Adeno-associated virus (AAV) clade F vector and uses therefor
Inventors
Wilson, James M. • Wang, Qiang • Tepe, April • Turner, Kevin
Assignees
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Publication Number
US-12644101-B2
Publication Date
2026-06-02
Expiration Date
Abstract
A recombinant adeno-associated virus (rAAV) vector comprising an AAVhu68 capsid produced in a production system comprising a nucleotide sequence of SEQ ID NO: 1, or a sequence at least 75% identical thereto which encodes SEQ ID NO:2. The AAVhu68 capsid comprises subpopulations of highly deamidated asparagine residues in asparagine-glycine pairs in the amino acid sequence of SEQ ID NO: 2. Also provided are compositions containing the rAAV and uses thereof. Additionally, rAAV having an engineered AAV capsid comprising at least one subpopulation of vp1 or vp2 proteins having a Val at amino acid position 157 with reference to the AAVhu68 vp1 numbering are provided.
Core Innovation
The invention relates to rAAVhu68 vectors and production-related constructs in which an AAVhu68 capsid nucleic acid sequence encodes the amino acid sequence of SEQ ID NO:2 and is operably linked to regulatory sequences that direct expression of AAVhu68 VP1 capsid proteins, AAVhu68 VP2 capsid proteins, and AAVhu68 VP3 capsid proteins that assemble into an AAVhu68 capsid in a packaging host cell culture. The vector genome used for packaging comprises a 5′ AAV inverted terminal repeat (ITR), a non-AAV nucleic acid sequence encoding a gene product operably linked to regulatory control sequences, and a 3′ AAV ITR.
The invention further includes nucleic acid sequences comprising AAV rep functions and nucleic acid sequences comprising helper functions for packaging of the vector genome into the AAVhu68 capsid. The disclosed production and vector design are associated with capsids containing heterogeneous deamidated asparagine-glycine subpopulations forming Asp/isoAsp, and optional additional modifications and PTMs are described in connection with the capsid subpopulations.
Engineered VP1/VP2 variants are described in which Val at a VP1 position 157 and, optionally, Glu at a VP1 position 67 are present (with AAVhu68 numbering). The described capsid population proportions (vp1:vp2:vp3) and the capsid protein composition are part of the claimed rAAVhu68 vector context, and purification and separation approaches are described to handle rAAVhu68 viral particles and genome-deficient AAVhu68 capsid intermediates.
The background problem being solved is addressed as the presence and impact of heterogeneous deamidation in capsids, including Asp/isoAsp-forming deamidated asparagine-glycine pairs, and the need for purification and separation of rAAVhu68 particles from genome-deficient AAVhu68 intermediates from an rAAVhu68 vector harvest. The invention therefore provides methods that incorporate downstream processing choices and separations intended to manage these harvested materials while producing bulk rAAVhu68.
Claims Coverage
The partial claim set includes two independent claims, each tied to downstream handling of rAAVhu68 derived from a packaging host cell. The inventive features emphasize two independent features: a rAAVhu68 purification method with selectable downstream processing steps and separation of full packaged particles from genome-deficient intermediates using fast performance liquid chromatography with strong anion exchange binding and monitoring.
Purifying a rAAVhu68 vector with selectable downstream processing steps
A method for purifying a rAAVhu68 vector by culturing a packaging host cell with an AAVhu68 capsid nucleic acid sequence encoding SEQ ID NO:2 under regulatory sequences for VP1/VP2/VP3 expression, providing a vector genome with a 5′ AAV ITR and a 3′ AAV ITR flanking a non-AAV gene product, supplying AAV rep functions and helper functions, harvesting vector-containing cells and/or culture media, and preparing bulk rAAVhu68 using one or more of concentrating, diafialtering, microfluidizing, nuclease digestion, filtering, crude chromatography, ultracentrifugation, tangential flow filtration buffer exchange, and/or formulation and/or filtration.
Separating full rAAVhu68 particles from genome-deficient intermediates by fast performance liquid chromatography and strong anion exchange at alkaline pH
A method for separating rAAVhu68 particles having packaged genomic sequences from genome-deficient AAVhu68 intermediates from an rAAVhu68 vector harvest by culturing a packaging host cell with an AAVhu68 capsid expression construct encoding SEQ ID NO:2 for VP1/VP2/VP3 assembly, providing a vector genome with 5′ and 3′ AAV ITRs around a non-AAV gene product, supplying AAV rep functions and helper functions, harvesting vector-containing cells and/or culture media, and subjecting a suspension comprising harvested recombinant AAVhu68 viral particles and AAVhu68 capsid intermediates to fast performance liquid chromatography with equilibration of a strong anion exchange resin at a pH of 10.0 to 10.4 for binding, salt-gradient elution with monitoring by ultraviolet absorbance at about 260 and about 280 and/or an A260/A280 inflection point, and collecting the full particles from the resulting fraction.
Across the independent claims, the core coverage is directed to producing rAAVhu68 using an AAVhu68 VP1/VP2/VP3 capsid expression system and an ITR-containing vector genome with rep and helper functions, and then purifying or separating rAAVhu68 from harvest material using selectable purification steps or strong anion exchange fast performance liquid chromatography with monitoring and collection of full particles.
Stated Advantages
Not explicitly described in patent.
Documented Applications
Not explicitly described in patent.
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