Dynamics of transgene expression in a neural stem cell line transduced with lentiviral vectors incorporating the cHS4 insulator

Transplantation of genetically manipulated cells to the central nervous system holds great promise for the treatment of several severe neurological disorders. The success of this strategy relies on sufficient levels of transgene expression after transplantation. This has been difficult to achieve, however, due to transgene silencing. In this study, we transduced the neural stem cell line RN33B with self-inactivating lentiviral vectors and analyzed transgenic expression of green fluorescent protein (GFP) in several different settings both in vitro and after transplantation to the brain. We found that the transgene was affected of silencing both when transduced cells were proliferating and after differentiation. To prevent silencing, the cHS4 insulator was incorporated into the lentiviral vector. We found that a vector carrying the cHS4 insulator was partially protected against differentiation-dependent downregulation in vitro and in vivo. However, in proliferating cells, we found evidence for variegation and positional effects that were not prevented by the cHS4 insulator, suggesting that the mechanism behind silencing in proliferating cells is not the same mechanism influencing differentiation-dependent silencing. Taken together, these findings favor vector optimization as a strategy for achieving efficient ex vivo gene transfer in the central nervous system.     

Retrovirus silencer blocking by the cHS4 insulator is CTCF independent

Silencing of retrovirus vectors poses a significant obstacle to genetic manipulation of stem cells and their use in gene therapy. We describe a mammalian silencer blocking assay using insulator elements positioned between retrovirus silencer elements and an LCRβ-globin reporter transgene. In transgenic mice, we show that retrovirus silencers are blocked by the cHS4 insulator. Silencer blocking is independent of the CTCF binding site and is most effective when flanking the internal reporter transgene. These data distinguish silencer blocking activity by cHS4 from its enhancer blocking activity. Retrovirus vectors can be created at high titer with one but not two internal dimer cHS4 cores. cHS4 in the LTRs has no effect on expression in transduced F9 cells, suggesting that position effect blocking is not sufficient to escape silencing. The Drosophila insulators gypsy and Scs fail to block silencing in transgenic mice, but gypsy stimulates vector expression 2-fold when located in the LTRs of an infectious retrovirus. The silencer blocking assay complements existing insulator assays in mammalian cells, provides new insight into mechanisms of insulation and is a valuable tool to identify additional silencer blocking insulators that cooperate with cHS4 to improve stem cell retrovirus vector design.     

Comparison of single regulated lentiviral vectors with rtTA expression driven by an autoregulatory loop or a constitutive promoter

Regulated expression of a therapeutic gene is crucial for safe and efficacious gene therapy. Many inducible regulatory systems use a constitutive promoter to express a regulatory protein, such as rtTA in the Tet-On system, which may restrict their use because of cytotoxicity and immunogenicity. Autoregulatory expression of rtTA provides extremely low levels of rtTA when transgene expression is off, with rapid transgene induction upon addition of doxycycline. Lentiviral vectors efficiently transfer genes to dividing and non-dividing cells with long-term gene expression both in vitro and in vivo. We compared regulatory function in a single lentiviral vector where rtTA was either expressed from a constitutive promoter or placed in an autoregulatory loop. Autoregulatory expression of rtTA was superior to constitutive promoter expression, resulting in higher viral titers, undetectable levels of both rtTA and transgene expression in the absence of doxycycline, improved induction kinetics and increased induction levels in all cells tested. We further expanded the utility of the autoregulatory vector by using an improved rtTA variant with an increased sensitivity to doxycycline. This lentiviral vector with doxycycline-regulated transgene expression may be useful for gene therapy applications and in experimental settings where strict temporal expression of a transgene is required.     

