Anton P McCaffrey

Beckman Research Institute, Duarte, California, United States

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Publications (24)216.52 Total impact

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    ABSTRACT: Optimization of small interfering RNAs (siRNAs) is important in RNA interference (RNAi)-based therapeutic development. Some specific chemical modifications can control which siRNA strand is selected by the RNA-induced silencing complex (RISC) for gene silencing. Intended strand selection will increase potency and reduce off-target effects from the unintended strand. Sometimes, blocking RISC loading of the unintended strand leads to improved intended strand-silencing potency, but the generality of this phenomenon is unclear. Specifically, unlocked nucleic acid (UNA) modification of the 5' end of canonical (i.e., 19+2) siRNAs abrogates gene silencing of the modified strand, but the fate and potency of the unmodified strand has not been investigated. Here, we show that 5' UNA-modified siRNAs show improved silencing potency of the unmodified strand. We harness this advantageous property in a therapeutic context, where a limited target region in a conserved HIV 5' long terminal repeat U5 region would otherwise yield siRNAs with undesired strand selection properties and poor silencing. Applying 5' UNA modification to the unintended sense (S) strand of these otherwise poorly targeted siRNAs dramatically improves on-target silencing by the intended antisense (AS) strand in pNL4-3.luciferase studies. This study highlights the utility of 5' UNA siRNA modification in therapeutic contexts where siRNA sequence selection is constrained.Molecular Therapy-Nucleic Acids (2013) 2, e103; doi:10.1038/mtna.2013.36; published online 2 July 2013.
    Molecular therapy. Nucleic acids. 01/2013; 2:e103.
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    ABSTRACT: Macrophages respond to external stimuli with rapid changes in expression of many genes. Different combinations of external stimuli lead to distinct polarized activation patterns, resulting in a spectrum of possible macrophage activation phenotypes. MicroRNAs (miRNAs) are small, noncoding RNAs that can repress the expression of many target genes. We hypothesized that miRNAs play a role in macrophage polarization. miRNA expression profiles were determined in monocyte-derived macrophages (MDMs) incubated in conditions causing activation toward M1, M2a, M2b, or M2c phenotypes. One miRNA guide strand and seven miRNA passenger strands were significantly altered. Changes were confirmed in MDMs from six separate donors. The amplitude of miRNA expression changes in MDMs was smaller than described studies of monocytes responding to inflammatory stimuli. Further investigation revealed this correlated with higher basal miRNA expression in MDMs compared with monocytes. The regulation of M1- and M2b-responsive miRNAs (miR-27a, miR-29b, miR-125a, miR-146a, miR-155, and miR-222) was similar in differentiated THP-1 cells and primary MDMs. Studies in this model revealed cross-talk between IFNγ- and LPS-associated pathways regulating miRNA expression. Furthermore, expression of M1-associated transcripts was increased in THP-1 cells transfected with mimics of miR-29b, miR-125a-5p, or miR-155. The apparent inflammatory property of miR-29b and miR-125a-5p can be at least partially explained by repression of TNFAIP3, a negative regulator of NF-κB signaling. Overall, these data suggest miRNAs can contribute to changes in macrophage gene expression that occur in different exogenous activating conditions.
    Journal of Biological Chemistry 05/2012; 287(26):21816-25. · 4.65 Impact Factor
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    ABSTRACT: Recognition of microbial products by members of the Toll-like receptor (TLR) family initiates intracellular signaling cascades that result in NF-κB activation and subsequent production of inflammatory cytokines. We explored the potential roles of microRNAs (miRNAs) in regulating TLR pathways. A target analysis approach to the TLR4 pathway adaptor molecules identified several putative targets of miR-200a, miR-200b and miR-200c. miRNA mimics were co-transfected with a NF-κB activity reporter plasmid into HEK293 cells stably expressing TLR4 (HEK293-TLR4). Mimics of both miR-200b and miR-200c, but not miR-200a, decreased NF-κB reporter activity in either untreated cells or in cells treated with endotoxin:MD2 as a TLR4 agonist. Transfection of HEK293-TLR4 cells with miR-200b or miR-200c significantly decreased expression of MyD88, whereas TLR4, IRAK-1 and TRAF-6 mRNAs were unaffected. When miR-200b or miR-200c mimics were transfected into the differentiated monocytic THP-1 cell line, the abundance of MyD88 transcripts, as well as LPS-induced expression of the pro-inflammatory molecules IL-6, CXCL9 and TNF-α were diminished. These data define miRNAs miR-200b and miR-200c as factors that modify the efficiency of TLR4 signaling through the MyD88-dependent pathway and can thus affect host innate defenses against microbial pathogens.
