Hydrodynamic Transfection Research Articles

Proteasome-dependent autoregulation of Bruton tyrosine kinase (Btk) promoter via NF-κB

AbstractBruton tyrosine kinase (Btk) is critical for B-cell development. Btk regulates a plethora of signaling proteins, among them nuclear factor-[κ]B (NF-κB). Activation of NF-κB is a hallmark of B cells, and NF-κB signaling is severely compromised in Btk deficiency. We here present strong evidence indicating that NF-κB is required for efficient transcription of the Btk gene. First, we found that proteasome blockers and inhibitors of NF-κB signaling suppress Btk transcription and intracellular expression. Similar to Btk, proteasome inhibitors also reduced the expression of other members of this family of kinases, Itk, Bmx, and Tec. Second, 2 functional NF-κB–binding sites were found in the Btk promoter. Moreover, in live mice, by hydrodynamic transfection, we show that bortezomib (a blocker of proteasomes and NF-κB signaling), as well as NF-κB binding sequence-oligonucleotide decoys block Btk transcription. We also demonstrate that Btk induces NF-κB activity in mice. Collectively, we show that Btk uses a positive autoregulatory feedback mechanism to stimulate transcription from its own promoter via NF-κB.

Long-term hepatitis C internal ribosome entry site-dependent gene expression mediated by phage φC31 integrase in mouse model

Background The lack of a robust small animal model for hepatitis C virus (HCV) has hindered the development of novel drugs, including internal ribosome entry site (IRES) inhibitors. Phage φC31 integrase has emerged as a potent tool for achieving long-term gene expression in vivo. This study utilized φC31 integrase to develop a stable, reproducible and easily accessible HCV IRES mouse model. Methods φC31 integrase plasmid and the reporter vector, HCV-IRES–luciferase expression cassette (containing an attB site), was codelivered to murine livers using high pressure tail vein injection. HCV IRES-dependent translation refected by luciferase expression was accurately monitored in vivo by bioluminescence imaging. Genomic integration of the transgene was confirmed by partial hepatectomy and nested PCR. An HCV IRES-targeted short hairpin RNA (shRNA) expression plasmid, sh184, was hydrodynamically transfected into mouse liver to study its inhibition efficacy in vivo. Results φC31 integrase mediated intramolecular recombination between wild-type attB and attP sites in mice. The expression of luciferase was stable after 30 days post-transfection and remained so for 300 days only in the livers of mice that were coinjected with the integrase-encoding plasmid. Luciferase levels reduced dramatically after hydrodynamic transfection of sh184. Conclusions These results indicate that this mouse model provides a powerful tool for accurate and long-term evaluation of potential anti-IRES compounds in vivo.

Human and mouse hepatocellular adenoma and carcinoma display similar tumorigenesis pathway alterations

Distinct pathways of genomic progression to benign and malignant tumors of the liver. Tward AD, Jones KD, Yant S, Cheung ST, Fan ST, Chen X, Kay MA, Wang R, Bishop JM. We used several of the genetic lesions commonly associated with human liver tumors to reconstruct genetic progression to hepatocellular carcinoma and adenoma in mouse models. We initiated tumorigenesis with a transgene of the protooncogene MET or by hydrodynamic transfection of MET in combination with other genes into the livers of adult animals. Hepatocellular carcinoma in both instances arose from cooperation between MET and constitutively active versions of β-catenin. In contrast, adenomas were produced by cooperation between MET and defective signaling through the transcription factor HNF1α. Prompted by these findings, we uncovered a coincidence between activation of the protein–tyrosine kinase encoded by MET and activating mutations of β-catenin in a subset of human hepatocellular carcinomas. Inactivation of MET transgenes led to regression of hepatocellular carcinomas despite the persistence of activated β-catenin. The tumors eventually recurred in the absence of MET expression, however, presumably after the occurrence of one or more events that cooperated with activated β-catenin in lieu of MET. These results offer insight into hepatic tumorigenesis, provide mouse models that should be useful in the further study of hepatic tumorigenesis and for pre-clinical testing, and identify a subset of human hepatocellular carcinomas that may be susceptible to combination therapy directed against Met and the Wnt signaling pathway. [Abstract reproduced by permission of Proc Natl Acad Sci USA 2007;104:14771–14776]

