Primary Human Cells Differ in Their Susceptibility to RAAV-2-mediated Gene Transfer and Duration of Reporter Gene Expression

Journal of Virological Methods. Sep, 2002  |  Pubmed ID: 12270659

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.

Production Methods for Gene Transfer Vectors Based on Adeno-associated Virus Serotypes

Methods (San Diego, Calif.). Oct, 2002  |  Pubmed ID: 12413413

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.

Helper Virus-free, Optically Controllable, and Two-plasmid-based Production of Adeno-associated Virus Vectors of Serotypes 1 to 6

Molecular Therapy : the Journal of the American Society of Gene Therapy. Jun, 2003  |  Pubmed ID: 12788658

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.

Preclinical in Vivo Evaluation of Pseudotyped Adeno-associated Virus Vectors for Liver Gene Therapy

Blood. Oct, 2003  |  Pubmed ID: 12791653

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 x 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.

Adeno-associated Virus Vectors for Short Hairpin RNA Expression

Methods in Enzymology. 2005  |  Pubmed ID: 15644194

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.

Increased Maintenance and Persistence of Transgenes by Excision of Expression Cassettes from Plasmid Sequences in Vivo

Human Gene Therapy. May, 2005  |  Pubmed ID: 15916481

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.

Liver Transduction with Recombinant Adeno-associated Virus is Primarily Restricted by Capsid Serotype Not Vector Genotype

Journal of Virology. Jan, 2006  |  Pubmed ID: 16352567

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 x 10(10) to 1.8 x 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 approximately 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.

Improved Cardiac Gene Transfer by Transcriptional and Transductional Targeting of Adeno-associated Viral Vectors

Cardiovascular Research. Apr, 2006  |  Pubmed ID: 16448634

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.

Fatality in Mice Due to Oversaturation of Cellular MicroRNA/short Hairpin RNA Pathways

Nature. May, 2006  |  Pubmed ID: 16724069

RNA interference (RNAi) is a universal and evolutionarily conserved phenomenon of post-transcriptional gene silencing by means of sequence-specific mRNA degradation, triggered by small double-stranded RNAs. Because this mechanism can be efficiently induced in vivo by expressing target-complementary short hairpin RNA (shRNA) from non-viral and viral vectors, RNAi is attractive for functional genomics and human therapeutics. Here we systematically investigate the long-term effects of sustained high-level shRNA expression in livers of adult mice. Robust shRNA expression in all the hepatocytes after intravenous infusion was achieved with an optimized shRNA delivery vector based on duplex-DNA-containing adeno-associated virus type 8 (AAV8). An evaluation of 49 distinct AAV/shRNA vectors, unique in length and sequence and directed against six targets, showed that 36 resulted in dose-dependent liver injury, with 23 ultimately causing death. Morbidity was associated with the downregulation of liver-derived microRNAs (miRNAs), indicating possible competition of the latter with shRNAs for limiting cellular factors required for the processing of various small RNAs. In vitro and in vivo shRNA transfection studies implied that one such factor, shared by the shRNA/miRNA pathways and readily saturated, is the nuclear karyopherin exportin-5. Our findings have fundamental consequences for future RNAi-based strategies in animals and humans, because controlling intracellular shRNA expression levels will be imperative. However, the risk of oversaturating endogenous small RNA pathways can be minimized by optimizing shRNA dose and sequence, as exemplified here by our report of persistent and therapeutic RNAi against human hepatitis B virus in vivo.

The 37/67-kilodalton Laminin Receptor is a Receptor for Adeno-associated Virus Serotypes 8, 2, 3, and 9

Journal of Virology. Oct, 2006  |  Pubmed ID: 16973587

Adeno-associated virus serotype 8 (AAV8) is currently emerging as a powerful gene transfer vector, owing to its capability to efficiently transduce many different tissues in vivo. While this is believed to be in part due to its ability to uncoat more readily than other AAV serotypes such as AAV2, understanding all the processes behind AAV8 transduction is important for its application and optimal use in human gene therapy. Here, we provide the first report of a cellular receptor for AAV8, the 37/67-kDa laminin receptor (LamR). We document binding of LamR to AAV8 capsid proteins and intact virions in vitro and demonstrate its contribution to AAV8 transduction of cultured cells and mouse liver in vivo. We also show that LamR plays a role in transduction by three other closely related serotypes (AAV2, -3, and -9). Sequence and deletion analysis allowed us to map LamR binding to two protein subdomains predicted to be exposed on the AAV capsid exterior. Use of LamR, which is constitutively expressed in many clinically relevant tissues and is overexpressed in numerous cancers, provides a molecular explanation for AAV8's broad tissue tropism. Along with its robust transduction efficiency, our findings support the continued development of AAV8-based vectors for clinical applications in humans, especially for tumor gene therapy.

