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.     

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.     

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.     

Tight control of transgene expression by lentivirus vectors containing second-generation tetracycline-responsive promoters

BACKGROUND: The goal of this study was to design improved regulatable lentivirus vector systems. The aim was to design tetracycline (tet)-regulatable lentivirus vectors based on the Tet-on system displaying low background expression in the absence of the doxycycline (DOX) inducer and high transgene expression levels in the presence of DOX. METHODS: We constructed a binary lentivirus vector system that is composed of a self-inactivating (SIN) lentivirus vector bearing inducible first- or second-generation tet-responsive promoter elements (TREs) driving expression of a transgene and a second lentivirus vector encoding a reverse tetracycline-controlled transactivator (rtTA) that activates transgene expression from the TRE in the presence of DOX. RESULTS: We evaluated a number of different rtTAs and found rtTA2S-M2 to induce the highest levels of transgene expression. Regulated transgene expression was stable in human breast carcinoma cells implanted into nude mice for up to 11 weeks. In an attempt to minimize background expression levels, the chicken beta-globin cHS4 insulator element was cloned into the 3' long terminal repeat (LTR) of the transgene transfer vector. The cHS4 insulator element reduced background expression but expression levels following DOX addition were lower than those observed with vectors lacking an insulator sequence. In a second strategy, vectors bearing second-generation TREs harboring repositioned tetracycline operator elements were used. Such vectors displayed greatly reduced leakiness in the absence of DOX and induced transgene expression levels were up to 522-fold above those seen in the absence of DOX. CONCLUSIONS: Inducible lentivirus vectors bearing insulators or second-generation TREs will likely prove useful for applications demanding the lowest levels of background expression.     

Erythroid-specific expression of β-globin from Sleeping Beauty-transduced human hematopoietic progenitor cells

Gene therapy for sickle cell disease will require efficient delivery of a tightly regulated and stably expressed gene product to provide an effective therapy. In this study we utilized the non-viral Sleeping Beauty (SB) transposon system using the SB100X hyperactive transposase to transduce human cord blood CD34(+) cells with DsRed and a hybrid IHK-β-globin transgene. IHK transduced cells were successfully differentiated into multiple lineages which all showed transgene integration. The mature erythroid cells had an increased β-globin to γ-globin ratio from 0.66±0.08 to 1.05±0.12 (p=0.05), indicating expression of β-globin from the integrated SB transgene. IHK-β-globin mRNA was found in non-erythroid cell types, similar to native β-globin mRNA that was also expressed at low levels. Additional studies in the hematopoietic K562 cell line confirmed the ability of cHS4 insulator elements to protect DsRed and IHK-β-globin transgenes from silencing in long-term culture studies. Insulated transgenes had statistically significant improvement in the maintenance of long term expression, while preserving transgene regulation. These results support the use of Sleeping Beauty vectors in carrying an insulated IHK-β-globin transgene for gene therapy of sickle cell disease.     

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.     

Production of transgenic rats via lentiviral transduction and xenogeneic transplantation of spermatogonial stem cells

Spermatogonial stem cells (SSCs) continue to proliferate in the testis to support spermatogenesis throughout life, which makes them ideal targets for germline modification. Although recent success in the production of transgenic and knockout animals using SSCs has opened up new experimental possibilities, several problems, including the low efficiency of germ cell transplantation and poor fertility rates, remain to be resolved. In the present study, we took advantage of the xenogeneic transplantation to resolve these problems. Rat SSCs were transduced in vitro with a lentiviral vector that expressed enhanced green fluorescent protein (EGFP), and then transplanted into the testes of immunodeficient mice. The transduced rat SSCs produced EGFP-expressing spermatogenic cells, and microinsemination using these cells was used to produce transgenic rats, which stably transmitted the transgene to the next generation. Thus, xenogeneic transplantation is a powerful strategy for transgenesis, and smaller xenogeneic surrogates can be used for male germline modification using SSCs.     

