Transduction of the inner mouse retina using AAVrh8 and AAVrh10 via intravitreal injection
Research highlights
► AAVrh8 and AAVrh10 can transduce several cell types of the inner retina. ► AAVrh10 strongly transduces horizontal cells. ► AAVrh8 and AAVrh10 are excellent candidates for gene delivery to the inner retina via intravitreal injections.
Introduction
Adeno-associated virus (AAV) vectors have proven to be exceptionally efficient for in vivo gene delivery to several tissues (Grimm, 2002). More than 100 different AAV capsids have been cloned from human and non-human primates and a considerable number have been engineered to pseudotype AAV2 vectors (Surace and Auricchio, 2008). Some of the newer serotypes have shown exceptional efficiency for in vivo gene delivery to certain tissues, e.g. AAV8 in brain (Broekman et al., 2006, Klein et al., 2006) and liver (Wang et al., 2005), and AAV9 in brain (Cearley and Wolfe, 2006) and heart (Inagaki et al., 2006, Pacak et al., 2006).
The retina has been a particularly attractive target for viral-mediated gene-therapy owing to its relatively small isolated environment, which facilitates specific delivery of therapeutic agents to that tissue, as well as an immune-privileged environment which shields it from the rest of the body (Allocca et al., 2006, Martin et al., 2002). For example, several studies have demonstrated the efficacy of AAV-mediated gene therapy for treating Leber congenital amaurosis (LCA) in various animal models, including non-human primates (Bennicelli et al., 2008, Jacobson et al., 2006, Pawlyk et al., 2005). In fact, subretinal injection of AAV vectors has shown great promise in several concluded phase I clinical studies in LCA patients (Bainbridge et al., 2008, Cai et al., 2009, Cideciyan et al., 2008, Hauswirth et al., 2008, Maguire et al., 2008).
AAV1 through 9 vectors have been tested for their transduction properties in the retinas of rodents and primates (Auricchio et al., 2001, Lebherz et al., 2008, Rabinowitz et al., 2002, Yang et al., 2002). AAV2 and AAV5 have been used extensively in the retina and have proven effective for gene delivery to photoreceptors, retinal pigment epithelium cells (RPE) and ganglion cells (Allocca et al., 2006, Dinculescu et al., 2005). AAVrh8 and AAVrh10 are two additional AAV capsids isolated from rhesus monkey (Gao et al., 2003), and recombinant vectors using these capsids have yet to be tested in retina. Generally, intravitreal injections of AAV vectors lead to transduction of ganglion cells while subretinal injections lead to transduction of photoreceptors and RPE. We undertook this study to identify new AAV vectors capable of efficiently transducing additional retinal cell types for use as new tools to rapidly manipulate the molecular properties of these cells for future functional studies. In this brief report, we evaluated the cellular tropism of AAVrh8 and AAVrh10 vectors encoding enhanced green fluorescent protein (EGFP) in the mouse retina after intravitreal delivery. We also assessed potential negative effects of the resultant transductions by observing changes in glial fibrillary accessory protein (GFAP) expression in retinal Müller cells.
Section snippets
Materials & methods
All reagents were purchased from Fisher Scientific (Waltham, MA) or Sigma (St. Louis, MO) unless otherwise indicated.
AAVrh8 and AAVrh10 transduction following intravitreal injection
In order to determine the cellular tropism of AAVrh8 and AAVrh10 vectors in the mouse retina, intravitreal injections were performed in the eyes of adult mice using 2 μl of 1 × 1013 gc/μl of either serotype and transduction was evaluated after a 4 week incubation period. This was chosen based on a pilot study that we conducted where we injected 2–3 μl of AAVrh8 at ∼1 × 1010 gc/μl and assessed transduction efficiency at 3, 4, 5, 8 and 9 weeks after injection. We found cell transduction patterns
Discussion
Our current study demonstrated that both AAVrh8 and AAVrh10 vectors can strongly transduce cells in the INL. Other more widely used AAV serotypes, such as AAV2 vectors have shown the most promise for transducing cells in the GCL and ONL (Allocca et al., 2006, Martin et al., 2002, Surace and Auricchio, 2008), while serotypes such as AAV8 (AAV vectors with an AAV2 genome and AAV8 capsid) have shown some ability to transduce cells in the inner retina (Lebherz et al., 2008). AAV2 vectors have
Acknowledgments
We would like to thank Zarmina Khankel and Felicitas B. Eldred for their technical assistance and Jamit Argawal for the creation of the custom Image J plugin used in determining our thresholds for normalization. This research was supported by NIH EY004785 to W.D. Eldred.
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Current address: Department of Neurology and Gene Therapy Center, University of Massachusetts Medical School, Worcester, MA 01605, USA.