AAV vector is used for in vivo delivery of mhFVIII of the present application. In this case, each AAV vector is constructed as described above and carries a portion of the mhFVIII coding sequence (e.g., one vector carries the coding sequence for the mhFVIII heavy chain and another vector carries the coding sequence for the mhFVIII light chain. ).

Suitable regulatory sequences for promoting tissue-specific expression of mutant hFVIII sequences in target cells are utilized for expression of mhFVIII in vitro or in vivo. Incorporation of tissue-specific regulatory elements in the expression constructs of the present application provides at least partial tissue tropism for expression of mhFVIII or a functional fragment thereof. For example, a nucleic acid sequence encoding mutant FVIII under the control of a cytomegalovirus (CMV) promoter or a CAG promoter can be used for skeletal muscle expression, or hAAT-ApoE, etc. can be used for liver-specific expression. It can be used for Hematopoietic-specific promoters in AAV and lentiviral vectors can also be utilized to drive expression of mhFVIII in vivo.

The viral capsid component of the packaged viral vector may be a parvovirus capsid. AAV Caps and chimeric capsids are preferred. An example of a suitable parvovirus capsid component is a capsid component from the Parvoviridae family, such as an autonomous parvovirus or a dependent virus. For example, the viral capsid may be an AAV capsid (e.g., an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV 9, AAV10, AAV11 or AAV12 capsid; one of ordinary skill in the art will know of other capsids that perform the same or similar functions. It may be known that other variants that have not been identified may exist) or may contain components derived from more than one AAV capsid. The complete complement of AAV Cap proteins includes VP1, VP2, and VP3. An ORF containing a nucleotide sequence encoding an AAV VP capsid protein may contain less than the full length complementary AAV Cap protein, or a full complement of AAV Cap protein may be provided.

One or more of the AAV Cap proteins may be a chimeric protein, which is a chimeric protein that is associated with two or more viruses, preferably two or more viruses, as described in Rabinowitz et al., US Pat. No. 6,491,907 AAV-derived amino acid sequence AAV Cap. For example, a chimeric viral capsid can include an AAV1 Cap protein or subunit and at least one AAV2 Cap or subunit. A chimeric capsid can include, for example, an AAV capsid with one or more B19 Cap subunits, eg, an AAV Cap protein or subunit can be replaced by a B19 Cap protein or subunit. For example, the Vp3 subunit of the AAV capsid can be replaced by the Vp2 subunit of B19.

Packaging cells can be cultured to produce the packaged viral vectors of the present application. A packaging cell may contain (1) viral vector functions, (2) packaging functions, and (3) helper functions. The viral vector function typically includes a portion of the parvovirus genome, such as the AAV genome, along with rep and cap and their associated expression control sequences, as described above, deleted and replaced by mutant FVIII sequences.

In certain embodiments, viral vector functions may be suitably provided as a dual vector template, as described in Samulski et al., US Patent Publication No. 2004/0029106. Duplex vectors are dimeric, self-complementary (sc) polynucleotides (typically DNA). For example, the DNA of a double vector can be selected to form a double-stranded hairpin structure due to intrastrand base pairing. Both strands of a double DNA vector can be packaged within a viral capsid. Dual vectors may provide functionality equivalent to double-stranded DNA viral vectors and reduce the need for target cells to synthesize complementary DNA to the single-stranded genome normally encapsidated by the virus.

The TRs (degradable and non-degradable) selected for use in viral vectors are preferably AAV sequences (from any AAV serotype). Degradable AAV ITRs need not have wild-type TR sequences (e.g., wild-type sequences are , insertions, deletions, truncations or missense mutations). The TR may be a synthetic sequence that functions as an AAV inverted terminal repeat sequence, such as the "double-D sequence" described in Samulski et al., US Pat. No. 5,478,745. Typically, but not necessarily, the TRs are from the same parvovirus, eg, both TR sequences are from AAV2.

The packaging function includes the capsid component. The capsid component is preferably derived from a parvovirus capsid, such as an AAV capsid or a chimeric AAV capsid function. An example of a suitable parvovirus capsid component is a capsid component from the Parvoviridae family, such as an autonomous parvovirus or a dependent virus. For example, the capsid component may be selected from AAV capsids, eg, AAV1-AAV12, and other novel capsids not yet identified or of non-human primate origin. The capsid component may include components derived from two or more AAV capsids.