A Chimeric HS4-SAR Insulator (IS2) That Prevents Silencing and Enhances Expression of Lentiviral Vectors in Pluripotent Stem Cells

Chromatin insulators, such as the chicken β-globin locus control region hypersensitive site 4 (HS4), and scaffold/matrix attachment regions (SARs/MARs) have been incorporated separately or in combination into retroviral vectors (RVs) in order to increase transgene expression levels, avoid silencing and reduce expression variability. However, their incorporation into RVs either produces a reduction on titer and/or expression levels or do not have sufficient effect on stem cells. In order to develop an improved insulator we decided to combine SAR elements with HS4 insulators. We designed several synthetic shorter SAR elements containing 4 or 5 MAR/SARs recognition signatures (MRS) and studied their effects on a lentiviral vector (LV) expressing eGFP through the SFFV promoter (SE). A 388 bp SAR element containing 5 MRS, named SAR2, was as efficient or superior to the other SARs analyzed. SAR2 enhanced transgene expression and reduced silencing and variability on human embryonic stem cells (hESCs). We next compared the effect of different HS4-based insulators, the HS4-Core (250 bp), the HS4-Ext (400 bp) and the HS4-650 (650 bp). All HS4 elements reduced silencing and expression variability but they also had a negative effect on transgene expression levels and titer. In general, the HS4-650 element had a better overall effect. Based on these data we developed a chimeric insulator, IS2, combining the SAR2 and the HS4-650. When incorporated into the 3′ LTR of the SE LV, the IS2 element was able to enhance expression, avoid silencing and reduce variability of expression on hESCs. Importantly, these effects were maintained after differentiation of the transduced hESCs toward the hematopoietic linage. Neither the HS4-650 nor the SAR2 elements had these effects. The IS2 element is therefore a novel insulator that confers expression stability and enhances expression of LVs on stem cells.     

Poly(ADP-ribose) Polymerase-1 (PARP-1) Contributes to the Barrier Function of a Vertebrate Chromatin Insulator

The prototypic chromatin insulator cHS4 has proven effective in reducing silencing chromosomal position effects in a variety of settings. Most of this barrier insulator activity has been mapped to a 250-bp core region, as well as to several proteins that bind this region. However, recent studies from our laboratory demonstrated that an extended 400-bp core region of the cHS4 element is necessary to achieve full barrier insulator activity when used as a single copy in the context of recombinant gammaretroviral and lentiviral vectors. In this study, electrophoretic gel mobility shift assays revealed specific DNA-protein binding activities associated with the distal portion of this extended core region. Affinity purification and tandem mass spectrometry studies led to the identification of one of these proteins as poly(ADP-ribose) polymerase-1 (PARP-1). The identity of this binding activity as PARP-1 was subsequently verified by a variety of biochemical studies in vitro and by chromatin immunoprecipitation studies in vivo. Functional studies with gammaretroviral reporter vectors in cell lines and primary mouse bone marrow progenitor cultures showed that cHS4 barrier activity was abrogated upon mutation of the putative PARP-1-binding site or upon treatment with a PARP inhibitor, respectively. The barrier activity of the cHS4 element was also found to be abrogated in studies using bone marrow from Parp1-null mice. Taken together, this study demonstrates that binding of PARP-1 plays a key functional role in the barrier activity of the extended cHS4 insulator core element.     

Ars insulator identified in sea urchin possesses an activity to ensure the transgene expression in mouse cells

Sea urchin arylsulfatase (Ars) gene locus has features of an insulator, i.e., blocking of enhancer and promoter interaction, and protection of a transgene against positional effects [Akasaka et al. (1999) Cell. Mol. Biol. 45, 555-565]. To examine the effect of Ars insulator on long-term expression of a transgene, the insulator was inserted into LTR of retrovirus vector harboring hrGFP gene as a reporter, and then introduced into mouse myoblast cells. The isolated clones transduced with the reporter gene with or without Ars insulator were cultured for more than 20 wk in the absence of a selection reagent, and the expression of hrGFP was periodically determined. Expression of hrGFP in four clones transduced with the reporter gene without Ars insulator was completely silenced after 20 wk of culture. On the other hand, hrGFP was expressed in all clones with Ars insulator inserted in one of the two different orientations. Histone H3 deacetylation and DNA methylation of the 5'LTR promoter region, signs for heterochromatin and silencing, were suppressed in the clones that were expressing hrGFP. Ars insulator is effective in maintaining a transgene in mouse cells in an orientation-dependent manner, and will be a useful tool to ensure stable expression of a transgene.     

