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Persistence of transgene expression is a major limitation for nonvirus-mediated gene therapy approaches. We have suggested that covalent linkage of bacterial DNA to the expression cassette plays a critical role in transcriptional silencing of transgenes in vivo. To gain insight into the role of the covalent linkage of plasmid DNA to the expression cassette and transcriptional repression, and whether this silencing effect could be alleviated by altering the molecular structure of vector DNAs in vivo, we generated a scheme for converting routine plasmids into a purified expression cassette, free of bacterial DNA after gene transfer in vivo. To do this, the human alpha-1-antitrypsin (hAAT) and human clotting factor IX (hfIX) reporter genes were flanked by two ISceI endonuclease recognition sites, and coinjected together with a plasmid encoding the I-SceI cDNA or a control plasmid into mouse liver. Two weeks after DNA administration, mice injected with the reporter gene alone or with the irrelevant control plasmid showed low serum levels of hAAT or hFIX, which remained low throughout the length of the experiment. However, animals that expressed I-SceI had a 5- to 10-fold increase in serum hAAT or hFIX that persisted for at least 8 months (length of study). Expression of I-SceI resulted in cleavage and excision of the expression cassettes from the plasmid backbone, forming mostly circles devoid of bacterial DNA sequences, as established by a battery of different Southern blot and polymerase chain reaction analyses in both C57BL/6 and scid treated mice. In contrast, only the input parental circular plasmid DNA band was detected in mice injected with the reporter gene alone, or an I-SceI plasmid together with the hAAT reporter plasmid lacking the I-SceI sites. Similar results were obtained when the Flp recombinase system was used to make mini-plasmids in mouse liver in vivo. This study presents further independent evidence that removing the covalent linkage between plasmid and transgene sequences leads to a marked increase in and persistence of transgene expression. Unraveling the mechanisms by which the covalent linkage of bacterial DNA to the expression cassette is connected to gene silencing is fundamental to establishing the mechanism of transcriptional regulation in mammalian systems and will be important for the development of versatile nonviral vectors that can be used to achieve persistent gene expression in different cell types.

We report the generation and use of pseudotyped adeno-associated viral (AAV) vectors for the liver-specific expression of human blood coagulation factor IX (hFIX). Therefore, an AAV-2 genome encoding the hfIX gene was cross-packaged into capsids of AAV types 1 to 6 using efficient, large-scale technology for particle production and purification. In immunocompetent mice, the resultant vector particles expressed high hFIX levels ranging from 36% (AAV-4) to more than 2000% of normal (AAV-1, -2, and -6), which would exceed curative levels in patients with hemophilia. Expression was dose- and time-dependent, with AAV-6 directing the fastest and strongest onset of hFIX expression at all doses. Interestingly, systemic administration of 2 × 1012 vector particles of AAV-1, -4, or -6 resulted in hFIX levels similar to those achieved by portal vein delivery. For all other serotypes and particle doses, hepatic vector administration yielded up to 84-fold more hFIX protein than tail vein delivery, corroborated by similarly increased vector DNA copy numbers in the liver, and elicited a reduced immune response against the viral capsids. Finally, neutralization assays showed variable immunologic cross-reactions between most of the AAV serotypes. Our technology and findings should facilitate the development of AAV pseudotype-based gene therapies for hemophilia B and other liver-related diseases. (Blood. 2003;102:2412-2419)

We present a simple and safe strategy for producing high-titer adeno-associated virus (AAV) vectors derived from six different AAV serotypes (AAV-1 to AAV-6). The method, referred to as "HOT," is helper virus free, optically controllable, and based on transfection of only two plasmids, i.e., an AAV vector construct and one of six novel AAV helper plasmids. The latter were engineered to carry AAV serotype rep and cap genes together with adenoviral helper functions, as well as unique fluorescent protein expression cassettes, allowing confirmation of successful transfection and identification of the transfected plasmid. Cross-packaging of vector DNA derived from AAV-2, -3, or -6 was up to 10-fold more efficient using our novel plasmids, compared to a conservative adenovirus-dependent method. We also identified a variety of useful antibodies, allowing detection of Rep or VP proteins, or assembled capsids, of all six AAV serotypes. Finally, we describe unique cell tropisms and kinetics of transgene expression for AAV serotype vectors in primary or transformed cells from four different species. In sum, the HOT strategy and the antibodies presented here, together with the reported findings, should facilitate and support the further development of AAV serotype vectors as powerful new tools for human gene therapy.

Roughly two decades after its discovery and cloning, hepatitis C virus (HCV)—a pathogen infecting up to 180 million people worldwide and capable of causing severe liver disease, including cirrhosis and hepatocellular carcinoma—remains a frustrating target for development of antiviral therapies. Two articles in this issue of Molecular Therapy now report combinatorial RNA interference (coRNAi) strategies1 against multiple HCV genotypes that aim to balance in vivo safety, efficiency, and specificity, and that promise to restrict viral escape mutants. In the first study, Chandra et al.2 used lipid nanoparticles called nanosomes to encapsulate two preselected potent anti-HCV small interfering RNAs (siRNAs), and show that six daily combined systemic injections sufficed to significantly block HCV replication in a liver xenograft mouse model. In the second report, Suhy et al.3 optimized an adeno-associated viral (AAV) vector of serotype 8 for robust and safe expression of three short hairpin RNAs (shRNAs) targeting conserved regions of the HCV genome, and demonstrate its sustained performance in the livers of nonhuman primates (NHPs) following a single intravenous injection. By validating the concept of nonviral- or viral vector–mediated coRNAi against rapidly evolving targets in different animal models, the studies represent an important step forward in our efforts to translate newer vector and RNAi designs into clinical therapies for HCV.

Item does not contain fulltext The ESF-EMBO conference on 'Antiviral RNAi: From Molecular Biology towards Applications' took place in June 2012 in Pultusk, Poland. It brought together scientists working at the interface of RNAi and virus infections in different organisms, covering the complete range from basic mechanisms of RNA silencing to RNAi-based antiviral therapy.

Vectors derived from adeno-associated virus serotype 2 (AAV-2) represent a most promising tool for human gene transfer because these vectors are neither pathogenic nor toxic to the target cell, and allow long-term gene expression in a large variety of tissues. However, they are rather inefficient at infecting a number of clinically relevant cell types, and transduction by these vectors is likely hampered by neutralizing antibodies that are highly prevalent in the human population. Therefore, an increasing number of researchers are currently turning their attention to the five other serotypes of AAV, to try and develop these as novel vectors for human gene transfer, hoping to overcome the problems associated with AAV-2 vectors. Here I describe and discuss the methodology to produce these alternative AAV vectors in tissue culture. In detail, two strategies are compared that rely on transfection of cells in culture with either two or three plasmids, containing the AAV vector genome and encoding AAV and adenoviral helper functions. Either of these protocols can be used to package a recombinant AAV genome into capsids of its own serotype (generation of "real" serotypes) or to "cross-package" this vector DNA into capsids derived from another AAV serotype ("pseudotyping"). As these approaches are still in their early stages, the existing limitations of current technology are discussed, and possible further improvements proposed.

Interferons (IFNs) are key players in the antiviral response. IFN sensing by the cell activates transcription of IFN-stimulated genes (ISGs) able to induce an antiviral state by affecting viral replication and release. IFN also induces the expression of ISGs that function as negative regulators to limit the strength and duration of IFN response. The ISGs identified so far belong to coding genes. However, only a small proportion of the transcriptome corresponds to coding transcripts and it has been estimated that there could be as many coding as long non-coding RNAs (lncRNAs). To address whether IFN can also regulate the expression of lncRNAs, we analyzed the transcriptome of HuH7 cells treated or not with IFNα2 by expression arrays. Analysis of the arrays showed increased levels of several well-characterized coding genes that respond to IFN both at early or late times. Furthermore, we identified several IFN-stimulated or -downregulated lncRNAs (ISRs and IDRs). Further validation showed that ISR2, 8, and 12 expression mimics that of their neighboring genes GBP1, IRF1, and IL6, respectively, all related to the IFN response. These genes are induced in response to different doses of IFNα2 in different cell lines at early (ISR2 or 8) or later (ISR12) time points. IFNβ also induced the expression of these lncRNAs. ISR2 and 8 were also induced by an influenza virus unable to block the IFN response but not by other wild-type lytic viruses tested. Surprisingly, both ISR2 and 8 were significantly upregulated in cultured cells and livers from patients infected with HCV. Increased levels of ISR2 were also detected in patients chronically infected with HIV. This is relevant as genome-wide guilt-by-association studies predict that ISR2, 8, and 12 may function in viral processes, in the IFN pathway and the antiviral response. Therefore, we propose that these lncRNAs could be induced by IFN to function as positive or negative regulators of the antiviral response.

