Redirection of tropism of AAV capsids
Inventors
Nonnenmacher, Mathieu E. • Hou, Jinzhao • Wang, Wei
Assignees
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Publication Number
US-12467046-B2
Publication Date
2025-11-11
Expiration Date
Abstract
The disclosure relates to compositions, methods, and processes for the preparation, use, and/or formulation of adeno-associated virus capsid proteins, wherein the capsid proteins comprise targeting peptide inserts for enhanced tropism to a target tissue.
Core Innovation
The disclosed methods and vector configurations generate adeno-associated virus (AAV) vector libraries encoding variant AAV capsid polypeptides with randomized amino-acid regions of 2 to 9 consecutive amino acids. These libraries utilize dual promoter configurations, comprising a P40 promoter alongside a cell-type-specific promoter or first and second promoters, to drive capsid mRNA expression in the absence of helper-virus co-infection. This design enables RNA-driven selection where recovery and analysis of capsid-encoding RNA serve as an indicator of complete transduction rather than relying solely on viral DNA recovery as the screening result.
The platform incorporates randomized peptide insertions into capsid loops to redirect viral tropism and evaluate variant capsid polypeptides through in vivo administration in animal models without helper-virus co-infection. Disclosed screening results demonstrate convergent variant enrichment, improved delivery, and enhanced central nervous system transduction across brain and spinal-cord populations relative to parental serotypes. Target-tissue analysis incorporates sequence determination, intronic barcode tracking, and measurement of capsid-encoding DNA or RNA while omitting operational details [procedural detail omitted for safety].
Claims Coverage
The provided relevant claims identify three independent claims: claims 1, 27, and 28, covering three inventive features.
AAV vector library with P40 and cell-type-specific promoters
Claim 1 covers generating an AAV vector library using nucleic acids comprising a P40 promoter and a cell-type-specific promoter that drives capsid mRNA expression in the absence of helper-virus co-infection, together with nucleic acids encoding variant AAV capsid polypeptides having a randomized region of 2 to 9 consecutive amino acids. [procedural detail omitted for safety]
AAV vector library with specified cell-type-specific promoters
Claim 27 covers generating an AAV vector library using nucleic acids comprising a first promoter and a second, cell-type-specific promoter that drives capsid mRNA expression in the absence of helper-virus co-infection, alongside nucleic acids encoding variant AAV capsid polypeptides with a randomized region of 2 to 9 consecutive amino acids, with promoter choices including neuron-, astrocyte-, blood cell-, eye-, heart-, muscle-, kidney-, lung-, pancreas-, and vasculature-specific promoters. [procedural detail omitted for safety]
Generation and evaluation of variant capsids in an animal
Claim 28 covers generating and evaluating variant AAV capsid polypeptides using an AAV vector library administered in an animal in the absence of helper virus, followed by recovery, sequence determination of capsid-encoding RNA, and measurement of capsid-encoding DNA or RNA in target cells or tissues. [procedural detail omitted for safety]
Together, the three independent claims cover promoter-based generation of AAV vector libraries encoding capsid variants and helper-virus-independent in vivo evaluation and recovery of variant capsids in target tissues.
Stated Advantages
The cell-type-specific promoter drives capsid mRNA expression without helper-virus co-infection, supporting RNA-driven in vivo selection and RNA recovery.
Recovery of capsid-encoding RNA provides a more stringent indicator of complete transduction than relying on viral DNA recovery as the screening result.
Randomized capsid peptide insertions redirect tropism and enhance transduction in target tissues.
Validated capsids improve central nervous system cell targeting, transduction, or viral-genome delivery to the brain and spinal cord relative to AAV9 or a parental capsid.
Selected capsid variants exhibit convergent enrichment or neuronal and astrocyte enrichment exceeding the PHP.eB benchmark.
Barcoded constructs retain high AAV production titers.
The drawing summary reports reduced diversity loss in the cloning-free library-construction approach.
Documented Applications
In vivo central nervous system-directed AAV capsid library screening and evaluation in animal models, including brain, spinal cord, and hippocampus compared to liver and heart.
Screening with neuron- and astrocyte-specific promoters to evaluate cell fitness, targeting, and tissue-selective capsid enrichment, including brain microvascular endothelial cells.
Use of intronic barcodes and tandem UMI barcode strategies in AAV vector designs for NGS-based identification, recovery, and analysis of full-length capsid variants.
Targeted viral vector delivery and transduction in the central nervous system and peripheral nervous system.
Delivery of therapeutic payloads, including proposed siRNA-encoding AAV particles for target-protein suppression and combination therapies in neurological disorders.
Targeting specific cell types and tissues using promoters for neurons, astrocytes, blood cells, eye, heart, muscle, kidney, lung, pancreas, and vasculature.
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