Tissue-specific Transgene Expression Research Articles

A Role for NF-κB Activity in Skin Hyperplasia and the Development of Keratoacanthomata in Mice

BackgroundPrevious studies have implicated NF-κB signaling in both cutaneous development and oncogenesis. However, these studies have been limited in part by the lethality that results from extreme over- or under-expression of NF-κB in available mouse models. Even cre-driven tissue specific expression of transgenes, or targeted deletion of NF-κB can cause cell death. Therefore, the present study was undertaken to evaluate a novel mouse model of enhanced NF-κB activity in the skin.MethodsA knock-in homologous recombination technique was utilized to develop a mouse model (referred to as PD mice) with increased NF-κB activity.ResultsThe data show that increased NF-κB activity leads to hyperproliferation and dysplasia of the mouse epidermis. Chemical carcinogenesis in the context of enhanced NF-κB activity promotes the development of keratoacanthomata.ConclusionOur findings support an important role for NF-κB in keratinocyte dysplasia. We have found that enhanced NF-κB activity renders keratinocytes susceptible to hyperproliferation and keratoacanthoma (KA) development but is not sufficient for transformation and SCC development. We therefore propose that NF-κB activation in the absence of additional oncogenic events can promote TNF-dependent, actinic keratosis-like dysplasia and TNF-independent, KAs upon chemical carcinogensis. These studies suggest that resolution of KA cannot occur when NF-κB activation is constitutively enforced.

Generation of a tumor- and tissue-specific episomal non-viral vector system

BackgroundA key issue for safe and reproducible gene therapy approaches is the autologous and tissue-specific expression of transgenes. Tissue-specific expression in vivo is either achieved by transfer vectors that deliver the gene of interest into a distinct cell type or by use of tissue-specific expression cassettes. Here we present the generation of non-viral, episomally replicating vectors that are able to replicate in a tissue specific manner thus allowing tissue specific transgene expression in combination with episomal replication. The episomal replication of the prototype vector pEPI-1 and its derivatives depends exclusively on a transcription unit starting from a constitutively active promoter extending into the scaffold/matrix attachment region (S/MAR).ResultsHere, we exchanged the constitutive promoter in the pEPI derivative pEPito by the tumor specific alpha fetoprotein (AFP) or the muscle specific smooth muscle 22 (SM22) promoter leading to specific transgene expression in AFP positive human hepatocellular carcinoma (HUH7) and in a SM22 positive cell line, respectively. The incorporation of the hCMV enhancer element into the expression cassette further boosted the expression levels with both promoters. Tissue specific-replication could be exemplary proven for the smooth muscle protein 22 (SM22) promoter in vitro. With the AFP promoter-driven pEPito vector hepatocellular carcinoma-specific expression could be achieved in vivo after systemic vector application together with polyethylenimine as transfection enhancer.ConclusionsIn this study we present an episomal plasmid system designed for tissue specific transgene expression and replication. The human AFP-promoter in combination with the hCMV enhancer element was demonstrated to be a valuable tissue-specific promoter for targeting hepatocellular carcinomas with non-viral gene delivery system, and tissue specific replication could be shown in vitro with the muscle specific SM22 promoter. In combination with appropriate delivery systems, the tissue specific pEPito vector system will allow higher tissue-specificity with less undesired side effects and is suitable for long term transgene expression in vivo within gene therapeutical approaches.