Its remarkable ease and efficiency make the CRISPR (clustered regularly interspaced short palindromic repeats) DNA editing machinery highly attractive as a new tool for experimental gene annotation and therapeutic genome engineering in eukaryotes. Here, we report a versatile set of plasmids and vectors derived from adeno-associated virus (AAV) that allow robust and specific delivery of the two essential CRISPR components - Cas9 and chimeric g(uide)RNA - either alone or in combination. All our constructs share a modular design that enables simple and stringent guide RNA (gRNA) cloning as well as rapid exchange of promoters driving Cas9 or gRNA. Packaging into potent synthetic AAV capsids permits CRISPR delivery even into hard-to-transfect targets, as shown for human T-cells. Moreover, we demonstrate the feasibility to direct Cas9 expression to or away from hepatocytes, using a liver-specific promoter or a hepatic miRNA binding site, respectively. We also report a streamlined and economical protocol for detection of CRISPR-induced mutations in less than 3 h. Finally, we provide original evidence that AAV/CRISPR vectors can be exploited for gene engineering in vivo, as exemplified in the liver of adult mice. Our new tools and protocols should foster the broad application of CRISPR technology in eukaryotic cells and organisms, and accelerate its clinical translation into humans.

To develop additional microsatellite (MS) markers in the region of the porcine skeletal muscle ryanodine receptor gene (RYR1), a microdissected genomic library was generated from the proximal half of the q arm of swine chromosome 6. Purified DNA was restriction enzyme-digested, ligated to oligonucleotide adaptors and amplified by PCR using primers complementary to the adaptor sequences. The purity of the amplified products and boundaries of the microdissected chromosomal region were verified by fluorescence in situ hybridization. (CA)n-containing sequences were then identified in a small insert genomic library generated from the PCR-amplified microdissected DNA. Oligonucleotide primers were developed for the PCR amplification of 30 of the 46 (CA)n repeat-containing clones, which were subsequently used to amplify DNA isolated from unrelated pigs of different breeds to determine the informativeness of these MS markers. Twenty-two of these MS markers were genotyped on the University of Illinois Yorkshire × Meishan swine reference population. These 22 markers were all assigned within a 50·7-c m region of the swine chromosome 6 linkage map, indicating the specificity of the microdissected library.

We demonstrate the rapid and reliable quantification of physical AAV-2 (adeno-associated virus type 2) particles via a novel ELISA based on a monoclonal antibody which selectively recognizes assembled AAV-2 capsids. Titration of a variety of recombinant AAV-2 (rAAV) preparations revealed that at least 80+percent of all particles were empty, compared with a maximum of 50percent in wild-type AAV-2 stocks, indicating that the recombinant genomes were less efficiently encapsidated. This finding was confirmed upon titration of CsCl gradient fractions from recombinant and wild-type AAV-2 stocks. ELISA-based measurement of capsid numbers revealed a large number of physical particles with low densities corresponding to empty capsids in the recombinant, but not in the wild-type AAV-2 preparations. Moreover, additional expression of VP proteins during rAAV production was found to result in an excessive capsid formation, whilst yielding only minor increases in DNA-containing or transducing rAAV particles. We conclude that encapsidation of viral genomes rather than capsid assembly can be limiting for rAAV production, provided that a critical level of VP expression is maintained. The feasibility of quantifying AAV-2 capsid numbers via the ELISA allows determination of physical to DNA-containing or infectious particle ratios. These are important parameters which should help to optimize and standardize the production and application of recombinant AAV-2.

Recombinant adeno-associated virus (rAAV) vectors have become attractive tools for in vivo gene transfer. The production and purification of high- titer rAAV vector stocks for experimental and therapeutic gene transfer continue to undergo improvement. Standard rAAV vector purification protocols include the purification of the vector by cesium chloride (CsCl)-density gradient centrifugation followed by extensive desalination via dialysis against a physiological buffer for in vivo use. These procedures are extremely time consuming and frequently result in a substantial loss of the infectious vector titer. As an alternative to CsCl we have investigated the use of Iodixanol, an X-ray contrast solution, as the density-gradient medium. Purification of rAAV vectors by Iodixanol shortened the centrifugation period to 3 hr and resulted in reproducible concentration and purification of rAAV- vector stocks. We show that injection of rAAV derived from an Iodixanol gradient can be used for in vivo gene transfer applications in the brain and spinal cord without detectable cytopathic effects and directing stable transgene expression for at least 2 months.

Standard protocols for the generation of adenoassociated virus type 2 (AAV-2)-based vectors for human gene therapy applications require cotransfection of cells with a recombinant AAV (rAAV) vector plasmid and a packaging plasmid that provides the AAV rep and cap genes. The transfected cells must also be overinfected with a helper virus, e.g., adenovirus (Ad), which delivers multiple helper functions necessary for rAAV production. Therefore, rAAV stocks produced using these protocols are contaminated with helper adenovirus. The generation of a novel packaging/helper plasmid, pDG, containing all AAV and Ad functions required for amplification and packaging of AAV vector plasmids, is described here. Cotransfection of cells with pDG and an AAV vector plasmid was sufficient for production of infectious rAAV, resulting in helper virus-free rAAV stocks. The rAAV titers obtained using pDG as packaging plasmid were up to 10-fold higher than those achieved using conventional protocols for rAAV production. Replacement of the AAV-2 p5 promoter by an MMTV-LTR promoter in pDG led to reduced expression of Rep78/68; however, expression of the VP proteins was significantly increased compared with VP levels from standard packaging plasmids. Immunofluorescence analyses showed that the strong accumulation of VP proteins in pDG-transfected cells resulted in enhanced AAV capsid assembly, which is limiting for efficient rAAV production. Furthermore, using a monoclonal antibody highly specific for AAV-2 capsids (A20), an rAAV affinity purification procedure protocol was established. The application of the tools described here led to a significant improvement in recombinant AAV vector production and purification.

Cytochrome P450 2D7 (CYP2D7) has long been considered a pseudogene. A recent report described an indel polymorphism (CYP2D7 138delT) that causes a frameshift generating an open reading frame and functional protein. This polymorphism was observed in 6 of 12 samples from an Indian population. Individuals with the 138delT polymorphism expressed CYP2D7 protein from a brain-specific, alternatively spliced transcript (J Biol Chem 279:27383-27389, 2004). The unexpectedly high frequency of the variant allele and resulting CYP2D7 expression could have important implications for brain-specific metabolism of CYP2D substrates including many psychoactive drugs. However, the 138delT variant has not been detected in other studies (Pharmacogenetics 11:45-55, 2001; Biochem Biophys Res Commun 336:1241-1250, 2005). Our goal was to determine the frequency of this variant in a larger, ethnically diverse population. CYP2D7 138delT genotypes for 163 Caucasians, 95 East Asians, 50 Indians, 68 Hispanic Latinos, and 68 African Americans were determined by Pyrosequencing. The 138delT allele was observed at a frequency of 1.0% in East Asians and 0.74% in Hispanic Latinos. The deletion was not observed in Indians or the other ethnic populations. In addition, in each of the three samples with 138delT, the putative brain-specific transcript contains a premature stop codon that would preclude protein expression. The low frequency of the CYP2D7 138delT polymorphism in our ethnically diverse sample, and particularly the absence from 50 Indian samples, is in contrast to the high frequency previously reported. Our results suggest that CYP2D7 138delT is unlikely to be highly relevant for population variation of pharmacokinetics or drug response.

