Fig 1: F4L5.13 specifically binds FZD4/LRP5 co-receptors F4L5.13 is specific for FZD4, as determined by biolayer interferometry (BLI). F4L5.13 or the isotype control molecule (100 nM) was tested for binding to immobilized recombinant FZD CRDs or the ectodomain of the unrelated receptor Her2. BLI sensors coated with Fc were used to determine the baseline response.Binding kinetics, as determined by BLI, for the binding of immobilized F4L5.13 to FZD4 CRD or LRP5. Experiments were performed in triplicate, and data are presented as the mean ± SD.F4L5.13 selectively binds to LRP5 over LRP6. LRP5/6 proteins coated on a maxisorp plate are recognized in a concentration-responsive manner by F4L5.13 but not by a non-targeting IgG control (4275). F4L5.13 binds to LRP5 with a low nanomolar EC50 value and is > 50-fold selective for LRP5 over LRP6. Data are presented as the mean ± SD from three independent experiments.Activation of ßcatenin signaling by WNT3A, F4L5.13, or recombinant NDP (30 nM each) in HEK293T cells transfected with plasmids encoding FZD4 and/or LRP5. Values represent fold activation of LEF/TCF reporter gene. Data are presented as mean ± SEM, n = 3.Binding of F4L5.13 to the cell surface of HEK293T cells overexpressing FZD4 and LRP5 by flow cytometry. Data are representative of two experiments.
Fig 2: Primate brain-enhanced AAVs gain interaction with LRP6.a Left: Arraying AAV capsid-specific hits by human brain endothelial cell expression levels reveals highly-conserved LRP6 as a potential receptor for BBB crossing. Right: the LRP6 extracellular domain contains 4 YWTD domains (E1-E4). b SPR confirms that the engineered capsids AAV9-X1.1 and CAP-Mac gained direct binding interactions with human LRP6. v.g.: viral genomes. c Representative AlphaFold models, from 5 structural models each of X1 and CAP-Mac peptides with up to 20 recycles, predict selective interaction with human LRP6 domain E1 (E1 and E2: teal, E3 and E4: yellow, AAV peptides: purple). d SPR of mouse LRP6-E1E2 and LRP6-E3E4 (the minimal stable extracellular domain fragments due to cooperative folding) confirms that AAV9-X1.1 and CAP-Mac bind only to LRP6-E1E2. Panel a created with BioRender.com released under a Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International license.
Fig 3: LRP6 enhances engineered AAV potency in primate BBB and neuronal cell culture.AAV9-X1.1 has enhanced potency in (a) macaque and (b) human primary brain microvascular endothelial cell culture (black data points), which decreases to AAV9 levels with Mesd inhibition of LRP6 (blue data points). 4 biological replicates were performed and quantified for each condition. Bars indicate the mean value. MOI: multiplicity of infection. c AAV9-X1.1 has enhanced potency in human pluripotent stem cell (hPSC)-derived midbrain dopaminergic neuronal culture (black data points), which decreases to AAV9 levels with Mesd inhibition of LRP6 (blue data points). 5 biological replicates were performed and quantified for each condition. d Quantification of LRP6 and NeuN immunohistochemistry confirms that LRP6 expression is largely restricted to mature neurons (20 biological replicates) and that the LRP6-dependent enhanced potency of AAV9-X1.1 is enriched in this population (5 biological replicates for each condition, Mesd inhibition condition in blue).
Fig 4: LRP6 modulates CNS function of engineered AAVs in mice.a Schematic of Lrp6 conditional knockout by sequential AAV injection. Cre-conditional Lrp6 knockout mice were systemically injected with AAV1-X1 packaging either Cre or mCherry, creating cohorts of mice that differ in their Lrp6 expression. After allowing time for expression, these cohorts were each injected with AAV9-PHP.eB or AAV9-X1.1 packaging eGFP. By switching serotypes, neutralizing antibodies are evaded and vector dependence on Lrp6 in vivo may be assessed. b Representative sagittal brain images (left) and liver images (right). Imaging parameters were optimized independently for AAV9-X1.1 and AAV9-PHP.eB second dose conditions. c Quantification of AAV potency demonstrating that conditional knockout of Lrp6 in mouse selectively and potently reduces AAV9-X1.1 brain and liver gene delivery. Data points are the average of two technical replicate sections per tissue region for each of 3 biological replicate animals, with consistent physiological regions of interest across the four experimental cohorts. Bars represent the mean value. Panel a created with BioRender.com released under a Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International license.
Supplier Page from R&D Systems, a Bio-Techne Brand for LRP-6 His Tagged Protein
Available conjugates: Sizes Available: 25 ug