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. 2011 Jun 22;3(88):88ra54.
doi: 10.1126/scitranslmed.3002103.

Dosage thresholds for AAV2 and AAV8 photoreceptor gene therapy in monkey

Luk H Vandenberghe  1 , Peter Bell, Albert M Maguire, Cassia N Cearley, Ru Xiao, Roberto Calcedo, Lili Wang, Michael J Castle, Alexandra C Maguire, Rebecca Grant, John H Wolfe, James M Wilson, Jean Bennett
Affiliations

V体育ios版 - Dosage thresholds for AAV2 and AAV8 photoreceptor gene therapy in monkey

Luk H Vandenberghe et al. Sci Transl Med. .

Erratum in

  • Sci Transl Med. 2011 Dec 7;3(112):112er9

"VSports" Abstract

Gene therapy is emerging as a therapeutic modality for treating disorders of the retina. Photoreceptor cells are the primary cell type affected in many inherited diseases of retinal degeneration VSports手机版. Successfully treating these diseases with gene therapy requires the identification of efficient and safe targeting vectors that can transduce photoreceptor cells. One serotype of adeno-associated virus, AAV2, has been used successfully in clinical trials to treat a form of congenital blindness that requires transduction of the supporting cells of the retina in the retinal pigment epithelium (RPE). Here, we determined the dose required to achieve targeting of AAV2 and AAV8 vectors to photoreceptors in nonhuman primates. Transgene expression in animals injected subretinally with various doses of AAV2 or AAV8 vectors carrying a green fluorescent protein transgene was correlated with surgical, clinical, and immunological observations. Both AAV2 and AAV8 demonstrated efficient transduction of RPE, but AAV8 was markedly better at targeting photoreceptor cells. These preclinical results provide guidance for optimal vector and dose selection in future human gene therapy trials to treat retinal diseases caused by loss of photoreceptors. .

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V体育官网 - Figures

Fig. 1
Fig. 1
GFP expression in monkey retina. Montages of photographs taken in vivo of monkey retinas 1 month after subretinal injection of AAV2 or AAV8 at 1010 or 1011 genome copy (GC) doses. Blue light was used for GFP excitation; GFP expression, green areas. Clockwise from top left: animal 18204, right eye; 18155, left eye; 18221, right eye; 18226, left eye.
Fig. 2
Fig. 2
Retinal pathology after highest-dose vector injection in monkey retina. (A) Correlating histology and live retinal imaging identifies heterogeneous GFP expression in the vector-exposed part of the retina. A halo-like GFP pattern (green rim) was observed by imaging of the retina (center inset) after a 1011 genome copy dose injection of the AAV2 vector subretinally (animal 18226, right eye). Histology along an axis (center inset, dotted lines) that traverses the bleb, the optic disc, and the halo pattern (I and II) shows that the rims of the GFP halo (see inset I) are defined by GFP-positive RPE (green), whereas adjacent RPE does not express GFP (inset II) (GFP, green; DAPI staining of nuclei, blue). (B) DAPI staining (blue) of a section from a monkey eye injected subretinally with AAV2 (animal 18144, right eye) showing normal outer and inner nuclear layers with only minimal GFP fluorescence (green; left). This section is adjacent to a region where the nuclear layers are disturbed (abnormal; right). (C) Retina from the right eye of monkey 18199 after subretinal injection of AAV8 showing DAPI-stained nuclei (blue) and GFP expression (green) illustrates loss of retinal architecture and GFP on the left while retaining some GFP expression but abnormal retinal structure on the right. (D) Retinal section from animal 18144 (right eye) showing the abnormal portion in (B) stained with H&E. (E) H&E-stained section corresponding to the right part of the retina shown in (C) (animal 18199, right eye). Scale bars, 500 μm [(A) to (C)] and 100 μm [(D) and (E)].
Fig. 3
Fig. 3
GFP transgene expression in the monkey retina. (A) Comparison of GFP expression from histological analysis of monkey retinas after subretinal injection with AAV2 or AAV8 with the designated number of genome copies. Clockwise from top left: animal 18168, left eye; animal 18238, right eye; animal 18155, right eye; animal 18226, right eye. (B) GFP expression after subretinal injection of AAV8 as a function of dose from 108 to 1011 genome copies (GC). Pictures are taken with equal exposure. Due to the intensity of GFP at the high dose, photoreceptor transduction in lower doses is less obvious. Clockwise from top left: animal 18204, left eye; animal 18217, left eye; animal 18208, right eye; animal 18238, right eye. (C) Cellular transduction characteristics after subretinal injection exposing the fovea to 1011 genome copies of AAV2 versus 1010 genome copies of AAV8. Both eyes show strong GFP expression in the RPE, but cone photoreceptors in the foveal pit of the AAV8-injected retina also are GFP-positive, as is the outer plexiform layer (OPL) in the AAV2-exposed retina (indicated by white arrowhead). (D) Transduction of Müller glial cells [with nuclei in the inner nuclear layer (INL)] after injection of AAV2 or AAV8. DAPI stain (blue) shows nuclear layers. Animal 18226, right eye (AAV2); animal 18204, right eye (AAV8). Scale bars, 100 mm. RPE, retinal pigment epithelium; ONL, outer nuclear layer; GCL, ganglion cell layer.
Fig. 4
Fig. 4
Quantitative analysis of retinal transduction with AAV2 and AAV8. (A and B) Whole-mount retinal fluorescence after necropsy. Eyeballs were fixed, and the cornea, lens, and vitreous humor were removed to expose the posterior eye cup. Relative fluorescence was measured in a Xenogen Lumina IVIS imager and normalized to the fluorescence signal from an uninjected control eye. Eyes injected with 150 μl of 1010 genome copies (A) and 1011 genome copies (B) of AAV2 or AAV8 vector are shown. (C and D) Morphometric analysis of RPE and photoreceptor (PR) transduction by AAV2 and AAV8. Relative intensity (C) and relative area (D) of the GFP expression signal in RPE and PR were established at doses of 109 and 1010 genome copies based on morphometric histological analysis within the vector-exposed area. Numbers shown identify the animal used. L, left eye; R, right eye.
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