    Innate Immunity 04/2012; · 2.68 Impact Factor
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    ABSTRACT: Engineered zinc finger nucleases (ZFNs) induce DNA double-strand breaks at specific recognition sequences and can promote efficient introduction of desired insertions, deletions or substitutions at or near the cut site via homology-directed repair (HDR) with a double- and/or single-stranded donor DNA template. However, mutagenic events caused by error-prone non-homologous end-joining (NHEJ)-mediated repair are introduced with equal or higher frequency at the nuclease cleavage site. Furthermore, unintended mutations can also result from NHEJ-mediated repair of off-target nuclease cleavage sites. Here, we describe a simple and general method for converting engineered ZFNs into zinc finger nickases (ZFNickases) by inactivating the catalytic activity of one monomer in a ZFN dimer. ZFNickases show robust strand-specific nicking activity in vitro. In addition, we demonstrate that ZFNickases can stimulate HDR at their nicking site in human cells, albeit at a lower frequency than by the ZFNs from which they were derived. Finally, we find that ZFNickases appear to induce greatly reduced levels of mutagenic NHEJ at their target nicking site. ZFNickases thus provide a promising means for inducing HDR-mediated gene modifications while reducing unwanted mutagenesis caused by error-prone NHEJ.
    Nucleic Acids Research 02/2012; 40(12):5560-8. · 8.81 Impact Factor
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    ABSTRACT: Chronic hepatitis C virus (HCV)-induced liver fibrosis involves upregulation of transforming growth factor (TGF)-β and subsequent hepatic stellate cell (HSC) activation. MicroRNAs (miRNAs) regulate HCV infection and HSC activation. TaqMan miRNA profiling identified 12 miRNA families differentially expressed between chronically HCV-infected human livers and uninfected controls. To identify pathways affected by miRNAs, we developed a new algorithm (pathway analysis of conserved targets), based on the probability of conserved targeting. This analysis suggested a role for miR-29 during HCV infection. Of interest, miR-29 was downregulated in most HCV-infected patients. miR-29 regulates expression of extracellular matrix proteins. In culture, HCV infection downregulated miR-29, and miR-29 overexpression reduced HCV RNA abundance. miR-29 also appears to play a role in HSCs. Hepatocytes and HSCs contribute similar amounts of miR-29 to whole liver. Both activation of primary HSCs and TGF-β treatment of immortalized HSCs downregulated miR-29. miR-29 overexpression in LX-2 cells decreased collagen expression and modestly decreased proliferation. miR-29 downregulation by HCV may derepress extracellular matrix synthesis during HSC activation. HCV infection downregulates miR-29 in hepatocytes and may potentiate collagen synthesis by reducing miR-29 levels in activated HSCs. Treatment with miR-29 mimics in vivo might inhibit HCV while reducing fibrosis.
