Subretinal delivery of adeno-associated virus serotype 2 results in minimal immune responses that allow repeat vector administration in immunocompetent mice

Background: Adeno-associated virus serotype 2 (AAV2) vectors show considerable promise for ocular gene transfer. However, one potential barrier to efficacious long-term therapy is the development of immune responses against the vector or transgene product. Methods: We evaluated cellular and humoural responses in mice following both single and repeated subretinal administration of AAV2, and examined their effects on RPE65 and green fluorescent protein transgene expression. Results: Following subretinal administration of vector, splenocytes and T-cells from draining lymph nodes showed minimal activation following stimulation by co-culture with AAV2. Neutralizing antibodies (NAbs) were not detected in the ocular fluids of any mice receiving AAV2 or in the serum of mice receiving a lower dose. NAbs were present in the serum of a proportion of mice receiving a higher dose of the vector. Furthermore, no differences in immunoglobulin titre in serum or ocular fluids against RPE65 protein or AAV2 capsid between treated and control mice were detected. Histological examination showed no evidence of retinal toxicity or leukocyte infiltration compared to uninjected eyes. Repeat administration of low-dose AAV.hRPE65.hRPE65 to both eyes of RPE65-/- mice resulted in transgene expression and functional rescue, but re-administration of high-dose AAV2 resulted in boosted NAb titres and variable transgene expression in the second injected eye. Conclusions: These data, which were obtained in mice, suggest that, following subretinal injection, immune responses to AAV2 are dose-dependent. Low-dose AAV2 is well tolerated in the eye, with minimal immune responses, and transgene expression after repeat administration of vector is achievable. Higher doses lead to the expression of NAbs that reduce the efficacy of repeated vector administration

A Partial Structural and Functional Rescue of a Retinitis Pigmentosa Model with Compacted DNA Nanoparticles

Previously we have shown that compacted DNA nanoparticles can drive high levels of transgene expression after subretinal injection in the mouse eye. Here we delivered compacted DNA nanoparticles containing a therapeutic gene to the retinas of a mouse model of retinitis pigmentosa. Nanoparticles containing the wild-type retinal degeneration slow (Rds) gene were injected into the subretinal space of rds+/− mice on postnatal day 5. Gene expression was sustained for up to four months at levels up to four times higher than in controls injected with saline or naked DNA. The nanoparticles were taken up into virtually all photoreceptors and mediated significant structural and biochemical rescue of the disease without histological or functional evidence of toxicity. Electroretinogram recordings showed that nanoparticle-mediated gene transfer restored cone function to a near-normal level in contrast to transfer of naked plasmid DNA. Rod function was also improved. These findings demonstrate that compacted DNA nanoparticles represent a viable option for development of gene-based interventions for ocular diseases and obviate major barriers commonly encountered with non-viral based therapies

Bicistronic Lentiviruses Containing a Viral 2A Cleavage Sequence Reliably Co-Express Two Proteins and Restore Vision to an Animal Model of LCA1

The disease processes underlying inherited retinal disease are complex and are not completely understood. Many of the corrective gene therapies designed to treat diseases linked to mutations in genes specifically expressed in photoreceptor cells restore function to these cells but fail to stop progression of the disease. There is growing consensus that effective treatments for these diseases will require delivery of multiple therapeutic proteins that will be selected to treat specific aspects of the disease process. The purpose of this study was to design a lentiviral transgene that reliably expresses all of the proteins it encodes and does so in a consistent manner among infected cells. We show, using both in vitro and in vivo analyses, that bicistronic lentiviral transgenes encoding two fluorescent proteins fused to a viral 2A-like cleavage peptide meet these expression criteria. To determine if this transgene design is suitable for therapeutic applications, we replaced one of the fluorescent protein genes with the gene encoding guanylate cyclase -1 (GC1) and delivered lentivirus carrying this transgene to the retinas of the GUCY1*B avian model of Leber congenital amaurosis – 1 (LCA1). GUCY1*B chickens carry a null mutation in the GC1 gene that disrupts photoreceptor function and causes blindness at hatching, a phenotype that closely matches that observed in humans with LCA1. We found that treatment of these animals with the 2A lentivector encoding GC1 restored vision to these animals as evidenced by the presence of optokinetic reflexes. We conclude that 2A-like peptides, with proper optimization, can be successfully incorporated into therapeutic vectors designed to deliver multiple proteins to neural retinal. These results highlight the potential of this vector design to serve as a platform for the development of combination therapies designed to enhance or prolong the benefits of corrective gene therapies

Protective Gene Expression Changes Elicited by an Inherited Defect in Photoreceptor Structure

