Adeno-associated virus compositions for restoring PAH gene function and methods of use thereof
Inventors
Seymour, Albert Barnes • Ahmed, Seemin Seher • Wright, Jason Boke • Dollive, Serena Nicole • McSwiggen, James Anthony • Prout, Jaime Michelle • Sookiasian, Danielle Lauren
Assignees
Interested in licensing this patent?
MTEC can help explore whether this patent might be available for licensing for your application.
Publication Number
US-12203094-B2
Publication Date
2025-01-21
Expiration Date
Abstract
Provided herein are adeno-associated virus (AAV) compositions that can restore phenylalanine hydroxylase (PAH) gene function in cell. Also provided are methods of use of the AAV compositions, and packaging systems for making the AAV compositions.
Core Innovation
The invention relates to a replication-defective adeno-associated virus (AAV) comprising an AAV capsid and a correction genome for editing a target locus in a human phenylalanine hydroxylase (PAH) gene. The correction genome includes an editing element comprising a nucleotide sequence that is at least 90% identical to specified SEQ ID numbers, together with a 5′ homology arm and a 3′ homology arm.
The 5′ homology arm is located 5′ of the editing element and is at least 100 nucleotides in length with at least 90% identity to SEQ ID-defined nucleic acid sequences, while the 3′ homology arm is located 3′ of the editing element with at least 90% identity to SEQ ID-defined nucleic acid sequences. The disclosed work further provides engineered correction-genome architectures for PAH locus editing and expression, including AAV correction vectors that differ in the 5′ homology arm sequence while preserving the overall vector framework.
These engineered variants include expression-enhancing sequence changes described as Kozak optimization, removal or disruption of 5′ UTR quadruplexes, cyclic AMP response element optimization, incorporation of SNP rs1522295 minor allele, and glucocorticoid/AP2 binding site modifications. The correction constructs also incorporate specified PAH coding and intron-inserted coding architectures, and may include intron designs such as HCR and HBB1 or splice-donor modifications intended to affect RNA processing or expression.
The disclosed work provides experimental support for targeted integration and expression using PAH-targeting AAV correction vectors, including in vitro human cell assays and in vivo mouse liver editing. The document describes ddPCR and LAM enrichment results for correct integration in human cell assays, ddPCR/PCR validation for liver edited alleles and mRNA levels for a specific in vivo construct, and phenotypic correction in PAH knockout mice with reduced serum phenylalanine, as well as durability and specificity in humanized or FRG mouse models.
Claims Coverage
The provided material includes two independent claims. Across these claims, three core inventive aspects are covered: targeted PAH-locus editing using an editing element flanked by 5′ and 3′ homology arms with sequence-identity thresholds; defined correction-genome architecture options; and specific polynucleotide sequences identified by SEQ IDs.
Replication-defective AAV with PAH editing element and homology arms
A replication-defective AAV comprising an AAV capsid and a correction genome with an editing element for editing a target locus in a human phenylalanine hydroxylase (PAH) gene, wherein the editing element comprises a nucleotide sequence at least 90% identical to a nucleotide sequence set forth in SEQ ID NO: 35, 83, or 84; a 5′ homology arm located 5′ of the editing element comprising a nucleotide sequence of at least 100 nucleotides in length that is at least 90% identical to a nucleic acid sequence contained within SEQ ID NO: 36, 37, 38, 39, 40, 41, 42, 43, 44, or 115; and a 3′ homology arm located 3′ of the editing element comprising a nucleotide sequence of at least 100 nucleotides in length that is at least 90% identical to a nucleic acid sequence contained within SEQ ID NO: 45 or 117.
Defined polynucleotide by SEQ ID membership
A polynucleotide comprising the nucleic acid sequence of any one of SEQ ID NO: 25, 46-63, 80-82, 116, 118, 119, 131, 132, 135-139, 143, 145, or 146, optionally wherein the polynucleotide is comprised within a vector or host cell.
Independent claim coverage focuses on a replication-defective AAV with a PAH-targeting correction genome defined by an editing element and 5′/3′ homology arms with specified length and ≥90% identity thresholds to SEQ ID-defined sequences, and on specific polynucleotides defined by membership in a list of SEQ ID sequences.
Stated Advantages
Not explicitly described in patent.
Documented Applications
Not explicitly described in patent.
Interested in licensing this patent?