    The Journal of Infectious Diseases 06/2011; 203(12):1753-62. · 5.85 Impact Factor
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    ABSTRACT: Zinc Finger Nucleases (ZFNs) are man-made restriction enzymes useful for manipulating genomes by cleaving target DNA sequences. ZFNs allow therapeutic gene correction or creation of genetically modified model organisms. ZFN specificity is not absolute; therefore, it is essential to select ZFN target sites without similar genomic off-target sites. It is important to assay for off-target cleavage events at sites similar to the target sequence. ZFN-Site is a web interface that searches multiple genomes for ZFN off-target sites. Queries can be based on the target sequence or can be expanded using degenerate specificity to account for known ZFN binding preferences. ZFN off-target sites are outputted with links to genome browsers, facilitating off-target cleavage site screening. We verified ZFN-Site using previously published ZFN half-sites and located their target sites and their previously described off-target sites. While we have tailored this tool to ZFNs, ZFN-Site can also be used to find potential off-target sites for other nucleases, such as TALE nucleases. ZFN-Site facilitates genome searches for possible ZFN cleavage sites based on user-defined stringency limits. ZFN-Site is an improvement over other methods because the FetchGWI search engine uses an indexed search of genome sequences for all ZFN target sites and possible off-target sites matching the half-sites and stringency limits. Therefore, ZFN-Site does not miss potential off-target sites.
    BMC Bioinformatics 01/2011; 12:152. · 3.02 Impact Factor
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    ABSTRACT: MicroRNAs (miRNAs) are small RNAs that regulate gene expression pathways. Previous studies have shown interactions between hepatitis C virus (HCV) and host miRNAs. We measured miR-122 and miR-21 levels in HCV-infected human liver biopsies relative to uninfected human livers and correlated these with clinical patient data. miR-122 is required for HCV replication in vitro, and miR-21 is involved in cellular proliferation and tumorigenesis. We found that miR-21 expression correlated with viral load, fibrosis and serum liver transaminase levels. miR-122 expression inversely correlated with fibrosis, liver transaminase levels and patient age. miR-21 was induced ∼twofold, and miR-122 was downregulated on infection of cultured cells with the HCV J6/JFH infectious clone, thus establishing a link to HCV. To further examine the relationship between fibrosis and the levels of miR-21 and miR-122, we measured their expression levels in a mouse carbon tetrachloride fibrosis model. As in the HCV-infected patient samples, fibrotic stage positively correlated with miR-21 and negatively correlated with miR-122 levels. Transforming growth factor β (TGF-β) is a critical mediator of fibrogenesis. We identified SMAD7 as a novel miR-21 target. SMAD7 is a negative regulator of TGF-β signaling, and its expression is induced by TGF-β. To confirm the relationship between miR-21 and the TGF-β signaling pathway, we measured the effect of miR-21 on a TGF-β-responsive reporter. We found that miR-21 enhanced TGF-β signaling, further supporting a relationship between miR-21 and fibrosis. We suggest a model in which miR-21 targeting of SMAD7 could increase TGF-β signaling, leading to increased fibrogenesis.
    Laboratory Investigation 12/2010; 90(12):1727-36. · 3.96 Impact Factor
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    ABSTRACT: Induction of heme oxygenase-1 (HO-1) inhibits hepatitis C virus (HCV) replication. Of the products of the reaction catalyzed by HO-1, iron has been shown to inhibit HCV ribonucleic acid (RNA) polymerase, but little is known about the antiviral activity of biliverdin (BV). Herein, we report that BV inhibits viral replication and viral protein expression in a dose-dependent manner in replicons and cells harboring the infectious J6/JFH construct. Using the SensoLyte 620 HCV Protease Assay with a wide wavelength excitation/emission (591 nm/622 nm) fluorescence energy transfer peptide, we found that both recombinant and endogenous nonstructural 3/4A (NS3/4A) protease from replicon microsomes are potently inhibited by BV. Of the tetrapyrroles tested, BV was the strongest inhibitor of NS3/4A activity, with a median inhibitory concentration (IC(50)) of 9 μM, similar to that of the commercial inhibitor, AnaSpec (Fremont, CA) #25346 (IC(50) 5 μM). Lineweaver-Burk plots indicated mixed competitive and noncompetitive inhibition of the protease by BV. In contrast, the effects of bilirubin (BR) on HCV replication and NS3/4A were much less potent. Because BV is rapidly converted to BR by biliverdin reductase (BVR) intracellularly, the effect of BVR knockdown on BV antiviral activity was assessed. After greater than 80% silencing of BVR, inhibition of viral replication by BV was enhanced. BV also increased the antiviral activity of α-interferon in replicons. Conclusion: BV is a potent inhibitor of HCV NS3/4A protease, which likely contributes to the antiviral activity of HO-1. These findings suggest that BV or its derivatives may be useful in future drug therapies targeting the NS3/4A protease.