Hepatic Fatty Acid Transporter Cd36 Is a Common Target of LXR, PXR, and PPARγ in Promoting Steatosis

Liver X receptor (LXR) is known to promote hepatic lipogenesis by activating the lipogenic transcriptional factor sterol regulatory element-binding protein (Srebp). Pregnane X receptor (PXR), a previously known "xenobiotic receptor," could mediate a Srebp-independent lipogenic pathway by activating the free fatty acid uptake transporter Cd36. The goal of this study is to investigate further the role of Cd36 in hepatic steatosis. Wild-type, LXR transgenic, PXR transgenic, and Cd36 null mice were used to study the regulation of Cd36 and other hepatic lipogenic genes and the implication of this regulation in hepatic steatosis. Promoter sequences of Cd36 and peroxisome proliferator-activated receptor (PPAR) gamma were cloned, and their respective regulation by LXR and PXR was investigated by combinations of receptor-DNA binding and reporter gene assays. We showed that genetic (transgene) or pharmacologic (ligands) activation of LXR induced Cd36. Promoter analysis established Cd36 as a novel transcription target of LXRalpha. Moreover, the hepatic steatosis induced by LXR agonists was largely abolished in Cd36 null mice. We also showed that PPARgamma, a positive regulator of Cd36, is a transcriptional target of PXR, suggesting that PXR can regulate Cd36 directly or through its activation of PPARgamma. Interestingly, both LXR-mediated Cd36 regulation and PXR-mediated PPARgamma regulation are liver specific. We conclude that Cd36 is a shared target of LXR, PXR, and PPARgamma. The network of CD36 regulation by LXR, PXR, and PPARgamma establishes this free fatty acid transporter as a common target of orphan nuclear receptors in their mediation of lipid homeostasis.

Abstract 1274: Syndecan-4 Pathway Interception Reduces Infarct Size After Myocardial Ischemia And Impacts On Remodelling

Objectives: Reperfusion injury after myocardial ischemia is governed by leukocyte recruitment. Syndecan-4 (SDC4) has been shown to be a major regulator of cell-cell and cell-matrix interactions. We tested if the SDC4-pathway impacts on myocardial necrosis, tissue loss and remodeling in mouse models of myocardial ischemia/infarction. In addition we analyzed the role of SDC4 in leukocyte recruitment in vitro and in mouse peritonitis. Methods: Syndecan4-deficient (SDC4 −/− ) or wildtype (WT) mice were subjected to transient (30 min; MI/R) or permanent (MI) LAD-ligation. In MI/R, after reperfusion for 24h, infarct size was measured by lack of TTC-staining. The extracellular SDC4-domain (ED-SDC4) was systemically overexpressed by hydrodynamic transfection (HDTF). Echocardiography revealed LV-function 4 days after MI(/R). Neutrophil extravasation was tested by lavaging mice 4h after IP-injection of thioglycollate. Adhesion of SDC4 −/− - or WT-PMN to resting or TNFα-activated, SDC4-siRNA-treated mouse endothelioma cells (EC) was tested in an adhesion assay. Results: Infarctions after MI/R were 43±7% larger in SDC4 −/− compared to WT (p<0.01, n=8). ED-SDC4-HDTF reduced infarct size by 27±4% (p<0.05, n=6). LV-function after MI was significantly worse in WT than SDC-4-KO (EF pre-post : −45±7% and −55±8%; p<0.05). In SDC4 −/− LV-dilation after MI was significantly ameliorated (EDV post-pre : 28±7μl vs. 39±8μl, SDC4 −/− vs WT, p<0.05). Emigration of GR-1 + -PMN of SDC4 −/− was increased by 2±0.5-fold in peritoneal lavages (n=6, p<0.05). SDC4 knock down (−80% in RT-PCR) in EC augmented adhesion of PMNs to resting EC by 41±8% and TNFα-activated EC by 20±5%. Adhesion of SDC-4-KO-PMN to resting and TNFα-activated EC was increased by 22±5% and 16±5%. SDC-4-KO-PMN adhesion to activated SDC4 knock down-EC was increased by 40±5% (p<0.05, n=3). Conclusion: SDC-4 is a powerful regulator of leukocyte-endothelial interactions and directly links molecular events driving myocardial necrosis and dilatative remodelling. Lack of SDC-4 increases, soluble SDC4 decreases infarct size. SDC4-deficiency reduces dilatative remodelling after MI. Therefore SDC4 beneficially influences early tissue necrosis after ischemia but adversely influences cardiac remodelling.