A Two-hybrid Screen Identifies Cathepsins B and L As Uncoating Factors for Adeno-associated Virus 2 and 8

Molecular Therapy : the Journal of the American Society of Gene Therapy. Feb, 2007  |  Pubmed ID: 17235311

Vectors based on different serotypes of adeno-associated virus hold great promise for human gene therapy, based on their unique tissue tropisms and distinct immunological profiles. A particularly interesting candidate is AAV8, which can efficiently and rapidly transduce a wide range of tissues in vivo. To further unravel the mechanisms behind AAV8 transduction, we used yeast two-hybrid analyses to screen a mouse liver complementary DNA library for cellular proteins capable of interacting with the viral capsid proteins. In total, we recovered approximately 700 clones, comprising over 300 independent genes. Sequence analyses revealed multiple hits for over 100 genes, including two encoding the endosomal cysteine proteases cathepsins B and L. Notably, these two proteases also physically interacted with the corresponding portion of the AAV2 capsid in yeast, but not with AAV5. We demonstrate that cathepsins B and L are essential for efficient AAV2- and AAV8-mediated transduction of mammalian cells, and document the ability of purified cathepsin B and L proteins to bind and cleave intact AAV2 and AAV8 particles in vitro. These data suggest that cathepsin-mediated cleavage could prime AAV capsids for subsequent nuclear uncoating, and indicate that analysis of additional genes recovered in our screen may help to further elucidate the mechanisms behind transduction by AAV8 and related serotypes.

Combinatorial RNAi: a Winning Strategy for the Race Against Evolving Targets?

Molecular Therapy : the Journal of the American Society of Gene Therapy. May, 2007  |  Pubmed ID: 17311009

The ability to use double-stranded RNA to inhibit gene expression sequence-specifically (RNA interference, or RNAi) is currently revolutionizing science and medicine alike. Numerous pre-clinical studies are evaluating RNAi as a novel therapeutic modality in the battle against gain-of-function autosomal dominant diseases, cancer, and viral infections. One emerging concern is that RNAi mono-therapies might ultimately fail to control viruses that can escape silencing by mutation and/or RNAi suppression. Thus, sophisticated strategies are being developed that aim to avert viral resistance by combining RNAi effectors with each other or with further gene expression inhibitors. Several reports already validate this new concept of "combinatorial RNAi" (coRNAi) and illustrate its versatility by describing co-expression of RNAi triggers directed against single or multiple, viral or cellular, targets. Other studies document the successful delivery of these triggers with additional RNA- or protein-based silencers. Moreover, vectors have been engineered to blend RNAi-mediated gene inhibition with conventional gene replacement strategies. Collectively, these efforts open up exciting new therapeutic avenues but could also augment the inherent risks of RNAi technology, including immune responses, off-targeting, and oversaturation of endogenous pathways. Here, we critically review all coRNAi strategies and discuss the requirements for their transition into clinical application.

Rapid and Stable Knockdown of an Endogenous Gene in Retinal Pigment Epithelium

Human Gene Therapy. Oct, 2007  |  Pubmed ID: 17892416

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.

RNAi and Gene Therapy: a Mutual Attraction

Hematology / the Education Program of the American Society of Hematology. American Society of Hematology. Education Program. 2007  |  Pubmed ID: 18024667

