Fig 1.
(A) A single-strand adeno-associated virus (AAV) genetic cassette was engineered by cloning a codon-optimized human ARSB (NM_000046.5) cDNA (opt ARSB) into a genetic cassette containing the ubiquitous EF1α human promoter and the bovine growth hormone polyadenylation [bGH poly(A)] sequence flanked by wild-type AAV2 inverted terminal repeats (ITR). (B) Arylsulfatase B activity was assessed using a colorimetric enzymatic activity assay. Protein harvested from HEK293 cells transfected with optARSB plasmid or GFP plasmid (control) was incubated with substrate, and the fluorescence intensity was measured and expressed as “nmol of 4-methylumbeliferyl sulfate (MUS)/µg of total protein/hour”. Graph is shown as mean with standard deviation. *p < 0.05, Student’s t-test. N.D., not detected. (C) An alamarBlue viability assay assessed cytotoxicity. HEK293 cells were transfected with the optARSB plasmid or GFP plasmid (control). Seventy-two hours after transfection, alamarBlue solution was added, and fluorescence was measured. A concentration of 20 µM etoposide was used as a cytotoxicity positive control. Graph is shown as mean with standard deviation. ns, no significant difference, Tukey’s HSD. ****p < 0.0001, GFP vs Etoposide and optARSB vs Etoposide, Tukey’s HSD.
Table 1.
Animals used in this study.
Fig 2.
Corneal clearing after AAV8-optARSB in MPS VI felines.
(A) Corneas from felines homozygous for the L476P mutation in the ARSB gene (ARSB-/-, affected MPS VI felines) were imaged 28 days after AAV8-optARSB intrastromal injection. Heterozygous feline cornea (ARSB+/-, not affected control felines) is shown for comparison. (B) Clinical corneal scores, consisting of extent and area of corneal opacity and corneal vascularization, were recorded over the experiment period (from 82 to 240 days of age). The individual cornea score for each eye is shown. Corneas were dosed with intrastromal AAV8-optARSB (1e9 vg) or saline injection at 152 days of age. One homozygous eye (Subject #2, OD) was dosed with AAV8-optARSB at 206 days of age following a preceding AAV8-optARSB injection on the contralateral eye (Subject #2, OS) at 152 days of age (sequential dosing). OD: right eye, OS: left eye.
Fig 3.
Confocal microscopy images of corneas in MPS VI felines.
Epithelium layer, superficial stroma, posterior stroma, and endothelium in heterozygote (non-affected) felines and in homozygote (affected MPS VI) felines with or without AAV8-optARSB treatment were imaged at 242 days of age. The sequentially dosed eye, which was injected with AAV8-optARSB on day 206 of age, was also imaged on day 242 of age. Images from untreated and sequentially dosed homozygote posterior stroma show diffuse plump cells with increased cytoplasmic volume.
Fig 4.
Histological evaluation of corneas in MPS VI felines.
Hematoxylin and eosin (A) and Masson’s Trichrome (B) stained sections from heterozygote (non-affected, ARSB+/-) and homozygote (affected MPS VI, ARSB-/-) felines with or without AAV8-optARSB intrastromal injection are imaged. Scale bars: 50 µm.
Fig 5.
Electron microscopy images of corneas in MPS VI felines.
Superficial and deep stroma on longitudinal sections (A) and cross sections (B) of MPS VI feline corneas were imaged with electron microscopy. Heterozygote (non-affected, ARSB+/-) and homozygote (affected MPS VI, ARSB-/-) felines with or without AAV8-optARSB intrastromal injection are imaged. Black arrows indicate corneal keratocytes, showing intracytoplasmic electron-lucent vacuoles in the corneal keratocytes in untreated homozygote feline cornea. Scale bars: 5 µm in (A) and 200 µm in (B).
Fig 6.
Immunofluorescence staining of corneas in MPS VI felines.
Heterozygote (non-affected, ARSB+/-) and homozygote (affected MPS VI, ARSB-/-) feline corneas with or without AAV8-optARSB intrastromal injection were stained with anti-α-smooth muscle actin (α-SMA, a marker of fibrosis, (A)) or with anti-human arylsulfatase B (ARSB, (B)). Corneal stroma close to the endothelial side (Endo) was imaged. Scale bars: 50 µm.