The CRISPR/Cas9 system currently revolutionizes the gene therapy field due to the unique ease and efficiency with which it can be exploited for targeted DNA editing in mammalian cells. It only requires a small g(uide)RNA that directs the Cas9 protein to a specific DNA sequence which is then cleaved by Cas9. In the absence of a homologous exogenous template, the ensuing double-stranded DNA break is repaired by non-homologous end joining, an error-prone cellular mechanism frequently resulting in insertions or deletions that disrupt the original sequence. While useful in principle to perturb the expression of disease-associated genes, rapidly evolving targets such as human viruses will require concurrent expression of multiple gRNAs to thwart adverse mutational escape. To fill in this gap, we created novel highly customizable AAV vectors permitting multiplexing of up to three gRNAs in a single self-complementary vector backbone. Notably, our constructs readily accommodate and deliver minimal expression cassettes only consisting of gRNA(s) and promoter(s) with total sizes as little as 250 bp, which reduces the amount of ectopic DNA and improves vector safety. We demonstrated the power of our vectors by their use for simultaneous knock-out of three key players in apoptosis signaling, exemplifying their usefulness for dissection of cellular pathways. In addition, we engineered corresponding AAV backbones for shRNA expression, to be able to eventually juxtapose targeting on the DNA (CRISPR) and the RNA (RNAi) level in a single vector. Therefore, we made all constructs compatible with Golden Gate Assembly which minimizes work while maximizing flexibility, in turn paving the way for high-throughput cloning and screening of combinatorial RNAi/CRISPR libraries. Notably, the vectors offer three different RNA polymerase III promoters – U6, H1 or 7SK – for expression of gRNA or shRNA, thus allowing users to fine-tune g/shRNA levels and to alleviate toxic effects from over-expression. Finally, we also assembled a library of promoters for Cas9 expression comprising viral, synthetic and mammalian variants, to further expand the options for customization of our AAV/TRISPR system according to specific targets and needs. Indeed, as described in more detail in a separate abstract (Borner et al.), the selection of optimal gRNA/Cas9 vector designs even permits potent disruption of very challenging targets, such as integrated proviral HIV-1 DNA in T cells. Likewise, the use of proper regulatory elements is crucial for potent in vivo CRISPR-mediated targeting of endogenous genes in the mouse liver, as noted for the miR-122 microRNA. Considering the high degree of versatility of our vector designs, as well as the additional options for in vivo re-targeting through AAV capsid engineering, we expect our novel AAV-TRISPR kit for combinatorial gRNA/shRNA expression to substantially foster future efforts to co-exploit RNAi and CRISPR, and to benefit a multitude of human gene therapy applications.

RNA interference (RNAi) has become an established tool for basic gene annotation and a promising option for therapeutic concepts that require specific inhibition of dysregulated or exogenous genes. However, we and others accumulated strong evidence that overexpression of short hairpin RNAs (shRNAs, an expressable and widely used form of RNAi trigger) can cause cytotoxicity leading up to organ damage and lethality in shRNA-treated animals. A meticulous dissection of the underlying mechanisms is critical as it will ultimately guide the design of safer RNAi therapies and thus foster their clinical translation. One informative finding was that co-expression of Ago2 (Slicer, as it cleaves targeted mRNA), a key component of the RNA-induced silencing complex RISC, increases shRNA efficiency in cells and animals, implying that Ago2 is a rate-limiting factor whose saturation may be involved in cytotoxicity. Moreover, over-expression of the three other mammalian Ago proteins – Ago1, Ago3 or Ago4 (all slicing-incompetent) – dampens RNAi potency, underscoring that a delicate balance of all four Ago variants is key to efficient and safe gene silencing. Here, we investigated three hypotheses that could explain how relative Ago1-4 levels may affect the functionality of shRNA-loaded RISC and hence the strength of target knockdown: over-abundance of non-Slicer Ago proteins could (i) dysregulate Ago2 expression (transcription and/or translation), (ii) re-localize intracellular Ago2 away from sites of RNAi activity, or (iii) quantitatively sequester shRNAs into slicing-incompetent RISC. Using codon-optimized Ago variants that are distinguishable by real-time PCR from the endogenous counterparts, as well as Western blotting, we could eliminate model (i). Likewise, wide-field and confocal microscopy analyses revealed no evidence for Ago2 re-localization upon Ago1/3/4 over-expression and vice versa. We therefore focused on hypothesis (iii) and fused the Ago1-4 cDNAs with GFP or RFP tags that enabled their specific immunoprecipitation, alone or in combination, using the TRAP system. Indeed, we found that Slicer and non-Slicers compete for shRNA loading and mRNA knockdown in a dose-dependent manner, readily explaining all prior observations of RNAi enhancement by Ago2 over-expression, or shRNA inhibition by Ago1/3/4, respectively. Importantly, the combination of GFP/RFP-Ago fusion constructs with the co-precipitation protocols newly established during this work should be widely useful to address other vital questions in the RNAi field, such as the selective processing of different cellular small RNAs by the four Ago proteins. Moreover, our notion that over-expression of non-Slicers has no adverse effect on endogenous Ago2 levels or localization paves the way for novel strategies to fine-tune therapeutic RNAi in a safe and potent manner. As a prototype, we present a new shRNA/Ago co-expression system whereby Ago1/2 levels can be regulated independently through exogenous macrolides, permitting a tight control of target knockdown efficiencies and providing an original avenue to reduce toxicity in future RNAi gene therapy applications.

Using immunofluorescence and in situ hybridization techniques, we studied the intracellular localization of adeno-associated virus type 2 (AAV-2) Rep proteins, VP proteins, and DNA during the course of an AAV-2/adenovirus type 2 coinfection. In an early stage, the Rep proteins showed a punctate distribution pattern over the nuclei of infected cells, reminiscent of replication foci. At this stage, no capsid proteins were detectable. At later stages, the Rep proteins were distributed more homogeneously over the nuclear interior and finally became redistributed into clusters slightly enriched at the nuclear periphery. During an intermediate stage, they also appeared at an interior part of the nucleolus for a short period, whereas most of the time the nucleoli were Rep negative. AAV-2 DNA colocalized with the Rep proteins. All three capsid proteins were strongly enriched in the nucleolus in a transient stage of infection, when the Rep proteins homogeneously filled the nucleoplasm. Thereafter, they became distributed over the whole nucleus and colocalized in nucleoplasmic clusters with the Rep proteins and AAV-2 DNA. While VP1 and VP2 strongly accumulated in the nucleus, VP3 was almost equally distributed between the nucleus and cytoplasm. Capsids, visualized by a conformation-specific antibody, were first detectable in the nucleoli and then spread over the whole nucleoplasm. This suggests that nucleolar components are involved in initiation of capsid assembly whereas DNA packaging occurs in the nucleoplasm. Expression of a transfected full-length AAV-2 genome followed by adenovirus infection showed all stages of an AAV-2/adenovirus coinfection, whereas after expression of the cap gene alone, capsids were restricted to the nucleoli and did not follow the nuclear redistribution observed in the presence of the whole AAV-2 genome. Coexpression of Rep proteins released the restriction of capsids to the nucleolus, suggesting that the Rep proteins are involved in nuclear redistribution of AAV capsids during viral infection. Capsid formation was dependent on the concentration of expressed capsid protein.

A paramount task in RNA interference research is to decipher the complex biology of cellular effectors, exemplified in humans by four pleiotropic Argonaute proteins (Ago1-Ago4). Here, we exploited DNA family shuffling, a molecular evolution technology, to generate chimeric Ago protein libraries for dissection of intricate phenotypes independently of prior structural knowledge. Through shuffling of human Ago2 and Ago3, we discovered two N-terminal motifs that govern RNA cleavage in concert with the PIWI domain. Structural modeling predicts an impact on protein rigidity and/or RNA-PIWI alignment, suggesting new mechanistic explanations for Ago3's slicing deficiency. Characterization of hybrids including Ago1 and Ago4 solidifies that slicing requires the juxtaposition and combined action of multiple disseminated modules. We also present a Gateway library of codon-optimized chimeras of human Ago1-Ago4 and molecular evolution analysis software as resources for future investigations into RNA interference sequence-structure-function relationships.