The phylogenetically conserved cellular phenomenon of RNA interference (RNAi)-the sequence-specific post-transcriptional silencing of gene expression mediated by small double-stranded RNAs-holds substantial promise for basic research and for drug development. Particularly attractive from a medical standpoint is the juxtaposition of new RNAi methodology with established gene transfer strategies, especially viral vectors for efficient and tissue-specific RNAi delivery to patients. Here, we summarize the latest experimental and clinical advances in RNAi-based gene therapy approaches. We briefly portray emerging nonviral strategies for siRNA transfer, before comparing the three viral vectors currently predominantly developed as shRNA delivery vehicles, adenovirus, lentivirus, and adeno-associated virus (AAV). Moreover, we describe the most clinically relevant genetic, acquired or infectious targets being pursued for therapeutic purposes. Specifically, we assess the use of vector-mediated RNAi for treatment of viral processes, solid cancers, lymphoproliferative disorders, and neurodegenerative and ocular diseases. In addition, we highlight further emerging applications, including stem cell therapies and animal transgenesis, as well as discuss some of the potential pitfalls and limitations inherent to the individual approaches. While we predict that eventual schemes will be shaped by our increasing understanding of the complexities of human RNAi biology, as well as by progressive refinements of viral shuttle designs, the potential scientific and medical benefits from a successful marriage of RNAi and gene therapy seem enormous.

Therapeutic Application of RNAi: is MRNA Targeting Finally Ready for Prime Time?

The Journal of Clinical Investigation. Dec, 2007  |  Pubmed ID: 18060021

With unprecedented speed, RNA interference (RNAi) has advanced from its basic discovery in lower organisms to becoming a powerful genetic tool and perhaps our single most promising biotherapeutic for a wide array of diseases. Numerous studies document RNAi efficacy in laboratory animals, and the first clinical trials are underway and thus far suggest that RNAi is safe to use in humans. Yet substantial hurdles have also surfaced and must be surmounted before therapeutic RNAi applications can become a standard therapy. Here we review the most critical roadblocks and concerns for clinical RNAi transition, delivery, and safety. We highlight emerging solutions and concurrently discuss novel therapeutic RNAi-based concepts. The current rapid advances create realistic optimism that the establishment of RNAi as a new and potent clinical modality in humans is near.

Hepatic Parenchymal Replacement in Mice by Transplanted Allogeneic Hepatocytes is Facilitated by Bone Marrow Transplantation and Mediated by CD4 Cells

Hepatology (Baltimore, Md.). Feb, 2008  |  Pubmed ID: 18220289

The lack of adequate donor organs is a major limitation to the successful widespread use of liver transplantation for numerous human hepatic diseases. A desirable alternative therapeutic option is hepatocyte transplantation (HT), but this approach is similarly restricted by a shortage of donor cells and by immunological barriers. Therefore, in vivo expansion of tolerized transplanted cells is emerging as a novel and clinically relevant potential alternative cellular therapy. Toward this aim, in the present study we established a new mouse model that combines HT with prior bone marrow transplantation (BMT). Donor hepatocytes were derived from human alpha(1)-antitrypsin (hAAT) transgenic mice of the FVB strain. Serial serum enzyme-linked immunosorbent assays for hAAT protein were used to monitor hepatocyte engraftment and expansion. In control recipient mice lacking BMT, we observed long-term yet modest hepatocyte engraftment. In contrast, animals undergoing additional syngeneic BMT prior to HT showed a 3- to 5-fold increase in serum hAAT levels after 24 weeks. Moreover, complete liver repopulation was observed in hepatocyte-transplanted Balb/C mice that had been transplanted with allogeneic FVB-derived bone marrow. These findings were validated by a comparison of hAAT levels between donor and recipient mice and by hAAT-specific immunostaining. Taken together, these findings suggest a synergistic effect of BMT on transplanted hepatocytes for expansion and tolerance induction. Livers of repopulated animals displayed substantial mononuclear infiltrates, consisting predominantly of CD4(+) cells. Blocking the latter prior to HT abrogated proliferation of transplanted hepatocytes, and this implied an essential role played by CD4(+) cells for in vivo hepatocyte selection following allogeneic BMT. CONCLUSION: The present mouse model provides a versatile platform for investigation of the mechanisms governing HT with direct relevance to the development of clinical strategies for the treatment of human hepatic failure.

In Vitro and in Vivo Gene Therapy Vector Evolution Via Multispecies Interbreeding and Retargeting of Adeno-associated Viruses

