Modified plasma clotting factor VIII and method of use thereof
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Publication Number
US-11795207-B2
Publication Date
2023-10-24
Expiration Date
Abstract
Modified hum1.an factor VIII polypeptides with enhanced factor VIII activity are described. In some embodiments, the modified human factor VIII polypeptides comprise one or more amino acid substitutions at positions A20, T21, P57, L69, I80, L178, R199, H212, I215, R269, I310, L318, S332, R378, I610 and/or I661. Such polypeptides and viral vectors encoding such polypeptides may be used for treatment of FVIII deficiencies, such as hemophilia A.
Core Innovation
The invention relates to a modified human factor VIII (mhFVIII) defined by specific amino acid substitution patterns. In particular, the mhFVIII comprises the amino acid sequence of SEQ ID NO:4 or the amino acid sequence of SEQ ID NO:6, each comprising four or more amino acid substitutions, including T21I, L69V, I80V, and L178F, where the substitution positions refer to amino acid residue positions in SEQ ID NO:3.
The engineered mhFVIII maintains the core substitution set while permitting additional substitutions, including A20K, H212Q, I215V, R269K, L318F, S332P, R378S, I610M, and I661V. The patent text further describes B-domain-deleted formats, including B-domainless/hBDDF8, and related single-chain, double-chain, and heterotrimer formats, as well as possible human/canine hybrid FVIII designs.
The invention is also directed to systems and compositions that encode and deliver the mhFVIII. These include encoding polynucleotides, expression cassettes and expression vectors, including viral vectors and recombinant AAV vectors, host cells, pharmaceutical compositions, and therapeutic methods that administer mhFVIII or mhFVIII-expressing cells to subjects with factor VIII deficiency.
Claims Coverage
The partial content includes three independent claims. Across these claims, the core inventive concept centers on mhFVIII variants defined by a shared set of substitutions, T21I, L69V, I80V, and L178F, referenced to SEQ ID NO:3, implemented as the mhFVIII protein itself, as a recombinant AAV vector encoding it, or as a method for expressing and secreting it via an expression vector.
Modified mhFVIII with core substitution set referenced to SEQ ID NO:3
A modified human factor VIII (mhFVIII) comprising the amino acid sequence of SEQ ID NO:4 or the amino acid sequence of SEQ ID NO:6, each comprising four or more amino acid substitutions, wherein the four or more amino acid substitutions comprise T21I, L69V, I80V, and L178F, and the positions of the substitutions refer to amino acid residue positions in SEQ ID NO:3.
Recombinant AAV vector encoding mhFVIII with core substitution set
A recombinant AAV vector comprising a nucleotide sequence encoding mhFVIII, wherein the mhFVIII comprises SEQ ID NO:4 or SEQ ID NO:6 with four or more amino acid substitutions comprising T21I, L69V, I80V, and L178F, and the AAV vector is capable of expressing the mhFVIII in a host cell.
Expression and secretion of mhFVIII using an expression vector
A method for expressing mhFVIII comprising introducing into host cells an expression vector comprising a polynucleotide encoding a signal peptide and a nucleotide sequence encoding the mhFVIII, together with a regulatory sequence operatively linked to the polynucleotides, growing the host cells under conditions suitable for expression and secretion, harvesting culture medium, and purifying the mhFVIII from the harvested culture medium.
Overall, the claim coverage protects mhFVIII proteins defined by the SEQ ID NO:4/SEQ ID NO:6 substitution set including T21I, L69V, I80V, and L178F, recombinant AAV vectors capable of expressing that mhFVIII in host cells, and an expression workflow using a signal peptide and a regulatory sequence, followed by harvesting and purification of the secreted mhFVIII.
Stated Advantages
Increased FVIII activity, as described in the patent text for rAAV transduction examples.
Documented Applications
Treating a subject with factor VIII deficiency by administering a pharmaceutical composition containing the mhFVIII.
Treating a subject with factor VIII deficiency by administering an effective amount of a recombinant AAV vector encoding the mhFVIII.
Expressing and secreting mhFVIII in host cells and purifying the mhFVIII from harvested culture medium.
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