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Retinal Physician

Article

Suprachoroidal Gene Transfer

A noninvasive approach to transduce photoreceptors and retinal pigment epithelium.

By: Peter A. Campochiaro, MD
Retinal Physician February 1, 2020 Vol 17, Issue Special Edition 2020 Page(s): 33-35

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Ocular gene therapy has tremendous potential for the treatment of inherited retinal degenerations (IRDs) and to provide sustained delivery of therapeutic proteins.1-5 Subretinal injection of adeno-associated viral (AAV) vectors provides strong, long-duration transgene expression and is currently the preferred approach. However, subretinal injection of viral vectors requires taking the patient to the operating room and performing a vitrectomy, which carries a 1% risk of postoperative retinal detachment and a high rate of cataract formation. The vector is injected through the retina into the subretinal space where the vector-containing fluid separates the photoreceptors from the retinal pigmented epithelium (RPE). The retinal detachment, or bleb, provides a confined space where many vector particles contact and enter photoreceptors and RPE, which has advantages and disadvantages. An advantage is that a high concentration of vector contacts and enters cells within the region of the bleb, resulting in high expression of transgene in those cells. A disadvantage is that there is little or no expression outside the region of the bleb, which is the majority of the retina. Subretinal injection of 250 µl of vector-containing fluid results in transduction of about 1/5 of photoreceptors and RPE. Another disadvantage is that separation of photoreceptor outer segments from the RPE can be damaging to photoreceptors, and while recovery is common, it does not always occur. This is particularly problematic when the bleb extends under the fovea, because reduction in visual acuity can occur.3 Eyes with retinal degeneration may have subretinal fibrosis, which increases adherence of photoreceptors to the RPE and makes it more difficult to perform a subretinal injection. High infusion pressure may be needed, and when fluid extends under the fovea, which is thin, the pressurized fluid may break through, creating a macular hole.

STUDY OF SUPRACHOROIDAL DELIVERY

It has recently been demonstrated that drugs can be delivered to the retina by injecting them into the suprachoroidal space.6 Fluorescent particles injected into the suprachoroidal space near the limbus are visualized around the entire circumference of the eye.7 Clinical trials using microneedles with a length that approximates the thickness of the sclera have demonstrated that suprachoroidal injections of triamcinolone acetonide are safe and result in prolonged improvement in macular edema due to uveitis or retinal vein occlusion.8,9 Ocular sections obtained immediately after suprachoroidal injection of 3 µl of ink 1 mm posterior to the limbus in a rat showed that the choroid was thickened and filled with ink on the side of the injection. Although the thickness gradually decreased, the ink extended all the way to the ora serrata on the side of the eye opposite the injection.10 The ink filled the entire choroid from the sclera to the basal surface of the RPE but did not enter the RPE or retina. In contrast, AAV8 vectors injected into the suprachoroidal space are not confined by the basal surface of the RPE, but instead enter the RPE and photoreceptors.10 Ocular sections 2 weeks after suprachoroidal injection of 2.85x1010 gene copies (GC) of AAV8.GFP in rats showed green fluorescence in the choroid, RPE, and photoreceptors throughout the entire eye.

Similar widespread expression of GFP was seen 2 weeks after suprachoroidal injection of 50 µl containing 4.75x1011 GC of AAV8.GFP in monkey and pig eyes, which are more similar in size to human eyes. As opposed to ocular sections for which magnification allows visualization of modest fluorescence, only very strong fluorescence can be visualized on flat mounts of the entire retina or RPE. Strong fluorescence is visualized throughout about 1/5 of the retina and RPE after suprachoroidal injection of 4.75x1011 GC of AAV8.GFP in monkeys and pigs, and this can be significantly increased by performing a second suprachoroidal injection of AAV8.GFP on the opposite side of the eye. Thus, with multiple suprachoroidal injections of an AAV8 vector, high-level transgene expression can be obtained in essentially all RPE and photoreceptors. This is ideal for treatment of IRDs, because the optimal approach is to intervene early and substitute a normal gene for a defective gene to prevent dysfunction and death of as many photoreceptors and/or RPE cells as possible. Early intervention means treatment of young, asymptomatic patients who have a lot to lose because they may have many years of functional vision remaining before visual disability occurs and thus the reduced invasiveness and increased safety of suprachoroidal gene transfer is important.

