AAV gene therapy approaches in the posterior eye resulted in the first FDA-approved gene therapy-based drug. However, application of AAV vectorology to the anterior eye has yet to enter even a Phase I trial. Furthermore, the simple and safe subconjunctival injection has been relatively unexplored in regard to AAV vector transduction. To determine the utility of this route for the treatment of various ocular disorders, a survey of gene delivery via natural AAV serotypes was performed and correlated to reported cellular attachment factors. AAV serotypes packaged with a self-complementary reporter were administered via subconjunctival injection to WT mice. Subconjunctival injection of AAV vectors was without incidence; however, vector shedding in tears was noted weeks following administration. AAV transduction was serotype dependent in anterior segment tissues including the eye lid, conjunctiva, and cornea, as well as the periocular tissues including muscle. Transgene product in the cornea was highest for AAV6 and AAV8, however, their corneal restriction was remarkably different; AAV6 appeared restricted to the endothelium layer while AAV8 efficiently transduced the stromal layer. Reported AAV cellular receptors were not well correlated to vector transduction; although, in some cases they were conserved among mouse and human ocular tissues. Subconjunctival administration of particular AAV serotypes may be a simple and safe targeted gene delivery route for ocular surface, muscular, corneal, and optic nerve diseases.
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This study was supported by grants from the NIH RO1AI072176-06A1 (MH), RO1AR064369-01A1 (MH), and Education Bureau of Hunan Province 14B112 (LJS). A portion of the imaging was done using the Neuroscience Center Microscopy Core Facility equipment, which is supported by funding from the NIH-NINDS Neuroscience Center Support Grant P30 NS045892 and the NIH-NICHD Intellectual and Developmental Disabilities Research Center Support Grant U54HD079124. The authors thank the Vector Core at the University of North Carolina for providing the AAV vectors used in this study, the CGIBD Histology Core and histology technician, Carolyn Suitt, for the work of tissue processing and sectioning, the Animal Histopathology and laboratory Medicine Core and Dr. Ling Wang for the clinical services, the Microscopy Core Facility of the Neuroscience Center and Dr. Michelle S. Itano for the valuable technical assistance in confocal imaging, Dr. Hua Mei for reviewing the data, and Jerry Wu for manuscript proofreading.
Conflict of interest
Matthew Hirsch has several licensed patents not related to this report and has received royalties from Fortress Biotech and Asklepios BioPharmaceutical. Matthew Hirsch is a consultant to Tamid Bio.
Electronic supplementary material
Figure S1:Vector Characterization. (A) Viral genome integrity assessment by alkaline electrophoresis followed by SYBR Gold staining. (B) Vector purity examination by silver staining of AAV capsid proteins.
Figure S2: Safety analysis. (A) Changes in body weight during the experiment; (B) Comparison of activity of ALT in serum obtained pre-, 2 weeks and 8 weeks post-injection. (C) Quantitative evaluation of clinical histopathology scores of H&E stained sections.
Figure S3: Southern blotting detection of mouse ß-actin in tears. Tear samples collected at 1 week (upper panel) and 4 week (lower panel) post-subconjunctival injection were subjected to two rounds of PCR using mouse ß-actin primer set and detected by mouse ß-actin specific probe via Southern blotting. Viral vector genome plus host cell gDNA was used as positive control template.
Figure S4: AAV receptor analysis in mouse conjunctiva (A) and retina (B) by immunofluorescence staining. Anti-HS antibody, anti-EGFR antibody, anti-67 KDa Lam R antibody (recognizes both 37KDa Lam R precursor and 67KDa Lam R) and anti-AAVR antibody were used for HSPG, EGFR, 37/67 KDa Lam R and AAVR, respectively. WGA, SNA and MAL I were used for staining of multivalent sialic acid, α2, 6 sialic acid and α2, 3 sialic acid, respectively. NC-1: Negative controls (no primary antibody) for EGFR, 37/67 KDa Lam R and AAVR staining; NC-2: Negative controls (no primary antibody) for HSPG staining; NC-3: Negative controls for sialic acid staining. Scale Bar=20 µm.
Figure S5: Representative negative control images. Negative controls for WGA staining in Fig. 5 (a, b, c & d). Negative controls for SNA staining in Fig. 5 (e, f, g & h). Negative controls (no primary antibody) for AAVR staining in Fig. 5 (i & k). Negative controls (no primary antibody) for longer exposure time used for the stromal layer of Lam R and AAVR staining (j). Negative controls (no primary antibody) for EGFR and 37/67 KDa Lam R staining in Fig. 5, (l, m, n & o). Negative controls for MAL I staining in Fig. 5 (p, q, r & s).
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Song, L., Llanga, T., Conatser, L.M. et al. Serotype survey of AAV gene delivery via subconjunctival injection in mice. Gene Ther 25, 402–414 (2018). https://doi.org/10.1038/s41434-018-0035-6
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