AAV capsid variants and uses thereof
Inventors
Nonnenmacher, Mathieu Emmanuel • Moyer, Tyler Christopher • Li, Jiangyu • Laks, Dan Richard
Assignees
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Publication Number
US-12296025-B2
Publication Date
2025-05-13
Expiration Date
Abstract
The disclosure relates to compositions and methods for the preparation, use, and/or formulation of adeno-associated virus capsid protein variants.
Core Innovation
The invention relates to adeno-associated virus (AAV) capsid variants comprising an amino acid sequence at least 95% identical to amino acids 203-742 of SEQ ID NO: 982, wherein the amino acid sequence comprises HDSPHK (SEQ ID NO: 2) in variable region IV (VR-IV). The disclosed work defines related peptide sequence specifications, motif variants, and positional context for HDSPHK in VR-IV, including sequence formulas and replacement rules in the loop IV region.
The document further discloses polynucleotides and AAV particles that encode and present the defined AAV capsid variants. It also describes AAV genome and cassette component options, including promoters, 5′ and 3′ ITRs, enhancers, introns, exons, miR binding sites, detargeting sites, polyA sequences, UTRs, stuffer or filler sequences, and miRNA/miR binding site configurations.
The described constructs include payloads such as proteins, antibodies, gene editing system components, and RNAi/siRNA. The content also summarizes generated and screened AAV capsid libraries using TRACER-based enrichment results with NGS for variant selection in non-human primates and mice, and reports matured AAV9 capsid variants including TTM-001 and TTM-002 with loop IV modifications.
Claims Coverage
The independent claims cover four related subject matters: an AAV capsid variant sequence definition, a capsid variant defined by the amino acid sequence of SEQ ID NO: 982, a polynucleotide encoding the defined capsid variant sequence, and an AAV particle comprising that capsid variant. In total, the independent set highlights three main inventive concept areas: the HDSPHK-in-VR-IV capsid motif with specified identity to a defined region of SEQ ID NO: 982, the corresponding polynucleotide encoding, and the incorporation of the capsid variant into AAV particles.
Hdsphk in variable region IV with at least 95% identity to amino acids 203-742 of SEQ ID NO: 982
An adeno-associated virus (AAV) capsid variant comprising an amino acid sequence at least 95% identical to amino acids 203-742 of SEQ ID NO: 982, wherein the amino acid sequence comprises HDSPHK (SEQ ID NO: 2) in variable region IV (VR-IV).
AAV capsid variant comprising SEQ ID NO: 982
An adeno-associated virus (AAV) capsid variant comprising the amino acid sequence of SEQ ID NO: 982.
Polynucleotide encoding Hdsphk-in-VR-IV capsid variant with at least 95% identity to amino acids 203-742 of SEQ ID NO: 982
A polynucleotide encoding an adeno-associated virus (AAV) capsid variant comprising an amino acid sequence at least 95% identical to amino acids 203-742 of SEQ ID NO: 982, wherein the amino acid sequence comprises HDSPHK (SEQ ID NO: 2) in variable region IV (VR-IV).
AAV particle comprising Hdsphk-in-VR-IV capsid variant
An adeno-associated virus (AAV) particle comprising an AAV capsid variant comprising an amino acid sequence at least 95% identical to amino acids 203-742 of SEQ ID NO: 982, wherein the amino acid sequence comprises HDSPHK (SEQ ID NO: 2) in variable region IV (VR-IV).
Overall, the claim coverage is directed to AAV capsid variants featuring HDSPHK (SEQ ID NO: 2) in variable region IV, with identity requirements to amino acids 203-742 of SEQ ID NO: 982, and to polynucleotides and AAV particles that encode and present these capsid variants.
Stated Advantages
Detargeting in peripheral tissues, including liver and DRG, together with enhanced CNS tropism.
Cross-species performance, including NHP biodistribution/transgene expression and mouse CNS targeting.
Cross-species central nervous system (CNS) tropism.
Peripheral de-targeting.
Organ-specific tropisms, including liver-specific tropisms, plus heart and muscle (quadriceps) enriched variants, including pan-tropic variants.
Decreased liver tropism.
Increased liver tropism.
Reduced CNS tropism.
Documented Applications
NGS/sequencing-based evaluation of matured AAV9 capsid variants TTM-001 and TTM-002 with loop IV modifications in non-human primates and mice.
NHP biodistribution measurement and transgene expression measurement for TTM-002 versus AAV9 across brain regions and spinal cord regions, including detargeting in DRG/liver and altered expression in heart.
Single-cell RNA-seq and IHC evidence of transduction of endothelial/astrocytic lineages, including Olig2+ astrocytes, to non-neuronal CNS cells.
Mouse studies including additional maturation showing the importance of an SPH motif for brain tropism.
Cross-species performance across multiple primate species, including cynomolgus macaques, African green monkeys, and marmosets, and mice.
Assessment of matured AAV9 capsid variants in mice, including brain expression fold-enrichment versus non-matured control, and assessment of tissue de-targeting including liver and DRG.
Assessment of matured AAV9 capsid variants in non-human primates using fold-enrichment panels and organ-specific tropisms, including liver, DRG, brain, heart, and muscle (quadriceps), with pan-tropic variants reported.
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