Engineered lentivector targeting of dendritic cells for in vivo immunization

We report a method of inducing antigen production in dendritic cells by in vivo targeting with lentiviral vectors that specifically bind to the dendritic cell-surface protein DC-SIGN. To target dendritic cells, we enveloped the lentivector with a viral glycoprotein from Sindbis virus engineered to be DC-SIGN-specific. In vitro, this lentivector specifically transduced dendritic cells and induced dendritic cell maturation. A high frequency (up to 12%) of ovalbumin (OVA)-specific CD8(+) T cells and a significant antibody response were observed 2 weeks after injection of a targeted lentiviral vector encoding an OVA transgene into naive mice. This approach also protected against the growth of OVA-expressing E.G7 tumors and induced regression of established tumors. Thus, lentiviral vectors targeting dendritic cells provide a simple method of producing effective immunity and may provide an alternative route for immunization with protein antigens.     

Epigenetic regulatory elements associate with specific histone modifications to prevent silencing of telomeric genes

In eukaryotic cells, transgene expression levels may be limited by an unfavourable chromatin structure at the integration site. Epigenetic regulators are DNA sequences which may protect transgenes from such position effect. We evaluated different epigenetic regulators for their ability to protect transgene expression at telomeres, which are commonly associated to low or inconsistent expression because of their repressive chromatin environment. Although to variable extents, matrix attachment regions (MARs), ubiquitous chromatin opening element (UCOE) and the chicken cHS4 insulator acted as barrier elements, protecting a telomeric-distal transgene from silencing. MARs also increased the probability of silent gene reactivation in time-course experiments. Additionally, all MARs improved the level of expression in non-silenced cells, unlike other elements. MARs were associated to histone marks usually linked to actively expressed genes, especially acetylation of histone H3 and H4, suggesting that they may prevent the spread of silencing chromatin by imposing acetylation marks on nearby nucleosomes. Alternatively, an UCOE was found to act by preventing deposition of repressive chromatin marks. We conclude that epigenetic DNA elements used to enhance and stabilize transgene expression all have specific epigenetic signature that might be at the basis of their mode of action.     

Capability of cultivated human hair-follicle cells to integrate with skin structure in vivo

In the present work, we labeled human epidermal keratinocytes and dermal papilla cells in order to study their behavior after intradermal transplantation. The cells were transduced by lentiviral vectors that bore a marker gene that encodes green fluorescent protein (copGFP) or red fluorescent protein (DsRed). A portion of the transgene expressing cells was evaluated by flow cytometry. The proposed genetic constructions have allowed one to achieve high efficiency (>95%) of the transduction of hair follicle cells. The in vitro transduced cells were injected under epidermis of human skin fragments, after which these fragments were transplanted under the skin of immunodeficient mice. The injected epidermal keratinocytes were found mainly in hair follicles and partially in the zone of interfollicular epidermis, while dermal papilla cells were found in the papilla of the derma. The results of the present study have shown that the chosen genetic constructions obtained based on human immunodeficiency lentivirus are capable of the effective and stable transduction of human skin cells. The injected cells survived and were found in the corresponding skin structures.     

Human amniotic fluid stem cells attract osteoprogenitor cells in bone healing

Current treatments of large bone defects are based on autologous or allogenic bone transplantation. Human amniotic fluid stem cells (hAFSCs) were evaluated for their potential in bone regenerative medicine. In this study, hAFSCs were transduced with lentiviral vector harboring red fluorescent protein to investigate their role in the regeneration of critical-size bone defects in calvarial mouse model. To distinguish donor versus recipient cells, a transgenic mouse model carrying GFP fluorescent reporter was used as recipient to follow the fate of hAFSCs transplanted in vivo into Healos® scaffold. Our results showed that transduced hAFSCs can be tracked in vivo directly at the site of transplantation. The presence of GFP positive cells in the scaffold at 3 and 6 weeks after transplantation indicates that donor hAFSCs can recruit host cells during the repair process. These observations help clarify the role of hAFSCs in bone tissue repair.