Fig 1: Impact of ALPL on VCAP-102 brain transduction in vivo(A) ALPL expression in young (6 week) and aged (18 months) mice relative to mTBP housekeeping gene. Values indicate mean ± SD (n = 9), normalized to young mouse data. (B and C) CNS transduction by VCAP-102 in young and aged mice. (B) Anti-HA staining 28 d after transduction with 1E13 VG/kg VCAP-102 containing scAAV-CAG-zsGreen-HA. Scale bars, 5 mm (whole brain), 1 mm (spinal cord), 200 μm (cortex, thalamus). (C) Transgene mRNA expression in the brain of young and aged mice. Data show mean ± SD (n = 3). (D) HeLa cells were treated with ALPL inhibitor SBI-425 and exposed to 1E4 VG/cell AAV9 or VCAP-102-luciferase. Data (mean ± SD) are normalized to untreated cells. (E) Brain transduction in mice treated with 35 mg/kg ALPL inhibitor SBI-425. VCAP-102 or 9P36 capsids with a luciferase transgene were administered i.v. at 2E13 VG/kg. In vivo luminescence shows luciferase activity in the brain 7 days after AAV administration. (F) Brain transgene mRNA in mice from (E). Values indicate mean ± SD (n = 5) normalized to vehicle control. All p values are derived from an unpaired two-tailed t test.
Fig 2: Identification of ALPL as the primary receptor of VCAP-102(A) Ectopic expression of ALPL increases VCAP-101 and VCAP-102 transduction. HEK293T cells transfected with indicated plasmid were treated with AAV9-mCherry or VCAP-102-GFP. Fluorescence was visualized after 24 h. (B) Functional interaction of VCAP-102 with ALPL across species. HEK293T cells transfected with indicated AAVR or ALPL plasmids were transduced with AAV9- or VCAP-102-luciferase. Data: mean ± SD luciferase activity normalized to control plasmid (n = 3). (C) Direct binding of VCAP-102 to human ALPL. Binding kinetics between VCAP-102 and human ALPL were analyzed by surface plasmon resonance (SPR). VCAP-102 or AAV9 capsids were immobilized and ALPL was used as an analyte. (D) pH-dependent dissociation of ALPL:VCAP-102 complex, measured by SPR. VCAP-102 capsid was immobilized and ALPL was injected at pH 7.4. The dissociation was then performed at pH 7.4 or pH 5.5. (E) Blocking of VCAP-102 transduction by ALPL antibody. HeLa cells were incubated with anti-ALPL antibody or an isotype control before adding 1E4 VG/cell AAV9 or VCAP-102 expressing luciferase. Luminescence data (mean ± SD) were normalized to untreated cells. (F) ALPL mediates capsid transcytosis. MCDK or MDCK-ALPL cells grown on a Transwell insert and AAV9 or VCAP-102 were added to the top chamber. AAV genomes in the bottom chamber was quantified 24 h later by qPCR. Data indicate mean ± SD normalized to AAV9 (n = 3). Insert shows detection of VCAP-102 capsid in the bottom chamber by dot blot with an anti-AAV9 antibody. p values are derived from an unpaired two-tailed t test.
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