In certain embodiments, one or more of the VP capsid proteins may comprise a chimeric protein comprising amino acid sequences from more than one virus, preferably more than one AAV. For example, a chimeric viral capsid can include a capsid region from an adeno-associated virus (AAV) and at least one capsid region from a B19 virus. A chimeric capsid includes, for example, an AAV capsid with one or more B19 capsid subunits, eg, an AAV capsid subunit can be replaced by a B19 capsid subunit. For example, the VP1, VP2 or VP3 subunit of the AAV capsid can be replaced by the VP1, VP2 or VP3 subunit of B19. As another example, the chimeric capsid has a type 2 VP1 subunit replaced by a VP1 subunit from an AAV type 1, 3, 4, 5, or 6 capsid, preferably a type 3, 4, or 5 capsid. , may contain AAV type 2 capsids. Alternatively, the chimeric parvovirus is an AAV2 parvovirus in which the type 2 VP2 subunit is replaced by a VP2 subunit from an AAV type 1, 3, 4, 5, or 6 capsid, preferably a type 3, 4, or 5 capsid. type capsid. Similarly, a chimeric parvovirus in which the VP3 subunit from AAV type 1, 3, 4, 5, or 6 (more preferably 3, 4, or 5) is replaced with the VP3 subunit of the AAV type 2 capsid. preferable. As a further alternative, chimeric parvoviruses are preferred in which two of the AAV type 2 subunits are replaced by subunits from AAV of a different serotype (eg, AAV types 3, 4, 5, or 6). In an exemplary chimeric parvovirus according to this embodiment, the VP1 and VP2, or VP1 and VP3, or VP2 and VP3 subunits of the AAV type 2 capsid are of different serotypes (e.g., AAV types 3, 4, 5, or 6). ) by the corresponding subunit of AAV. Similarly, in other preferred embodiments, the chimeric parvovirus is an AAV1, 3, 4 subunit in which one or two subunits are replaced with those from a different serotype of AAV, as described above for AAV type 2. , 5, or 6 type capsid (preferably type 2, 3, or 5 capsid).

The packaged viral vector generally contains a mutant FVIII sequence and an expression control flanking sufficient TR elements to effect packaging of the vector DNA and subsequent expression of the mutant FVIII sequence in transduced cells. Contains arrays. Viral vector functions may be supplied to cells as components of, for example, plasmids or amplicons. Viral vector functions may reside extrachromosomally within the cell line and/or may be integrated into the chromosomal DNA of the cell.

IV. mhFVIII Protein Production Methods and Cell Lines Another aspect of the present application relates to methods of producing mhFVIII of the present application. This involves transforming host cells with an expression vector and growing the host cells of the present application under conditions that express mhFVIII. The expressed mhFVIII is then isolated.

A further aspect of the present application relates to a host cell comprising an isolated nucleic acid molecule encoding mhFVIII of the present application. A host cell can contain the isolated nucleic acid molecule as a DNA molecule in the form of an episomal plasmid, or it can be stably integrated into the host cell genome. Additionally, the host cell can constitute an expression system for producing mhFVIII protein. Suitable host cells include, but are not limited to, animal cells (e.g., human HuH7 and HEK293 cells, Chinese hamster ovary cells (“CHO”), baby hamster kidney (“BHK”) cells), bacterial cells (e.g., E. coli ( E. coli), insect cells (e.g. Sf9 cells), fungal cells, yeast cells (e.g. Saccharomyces or Schizosaccharomyces), plant cells (e.g. Arabidopsis or tobacco cells), algae. It can be a cell, etc. Suitable mammalian cells for carrying out this application include, among others, COS (e.g. ATCC No. CRL 1650 or 1651), BHK (e.g. ATCC No. CRL 6281), CHO (ATCC No. CCL 61), HeLa ( Examples include ATCC No. CCL 2), 293 (ATCC No. 1573), CHOP, HuH7, HEK293 and NS-1 cells.

Another aspect of the present application relates to a method of producing mhFVIII from cultured cells. In some embodiments, the method provides a polynucleotide comprising (a) a nucleotide sequence encoding a signal peptide and a nucleotide sequence encoding mhFVIII, wherein mhFVIII is A20K, T21I, T21V, F57L, L69V, I80V, a polynucleotide comprising one or more amino acid substitutions at a position selected from the group consisting of L178F, R199K, H212Q, I215V, R269K, I310V, L318F, S332P, R378S, I610M and I661V; and operably linked to the polynucleotide; (b) growing the host cell carrying the expression vector under conditions suitable for expression and secretion of mhFVIII; and (c) introducing the expression vector into the host cell. harvesting the culture medium and/or host cells; and (d) purifying mhFVIII from the harvested culture medium and/or host cells.

