Tyrosine capsid-mutant AAV vectors for gene delivery to the canine retina from a subretinal or intravitreal approach.

Recombinant adeno-associated viruses are important vectors for retinal gene delivery. Currently utilized vectors have relatively slow onset, and for efficient transduction it is necessary to deliver treatment subretinally, with the potential for damage to the retina. Amino-acid substitutions in the viral capsid improve efficiency in rodent eyes by evading host responses. As dogs are important large animal models for human retinitis pigmentosa, we evaluated the speed and efficiency of retinal transduction using capsid-mutant vectors injected both subretinally and intravitreally. We evaluated AAV serotypes 2 and 8 with amino-acid substitutions of surface-exposed capsid tyrosine residues. The chicken beta-actin promoter was used to drive green fluorescent protein expression. Twelve normal adult beagles were injected; four dogs received intravitreal injections and eight dogs received subretinal injections. Capsid-mutant viruses tested included AAV2(quad Y-F) (intravitreal and subretinal) and self-complementary scAAV8(Y733F) (subretinal only). Contralateral control eyes received injections of scAAV5 (subretinal) or scAAV2 (intravitreal). Subretinally delivered vectors had a faster expression onset than intravitreally delivered vectors. Subretinally delivered scAAV8(Y733F) had a faster onset of expression than scAAV5. All subretinally injected vector types transduced the outer retina with high efficiency and the inner retina with moderate efficiency. Intravitreally delivered AAV2(quad Y-F) had a marginally higher efficiency of transduction of both outer retinal and inner retinal cells than scAAV2. Because of their rapid expression onset and efficient transduction, subretinally delivered capsid-mutant AAV8 vectors may increase the efficacy of gene therapy treatment for rapid photoreceptor degenerative diseases. With further refinement, capsid-mutant AAV2 vectors show promise for retinal gene delivery from an intravitreal approach.

AAV-mediated gene therapy in mouse models of recessive retinal degeneration.

In recent years, more and more mutant genes that cause retinal diseases have been detected. At the same time, many naturally occurring mouse models of retinal degeneration have also been found, which show similar changes to human retinal diseases. These, together with improved viral vector quality allow more and more traditionally incurable inherited retinal disorders to become potential candidates for gene therapy. Currently, the most common vehicle to deliver the therapeutic gene into target retinal cells is the adenoassociated viral vector (AAV). Following delivery to the immuno-privileged subretinal space, AAV-vectors can efficiently target both retinal pigment epithelium and photoreceptor cells, the origin of most retinal degenerations. This review focuses on the AAV-based gene therapy in mouse models of recessive retinal degenerations, especially those in which delivery of the correct copy of the wild-type gene has led to significant beneficial effects on visual function, as determined by morphological, biochemical, electroretinographic and behavioral analysis. The past studies in animal models and ongoing successful LCA2 clinical trials, predict a bright future for AAV gene replacement treatment for inherited recessive retinal diseases.

Anti-clarin-1 AAV-delivered ribozyme induced apoptosis in the mouse cochlea.

Usher syndrome type 3 is caused by mutations in the USH3A gene, which encodes the protein clarin-1. Clarin-1 is a member of the tetraspanin superfamily (TM4SF) of transmembrane proteins, expressed in the organ of Corti and spiral ganglion cells of the mouse ear. We have examined whether the AAV-mediated anti-clarin ribozyme delivery causes apoptotic cell death in vivo in the organ of Corti. We used an AAV-2 vector delivered hammerhead ribozyme, AAV-CBA-Rz, which specifically recognizes and cleaves wild type mouse clarin-1 mRNA. Cochleae of CD-1 mice were injected either with 1mul of the AAV-CBA-Rz, or control AAV vectors containing the green fluorescent protein (GFP) marker gene (AAV-CBA-GFP). Additional controls were performed with saline only. At one-week and one-month post-injection, the animals were sacrificed and the cochleae were studied by histology and fluorescence imaging. Mice injected with AAV-CBA-GFP displayed GFP reporter expression of varying fluorescence intensity throughout the length of the cochlea in the outer and inner hair cells and stria vascularis, and to a lesser extent, in vestibular epithelial cells. GFP expression was not detectable in the spiral ganglion. The pro-apoptotic effect of AAV-CBA-delivered anti-clarin-1 ribozymes was evaluated by TUNEL-staining. We observed in the AAV-CBA-Rz, AAV-CBA-GFP and saline control groups apoptotic nuclei in the outer and inner hair cells and in the stria vascularis one week after the microinjection. The vestibular epithelium was also observed to contain apoptotic cells. No TUNEL-positive spiral ganglion neurons were detected. After one-month post-injection, the AAV-CBA-Rz-injected group had significantly more apoptotic outer and inner hair cells and cells of the stria vascularis than the AAV-CBA-GFP group. In this study, we demonstrate that AAV-CBA mediated clarin-1 ribozyme may induce apoptosis of the cochlear hair cells and cells of the stria vascularis. Surprisingly, we did not observe apoptosis in spiral ganglion cells, which should also be susceptible to clarin-1 mRNA cleavage. This result may be due to the injection technique, the promoter used, or tropism of the AAV serotype 2 viral vector. These results suggest the role of apoptosis in the progression of USH3A hearing loss warrants further evaluation.

The effect of dihydronicotinate N-substitution on the brain-targeting efficacy of a zidovudine chemical delivery system.

Enhanced brain delivery of zidovudine (AZT) has been demonstrated using a redox-based chemical delivery system (CDS). Optimization of the prototype AZT-CDS (5'-[(1-methyl-1,4-dihydropyridin-3-yl)carbonyl]-3'-azido-3'-deoxy thymidine ) was investigated by manipulation of the N-methyl group present on the dihydronicotinate portion of the molecule and examining the release of AZT in vivo in a rat model. Of the five compounds examined, all produced higher brain levels and lower blood levels of AZT than did AZT itself. In comparing the novel AZT-CDS analogues to the N-methyl benchmark, the N-propyl system proved to be the most efficient of the compounds tested.

Carbonic anhydrase inhibitors. V: Pyrylium salts in the synthesis of isozyme-specific inhibitors.

Syntheses and physicochemical properties of 2,4,6-tri-, 2,3,4,6-tetra-, or 2,3,4,5,6-pentasubstituted 1-(2-sulfonamido-1,3,4-thiadiazol-5-yl)pyridinium perchlorates are presented. The new compounds, putative inhibitors of membrane-bound carbonic anhydrase, were tested for inhibitory action on the bovine red cell enzyme.