Novel properties of tyrosine-mutant AAV2 vectors in the mouse retina

Abstract

Vectors based on adeno-associated virus serotype 2 (AAV2) have been used extensively in many gene-delivery applications, including several successful clinical trials for one type of Leber congenital amaurosis in the retina. Many studies have focused on improving AAV2 transduction efficiency and cellular specificity by genetically engineering its capsid. We have previously shown that vectors-containing single-point mutations of capsid surface tyrosines in serotypes AAV2, AAV8, and AAV9 displayed significantly increased transduction efficiency in the retina compared with their wild-type counterparts. In the present study, we evaluated the transduction characteristics of AAV2 vectors containing combinations of multiple tyrosine to phenylalanine mutations in seven highly conserved surface-exposed capsid tyrosine residues following subretinal or intravitreal delivery in adult mice. The multiply mutated vectors exhibited different in vivo transduction properties, with some having a unique ability of transgene expression in all retinal layers. Such novel vectors may be useful in developing valuable new therapeutic strategies for the treatment of many genetic diseases.

Figure 1

Distribution of mutated tyrosine residues (depicted in red) on the AAV2 capsid surface. (a) Molecular model of the complete AAV2 capsid assembled from 60 VP3 monomers generated with the Pymol software (www.pymol.org). (b) A “roadmap” showing the position of the mutated tyrosine residues (red), heparan sulfate binding sites (blue), NGR motif (green) known to bind the α5β1 coreceptor, and neutralizing peptides residues (gray) on the surface of the AAV2 capsid. An icosahedral viral asymmetric unit is depicted by the large triangle bounded by two threefold axes (filled triangles) separated by a twofold axis (filled oval) and a fivefold (filled pentagon). A close-up view is shown to the right. This figure was produced with the RIVEM program.⁵⁰ Note that mutated residues 700, 704, and 730 are located close to the positively charged residues involved in the interaction with the heparan sulfate receptor, and tyrosines 700 and 704 are part of a neutralizing epitope region. AAV2, adeno-associated virus serotype 2.

Figure 2

Representative fundus photographs showing GFP expression in vivo after subretinal delivery of multiple Y-F mutant AAV2 vectors in adult mouse retinas. (a) Strong fluorescence was detectable as early as 3 weeks postinjection in eyes treated with the (Y444,500,730F) triple mutant, and remained higher for this mutant (d) than for other vectors at 10 weeks postinjection, (b) AAV2 wild type, (c) double mutant (Y444, 730F), (e) quadruple mutant (Y272,444,500,730F), (f) pentuple mutant (Y272,444,500,704,730F), (g) sextuple mutant (Y252,272,444,500,704,730F), (h) septuple mutant (Y252,272,444,500,700,704,730F). AAV2, adeno-associated virus serotype 2; GFP, green fluorescent protein.

Figure 3

Representative images of GFP expression in retinal sections by immunohistochemistry at 1 month after subretinal vector injection with: (a) double mutant (Y444,730F), (b) triple mutant (Y444,500,730F), (c) quadruple mutant (Y272,444,500,730F), (d) pentuple mutant (Y272,444,500,704,730F), (e) sextuple mutant (Y252,272,444,500,704,730F), and (f) septuple mutant (Y252,272,444,500,700,704,730F). Images were intentionally overexposed to allow for visualization of all transduced inner retinal cells. AAV2, adeno-associated virus serotype 2; gcl, ganglion cell layer; GFP, green fluorescent protein; inl, inner nuclear layer; onl, outer nuclear layer; PR, photoreceptor layer; rpe, retinal pigmented epithelium.

Figure 4

Detection of vector-expressed GFP in rod bipolar or ganglion cells in frozen retinal sections by immunohistochemistry at 1 month following subretinal injections with different AAV2 Y-F mutant vectors. (a,b) Arrows indicate colocalization of GFP (green) and PKC-α positive rod bipolar cells (red) in the retinas treated with the quadruple and pentuple mutant, respectively; (c,d) Colocalization of GFP (green) with TUJ-1 positive (red) ganglion cells for pentuple and sextuple mutant-treated retinas. Bar = 10 µm. AAV2, adeno-associated virus serotype 2; gcl, ganglion cell layer; GFP, green fluorescent protein; ipl, inner plexiform layer; inl, inner nuclear layer; onl, outer nuclear layer; os, outer segment; rpe, retinal pigment epithelium.

Figure 5

Immunohistochemistry for GFP protein expression in flat-mount whole retinas at 1 month following intravitreal delivery. (a) WT AAV2, (b) triple mutant (Y730,500,444F), (c) Comparison of GFP intensity of wild-type AAV2 vector and triple mutant in retinal flatmounts. Values indicate percent GFP intensity relative to treatment with wild-type AAV2 vector. All pictures were taken with the same exposure time to evaluate GFP intensity using ImageJ. AAV2, adeno-associated virus serotype 2; GFP, green fluorescent protein; WT, wild type.

Figure 6

Immunostaining for GFP protein expression in retinal sections at 1 month following intravitreal delivery of (a) double mutant (Y444,730F), (b) triple mutant (Y730,500,444F), (c) quadruple mutant (Y272,444,500,730F), (d) pentuple mutant (Y272,444,500,704,730F), (e) sextuple mutant (Y252,272,444,500,704,730F), and (f) septuple mutant (Y252,272,444,500,700,704,730F). Note the GFP expression throughout all retinal layers with the quadruple and pentuple mutant, and intense labeling of Müller cell outward processes terminating at the outer limiting membrane (arrows), in all mutants analyzed. Bar = 100 µm. AAV2, adeno-associated virus serotype 2; gcl, ganglion cell layer; GFP, green fluorescent protein; inl, inner nuclear layer; onl, outer nuclear layer; PR, photoreceptor layer; rpe, retinal pigmented epithelial layer.

Figure 7

Evaluation of colocalization of GFP (green) with cell-marker proteins (red), as shown by immunohistochemistry of frozen retinal sections at 1 month following intravitreal injections with different AAV2 Y-F mutant vectors in adult mice. (a) Colocalization (yellow) of GFP and some calretinin (red) positive ganglion or amacrine cells after injection of the quadruple mutant. (b,d) Colocalization of GFP (green) and PKC-α (red) positive rod bipolar in the eyes treated with the quadruple or pentuple mutant vector, respectively; (c,e) Insets of panels (b) and (d) at higher magnification showing colocalization of GFP and PKC-α in the inner nuclear layer. Bar = 10 µm (a;b;d) and 5 µm (c;e). AAV2, adeno-associated virus serotype 2; gcl, ganglion cell layer; GFP, green fluorescent protein; ipl, inner plexiform layer; inl, inner nuclear layer; onl, outer nuclear layer; os, outer segments; rpe, retinal pigmented epithelial layer.