In-depth comparison of Anc80L65 and AAV9 retinal targeting and characterization of cross-reactivity to multiple AAV serotypes in humans

Anc80L65 is a synthetic, ancestral adeno-associated virus that has high tropism toward retinal photoreceptors after subretinal injection in mice and non-human primates. We characterized, for the first time, post-intravitreal cell-specific transduction profile of compared with AAV9. Here we use AAV9 to intravitreally deliver copy gene encoding GFP into WT C57Bl/6J mice. expression was driven by one two clinically relevant promoters, chicken β actin (CB) or truncated MECP2 (P546). After qualitative assessment relative expression, found have similar profiles. Through development novel method quantifying GFP-positive cells, higher Müller glia horizontal cells. In addition, P546 promote at more moderate level levels seen under CB promoter. Finally, characterized cross-reactivity human sera; 83% patients AAV2 pre-existing antibodies were be seropositive Anc80L65. This study demonstrates expanded therapeutic applications treat disease provides insights immunity humans. 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However, evaluate pattern retina. show overall slightly affinities nuclear layer reaffirm promoters modulate specific subsets types. because risks greater innate cross-react investigated conventional AAV1, serum samples.9Ladha Scholar,24Zinn likely finding suggests profiles should thoroughly prior translation confirm published reports superior differences promoter-driven subretinally injected four consisting cassette either CMV enhancer β-actin (546) packaged Anc80. respective doses each follows: AAV9.CB.GFP (2.03E+10vg), Anc80.CB.GFP (1.87E+10vg), AAV9.546.GFP (1.96E+10vg), Anc80.546.GFP (1.28E+10vg). Vector concentration transgene-specific digital droplet PCR (Table While three had very concentrations assay variability range, fourth (Anc80.546.GFP) lower concentration. Thus, volume restriction could inject exact same all All results distinction mind. 2–4 months age killed 4–8 weeks post-injection. During period, expect reach peak without any loss signal, allowing direct comparison efficacy. immunofluorescent staining cryosections anti-GFP anti-recoverin, comparable signal (Figures 1B 1C ). AAV9.546.GFP-injected eyes, signals Anc80.546.GFP-injected eyes increased co-localization recoverin (photoreceptors/RPE) counterstain 1C). despite Anc80.546 being AAV9.546.GFP. administration. general, our constructs 1C), line leading spread AAV9- Anc80-injected 1A). Similar 2- 4-month-old (1.28E+10vg) μL. Four 8 post-injection, period reached expected, qualitatively assessed interest cryosections. Samples labeled antibody counterstained cell-type-specific antibodies: anti-Brn3a (ganglion), anti-AP-2α (amacrine), anti-Sox2 (Müller glia), anti-Otx2 (bipolar), anti-Calb (horizontal). z stack images obtained Zeiss 800 Laser Scanning Confocal Microscope maximum intensity projections acquired orthogonal views. Transduction perceived individually assessments, degree between cell-type markers, fluorescent Figure 2A shows whole along ×30 zoom insets 2B) taken regions interest. Each displays own unique 2B). highest ganglion significant GFP, Brn3a, Calb 2C 2G). Comparatively, less AP-2α-, Sox2-, Otx2-positive indicating amacrine glia, bipolar respectively 2D–2F). Orthogonal views indicate predict confounded overlap Brn3a Sox2, 2E). Moderate observed, positive AP-2α, Otx2, 2D). GFP- Calb-positive rates 2F suggest processes 2F). A co-localization, retinas, administration, Brn3a-, Calb-labeled 2C, 2D, Co-localization Sox2 Otx2 2E low except comparatively 2C). again (horizontal), positively transduced custom workflow blinded fashion Nikon NIS-Elements software platform automatically detect individual SegmentObjects.ai convolutional neural network module, empirically calculated threshold operator input avoid bias 3A confirming accuracy AI-based quantification comparing hand counts S2), quantify number type 3B 3C). allowed us increase validity rigor visualizations 2. Overall, aligned assessments. 3D) almost 50% 20% only about 13% statistically significantly rates. 3E) 31% 23% targeted, respectively. rate 18%, 15% 9% Significant average 3F) maintained revealing 34% positivity, (15%), (2%), (4%). 3G) resulted numbers (41%) percentage amacrine, bipolar, below 10%. retinas delivered time Fin