paav itr mcs plasmid  (TaKaRa)

Journal: iScience

Article Title: Computationally guided discovery of Ly6e/LY6E-dependent AAV capsid variants

doi: 10.1016/j.isci.2026.115554

Figure Lengend Snippet: Workflow of EvoPRAISE and its application in generating a peptide binder for Ly6e (A) Workflow of EvoPRAISE. Firstly, the extracellular domain of the target membrane protein and a pool of randomly generated peptides were prepared. Secondly, these inputs were processed by APPRAISE, which calculates an energetic binding score (B) for each peptide based on atom counting. Peptides were ranked according to binding score, and the top candidate was selected (Round 0). For the top-ranked peptide, a saturation mutagenesis library was generated by substituting each residue with one of the 19 other common natural amino acids. This library was then evaluated by APPRAISE to determine a new top-ranking peptide (Round 1). In subsequent rounds, residues that had already evolved were fixed, while saturation mutagenesis was applied to the remaining residues. This iterative process was repeated until all residues had been evolved. (B) Structural model of AAV-PHP.eB, which highlights the peptide insertion site in blue. The left panel shows the AAV capsid composed of 60 structurally identical subunits (PDB ID: 7WQO ). The middle panels show top views around the 3-fold symmetry axis, with the three subunits forming the trimer displayed. A single VP3 subunit is highlighted in green, and the inserted peptide sequence is shown in blue. Peptide sequence used as the EvoPRAISE input. Seven-residue peptide binders were inserted between residues 588 and 589 (VP1 numbering) in a surface-exposed variable region of AAV9. (C) Binding scores of 100 randomly generated peptides were compared with that of the AAV9 peptide (AQAQAQTG) and plotted as ΔB in ranking order. In Round 0, the RLPAYEI peptide (red) ranked first. The peptide pool also included the PHP.eB peptide (green) and the AAV9 peptide (blue). (D) Amino acid sequences at the AAV9 VP1 peptide-insertion site are shown for each variant along the directed-evolution trajectory (arrow). The 7-mer insert sequence (blue; residues 588–594, VP1 numbering) was iteratively optimized from RLPAYEI (Cap-PF1.0) to WMDQIIY (Cap-PF1.7). Red letters indicate the amino acid substitutions that emerged in that round relative to the preceding variant. Numbers denote the flanking VP1 residue positions (587 and 594). (E) Changes in binding scores of the top-ranked peptides across rounds. Red plots indicate the top peptide of each round. In the subsequent round, the same peptide was used as the reference for comparison against its variants (blue). (F) In vitro infectivity assay. AAV.Cap-PF1.7 showed Ly6e-dependent enhancement of transduction in HEK293T LY6E-KO cells overexpressing Ly6e , whereas the negative-control AAV9 did not. AAV capsids carrying a fluorescent protein expression cassette were applied at 5 × 10 9 viral genomes (v.g.) per well to HEK293T cells transfected or not with Ly6e in a 96-well plate format. Images were taken 24 h after transduction ( n = 3 per condition). Scale bars, 200 μm. (G) Bright-field and mNeonGreen images were quantified to calculate extent of infection (infection rate, %; left) and intensity (brightness per transduced area, a.u.; right) for AAV9 and Cap-PF1.7 under LY6E-KO ( None ) or LY6E-expressing ( Ly6e ) conditions. Bars represent mean ± SD; open circles denote individual image measurements. Asterisks indicate comparisons of Cap-PF1.7 under Ly6e-expressing conditions versus each of the other indicated groups (∗ p < 0.05, ∗∗ p < 0.01; Welch’s two-sided t test with Holm-Bonferroni correction).

Article Snippet: AAVpro Helper Free System (AAV9) , TaKaRa Bio Inc , Cat# 6690.