Serotonin 1A auto-receptors are not sufficient to modulate anxiety in mice

The neurotransmitter serotonin plays an important role in modulating diverse behavioral traits. Mice lacking the serotonin 1A receptor (Htr1a) show elevated avoidance of novel open spaces, suggesting that it has a role in modulating anxiety behavior. Htr1a is a Gαi -coupled G-protein-coupled receptor expressed on serotonin neurons (auto-receptor), where it mediates negative feedback of serotonin neuron firing. Htr1a is also expressed on non-serotonin neurons (hetero-receptor) in diverse brain regions, where it mediates an inhibitory effect of serotonin on neuronal activity. Debate exists about which of these receptor populations is responsible for the modulatory effects of Htr1a on anxiety. Studies using tissue-specific transgenic expression have suggested that forebrain Htr1a hetero-receptors are sufficient to restore normal anxiety behavior to Htr1a knockout mice. At the same time, experiments using tissue-specific transgenic suppression of Htr1a expression have demonstrated that Htr1a auto-receptors, but not forebrain hetero-receptors, are necessary for normal anxiety behavior. One interpretation of these data is that multiple Htr1a receptor populations are involved in modulating anxiety. Here, we aimed to test this hypothesis by determining whether Htr1a auto-receptors are sufficient to restore normal anxiety to Htr1a knockout animals. Transgenic mice expressing Htr1a under the control of the tryptophan hydroxylase 2 (Tph2) promoter showed restored Htr1a-mediated serotonin negative feedback and hypothermia, but anxiety behavior indistinguishable from that of knockout mice. These data show that, in the absence of Htr1a hetero-receptors, auto-receptors are unable to have an impact on anxiety. When combined with previous data, these findings support the hypothesis that Htr1a auto-receptors are necessary, but not sufficient, to modulate anxiety.

Foamy virus for efficient gene transfer in regeneration studies

BackgroundMolecular studies of appendage regeneration have been hindered by the lack of a stable and efficient means of transferring exogenous genes. We therefore sought an efficient integrating virus system that could be used to study limb and tail regeneration in salamanders.ResultsWe show that replication-deficient foamy virus (FV) vectors efficiently transduce cells in two different regeneration models in cell culture and in vivo. Injection of EGFP-expressing FV but not lentivirus vector particles into regenerating limbs and tail resulted in widespread expression that persisted throughout regeneration and reamputation pointing to the utility of FV for analyzing adult phenotypes in non-mammalian models. Furthermore, tissue specific transgene expression is achieved using FV vectors during limb regeneration.ConclusionsFV vectors are efficient mean of transferring genes into axolotl limb/tail and infection persists throughout regeneration and reamputation. This is a nontoxic method of delivering genes into axolotls in vivo/ in vitro and can potentially be applied to other salamander species.

An E8 promoter–HSP terminator cassette promotes the high-level accumulation of recombinant protein predominantly in transgenic tomato fruits: a case study of miraculin

The E8 promoter-HSP terminator expression cassette is a powerful tool for increasing the accumulation of recombinant protein in a ripening tomato fruit. Strong, tissue-specific transgene expression is a desirable feature in transgenic plants to allow the production of variable recombinant proteins. The expression vector is a key tool to control the expression level and site of transgene and recombinant protein expression in transgenic plants. The combination of the E8 promoter, a fruit-ripening specific promoter, and a heat shock protein (HSP) terminator, derived from heat shock protein 18.2 of Arabidopsis thaliana, produces the strong and fruit-specific accumulation of recombinant miraculin in transgenic tomato. Miraculin gene expression was driven by an E8 promoter and HSP terminator cassette (E8-MIR-HSP) in transgenic tomato plants, and the miraculin concentration was the highest in the ripening fruits, representing 30-630μg miraculin of the gram fresh weight. The highest level of miraculin concentration among the transgenic tomato plant lines containing the E8-MIR-HSP cassette was approximately four times higher than those observed in a previous study using a constitutive 35S promoter and NOS terminator cassette (Hiwasa-Tanase et al. in Plant Cell Rep 30:113-124, 2011). These results demonstrate that the combination of the E8 promoter and HSP terminator cassette is a useful tool to increase markedly the accumulation of recombinant proteins in a ripening fruit-specific manner.