Distinct pathways of genomic progression to benign and malignant tumors of the liver

We used several of the genetic lesions commonly associated with human liver tumors to reconstruct genetic progression to hepatocellular carcinoma and adenoma in mouse models. We initiated tumorigenesis with a transgene of the protooncogene MET or by hydrodynamic transfection of MET in combination with other genes into the livers of adult animals. Hepatocellular carcinoma in both instances arose from cooperation between MET and constitutively active versions of beta-catenin. In contrast, adenomas were produced by cooperation between MET and defective signaling through the transcription factor HNF1alpha. Prompted by these findings, we uncovered a coincidence between activation of the protein-tyrosine kinase encoded by MET and activating mutations of beta-catenin in a subset of human hepatocellular carcinomas. Inactivation of MET transgenes led to regression of hepatocellular carcinomas despite the persistence of activated beta-catenin. The tumors eventually recurred in the absence of MET expression, however, presumably after the occurrence of one or more events that cooperated with activated beta-catenin in lieu of MET. These results offer insight into hepatic tumorigenesis, provide mouse models that should be useful in the further study of hepatic tumorigenesis and for preclinical testing, and identify a subset of human hepatocellular carcinomas that may be susceptible to combination therapy directed against Met and the Wnt signaling pathway.

Ultrastructural demonstration of hepatitis B virus production in a mouse model produced by hydrodynamic transfection

A mouse model of hepatitis B virus (HBV) infection produced by hydrodynamic injection of HBV DNA has been recently established. However, the ultrastructural demonstration of HBV particles in this mouse model has not as yet been reported. In our study, plasmid DNA containing wild-type HBV DNA was rapidly injected into 8-week-old female SCID mice via the tail vein. Serum levels of HBsAg were measured by ELISA kit. Intrahepatic HBV protein expression was detected by immunohistochemistry of HBcAg. Ultrastructural study of the serum samples was performed by transmission electron microscopy and immunogold electron microscopy. Serum HBsAg and intrahepatic HBcAg were detected in HBV DNA-injected mice for at least 14 days. Spherical and filamentous particles 22 nm in diameter and double-shelled Dane-like particles 42 nm in diameter were detected in the sera of mice. The ultrastructural features of these particles were identical to HBV particles observed in serum from chronic hepatitis B patients. These particles were confirmed to be HBV particles by immunogold electron microscopy. We conclude that our present HBV mouse model using hydrodynamic transfection of HBV DNA is appropriate for production of HBV virions including Dane particles. This mouse model may be useful for screening in vivo the efficacy of antiviral drugs.

In vivo imaging of hepcidin promoter stimulation by iron and inflammation

Hepcidin is an acute-phase response antimicrobial peptide that has emerged as a central regulator of iron absorption. Circulating hepcidin levels have been shown to affect iron uptake, release and storage. Hepcidin is mainly liver-derived and regulated, at least in part, transcriptionally. Hypoxia, erythroid demand, iron content and inflammation each have been shown to influence hepcidin mRNA expression in intact animals. In vitro, regulation of hepcidin by cytokines and by hypoxia is readily demonstrated in primary hepatocytes or in hepatocyte lines, but incubating the same cell lines with iron does not increase transcription of hepcidin. Thus, how iron excess stimulates hepcidin production in hepatocytes remains unknown. In addition, there is no current technique available that can investigate how iron induces hepcidin expression. To provide a better understanding of hepcidin gene expression in response to these regulatory stimuli, we have established a whole animal in vivo bioluminescence imaging assay to measure the activity of hepcidin promoter constructs in the animals' liver after hydrodynamic transfection of hepcidin promoter/luciferase constructs into mice. Transfected hepcidin promoter constructs were shown to respond to both inflammatory and iron stimuli in vivo. This work highlights the ability of this new imaging technique to investigate the key regions of the hepcidin promoter involved in iron induction of hepcidin expression.