    Hepatology 12/2010; 52(6):1897-905. · 12.00 Impact Factor
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    ABSTRACT: Hepatitis B virus (HBV) chronically infects 350-400 million people worldwide and causes >1 million deaths yearly. Current therapies prevent new viral genome formation, but do not target pre-existing viral genomic DNA, thus curing only approximately 1/2 of patients. We targeted HBV DNA for cleavage using zinc-finger nucleases (ZFNs), which cleave as dimers. Co-transfection of our ZFN pair with a target plasmid containing the HBV genome resulted in specific cleavage. After 3 days in culture, 26% of the target remained linear, whereas approximately10% was cleaved and misjoined tail-to-tail. Notably, ZFN treatment decreased levels of the hepatitis C virus pregenomic RNA by 29%. A portion of cleaved plasmids are repaired in cells, often with deletions and insertions. To track misrepair, we introduced an XbaI restriction site in the spacer between the ZFN sites. Targeted cleavage and misrepair destroys the XbaI site. After 3 days in culture, approximately 6% of plasmids were XbaI-resistant. Thirteen of 16 clones sequenced contained frameshift mutations that would lead to truncations of the viral core protein. These results demonstrate, for the first time, the possibility of targeting episomal viral DNA genomes using ZFNs.
    Molecular Therapy 02/2010; 18(5):947-54. · 7.04 Impact Factor
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    ABSTRACT: During liver regeneration, normally quiescent liver cells reenter the cell cycle, nonparenchymal and parenchymal cells divide, and proper liver architecture is restored. The gene expression programs regulating these transitions are not completely understood. MicroRNAs are a newly discovered class of small regulatory RNAs that silence messenger RNAs by binding to their 3'-untranslated regions (UTRs). A number of microRNAs, including miR-21, have been shown to be involved in regulation of cell proliferation. We performed partial hepatectomies on mice and allowed the liver to regenerate for 1, 6, 12, 24, and 48 h and 4 and 7 days. We compared the expression of miR-21 in the posthepatectomy liver to the prehepatectomy liver by Northern blot and found that miR-21 was upregulated during the early stages of liver regeneration. NF-kappaB signaling is also activated very early during liver regeneration. It has been previously reported that NF-kappaB upregulates the miR-21 precursor transcript. The predicted miR-21 target, Pellino (Peli1), is a ubiquitin ligase involved in activating NF-kappaB signaling. We observed an inverse correlation between miR-21 and Peli1 mRNA levels during liver regeneration. miR-21 overexpression in cultured cells inhibited a Peli1 3'-UTR luciferase reporter. Using NF-kappaB reporter assays, we determined that miR-21 overexpression inhibits NF-kappaB signaling. In conclusion, miR-21 expression was upregulated during early stages of liver regeneration. Targeting of Peli1 by miR-21 could potentially provide the basis for a negative feedback cycle regulating NF-kappaB signaling.
