Transgenic animals with inducible, targeted gene expression in brain.

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. 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. 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. Inducible lentivirus vectors bearing insulators or second-generation TREs will likely prove useful for applications demanding the lowest levels of background expression.

Apolipoprotein B (apo-B) plays a crucial role in the assembly of lipoproteins in the liver and the intestine. Here, we review how transgenic mouse expression studies with large genomic clones have been used to define distant cis-acting regulatory DNA sequences that control the expression of the apo-B gene. In early studies, apo-B transgenic mice were generated with approximately 80-kb P1 bacteriophage clones spanning either the human or the mouse apo-B genes. Both the human and mouse clones directed high levels of transgene expression in the liver, but transgene expression was absent in the intestine. The absence of transgene expression in the intestine was surprising because both P1 clones contained more than 11 kb of flanking sequences both 5' and 3' to the gene. Subsequently, we isolated and characterized 145-kb and 207-kb bacterial artificial chromosome (BAC) clones that spanned the human apo-B gene. Each of these BAC's contained extensive 5 and 3' flanking sequences and each directed spatially and physiologically appropriate apo-B gene expression in the intestines of transgenic mice. To define the location of the sequences that control intestinal expression of the apo-B gene, we generated transgenic mice by co-microinjecting the approximately 80-kb P1 bacteriophage clone (which did not confer intestinal expression of apo-B) with either the 5' sequences or the 3' sequences from the 145-kb BAC. Analysis of the apo-B expression pattern in those mice revealed that the DNA sequences controlling intestinal expression were located 5' to the apo-B gene. Next, we used recA-assisted restriction endonuclease (RARE) cleavage to truncate specific segments of the 5' and 3' flanking sequences from the 145-kb BAC. A series of the truncated BAC's containing different lengths of 5' and 3' sequences was used to generate more than 40 additional lines of human apo-B transgenic mice. Analysis of human apo-B gene expression in those mice demonstrated that the sequences controlling the expression of the apo-B gene in the intestine are located more than 50 kb 5' to the apo-B gene. Our studies demonstrate that the RARE cleavage/transgenic expression strategy is a powerful approach for examining gene regulation by distant gene-regulatory elements.

Fast onset and high-level neurospecific transgene expression in vivo is of importance for many areas in neuroscience, from basic to translational, and can significantly reduce the amount of vector load required to maintain transgene expression in vivo. In this study, we tested various cis elements to optimize transgene expression at transcriptional, posttranscriptional, and posttranslational levels and combined them together to create the high-level neuronal transgene expression cassette pUNISHER. Using a second-generation adenoviral vector system in combination with the pUNISHER cassette, we characterized its rate of onset of detectable expression and levels of expression compared with a neurospecific expression cassette driven by the 470-bp human synapsin promoter in vitro and in vivo. Our results demonstrate in primary neurons that the pUNISHER cassette, in a recombinant adenovirus type 5 background, led to a faster rate of onset of detectable transgene expression and higher level of transgene expression. More importantly, this cassette led to highly correlated neuronal expression in vivo and to stable transgene expression up to 30 days in the auditory brain stem with no toxicity on the characteristics of synaptic transmission and plasticity at the calyx of Held synapse. Thus the pUNISHER cassette is an ideal high-level neuronal expression cassette for use in vivo for neuroscience applications.

