CpG reduced factor VIII variants, compositions and methods and uses for treatment of hemostasis disorders
Inventors
Anguela, Xavier • SHEN, Sam Hsien-I
Assignees
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Publication Number
US-11168124-B2
Publication Date
2021-11-09
Expiration Date
Abstract
CpG reduced nucleic acid variants encoding FVIII protein and methods of use thereof are disclosed. In particular embodiments, CpG reduced nucleic acid variants encoding FVIII are expressed more efficiently by cells, are secreted at increased levels by cells over wild-type Factor VIII proteins, exhibit enhanced expression and/or activity over wild-type Factor VIII proteins or are packaged more efficiently into viral vectors.
Core Innovation
The disclosure provides CpG-reduced nucleic acid variants encoding Factor VIII (FVIII) having a B domain deletion (FVIII-BDD). The disclosed nucleic acid variant encodes FVIII-BDD in a form having 95% or greater sequence identity to SEQ ID NO:7, and the variants are characterized by CpG reduction quantified by cytosine-guanine dinucleotide (CpG) count limits.
The disclosure also provides an adeno-associated virus (AAV) vector for delivering the FVIII-BDD nucleic acid variant. The AAV vector comprises an expression control element operably linked to the nucleic acid variant, where the expression control element comprises an element that confers expression in liver, and an AAV capsid serotype having the sequence of SEQ ID NO:27. In some embodiments, the expression control element includes a liver-expression control element, including a mutant transthyretin (TTRmut) promoter (SEQ ID NO:22), and additional defined expression cassette elements referenced in SEQ ID NO:23.
The patent frames the invention around improving expression, secretion, activity, and/or viral packaging efficiency relative to wild-type or Codon-optimized controls, using CpG-reduced nucleic acid variants. The disclosed approach is presented for treating a human in need of FVIII by intravenous administration so that FVIII is expressed in hepatocytes, reducing bleeding episodes and reducing the need to administer recombinant FVIII.
Claims Coverage
The provided material includes two independent claims: one directed to a nucleic acid variant encoding FVIII-BDD, and one directed to a method of treating a human with an intravenously administered liver-expressing FVIII-BDD AAV vector. Across these independent claims, the main inventive features center on defined FVIII-BDD sequence identity, liver-expression control elements, and a specified AAV capsid serotype.
FVIII-BDD nucleic acid variant with specified sequence identity
A nucleic acid variant encoding Factor VIII (FVIII) having a B domain deletion (FVIII-BDD), wherein said nucleic acid variant has 95% or greater sequence identity to the sequence of SEQ ID NO:7.
Intravenous AAV gene-transfer treatment for FVIII deficiency with liver expression
A method of treating a human in need of FVIII comprising providing a pharmaceutical composition comprising an adeno-associated virus (AAV) vector, wherein the AAV vector comprises a nucleic acid variant encoding Factor VIII (FVIII) having a B domain deletion (FVIII-BDD) with the sequence of SEQ ID NO:7, an expression control element operably linked to the nucleic acid variant comprising an element that confers expression in liver, and an AAV capsid serotype having the sequence of SEQ ID NO:27; and intravenously administering the pharmaceutical composition to the human so that FVIII is expressed in hepatocytes, reducing bleeding episodes and resulting in reduced need to administer recombinant FVIII.
Overall, the independent claim set ties a specific FVIII-BDD nucleic acid sequence identity (SEQ ID NO:7) to an AAV vector format that drives liver expression using a specified capsid serotype (SEQ ID NO:27), with the treatment outcome of reduced bleeding episodes and reduced recombinant FVIII needs.
Stated Advantages
Reduces bleeding episodes in the human.
Results in reduced need to administer recombinant FVIII to the human.
Improves expression, secretion, activity, and/or viral packaging efficiency over wild-type or Codon-optimized controls.
Markedly limited semen dissemination despite similar liver gene transfer exposure.
Documented Applications
Preclinical evaluation in rabbits comparing AAV-SPK and AAV-LK03 for germline/semen dissemination and systemic expression, including semen hFIX DNA detection and plasma human FIX antigen kinetics, and anti-human FIX antibody development.
Use of an intravenously administered AAV vector pharmaceutical composition for treating a human in need of FVIII, producing FVIII expression in hepatocytes and reducing bleeding episodes with reduced need for recombinant FVIII.
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