    AJP Gastrointestinal and Liver Physiology 02/2010; 298(4):G535-41. · 3.65 Impact Factor
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    ABSTRACT: Prostate cancer cells expressing prostate-specific membrane antigen (PSMA) have been targeted with RNA aptamer-small interfering (si)RNA chimeras, but therapeutic efficacy in vivo was demonstrated only with intratumoral injection. Clinical translation of this approach will require chimeras that are effective when administered systemically and are amenable to chemical synthesis. To these ends, we enhanced the silencing activity and specificity of aptamer-siRNA chimeras by incorporating modifications that enable more efficient processing of the siRNA by the cellular machinery. These included adding 2-nucleotide 3'-overhangs and optimizing the thermodynamic profile and structure of the duplex to favor processing of the siRNA guide strand. We also truncated the aptamer portion of the chimeras to facilitate large-scale chemical synthesis. The optimized chimeras resulted in pronounced regression of PSMA-expressing tumors in athymic mice after systemic administration. Anti-tumor activity was further enhanced by appending a polyethylene glycol moiety, which increased the chimeras' circulating half-life.
    Nature Biotechnology 09/2009; 27(9):839-49. · 32.44 Impact Factor
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    Anton P McCaffrey
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    ABSTRACT: Previously, we showed that short hairpin RNAs (shRNAs) targeting hepatitis B virus (HBV) potently inhibit the virus in a transient mouse model. However, subsequent studies showed that expression of these hairpins (as well as hairpins targeting human alpha-1 antitrypsin) from adeno-associated virus vectors (AAV) cause fatality in mice. We used rational design to develop significantly more potent second-generation HBV RNAi triggers embedded within the endogenous microRNA (miRNA) miR-30. A statistical analysis of thermodynamic parameters revealed characteristics important for RNAi potency. Small interfering RNAs (siRNAs) and shRNAs are known to compete with each other and with endogenous miRNAs for the miRNA machinery. We show that exogenous miRNA expression cassettes can compete with exogenous siRNAs, shRNA, and miRNAs as well as with endogenous miRNAs. Preliminary studies demonstrate that miRNA-based HBV RNAi expression from AAV vectors is well tolerated in mice.
    Annals of the New York Academy of Sciences 09/2009; 1175:15-23. · 4.38 Impact Factor
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    ABSTRACT: RNA interference (RNAi) can be an effective antiviral agent; however, overexpression of RNAi can be toxic through competition with the endogenous microRNA (miRNA) machinery. We used rational design to identify highly potent RNAi that is effective at nontoxic doses. A statistical analysis was conducted to pinpoint thermodynamic characteristics correlated with activity. Sequences were selected that conformed to a consensus internal stability profile (ISP) associated with active RNAi, and RNAi triggers were expressed in the context of an endogenous miRNA. These approaches yielded highly active hepatitis B virus (HBV) RNAi. A statistical analysis found a correlation between activity and nucleation by binding within the seed sequence to accessible regions in the target RNA. Guide strands were selected for favorable strand biasing, but increased strand biasing did not correlate with potency, suggesting a threshold effect. Exogenous short hairpin RNAs (shRNAs), but not miRNAs were previously reported to compete with miRNAs for the miRNA/RNAi machinery. In contrast, we show that exogenous Polymerase III- but not Polymerase II-driven miRNAs compete with exogenous miRNAs, at multiple steps in the miRNA pathway. Exogenous miRNAs also compete with endogenous miR-21. Thus, competition with endogenous miRNAs should be monitored even when using miRNA-based therapeutics. However, potent silencing was achieved at doses where competition was not observed.
    Molecular Therapy 01/2009; 17(3):538-47. · 7.04 Impact Factor
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    ABSTRACT: Understanding host responses to viral gene therapy vectors is necessary for the development of safe and efficacious in vivo gene transfer agents. We describe the use of high-density spotted complementary DNA microarrays in monitoring the in vivo host transcriptional responses in mouse liver upon administration of either a "first-generation"adenoviral (Ad) vector, a helper-dependent "gutless" adenoviral (HD) vector, or an adeno-associated viral (AAV) vector containing human factor IX (hFIX) expression cassettes. Since HD and AAV do not contain any viral genes, they allow us to assess the host response to the viral capsid and packaged nonviral DNA in whole animals. Comparison of the host response to Ad and HD helps assess the importance of leaky adenoviral gene expression. While all three vectors induced characteristic temporally sequenced programs of gene expression, the gene expression programs induced by the Ad and HD adenovirus vectors were remarkably similar, including the induction of a prominent type I interferon (IFN)-dependent cluster within 6 hours of administration. In contrast, the AAV-based vector caused far fewer alterations of host-gene expression. Our results indicate that recognition of the Ad capsid or double-stranded DNA (of nonviral origin) in the vector elicits a robust type I IFN response that is, however, not elicited by AAV-derived vector transduction.
