Vectors derived from adeno-associated virus (AAV) are promising for human gene therapy, including treatment for retinal blindness. One major limitation of AAVs as vectors is that AAV cargo capacity has been considered to be restricted to 4.7 kb. Here we demonstrate that vectors with an AAV5 capsid (i.e., rAAV2/5) incorporated up to 8.9 kb of genome more efficiently than 6 other serotypes tested, independent of the efficiency of the rAAV2/5 production process. Efficient packaging of the large murine Abca4 and human MYO7A and CEP290 genes, which are mutated in common blinding diseases, was obtained, suggesting that this packaging efficiency is independent of the specific sequence packaged. Expression of proteins of the appropriate size and function was observed following transduction with rAAV2/5 carrying large genes. Intraocular administration of rAAV2/5 encoding ABCA4 resulted in protein localization to rod outer segments and significant and stable morphological and functional improvement of the retina in Abca4 -/-mice. This use of rAAV2/5 may be a promising therapeutic strategy for recessive Stargardt disease, the most common form of inherited macular degeneration. The possibility of packaging large genes in AAV greatly expands the therapeutic potential of this vector system.

Severe inherited retinal diseases, such as retinitis pigmentosa and Leber congenital amaurosis, are caused by mutations in genes preferentially expressed in photoreceptors. While adeno-associated virus (AAV)-mediated gene transfer can correct retinal pigment epithelium (RPE) defects in animal models, approaches for the correction of photoreceptor-specific diseases are less efficient. We evaluated the ability of novel AAV serotypes (AAV2/7, AAV2/8, AAV2/9, AAV2rh.43, AAV2rh.64R1, and AAV2hu.29R) in combination with constitutive or photoreceptor-specific promoters to improve photoreceptor transduction, a limiting step in photoreceptor rescue. Based on a qualitative analysis, all AAV serotypes tested efficiently transduce the RPE as well as rod and cone photoreceptors after subretinal administration in mice. Interestingly, AAV2/9 efficiently transduces Müller cells. To compare photoreceptor transduction from different AAVs and promoters in both a qualitative and quantitative manner, we designed a strategy based on the use of a bicistronic construct expressing both enhanced green fluorescent protein and luciferase. We found that AAV2/8 and AAV2/7 mediate six-to eightfold higher levels of in vivo photoreceptor transduction than AAV2/5, considered so far the most efficient AAV serotype for photoreceptor targeting. In addition, following subretinal administration of AAV, the rhodopsin promoter allows significantly higher levels of photoreceptor expression than the other ubiquitous or photoreceptor-specific promoters tested. Finally, we show that AAV2/7, AAV2/8, and AAV2/9 outperform AAV2/5 following ex vivo transduction of retinal progenitor cells differentiated into photoreceptors. We conclude that AAV2/7 or AAV2/8 and the rhodopsin promoter provide the highest levels of photoreceptor transduction both in and ex vivo and that this may overcome the limitation to therapeutic success observed so far in models of inherited severe photoreceptor diseases.

Duchenne muscular dystrophy is an X-linked muscle disease characterized by mutations in the dystrophin gene. Many of these can be corrected at the posttranscriptional level by skipping the mutated exon. We have obtained persistent exon skipping in mdx mice by tail vein injection with an adeno-associated viral (AAV) vector expressing antisense sequences as part of the stable cellular U1 small nuclear RNA. Systemic delivery of the AAV construct resulted in effective body-wide colonization, significant recovery of the functional properties in vivo, and lower creatine kinase serum levels, suggesting an overall decrease in muscle wasting. The transduced muscles rescued dystrophin expression and displayed a significant recovery of function toward the normal values at single muscle fiber level. This approach provides solid bases for a systemic use of AAV-mediated antisense-U1 small nuclear RNA expression for the therapeutic treatment of Duchenne muscular dystrophy.exon skipping ͉ adeno-associated virus vectors ͉ antisense ͉ small nuclear RNA ͉ dystrophin D eletions and point mutations in the human 2.5-Mb-long dystrophin gene cause either the severe progressive myopathy Duchenne muscular dystrophy (DMD) or the milder Becker muscular dystrophy (BMD), depending on whether the translational reading frame is lost or maintained (1).The mdx mouse, carrying a stop codon inside exon 23 of the dystrophin gene, provides a useful system to study the effectiveness of different therapeutic strategies for the cure of this disease (2). The two challenging issues in the gene therapy of DMD are related on one side to the type of therapeutic gene to use and on the other to the delivery system suitable for efficient muscular transduction. Due to the large size of the protein, a traditional gene replacement approach is difficult: only adenoviral vectors could allow the expression of a full-length dystrophin cDNA. However, treatments of mdx mice with ''gutted'' vectors carrying the full-length dystrophin cDNA induced a weak immune reaction and transduction was not very effective (3, 4).A different approach took advantage of adeno-associated viral (AAV) vectors for the delivery of microdystrophin gene (5). AAV vectors with an AAV8 capsid (or the very similar but not identical AAV1, or AAV6) have been shown to transduce efficiently and widely murine muscles after administration in the tail vein of WT mice (6). Those with an AAV6 capsid have been shown to correct the phenotype of dystrophic mdx mice when carrying microdystrophin (7).Recently, other alternative ways of correcting the DMD phenotype have been achieved that consist in the delivery of antisense sequences able to induce exon skipping and cure the genetic alteration at the posttranscriptional level. Many of the internal in-frame deletions fall in the region encoding the spectrin-like central rod domain that is largely dispensable and produce only mild myopathic symptoms; therefore, for the out-of-frame mutations, it should be possible, by preventing the inclusion of specific mutated ...

Molecules with neurotrophic activity are being evaluated for treatment of retinitis pigmentosa in animal models. In particular, great interest has been focused recently on erythropoietin (Epo). Evidence of its neurotrophic activity comes mainly from data demonstrating photoreceptor protection in a rodent light-damage model through systemic administration of a recombinant form of this hormone. Our goal was to test whether Epo retinal gene transfer can rescue or delay photoreceptor cell death. We delivered adeno-associated viral vectors encoding Epo intraocularly and, for comparison, intramuscularly to one light-induced and two genetic models of retinal degeneration. Intraocular Epo gene transfer resulted in sustained hormone expression in the eye, which was undetectable systemically. In contrast, Epo intramuscular gene transfer resulted in hormone secretion in the circulation, which was not detected in ocular fluids. The protein secreted from muscle and retina is of the same molecular weight as a commercial recombinant human Epo. Interestingly, following systemic but not intraocular Epo delivery, morphological photoreceptor protection was observed in the light-damage and rds/peripherin (Prph2) models of retinal degeneration. In the light-damage model, the morphological rescue was accompanied by a significant electrophysiological improvement of photoreceptor function. In contrast, no photoreceptor rescue was observed following Epo gene transfer in the rd10 model. This suggests that different apoptotic mechanisms, with varying sensitivities to Epo, occur in different retinal degeneration models. In conclusion, our data support Epo as a neuroprotective agent in some, but not all, retinal degenerations. Further, rescue is observed in specific models after systemic but not intraocular Epo gene transfer.