Successful RNAi applications depend on strategies allowing robust and persistent expression of minimal gene silencing triggers without perturbing endogenous gene expression. Here, we propose a novel avenue which is integration of a promoterless shmiRNA, i.e. a shRNA embedded in a micro-RNA (miRNA) scaffold, into an engineered genomic miRNA locus. For proof-of-concept, we used TALE or CRISPR/Cas9 nucleases to site-specifically integrate an anti-hepatitis C virus (HCV) shmiRNA into the liver-specific miR-122/hcr locus in hepatoma cells, with the aim to obtain cellular clones that are genetically protected against HCV infection. Using reporter assays, Northern blotting and qRT-PCR, we confirmed anti-HCV shmiRNA expression as well as miR-122 integrity and functionality in selected cellular progeny. Moreover, we employed a comprehensive battery of PCR, cDNA/miRNA profiling and whole genome sequencing analyses to validate targeted integration of a single shmiRNA molecule at the expected position, and to rule out deleterious effects on the genomes or transcriptomes of the engineered cells. Importantly, a subgenomic HCV replicon and a full-length reporter virus, but not a Dengue virus control, were significantly impaired in the modified cells. Our original combination of DNA engineering and RNAi expression technologies benefits numerous applications, from miRNA, genome and transgenesis research, to human gene therapy.

Comprehensive analysis of deep sequencing data originating from the newly introduced Targeted Enrichment Sequencing (TES) indicates so far undescribed recombinations within the Inverted Terminal Repeats of recombinant Adeno-Associated Viruses (rAAVs). For the detection of vector integration sites into the host genome we routinely apply LAM-PCR. However, TES, in which we enrich for genomic regions that include vector sequences, has major advantages over LAM-PCR, as it neither depends on the existence of a vector-specific primer binding site (often lost during rAAV integration) nor on a restriction site in the vicinity of the vector insertion site. Furthermore, regions that are captured together with the vector allow for relative quantification of vector copies per genome. As the entire vector can be sequenced by TES, mutations within the vector and transgene can be detected. In summary, we here provide new and so far unpublished information about rAAV integration patterns and show how we optimized TES to become an important complementary tool to primer-based approaches (like LAM-PCR) for mapping of vector/viral integration sites.

A 2010 report that Adeno-associated virus 2 (AAV2) encodes an additional protein in the second open reading frame of the cap(sid) gene significantly changed our perception of the biology of this virus. This so-called assembly-activating protein or AAP appears to interact with the major viral capsid protein 3 (VP3) and to chaperone its assembly into virus-like particles, via mechanisms that remain unknown. Moreover, it was shown that deliberate mutation of the AAP gene diminishes AAV particle production.As the AAP open reading frame fully overlaps with that of the AAV capsid proteins VP1/VP2 (and partially VP3), it is likely that the AAP gene is inadvertently disrupted or mutated during molecular AAV evolution protocols that involve capsid gene shuffling (homology-directed recombination of cap gene fragments). To study this possibility comprehensively and to provide solutions, if needed, we first investigated the role of AAP for 9 further AAV serotypes other than AAV2. Therefore, we knocked out AAP expression in serotypes 1, 3-9 and rh10 while leaving the rep and cap genes intact. Indeed, all mutants were markedly impaired in particle production, confirming that functional AAP is critically required for at least 10 different AAV serotypes.Notably, all vectors were fully restored upon co-delivery of the corresponding AAP via an extra plasmid during particle production. Furthermore, when testing the cross-reactivity of all AAPs with the AAV2-AAP mutant (AAV2mut), we observed a broad interchangeability except for AAP4 and 5 which predominantly rescued their cognate serotype.These results were confirmed independently in a baculovirus AAV2 vector production system where we also observed a dramatic reduction in particle yields upon AAP mutation. Interestingly, in both, mammalian and insect cells, we consistently noted that absence of AAP reduced the steady-state levels of VP proteins, suggesting a similar biological function of AAP in the two heterologous cellular systems. As we could rule out an effect on the transcriptional level, we postulate that AAP-mediated chaperoning of AAV virion assembly indirectly stabilizes VP proteins in cells of different species.To explicitly dissect the role of AAP in the context of molecular AAV evolution, we produced five different AAV capsid libraries with increasing complexities, subcloned 46 randomly selected shuffled AAPs from these and tested them with the AAV-AAP mutants. Out of these 46 shuffled AAPs, 37 could rescue AAV2mut, and two more efficiently complemented an AAV5-AAP mutant. In total, 84.8 % of all tested AAPs were functional.Of note, the highest proportion of defect AAPs was found in libraries that contained the diverse capsids of AAV4 and 5. We thus shuffled serotypes 2, 4, 5, 8 and 9 and produced viral libraries with or without a cocktail of excess AAP from all five serotypes. Strikingly, albeit up to 40% of all AAP genes were predicted to be non-functional as a result of inadvertent shuffling, AAP trans-complementation had no significant effect on particle yields.Collectively, our data suggest that albeit AAP is disrupted to some extent during AAV capsid gene shuffling, this is likely not a major restriction for the efficiency and success of this technology.

Parvoviruses have long been developed as safe, efficient and versatile DNA delivery vectors for gene therapy applications. In particular adeno-associated viral (AAV) vectors have emerged as lead candidates owing to their apathogenicity and amenability to genetic modifications. Yet, a drawback is their limited cargo capacity of 4.9 kb, which is insufficient to accommodate larger genes. Intriguingly, it was shown recently that oversized single-stranded AAV genomes can be packaged into capsids of human bocavirus 1 (HboV1, also a parvovirus), yielding HboV1/AAV chimeras that specifically and efficiently transduce human airway epithelia (HAE). Motivated by this pioneering work, we aimed to expand the repertoire of HboV/AAV chimeras by vectorizing four additional primate bocaviruses known to infect the gastrointestinal (GI) tract. We thus assembled and cloned the VP1/VP2 ORFs from three human variants (HboV2-4) and gorilla bocavirus into a helper plasmid derived from HboV1, carrying the genes required for HboV1 replication and packaging. To assess viral particle assembly, HEK293T cells were transfected with three plasmids: (i) one of our new HboV helpers; (ii) a self-complementary AAV-YFP vector; and (iii) pDG, a plasmid encoding all genes for AAV packaging and replication. In all cases, correct expression of VP1/VP2 proteins was confirmed by Western blot analyses of cell lysates. Also, following large-scale production and iodixanol gradient purification, qPCR analyses of the 40% phase showed the presence of DNase-resistant particles for all five bocaviral serotypes. Most importantly, titration of these particles revealed comparable quantities, demonstrating that expression of NS and NP1 proteins from HboV1 supports assembly of the four other bocaviruses. Analysis in primary HAE showed YFP transgene expression for all chimeric vectors except for HboV2/AAV, congruent with prior detection of the cognate wild-type viruses in nasopharyngeal aspirates. Further in line with epidemiological data, we noted a marked difference in infectivity, from 15% for HboV1, to below 1% for the others. In looming experiments, the new vectors will be studied in primary epithelial cells from the GI tract, which are the putative natural target cells for HboV2-4 and gorilla bocavirus. Interestingly, infectivity of all chimeras could be boosted by adding proteasome inhibitors, reminiscent of data with AAV. Hence, to enhance escape from the proteasome degradation pathway and improve transduction, we mutated surface tyrosines in the VP2 protein of HboV1. Functional assessment of the resulting mutants is currently ongoing. Collectively, the large capacity, unique cell specificities and ability to cross-package AAV DNA make this novel vector set highly attractive for human gene therapy applications. In the future, it should be moreover rewarding to attempt molecular evolution of bocavirus capsids, taking advantage of the profound experience with AAV vectors.