Figure 1. Widespread expression of green fluorescent protein (GFP) in retinal pigmented epithelium (RPE) and photoreceptors after suprachoroidal injection of AAV8.GFP. Two weeks after suprachoroidal injection of 3 µl containing 2.85 x1010 genome copies of AAV8.GFP, a transverse ocular section at the level of the equator showed fluorescence from GFP expression in the retina and RPE. The fluorescence was very strong on the side of the eye in which the injection was done, but fluorescence was detectable around the entire circumference of the eye. High magnification view of the boxed region of the section that is stained with an antibody directed against GFP (red) showed that the GFP was present in the RPE (partially separated in this location), photoreceptor inner segments (IS) and outer segments (OS) and some photoreceptor cell bodies in the outer nuclear layer (ONL). The nuclei of the photoreceptors are stained blue.
Figure 1. Widespread expression of green fluorescent protein (GFP) in retinal pigmented epithelium (RPE) and photoreceptors after suprachoroidal injection of AAV8.GFP. Two weeks after suprachoroidal injection of 3 µl containing 2.85 x1010 genome copies of AAV8.GFP, a transverse ocular section at the level of the equator showed fluorescence from GFP expression in the retina and RPE. The fluorescence was very strong on the side of the eye in which the injection was done, but fluorescence was detectable around the entire circumference of the eye. High magnification view of the boxed region of the section that is stained with an antibody directed against GFP (red) showed that the GFP was present in the RPE (partially separated in this location), photoreceptor inner segments (IS) and outer segments (OS) and some photoreceptor cell bodies in the outer nuclear layer (ONL). The nuclei of the photoreceptors are stained blue.

SUPRACHOROIDAL DELIVERY IN GENE THERAPY

The potential of using suprachoroidal gene transfer for sustained delivery of a therapeutic protein was tested using RGX-314 (REGENXBIO), an AAV8 vector expressing a vascular endothelial growth factor (VEGF)-neutralizing protein. Suprachoroidal injection of RGX-314 strongly suppressed VEGF-induced vascular leakage in a rat model relevant to retinal/choroidal vascular diseases, and efficacy and level of expression were similar to that seen with subretinal injection of the same dose of RGX-314.10 This suggests that it may be feasible to deliver RGX-314 noninvasively in an outpatient setting to obtain sustained suppression of VEGF. This could revolutionize the treatment of highly prevalent retinal and choroidal vascular diseases.

Although suprachoroidal gene transfer appears promising for IRDs and sustained delivery of therapeutic proteins, there are still gaps in our knowledge regarding this approach. Suprachoroidal injection of AAV9.GFP resulted in widespread expression of GFP in photoreceptors and RPE similar to that seen with AAV8.GFP, but expression was weak and limited after suprachoroidal injection of AAV2.GFP.10 Thus, there are differences among AAV vectors regarding transduction efficiency after suprachoroidal injection, and more work is needed to determine the best vectors for this route of delivery. Pre-existing anti-AAV antibodies do not appear to have a negative impact on subretinal injection of AAV vectors, but they may reduce transgene expression after intravitreous injections of AAV vectors.11-13 It is not yet known if high anti-AAV8 antibody titers negatively affect expression after suprachoroidal injection of AAV8 vectors, so more work is needed in this area as well.

Similar to suprachoroidal injections, intravitreous injections of viral vectors are relatively noninvasive and can be done in an outpatient setting. However, the internal limiting membrane (ILM) binds AAV2, AAV8, and other wild-type AAV vectors, markedly reducing penetration into the retina and level of expression after intravitreous injection of AAV vectors. Mutant AAV vectors, in which surface tyrosine residues involved in ubiquitination and degradation are replaced with phenylalanines, increase transgene expression at lower vector doses, which increases effectiveness of small amounts of vector that penetrate the ILM.14,15 Directed evolution by creation of mutant AAV vector libraries and selecting for vectors with reduced binding to the ILM has identified novel AAV vectors with increased expression after intravitreous injection,16 but it is not yet known if expression levels comparable to those obtained after subretinal or suprachoroidal injection can be obtained.

CONCLUSION

Preclinical studies have demonstrated widespread, high-level transgene expression after suprachoroidal injection of AAV8 and AAV9, but not AAV2 vectors, in rats, nonhuman primates, and pigs. Total transgene expression after a single suprachoroidal injection of AAV8 vector is comparable to that seen after subretinal injection of the same vector dose, and can be increased by multiple suprachoroidal vector injections. Studies are needed to determine if these findings are translatable into the clinic, and if so, suprachoroidal gene transfer may provide a noninvasive approach for ocular gene therapy that can be done in an outpatient setting. RP

Peter A. Campochiaro, MD, is the Eccles Professor of Ophthalmology and Neuroscience at the Wilmer Eye Institute, Johns Hopkins University School of Medicine, in Baltimore, Maryland. Dr. Campochiaro reports, related to the current manuscript, that he is an investigator for and has received grants from REGENXBIO Inc. Reach Dr. Campochiaro at pcampo@jhmi.edu.

REFERENCES

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Editor’s note: This article is part of a special edition of Retinal Physician that was supported by REGENXBIO.

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