Journal of Virology. Jun, 2008  |  Pubmed ID: 18400866

Adeno-associated virus (AAV) serotypes differ broadly in transduction efficacies and tissue tropisms and thus hold enormous potential as vectors for human gene therapy. In reality, however, their use in patients is restricted by prevalent anti-AAV immunity or by their inadequate performance in specific targets, exemplified by the AAV type 2 (AAV-2) prototype in the liver. Here, we attempted to merge desirable qualities of multiple natural AAV isolates by an adapted DNA family shuffling technology to create a complex library of hybrid capsids from eight different wild-type viruses. Selection on primary or transformed human hepatocytes yielded pools of hybrids from five of the starting serotypes: 2, 4, 5, 8, and 9. More stringent selection with pooled human antisera (intravenous immunoglobulin [IVIG]) then led to the selection of a single type 2/type 8/type 9 chimera, AAV-DJ, distinguished from its closest natural relative (AAV-2) by 60 capsid amino acids. Recombinant AAV-DJ vectors outperformed eight standard AAV serotypes in culture and greatly surpassed AAV-2 in livers of naïve and IVIG-immunized mice. A heparin binding domain in AAV-DJ was found to limit biodistribution to the liver (and a few other tissues) and to affect vector dose response and antibody neutralization. Moreover, we report the first successful in vivo biopanning of AAV capsids by using a new AAV-DJ-derived viral peptide display library. Two peptides enriched after serial passaging in mouse lungs mediated the retargeting of AAV-DJ vectors to distinct alveolar cells. Our study validates DNA family shuffling and viral peptide display as two powerful and compatible approaches to the molecular evolution of novel AAV vectors for human gene therapy applications.

Silencing of Hepatic Fatty Acid Transporter Protein 5 in Vivo Reverses Diet-induced Non-alcoholic Fatty Liver Disease and Improves Hyperglycemia

The Journal of Biological Chemistry. Aug, 2008  |  Pubmed ID: 18524776

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.

Expression of ShRNA from a Tissue-specific Pol II Promoter is an Effective and Safe RNAi Therapeutic

Molecular Therapy : the Journal of the American Society of Gene Therapy. Sep, 2008  |  Pubmed ID: 18665161

It has been observed that overexpression of some short-hairpin RNAs (shRNAs) can induce acute cytotoxicity. This has raised concerns about the safety of using RNA interference (RNAi) technology as a potential therapeutic tool. We have sought to address this challenge of expression control by developing a mono-cistronic vector for the tissue-specific expression of an shRNA from a liver-derived polymerase (pol) II promoter. This new construct efficiently induces target silencing in hepatoma cells in vitro and in mouse livers in vivo. In order to demonstrate the therapeutic potential and improved safety of this approach, we selected an shRNA targeting the envelope surface antigen (sAg) of hepatitis B virus (HBV), which is among the most toxic when expressed from the commonly used U6 promoter. Packaging it as a double-stranded DNA into an adeno-associated virus (AAV) pseudotype 8 and delivering it at a high particle dose (1 x 10(12)) to HBV transgenic mice resulted in the stable reduction of serum sAg to 85% of starting levels, without any concomitant sign of liver damage. With this improved tolerability, the liver-specific pol II shRNA expression persisted for more than one year after the injection. We conclude that this pol II shRNA expression system combined with a potent delivery vector represents an effective alternative to either U6-based strategies or systems that achieve tissue specificity through the use of additional elements.

Small Silencing RNAs: State-of-the-art

Advanced Drug Delivery Reviews. Jul, 2009  |  Pubmed ID: 19427885

Over just a single decade, we have witnessed the rapid maturation of the field of RNA interference - the sequence-specific gene silencing mediated by small double-stranded RNAs - directly from its infancy into adulthood. With exciting data currently emerging from first clinical trials, it is now more likely than ever that RNAi drugs will soon provide another potent class of agents in our battle against infectious and genetic diseases. Accelerating this process and adding to RNAi's promise is our steadily expanding arsenal of innovative RNAi-based experimental tools and clinically applicable technologies. This article will critically review a selection of relevant recent advances in RNAi therapeutics, from novel asymmetric or bi-functional siRNA designs, deliberate use of small RNAs to regulate nuclear transcription, engineering of potent adeno-associated viral vectors for shRNA expression, exploitation of endogenous miRNAs to control transgene expression or vector tropism, to elegant attempts to inhibit cellular miRNAs involved in human disease. This review will also present cautionary notes on the potential risks inherent to in vivo RNAi applications, before discussing the latest surprising findings on circulating miRNAs in human body fluids, and concluding with an outlook into the possible future of RNAi as an increasingly powerful biomedical tool.