Adeno-associated viral (AAV) vectors represent some of the most potent and promising vehicles for therapeutic human gene transfer due to a unique combination of beneficial properties(1). These include the apathogenicity of the underlying wildtype viruses and the highly advanced methodologies for production of high-titer, high-purity and clinical-grade recombinant vectors(2). A further particular advantage of the AAV system over other viruses is the availability of a wealth of naturally occurring serotypes which differ in essential properties yet can all be easily engineered as vectors using a common protocol(1,2). Moreover, a number of groups including our own have recently devised strategies to use these natural viruses as templates for the creation of synthetic vectors which either combine the assets of multiple input serotypes, or which enhance the properties of a single isolate. The respective technologies to achieve these goals are either DNA family shuffling(3), i.e. fragmentation of various AAV capsid genes followed by their re-assembly based on partial homologies (typically80% for most AAV serotypes), or peptide display(4,5), i.e. insertion of usually seven amino acids into an exposed loop of the viral capsid where the peptide ideally mediates re-targeting to a desired cell type. For maximum success, both methods are applied in a high-throughput fashion whereby the protocols are up-scaled to yield libraries of around one million distinct capsid variants. Each clone is then comprised of a unique combination of numerous parental viruses (DNA shuffling approach) or contains a distinctive peptide within the same viral backbone (peptide display approach). The subsequent final step is iterative selection of such a library on target cells in order to enrich for individual capsids fulfilling most or ideally all requirements of the selection process. The latter preferably combines positive pressure, such as growth on a certain cell type of interest, with negative selection, for instance elimination of all capsids reacting with anti-AAV antibodies. This combination increases chances that synthetic capsids surviving the selection match the needs of the given application in a manner that would probably not have been found in any naturally occurring AAV isolate. Here, we focus on the DNA family shuffling method as the theoretically and experimentally more challenging of the two technologies. We describe and demonstrate all essential steps for the generation and selection of shuffled AAV libraries (Fig. 1), and then discuss the pitfalls and critical aspects of the protocols that one needs to be aware of in order to succeed with molecular AAV evolution.

RNA interference (RNAi) is an evolutionarily conserved fundamental cellular mechanism of potent gene and genome regulation whose misfunction is associated with numerous major human pathologies, from metabolic disorders and viral infections to cancers. Over the past 5 years, compelling evidence has been accumulated that this association is provided by dysregulations of specific mi(cro)RNAs and the ensuing aberrant expression of their target genes. Moreover, a string of interesting reports has now added proof that human disorders are also frequently characterized by global alterations in the RNAi machinery, comprising irregular expression and function of the key protein players Drosha, DGCR8, Exportin-5, Dicer, TRBP, and Argonaute. Here, we comprehensively review these emerging findings in the specific contexts of cancers and infections with viral pathogens and, in addition, describe related observations in preclinical gene/RNAi therapy studies. Finally, we also thoroughly discuss the relevance of these results for future basic RNAi research as well as for the looming clinical translation of RNAi-based technologies and therapeutic concepts.

MicroRNAs and siRNAs interact with target sequences in mRNAs, inducing cleavage- and non-cleavage–based gene repression through the RNA-induced silencing complex (RISC) that consists of one of four mammalian Argonaute proteins, Ago1–Ago4. The process of how Dicer substrate small hairpin RNAs (shRNAs) are loaded into different mammalian Agos in vivo is not well established. Here we report that shRNAs are loaded into mammalian Agos in two stepwise processes, physical association and activation, with the latter being the rate-limiting step with noncleaving RISC. We establish that, although RNA duplexes processed from shRNAs bind to Agos in cells with similar affinity, the degree by which the complexes are activated (coupled with the removal of the passenger strand) correlates with the thermodynamic instability of RNA duplexes being loaded rather than the structure of the RNA, as was previously demonstrated in Drosophila . Interestingly, Ago loading of siRNAs is less sensitive to thermostability than that of their shRNA equivalents. These results may have important implications for the future design of RNAi-based therapeutics.

RNA interference (RNAi) is an evolutionarily conserved fundamental cellular mechanism of potent gene and genome regulation whose misfunction is associated with numerous major human pathologies, from metabolic disorders and viral infections to cancers. Over the past 5 years, compelling evidence has been accumulated that this association is provided by dysregulations of specific mi(cro)RNAs and the ensuing aberrant expression of their target genes. Moreover, a string of interesting reports has now added proof that human disorders are also frequently characterized by global alterations in the RNAi machinery, comprising irregular expression and function of the key protein players Drosha, DGCR8, Exportin-5, Dicer, TRBP, and Argonaute. Here, we comprehensively review these emerging findings in the specific contexts of cancers and infections with viral pathogens and, in addition, describe related observations in preclinical gene/RNAi therapy studies. Finally, we also thoroughly discuss the relevance of these results for future basic RNAi research as well as for the looming clinical translation of RNAi-based technologies and therapeutic concepts.

Sinclair miniature swine represent a breed of miniature swine which display a significant incidence of inheritable melanoma which undergo a developmentally regulated spontaneous regression. In an attempt to characterize the host cellular immune response to the melanoma, lymphocyte cell lines have been generated from peripheral blood and designated as peripheral blood lymphocyte cell lines (PBLCLs). The cell lines were expanded in vitro without the addition of exogenous mediators, cloned by limiting dilution, and characterized by flow microfluorimetry, Western, and Northern blot analysis. The cell lines were shown to be CD2-, CD4-, CD8-, and slg-, a phenotype consistent with a null cell population described in swine. The null cell population in swine has been reported to consist of a subpopulation of cells which express the gamma delta T cell receptor (TCR) heterodimer, swine gamma delta T lymphocytes. The PBLCLs were further analyzed by flow microfluorimetry and observed to express the IL-2R, swine MHC Class II antigens, and the endothelial lymphocyte adhesion marker (CD44), which can function as a homing receptor for the skin. In addition, the PBLCLs were observed to express the antigen which is recognized by mAb 86D, an antibody that has been reported to recognize an external epitope on a subset of gamma delta TCR bearing swine T lymphocytes. Western blot analysis of Triton X-114 phase fractions of a PBLCL revealed a protein recognized by the W6 antibody, an antibody which recognizes a conserved region of the C delta chain. Furthermore, Southern and Northern blot analysis indicated that the PBLCL have rearranged the TCR gamma chain gene and express mRNA from the TCR gamma and delta chain genes prior to and following treatment with ionomycin or Concanavalin A. Therefore, the data indicates that the PBLCLs represent swine gamma delta T lymphocyte cell lines which should enable us to enhance our understanding of the role of gamma delta T lymphocytes in the porcine immune system.

Within one decade, ribonucleic acid interference (RNAi), that is, the sequence-specific knockdown of gene expression triggered by small silencing RNAs, has rapidly matured from a biological curiosity into our single most promising biotherapeutic for a wide array of human diseases. Its exciting looming clinical translation is particularly accelerated by the increasingly pursued juxtaposition of RNAi technologies with established gene therapy methodologies. Fostering this mutual attraction of two potent clinical modalities and paving the way to fully harness their therapeutic power are the abilities of viral gene transfer vectors to mediate stable, efficient and tailored transduction of RNAi into recipient cells. Finally, moreover adding to the enormous promise of combining small silencing RNAs and gene therapy are latest findings on the role of endogenous microRNAs for various human diseases, further enlarging our already fertile chest of tools and targets for intervention and fortifying the optimism that RNAi gene therapies will soon become a clinical reality. Key Concepts: Expression of RNAi triggers from viral gene transfer vehicles is a potent strategy to optimise the issues of in vivo delivery and specificity. Vice versa, the development of expressible, vector-compatible RNAi triggers adds a crucial therapeutically relevant template to our arsenal of gene therapy vectors. The combination of RNAi and gene therapy thus synergistically enhances the power and promise of each individual approach. Beyond exploiting RNAi to directly target and destroy disease-associated genes, cellular miRNAs can also be used to segregate vectors or viruses. This allows to either direct transgene expression to a certain subset of cells in the body, or to increase the specificity and safety of oncolytic viruses. Further improvements of RNAi gene therapy vectors concern the promoter, which can be constitutive or inducible, weak or strong, or specific or ubiquitous. Concurrent advances in viral vector genome designs and structures help to maintain a threshold of RNAi expression required for potent gene silencing. Latest improvements in the field of RNAi gene therapy vectors comprise combinatorial approaches either aiming at enhancing custom vector properties or juxtaposing various RNAi triggers and other gene silencers, or co-targeting different viral or cellular genes, to avert target viral escape by mutation. Keywords: RNA interference; RNAi; small silencing RNAs; short hairpin RNA; microRNA; gene therapy; AAV vectors; posttranscriptional regulation; molecular evolution; combinatorial RNAi