Blocking Monoclonal Antibodies Specific for Mouse IFN-α/βReceptor Subunit 1 (IFNAR-1) from Mice Immunized byIn VivoHydrodynamic Transfection

Herein we report the generation of mouse monoclonal antibodies (mAbs) specific for the IFNAR-1 subunit of the mouse interferon-alpha/beta (IFN-alpha/beta) receptor (MAR1 mAbs) that block type I IFN receptor signaling and biologic response induction in vitro and in vivo. These mAbs were generated from Ifnar1 (/) mice immunized by in vivo hydrodynamic transfection with a plasmid encoding the extracellular domain (ECD) of murine IFNAR-1. All MAR1 mAbs bound native receptor expressed on cell surfaces and immunoprecipitated IFNAR-1 from solubilized cells, and two mAbs also detected IFNAR-1 by Western blot analysis. in vitro, the mAbs prevented ligand-induced intracellular signaling and induction of a variety of type I IFN-induced biologic responses but had no effect on IFN-gamma-induced responses. The most effective in vitro blocker, MAR1-5A3, also blocked type I IFN-induced antiviral, antimicrobial, and antitumor responses in vivo. We also explored whether murine IFNAR-1 surface expression required the presence of Tyk2. In contrast to Tyk2-deficient human cell lines, comparable IFNAR-1 expression was found on primary cells derived either from wild-type or Tyk2 (/) mice. These mAbs represent much needed tools to more clearly elucidate the biochemistry, cell biology, and physiologic function of the type I IFNs and their receptor in mediating host-protective immunity and immunopathology.

Increasing numbers of hepatic dendritic cells promote HMGB1-mediated ischemia-reperfusion injury

Endogenous ligands released from damaged cells, so-called damage-associated molecular pattern molecules (DAMPs), activate innate signaling pathways including the TLRs. We have shown that hepatic, warm ischemia and reperfusion (I/R) injury, generating local, noninfectious DAMPs, promotes inflammation, which is largely TLR4-dependent. Here, we demonstrate that increasing dendritic cell (DC) numbers enhance inflammation and organ injury after hepatic I/R. High-mobility group box 1 (HMGB1), a NF released by necrotic cells or secreted by stimulated cells, is one of a number of ligands promoting TLR4 reactivity. Augmentation of DC numbers in the liver with GM-CSF hydrodynamic transfection significantly increased liver damage after I/R when compared with controls. TLR4 engagement on hepatic DC was required for the I/R-induced injury, as augmentation of DC numbers in TLR4 mutant (C3H/HeJ) mice did not worsen hepatic damage. It is interesting that TLR4 expression was increased in hepatic DC following HMGB1 stimulation in vitro, suggesting a mechanism for the increased liver injury following I/R. It thus appears that functional TLR4 on DC is required for I/R-induced injury. Furthermore, HMGB1 may direct the inflammatory responses mediated by DC, at least in part, by enhancing TLR4 expression and reactivity to it and other DAMPs.

Inhibition of hepatitis C virus gene expression by small interfering RNAs using a tri-cistronic full-length viral replicon and a transient mouse model

HCV is an ideal target for siRNA as its genome, a single-stranded RNA, is translated into a single viral polyprotein and replicated into negative-stranded RNA. In the present study, we monitored the effects of 36 different small interfering RNAs (siRNAs) transcribed from a plasmid-derived expression system on the luciferase activities expressed from a full-length HCV replicon, to identify potent siRNA target sites. Delivery of nine selected siRNA expression vectors into human hepatoma cells (Huh7) carrying a genomic HCV replicon resulted in a significant reduction in viral protein and RNA levels. Moreover, synthetic siRNAs directed to target sites (core, NS3, NS4A and NS4B coding regions) in the HCV genome efficiently suppressed viral replication in a dose-dependent manner. A transient mouse model system expressing viral structural proteins in the liver was constructed using the hydrodynamic transfection method to confirm in vivo anti-HCV activity of the selected siRNAs. A 21-nucleotide siRNA, which can hybridize to the HCV core coding region with a single G-U base pair, suppressed weakly transgene expression in mice. However, this anti-viral effect was enhanced upon substitution with a 27-mer duplex RNA. Our results will provide useful information about designing potent siRNAs against variable target sites.