Inherited defects in retinal photoreceptor structure impair visual transduction, disrupt relationship with the retinal pigment epithelium (RPE), and compromise cell viability. A variety of progressive retinal degenerative diseases can result, and knowledge of disease etiology remains incomplete. To investigate pathogenic mechanisms in such instances, we have characterized rod photoreceptor and retinal gene expression changes in response to a defined insult to photoreceptor structure, using the retinal degeneration slow (rds) mouse model. Global gene expression profiling was performed on flow-sorted rds and wild-type rod photoreceptors immediately prior and subsequent to times at which OSs are normally elaborated. Dysregulated genes were identified via microarray hybridization, and selected candidates were validated using quantitative PCR analyses. Both the array and qPCR data revealed that gene expression changes were generally modest and dispersed amongst a variety of known functional networks. Although genes showing major (>5-fold) differential expression were identified in a few instances, nearly all displayed transient temporal profiles, returning to WT levels by postnatal day (P) 21. These observations suggest that major defects in photoreceptor cell structure may induce early homeostatic responses, which function in a protective manner to promote cell viability. We identified a single key gene, Egr1, that was dysregulated in a sustained fashion in rds rod photoreceptors and retina. Egr1 upregulation was associated with microglial activation and migration into the outer retina at times subsequent to the major peak of photoreceptor cell death. Interestingly, this response was accompanied by neurotrophic factor upregulation. We hypothesize that activation of Egr1 and neurotrophic factors may represent a protective immune mechanism which contributes to the characteristically slow retinal degeneration of the rds mouse model

Gene therapy for retinitis pigmentosa and Leber congenital amaurosis caused by defects in AIPL1: effective rescue of mouse models of partial and complete Aipl1 deficiency using AAV2/2 and AAV2/8 vectors

Defects in the photoreceptor-specific gene encoding aryl hydrocarbon receptor-interacting protein-like 1 (AIPL1) are clinically heterogeneous and present as Leber Congenital Amaurosis, the severest form of early-onset retinal dystrophy and milder forms of retinal dystrophies such as juvenile retinitis pigmentosa and dominant cone-rod dystrophy. [Perrault, I., Rozet, J.M., Gerber, S., Ghazi, I., Leowski, C., Ducroq, D., Souied, E., Dufier, J.L., Munnich, A. and Kaplan, J. (1999) Leber congenital amaurosis. Mol. Genet. Metab., 68, 200–208.] Although not yet fully elucidated, AIPL1 is likely to function as a specialized chaperone for rod phosphodiesterase (PDE). We evaluate whether AAV-mediated gene replacement therapy is able to improve photoreceptor function and survival in retinal degeneration associated with AIPL1 defects. We used two mouse models of AIPL1 deficiency simulating three different rates of photoreceptor degeneration. The Aipl1 hypomorphic (h/h) mouse has reduced Aipl1 levels and a relatively slow degeneration. Under light acceleration, the rate of degeneration in the Aipl1 h/h mouse is increased by 2–3-fold. The Aipl1–/– mouse has no functional Aipl1 and has a very rapid retinal degeneration. To treat the different rates of degeneration, two pseudotypes of recombinant adeno-associated virus (AAV) exhibiting different transduction kinetics are used for gene transfer. We demonstrate restoration of cellular function and preservation of photoreceptor cells and retinal function in Aipl1 h/h mice following gene replacement therapy using an AAV2/2 vector and in the light accelerated Aipl1 h/h model and Aipl1–/– mice using an AAV2/8 vector. We have thus established the potential of gene replacement therapy in varying rates of degeneration that reflect the clinical spectrum of disease. This is the first gene replacement study to report long-term rescue of a photoreceptor-specific defect and to demonstrate effective rescue of a rapid photoreceptor degeneration

INRAVITREAL BEVACIZUMAB COMBINED WITH CATARACT SURGERY FOR TREATMENT OF EXUDATIVE MACULAR DEGENERATION

PURPOSE: The aim of this study was to report on the combination of an intravitreal injection of bevacizumab and cataract surgery in patients with exudative age-related macular degeneration (AMD). METHODS: The interventional case series study included 11 patients (11 eyes) who received an intravitreal injection of 1.5 mg bevacizumab as treatment of exudative AMD (n = 10) or exudative myopic macular degeneration (n = 1), combined with a routine phacoemulsification and posterior chamber lens implantation for treatment of cataract. RESULTS: Intraoperatively and during the follow-up of 150 +/- 77.5 days, there were no complications related to the intravitreal application of bevacizumab combined with cataract surgery, such as wound dehiscence and leakage, delayed wound healing, corneal edema, dislocation of the pseudophakos, rupture of the posterior lens capsule, or rhegmatogenous retinal detachment. CONCLUSIONS: The results of this pilot study suggest that from a safety point of view, intravitreal injections of bevacizumab may be combined with routine cataract surgery