Recombinant adeno-associated virus (rAAV) is produced by transfecting cells with two constructs: the rAAV vector plasmid and the rep-cap plasmid. After subsequent adenoviral infection, needed for rAAV replication and assembly, the virus is purified from total cell lysates through CsCl gradients. Because this is a long and complex procedure, the precise titration of rAAV stocks, as well as the measure of the level of contamination with adenovirus and rep-positive AAV, are essential to evaluate the transduction efficiency of these vectors in vitro and in vivo. Our vector core is in charge of producing rAAV for outside investigators as part of a national network promoted by the Association Française contre les Myopathies/Généthon. We report here the characterization of 18 large-scale rAAV stocks produced during the past year. Three major improvements were introduced and combined in the rAAV production procedure: (i) the titration and characterization of rAAV stocks using a stable rep-cap HeLa cell line in a modified Replication Center Assay (RCA); (ii) the use of different rep-cap constructs to provide AAV regulatory and structural proteins; (iii) the use of an adenoviral plasmid to provide helper functions needed for rAAV replication and assembly. Our results indicate that: (i) rAAV yields ranged between 10(11) to 5 x 10(12) total particles; (ii) the physical particle to infectious particle (measured by RCA) ratios were consistently below 50 when using a rep-cap plasmid harboring an ITR-deleted AAV genome; the physical particle to transducing particle ratios ranged between 400 and 600; (iii) the use of an adenoviral plasmid instead of an infectious virion did not affect the particles or the infectious particles yields nor the above ratio. Most of large-scale rAAV stocks (7/9) produced using this plasmid were free of detectable infectious adenovirus as determined by RCA; (iv) all the rAAV stocks were contaminated with rep-positive AAV as detected by RCA. In summary, this study describes a general method to titrate rAAV, independently of the transgene and its expression, and to measure the level of contamination with adenovirus and rep-positive AAV. Furthermore, we report a new production procedure using adenoviral plasmids instead of virions and resulting in rAAV stocks with undetectable adenovirus contamination.

Recombinant adeno-associated virus vectors (rAAV) have been successfully used for long-term gene expression in animal models and in patients. However, while the therapeutic potential of rAAV appears promising, safety issues, including contaminants found in vector stocks, must be further evaluated. We previously reported that a cis-acting replication element present within the AAV-2 p5 promoter was responsible for the encapsidation of rep-cap sequences observed during rAAV production. In that study, we also noticed that plasmid-derived prokaryotic sequences (such as the ampicillin resistance gene) could be found packaged into AAV capsids. In this report, first we confirmed and extended the latter observation by analyzing rAAV stocks produced using different procedures. Second, we demonstrated that these plasmid-derived sequences were transferred and persisted in vivo after rAAV injection into different tissues. Third, our data showed that at least some of these packaged plasmid molecules were linked to the AAV ITRs and were present in vivo in a form that could be rescued through bacterial transformation. This study highlights the need for more stringent characterization of rAAV stocks and provides useful information on the development of rAAV production methods that are able to circumvent or limit the generation of such undesirable particles.

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Electrostatic charges play a critical role during the first step in mammalian cell transduction mediated by a recombinant baculovirus.

This study identifies a region of the adeno-associated virus type 2 (AAV-2) rep gene (nucleotides 190 to 540 of wild-type AAV-2) as a cis-acting Rep-dependent element able to promote the replication of transiently transfected plasmids. This viral element is also shown to be involved in the amplification of integrated sequences in the presence of adenovirus and Rep proteins.It was previously reported that efficient recombinant adenoassociated virus (AAV) production using stable rep-cap cell lines correlated with a 100-fold amplification of the AAV-2 genes upon adenovirus infection (3,9). This phenomenon, which occurred despite the absence of inverted terminal repeats (ITRs) generated extrachromosomal double-stranded DNA molecules harboring the rep-cap genes and required the activity of the adenovirus DNA binding protein, cellular polymerases, and Rep proteins (9). A question that remained unanswered was whether the rep-cap amplification was dependent on the activity of an as-yet-unidentified viral origin of replication present within the viral genome.To answer this question, we investigated if a rep-cap-containing plasmid was able to replicate following transient transfection into adenovirus-infected cells. 293 cells were transfected with plasmid pRCtag containing the rep-cap genome with the ITRs deleted, ligated to a 3Ј tag sequence, and then mock or adenovirally infected. After DNA extraction, replication was assessed by digestion with DpnI or MboI followed by Southern blot analysis using a tag probe (Fig. 1). Cleavage by DpnI indicates that both strands are methylated in the absence of replication of the transfected DNA; cleavage by MboI occurs only if both strands are unmethylated as a result of two rounds of replication. In the absence of adenovirus, the pRCtag plasmid did not replicate (Fig. 1B, lanes 5 and 6). In contrast, upon adenoviral infection, a fraction of the plasmid DNA was susceptible to MboI digestion (Fig. 1B, lane 9), indicating that some input rep-cap molecules had replicated. After DpnI digestion, high-molecular-weight resistant bands were detected as weak signals, suggesting that pRCtag replication generated products which are heterogenous in size (Fig. 1B, lane 8).To identify the cis element(s) involved in pRCtag replica- , and analyzed on a Southern blot by using a tag probe. As a control (lanes 1, 2, and 3), untransfected pRCtag plasmid DNA mixed with 10 g of total DNA from 293 cells was digested with DpnI or MboI and similarly analyzed using the tag probe. The expected 1,430-bp DpnI-MboI fragment hybridizing to the tag probe is indicated. 9991on May 12, 2018 by guest