796. Pol II-Driven shRNA as an Effective Hepatitis B Virus Therapeutic

The majority of attempts to employ short-hairpin RNA (shRNA) expression vectors as therapeutics to induce the gene-specific silencing effects of RNA interference (RNAi) utilized ubiquitously recognized, highly active pol III promoters like U6 and H1. While silencing efficiency is, at least initially, extremely high, such constructs have the potential to cause serious side effects, resulting either from widespread off-target effects or from saturating the RNAi machinery, which is proving more central to cellular function. We aim to address both of these issues by using pol II promoters instead, which offer a greater means of control, including tissue specificity, chemical inducibility, and more moderate expression levels. Several structural variations were tested in vitro to optimize RNAi induction by pol II-driven shRNAs. The most successful version, selected for in vivo testing, includes the liver-specific fusion promoter containing the human ApoE-hepatic control region enhancer and human alpha-1 antitrypsin promoter (ApoE/hAAT), a sequence for the expression of a simple shRNA with 7nt loop, and the U1 snRNA 3' termination box. When designed to target luciferase and cotransfected with a luciferase expression plasmid into mouse liver by hydrodynamic transfection, this construct induced 90% silencing relative to a random-hairpin control. The construct was next adapted to target hepatitis B virus (HBV) s-antigen (sAg), and packaged as a double-strand in pseudotyped adeno-associated virus serotype 8 (dsAAV8). The hairpin sequence used was for the expression of a 25mer shRNA that had proven to be toxic when expressed from a U6 promoter. Previous work demonstrated that the toxic effect observed for this U6 construct was such that the ultimate result was either loss of the RNAi- induced silencing effect or the death of recipient mice due to liver failure less than a month following injection of expression cassette-harboring AAV. We tested the ApoE/hAAT expression construct in transgenic mice harboring an integrated HBV genome. Over the course of three months, the ApoE/hAAT construct was well tolerated and induced a stable level of sAg silencing of 80% of starting levels. Healthy FVB mice were used in a separate toxicity study for direct comparison of U6- and ApoE/hAAT-driven 25mers. Serum ALT levels were followed over three months as an indicator of liver toxicity. Mice that received the ApoE/hAAT-driven construct had stable, normal ALT levels, while those of mice that received the U6-driven construct spiked to peak around three weeks before gradually returning to normal. While we continue to optimize its efficacy, we conclude that this pol II system is an effective and safer HBV therapeutic in a mouse model of human disease.

Analysis of the Effect of Soluble Transferrin Receptor 1 (sTfR1) on Iron Metabolism in Mice.

Transferrin receptor 1 (TfR1) is a cell surface-expressed protein that mediates cellular uptake of iron. The TfR1 molecule may be shed from cells by proteolytic cleavage at Arg100 - Leu101, resulting in a cleaved form called soluble transferrin receptor 1 (sTfR1), which circulates in the blood as a 74 kDa monomer bound to transferrin. Measurement of soluble transferrin receptor has been widely used for the diagnosis of iron deficiency, but the levels of the receptor are also increased in patients with ineffective erythropoiesis, because the amount of circulating sTfR1 is directly proportional to the total amount of cell-associated TfR1. The key factors that stimulate increased sTfR1 levels are the integrated effects of reduced iron availability and increased erythropoietic stimulation. It is notable that both in iron deficiency and in diseases such as thalassemia, in which there is active ineffective erthropoiesis, iron absorption is increased This raises the question of whether sTfR1 is merely an intermediate in the degradation of the protein or whether it is a physiological regulator of iron absorption. To investigate this potential signaling function of sTfR1, a hydrodynamic gene transfer technique was established to express transfected plasmid constructs of human sTfR1 (hsTfR1, aa89–759) and murine sTfR1 (MsTfR1, 86–763) from the livers of C57Bl6 mice. To assess the effect that hsTfR1 and MsTfR1 might have on iron metabolism, iron absorption, serum iron and gene expression of hepcidin was measured in hydrodynamically transfected mice. The efficacy of the hydrodynamic technique was demonstrated by sustained expression of hsTfR1 in mice, at a level six fold higher than the normal level of hsTfR1 in humans (Fig. 1). Iron absorption was determined in transfected mice by measuring the retention of gavage fed 59Fe in a carrier iron solution, using a whole animal counting technique. Repeated experiments showed neither hsTfR1 nor MsTfR1 had any effect on the amount of iron absorbed. In agreement with these data, hepcidin levels were found by real time PCR to be unaltered in the same transfected mice. When serum iron levels were measured, expression of either hsTfR1 or MsTfR1 in mice resulted in significantly higher serum iron levels versus control mice (P=0.0141 and P=0.0192 respectively by t-test). We conclude that despite its attractiveness as a potential modifier of iron absorption, sTfR1 does not have any effect on hepcidin expression or iron absorption in mice. Mice transfected with hsTfR1 or MsTfR1 do have significantly higher serum iron levels, which may reflect an increased capability of these mice to retain transferrin in the serum. We have found that hydrodynamic transfection is a useful method to increase the levels of putative biologically active compounds in mice; this technique can be valuable in investigating the roles of serum or liver specific proteins in mice. [Display omitted]

