E of SMA. The basis of this robust transduction of central

E of SMA. The basis of this robust transduction of central nervous system (CNS) cells after the systemic delivery of scAAV9 remains unclear. It is thought to involve differential BBB transport, but it remains unclear how AAV9 crosses the BBB and whether this mechanism is different from that of other serotypes in vivo. The superiority of scAAV9 for the systemic transduction of nerve cells may be due to various factors, including capsid-interacting blood factors, strong neural cell tropism or intracellular trafficking, the rapid uncoating of virion shells in cells and delayed blood clearance [31,32]. In this study, we investigated whether the systemic injection of scAAV9 could mediate transduction of the retina in adult mice despite the presence of functional blood-eye barriers. We found that the intravenous injection of scAAV9 into adult mice resulted in gene transfer to all cell layers of the retina, with the predominant transduction of RGC and ciliary bodies. This study suggests that this vector could cross mature blood-eye barriers, and constitutes the basis for future development of a non invasive alternative to the current methods of viral gene delivery to the retina.The production of serotype 9 AAV has been described elsewhere [24]. Briefly, pseudotyped AAV9 and AAV2 vectors were generated by packaging AAV2-based recombinant single-stranded (ss) or self-complementary (sc) genomes into the AAV9 or AAV2 capsids. Virions were produced by transfecting HEK293 cells with (i) the adenovirus helper plasmid (pXX6-80), (ii) the AAV packaging plasmid encoding the rep2 and the cap2 or the cap9 genes, and (iii) the AAV2 shuttle plasmid containing the gene encoding GFP or mSEAP in a ss or sc genome. Recombinant vectors (rAAV) were purified by double-CsCl ultracentrifugation followed by dialysis against the formulation buffer of the vector stocks, namely phosphate-buffered saline containing 0.5 mM MgCl2 and 1.25 mM KCl (PBS-MK; five buffer changes, 3 hours per round of dialysis). Physical particles were quantified by realtime PCR. Vector titers are expressed as viral genomes per milliliter (vg/ml).Peripheral Administration of AAV VectorsIn adults, AAV vectors were administered peripherally by injection into the tail vein at 8 weeks of age. The animals were restrained in a tube, facilitating manipulation of the tail. A 30gauge Triptorelin needle attached to a 1 ml syringe was inserted into the tail vein and 500 ml of the viral solution was injected over a period of about 30 15755315 seconds.Real-time PCR Quantification of Vector Genome Copy Number in the RetinaEnucleated whole eyes were snap-frozen in liquid nitrogen and stored at 280uC until further processing. Frozen tissues were lysed in 700 ml of nuclear lysis buffer (Wizard genomic DNA extraction kit, Promega, Charbonnieres-les-Bains, France). Tissues were ` homogenized by four 30-second pulses with an Ultra-Turrax homogenizer, to ensure complete lysis. Cell membranes and order 370-86-5 debris were pelleted by centrifugation for 2 minutes at 10,0006g and 4uC. A sample of the supernatant was collected for the mSEAP quantification assay, and genomic DNA containing the AAV vector genome was purified according to the manufacturer’s instructions. For each sample, we used 72 ng of genomic DNA as the template. Vector genome copy number was determined by a real-time PCR assay with primers and a probe corresponding to the inverted terminal repeat region (ITR) of the AAV vector genome common to ss and scAAV vectors. The sequences of the.E of SMA. The basis of this robust transduction of central nervous system (CNS) cells after the systemic delivery of scAAV9 remains unclear. It is thought to involve differential BBB transport, but it remains unclear how AAV9 crosses the BBB and whether this mechanism is different from that of other serotypes in vivo. The superiority of scAAV9 for the systemic transduction of nerve cells may be due to various factors, including capsid-interacting blood factors, strong neural cell tropism or intracellular trafficking, the rapid uncoating of virion shells in cells and delayed blood clearance [31,32]. In this study, we investigated whether the systemic injection of scAAV9 could mediate transduction of the retina in adult mice despite the presence of functional blood-eye barriers. We found that the intravenous injection of scAAV9 into adult mice resulted in gene transfer to all cell layers of the retina, with the predominant transduction of RGC and ciliary bodies. This study suggests that this vector could cross mature blood-eye barriers, and constitutes the basis for future development of a non invasive alternative to the current methods of viral gene delivery to the retina.The production of serotype 9 AAV has been described elsewhere [24]. Briefly, pseudotyped AAV9 and AAV2 vectors were generated by packaging AAV2-based recombinant single-stranded (ss) or self-complementary (sc) genomes into the AAV9 or AAV2 capsids. Virions were produced by transfecting HEK293 cells with (i) the adenovirus helper plasmid (pXX6-80), (ii) the AAV packaging plasmid encoding the rep2 and the cap2 or the cap9 genes, and (iii) the AAV2 shuttle plasmid containing the gene encoding GFP or mSEAP in a ss or sc genome. Recombinant vectors (rAAV) were purified by double-CsCl ultracentrifugation followed by dialysis against the formulation buffer of the vector stocks, namely phosphate-buffered saline containing 0.5 mM MgCl2 and 1.25 mM KCl (PBS-MK; five buffer changes, 3 hours per round of dialysis). Physical particles were quantified by realtime PCR. Vector titers are expressed as viral genomes per milliliter (vg/ml).Peripheral Administration of AAV VectorsIn adults, AAV vectors were administered peripherally by injection into the tail vein at 8 weeks of age. The animals were restrained in a tube, facilitating manipulation of the tail. A 30gauge needle attached to a 1 ml syringe was inserted into the tail vein and 500 ml of the viral solution was injected over a period of about 30 15755315 seconds.Real-time PCR Quantification of Vector Genome Copy Number in the RetinaEnucleated whole eyes were snap-frozen in liquid nitrogen and stored at 280uC until further processing. Frozen tissues were lysed in 700 ml of nuclear lysis buffer (Wizard genomic DNA extraction kit, Promega, Charbonnieres-les-Bains, France). Tissues were ` homogenized by four 30-second pulses with an Ultra-Turrax homogenizer, to ensure complete lysis. Cell membranes and debris were pelleted by centrifugation for 2 minutes at 10,0006g and 4uC. A sample of the supernatant was collected for the mSEAP quantification assay, and genomic DNA containing the AAV vector genome was purified according to the manufacturer’s instructions. For each sample, we used 72 ng of genomic DNA as the template. Vector genome copy number was determined by a real-time PCR assay with primers and a probe corresponding to the inverted terminal repeat region (ITR) of the AAV vector genome common to ss and scAAV vectors. The sequences of the.