In one embodiment, the host cells are grown in vitro in a growth medium. Suitable growth media may include, but are not limited to, growth media containing von Willebrand factor (referred to herein as "VWF"). In this embodiment, the host cell may contain a transgene encoding VWF, or VWF may be introduced into the growth medium as a supplement. VWF in the growth medium allows for higher expression levels of mhFVIII. Once the recombinant FVIII is secreted into the growth medium, it is then isolated using techniques well known to those skilled in the relevant recombinant DNA and protein arts, including those described herein. , can be isolated from the growth medium. In another embodiment, the method of producing mhFVIII of the present application further comprises the step of disrupting the host cell prior to isolation of mhFVIII. In this embodiment, mhFVIII is isolated from cell debris.

mhFVIII is preferably produced in substantially pure form. In certain embodiments, substantially pure recombinant FVIII is at least about 80% pure, more preferably at least 90% pure, most preferably at least 95% pure, 98% pure, 99% pure, or 99% pure. .9% pure. Substantially pure recombinant FVIII can be obtained by conventional techniques well known in the art. Typically, substantially pure mhFVIII is secreted into the growth medium of recombinant host cells. Alternatively, substantially pure mhFVIII is produced but not secreted into the growth medium. In such cases, to isolate substantially pure mhFVIII, host cells carrying the recombinant plasmid are expanded, lysed by sonication, heated, or chemically treated, and the homogenate is centrifuged. Separation is performed to remove cell debris. The supernatant is then subjected to sequential ammonium sulfate precipitation. Fractions containing substantially pure mhFVIII are subjected to gel filtration on an appropriately sized dextran or polyacrylamide column to separate mhFVIII. If desired, the protein fraction (containing substantially pure mhFVIII) may be further purified by high performance liquid chromatography ("HPLC").

V. Methods of Treatment Another aspect of the present application relates to methods of treating patients with FVIII deficiency.

In some embodiments, the method comprises administering an effective amount of mhFVIII of the present application to a patient in need thereof. In some embodiments, mhFVIII is administered intravenously in purified form.

In other embodiments, the method comprises administering to a patient in need thereof an expression vector comprising an effective amount of the mhFVIII coding sequence of the present application, wherein the expression vector is capable of expressing mhFVIII in the patient. can.

In other embodiments, the method comprises administering to a patient in need thereof an effective amount of cells comprising an mhFVIII coding sequence of the present application, wherein the cells express mhFVIII in the patient after transplantation. I can do it. In some embodiments, the cells are dermal fibroblasts. In some embodiments, the cells are autologous cells. In some embodiments, the method comprises introducing an mhFVIII coding sequence into a population of target cells, wherein the target cells are isolated from a subject in need of such treatment. and injecting into the subject an effective amount of cells expressing mhFVIII.

In some embodiments, the FVIII deficiency is hemophilia A. In this case, the expression of mhFVIII of the present application is associated with uncontrolled bleeding that would otherwise result from FVIII deficiency (e.g., intra-articular, intracranial, or gastrointestinal bleeding), including hemophilia that develops antibodies against human FVIII. coagulation in susceptible patients. Target cells for the vectors include cells capable of expressing polypeptides having FVIII activity, such as cells of the mammalian liver lineage, endothelial cells, and precursors, which are treated with suitable cells to produce proteins with FVIII activity. It is another cell with cellular machinery.

Administration of mhFVIII protein, or an expression vector encoding mhFVIII, or cells expressing mhFVIII to a patient with FVIII deficiency can functionally reconstitute the coagulation cascade. The mhFVIII protein, or the expression vector encoding mhFVIII, or the cell expressing mhFVIII, can be administered alone or in combination with other therapeutic agents in a pharmaceutically acceptable or biologically compatible composition. I can do it.

In some embodiments, the method comprises intravenously administering to the patient a pharmaceutical composition comprising mhFVIII protein following the same procedure used for human or animal FVIII infusion. Suitable effective amounts of mhFVIII can include, but are not limited to, about 10 to about 500 units/kg of patient body weight.

The treatment dosage of the expression vector encoding mhFVIII, or the mhFVIII protein, or the cells expressing mhFVIII, will vary depending on the severity of the FVIII deficiency. Generally, dosage levels are adjusted in frequency, duration, and unit according to the severity and duration of each patient's bleeding episodes. Therefore, an expression vector encoding mhFVIII, or an mhFVIII protein, or a cell expressing mhFVIII, is capable of delivering a therapeutically effective amount of protein to a patient to stop bleeding, as measured by standard coagulation assays. in a pharmaceutically acceptable carrier, delivery vehicle, or stabilizer in an amount sufficient to

FVIII is classically defined as a substance present in normal plasma that corrects the coagulation defect in plasma from individuals with hemophilia A. The in vitro clotting activity of purified and partially purified forms of FVIII is used to calculate the dose of mhFVIII for infusion in human patients, and the activity recovered from patient plasma and in vivo bleeding is a reliable indicator of defect correction. No discrepancies between standard assays of the novel FVIII molecules in vitro and their behavior in canine injection models or human patients have been reported.