Non-alcoholic fatty liver disease is a serious health problem linked to obesity and type 2 diabetes. To investigate the biological outcome and therapeutic potential of hepatic fatty acid uptake inhibition, we utilized an adeno-associated virus-mediated RNA interference technique to knock down the expression of hepatic fatty acid transport protein 5 in vivo prior to or after establishing non-alcoholic fatty liver disease in mice. Using this approach, we demonstrate here the ability to achieve specific, non-toxic, and persistent knockdown of fatty acid transport protein 5 in mouse livers from a single adeno-associated virus injection, resulting in a marked reduction of hepatic dietary fatty acid uptake, reduced caloric uptake, and concomitant protection from diet-induced non-alcoholic fatty liver disease. Importantly, knockdown of fatty acid transport protein 5 was also able to reverse already established non-alcoholic fatty liver disease, resulting in significantly improved whole-body glucose homeostasis. Thus, continued activity of hepatic fatty acid transport protein 5 is required to sustain caloric uptake and fatty acid flux into the liver during high fat feeding and may present a novel avenue for the treatment of non-alcoholic fatty liver disease.

The selective silencing of target genes in specific cell types by RNA interference (RNAi) represents a powerful approach both to gene therapy of dominantly active mutant alleles, and to the investigation of normal gene function in animal models in vivo. We established a simple and versatile in vitro method for screening the efficacy of DNA-based short hairpin RNAs (shRNAs), and identified a highly effective shRNA targeting basic fibroblast growth factor (bFGF), a gene thought to play important roles in endogenous neuroprotective responses in the rat retina. We used two viral vectors, based on lentivirus and adeno-associated virus (AAV), to deliver shRNAs and silence bFGF in retinal pigment epithelial cells in vivo. The AAV experiments made use of a "stabilized double-stranded" version of these vectors with rapid onset of gene expression. In the rat retinal pigment epithelium, shRNAs delivered by either vector reduced bFGF immunoreactivity to undetectable levels in transduced cells, whereas a nonfunctional control construct incorporating a two-base pair mutation had no measurable effect on bFGF expression. Silencing commenced within a few days after injection of virus and remained stable throughout the period of observation, as long as 60 days. Viral delivery of RNAi constructs offers a powerful and versatile approach for both gene therapy and the analysis of fundamental questions in retinal biology.

Over 500 million people worldwide are infected with one or more different and unrelated types of human hepatitis virus. Such individuals are at a high risk of developing acute or chronic hepatic disease, and ultimately dying from sequelae. Although a vaccine is available for hepatitis A and B virus, treatment options for chronically infected patients are limited, and particularly ineffective in case of hepatitis C virus (HCV) infection. A promising new avenue currently being explored is to harness the power of RNA interference for development of an antiviral therapy. The timing to pursue this particular approach is excellent, with the first in vivo animal models for HCV infection becoming available, and the technology for liver-specific expression of short hairpin RNAs advancing at a rapid pace. Here, we critically review these important current developments, and discuss the next steps to bring this novel approach into the clinics.

Gene transfer vectors based on naturally occurring serotypes of Adeno-associated virus (AAV) are gaining increasing attention due to their numerous advantages over other systems, including safety and capability to transduce a wide variety of cell types and tissues. However, remaining problems with AAV vectors include the high prevalence of neutralizing antibodies to at least some of the wildtype viruses in humans, and the fact that many relevant cell types are largely refractory to most AAV serotypes. Several strategies were proposed to overcome these hurdles, such as peptide insertion into the AAV-2 capsid or its site-directed mutagenesis, but required knowledge of the capsid structure and were thus restricted. Here, we pursued a most powerful approach to functional diversification of the AAV virion, by in vitro recombining the capsid genes of eight divergent primate and non-primate serotypes. In detail, we |[ldquo]|shuffled|[rdquo]| the capsid genes of AAV serotypes 2, 4, 5, 8 and 9, as well as of avian, bovine and caprine AAV, by DNase-fragmenting the respective sequences, followed by primer-less PCR amplification. The resulting hybrid capsid genes were fused with the rep gene and cloned between the ITRs from AAV-2, thus yielding a library of |[sim]|3|[times]|104 shuffled AAV variants. Sequencing of 24 individual clones confirmed that each capsid gene consisted of domains from at least 2, and often 6 or more, distinct serotypes. Library functionality was initially demonstrated by its successful sequential amplification in kidney, liver and fibroblast cell lines. Our main goal was however to isolate novel AAVs from the library, able to efficiently transduce hepatocytes or entire livers in the presence of neutralizing anti-AAV antibodies. We therefore subjected the virions to a respective selection pressure, by mixing them with high doses of human sera containing such antibodies, prior to inoculation on human Huh-7 liver cells together with helper Adenovirus. Following five consecutive amplification rounds under these conditions, we indeed succeeded in isolating a shuffled virion able to escape serum inactivation, while maintaining a high potential for liver cell infection. Sequence analyses showed that this mutant consisted of domains from serotypes 2, 8 and 9, with the latter two particularly dominant on the exterior of the capsid, and AAV-2 sequences prevailing in the VP1 protein N-terminus. A direct comparison of vectors based on this hybrid with genuine serotypes 2, 8 or 9 showed that the novel clone was indeed at least 5-fold more efficient on Huh-7 cells, and also the only one next to AAV-8 able to escape serum inactivation, thus exemplifying the power of our approach. We therefore extended our strategy to even more challenging targets, primary human hepatocytes and intact murine livers. At this point, we have isolated a subset of shuffled AAV virions consisting of sequences from serotypes 2, 4, 8 and 9, and mediating efficient infection of these targets. Ongoing long-term in vivo studies will resolve the potential of the novel capsids for liver transduction, and will hopefully identify them as promising candidates to replace or complement genuine AAV serotypes in clinical vector applications.

In recent years, the increasing prevalence of obesity and obesity-related co-morbidities fostered intensive research in the field of adipose tissue biology. To further unravel molecular mechanisms of adipose tissue function, genetic tools enabling functional studies in vitro and in vivo are essential. While the use of transgenic animals is well established, attempts using viral and non-viral vectors to genetically modify adipocytes in vivo are rare. Therefore, we here characterized recombinant Adeno-associated virus (rAAV) vectors regarding their potency as gene transfer vehicles for adipose tissue. Our results demonstrate that a single dose of systemically applied rAAV8-CMV-eGFP can give rise to remarkable transgene expression in murine adipose tissues. Upon transcriptional targeting of the rAAV8 vector to adipocytes using a 2.2 kb fragment of the murine adiponectin (mAP2.2) promoter, eGFP expression was significantly decreased in off-target tissues while efficient transduction was maintained in subcutaneous and visceral fat depots. Moreover, rAAV8-mAP2.2-mediated expression of perilipin A – a lipid-droplet-associated protein – resulted in significant changes in metabolic parameters only three weeks post vector administration. Taken together, our findings indicate that rAAV vector technology is applicable as a flexible tool to genetically modify adipocytes for functional proof-of-concept studies and the assessment of putative therapeutic targets in vivo. ISSN:1932-6203

We and others have recently reported highly efficient liver gene transfer with adeno-associated virus 8 (AAV-8) pseudotypes, i.e., AAV-2 genomes packaged into AAV-8 capsids. Here we studied whether liver transduction could be further enhanced by using viral DNA packaging sequences (inverted terminal repeats [ITRs]) derived from AAV genotypes other than 2. To this end, we generated two sets of vector constructs carrying expression cassettes embedding a gfp gene or the human factor IX ( hfIX ) gene flanked by ITRs from AAV genotypes 1 through 6. Initial in vitro analyses of gfp vector DNA replication, encapsidation, and cell transduction revealed a surprisingly high degree of interchangeability among the six genotypes. For subsequent in vivo studies, we cross-packaged the six hfIX variants into AAV-8 and infused mice via the portal vein with doses of 5 × 10 10 to 1.8 × 10 12 particles. Notably, all vectors expressed comparably high plasma hFIX levels within a dose cohort over the following 6 months, concurrent with the finding of equivalent vector DNA copy numbers per cell. Partial hepatectomies resulted in ∼80% drops of hFIX levels and vector DNA copy numbers in all groups, indicating genotype-independent persistence of predominantly episomal vector DNA. Southern blot analyses of total liver DNA in fact confirmed the presence of identical and mostly nonintegrated molecular vector forms for all genotypes. We conclude that, unlike serotypes, AAV genotypes are not critical for efficient hepatocyte transduction and can be freely substituted. This corroborates our current model for AAV vector persistence in the liver and provides useful information for the future design and application of recombinant AAV.

