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Publication Number
US-11981967-B2
Publication Date
2024-05-14
Expiration Date
Abstract
The present disclosure provides methods and compositions to develop AAV capsids with a desired characteristic compared to a natural AAV serotype. These capsids are useful, for example, for the delivery of genome engineering molecules and gene therapy molecules for the treatment of a subject in need thereof.
Core Innovation
The invention relates to AAV capsid protein variants based on AAV VP1 capsid protein sequences, including AAV9 VP1 capsid variants. The document describes inserting peptide fragments or amino-acid sequences into hypervariable and/or surface-exposed capsid loop regions across multiple AAV serotypes, with serotype-specific insertion-site mapping provided for different reference capsid sequences.
AAV capsid variant libraries are described in which the inserted sequences are linked to barcodes and reporter genes or reporter/localization cassettes, including nuclear localization components such as SV40 NLS, KASH, and histone H2B. The libraries are implemented with packaging in producer cells with helper functions in trans, and the resulting AAV variant particles support downstream functional selection and identification by barcode readout.
Selection and readout are described using transduction followed by recovery and barcode identification, including barcode sequencing and NGS-based enrichment, as well as reporter expression and reporter expression in isolated nuclei. The document describes enrichment of peptide-inserted variants and effects on tropism and intracellular and/or nuclear localization, with evaluations in cells and tissues including CNS and liver.
Claims Coverage
The independent claims cover engineered AAV capsid proteins in which amino-acid sequences from specified SEQ ID ranges are inserted into AAV capsid proteins at defined positions. Across these independent claims, the inventive features include sequence selection from SEQ ID NO: 1-32 or SEQ ID NO: 68-110 and insertion site constraints within the capsid protein.
Inserted contiguous amino acids from specified SEQ ID ranges
An AAV capsid protein comprising at least 4 contiguous amino acids of an amino acid sequence set forth in any one of SEQ ID NO: 1-32 or at least 4 contiguous amino acids of an amino acid sequence set forth in any one of SEQ ID NO: 68-110.
Inserted between amino acid positions 450 and 600
An AAV capsid protein comprising an amino acid sequence set forth in any one of SEQ ID NO: 1-32 or in any one of SEQ ID NO: 68-110, wherein the amino acid sequence is inserted into the AAV capsid protein between amino acid positions 450 and 600.
Inserted at enumerated amino acid positions
An AAV capsid protein comprising an amino acid sequence set forth in any one of SEQ ID NO: 1-32 or in any one of SEQ ID NO: 68-110, wherein the amino acid sequence is inserted into the AAV capsid protein at amino acid position 266, 454, 455, 578, 588, 589, 590, or 591.
Overall, the claim coverage centers on AAV capsid proteins engineered by inserting amino-acid sequences selected from SEQ ID NO: 1-32 or SEQ ID NO: 68-110 into defined regions or specific positions of the capsid protein. Dependent claims further narrow insertion location, including hypervariable and/or surface-exposed loop regions and specific amino-acid positions, and constrain inserted sequences and permitted AAV serotypes.
Stated Advantages
selection/enrichment of peptide-inserted AAV capsid variants.
demonstrated tropism effects.
effects on intracellular and/or nuclear localization.
functional testing across cells and tissues, including CNS and liver.
evaluation across species including mouse, NHP, and human-derived neurons.
enrichment and improved performance in selected tissues and cell types, including comparisons across species and cell types.
head-to-head comparisons and identification of enriched peptide-inserted variants.
Documented Applications
Functional selection/enrichment of engineered AAV capsid variants based on transduction, followed by barcode/NGS identification and analysis.
Transduction studies assessing tropism and intracellular/nuclear localization using fluorescent reporters with FACS/IHC across cells and tissues, including CNS and liver.
Application testing in species models including mouse and NHP, and in human-derived neurons.
Payload/transgene delivery and reporter/localization cassette evaluation using AAV vectors and associated compositions.
In vitro enrichment in primary neurons and multiple cell lines.
In vivo selections in mice in the CNS using neuron-specific promoters.
In vivo selections in non-human primates, including liver targeting using a promoter.
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