The expansion of CAG/CTG repeats is responsible for many diseases, including Huntington's disease (HD) and myotonic dystrophy 1. CAG/CTG expansions are unstable in selective somatic tissues, which accelerates disease progression. The mechanisms underlying repeat instability are complex, and it remains unclear whether chromatin structure and/or transcription contribute to somatic CAG/CTG instability in vivo. To address these issues, we investigated the relationship between CAG instability, chromatin structure, and transcription at the HD locus using the R6/1 and R6/2 HD transgenic mouse lines. These mice express a similar transgene, albeit integrated at a different site, and recapitulate HD tissue-specific instability. We show that instability rates are increased in R6/2 tissues as compared to R6/1 matched-samples. High transgene expression levels and chromatin accessibility correlated with the increased CAG instability of R6/2 mice. Transgene mRNA and H3K4 trimethylation at the HD locus were increased, whereas H3K9 dimethylation was reduced in R6/2 tissues relative to R6/1 matched-tissues. However, the levels of transgene expression and these specific histone marks were similar in the striatum and cerebellum, two tissues showing very different CAG instability levels, irrespective of mouse line. Interestingly, the levels of elongating RNA Pol II at the HD locus, but not the initiating form of RNA Pol II, were tissue-specific and correlated with CAG instability levels. Similarly, H3K36 trimethylation, a mark associated with transcription elongation, was specifically increased at the HD locus in the striatum and not in the cerebellum. Together, our data support the view that transcription modulates somatic CAG instability in vivo. More specifically, our results suggest for the first time that transcription elongation is regulated in a tissue-dependent manner, contributing to tissue-selective CAG instability.

Analysis of the Human Ferrochelatase Promoter in Transgenic Mice

We, and others, have previously achieved high and sustained levels of transgene expression from viral vectors, such as recombinant adeno-associated virus (rAAV). However, regulatable transgene expression may be preferred in gene therapy for diseases, such as type 1 diabetes (T1D) and rheumatoid arthritis (RA), in which the timing and dosing of the therapeutic gene product play critical roles. In the present study, we generated a positive feedback regulation system for human alpha 1 antitrypsin (hAAT) expression in the rAAV vector. We performed quantitative kinetics studies in vitro and in vivo demonstrating that this vector system can mediate high levels of inducible transgene expression. Transgene induction could be tailored to occur rapidly or gradually, depending on the dose of the inducing drug, doxycycline (Dox). Conversely, after withdrawal of Dox, the silencing of transgene expression occurred slowly over the course of several weeks. Importantly, rAAV delivery of inducible hAAT significantly prevented T1D development in non-obese diabetic (NOD) mice. These results indicate that this Dox-inducible vector system may facilitate the fine-tuning of transgene expression, particularly for hAAT treatment of human autoimmune diseases, including T1D.

Two missense mutations (A53T and A30P) in the gene encoding the presynaptic protein α-synuclein (asyn) are associated with rare, dominantly inherited forms of Parkinson’s disease (PD) and its accumulation in Lewy bodies and Lewy neurites. As an initial step in investigating the role of asyn in the pathogenesis of PD, we have generated C57BL/6 transgenic mice overexpressing the doubly mutated human asyn under the control of three different promoters; the chicken β-actin (chβactin), the mouse tyrosine hydroxylase 9.6 kb (msTH) and the mouse prion protein (msprp). In this study we compared the regional and cellular expression pattern of the transgenic protein in the brain and peripheral organs of various transgenic mouse lines. Western blot analysis and immunohistochemistry consistently showed that all three promoters successfully drive the expression of the transgene. The msprp promoter was found to give the highest level of transgene expression. All promoters directed the expression into the brain and specific neuron types. However, the promoters differed with respect to (i) the expression pattern in peripheral organs, (ii) the number and (iii) the regional distribution of expressing cells in the brain. Furthermore, remarkable line-to-line variation of expression patterns was observed in mouse lines carrying the same construct. Future studies will analyze how the variations in transgene expression affect the pathogenesis in the animals.

Both oncoretroviral and lentiviral vectors have been shown to transduce CD34(+) human hematopoietic stem cells (HSC) capable of establishing human hematopoiesis in nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mice that support partially human hematopoiesis. We and others have reported that murine stem cell virus (MSCV)-based oncoretroviral vectors efficiently transduced HSC that had been cultured ex vivo for 4-7 days with cytokines, resulting in transgene expression in lymphoid and myeloid progenies of SCID-engrafting cells 4-8 weeks post-transplantation. Although lentiviral vectors have been demonstrated to transduce HSC under minimal ex vivo culture conditions, concerns exist regarding the level of transgene expression mediated by these vectors. We therefore evaluated a novel hybrid lentiviral vector (GIN-MU3), in which the U3 region of the HIV-1 long terminal repeat was replaced by the MSCV U3 region (MU3). Human cord blood CD34(+) cells were transduced with vesicular stomatitis virus G envelope protein-pseudotyped lentiviruses during a 48-hour culture period. After a total of 4 days in culture, transduced cells were transplanted into NOD/SCID mice to examine gene transfer and expression in engrafting human cells. Fifteen weeks post-transplantation, 37% +/- 12% of engrafted human cells expressed the green fluorescence protein (GFP) gene introduced by the lentiviral vector. High levels of GFP expression were observed in lymphoid, myeloid and erythroid progenies, and in engrafted human cells that retained the CD34(+) phenotype 15 weeks post-transplantation. This study provides evidence that lentiviral vectors transduced both short-term and long-term engrafting human cells, and mediated persistent transgene expression at high levels in multiple lineages of hematopoietic cells.