Typically, the desired plasma FVIII activity level achieved in a patient by administration of FVIII is within the range of 30-200% of normal. In one embodiment, the administration of therapeutic mhFVIII is at a preferred dosage within the range of about 5-500 units/kg of body weight, and particularly within the range of 10-100 units/kg of body weight, and even more particularly 20-40 units. given intravenously at a preferred dosage of /kg body weight; the frequency of intervals is within the range of approximately 8-24 hours (in severe hemophilia); the duration of treatment (days) is 1-10 within days or until the bleeding episode resolves. Patients with inhibitors may require different amounts of mhFVIII than their previous forms of FVIII. For example, a patient may require less mhFVIII due to its higher specific activity and decreased antibody reactivity than wild-type VIII. As in treatment with human or plasma-derived FVIII, the amount of therapeutic mhFVIII infused is defined by a one-step FVIII coagulation assay and, in selected instances, in vivo recovery in the patient's plasma after infusion. Determined by measuring FVIII. For any particular subject, the specific dosing regimen should be adjusted over time according to the individual's needs and the professional judgment of the person administering the composition or those supervising its administration, and herein It should be understood that the concentration ranges set forth in are merely exemplary and are not intended to limit the scope or practice of mhFVIII in the claims.

Treatment can take the form of a single intravenous administration of mtFVIII, or periodic or continuous administration over an extended period of time, as appropriate. Alternatively, therapeutic mhFVIII can be administered subcutaneously or orally in one or several doses of liposomes at various time intervals.

Administration of the expression vector to a human subject or animal in need thereof can be by any means known in the art for administering viral vectors. Exemplary modes of administration include rectal, transmucosal, topical, transdermal, inhalation, parenteral (e.g., intravenous, subcutaneous, intradermal, intramuscular, and intraarticular) administration, and administration into tissues or organs. Direct injections include intrathecal, direct intramuscular, intraventricular, intravenous, intraperitoneal, intranasal, or intraocular injection. Injectables can be prepared in conventional forms, either as liquid solutions or suspensions, solid forms suitable for solution or suspension in liquid prior to injection, or as emulsions. Alternatively, the virus may be administered in a local rather than systemic manner, eg, in a depot or sustained release formulation.

In certain preferred embodiments, the expression vector is administered by intramuscular, more preferably intramuscular injection, or local administration. The vectors disclosed herein may be administered to a subject's lungs by any suitable means, but preferably by an aerosol of respirable particles composed of a parvovirus vector of the invention that the subject inhales. Administered by administering a suspension. Respirable particles can be liquid or solid. Aerosols of liquid particles containing parvovirus vectors (e.g. AAV) of the invention can be produced by any suitable means, such as a pressure-driven aerosol spray inhaler or an ultrasonic spray inhaler, as known to those skilled in the art. may be generated. See, eg, US Pat. No. 4,501,729.

The dosage of a viral vector expressing mhFVIII depends on the mode of administration, the disease or condition being treated, the condition of the individual subject, the particular viral vector, and the gene being delivered, and can be determined in a routine manner. I can do it. Exemplary doses to achieve a therapeutic effect include at least about 10 ⁵ , 10 ⁶ , 10 ⁷ , 10 ⁸ , 10 ⁹ , 10 ¹⁰ , 10 ¹¹ , 10 ¹² , 10 ¹³ , 10 ¹⁴ , 10 ¹⁵ transducing units. Preferably, the virus titer is between about 10 ⁸ and 10 ¹³ transducing units, even more preferably 10 ¹² transducing units. Polynucleotides encoding mtFVIII of the present application may be administered as a component of a DNA molecule with appropriate regulatory elements for expression in target cells. Polynucleotides encoding mtFVIII of the present application may be administered as a component of a viral plasmid or viral particle, eg, an AAV particle. Viral particles can be delivered either as viral particles alone by direct in vivo delivery to the portal blood vessels of a subject in need thereof, or by transduction of cells with the viral particles ex vivo followed by in vivo The transduced cells may be administered as an ex vivo treatment involving the introduction of the transduced cells back into the subject.

The polynucleotide encoding mtFVIII can be expressed as a single chain molecule containing both heavy and light chain portions, or as two or more polynucleotides in multiple independent viral or non-viral vectors for delivery to patient host cells. It can be used by dividing into molecules (eg, heavy chain and light chain).

