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Publication Number
US-11787851-B2
Publication Date
2023-10-17
Expiration Date
Abstract
The present invention provides codon optimized Factor VIII sequences, vectors and host cells comprising codon optimized Factor VIII sequences, polypeptides encoded by codon optimized Factor VIII sequences, and methods of producing such polypeptides.The present invention also provides methods of treating bleeding disorders such as hemophilia comprising administering to the subject a codon optimized Factor VIII nucleic acid sequence or the polypeptide encoded thereby.
Core Innovation
The invention relates to isolated nucleic acid molecules comprising the nucleotide sequence of SEQ ID NO:1, wherein the nucleotide sequence encodes a polypeptide with Factor VIII activity. The codon-optimized B-domain-deleted human Factor VIII coding sequences are described relative to a parental sequence of SEQ ID NO:3, with increased human codon adaptation index and sequence feature modifications.
The sequence modifications include changes in G/C content and reductions in MARS/ARS and destabilizing elements, along with removal or absence of splice site GGTGAT, poly-T, poly-A, and promoter/TATA-like inhibitory sites. Additional modifications include reduced AU-rich elements and related cis-acting negative regulatory elements.
Nucleic acids, vectors, host cells, and FVIII polypeptides corresponding to the optimized coding sequences are provided. The disclosure further includes optional heterologous half-life-extender moieties and structural formats such as monomer-dimer hybrid constructs for FVIII, in a gene therapy context for hemophilia A and other bleeding disorders.
Claims Coverage
The provided independent claim set is anchored by one independent claim covering an isolated nucleic acid molecule encoding a Factor VIII activity polypeptide. The independent claim is complemented by dependent claims that add inventive structural and compositional features and additional claim limits tied to production and use.
Isolated nucleic acid encoding SEQ ID NO:1 Factor VIII activity polypeptide
An isolated nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO:1, wherein the nucleotide sequence encodes a polypeptide with Factor VIII activity.
Half-life-extending heterologous nucleotide sequence linked into the FVIII encoding construct
A nucleic acid wherein the heterologous nucleotide sequence encodes a half-life-extending heterologous amino acid sequence, optionally as an immunoglobulin constant region or portion, transferrin, albumin, a PAS sequence, or an Fc region linked at the N-terminus or C-terminus or inserted between two amino acids.
Monomer-dimer hybrid Factor VIII molecule encoding format
The nucleic acid encodes a monomer-dimer hybrid molecule comprising Factor VIII.
Selected recombinant expression host cells
The host cell is selected from CHO cell, HEK293 cell, BHK21 cell, PER.C6 cell, NS0 cell, and CAP cell.
Increased expression or improved yield relative to a reference SEQ ID NO:3 nucleic acid
A method increases expression or improves yield of a Factor VIII activity polypeptide by culturing the host cell under conditions that yield a higher expression or yield than a reference nucleic acid sequence comprising SEQ ID NO:3, under the same conditions.
Treatment of hemophilia A bleeding disorder
The bleeding disorder is hemophilia A.
Across the provided claims, coverage centers on an isolated nucleic acid comprising SEQ ID NO:1 that encodes a Factor VIII activity polypeptide, with dependent claim coverage extending to half-life-extender heterologous sequences, monomer-dimer hybrid FVIII format, specified host cell selections, comparative expression or yield improvement relative to SEQ ID NO:3, and treatment of hemophilia A.
Stated Advantages
Increased plasma FVIII activity after administration.
Increased expression or improved yield of a Factor VIII activity polypeptide relative to a reference nucleic acid sequence comprising SEQ ID NO:3 under the same conditions.
Factor VIII activity expression or plasma BDD FVIII activity is increased compared with a reference nucleotide sequence (SEQ ID NO:3/wild-type reference).
Documented Applications
Producing recombinant FVIII using the isolated nucleic acid molecules and associated vectors or host cells, including methods that increase expression or improve yield versus a reference sequence.
Treating bleeding disorders, including hemophilia A, by administering a nucleic acid, vector, or polypeptide with increased plasma FVIII activity.
Gene therapy of hemophilia A using FVIII polypeptides encoded by codon-optimized nucleic acids and expressed in vivo using viral or non-viral vectors, including AAV, adenoviral vectors, and lentiviral vectors.
Producing and assessing plasma FVIII activity using codon-optimized BDD FVIII sequences expressed in HemA mice, with comparison to a reference nucleotide sequence comprising SEQ ID NO:3.
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