Highlights d Seipin is enriched at ER-MAMs d Seipin interacts with MAM calcium regulators in a nutritionally regulated manner d Adipocyte seipin deficiency impairs mitochondrial calcium import and ATP production d Inducible seipin removal from adipose tissue leads to rapid mitochondrial dysfunction

We previously reported that a 350-bp region of the adeno-associated virus (AAV) type 2 rep gene contains a cis-acting element responsible for the Rep-dependent replication of a transiently transfected rep-cap plasmid. In this study, we further report that replicated rep-cap sequences can be packaged into AAV capsids in the absence of the inverted terminal repeats.The usual procedure for recombinant adeno-associated virus (rAAV) assembly involves transfection of the vector and the rep-cap plasmid into cells which are either infected with adenovirus or cotransfected with an adenoviral helper plasmid (4,11,15). Despite the lack of homologous sequences between the rep-cap and vector sequences, the precise characterization of rAAV preparations indicated that they were contaminated to various extents with particles containing rep-cap AAV sequences. We designated these contaminating particles rep positive (rep ϩ ), because they were able to transfer a Rep function, as detected by a replication center assay (RCA) (10). Previous studies have indicated that most of these particles were replication competent and that they arose from nonhomologous recombination events between the rep-cap plasmid and the inverted terminal repeats (ITRs) in the rAAV vector (1, 14). Deletion of critical ITR sequences involved in the nonhomologous recombination events prevented the formation of such replication-competent particles. However, rAAV preparations remained contaminated by replication-defective AAV particles containing rep-cap genomes, further suggesting that these sequences had been packaged in the absence of the ITRs (14).Altogether, these observations suggested that some additional cis-acting elements were present in the rep-cap sequences, allowing their replication and encapsidation. We and others have previously reported the presence of a cis-acting replication element (CARE) located in the 5Ј portion of the rep gene (9, 12). The CARE was localized in a 350-bp region that included the p5 promoter and the 5Ј portion of the rep coding sequence (nucleotides 190 to 540 of wild-type AAV), and it was demonstrated that this element behaved in vitro and in vivo as a Rep-dependent origin of replication in the absence of both ITRs (9).In the present study, we investigated whether the presence of CARE could also lead to the packaging of rep-cap sequences into AAV capsids in the absence of the viral ITRs. A critical element in this study was the need to distinguish between DNA truly packaged inside the particles and that simply contaminating the preparations. The conventional procedure to extract viral DNA from a cell lysate or a purified rAAV stock relies on the use of DNase I to digest contaminating DNA before extraction of packaged sequences. The activity of DNase I was checked by mixing purified rAAVLZ particles (rAAV encoding the nucleus-localized ␤-galactosidase) with up to 400 ng of X174 DNA (either double or single stranded). After digestion for 1 h at 37°C with 50 U of DNase I (Roche) in 500 l of Dulbecco modified Eagle medium, pack...

Despite the existence of a preventive vaccine, chronic infection with Hepatitis B virus (HBV) affects more than 250 million people and represents a major global cause of hepatocellular carcinoma (HCC) worldwide. Current clinical treatments, in most of cases, do not eliminate viral genome that persists as a DNA episome in the nucleus of hepatocytes and constitutes a stable template for the continuous expression of viral genes. Several studies suggest that, among viral factors, the HBV core protein (HBc), well-known for its structural role in the cytoplasm, could have critical regulatory functions in the nucleus of infected hepatocytes. To elucidate these functions, we performed a proteomic analysis of HBc-interacting host-factors in the nucleus of differentiated HepaRG, a surrogate model of human hepatocytes. The HBc interactome was found to consist primarily of RNA-binding proteins (RBPs), which are involved in various aspects of mRNA metabolism. Among them, we focused our studies on SRSF10, a RBP that was previously shown to regulate alternative splicing (AS) in a phosphorylation-dependent manner and to control stress and DNA damage responses, as well as viral replication. Functional studies combining SRSF10 knockdown and a pharmacological inhibitor of SRSF10 phosphorylation (1C8) showed that SRSF10 behaves as a restriction factor that regulates HBV RNAs levels and that its dephosphorylated form is likely responsible for the anti-viral effect. Surprisingly, neither SRSF10 knock-down nor 1C8 treatment modified the splicing of HBV RNAs but rather modulated the level of nascent HBV RNA. Altogether, our work suggests that in the nucleus of infected cells HBc interacts with multiple RBPs that regulate viral RNA metabolism. Our identification of SRSF10 as a new anti-HBV restriction factor offers new perspectives for the development of new host-targeted antiviral strategies.