In some embodiments, the expression vector is a viral vector. Viral vectors that may be used in this application include, but are not limited to, adeno-associated virus (AAV) vectors of multiple serotypes (e.g., AAV-1 to AAV-12, and pseudotyped vectors thereof); Hybrid AAV vectors, retroviral vectors include lentiviral vectors and pseudotyped lentiviral vectors (e.g., human immunodeficiency virus (HIV) and feline immunodeficiency virus (FIV)); adenoviral vectors, herpes simplex virus vectors, vaccinia. Including viral vectors, non-viral vectors, etc. In addition, any of the viral vectors may be modified to include tissue-specific promoters/enhancers and the like.

VI. Pharmaceutical Compositions Another aspect of the present application is a pharmaceutical composition comprising (1) an mhFVIII polypeptide of the present application, an expression vector encoding mhFVIII, or a cell expressing mhFVIII, and (2) a pharmaceutically acceptable carrier. relating to things.

Exemplary pharmaceutically acceptable carriers include sterile pyrogen-free water and sterile pyrogen-free phosphate buffered saline. Physiologically acceptable carriers include pharmaceutically acceptable carriers. A pharmaceutically acceptable carrier is one that is not biologically harmful, i.e., a material that can be administered to a subject without causing adverse biological effects that outweigh the material's beneficial biological effects. It is. In some embodiments, the pharmaceutical composition is formulated for injection.

For injections, the carrier is typically a liquid. As injection medium it is preferable to use water, which contains additives useful for injection solutions, such as stabilizers, salts or saline, and/or buffers. For other methods of administration, the carrier can be either solid or liquid. For inhalation administration, the carrier is respirable and is preferably in solid or liquid particulate form.

The present application is further illustrated by the following examples, which should not be construed as limiting the present application. The contents of all references, patents and published patent applications cited throughout this application are incorporated herein by reference.

Example 1 Construction, Expression and Characterization of Factor VIII Variants Template: Plasmid pANG-CAG-hBDDF8 was used as a template to introduce multiple hFVIII mutations into the coding region of the hFVIII heavy chain. As shown in Figure 1, pANG-CAG-hBDDF8 (SEQ ID NO: 15) containing human factor VIII (hBDDF8) cDNA is under the control of the CAG promoter. In addition, pANG-CAG-hBDDF8 has a deletion in the B domain resulting in a functionally defective B domain. Figure 2 identifies hFVIII mutations constructed in pANG-CAG-hBDDF8. Figure 3 shows both hFVIII mutations analyzed in this study, including both single amino acid substitutions and combinations thereof.

Single mutations: A20K, T21I, T21V, F57L, L69V, I80V, L178F, R199K, H212Q, I215V, R269K, I310V, L318F, S332P of human factor VIII using the GIBSON ASSEMBLY® method. Individual mutations corresponding to R378S, I610M and I661V were introduced into the pANG-CAG-hBDDF8 plasmid. The resulting plasmids include pANG-CAG-hBDDF8-A20K, pANG-CAG-hBDDF8, pANG-CAG-hBDDF8-T21I, pANG-CAG-hBDDF8-T21V, pANG-CAG-hBDDF8-F57L, pANG-CAG-hBDDF8. -L69V, pANG-CAG-hBDDF8-I80V, pANG-CAG-hBDDF8-L178F, pANG-CAG-hBDDF8-R199K, pANG-CAG-hBDDF8-H212Q, pANG-CAG-hBDDF8-I215V, pANG-CAG -hBDDF8-R269K , pANG-CAG-hBDDF8-I310V, pANG-CAG-hBDDF8-L318F, pANG-CAG-hBDDF8-S332P, pANG-CAG-hBDDF8-R378S, pANG-CAG-hBDDF8-I610M, pANG-CAG-hBD Includes DF8-I661V It will be done.

T21 variant: An AvrII restriction site was introduced into pANG-CAG-hBDDF8 by replacing T21 with P. The resulting plasmid pANG-CAG-hBDDF8-T21P was used as a template to create multiple point mutations at amino acid position 21. pANG-CAG-hBDDF8-T21P was digested with AvrII and used as a template for the mutant constructs described herein. Nineteen oligonucleotides with NNN corresponding to position T21 were recombined into pANG-CAG-hBDDF8 by HIFI assembly. Mutant plasmids include pANG-CAG-hBDDF8-T21V, pANG-CAG-hBDDF8-T21I...pANG-CAG-hBDDF8-T21G, and the like. The last letter indicates the amino acid substitution at that particular position. Similar strategies can be used to make other substitutions according to the invention.