Five recent publications have documented the successful development and use of gene transfer vectors based on adeno-associated virus (AAV) for expressing short hairpin RNA (shRNA). In cultured mammalian cells and in whole animals, infection with these vectors was shown to result in specific, efficient, and stable knockdown of various targeted endo- or exogenous genes. Here we review this exciting approach, to trigger RNA interference in vitro and in vivo by shRNA expressed from AAV vectors, and describe the state-of-the-art technology for vector particle generation. In particular, we present a set of novel AAV vector plasmids that were specifically designed for the easy and rapid cloning of shRNA expression cassettes into AAV. The plasmids contain alternative RNA polymerase III promoters (U6, H1, or 7SK) together with a respective terminator sequence, as well as stuffer DNA to guarantee an optimal vector size for efficient packaging into AAV capsids. To provide maximum versatility and user-friendliness, the constructs were also engineered to contain a set of unique restriction enzyme recognition sites, allowing the simple and straightforward replacement of the shRNA cassette or other vector components with customized sequences. Our novel vector plasmids complement existing AAV vector technology and should help further establish AAV as a most promising alternative to using adeno- or retro-?lentiviral vectors as shRNA delivery vehicles.

Five recent publications have documented the successful development and use of gene transfer vectors based on adeno-associated virus (AAV) for expressing short hairpin RNA (shRNA). In cultured mammalian cells and in whole animals, infection with these vectors was shown to result in specific, efficient, and stable knockdown of various targeted endo- or exogenous genes. Here we review this exciting approach, to trigger RNA interference in vitro and in vivo by shRNA expressed from AAV vectors, and describe the state-of-the-art technology for vector particle generation. In particular, we present a set of novel AAV vector plasmids that were specifically designed for the easy and rapid cloning of shRNA expression cassettes into AAV. The plasmids contain alternative RNA polymerase III promoters (U6, H1, or 7SK) together with a respective terminator sequence, as well as stuffer DNA to guarantee an optimal vector size for efficient packaging into AAV capsids. To provide maximum versatility and user-friendliness, the constructs were also engineered to contain a set of unique restriction enzyme recognition sites, allowing the simple and straightforward replacement of the shRNA cassette or other vector components with customized sequences. Our novel vector plasmids complement existing AAV vector technology and should help further establish AAV as a most promising alternative to using adeno- or retro-⧸lentiviral vectors as shRNA delivery vehicles.

Background The metabolizing enzyme cytochrome P450 (CYP) 3A5 is polymorphically expressed as a result of genetic variants that do not encode functional protein. Because of overlapping substrate specificity with CYP3A4 and the multidrug efflux pump P-glycoprotein, the importance of CYP3A5 genetic polymorphism for pharmacokinetics is controversial. Objective Our objective was to determine whether genetic polymorphisms in CYP3A5 or MDR-1 (which encodes P-glycoprotein) influence the drug levels of ABT-773, a ketolide antibiotic that is a substrate for both CYP3A and P-glycoprotein. Methods Healthy volunteers given 3 different oral dose levels of ABT-773 were genotyped at 2 common CYP3A5 and 7 common MDR-1 polymorphisms. Individuals were categorized as CYP3A5-positive if they carried at least 1 functional CYP3A5*1 allele and as CYP3A5-negative if they did not. Area under the plasma concentration–time curves (AUCs) from 0 to 6 hours (AUCt) and maximum postdose plasma concentration (Cmax) after a single dose and on day 5 of a twice-daily regimen were calculated and correlated with genotypes. Results ABT-773 AUCt and Cmax were, on average, higher in CYP3A5-negative subjects given 450 mg ABT-773 (n = 9) than in CYP3A5-positive subjects with identical doses (n = 8). The relationship for AUCt was statistically significant both after a single dose (geometric mean and 95% confidence interval [CI], 5.0 μg · h/mL [3.9–6.4 μg · h/mL] versus 2.8 μg · h/mL [1.8–4.3 μg · h/mL]; P = .03) and on the fifth day of twice-daily dosing (12.4 μg · h/mL [8.7–17.6 μg · h/mL] versus 7.4 μg · h/mL [5.5–9.8 μg · h/mL], P = .04). The relationship for Cmax was statistically significant after a single dose (1220 μg/mL [867–1167 μg/mL] versus 727 μg/mL [506–1044 μg/mL], P = .04) and showed a trend in the same direction on the fifth day of twice-daily dosing (2566 μg/mL [1813–3631 μg/mL] versus 1621 μg/mL [1122–2343 μg/mL], P = .07). In contrast, AUCt and Cmax were not significantly different between CYP3A5-positive and CYP3A5-negative individuals given 150 mg or 300 mg ABT-773. ABT-773 plasma levels did not trend with MDR-1 genotypes. Conclusions These results suggest that CYP3A5 genotype may be an important determinant of in vivo drug disposition and that this effect may be dose-dependent. Clinical Pharmacology & Therapeutics (2004) 75, 516–528; doi: 10.1016/j.clpt.2004.01.013

Objective Vectors based on recombinant adeno-associated virus 2 (AAV-2) are a promising tool for cardiac gene transfer. However, potential therapeutic applications need to consider the predominant transduction of the liver once AAV-2 vectors enter the systemic circulation. We therefore aimed to increase efficiency and specificity of cardiac vector delivery by combining transcriptional and cell surface targeting. Methods For analysis of transcriptional targeting, recombinant AAV vectors were generated harboring a luciferase reporter gene under control of the cytomegalovirus (CMV) promoter or the 1.5-kb cardiac myosin light chain promoter fused to the CMV immediate-early enhancer (CMVenh/MLC1.5). Luciferase activities were determined in representative organs three weeks after intravenous injection of the vector into adult mice. Transductional targeting was studied using luciferase-reporter constructs crosspackaged into capsids of AAV serotypes 1 to 6 and modified AAV-2 capsids devoid of binding their primary receptor heparan sulfate proteoglycan. Results Intravenous injections of AAV-2 vectors harboring the CMVenh/MLC1.5 promoter enabled a specific and 50-fold higher reporter gene expression in left ventricular myocardium of adult mice compared to vectors containing the CMV promoter. Comparison of AAV-2 vector genomes crosspackaged into capsids of AAV-1 to -6 showed that AAV-1, -4, -5, and -6 capsids increased cardiac transduction efficiency by about 10-fold. However, transduction of other organs such as the liver was also increased after systemic administration. In contrast, AAV-2-based vectors with ablated binding to their primary receptor heparan sulfate proteoglycan enabled a significantly increased efficiency of cardiac gene transfer and reduced transduction of the liver. Conclusions Combining transcriptional targeting by the CMVenh/MLC1.5 promoter and AAV vectors devoid of binding the AAV-2 primary receptor results in an efficient cardiac gene transfer with a significantly reduced hepatic transduction.