    Molecular Therapy 06/2008; 16(5):931-41. · 7.04 Impact Factor
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    Rebecca T Marquez, Anton P McCaffrey
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    ABSTRACT: MicroRNAs (miRNAs) are a class of small regulatory RNAs that are thought to regulate the expression of as many as one-third of all human messenger RNAs (mRNAs). miRNAs are thought to be involved in diverse biological processes, including tumorigenesis. Analysis of miRNA levels may have diagnostic implications. Evidence shows that numerous viruses interact with the miRNA machinery, and that a number of viruses encode their own miRNAs. It seems likely that miRNAs will be implicated in many human diseases. Manipulation of miRNA levels by gene therapy provides an attractive new approach for therapeutic development. This review focuses on approaches to manipulate miRNA levels in cells and in vivo, and the implications for gene therapy. Furthermore, we discuss the use of endogenous miRNAs as scaffolds for the expression of RNA interference (RNAi) as well as competition between exogenous RNAi triggers and endogenous miRNAs. Because short interfering RNAs can also act as miRNAs, seed matches with the 3' untranslated regions of genes should be avoided to prevent off-target effects. Last, we discuss the use of miRNAs to avoid immune responses to viral vectors.
    Human Gene Therapy 02/2008; 19(1):27-38. · 4.02 Impact Factor
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    ABSTRACT: Chemical modifications have been incorporated into short interfering RNAs (siRNAs) without reducing their ability to inhibit gene expression in mammalian cells grown in vitro. In this study, we begin to assess the potential utility of 2'-modified siRNAs in mammals. We demonstrate that siRNA modified with 2'-fluoro (2'-F) pyrimidines are functional in cell culture and have a greatly increased stability and a prolonged half-life in human plasma as compared to 2'-OH containing siRNAs. Moreover, we show that the 2'-F containing siRNAs are functional in mice and can inhibit the expression of a target gene in vivo. However, even though the modified siRNAs have greatly increased resistance to nuclease degradation in plasma, this increase in stability did not translate into enhanced or prolonged inhibitory activity of target gene reduction in mice following tail vein injection. Thus, this study shows that 2'-F modified siRNAs are functional in vivo, but that they are not necessarily more potent than unmodified siRNAs in animals.
    RNA 06/2004; 10(5):766-71. · 5.09 Impact Factor
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    ABSTRACT: The RNA interference (RNAi) pathway specifically silences genes in response to double stranded RNA (dsRNA) triggers that share homology with the targeted gene. Short hairpin RNAs (shRNAs) expressed from transcription templates can act as the dsRNA RNAi trigger. Recently we showed that shRNA expression plasmids could silence luciferase gene expression in adult mice. Furthermore, we showed that shRNA expression plasmids directed against hepatitis B virus (HBV) could reduce the production of HBV RNAs, DNAs and proteins in mice when introduced by hydrodynamic transfection. However, if these promising new therapeutics are to be useful in humans, delivery methods that can safely introduce RNAi triggers into the majority of hepatocytes in humans may be required.Adenoviral vectors efficiently transduce hepatocytes in vivo, and transduction of nearly all hepatocytes may be feasible using these vectors. It has been previously reported that shRNAs expressed from adenoviral vectors can direct RNAi in cultured cells and in vivo. In order to follow the kinetics of adenoviral mediated RNAi in cultured cells and in vivo, we constructed E1/E3 deleted adenoviruses that express luciferase shRNAs or control shRNAs. Expression of transfected luciferase expression plasmids and kinetics of RNAi targeting luciferase was monitored using real time bioluminescence imaging.Cells transduced with control adenoviruses that did not express shRNAs or expressed shRNAs that did not express luciferase shRNAs had increased luciferase expression compared to cells that received no adenovirus. This general increase in gene expression may be due to stimulation of the SV40 promoter driving luciferase expression in the reporter