A20 mutation with T21I: 19 oligonucleotides with NNN corresponding to T21I and alanine 20 were recombined by HIFI assembly into pANG-CAG-hBDDF8-T21P, which was digested by AvrII. The resulting mutant plasmids include pANG-CAG-hBDDF8-A20K/T21I (2M1), pANG-CAG-hBDDF8-A20E/T21I...pANG-CAG-hBDDF8-A20V/T21I, and the like. A similar strategy can be used to create other substitutions according to the invention, eg pANG-CAG-hBDDF8-A20K/T21V (2M2).

Combination of mutations: hFVIII mutants with multiple HC mutations (as shown in Figure 3) were constructed in pANG-CAG-hBDDF8. In one embodiment, DNA fragments encoding substitution mutations A20K, T21V, L69V, I80V, L178F, R199K, H212Q, I215V, R269K, I310V, L318F, S332P and I661V are chemically synthesized and the corresponding used to replace the region. The resulting plasmid pANG-CAG-BDDF8-13M1 expresses a mutant factor VIII protein (13M1) having the 13 mutations described above.

In another embodiment, DNA fragments encoding substitution mutations T21I, L69V, I80V, L178F, R199K, H212Q, I215V, R269K, I310V, L318F, S332P and I661V are chemically synthesized and the corresponding regions of pANG-CAG-hBDDF8 are used to replace . The resulting plasmid pANG-CAG-BDDF8-12M1 expresses a mutant factor VIII protein (12M1) having the above 12 mutations.

In another embodiment, DNA fragments encoding substitution mutations A20K, T21V, L69V, I80V, L178F, H212Q, I215V, R269K, L318F, and I661V are chemically synthesized to replace the corresponding regions of pANG-CAG-hBDDF8. used for. The resulting plasmid pANG-CAG-BDDF8-10M1 expresses a mutant factor VIII protein (10M1) having the above-mentioned 10 mutations.

In another embodiment, DNA fragments encoding substitution mutations T21I, L69V, I80V, L178F, H212Q, I215V, R269K, L318F, and I661V are chemically synthesized to replace the corresponding regions of pANG-CAG-hBDDF8. used. The resulting plasmid pANG-CAG-BDDF8-9M1 expresses a mutant factor VIII protein (9M1) having the above-mentioned nine mutations.

In another embodiment, DNA fragments encoding substitution mutations R199K, H212Q, I215V, R269K, I310V, L318F, and S332P were chemically synthesized and used to replace the corresponding regions of pANG-CAG-hBDDF8. The resulting plasmid pANG-CAG-BDDF8-7M2 expresses a mutant factor VIII protein (7M2) having the above seven mutations.

In another embodiment, DNA fragments encoding substitution mutations T21I, L69V, I80V, L178F, and I661V were chemically synthesized and used to replace the corresponding regions of pANG-CAG-hBDDF8. The resulting plasmid pANG-CAG-BDDF8-5M4 expresses a mutant factor VIII protein (5M4) having the above five mutations.

In another embodiment, DNA fragments encoding substitution mutations T21V, L69V, I80V and I661V were chemically synthesized and used to replace the corresponding regions of pANG-CAG-hBDDF8. The resulting plasmid pANG-CAG-BDDF8-4M3 expresses a mutant factor VIII protein (4M3) having the four mutations described above.

In another embodiment, DNA fragments encoding substitution mutations T21I, L69V, I80V and L178F were chemically synthesized and used to replace the corresponding regions of pANG-CAG-hBDDF8. The resulting plasmid pANG-CAG-BDDF8-4M1 expresses a mutant factor VIII protein (4M1) having the four mutations described above.

In another embodiment, DNA fragments encoding substitution mutations T21I, L69V and I80V were chemically synthesized and used to replace the corresponding regions of pANG-CAG-hBDDF8. The resulting plasmid pANG-CAG-BDDF8-3M1 expresses a mutant factor VIII protein (3M1) having the three mutations described above.

To test the functional activity of the mutant constructs, HEK 293T, HuH7 and CHO cells were cultured in DMEM with 10% fetal bovine serum, penicillin (100 U/ml) and streptomycin (100 μg/ml) in 5% CO The cells were cultured at 37°C in a humid environment supplied with ₂ . HEK 293T, HuH7 and CHO cells were transfected with wild type and mutant expression constructs in pANG-CAG-hDDF8. After transfection, cells were maintained in RPMI-1640 medium with 2% inactivated fetal bovine serum. Cell culture medium was collected at different times (24 hours, 48 hours, 72 hours) after transfection. The activity of secreted FVIII was analyzed using an activated partial thromboplastin time (APTT) assay. Normal human plasma was used as a standard.