Cationic Lipid Formulations Alter the In Vivo Tropism of AAV2/9 Vector in Lung

Background: Mesenchymal stem cells (MSCs) and their chondrogenic differentiation have been extensively investigated in vitro as MSCs provide an attractive source besides chondrocytes for cartilage repair therapies. Here we established prototype foamyviral vectors (FVV) that are derived from apathogenic parent viruses and are characterized by a broad host range and a favorable integration pattern into the cellular genome. As the inflammatory cytokine interleukin 1 beta (IL1β) is frequently present in diseased joints, the protective effects of FVV expressing the human interleukin 1 receptor antagonist protein (IL1RA) were studied in an established in vitro model (aggregate culture system) of chondrogenesis in the presence of IL1β.Materials and Methods: We generated different recombinant FVVs encoding enhanced green fluorescent protein (EGFP) or IL1RA and examined their transduction efficiencies and transgene expression profiles using different cell lines and human primary MSCs derived from bone marrow-aspirates. Transgene expression was evaluated by fluorescence microscopy (EGFP), flow cytometry (EGFP), and ELISA (IL1RA). For evaluation of the functionality of the IL1RA transgene to block the inhibitory effects of IL1β on chondrogenesis of primary MSCs and an immortalized MSC cell line (TERT4 cells), the cells were maintained following transduction as aggregate cultures in standard chondrogenic media in the presence or absence of IL1β. After 3 weeks of culture, pellets were harvested and analyzed by histology and immunohistochemistry for chondrogenic phenotypes.Results: The different FVV efficiently transduced cell lines as well as primary MSCs, thereby reaching high transgene expression levels in 6-well plates with levels of around 100 ng/ml IL1RA. MSC aggregate cultures which were maintained in chondrogenic media without IL1β supplementation revealed a chondrogenic phenotype by means of strong positive staining for collagen type II and matrix proteoglycan (Alcian blue). Addition of IL1β was inhibitory to chondrogenesis in untreated control pellets. In contrast, foamyviral mediated IL1RA expression rescued the chondrogenesis in pellets cultured in the presence of IL1β. Transduced MSC pellets reached thereby very high IL1RA transgene expression levels with a peak of 1087 ng/ml after day 7, followed by a decrease to 194 ng/ml after day 21, while IL1RA concentrations of controls were permanently below 200 pg/ml.Conclusion: Our results indicate that FVV are capable of efficient gene transfer to MSCs, while reaching IL1RA transgene expression levels, that were able to efficiently block the impacts of IL1β in vitro. FVV merit further investigation as a means to study the potential as a gene transfer tool for MSC based therapies for cartilage repair.

High transgene expression levels in sugarcane ( Saccharum officinarum L.) driven by the rice ubiquitin promoter RUBQ2