construct. In contrast, treatment of these cells with an adenovirus that expressed luciferase shRNAs resulted in an ∼80 % decrease in luciferase activity compared to cells transduced with control adenoviruses. This level of inhibition is comparable to that observed by treatment of the cells with plasmids expressing the same luciferase shRNA.Similar experiments were conducted in mice. Mice were hydrodynamically transfected with luciferase plasmids. The subsequently, the mice were transduced with adenoviruses expressing luciferase shRNAs or control viruses. As seen in the tissue culture experiments, infection with control adenoviruses stimulated luciferase activity. Treatment with an adenovirus expressing luciferase shRNAs significantly decreased luciferase expression.The ultimate goal of these experiments is to use adenoviral vectors to inhibit HBV replication in vivo. Transgenic mice with an integrated copy of the HBV genome were infected with adenoviral vectors expressing control shRNAs or shRNAs targeting HBV RNAs at high multiplicities of infection. Cleavage of viral RNAs, decreases in viral genomes and reduction in viral protein levels are being assessed.
    Molecular Therapy - MOL THER. 01/2004; 9.
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    ABSTRACT: We report the generation and in vivo evaluation of novel Adeno-Associated Viral (AAV) vectors for the expression of short hairpin RNA (shRNA) against human Hepatitis B Virus (HBV), and demonstrate their superior efficiency over conventional AAV vectors.Our approach combines two recent major advances in the AAV field: pseudotyping of AAV-2-based vector genomes with capsids from another serotype, and development of “double-stranded” (ds) vectors, that express faster and more strongly than standard, single-stranded (ss) recombinant AAV. In detail, we cross-packaged AAV genomes comprising our most robust anti-HBV shRNA, or a control shRNA against luciferase, into capsids from AAV-8, which is the AAV serotype with the highest known transduction rate in liver. To force their encapsidation as ds genomes, we replaced one AAV-2 packaging signal with a mutant derived from AAV-4 or -5, which concurrently improved vector genome stability and particle titers. The small size of the shRNA inserts enabled us to encapsidate a second cistron, encoding a fluorescence protein for easy detection of shRNA-expressing cells.Specificity and efficiency of all vectors was first analysed in a human HBV-transformed cell line. As hoped for, only the two anti-HBV vectors significantly reduced HBV surface antigen (sAg) expression. Importantly, the novel dsAAV showed a much stronger effect, and more rapidly, than the standard ssAAV.All vectors were next evaluated in transgenic mice carrying and expressing an integrated HBV genome. Particles were given intraportally or by peripheral intravenous infusion, at doses from 5e10 up to 7e12 vector genomes (vg) per animal. Most remarkably, analyses of serum before and after systemic delivery of the ds anti-HBV vector showed complete elimination of sAg between day 4 and 11 after injection. This was found at doses as low as 3e11 vg per animal, whereas to get the same effect with the ss vector, 20-fold higher doses and longer incubation were required. No significant change in sAg was seen with the anti-luciferase control vectors or after PBS injection. Those results were corroborated by HBV mRNA and DNA analyses, and were confirmed in a second lineage of HBV-transgenic mice.Interestingly, we noted a dose-dependent toxicity of the anti-HBV vectors, resulting in hypoproteinemia and ultimately death of animals given high particle doses (1.8e12 vg and greater). This was seen as early as 5 days after injection, and in both strains of HBV-transgenic as well as in normal mice, ruling out a role for the integrated HBV target genome. The fact that toxicity was absent from the control shRNA vectors indicated an adverse off-target effect specific for the anti-HBV shRNA used here, which is a current matter of further investigation.In sum, our novel dsAAV-8 vector system provides an utmost efficient means for delivery of shRNA to the liver, allowing the use of minimal vector doses and non-invasive routes of administration. This makes it highly interesting for further development towards clinical exploitation of RNA interference to treat hepatitis and other liver diseases.