Representative results of these assays are shown in Figures 4-10, 11B and 13. Briefly, Figures 4-6 show specific single amino acid substitution mutants in HuH7 cells (Figure 4) and HEK 293T cells (Figures 5, 6) compared to wild-type hFVIII at 48 hours post-transfection. shows increased functional activity. Figure 6 shows that T21I and T21V mutants significantly increased FVIII activity in HEK 293T cells 48 hours after transfection. Figure 7 shows that double mutants harboring A20K (A20K/T21I and A20K/T21V) exhibit further FVIII activity in HEK 293T cells relative to their T21 and T21V single substitution counterparts at 48 hours post-transfection. Indicates that the value has been increased. Figures 8-10 show that the combination of multiple mutations compared to wild type, single substitution and double substitution mutants in HuH7, HEK 293T, and CHO cells at 24 and 48 hours post-transfection, respectively. shows that FVIII functional activity can be greatly increased.

Example 2 Construction, expression and functional activity of hybrid human/canine factor VIII mutants Comparison of mutant hybrid human/canine FVIII consisting of mutant hVIII heavy chain (hHC) and canine FVIII light chain (cLC) To assess the functional activity of mutant hVIII, a series of B domain-less FVIII constructs were prepared and expressed in HEK 293T cells, as shown in Figures 11A and 11B.

Briefly, a mutant FVIII was constructed containing a mutant human FVIII heavy chain and a canine FVIII light chain. Briefly, pANG-CAG-hBDDF8 was digested with CspCI and XhoI. A DNA fragment encoding canine light chain (cLC) was chemically synthesized and used to replace the corresponding human light chain (hLC) region in pANG-CAG-hBDDF8 by Gibson assembly. The resulting plasmid pANG-CAG-hHC-cLC expresses a hybrid human/canine factor VIII polypeptide without the B domain, composed of hHC and cLC (ie, hHC-cLC). SEQ ID NO: 10 shows the amino acid sequence of hHC-cLC protein with native hFVIII signal peptide. SEQ ID NO: 11 shows the cDNA sequence encoding the protein in SEQ ID NO: 10. SEQ ID NO: 12 shows the amino acid sequence of hHC-cLC protein without hFVIII signal peptide. SEQ ID NO: 13 shows the amino acid sequence of the truncated B domain in hHC-cLC protein, and SEQ ID NO: 14 shows the amino acid sequence of canine FVIII light chain (cLC).

Using a similar strategy, pANG-CAG-qwHC-2M1-cLC (2M1 mutant) and pANG-CAG-qwHC-9M1-cLC (9M1 Mutant) plasmid was created. The activity of secreted FVIII was analyzed using an activated partial thromboplastin time (APTT) assay. FIG. 11B shows the functional activity of various human/dog hybrid FVIII variants in HEK 293T cells compared to that of hFVIII (ie, unmodified hBDDF8 protein) 48 hours after transfection. As shown in FIG. 11B, replacement of hLC with cLC in this system resulted in increased FVIII activity compared to hFVIII expressed from the parental plasmid hBDDF8.

In another aspect, the functional activity of rAAV vectors expressing mutant hVIII of the present application was compared to rAAV vectors expressing parental hBDDF8. In this case, a series of rAAVs expressing hBDDF8 protein and modified hBDDF8 proteins were constructed and produced. The obtained rAAV was used to infect HuH7 cells, and hVIII activity in the infected cells was analyzed.

In this case, a series of rAAVs were constructed expressing the first variant hVIII of the present application in which the CAG promoter in hBDDF8 was replaced with the human TTR promoter. Briefly, pANG-CAG-hBDDF8 was digested with SnaBI and MluI. A DNA fragment encoding the TTR promoter and intron was chemically synthesized and used to replace the CAG promoter region in pANG-CAG-hBDDF8 by Gibson assembly.

As shown in FIG. 12, the resulting plasmid pANG-TTR-hBDDF8 (SEQ ID NO: 18) expresses the hBDDF8 protein under the control of the TTR promoter. Using a similar strategy, pANG-TTR-qwBDDF8-2M1, pANG-TTR-qwBDDF8-2M2, pANG-TTR-qwBDDF8-9M1, pANG-TTR-qwBDDF8-10M1, pANG-TTR-qwBDDF8-12M1 , pANG- A TTR-qwBDDF8-13M1 plasmid was created.

pANG-TTR-hBDDF8 and its modified variant plasmids were packaged in AAV2 capsids to produce rAAV from them. Briefly, pAAV-Rep and Cap (serotype 2), pAd helper, and transgene plasmids were co-transfected into HEK 293T cells cultured in roller bottles at a 1:1:1 ratio. rAAV from transfected cell media was harvested 72 hours post-transfection and purified by two rounds of CsCl gradient ultracentrifugation. Each of the rAAVs was collected and extensively exchanged into PBS with 5% D-sorbitol.