Prototypic chromatin insulator cHS4 protects retroviral transgene from silencing in Schistosoma mansoni

Vesicular stomatitis virus glycoprotein (VSVG) pseudotyped murine leukemia virus (MLV) virions can transduce schistosomes, leading to chromosomal integration of reporter transgenes. To develop VSVG-MLV for functional genomics in schistosomes, the influence of the chicken β-globin cHS4 element, a prototypic chromatin insulator, on transgene expression was examined. Plasmid pLNHX encoding the MLV 5'- and 3'-Long Terminal Repeats flanking the neomycin phosphotransferase gene (neo) was modified to include, within the U3 region of the 3'-LTR, active components of cHS4 insulator, the 250 bp core fused to the 400 bp 3'-region. Cultured larvae of Schistosoma mansoni were transduced with virions from producer cells transfected with control or cHS4-bearing plasmids. Schistosomules transduced with cHS4 virions expressed 2-20 times higher levels of neo than controls, while carrying comparable numbers of integrated proviral transgenes. The findings not only demonstrated that cHS4 was active in schistosomes but also they represent the first report of activity of cHS4 in any Lophotrochozoan species, which has significant implications for evolutionary conservation of heterochromatin regulation. The findings advance prospects for transgenesis in functional genomics of the schistosome genome to discover intervention targets because they provide the means to enhance and extend transgene activity including for vector based RNA interference.     

Chicken hypersensitive site-4 insulator increases human serum albumin expression in bovine mammary epithelial cells modified with phiC31 integrase

Achieving high expression levels of recombinant human serum albumin (HSA) for purification is a solution for the large amount of plasma-derived HSA needed in therapeutic applications. Here, we employed phiC31 integrase system and chicken hypersensitive site-4 (cHS4) insulators to construct a HSA expression vector for high-level HSA expression. The phiC31 integrase system mediated efficient transgene integration in bovine mammary epithelial cells (bMECs). A preferred pseudo attP site, which had 38 % identity with the 39 bp wild-type attP sequence, was detected in six out of 55 bMEC colonies. Addition of the cHS4 insulator to the phiC31 integrase system resulted in 8–20-fold increases of HSA expression compared with that of using integrase alone. Moreover, the reverse-oriented cHS4 insulator in the phiC31 integrase system provided the optimal level of HSA expression in bMECs.     

Identification and Characterization of Enhancer-Blocking Insulators to Reduce Retroviral Vector Genotoxicity

The chromatin insulator cHS4 can reduce silencing chromosomal position effects and genotoxicity associated with integrating viral vectors. However, the fully active version of this element can also reduce vector titers and is only partially effective. In order to identify alternatives to cHS4, we developed a functional lentiviral vector-based reporter screen for enhancer-blocking insulators. Using this system, we screened candidate sequences that were initially identified by chromatin profiling for binding by CTCF and for DNase hypersensitivity. All 12 analyzed candidates blocked enhancer-promoter activity. The enhancer-blocking activity of the top two candidates was confirmed in two complementary plasmid-based assays. Studies in a gammaretroviral reporter vector indicated these two candidates have little to no effect on vector titers, and do not diminish vector expression in primary mouse bone marrow cultures. Subsequent assessment in a mouse in vivo tumor formation model demonstrated that both candidates reduced the rate of gammaretroviral vector-mediated genotoxicity as effectively as the cHS4 insulator. In summary, we have developed a novel lentiviral vector-based method of screening candidate elements for insulator activity, and have used this method to identify two new insulator elements capable of improving the safety of retroviral vectors without diminishing vector titers or expression. These findings expand the limited arsenal of insulators functionally validated to reduce the rate of retroviral vector-mediated genotoxicity.     

Lack of an immune response against the tetracycline-dependent transactivator correlates with long-term doxycycline-regulated transgene expression in nonhuman primates after intramuscular injection of recombinant adeno-associated virus