MULTI-TRANSGENIC PIGS FOR XENOTRANSPLANTATION

Successful development of somatic cell nuclear transfer (cloning) technology in pigs has allowed for precise genetic manipulation of the pig genome. For xenotransplantation applications, pigs have been produced in which both copies of the α1,3-galactosyl transferase (GT) gene were inactivated (GTKO pigs). Analysis of tissues from GTKO pigs demonstrated a complete lack of immunogenic Galα1,3Gal (Gal) sugars, while in vivo pre-clinical studies in nonhuman primates, using cells (i.e. pancreatic islets) or whole organs (heart, kidney, liver, lung), demonstrated the elimination of hyperacute rejection, and prolonged survival compared to wild-type controls. While survival of GTKO xenografts was extended, challenges including induced antibody responses to non-gal antigens, thrombosis, inflammation, and cell-mediated rejection remained, pointing to the need for further genetic modification of the source pig. Towards this goal, through a combination of cloning and breeding, in combination with GTKO, we have produced multi-transgenic pigs (some with 5 different transgenes) with controlled expression of genes for (1) complement regulation to address the humoral response to anti-non-gal targets (DAF, CD46); (2) inhibition of inflammation and thrombosis (TFPI, CD39, thrombomodulin, EPCR); and (3) local protection against the human cellular response (CTLA4Ig, CIITA-DN). For some transgenes a constitutive promoter system can be used for expression in all tissues, such that one animal can be used for multiple transplant applications, however, our results have shown that for certain transgenes, tissue-specific gene expression is preferred. Since inhibition of thrombosis, complement modulation, and suppression of T-cell responses are important to delayed xenograft rejection of both whole organs and islet cell xenografts, pigs have been produced with tissue-specific transgene expression in either the vascular endothelium or endocrine pancreas compartments, or constitutively in all tissues. In vivo results in nonhuman primates have demonstrated complete normalization of blood glucose for up to 1 year in diabetic monkeys, and 8-month survival of multigenic pig hearts in baboons, evidence for the promise of this technology for human clinical applications.

Tissue-Specific Transgene Expression in Induced Pluripotent Stem (iPS) Cell-Derived Megakaryocytes: Correction of Glanzmann Thrombasthenia (GT)

Abstract 387Megakaryocyte-specific transgene expression in patient-derived iPS cells offers a new approach to study and potentially treat disorders that affect megakaryocytes and platelets. The feasibility of this method was demonstrated in GT, a disorder resulting from an absence of functional platelet integrin alphaIIb/beta3, leading to impaired platelet aggregation and clinically presenting with severe bleeding. While rare, GT can serve as a model of an inherited platelet disorder that would benefit from corrective cell therapy. For this study, iPS cells were generated from the peripheral blood of healthy controls and two patients with GT. Patient 1 is homozygous for a novel alphaIIb exon/intron 4 splice site mutation, leading to an alternatively spliced transcript and premature stop codon. Patient 2 is homozygous for a previously reported alphaIIb missense mutation in exon 8 (p.G273D), leading to impaired intracellular transport and surface expression of the heterodimer. Both patients express <5% alphaIIb/beta3 on the surface of their platelets, which was confirmed in IPS cell-derived megakaryocytes. To achieve megakaryocyte-specific expression, transgene constructs were created using the murine proximal GPIbalpha promoter driving either an eGFP reporter or wild type alphaIIb cDNA. These constructs were inserted into the AAVS1 safe harbor locus using zinc finger nuclease-mediated homologous recombination. When targeted to a single allele of the AAVS1 locus in human ES cells, the GPIbalpha promoter driving eGFP showed a high-level of expression in ES cell-derived megakaryocytes. Inserting the GPIbalpha promoter driving alphaIIb into a single allele of the AAVS1 locus in GT iPS cells restored ∼60% surface expression of integrin alphaIIb/beta3 when compared to control iPS cell-derived megakaryocytes. Transgenic alphaIIb mRNA levels were similar to endogenous alphaIIb mRNA levels in differentiated megakaryocytes. When stimulated with thrombin, corrected GT iPS cell-derived megakaryocytes demonstrated both PAC-1 antibody binding and fibrinogen binding. This study shows the GPIbalpha promoter construct targeted to the AAVS1 locus drives a high-level of expression in iPS cell-derived megakaryocytes, which offers a novel tool to study megakaryocyte and platelet biology in samples obtained from affected individuals. For GT, this study demonstrates a higher level of correction than seen with a lentiviral approach. To our knowledge, this is the first report of the generation and correction of iPS cell lines from patients with a disease affecting platelet function. Disclosures:No relevant conflicts of interest to declare.

