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Stargardt disease (STGD1) due to mutations in the large ABCA4 gene is the most common inherited macular degeneration in humans. We have shown that dual adeno-associated viral (AAV) vectors effectively transfer ABCA4 to the retina of Abca4-/- mice. However, they express both lower levels of transgene compared with a single AAV and truncated proteins. To increase productive dual AAV concatemerization, which would overcome these limitations, we have explored the use of either various regions of homology or heterologous inverted terminal repeats (ITR). In addition, we tested the ability of various degradation signals to decrease the expression of truncated proteins. We found the highest levels of transgene expression using regions of homology based on either alkaline phosphatase or the F1 phage (AK). The use of heterologous ITR does not decrease the levels of truncated proteins relative to full-length ABCA4 and impairs AAV vector production. Conversely, the inclusion of the CL1 degradation signal results in the selective degradation of truncated proteins from the 5'-half without affecting full-length protein production. Therefore, we developed dual AAV hybrid ABCA4 vectors including homologous ITR2, the photoreceptor-specific G protein-coupled receptor kinase 1 promoter, the AK region of homology and the CL1 degradation signal. We show that upon subretinal administration these vectors are both safe in pigs and effective in Abca4-/- mice. Our data support the use of improved dual AAV vectors for gene therapy of STGD1.

Citrus aurantium L. dry extracts (CAde) improve adipogenesis in vitro. These effects are dependent from an early modulation of CCAAT/enhancer-binding protein beta (C/Ebp&beta, ) expression and cyclic Adenosine Monophosphate (cAMP) response element-binding protein (CREB) activation. C/Ebp&beta, and Creb are also targets of miR-155. This study investigated whether CAde regulates miR-155 expression in the early stages of adipogenesis and whether it ameliorates adipocyte differentiation of cells exposed to tumor necrosis factor-alpha (TNF&alpha, ). Adipogenic stimuli (AS) were performed in 3T3-L1 pre-adipocytes treated with CAde, TNF&alpha, or both. Gene and miRNA expression were determined by quantitative real-time PCR. Adipogenesis was evaluated by Oil-Red O staining. CAde treatment enhanced AS effects during the early adipogenesis phases by further down-regulating miR-155 expression and increasing both C/Ebp&beta, and Creb mRNA and protein levels. At variance, TNF&alpha, inhibited 3T3-L1 adipogenesis and abolished AS effects on miR-155, C/Ebp&beta, and Creb expression. However, in cells exposed to TNF&alpha, CAde improved adipocyte differentiation and restored the AS effects on miRNA and gene expression at early time points. In conclusion, this study identified miR-155 down-regulation as part of the mechanism through which CAde enhances adipogenesis of pre-adipocytes in vitro. Furthermore, it provides evidence of CAde efficacy against TNF&alpha, negative effects on adipogenesis.

Retinal gene therapy with adeno-associated viral (AAV) vectors holds promises for treating inherited and noninherited diseases of the eye. Although clinical data suggest that retinal gene therapy is safe and effective, delivery of large genes is hindered by the limited AAV cargo capacity. Protein trans-splicing mediated by split inteins is used by single-cell organisms to reconstitute proteins. Here, we show that delivery of multiple AAV vectors each encoding one of the fragments of target proteins flanked by short split inteins results in protein trans-splicing and full-length protein reconstitution in the retina of mice and pigs and in human retinal organoids. The reconstitution of large therapeutic proteins using this approach improved the phenotype of two mouse models of inherited retinal diseases. Our data support the use of split intein–mediated protein trans-splicing in combination with AAV subretinal delivery for gene therapy of inherited blindness due to mutations in large genes.