Journal: Human Gene Therapy

Article Title: Direct and Retrograde Transduction of Nigral Neurons with AAV6, 8, and 9 and Intraneuronal Persistence of Viral Particles

doi: 10.1089/hum.2012.174

Figure Lengend Snippet: Intact viral capsids and DNase I-protected viral genomes remained present in the nigrostriatal system, weeks after AAV injections. (a–d) Antibody ADK1a, recognizing assembled native AAV1 capsid was tested for its capacity to detect AAV6 intact capsid in HEK-293T cells, 48 h after infection with recombinant AAV6 CMV EGFP. (a) native EGFP fluorescence in transduced HEK293T cells. (b) Immunofluorescence labeling with ADK1a antibody. (c) Overlay of (a) and (b) demonstrating AAV6 viral particle detection in the periphery of transduced cells by antibody ADK1a. No signal was detected when infected cells were incubated with secondary antibody only (d). (e–h) Coronal sections through rat SN, 4 weeks after injection with 1.9×1010 vg of AAV6 CMV EGFP. Co-immunofluorescence labeling with anti-GFP antibody (e), ADK1a antibody (f), and the nuclear marker DAPI (g) showed accumulation of AAV6 intact capsid in the nucleus of nigral neurons [see overlay in (h)]. No signal was detected with ADK1a antibody in the noninjected SN (m). Likewise, intact AAV6 particles persist in striatal neurons, 4 weeks after striatal AAV6 injections (i–l). Nuclear accumulation of AAV6 assembled capsid was demonstrated in overlays (l) of GFP (i), ADK1a (j), and DAPI (k) immunofluorescence labelings. No ADK1a signal was detected in the uninjected side (n). (o) Detection of DNase I-protected AAV genomes in the rat SN, 8 weeks after injection with 1.8×107 TU of AAV6 (n=3). In parallel, noninjected SN was mixed with 1.8×107 TU of the same viral preparation, either intact (n=3) or after capsid denaturation through heat treatment (n=3). The graph indicates the ratio of vg measured in DNase I–treated samples to untreated samples, indicative of the proportion of DNase-resistant, encapsidated vg (ANOVA, Fisher test, *p<0.05; **p<0.01). DAPI, 4′,6-diamidino-2-phenylindole.

Article Snippet: Optimal DNase I digestion of AAV decapsidated genomes, with minimal degradation of encapsidated genomes, was obtained with 30-min digests at 37°C using 0.3 U DNase I (M6101, Promega).

Techniques: Infection, Recombinant, Fluorescence, Immunofluorescence, Labeling, Incubation, Injection, Marker