Gene Carrier Showing All-or-None Response to Cancer Cell Signaling

In this work we designed a novel nano carrier, a linear polyethylenimine (LPEI)-peptide conjugate, for cancer-specific expression of transgenes. The conjugate was easily synthesized by using a click chemistry scheme orthogonal to the reactive side groups of the peptide, which is the substrate of protein kinase Cα (PKCα). Polyplexes of the conjugates with plasmid DNA (pDNA) were intact and stably dispersed even in the presence of cell lysate. Despite this stability, the polyplexes readily dissociated upon phosphorylation of the grafted peptides by PKCα. Because of its endosomal escape ability and adequate susceptibility to PKCα, the polyplexes showed an all-or-none type response to PKCα activity in transgene expression in vitro. The polyplexes achieved cancer tissue-specific transgene expression even for a tumor with a relatively low PKCα activity. Thus the LPEI-peptide conjugate has high potential as a nanocarrier for cancer-targeted gene therapy.

Simple, fast, tissue‐specific bacterial artificial chromosome transgenesis in Xenopus

We have developed a method of injecting bacterial artificial chromosome (BAC) DNA into Xenopus embryos that is simple and efficient, and results in consistent and tissue-specific expression of transgenes cloned into BAC vectors. Working with large pieces of DNA, as can be accommodated by BACs, is necessary when studying large or complex genes and conducive to studying the function of long-range regulatory elements that act to control developmentally restricted gene expression. We recombineered fluorescent reporters into three Xenopus tropicalis BAC clones targeting three different genes and report that up to 60% of injected embryos express the reporter in a manner consistent with endogenous expression. The behavior of these BACs, which are replicated after injection, contrasts with that of smaller plasmids, which degrade relatively quickly when injected as circular molecules and generally fail to recapitulate endogenous expression when not integrated into the Xenopus genome.

Gene therapy for primary immunodeficiency

Haematopoietic stem cell transplantation (HSCT) is the mainstay of definitive treatment for children with a wide spectrum of primary immunodeficiencies (PIDs), but outcome is heavily dependent on the availability of a human leukocyte antigen-matched donor. Gene therapy using autologous gene-corrected haematopoietic stem cells is an alternative for patients who lack an appropriate donor and has been used to treat children and adults with specific forms of PID, such as severe combined immunodeficiency, for over 10 years. This review summarizes the encouraging long-term outcome data available from these clinical trials and considers the important adverse events that have arisen. Current strategies directed towards improving the efficacy and safety profile of gene therapy will be discussed. Effective clinical trials have been conducted for other forms of PID including chronic granulomatous disease and Wiskott-Aldrich syndrome. Preclinical and clinical studies are now focussed on the development of improved viral vectors giving more regulated or tissue-specific transgene expression with reduced mutagenic potential. Gene therapy offers a valuable alternative management option for selected immunodeficiency patients who lack a suitable donor for HSCT. Clinical trials have confirmed proof-of-principle in terms of stem cell transduction and subsequent immune reconstitution, but have also highlighted the potential for clonal disturbances related to semi-random vector insertion within the genome.

Selective Targeting of Genetically Engineered Mesenchymal Stem Cells to Tumor Stroma Microenvironments Using Tissue-Specific Suicide Gene Expression Suppresses Growth of Hepatocellular Carcinoma

