Adeno-associated virus factor VIII vectors
Inventors
Colosi, Peter Cameron • Nathwani, Amit • McIntosh, Jenny • Tuddenham, Edward • Davidoff, Andrew
Assignees
UCL Business Ltd • St Jude Childrens Research Hospital • Biomarin Pharmaceutical Inc
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Publication Number
US-12239692-B2
Publication Date
2025-03-04
Expiration Date
Abstract
The invention provides improved adeno-associated virus (AAV) Factor VIII (FVIII) vectors, including AAV FVIII vectors that produce a functional Factor VIII polypeptide and AAV FVIII vectors with high expression activity.
Core Innovation
The invention relates to an adeno-associated virus (AAV) vector comprising a 59 inverted terminal repeat (ITR), a liver-specific transcription regulatory region operably linked to a codon-optimized nucleic acid sequence encoding a functionally active Factor VIII (FVIII) protein, and a 39 ITR. The FVIII protein has the A3 and B domain deleted, and the nucleic acid sequence comprises nucleotides 646-4611 of SEQ ID NO: 5.
The disclosed vector uses multiple AAV genome sizes and architectures, including completely packaged smaller AAV genomes and variant genomes within the described size ranges, while encoding codon-optimized SQ FVIII variants. The disclosure includes AAV vector designs in which the B domain is replaced by 14 aa SQ, and optionally includes A3 deletion and/or B/A3 deletion or truncation of FVIII.
Production and evaluation are described for recombinant AAV vectors, including transfected producer cells and recovering virions from the supernatant. The document describes expression and activity testing, assessment of AAV virion packaging in producer cell systems, and FVIII expression/activity testing in Rag2 mice.
Claims Coverage
The independent claim covers an AAV vector defined by inventive features in its genome elements and encoded FVIII protein design. Dependent claims add further sequence constraints and cover production and composition aspects.
AAV vector with 59 and 39 ITRs
An adeno-associated virus (AAV) vector comprising a 59 inverted terminal repeat (ITR) and a 39 ITR.
Liver-specific transcription regulatory region linked to codon-optimized FVIII
A liver-specific transcription regulatory region operably linked to a codon-optimized nucleic acid sequence encoding a functionally active Factor VIII (FVIII) protein.
FVIII with A3 and B domain deleted encoded by a specific sequence window
The codon-optimized nucleic acid sequence comprises nucleotides 646-4611 of SEQ ID NO: 5, wherein the functionally active FVIII protein has the A3 and B domain deleted.
Recombinant AAV production by culturing transfected cells and recovering supernatant virions
Producing a recombinant adeno-associated virus (AAV) particle by culturing a cell transfected with the AAV vector and recovering recombinant AAV particle from the supernatant of the transfected cell.
Viral particle comprising the claimed AAV vector
A viral particle comprising the AAV vector.
Isolated host cell comprising the claimed AAV vector
An isolated host cell comprising the AAV vector.
Sequence constrained to specified SEQ ID NO alternatives
The AAV vector wherein the nucleic acid sequence comprises nucleotide sequence of SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, or SEQ ID NO: 8.
Overall, the claim coverage is grounded on a specifically defined AAV genome architecture and a codon-optimized, functionally active FVIII design with A3 and B domains deleted, tied to a defined nucleotide range of SEQ ID NO: 5. Dependent claims narrow the FVIII nucleic acid options to specified SEQ ID NOs and additionally cover recombinant AAV production and related compositions.
Stated Advantages
Maintained or sometimes increased FVIII expression/activity.
Improved vector yield.
Reduced fragmentary genome contaminants.
Documented Applications
Hemophilia A use case involving delivery/expression of functional Factor VIII (FVIII) using the improved AAV vectors.
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