High-Resolution Histological Landscape of AAV DNA Distribution in Cellular Compartments and Tissues following Local and Systemic Injection

Figure 1. AAV DNA Distribution in the TA Muscle at Different Time Points after i.v. Injection AAV DNA distribution in the TA muscle at 2 and 24 h, 5 and 8 days, and 2, 4, 12, and 24 weeks after i.v. injection. Cyan arrowhead points to sarcolemma; red arrow points to cytoplasmic AAV DNA; yellow arrow points to nuclei without AAV DNA; blue arrow points to nuclei with AAV DNA. Bottom rows are the high-magnification images of the green boxed areas in the corresponding top row images. Scale bars, 50 μm (for both ×20 and ×60 original magnification).

Figure 2. AAV DNA Distribution in the Heart at Different Time Points after i.v. Injection AAV DNA distribution in the heart at 2 and 24 h, 5 and 8 days, and 2, 4, 12, and 24 weeks after i.v. injection. Cyan arrowhead points to sarcolemma; red arrow points to cytoplasmic AAV DNA; yellow arrow points to nuclei without AAV DNA; blue arrow points to nuclei with AAV DNA. Bottom rows are the high-magnification images of the green boxed areas in the corresponding top row images. Scale bars, 50 μm (for both ×20 and ×60 original magnification).

Figure 3. AAV DNA Distribution in the Liver at Different Time Points after i.v. Injection At 5 days after i.v. injection, a massive amount of the AAV DNA accumulated in the nuclei, with spotty signals in the cytoplasm of hepatocytes. From 4 to 24 weeks after i.v. injection, the AAV DNA exclusively resided in the nuclei. Scale bars, 100 μm (for ×10 original magnification), 50 μm (for both ×20 and ×60 original magnification).

Figure 4. AAV DNA Distribution in the Kidney at 5 Days after i.v. Injection The distribution features of AAV are presented for both the cortex and medulla of the kidney. Clearly, the AAV DNA accumulated in the cortical glomerulus (blue arrow). Almost all glomeruli in the cortex contain a massive amount of the AAV DNA. However, no AAV DNA was observed in the Bowman’s capsule (green arrow). The AAV DNA was scattered in the medulla of the kidney, with spotty signals in the epithelium of proximal and distal convoluted tubules. OS, outer stripe; IS, inner stripe. The yellow box indicates the region amplified in higher magnification in the row below. Scale bars, 100 μm (for ×10 original magnification), 50 μm (for both ×20 and ×60 original magnification).

Figure 5. AAV DNA Distribution in the Lymphoid Tissues at 5 Days after i.v. Injection (A) AAV DNA in the spleen. Massive distribution in the spleen was observed, accompanied by highly concentrated localization in the white pulp (region II) and scattered cluster distribution in the red pulp (region I). (B) AAV DNA in the lymph node (LN). The accessory mandibular LN, which is located near the submandibular salivary glands, was harvested and the AAV DNA was examined by RNAscope. Substantial quantities of the AAV DNA were found in the LN, with distinguished accumulation in the germinal centers of the lymphoid nodules on the periphery of the LN (region II) and scattered cluster distribution in the interfollicular zone (region I). The border of lymphoid nodules, which contains germinal centers (mainly B cells), is marked with blue lines. Scale bar, 50 μm (for ×20 original magnification).

Figure 6. AAV DNA Distribution in the Teste at 5 Days and 4 Weeks after i.v. Injection The seminiferous tubules (S) are separated by interstitium, where Leydig cells and blood vessels are located. At 5 days after injection, AAV DNA was confined to interstitium and there was no AAV DNA inside the seminiferous tubules, which are lined with Sertoli cells and germ cells. The same distribution pattern was seen at 4 weeks after i.v. injection. Green arrow points to AAV DNA in interstitial cells; red arrow points to no AAV DNA in Sertoli and germ cells. Scale bars, 100 μm (for ×10 original magnification), 50 μm (for both ×20 and ×60 original magnification).

Figure 7. AAV DNA Distribution in the Ovary at 5 Days and 4 Weeks after i.v. Injection (A) AAV DNA distribution in the ovary at 5 days after i.v. injection. The AAV DNA was scattered in the medulla but highly accumulated in the corpus luteum. No AAV DNA was seen in follicles. The AAV DNA was found in lutein cells and thecal cells but not in granulosa cells and oocytes. (B) AAV DNA distribution in the ovary at 4 weeks after i.v. injection. The AAV DNA in the corpus luteum and thecal cells disappeared, but the AAV DNA in the medulla persisted. No AAV was seen inside the follicles. CL, corpus luteum; O, oocyte; ZG, zona granulosa; TF, theca folliculi. Scale bars, 50 μm (for both ×20 and ×60 original magnification).

Figure 8. AAV DNA Distribution in the TA Muscle at Different Time Points after i.m. Injection At 2 and 24 h after i.m. injection, a considerable amount of the AAV DNA was seen along the muscle membrane, with occasional cytoplasmic and nuclear localization at 24 h after injection. From 5 days to four weeks after i.m. injection, most of the AAV DNA resided in the nuclei (blue arrow), with isolated cytoplasmic distribution (red arrow). From 12 to 24 weeks, no viral DNA was seen intercellularly or intracellularly. Arrowhead points to AAV DNA at the membrane; red arrow points to AAV DNA in the cytoplasm; yellow arrow points to nuclei without AAV DNA; blue arrow points to nuclei with AAV DNA. Scale bars, 50 μm (for both ×20 and ×60 original magnification).

Figure 9. AAV DNA Distribution in the Liver at 5 Days after Bilateral and Unilateral i.m. Injections (A) Distribution of the AAV DNA in the liver after bilateral tibialis anterior (TA) injection. A significant amount of the AAV DNA was found in the nuclei of hepatocytes. (B) The AAV DNA in the liver after unilateral TA injection. Pronounced amount of the AAV DNA was detected in the liver, although to a lesser extent than that of bilateral TA injection. Scale bars, 100 μm (for ×10 original magnification), 50 μm (for both ×20 and ×60 original magnification).

Figure 10. AAV DNA Distribution in the Kidney, LN, Spleen, Tibia Bone Marrow, and Testis at 5 Days after Bilateral TA Injections AAV DNA distribution in the kidney, accessory mandibular LN, spleen, tibia bone marrow, and testis at 5 days after bilateral TA injections. Scale bars, 100 μm (for ×10 original magnification), 50 μm (for both ×20 and ×60 original magnification).

Figure 11. AAV DNA Distribution in the Ovary at 5 days after Bilateral TA Injections Distribution of the AAV DNA in the ovary at 5 days after bilateral TA injection. CL, corpus luteum; O, oocyte; ZG, zona granulosa; TF, theca folliculi. Scale bars, 50 μm (for both ×20 and ×60 original magnification).