The use of engineered mesenchymal stem cells (MSCs) as therapeutic vehicles for the treatment of experimental pancreatic and breast cancer has been previously demonstrated. The potential application of MSCs for the treatment of hepatocellular carcinoma (HCC) has been controversial. The general approach uses engineered MSCs to target different aspects of tumor biology, including angiogenesis or the fibroblast-like stromal compartment, through the use of tissue-specific expression of therapeutic transgenes. The aim of the present study was (1) to evaluate the effect of exogenously added MSCs on the growth of HCC and (2) the establishment of an MSC-based suicide gene therapy for experimental HCC. Mesenchymal stem cells were isolated from bone marrow of C57/Bl6 p53(-/-) mice. The cells were injected into mice with HCC xenografts and the effect on tumor proliferation and angiogenesis was evaluated. The cells were then stably transfected with red fluorescent protein (RFP) or Herpes simplex virus thymidine kinase (HSV-Tk) gene under control of the Tie2 promoter/enhancer or the CCL5 promoter. Mesenchymal stem cells were injected intravenously into mice with orthotopically growing xenografts of HCC and treated with ganciclovir (GCV). Ex vivo examination of hepatic tumors revealed tumor-specific recruitment, enhanced tumor growth, and increased microvessel density after nontherapeutic MSC injections. After their homing to the hepatic xenografts, engineered MSCs demonstrated activation of the Tie2 or CCL5 promoter as shown by RFP expression. Application of CCL5/HSV-TK transfected MSCs in combination with GCV significantly reduced tumor growth by 56.4% as compared with the control group and by 71.6% as compared with nontherapeutic MSC injections. CCL5/HSV-TK(+) transfected MSCs proved more potent in tumor inhibition as compared with Tie2/HSV-TK(+) MSCs. Exogenously added MSCs are recruited to growing HCC xenografts with concomitant activation of the CCL5 or Tie2 promoters within the MSCs. Stem cell-mediated introduction of suicide genes into the tumor followed by prodrug administration was effective for treatment of experimental HCC and thus may help fill the existing gap in bridging therapies for patients suffering from advanced HCCs.

The Anopheles gambiae alpha-tubulin-1b promoter directs neuronal, testes and developing imaginal tissue specific expression and is a sensitive enhancer detector

A knowledge gap in mosquito functional genetic analysis is the dearth of characterized regulatory regions that can target tissue specific transgene expression. To broaden the tools available, a promoter region of the Anopheles gambiaeα-tubulin1b gene has been assayed following fusion to the green fluorescent protein (GFP) reporter gene and stable transformation of An. gambiae. In eight transgenic lines, the Angtub α1b regulatory region directed a core profile of tissue specific expression in the head, chordotonal organs, ventral nerve cord and testes. This profile overlaps those seen for α2-tubulin expression in Drosophila melanogaster and Bombyx mori. In addition, widespread position dependant expression was observed in other specific tissues that were unique to each line. For example, in different lines, expression was observed in larval and adult muscles, fatbody, cuticle and midgut secretory cells. The majority of genomic transgene insertions were mapped to within 10 kb of a gene, suggesting that the Angtub α1b basal promoter is particularly sensitive to enhancers and may be suitable to form the basis of a sensitive enhancer trapping construct, in combination with a binary expression system such as Gal4-UAS.

Development of a Liver-specific Tet-On Inducible System for AAV Vectors and Its Application in the Treatment of Liver Cancer

Recombinant adeno-associated virus (rAAV) are effective gene delivery vehicles that can mediate long-lasting transgene expression. However, tight regulation and tissue-specific transgene expression is required for certain therapeutic applications. For regulatable expression from the liver we designed a hepatospecific bidirectional and autoregulatory tetracycline (Tet)-On system (Tet(bidir)Alb) flanked by AAV inverted terminal repeats (ITRs). We characterized the inducible hepatospecific system in comparison with an inducible ubiquitous expression system (Tet(bidir)CMV) using luciferase (luc). Although the ubiquitous system led to luc expression throughout the mouse, luc expression derived from the hepatospecific system was restricted to the liver. Interestingly, the induction rate of the Tet(bidir)Alb was significantly higher than that of Tet(bidir)CMV, whereas leakage of Tet(bidir)Alb was significantly lower. To evaluate the therapeutic potential of this vector, an AAV-Tet(bidir)-Alb-expressing interleukin-12 (IL-12) was tested in a murine model for hepatic colorectal metastasis. The vector induced dose-dependent levels of IL-12 and interferon-γ (IFN-γ), showing no significant toxicity. AAV-Tet(bidir)-Alb-IL-12 was highly efficient in preventing establishment of metastasis in the liver and induced an efficient T-cell memory response to tumor cells. Thus, we have demonstrated persistent, and inducible in vivo expression of a gene from a liver-specific Tet-On inducible construct delivered via an AAV vector and proved to be an efficient tool for treating liver cancer.