Techniques: Membrane, Generated, Binding Assay, Mutagenesis, Residue, Sequencing, Variant Assay, Comparison, In Vitro, Infection, Transduction, Negative Control, Expressing, Transfection

Journal: iScience

Article Title: Computationally guided discovery of Ly6e/LY6E-dependent AAV capsid variants

doi: 10.1016/j.isci.2026.115554

Figure Lengend Snippet: AAV.Cap-PF1.7 crosses the blood-brain barrier (BBB) of the Syrian hamster (A) Experimental scheme to assess capsid tropism for the central nervous system (CNS) in vivo . The AAV genome was engineered to express mNeonGreen under the control of a CAG promoter. Vectors were administered systemically to weaning Syrian hamsters via the retro-orbital sinus. Animals were sampled ≥4 weeks post-injection, and mNeonGreen expression in the CNS was examined by fluorescence microscopy. (B) Evaluation of the impact of peptide insertion on capsid fitness based on production yield (v.g./mL/20 cm dish). The color bar indicates the mean vector yield from 2 to 4 independent preparations per variant. (C) Fluorescence imaging of Syrian hamster brains. AAV9, AAV-PHP.eB, AAV.CAP-B10, and Cap-PF1.7 packaging CAG–mNeonGreen were administered intravenously at 1 × 10 13 v.g. per animal ( n = 3 per condition). Scale bars: 3 mm for whole brain images and 1 mm for higher-magnification images. (D) Fluorescence intensity was quantified in the indicated brain regions (cortex, thalamus, hippocampus, and whole brain) following administration of AAV vectors packaged with the indicated capsids. Each dot represents an individual animal; bars represent mean ± SD. Asterisks indicate comparisons of Cap-PF1.7 versus each of the other capsids within each brain region (∗ p < 0.05, ∗∗ p < 0.01; Welch’s two-sided t test).

Article Snippet: AAVpro Helper Free System (AAV9) , TaKaRa Bio Inc , Cat# 6690.

Techniques: In Vivo, Control, Injection, Expressing, Fluorescence, Microscopy, Plasmid Preparation, Variant Assay, Imaging

Journal: iScience

Article Title: Computationally guided discovery of Ly6e/LY6E-dependent AAV capsid variants

doi: 10.1016/j.isci.2026.115554

Figure Lengend Snippet: AAV capsid variant that interacts with human LY6E (A) Comparison of SLC2A1 , TFRC , and LY6E mRNA expression levels across human brain regions using RNA expression data from the human protein atlas ( https://www.proteinatlas.org ). SLC2A1 and TFRC are representative marker genes of the blood-brain barrier (BBB). Asterisks indicate comparisons between LY6E and each of the other genes within the same brain region (∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001; Mann-Whitney U test with Benjamini-Hochberg correction). (B) Impact of peptide insertion on capsid fitness, assessed by production yield (v.g./mL/20 cm dish). The color bar indicates the mean vector yield from 2 to 4 independent preparations per variant. (C) Bright-field and mNeonGreen images were quantified to calculate extent of infection (infection rate, %; left) and intensity (brightness per transduced area, a.u.; right) for AAV9 and Cap-PF.h variants under LY6E-KO (LY6E−) or LY6E-expressing (LY6E+) conditions. Bars represent mean ± SD; open circles denote individual image measurements. Asterisks indicate comparisons between LY6E− and LY6E+ within the same capsid ( p < 0.05 (∗), p < 0.01 (∗∗), and p < 0.001 (∗∗∗)). Daggers (†) indicate comparisons of each capsid under LY6E + conditions versus AAV9 (LY6E+) (ns, not significant ; p < 0.05 (†), p < 0.01 (††); Welch’s two-sided t test with Holm-Bonferroni correction).

Article Snippet: AAVpro Helper Free System (AAV9) , TaKaRa Bio Inc , Cat# 6690.

Techniques: Variant Assay, Comparison, Expressing, RNA Expression, Marker, MANN-WHITNEY, Plasmid Preparation, Infection