IntroductionAdeno-associated viral (AAV) vectors are widely used for stable in vivo gene transfer to terminally differentiated or quiescent cells such as muscle fibers, hepatocytes, neurons, retinal cells, and others. These vectors, derived from a nonpathogenic, replicationdefective parvovirus with a small single-stranded (ss) DNA genome, have recently been successfully used in clinical gene transfer for inherited blindness and also show promise for other diseases. 1,2 Eight years ago, Zaiss et al 3 found that ssAAV serotype-2 vectors caused only a weak and highly transient innate immune response in the liver, suggesting that inflammatory reactions to AAV are negligible. Numerous animal studies have shown stable correction of genetic diseases by hepatic AAV gene transfer that may, in part, be because of the low innate immune profile of the vector, avoiding inflammatory signals. 4 In humans, hepatic gene transfer with AAV2 has been hampered by pre-existing adaptive immunity after natural infection in the form of neutralizing antibodies and capsid-specific CD8 ϩ T cells. 5 Numerous changes to capsid and vector genomes have been developed in recent years in attempts to improve gene transfer efficacy and possibly evade immunity. For example, AAV8 shows substantially higher transduction efficiency in mouse liver and reduced activation of capsid-specific T cells, and it facilitates tolerance induction to transgenes. 6,7 Furthermore, prevalence for neutralizing antibodies in humans is markedly lower to AAV8 than to AAV2. 8 In another set of investigations, replacing surfaceexposed tyrosine residues to phenylalanine has been shown to improve gene transfer for several serotypes. The resulting reduction in capsid phosphorylation in turn reduces accumulation in the cytoplasm (in favor of trafficking to the nucleus) and ubiquitination of capsid. 9 AAV2 gene transfer to hepatocytes was most improved by a combination of 3 Tyr-Phe changes in amino acid residues 444, 500, and 730. 10 Modifications of the recombinant AAV genome also can improve transduction rates. Being ss, the ssAAV genome has to be converted to a double-stranded form in the nucleus of an infected cell for transgene expression to occur. To overcome this ratelimiting step, self-complementary (sc)AAV vectors were developed by elimination of the terminal resolution site in one of the inverted terminal repeats (ITRs). 11 For such a genome to be packaged into capsid, the size of the expression cassette has to be further reduced to not exceed the packaging limit. Two groups reported optimized scAAV vectors for treatment of the X-linked bleeding disorder hemophilia B (coagulation factor IX deficiency) by liver gene transfer. 12,13 The hepatic microenvironment is more tolerogenic than that of many other tissues. 14 For example, we were able to tolerize hemophilia B mice to human factor IX (hF.IX) by hepatic ssAAV2 gene transfer. This protocol was successful in several strains, with the exception of C3H. 15 Nonetheless, AAV8 and AAV2(Y444/500/ 730F) vectors were able to...

Development of viral vectors capable of transducing photoreceptors by less invasive methods than subretinal injection would provide a major advancement in retinal gene therapy. We sought to develop novel AAV vectors optimized for photoreceptor transduction following intravitreal delivery and to develop methodology for quantifying this transduction in vivo. Surface exposed tyrosine (Y) and threonine (T) residues on the capsids of AAV2, AAV5 and AAV8 were changed to phenylalanine (F) and valine (V), respectively. Transduction efficiencies of self-complimentary, capsid-mutant and unmodified AAV vectors containing the smCBA promoter and mCherry cDNA were initially scored in vitro using a cone photoreceptor cell line. Capsid mutants exhibiting the highest transduction efficiencies relative to unmodified vectors were then injected intravitreally into transgenic mice constitutively expressing a Rhodopsin-GFP fusion protein in rod photoreceptors (Rho-GFP mice). Photoreceptor transduction was quantified by fluorescent activated cell sorting (FACS) by counting cells positive for both GFP and mCherry. To explore the utility of the capsid mutants, standard, (non-self-complementary) AAV vectors containing the human rhodopsin kinase promoter (hGRK1) were made. Vectors were intravitreally injected in wildtype mice to assess whether efficient expression exclusive to photoreceptors was achievable. To restrict off-target expression in cells of the inner and middle retina, subsequent vectors incorporated multiple target sequences for miR181, an miRNA endogenously expressed in the inner and middle retina. Results showed that AAV2 containing four Y to F mutations combined with a single T to V mutation (quadY−F+T−V) transduced photoreceptors most efficiently. Robust photoreceptor expression was mediated by AAV2(quadY−F+T−V) −hGRK1−GFP. Observed off-target expression was reduced by incorporating target sequence for a miRNA highly expressed in inner/middle retina, miR181c. Thus we have identified a novel AAV vector capable of transducing photoreceptors following intravitreal delivery to mouse. Furthermore, we describe a robust methodology for quantifying photoreceptor transduction from intravitreally delivered AAV vectors.

Adeno-associated virus (AAV) has proven an effective gene delivery vehicle for the treatment of retinal disease. Ongoing clinical trials using a serotype 2 AAV vector to express RPE65 in the retinal pigment epithelium have proven safe and effective. While many proof-of-concept studies in animal models of retinal disease have suggested that gene transfer to the neural retina will also be effective, a photoreceptor-targeting AAV vector has yet to be used in the clinic, principally because a vector that efficiently but exclusively targets all primate photoreceptors has yet to be demonstrated. Here, we evaluate a serotype 5 AAV vector containing the human rhodopsin kinase (hGRK1) promoter for its ability to target transgene expression to rod and cone photoreceptors when delivered subretinally in a nonhuman primate (NHP). In vivo fluorescent fundus imaging confirmed that AAV5-hGRK1-mediated green fluorescent protein (GFP) expression was restricted to the injection blebs of treated eyes. Optical coherence tomography (OCT) revealed a lack of gross pathology after injection. Neutralizing antibodies against AAV5 were undetectable in post-injection serum samples from subjects receiving uncomplicated subretinal injections (i.e., no hemorrhage). Immunohistochemistry of retinal sections confirmed hGRK1 was active in, and specific for, both rods and cones of NHP retina. Biodistribution studies revealed minimal spread of vector genomes to peripheral tissues. These results suggest that AAV5-hGRK1 is a safe and effective AAV serotype/promoter combination for targeting therapeutic transgene expression protein to rods and cones in a clinical setting.