Abstract 829: Targeted antizyme expression impairs HER2/neu-induced tumorigenesis but not normal mammary gland development

Abstract The objective of these studies was to utilize a novel transgenic mouse model with inducible and mammary gland-specific antizyme (AZ) expression to test the hypothesis that AZ will prevent mammary tumorigenesis and restrict the growth of established mammary tumors. AZ is a negative regulator of cellular polyamine levels that inhibits ornithine decarboxylase (ODC) activity, stimulates ODC degradation and suppresses exogenous polyamine uptake. Mouse lines were generated harboring a transgene construct (tetO-AZ) containing Tet operon repeat sequences upstream of an AZ cDNA followed by an internal ribosome entry site and a luciferase coding region. The tetO-AZ mice were crossed with MMTV-rtTA (MTB) mice to achieve AZ expression within the mammary gland that is induced by doxycycline (Dox). Bioluminescent imaging and in vitro assays of luciferase activity and AZ protein demonstrate tissue-specific and Dox-dependent transgene expression. Morphometric and immunohistochemical analysis of the mammary epithelial tree verified that normal development is unaffected by elevated levels of AZ. Next, we examined the ability of AZ to prevent tumor appearance in a widely used model of human breast cancer. TetO-AZ/MTB mice were crossed with MMTV-neu mice that develop spontaneous mammary tumors and Dox-treated animals were monitored for up to 1 year. Tumor-free survival was greatly extended in tetO-AZ/MTB/neu mice relative to controls (348 vs. 234 days, p=0.0003). In vivo imaging of the luciferase reporter revealed a pronounced absence of transgene expression within all 19 tumors detected in Dox-treated tetO-AZ/MTB/neu mice, therefore these tumors developed from cells that initially lacked AZ expression or lost AZ expression at a very early stage of tumor growth. The Dox-regulated transgenic model also allowed us to examine the effect of AZ on the maintenance and growth of established tumors in Dox-naïve mice. Once tumors reached 5 mm in diameter, Dox treatment was initiated to induce AZ expression in tetO-AZ/MTB/neu mice and tumor growth was evaluated. The established primary tumors from Dox-treated controls grew very rapidly and 34/45 (76%) exceeded 2000 mm3 within the monitoring period of 8 weeks, whereas only 6/16 (38%) of the tumors from tetO-AZ/MTB/neu mice reached that size. In vivo imaging of transgene-derived luciferase reporter expression allows us to compare transgene expression levels with tumor growth rate over time. This analysis has revealed several classes of response including a) reduced tumor growth in the presence of strong AZ expression, b) evolutionary loss of AZ expression and subsequent increased growth rate and c) occasional rapidly growing tumors that tolerate high levels of AZ expression. In summary, our studies indicate that elevated AZ expression prevents the appearance of HER2/neu-induced mammary tumors and suppresses the growth of established mammary tumors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 829. doi:10.1158/1538-7445.AM2011-829

ChemInform Abstract: Development of Recombinant Adenovirus Carrying MicroRNA‐Regulated Gene Expression System

AbstractReview: 23 refs.

Inactivation of the Mitotic Checkpoint Protein UBE2Q2 to Identify Its Function In Oncogenesis and Development

