Adeno-associated virus (AAV) vectors, AAV vectors having reduced capsid deamidation and uses therefor
Inventors
Wilson, James M. • Tepe, April • Turner, Kevin • Sims, Joshua Joyner • Wang, Qiang
Assignees
University of Pennsylvania Penn
Publication Number
US-12416016-B2
Publication Date
2025-09-16
Expiration Date
Interested in licensing this patent?
MTEC can help explore whether this patent might be available for licensing for your application.
Abstract
A recombinant adeno-associated virus (rAAV) vector comprising an AAV capsid having a heterogeneous population of vp1 proteins, a heterogeneous population of vp2 protein and a heterogeneous population of vp3 proteins. The capsid contains modified amino acids as compared to the encoded VP1 amino acid sequence, the capsid containing highly deamidated asparagine residues at asparagine-glycine pair, and further comprising multiple other, less deamidated asparagine and optionally glutamine residues.
Core Innovation
A recombinant adeno-associated virus (rAAV) vector comprising an AAV capsid having a heterogeneous population of vp1 proteins, a heterogeneous population of vp2 proteins and a heterogeneous population of vp3 proteins is provided. The capsid contains modified amino acids as compared to the encoded VP1 amino acid sequence, the capsid containing highly deamidated asparagine residues at asparagine-glycine pair, and further comprising multiple other, less deamidated asparagine and optionally glutamine residues. The composition further comprises a vector genome in the AAV capsid, the vector genome comprising a nucleic acid molecule comprising AAV inverted terminal repeat sequences and a non-AAV nucleic acid sequence encoding a product operably linked to sequences which direct expression of the product in a host cell.
The patent addresses a need described in the background for compositions comprising AAV-based constructs that have stable receptor binding and/or stable capsids, avoid neutralizing antibodies and/or retain purity on storage. The disclosure provides rAAVs and modified rAAVs having decreased modifications and methods for reducing deamidation — including producing capsids from nucleic acid sequences containing modified glycine codons at asparagine-glycine pairs — to provide rAAV having capsids which retain greater stability, potency, and/or purity.
Claims Coverage
This section identifies five independent claims and extracts their main inventive features.
rAAV comprising an AAVrh79 capsid with highly deamidated NG residues
An rAAV comprising an AAVrh79 capsid having AAVrh79 vp1, vp2, and vp3 proteins that (a) comprise glutamic acid (E) at position 67 in the AAVrh79 vp1 proteins and arginine (R) at position 169 in the AAVrh79 vp1 and vp2 proteins; (b) contain amino acid modifications comprising at least 50% to 100% deamidated asparagines (N) in asparagine-glycine pairs at positions N57, N263, N385 and N514 in vp1 (and corresponding positions in vp2/vp3 as recited); (c) the deamidated asparagines are deamidated to aspartic acid, isoaspartic acid, an interconverting aspartic/isoaspartic pair, or combinations thereof; and (d) further comprise a vector genome with AAV inverted terminal repeats (ITRs) and a non-AAV nucleic acid sequence encoding a product operably linked to expression sequences.
rAAV production system comprising capsid nucleic acid encoding SEQ ID NO:2
A production system useful for producing an rAAV in a production cell, comprising: (a) an AAVrh79 capsid nucleic acid sequence encoding the amino acid sequence of SEQ ID NO:2; (b) a nucleic acid molecule suitable for packaging into the AAV capsid comprising at least one AAV ITR, a functional rep gene and a non-AAV nucleic acid sequence encoding a gene product operably linked to expression sequences; and (c) sufficient AAV rep functions and helper functions to permit packaging of the nucleic acid molecule into the AAV capsid.
rAAV comprising AAVrh79 capsid produced by expression of 1–738 of SEQ ID NO:2
An rAAV comprising an AAVrh79 capsid produced by expression from a nucleic acid sequence which encodes the predicted amino acid sequence of 1 to 738 of SEQ ID NO:2, wherein the vp1, vp2 and vp3 proteins contain subpopulations with amino acid modifications comprising at least two highly deamidated asparagines in asparagine-glycine pairs in SEQ ID NO:2, and wherein the rAAV further comprises a vector genome with AAV ITRs and a non-AAV nucleic acid sequence encoding a product operably linked to expression sequences.
method of generating a recombinant AAV by culturing a host cell containing specified nucleic acids
A method of generating a recombinant AAV comprising culturing a host cell containing: (a) a nucleic acid sequence encoding an AAVrh79 vp1 capsid protein having amino acids 1 to 738 of SEQ ID NO:2 operably linked to regulatory sequences permitting expression of vp1/vp2/vp3 proteins; (b) a functional rep gene; (c) a vector genome nucleic acid comprising AAV 5' and 3' ITRs and an expression cassette with a non-AAV nucleic acid sequence encoding a product; and (d) sufficient helper functions to permit packaging of the vector genome into the AAV capsid.
cultured host cell comprising a recombinant nucleic acid molecule encoding AAVrh79 vp1 1–738 and other elements
A cultured host cell comprising a recombinant nucleic acid molecule comprising: (a) a nucleic acid sequence encoding an AAVrh79 vp1 capsid protein of amino acids 1 to 738 of SEQ ID NO:2 operably linked to regulatory sequences for expression of vp1/vp2/vp3; (b) a functional rep gene; (c) a vector genome with AAV 5' and 3' ITRs and a non-AAV nucleic acid sequence encoding a product; and (d) sufficient helper functions to permit packaging of the vector genome into an AAV capsid.
The independent claims focus on (i) rAAV particles with AAVrh79 capsids characterized by high-frequency deamidation at specified asparagine-glycine sites and specified residue substitutions, combined with packaged vector genomes containing AAV ITRs and non-AAV expression cassettes; (ii) production systems, host cells, and methods for producing such rAAVs using nucleic acids encoding SEQ ID NO:2 and requisite rep and helper functions.
Stated Advantages
Capsids which retain greater stability, potency, and/or purity.
Mutant rAAV having reduced deamidation may have reduced immunogenicity and/or increased stability on storage, particularly storage in suspension form.
Engineering modifications (e.g., modifying glycine codons at asparagine-glycine pairs) to reduce deamidation can improve vector transduction and reduce lot-to-lot molecular variability.
AAV variants with altered capsids (e.g., AAV8.AR2.08, AAVrh79, AAV5.5.9) offer distinct tissue transduction properties such as increased liver transduction or tissue specificity as described.
Documented Applications
Delivery of a desired gene product to a subject in need thereof using recombinant AAV vectors comprising the described capsids and packaged vector genomes.
Gene therapy vectors, antisense delivery vectors, or vaccine vectors for therapeutic purposes or immunization, as explicitly described for rAAV vectors.
Methods of transducing target tissues including administering an AAV having an AAVrh79 capsid or an AAV8.AR2.08 capsid for transducing liver tissue and for transducing muscle tissue.
Producing a desired gene product in vitro by transfection of host cells with a rAAV vector containing a coding sequence and recovering the expressed product.
Treatment applications explicitly described include delivery of transgenes for disorders such as hemophilia (Factor IX, Factor VIII), spinal muscular atrophy, muscular dystrophy, cystic fibrosis, rare or orphan diseases, and numerous other therapeutic transgenes enumerated in the specification.
Interested in licensing this patent?