This study utilized cytochrome P450 2D6 (CYP2D6) genotypes to explain variability of desipramine pharmacokinetics in a cohort of non-poor metabolizer individuals. In an interaction study utilizing desipramine as a probe, genotyping for the CYP2D6*3, *4, *5 and *6 alleles was used to screen out CYP2D6 poor metabolizers. Individuals were categorized according to these and additional alleles (CYP2D6*2, *9, *10, *17, *41 and x2). Genotypes of individuals heterozygous for two or three of *2, *17 and *41 alleles were confirmed by molecular haplotyping. Pharmacokinetic parameters of desipramine were analysed according to CYP2D6 category. Molecular haplotyping was necessary to definitively categorize four of 16 individuals. A subject who had unusually high plasma elimination half-time, exposure and metabolic ratios carried an intermediate metabolizer (IM) *9 allele in combination with a non-functional allele. This combination has a population frequency of less than 1 : 200. Individuals with *1/*1, *1/*2 and *2/*2 genotypes had lower than average plasma elimination half-time, exposure and metabolic ratios. For desipramine, additional genotyping of CYP2D6 IM alleles helped define subgroups of the CYP2D6-positive cohort. This suggests that genotyping for IM alleles will aid in interpretation of clinical trials involving CYP2D6 substrates. Due to the diversity of IM alleles, molecular haplotyping may be necessary to fully characterize CYP2D6 genotype-phenotype relationships.

Gene transfer vectors based on the human adeno-associated virus serotype 2 (AAV-2) have been developed and tested in pre-clinical studies for almost 20 years, and are currently being evaluated in clinical trials. So far, all these studies have provided evidence that AAV-2 vectors possess many properties making them very attractive for therapeutic gene delivery to humans, such as a lack of pathogenicity or toxicity, and the ability to confer long-term gene expression. However, there is concern that two restrictions of AAV-2 vectors might limit their clinical use in humans. First, these vectors are rather inefficient at transducing some cells of therapeutic interest, such as liver and muscle cells. Second, gene transfer might be hampered by neutralizing anti-AAV-2 antibodies, which are highly prevalent in the human population. In efforts to overcome both limitations, an increasing number of researchers are now focusing on the seven other naturally occurring serotypes of AAV (AAV-1 and AAV-3 to -8), which are structurally and functionally different from AAV-2. To this end, several strategies have been devised to cross-package an AAV-2 vector genome into the capsids of the other AAV serotypes, resulting in a new generation of "pseudotyped" AAV vectors. In vitro and in vivo, these novel vectors were shown to have a host range different from AAV-2, and to escape the anti-AAV-2 immune response, thus underscoring the great potential of this approach. Here the biology of the eight AAV serotypes is summarized, existing technology for pseudotyped AAV vector production is described, initial results from pre-clinical evaluation of the vectors are reviewed, and finally, the prospects of these promising novel tools for human gene therapy are discussed.

The susceptibility of a variety of different primary tissues was examined to long-term transduction with recombinant adeno-associated virus type 2 (rAAV-2) and factors influencing the transduction efficiency. In contrast to others using cell lines and animal models, emphasis was placed on the use of primary human cells. Enhanced green fluorescent protein (EGFP) marker gene expression was examined using fluorescence-activated cell sorting analysis. The most effective target cells for rAAV-2-mediated gene transfer were bronchial epithelial, artery endothelial as well as smooth and skeletal muscle cells with mean transduction rates ranging from 34.3 to 81.6%. Lower transduction rates between 4.3 and 19.5% were found in chondrocytes, dermal papilla follicle epithelial cells and fibroblasts. No transduction was observed in melanocytes, granulocyte colony-stimulating factor (G-CSF)-mobilized CD34(+) cells or malignant CD19(+) cells from patients with chronic lymphocytic leukemia. A proportion of EGFP-expressing skeletal muscle and smooth muscle cells was maintained over a period of 6 weeks after transduction (42.7+/-5.4 and 67.1+/-0.9%, respectively). Interestingly, among hair follicle epithelial cells the proportion of transduced cells increased from 8+/-0.5 to 36+/-7.7% in the course of 6 weeks. In contrast, for endothelial cells, bronchial epithelial cells and fibroblasts, a rapid decline in the number of EGFP expressing cells were noted. An inverse relationship between the proportion of cells in G2/M phase of cell cycle and long-term gene expression was observed. All rAAV-2 susceptible primary cells expressed FGFR-1 and the alphaV integrin consistent with their role as co-receptors for AAV-2. In conclusion, AAV-2 is a suitable vector system for transduction and evaluation of functional effects of long-term gene expression in primary human muscle and hair follicle cells.

Extensive experimental and limited epidemiologic data suggest that adeno-associated viruses (AAV) can have antioncogenic activity and may be protective factors for the development of cervical cancer. To examine the association between AAV-2 IgG antibodies and cervical neoplasia in Spain and Colombia, we tested for AAV-2 antibodies using an ELISA assay for 109 women with invasive cervical cancer, 100 population-based controls age-matched to the invasive cases, 77 women with carcinoma in situ (CIN III) and 100 clinic-based controls age-matched to the CIN III cases. Human papillomavirus (HPV) DNA was detected in cervical exfoliated cells by polymerase chain reaction using HPV-L1 and GP5+/6+ consensus primers. The prevalence of AAV-2 antibody titers >100 was significantly lower in invasive cervical cancer cases than control participants. When comparing women with invasive cancer with controls or with CIN III cases, a pattern of decreasing cervical cancer risk with increasing AAV-2 titers was observed. Elevated AAV antibody titers (>100) were inversely associated with invasive cervical cancer (OR 0.3; 95% CI 0.1–0.7), although results were not statistically significant after controlling for HPV (OR 0.4; 95% CI 0.1–1.6). In contrast, AAV-2 antibodies were not significantly associated with the risk of CIN III (OR 1.4; 95% CI 0.3–6.8). These results provide supportive evidence that AAV infection may be a protective factor for the development of invasive cervical cancer. Alternatively, the lower AAV-2 seroprevalence in invasive cervical cancer cases may be due to an immunosuppressive effect of cervical cancer on AAV antibody response. To investigate whether a direct viral interaction is occurring, future studies should aim to resolve at what frequency AAV is found in the genital tract and to clarify further whether AAV may infect the same HPV-positive cells in the cervix. © 2001 Wiley-Liss, Inc.

Helicases not only catalyse the disruption of hydrogen bonding between complementary regions of nucleic acids, but also move along nucleic acid strands in a polar fashion. Here we show that the Rep52 and Rep40 proteins of adeno-associated virus type 2 (AAV-2) are required to translocate capsid-associated, single-stranded DNA genomes into preformed empty AAV-2 capsids, and that the DNA helicase function of Rep52/40 is essential for this process. Furthermore, DNase protection experiments suggest that insertion of AAV-2 genomes proceeds from the 3' end, which correlates with the 3'-->5' processivity demonstrated for the Rep52/40 helicase. A model is proposed in which capsid-immobilized helicase complexes act as molecular motors to 'pump' single-stranded DNA across the capsid boundary.

Approximately 90% of cervical carcinomas are causally linked to infections with high-risk human papillomaviruses (HPVs), whose oncogenicity has been assigned to the continued expression of two early genes, E6 and E7. Reversal of the transformed phenotype by inhibiting E6/E7 gene expression therefore provides a suitable goal for future tumor therapy. Using recombinant adeno-associated virus type 2 (AAV-2) vectors, two types of therapeutic genes were expressed in cervical carcinoma cells with the aim of suppressing the E6/E7 oncogenes: (a) antisense E6/E7 and ribozyme genes and (b) the monocyte chemoattractant protein-1 (MCP-1) gene encoding MCP-1. Previous studies have shown that the MCP-1 protein is able to indirectly repress E6/E7 gene expression and is consistently absent in tumorigenic HPV-positive cervical carcinoma cell lines. Here, the effect of these therapeutic genes on tumor formation is analyzed in nude mice after ex vivo gene transfer into a HPV16- or HPV18-positive cervical carcinoma cell line (HeLa or SiHa, respectively). Whereas AAV-2 vector-mediated transfer of antisense or even ribozyme genes did not significantly influence tumor formation from implanted SiHa cells, the transfer and expression of human MCP-1 strongly inhibited the development of tumors derived from either HeLa or SiHa cells. Similar results were also obtained after in vivo delivery of these genes into SiHa-derived tumors. This suggests that transfer of therapeutic genes mediating a systemic effect via recombinant AAV-2 vectors offers a promising approach for the development of gene therapies directed against papillomavirus-induced human cancers.