AbstractAbstract 5115UBE2Q2 is a ubiquitin conjugating enzyme that was first identified in a microarray screen for mitotic regulatory proteins. Since UBE2Q2 is expressed in G2- and M-phases, we evaluated it as a possible regulator of mitotic entry or progression. While inactivation of the protein using either siRNA or dominant-negative protein expression caused no overt change in cell cycle progression in unperturbed cultured human cells, it induced a prolonged early mitotic arrest after cells were treated with vincristine or other microtubule inhibitors (MIs). In addition, inhibition of UBE2Q2 caused a pronounced and specific sensitization to the cytotoxicity of MIs, an effect mediated by apoptosis (Banerjee et al, Oncogene 26;6509-17). The cell cycle effect caused by UBE2Q2 inactivation was indistinguishable from that produced by over-expression of the checkpoint protein CHFR, suggesting that the two proteins function in the same mitotic checkpoint, the mitotic stress or prophase checkpoint. Although UBE2Q2 and CHFR appear to function in the same checkpoint, they do so in opposition to one another. In this study, we have explored possible mechanisms for UBE2Q2-mediated cell cycle arrest by assessing the effect of its inhibition on the accumulation of candidate gene products after checkpoint activation. We have found that prophase checkpoint activation in cells lacking functional UBE2Q2 is accompanied by accumulation of several mitotic regulatory proteins including Securin and Aurora-A. Studies are underway to determine if UBE2Q2 inactivation exerts this effect by inhibiting the ubiquitination of these proteins and to determine if proteins upregulated by UBE2Q2 inactivation are mediators of checkpoint function. Since CHFR functions as a tumor suppressor (Yu et al, Nat Genet 37;401-6) and in opposition to UBE2Q2, we hypothesize that UBE2Q2 will be oncogenic when constitutively over-expressed in vivo. In order to test this hypothesis, we have generated transgenic mice using an inducible system that allows expression of the FLAG-tagged UBE2Q2 transgene after Cre-mediated recombination, shown schematically in the accompanying figure. Several independent transgenic lines were generated and screened for expression of β-galactosidase (β-gal), the protein that serves as a marker for transgene expression. This system provides several advantages, allowing us to circumvent potential embryonic lethality in transgenics, providing an opportunity for tissue specific transgene expression, and allowing activation at a specific age or developmental stage. Two founders with abundant β-gal expression have been bred with mice expressing EIIa-Cre, which is constitutively expressed in all tissues. We have confirmed that Cre+-offspring from these matings have high level expression of UBE2Q2-FLAG. We are now breeding mice with the activated allele to generate a cohort of UBE2Q2-transgenic mice for evaluation. Since we hypothesize that UBE2Q2 over-expression will promote the development of malignancies, we will initially evaluate mice separately for the development of (1) spontaneous malignancies and (2) carcinogen-induced (DMBA) skin tumors. A group of about 100 transgenic mice and 100 littermate controls will be monitored for the spontaneous development of malignancies and submitted for necropsy in the case of death or an obvious malignancy. Since UBE2Q2 over-expression might have effects other than the promotion of tumors, we will also monitor the mice in these groups for weight gain (by weighing monthly), and metabolic and hematologic abnormalities (by laboratory evaluation). To assess the ability of UBE2Q2 over-expression to promote the development of DMBA-induced malignancies, we will give a single application of DMBA to the skin of about 25 transgenic mice and 25 littermate controls. Using this approach, there should be a low frequency of skin tumors in control mice (Yu et al, Nat Genet 37;401-6). In contrast, we expect a much higher frequency of skin tumors in UBE2Q2-transgenic mice. If UBE2Q2 over-expression promotes malignancies, we can evaluate the tissue specificity of this property by breeding with mice expressing a tissue-specific Cre. Finally, our data suggest that UBE2Q2 is a potentially useful target for the treatment of malignancies (Banerjee et al, Oncogene 26;6509-17). If our transgenic mice develop tumors, these mice will be a very useful tool for testing UBE2Q2 inhibitors that are developed. Disclosures:No relevant conflicts of interest to declare.

Development of Recombinant Adenovirus Carrying MicroRNA-regulated Gene Expression System

Target tissue-specific delivery and transcription of foreign genes are desirable for safe and effective gene therapy. Two approaches for this purpose, "Targeted Delivery" and "Targeted Expression", have been mainly reported. Among "Targeted Expression" approaches, microRNA (miRNA)-mediated "post-transcriptional de-targeting" has been recently demonstrated, and much attention has been focused on this approach. MiRNAs are an approximately 22-nt length non-coding RNA, and bind to imperfectly complementary sequences in the 3'-untranslated region (UTR) of target mRNA, leading to suppression of gene expression via post-transcriptional regulation. First, in order to reduce the hepatic transduction by Ad vectors, complementary sequences of liver-specific miRNA, miR-122a, were inserted into the 3'-UTR of the transgene expression cassette. Intratumor injection of this Ad vector resulted in approximately 100-fold lower hepatic expression than that of the conventional Ad vector, without reducing gene expression in the tumor. Second, complementary sequences for miRNAs selectively down-regulated in tumor cells were inserted into the E1 gene expression cassette in oncolytic Ads, which exhibit tumor cell-specific replication and antitumor effects. Recent studies demonstrated that expression of several miRNAs is exclusively reduced in tumor cells. Oncolytic Ads containing the miRNA complementary sequences showed reduced replication in the normal cells, without altering the antitumor effects. MiRNA-regulated gene expression system mediates "post-transcriptional de-targeting", in which translation of transgene is suppressed in a tissue-specific manner; however, tissue-specific transgene expression can be achieved by taking tropism of gene delivery vehicles into consideration and reducing the transgene expression in untargeted organs via miRNA-regulated gene expression system.

