rAAV-CAscFv59 injection reduces Aβdeposits in the brain of Tg2576 mice. Aβ deposits were detected with 6E10 antibody. (A) rAAV-CAscFv59-injected brain. (B) PBS-injected brain. (C) Morphometric analysis of Aβload. Average percentages of area showing Aβimmunoreactivity are indicated (n = 5 for each group). Scale bars 250 μm (A and B).

Construction of pAAV-CA-scFv59 vector. ITR: inverted terminal repeats; CMV: cytomegalovirus enhancer; β-actin: β-actin promoter; SS: signal sequence; scFv: cDNA for single chain antibody 59; WPRE: woodchuck hepatitis virus post-transcriptional regulatory element; hGH polyA: human growth hormone polyadenylation signal.

Prussian blue staining for detection of hemorrhages in Tg2576 mice. (A) and (B) Blue staining spots scattered through the needle track are seen in the boxed area of the hippocampus of a PBS-injected Tg2576 mouse. (B) is a higher magnification of the boxed area in (A). In order to show iron staining clearly, the picture was taken before counter staining with pararosaniline. Arrows indicate iron staining associated with phagocytic cells. (C) A brain section of a Tg2576 mouse subjected to rAAV-CAscFv59 injection shows no staining. Scale bars 50 μm (A and C) and 10 μm (B).

scFv59 is readily detectable in the brain three weeks and one year after injection of rAAV-CAscFv59 without showing cytotoxicity. (A) rAAV-CAscFv59 (5 × 10⁹ vg/hemisphere) was injected into the hippocampal regions of C57BL/6 mice. Three weeks later, expressed scFv59 was detected in the hippocampus using anti-FLAG M2 antibody.
The arrowheads indicate heavy neuropil staining in the dentate gyrus. (B) As a control, PBS-injected mouse brain is shown. (C) rAAV-CAscFv59 was similarly injected into the hippocampal regions of mice. One year after injection, expression of scFv59 was similarly detected. (D) As a control, PBS-injected mouse brain is shown. (E) Possible cell death (apoptosis) associated with long-term expression of scFv59 in the brain of the mice was investigated by TUNEL staining. (F) As a control, PBS-injected mouse brain was used. The insets are higher magnifications of the boxed area indicated by the arrows. Scale bars 200 μm (A through F) and 40 μm (insets).

Secretion and Aβ-immunoreactivity of scFv59. (A) Expression and secretion of scFv59 in cultured COS cells. COS cells were transfected with pAAV-CAscFv59, or pAAV-CAWPRE (no cDNA insert). Three days after transfection, the media were collected and cells were harvested. Twenty ng protein/lane of cell lysate was loaded on 4–15% Tris-HCl-polyacrylamide gel. For media, 10nl of medium from each sample was mixed with 10 μl of 2× Laemmli buffer and loaded on the same gel. After electrophoresis and electroblotting to a PVDF membrane, scFv59 was visualized by anti-FLAG M2 antibody. (B) scFv59 secreted from COS cells reacts with Aβ deposits in blood vessels as well as in neuritic and diffuse plaques in a brain section from a patient with AD. (C) A serial section of (B) stained with 6E10 anti-Aβ antibody for comparison. (D) A serial section of (B) immunostained with scFv-Gag secreted from COS cells shows no staining. COS cells were transfected with pAAV-CAscFv59 or pAAV-CAscFv-Gag and, 3 days after transfection, media were harvested. The media were diluted to 1:4 with 5% horse serum in 0.1 M Tris-saline (pH 7.4) and used as primary antibodies. For detection of scFvs, the mouse monoclonal anti-FLAG M2 antibody and biotinylated anti-mouse IgG were used as secondary and tertiary antibody, respectively. Biotinylated anti-mouse IgG was used as secondary antibody for 6E10 antibody. The arrowheads, arrows, and asterisks indicate Aβ deposits in blood vessels, neuritic, and diffuse plaques, respectively. Scale bars 200 μm (B through D).