    Molecular Therapy 01/2004; 9. · 7.04 Impact Factor
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    ABSTRACT: Hepatitis C virus (HCV) is an RNA virus infecting one in every 40 people worldwide. Current treatments are ineffective and HCV is the leading cause of liver failure leading to transplantation in the United States and Europe. Translational control of HCV is a prime therapeutic target. We assessed the inhibitory potential of morpholino phosphoramidate antisense oligonucleotides (morpholinos) on HCV translation by codelivering them with reporter plasmids expressing firefly luciferase under the translational control of the HCV internal ribosome entry site (IRES) into the livers of mice. Real-time imaging of HCV IRES luciferase reporter messenger RNA (mRNA) translation in living mice showed that a 20-mer complementary to nucleotides 345-365 of the IRES inhibited translation by greater than 95% for at least 6 days and showed mismatch specificity. No significant nonspecific inhibition of a cap-dependent luciferase or encephalomyocarditis virus (EMCV) IRES luciferase reporter translation was observed. Inhibition by the 20-mer morpholino was dose dependent, with 1 nmol/mouse giving the highest inhibition. In conclusion, morpholino antisense oligonucleotides are potent inhibitors of HCV IRES translation in a preclinical mouse model; morpholinos have potential as molecular therapeutics for treating HCV and other viral infections. The in vivo model described is a broadly applicable, straightforward, and rapid readout for inhibitor efficacy. As such, it will greatly facilitate the development of novel therapeutic strategies for viral hepatitis. Notably, the level of antisense inhibition observed in this in vivo model is similar to the maximal inhibition we have obtained previously with RNA interference in mice.
    Hepatology 09/2003; 38(2):503-8. · 12.00 Impact Factor
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    ABSTRACT: Hepatitis B virus (HBV) infection substantially increases the risk of chronic liver disease and hepatocellular carcinoma in humans. RNA interference (RNAi) of virus-specific genes has emerged as a potential antiviral mechanism. Here we show that RNAi can be applied to inhibit production of HBV replicative intermediates in cell culture and in immunocompetent and immunodeficient mice transfected with an HBV plasmid. Cotransfection with plasmids expressing short hairpin RNAs (shRNAs) homologous to HBV mRNAs induced an RNAi response. Northern and Southern analyses of mouse liver RNA and DNA showed substantially reduced levels of HBV RNAs and replicated HBV genomes upon RNAi treatment. Secreted HBV surface antigen (HBsAg) was reduced by 94.2% in cell culture and 84.5% in mouse serum, whereas immunohistochemical detection of HBV core antigen (HBcAg) revealed >99% reduction in stained hepatocytes upon RNAi treatment. Thus, RNAi effectively inhibited replication initiation in cultured cells and mammalian liver, showing that such an approach could be useful in the treatment of viral diseases.
    Nature Biotechnology 06/2003; 21(6):639-44. · 32.44 Impact Factor

Publication Stats

2k Citations
216.52 Total Impact Points

Institutions

  • 2013
    • Beckman Research Institute
      Duarte, California, United States
    • TriLink BioTechnologies
      San Diego Country Estates, California, United States
  • 2008–2011
    • University of Iowa
      • Department of Internal Medicine
      Iowa City, IA, United States
  • 2010
    • Coe College
      • Biology
      Cedar Rapids, Iowa, United States
  • 2009
    • Molecular and Cellular Biology Program
      Seattle, Washington, United States
  • 2003–2004
    • Stanford Medicine
      • Program in Human Gene Therapy
      Stanford, California, United States
  • 2002–2004
    • Stanford University
      • Department of Pediatrics
      Palo Alto, California, United States