Low-level ShRNA Cytotoxicity Can Contribute to MYC-induced Hepatocellular Carcinoma in Adult Mice

Molecular Therapy : the Journal of the American Society of Gene Therapy. Jan, 2010  |  Pubmed ID: 19844192

Short hairpin RNAs (shRNAs) have emerged as a novel therapeutic modality, but there is increasing concern over nonspecific effects in vivo. Here, we used viral vectors to express shRNAs against endogenous p53 in livers of conditional MYC-transgenic mice. As expected, the shRNAs silenced hepatic p53 and accelerated liver tumorigenesis when MYC was concurrently expressed. Surprisingly, various irrelevant control shRNAs similarly induced a rapid onset of tumorigenesis, comparable to carbon tetrachloride (CCl4), a potent carcinogen. We found that even marginal shRNA doses can already trigger histologically detectable hepatoxicity and increased hepatocyte apoptosis. Moreover, we noted that shRNA expression globally dysregulated hepatic microRNA (miRNA) expression, and that shRNA levels and activity further increased in the presence of MYC. In MYC-expressing transgenic mice, the marginal shRNA-induced liver injury sufficed to further stimulate hepatocellular division that was in turn associated with markedly increased expression of the mitotic cyclin B1. Hence, even at low doses, shRNAs can cause low-level hepatoxicity that can facilitate the ability of the MYC oncogene to induce liver tumorigenesis. Our data warrant caution regarding the possible carcinogenic potential of shRNAs when used as clinical agent, particularly in circumstances where tissues are genetically predisposed to cellular transformation and proliferation.

Six RNA Viruses and Forty-one Hosts: Viral Small RNAs and Modulation of Small RNA Repertoires in Vertebrate and Invertebrate Systems

PLoS Pathogens. Feb, 2010  |  Pubmed ID: 20169186

We have used multiplexed high-throughput sequencing to characterize changes in small RNA populations that occur during viral infection in animal cells. Small RNA-based mechanisms such as RNA interference (RNAi) have been shown in plant and invertebrate systems to play a key role in host responses to viral infection. Although homologs of the key RNAi effector pathways are present in mammalian cells, and can launch an RNAi-mediated degradation of experimentally targeted mRNAs, any role for such responses in mammalian host-virus interactions remains to be characterized. Six different viruses were examined in 41 experimentally susceptible and resistant host systems. We identified virus-derived small RNAs (vsRNAs) from all six viruses, with total abundance varying from "vanishingly rare" (less than 0.1% of cellular small RNA) to highly abundant (comparable to abundant micro-RNAs "miRNAs"). In addition to the appearance of vsRNAs during infection, we saw a number of specific changes in host miRNA profiles. For several infection models investigated in more detail, the RNAi and Interferon pathways modulated the abundance of vsRNAs. We also found evidence for populations of vsRNAs that exist as duplexed siRNAs with zero to three nucleotide 3' overhangs. Using populations of cells carrying a Hepatitis C replicon, we observed strand-selective loading of siRNAs onto Argonaute complexes. These experiments define vsRNAs as one possible component of the interplay between animal viruses and their hosts.

Argonaute Proteins Are Key Determinants of RNAi Efficacy, Toxicity, and Persistence in the Adult Mouse Liver

The Journal of Clinical Investigation. Sep, 2010  |  Pubmed ID: 20697157

shRNA overexpression from viral gene therapy vectors can trigger cytotoxicity leading to organ failure and lethality in mice and rats. This process likely involves saturation of endogenous cellular RNAi factors including exportin-5 (Xpo-5). Here, we have shown that Xpo-5 overexpression enhanced shRNA efficiency in the liver of adult mice but increased hepatotoxicity. We identified the 4 members of the human Argonaute (Ago) protein family as downstream factors involved in saturation of endogenous cellular RNAi, all of which were able to interact with shRNAs in cells and mice. In Ago/shRNA coexpression studies, Ago-2 (Slicer) was the primary rate-limiting determinant of both in vitro and in vivo RNAi efficacy, toxicity, and persistence. In adult mice, vector-based Ago-2/Xpo-5 coexpression enhanced U6-driven shRNA silencing of exogenous and endogenous hepatic targets, reduced hepatotoxicity, and extended RNAi stability by more than 3 months. Use of weaker RNA polymerase III promoters to minimize shRNA expression likewise alleviated in vivo toxicity and permitted greater than 95% persistent knockdown of hepatitis B virus and other transgenes in mouse liver for more than 1 year. Our studies substantiate that abundant small RNAs can overload the endogenous RNAi pathway and reveal possible strategies for reducing hepatotoxicity of short- and long-term clinical gene silencing in humans.