HuH7 cells were grown in DMEM (Invitrogen, Carlsbad, CA) with 10% FBS, penicillin (100 U/ml), and streptomycin (100 μg/ml) at 37°C in a humidified environment supplied with 5% _CO2 . I grew it in For each transduction experiment, 50,000 viable cells were seeded in 24-well plates 24 hours prior to transduction. rAAV was added directly to each well at 100,000 vg/cell. The activity of secreted FVIII was analyzed 72 hours after transfection using an activated partial thromboplastin time (APTT) assay.

As shown in Figure 13, modified hBDDF8 showed increased FVIII activity when expressed by rAAV vector in HuH7 cells compared to unmodified hBDDF8.

The above examples demonstrate that the mutant Factor VIII products of the present application exhibit increased functional activity compared to wild-type Factor VIII. Therefore, use of the variants described herein can reduce the required production costs and levels of FVIII expression compared to existing constructs. They also allow lower vector doses to be administered by providing a more highly active FVIII product.

The above description is for the purpose of teaching those skilled in the art how to practice the present application. It is not intended to detail all obvious modifications and variations thereof that will become apparent to those skilled in the art upon reading the description. However, all such obvious modifications and variations are intended to be included within the scope of this application as defined by the following claims. The claims are intended to include the claimed elements and steps in any order effective to meet their intended purpose, unless the context specifically indicates otherwise. intend.

Claims (15)

An isolated compound containing one or more amino acid substitutions at a position selected from the group consisting of A20, T21, F57, L69, I80, L178, R199, H212, I215, R269, I310, L318, S332, R378, I610 and I661. modified human factor VIII (mhFVIII). 2. The isolated mhFVIII of claim 1, comprising one or more amino acid substitutions selected from the group consisting of the amino acid substitutions set forth in Table 1. A claim comprising one or more amino acid substitutions selected from the group consisting of A20K, T21I, T21V, F57L, L69V, I80V, L178F, R199K, H212Q, I215V, R269K, I310V, L318F, S332P, R378S, I610M and I661V. The isolated mhFVIII according to 1 or 2. (1) A20K and T21I, or (2) A20K and T21V, or (3) T21I, L69V and I80V, or (4) T21I, L69V, I80 and L178F, or (5) T21I, L69V, I80V, and I661V, or (6) T21I, L69V, I80, L178F and I661V, or (7) R199K, H212Q, I215V, R269K, I310V, L318F and S332P, or (8) T21I, L69V, I80V, L178F, H212Q, I215V, R269K, L318F and I661V, or (9) A20K, T21I, L69V, I80V, L178F, H212Q, I215V, R269K, L318F and I661V, or (10) T21I, L69V, I80V, L178F, R199K, H212Q, I215V, R269K, I 310V, L318F, S332P and I661V, or (11) A20K, T21V, L69V, I80V, L178F, R199K, H212Q, I215V, R269K, I310V, L318F, S332P and I661V
mhFVIII according to any one of claims 1 to 3, comprising an amino acid substitution of. mhFVIII according to any one of claims 1 to 4, consisting of a single polypeptide. mhFVIII according to any one of claims 1 to 5, comprising a truncated B domain. mhFVIII according to any one of claims 1 to 6, comprising (1) the A1 and A2 domains of wild-type hFVIII or mhFVIII, and (2) the A3, C1 and C2 domains of FVIII from a non-human species. mhFVIII according to claim 7, wherein the A3, C1 and C2 domains are derived from canine factor VIII. 9. An isolated polynucleotide encoding mhFVIII according to any one of claims 1 to 8, optionally containing a regulatory sequence operably linked to the polynucleotide. nucleotide. An expression vector comprising the polynucleotide according to claim 9. The expression vector according to claim 10, which is a viral vector. The expression vector according to claim 11, wherein the viral vector is an AAV vector. A host cell comprising an expression vector according to claim 10. (1) an isolated mhFVIII according to any one of claims 1 to 8, an expression vector according to any one of claims 10 to 12, or a host cell according to claim 13; and ( 2) A pharmaceutical composition comprising a pharmaceutically acceptable carrier. 15. A pharmaceutical composition according to claim 14 for use in medicine.