Physiological and tissue-specific vectors for treatment of inherited diseases

After more than 1500 gene therapy clinical trials in the past two decades, the overall conclusion is that for gene therapy (GT) to be successful, the vector systems must still be improved in terms of delivery, expression and safety. The recent development of more efficient and stable vector systems has created great expectations for the future of GT. Impressive results were obtained in three primary immunodeficiencies and other inherited diseases such as congenital blindness, adrenoleukodystrophy or junctional epidermolysis bullosa. However, the development of leukemia in five children included in the GT clinical trials for X-linked severe combined immunodeficiency and the silencing of the therapeutic gene in the chronic granulomatous disease clearly showed the importance of improving safety and efficiency. In this review, we focus on the main strategies available to achieve physiological or tissue-specific expression of therapeutic transgenes and discuss the importance of controlling transgene expression to improve safety. We propose that tissue-specific and/or physiological viral vectors offer the best balance between efficiency and safety and will be the tools of choice for future clinical trials in GT of inherited diseases.

Promoter-specific lentivectors for long-term, cardiac-directed therapy of Fabry disease

In Fabry disease a deficiency of α-galactosidase A (α-gal A) activity leads to accumulation of globotriaosylceramide (Gb3) in various tissues including the heart. A specific cardiac variant of Fabry disease has also been described. Previously we have demonstrated the feasibility of gene therapy for Fabry disease. Here, to provide efficient transfer and increased specificity of transgene expression, we synthesized lentiviral vectors (LVs) with myocardial-specific promoters including: α-myosin heavy chain (α-MHC), myosin light chain (MLC2v), and cardiac troponin T (cTnT). Initially, neonatal Balb/c mice were injected with such LV constructs engineering expression of luciferase. One month post-injection, we found specific expression of luciferase in hearts of recipient animals when compared with transgene expression driven by the standard EF1-α promoter. To examine the feasibility of long-term therapy specifically targeting the heart, recombinant LV/α-gal A therapeutic vectors with analogous cardiac promoters were generated and injected into numerous neonatal Fabry mice. No immune response against the corrective α-gal A hydrolase was observed in the treated mice. Serum α-gal A activity of 10-week-old Fabry mice was increased in LV/α-gal A-injected animals compared to controls. In 28-week-old Fabry mice we observed significantly decreased Gb3 accumulation. Neonatal injections with LVs harboring cardiac-specific promoters may thus be an effective long-term treatment strategy for heart manifestations and cardiac variant Fabry disease. These results can be also extended to other progressive pathologies of the heart.

Seasonal and tissue-specific transgene expression and resveratrol-3-glucoside (piceid) accumulation in genetically modified white poplars carrying the grapevine StSy gene

Unexpected responses may occur in trees at specific stages after extensive cultivation time or they may be induced by specific environmental signals. The present work reports on a greenhouse trial carried out with transgenic white poplars (Populus alba L. cv. ‘Villafranca’) expressing the StSy gene from Vitis vinifera L., responsible for resveratrol biosynthesis. Notwithstanding the presence of the constitutive 35SCaMV promoter, significant fluctuations in the amount of the StSy transcript were observed in relation to season, tissue/organ type and position. High-Performance Liquid Chromatography with Diode-Array Detection revealed that the trans-isomer of resveratrol-3-glucoside (trans-piceid) accumulated in highly variable concentrations in leaves while reaching more stable values in stems and roots. Substantial amounts were observed in leaves (up to 717 mg kg−1 fresh weight), stems (up to 226 mg kg−1 fresh weight) and roots (up to 330 mg kg−1 fresh weight) while the cis-isomer was present only in leaves (up to 321 mg kg−1 fresh weight).