BackgroundRecessive mutations in guanylate cyclase-1 (Gucy2d) are associated with severe, early onset Leber congenital amaurosis-1(LCA1). Gucy2d encodes guanylate cyclase (GC1) is expressed in photoreceptor outer segment membranes and produces cGMP in these cells. LCA1 patients present in infancy with severely impaired vision and extinguished electroretinogram (ERG) but retain some photoreceptors in both their macular and peripheral retina for years. Like LCA1 patients, loss of cone function in the GC1 knockout (GC1KO) mouse precedes cone degeneration. The purpose of this study was to test whether delivery of functional GC1 to cone cells of the postnatal GC1KO mouse could restore function to these cells.Methodology/Principal FindingsSerotype 5 AAV vectors containing either a photoreceptor-specific, rhodopsin kinase (hGRK1) or ubiquitous (smCBA) promoter driving expression of wild type murine GC1 were subretinally delivered to one eye of P14 GC1KO mice. Visual function (ERG) was analyzed in treated and untreated eyes until 3 months post injection. AAV-treated, isogenic wild type and uninjected control mice were evaluated for restoration of visual behavior using optomotor testing. At 3 months post injection, all animals were sacrificed, and their treated and untreated retinas assayed for expression of GC1 and localization of cone arrestin. Cone-mediated function was restored to treated eyes of GC1KO mice (ERG amplitudes were ∼45% of normal). Treatment effect was stable for at least 3 months. Robust improvements in cone-mediated visual behavior were also observed, with responses of treated mice being similar or identical to that of wild type mice. AAV-vectored GC1 expression was found in photoreceptors and cone cells were preserved in treated retinas.Conclusions/SignificanceThis is the first demonstration of gene-based restoration of both visual function/vision-elicited behavior and cone preservation in a mammalian model of GC1 deficiency. Importantly, results were obtained using a well characterized, clinically relevant AAV vector. These results lay the ground work for the development of an AAV-based gene therapy vector for the treatment of LCA1.

Retinal gene therapy has had unprecedented success in generating treatments that can halt vision loss. However, immunogenic response and long-term toxicity with the use of viral vectors remain a concern. Non-viral vectors are relatively non-immunogenic, scalable platforms that have limited success with DNA delivery to the eye. Messenger RNA (mRNA) therapeutics has expanded the ability to achieve high gene expression while eliminating unintended genomic integration or the need to cross the restrictive nuclear barrier. Lipid-based nanoparticles (LNPs) remain at the forefront of potent delivery vectors for nucleic acids. Herein, we tested eleven different LNP variants for their ability to deliver mRNA to the back of the eye. LNPs that contained ionizable lipids with low pKa and unsaturated hydrocarbon chains showed the highest amount of a reporter gene transfection in the retina. The kinetics of gene expression showed a rapid onset (within 4 h) that persisted for 96 h. The gene delivery was cell-type specific with majority of the expression in the retinal pigmented epithelium (RPE) and limited expression in the Müller glia. LNP-delivered mRNA can be used to treat monogenic retinal degenerative disorders of the RPE. The transient nature of mRNA-based therapeutics makes it desirable for applications that are directed towards retinal reprogramming or genome editing. Overall, non-viral delivery of RNA therapeutics to diverse cell types within the retina can provide transformative new approaches to prevent blindness.

The authors demonstrate for the first time that long-term therapy (∼1 year) is achievable in a mammalian model of GC1 deficiency. These data provide additional justification for the development of an AAV-based gene therapy vector for the clinical treatment of Leber congenital amaurosis-1.

Gene therapy is now an effective approach to treat many forms of retinal degeneration. Delivery agents that are cell-specific, allow for multiple dosing regimens, and have low immunogenicity are needed to expand the utility of gene therapy for the retina. We generated eight novel lipid nanoparticles (LNPs) ranging in size from 50 nm to 150 nm by changing the PEG content from 5% to 0.5%, respectively. Subretinal injections of LNP-mRNA encoding luciferase revealed that 0.5% PEG content within nanoparticles elicits the highest expression. Similar injections of LNP delivered cre mRNA into Ai9 mice revealed cell-specific protein expression in the retinal pigment epithelium (RPE), confirmed by fundus photography and immunohistochemistry of whole globe cross-sections. To investigate mechanisms of LNP delivery to the eye, we injected mCherry mRNA using the subretinal approach in apoE -/- and Mertk -/- mice. RPE transfection was observed in both mouse models suggesting that LNP intracellular delivery is not solely dependent on apolipoprotein adsorption or phagocytosis. To investigate LNP penetration, particles were delivered to the vitreous chamber via an intravitreal injection. The 0.5% PEG particles mediated the highest luciferase activity and expression was observed in the Müller glia, the optic nerve head and the trabecular meshwork, but failed to reach the RPE. Overall, particles containing less PEG (~150 nm in size) mediated the highest expression in the eye. Thus far, these particles successfully transfect RPE, Müller cells, the optic nerve head and the trabecular meshwork based on route of administration which can expand the utility of LNP-mediated gene therapies for the eye.

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