We previously documented persistent regulation of erythropoietin (Epo) secretion in mice after a single intramuscular (i.m.) injection of a recombinant adeno-associated virus (rAAV) vector harboring both the tetracycline-dependent transactivator (rtTA) and the Epo cDNA (D. Bohl, A. Salvetti, P. Moullier, and J. M. Heard, Blood 92:1512-1517, 1998). Using the same vector harboring the cynomolgus macaque Epo cDNA instead, the present study evaluated the ability of the tetracycline-regulatable (tetR) system to establish long-term transgene regulation in nonhuman primates. The vector was administered i.m., after which 5-day induction pulses were performed monthly for up to 13 months by using doxycycline (DOX), a tetracycline analog. We show that initial inductions were successful in all individuals and that there was a tight regulation and a rapid deinduction pattern upon DOX withdrawal. For one macaque, regulation of Epo secretion was maintained during the entire experimental period; for the five remaining macaques, secreted Epo became indistinguishable from endogenous Epo upon repeated DOX inductions. We investigated the mechanism involved and showed that, except in the animal in which secretion persisted, delayed humoral and cellular immune responses were directed against the rtTA transactivator protein associated with the reduction of vector DNA in transduced muscles. This study provides some evidence that, when the immune system is not mobilized against the rtTA transactivator, the tetR-regulatable system is able to support long-term transgene regulation in the context of an rAAV in nonhuman primates. In addition, our results suggest potential improvements for vector design.     

A tetracycline‐inducible and skeletal muscle‐specific Cre recombinase transgenic mouse

We have generated a transgenic mouse that expresses Cre recombinase only in skeletal muscle and only following tetracycline treatment. This spatiotemporal specificity is achieved using two transgenes. The first transgene uses the human skeletal actin (HSA) promoter to drive expression of the reverse tetracycline‐controlled transactivator (rtTA). The second transgene uses a tetracycline responsive promoter to drive the expression of Cre recombinase. We monitored transgene expression in these mice by crossing them with ROSA26 loxP‐LacZ reporter mice, which express β‐galactosidase when activated by Cre. We find that the expression of this transgene is only detectable within skeletal muscle and that Cre expression in the absence of tetracycline is negligible. Cre is readily induced in this model with tetracycline analogs at a range of embryonic and postnatal ages and in a pattern consistent with other HSA transgenic mice. This mouse improves upon existing transgenic mice in which skeletal muscle Cre is expressed throughout development by allowing Cre expression to begin at later developmental stages. This temporal control of transgene expression has several applications, including overcoming embryonic or perinatal lethality due to transgene expression. This mouse is especially suited for studies of steroid hormone action, as it uses tetracycline, rather than tamoxifen, to activate Cre expression. In summary, we find that this transgenic induction system is suitable for studies of gene function in the context of hormonal regulation of skeletal muscle or interactions between muscle and motoneurons in mice. © 2009 Wiley Periodicals, Inc. Develop Neurobiol, 2009     

Construction of a Single Lentiviral Vector Containing Tetracycline-Inducible Alb-uPA for Transduction of uPA Expression in Murine Hepatocytes

The SCID-beige/Alb-uPA mouse model is currently the best small animal model available for viral hepatitis infection studies [1]. But the construction procedure is often costly and time-consuming due to logistic and technical difficulties. Thus, the widespread application of these chimeric mice has been hampered [2]. In order to optimize the procedure, we constructed a single lentiviral vector containing modified tetracycline-regulated system to control Alb-uPA gene expression in the cultured hepatocytes. The modified albumin promoter controlled by tetracycline (Tet)-dependent transactivator rtTA2S-M2 was integrated into a lentiviral vector. The full-length uPA cDNA was inserted into another lentiviral vector containing PTight, a modified Tet-responsive promoter. Two vectors were then digested by specific enzymes and ligated by DNA ligase 4. The ligated DNA fragment was inserted into a modified pLKO.1 cloning vector and the final lentiviral vector was then successfully constructed. H2.35 cell, Lewis lung carcinoma, primary kidney, primary hepatic interstitial and CT26 cells were infected with recombinant lentivirus at selected MOI. The expression of uPA induced by DOX was detectable only in the infected H2.35 cells, which was confirmed by real-time PCR and Western blot analysis. Moreover, DOX induced uPA expression on the infected H2.35 cells in a dose-dependent manner. The constructed single lentiviral vector has many biological advantages, including that the interested gene expression under “Tet-on/off” system is controlled by DOX in a dose-depending fashion only in murine liver cells, which provides an advantage for simplifying generation of conditional transgenic animals.