Herpes simplex virus type-1 (HSV-1) amplicon vectors are attractive tools for gene transfer because of their large DNA insert capacity, their broad host range of vector transduction and a minimal immune response as a result of the absence of helper viruses during viral packaging. However, the transient gene expression remains a challenge for the translation of HSV-1 amplicon based therapeutic strategies to a clinical setting. Although oriP/EBV nuclear antigen (EBNA)-1 elements of Epstein-Barr virus (EBV) have been successfully employed to achieve prolonged transgene expression, little is known about the stability of the EBNA-1 elements in the context of HSV-1 amplicon viral vectors. We have generated HSV/EBV hybrid vectors expressing the mutant EBNA-1 gene with the luciferase reporter gene bicistronically to enable monitoring of EBNA-1 expression in real-time, both in vitro and in vivo. The results obtained showed that the HSV/EBV hybrid vectors could mediate high levels of transgene expression (ranging from approximately two-fold to nine-fold) in primary human tumor cells and human bone marrow-derived mesenchymal stem cells compared to the control vector. Prolonged transgene expression could also be observed in primary patient-derived human hepatocellular carcinoma xenografts and in the mouse brain parenchyma up to a period of 17 and 365 days, respectively. Taken together, we have demonstrated that these hybrid vectors could be promising tools as carriers of therapeutic genes in mesenchymal stem cells or even provide an alternative non-integrating platform for the generation of induced pluripotent stem cells.

Supramolecular polypseudorotaxane gels for controlled delivery of rAAV vectors in human mesenchymal stem cells for regenerative medicine

The persistence of transgene expression has become a hallmark for adenovirus vector evaluation in vivo. Although not all therapeutic benefit in gene therapy is reliant on long-term transgene expression, it is assumed that the treatment of chronic diseases will require significant persistence of expression. To understand the mechanisms involved in transgene persistence, a number of adenovirus vectors were evaluated in vivo in different strains of mice. Interestingly, the rate of vector genome clearance was not altered by the complete deletion of early region 4 (E4) in our vectors. The GV11 (E1- E4-) vector genome cleared with a similar kinetic profile as the GV10 (E1-) vector genome in immunocompetent and immunocompromised mice. These results suggest that the majority of adenovirus vector genomes are eliminated from transduced tissue via a mechanism(s) independent of T-cell, B-cell, and NK cell immune mechanisms. While the levels of persistence of transgene expression in liver or lung transduced with GV10 and GV11 vectors expressing beta-galactosidase, cystic fibrosis transmembrane conductance regulator, or secretory alkaline phosphatase were similar in immunocompetent mice, a marked difference was observed in immunocompromised animals. Levels of transgene expression initially from both GV10 and GV11 vectors were the same. However, GV11 transgene expression correlated with loss of vector genome, while GV10 transgene expression persisted at a high level. Coadministration and readministration of GV10 vectors showed that E4 provided in trans could activate transgene expression from the GV11 vector genome. While transgene expression activity per genome from the GV10 vector is clearly activated, expression from a cytomegalovirus promoter expression cassette in a GV11 vector appeared to be further inactivated as a function of time. Understanding the molecular mechanisms underlying these expression effects will be important for developing persistent adenovirus vectors for chronic applications.

BackgroundTechniques that enable high levels of transgene expression in plants are attractive for the commercial production of plant-made recombinant pharmaceutical proteins or other gene transfer related strategies. The conventional way to increase the yield of desired transgenic products is to use strong promoters to control the expression of the transgene. Although many such promoters have been identified and characterized, the increase obtainable from a single promoter is ultimately limited to a certain extent.ResultsIn this study, we report a method to magnify the effect of a single promoter by using a weak promoter-based selection system in transgenic rice. tCUP1, a fragment derived from the tobacco cryptic promoter (tCUP), was tested for its activity in rice by fusion to both a β-glucuronidase (GUS) reporter and a hygromycin phosphotransferase (HPT) selectable marker. The tCUP1 promoter allowed the recovery of transformed rice plants and conferred tissue specific expression of the GUS reporter, but was much weaker than the CaMV 35S promoter in driving a selectable marker for growth of resistant calli. However, in the resistant calli and regenerated transgenic plants selected by the use of tCUP1, the constitutive expression of green fluorescent protein (GFP) was dramatically increased as a result of the additive effect of multiple T-DNA insertions. The correlation between attenuated selection by a weak promoter and elevation of copy number and foreign gene expression was confirmed by using another relatively weak promoter from nopaline synthase (Nos).ConclusionsThe use of weak promoter derived selectable markers leads to a high T-DNA copy number and then greatly increases the expression of the foreign gene. The method described here provides an effective approach to robustly enhance the expression of heterogenous transgenes through copy number manipulation in rice.