Small Hairpin RNAs Efficiently Inhibit Hepatitis C IRES–Mediated Gene Expression in Human Tissue Culture Cells and a Mouse Model

Treatment and prevention of hepatitis C virus (HCV) infections remain a major challenge for controlling this worldwide health problem; existing therapies are only partially effective and no vaccine is currently available. RNA interference offers the potential of a novel therapeutic approach for treating HCV infections. Toward this end, we evaluated small hairpin interfering RNAs (shRNAs) targeting the conserved internal ribosome entry site (IRES) element of the HCV genome for their ability to control gene expression in human cells and animals. We used a reporter gene plasmid in which firefly luciferase (fLuc) expression is dependent on the HCV IRES. Direct delivery of HCV IRES shRNAs efficiently blocked HCV IRES-mediated fLuc expression in transfected human 293FT cells as well as in a mouse model in which nucleic acids were delivered to liver cells by hydrodynamic transfection via the tail vein. These results indicate that shRNAs, delivered as RNA or expressed from viral or nonviral vectors, may be effective agents for the control of HCV and related viruses.

1003. Inhibition of HBV Replication In Vivo by HBx-Specific siRNA

Top of pageAbstract Hepatitis B virus (HBV) causes acute and chronic hepatitis that could result in severe liver diseases such as cirrhosis and hepatocellular carcinoma. Because of the restricted host range of HBV, it has been difficult to study HBV replication in vivo and to test in vivo efficacy of possible antiviral therapeutics. In this study, we reproduced a naturally occurring HBV infection in mice by simply injecting HBV genome and also studied the effects of an HBV-specific short interfering RNA (siRNA) targeted to the X gene using this mouse model system. The viral replication cycle of HBV was resulted from the hydrodynamic transfection with the fragmented form of infectious HBV genome in immunocompetent mice. HBV envelope (HBe), HBV surface (HBs) antigens and viral DNA could be detected in the serum of the animals, followed by the induction of HBV-specific immune responses which mimics acute infection of HBV in humans. Also, the immunocompromized mice injected with the infectious HBV genome supported the prolonged replication of HBV mimicking the chronic infection of HBV in humans. In both systems, injection of siRNA targeted to the X gene region, where four major HBV transcript overlap, led to the dramatic inhibition of HBV replication up to 99% in the treated mice demonstrating the potential utility of HBx-specific siRNAs as anti-viral inhibitors for the treatment of chronic viral hepatitis. In summary, these mouse model systems offered opportunities to test in vivo efficacy of HBx-specific siRNAs for control of HBV replication.