Thermodynamic Stability of Small Hairpin RNAs Highly Influences the Loading Process of Different Mammalian Argonautes

Proceedings of the National Academy of Sciences of the United States of America. May, 2011  |  Pubmed ID: 21576459

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.

Cellular RNA Interference Mechanisms. Preface

Progress in Molecular Biology and Translational Science. 2011  |  Pubmed ID: 21846566

When Cellular Networks Run out of Control: Global Dysregulation of the RNAi Machinery in Human Pathology and Therapy

Progress in Molecular Biology and Translational Science. 2011  |  Pubmed ID: 21846572

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.

To Go, or Not to Go, That is the Question - Six Personal Reflections on How Geographic Mobility May Affect Your Career and Life

BioEssays : News and Reviews in Molecular, Cellular and Developmental Biology. Oct, 2011  |  Pubmed ID: 21858845

The Dose Can Make the Poison: Lessons Learned from Adverse in Vivo Toxicities Caused by RNAi Overexpression

Silence. 2011  |  Pubmed ID: 22029761

ABSTRACT: For the past five years, evidence has accumulated that vector-mediated robust RNA interference (RNAi) expression can trigger severe side effects in small and large animals, from cytotoxicity and accelerated tumorigenesis to organ failure and death. The recurring notions in these studies that a critical parameter is the strength of RNAi expression and that Exportin-5 and the Argonaute proteins are rate-limiting mammalian RNAi, strongly imply dose-dependent saturation of the endogenous miRNA pathway as one of the underlying mechanisms. This minireview summarizes the relevant work and data leading to this intriguing model and highlights potential avenues by which to alleviate RNAi-induced toxicities in future clinical applications.

Fate Tracing of Mature Hepatocytes in Mouse Liver Homeostasis and Regeneration

The Journal of Clinical Investigation. Dec, 2011  |  Pubmed ID: 22105172

Recent evidence has contradicted the prevailing view that homeostasis and regeneration of the adult liver are mediated by self duplication of lineage-restricted hepatocytes and biliary epithelial cells. These new data suggest that liver progenitor cells do not function solely as a backup system in chronic liver injury; rather, they also produce hepatocytes after acute injury and are in fact the main source of new hepatocytes during normal hepatocyte turnover. In addition, other evidence suggests that hepatocytes are capable of lineage conversion, acting as precursors of biliary epithelial cells during biliary injury. To test these concepts, we generated a hepatocyte fate-tracing model based on timed and specific Cre recombinase expression and marker gene activation in all hepatocytes of adult Rosa26 reporter mice with an adenoassociated viral vector. We found that newly formed hepatocytes derived from preexisting hepatocytes in the normal liver and that liver progenitor cells contributed minimally to acute hepatocyte regeneration. Further, we found no evidence that biliary injury induced conversion of hepatocytes into biliary epithelial cells. These results therefore restore the previously prevailing paradigms of liver homeostasis and regeneration. In addition, our new vector system will be a valuable tool for timed, efficient, and specific loop out of floxed sequences in hepatocytes.

Expression Determinants of Mammalian Argonaute Proteins in Mediating Gene Silencing

Nucleic Acids Research. Dec, 2011  |  Pubmed ID: 22210886

RNA interference occurs by two main processes: mRNA site-specific cleavage and non-cleavage-based mRNA degradation or translational repression. Site-specific cleavage is carried out by argonaute-2 (Ago2), while all four mammalian argonaute proteins (Ago1-Ago4) can carry out non-cleavage-mediated inhibition, suggesting that Ago1, Ago3 and Ago4 may have similar but potentially redundant functions. It has been observed that in mammalian tissues, expression of Ago3 and Ago4 is dramatically lower compared with Ago1; however, an optimization of the Ago3 and Ago4 coding sequences to include only the most common codon at each amino acid position was able to augment the expression of Ago3 and Ago4 to levels comparable to that of Ago1 and Ago2. Thus, we examined whether particular sequence features exist in the coding region of Ago3 and Ago4 that may prevent a high level of expression. Swapping specific sub-regions of wild-type and optimized Ago sequence identified the portion of the coding region (nucleotides 1-1163 for Ago-3 and 1-1494 for Ago-4) that is most influential for expression. This finding has implications for the evolutionary conservation of Ago proteins in the mammalian lineage and the biological role that potentially redundant Ago proteins may have.