88. Driving shRNA Transcription from PolII Promoters in Mammalian Cells

The use of RNA interference (RNAi) for sequence-specific post-transcriptional gene silencing has demonstrated clear therapeutic potential, especially as treatment for viral infection. However, current systems driving shRNA expression from ubiquitous promoters, such as U6 and H1, increase the threat of off-target effects by inducing RNAi in uninfected tissues. A much wider array of polII promoters offers greater control over expression, including tissue specificity and regulation. Relatively little is known about how to achieve optimized expression of functional shRNAs. Some suggested parameters include nucleotide modifications at the 5|[prime]| and 3|[prime]| positions, RNA length, and structure of the overhangs required for robust activity. Using these as guidelines, we developed constructs that effectively induce RNAi in mammalian cells by shRNAs expressed from mammalian polII promoters.The various constructs evaluated utilized either the human ubiquitin C promoter (hUbC) for ubiquitous expression or a fusion promoter containing the apolipoprotein E hepatic control region and the human alpha-1 antitrypsin promoter (AhAAT) for hepatocyte-specific expression. Multiple variations using these promoters, a hairpin against firefly luciferase, and one of two termination sequences were constructed. We evaluated shRNA-induced silencing of a luciferase target for each construct in mouse hepatoma cells by the Dual-Luciferase Assay (Promega). While the gap between the end of the promoter and the start of shRNA transcription was found to render a hairpin completely ineffective if too large, elimination of the entire gap was detrimental as well. On the other hand, shRNA activity was improved by replacing a minimal polyadenylation sequence with the termination sequence from the U1 snRNA. The most effective hUbC and AhAAT constructs induced silencing of approximately 72% and 32%, respectively, relative to an empty BlueScript control. These constructs were also evaluated in vivo by hydrodynamic transfection of mouse liver followed by imaging with a charge-coupled device camera. shRNA-induced silencing by as much as 80% was obtained for the hUbC and AhAAT constructs. Thus, we conclude that optimal 5|[prime]|-initiation and 3|[prime]|-termination sites are required for robust shRNA expression from polII promoters.

58. Toward Adenoviral Mediated RNA Interference for the Treatment of Hepatitis B Virus Infection

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.

Intrahepatic gene silencing by RNA interference

Intrahepatic gene silencing by RNA interference

Optimization of cis-acting elements for gene expression from nonviral vectors in vivo.

While naked DNA gene transfer in vivo usually results in transient gene expression, in some cases long-term transgene expression can be achieved. Here we demonstrate that cis-acting DNA elements flanking the transgene expression cassette and components in the plasmid backbone can significantly influence expression levels from nonviral vectors. To demonstrate this, we administered our most robust human coagulation factor IX (hFIX) expression cassette placed in two different plasmid backbones, into the livers of mice, by hydrodynamic transfection. We found that placing the expression cassette within a minimal plasmid vector pHM5, a modified version of pUC19, resulted in 10 times higher serum hFIX expression levels (up to 20000 ng/ml, 400% of normal hFIX serum levels), compared to a pBluescript backbone. To optimally increase expression levels from a nonviral vector, we added matrix attachment regions (MARs) as cis-acting DNA elements flanking the hFIX expression cassette. We detected five fold higher hFIX expression levels in vivo for up to 1-year posttransfection from a vector that contained the chicken MAR from the lysozyme locus. Together, the present work demonstrates that in addition to the transgene expression cassette, cis-acting DNA elements within and outside of the plasmid backbone need to be evaluated to achieve optimal expression levels in a nonviral gene therapy approach.

Development of catheter-based procedures for transducing the isolated rabbit liver with plasmid DNA.

Rapid systemic injection of naked plasmid DNA (pDNA) in a large volume into a mouse tail vein has been shown to result in a high level of gene expression in the liver. However, the potential therapeutic benefit to humans embodied in hydrodynamic transfection of the liver cannot be realized until a clinically viable method for gene delivery is developed. In light of this fact, we have devised and evaluated several methods for delivering pDNA to the isolated rabbit liver using minimally invasive catheter-based techniques. Using a lobar technique, pDNA was delivered hydrodynamically to an isolated hepatic lobe using a balloon occlusion balloon catheter to occlude a selected hepatic vein. A whole organ technique was used wherein the entire hepatic venous system was isolated and the pDNA solution injected hydrodynamically into the vena cava between two balloons used to block hepatic venous outflow. Lobar delivery of a plasmid encoding a secreted alkaline phosphatase (SEAP) reporter gene resulted in significant levels of transgene product in the serum. A nonsecreted transgene product, chloramphenicol acetyltransferase (CAT), showed the highest levels of expression in the injected lobe distal to the injection site. Compared to lobar delivery, whole organ delivery yielded much higher serum levels of SEAP expression and a significantly broader hepatic parenchymal distribution of CAT expression. These preliminary studies suggest that catheter-mediated hydrodynamic delivery of pDNA to the isolated liver may provide a method for human gene therapy that is both therapeutically significant and clinically practicable.