Modification of a Constitutive to Glucose-Responsive Liver-Specific Promoter Resulted in Increased Efficacy of Adeno-Associated Virus Serotype 8-Insulin Gene Therapy of Diabetic Mice.

We have previously used a hepatotropic adeno-associated viral (AAV) vector with a modified human insulin gene to treat diabetic mice. The HLP (hybrid liver-specific promoter) used was constitutively active and non-responsive to glucose. In this study, we examined the effects of addition of glucose responsive elements (R3G) and incorporation of a 3' albumin enhancer (3'iALB) on insulin expression. In comparison with the original promoter, glucose responsiveness was only observed in the modified promoters in vitro with a 36 h lag time before the peak expression. A 50% decrease in the number of viral particles at 5 × 109 vector genome (vg)/mouse was required by AAV8-R3GHLP-hINSco to reduce the blood sugar level to near normoglycemia when compared to the original AAV8-HLP-hINSco that needed 1 × 1010 vg/mouse. The further inclusion of an 860 base-pairs 3'iALB enhancer component in the 3' untranslated region increased the in vitro gene expression significantly but this increase was not observed when the packaged virus was systemically injected in vivo. The addition of R3G to the HLP promoter in the AAV8-human insulin vector increased the insulin expression and secretion, thereby lowering the required dosage for basal insulin treatment. This in turn reduces the risk of liver toxicity and cost of vector production.

Magnetically triggered transgene expression in mammalian cells by localized cellular heating of magnetic nanoparticles.

To develop a remote control system of transgene expression through localized cellular heating of magnetic nanoparticles, a heat-inducible transgene expression system was introduced into mammalian cells. Cells were labeled with magnetic nanoparticles and exposed to an alternating magnetic field. The magnetically labeled cells expressed the transgene in a monolayer and multilayered cell sheets in which cells were heated around the magnetic nanoparticles without an apparent temperature increase in the culture medium. Magnetic cells were also generated by genetically engineering with a ferritin gene, and transgene expression could be induced by exposure to an alternating magnetic field. This approach may be applicable to the development of novel gene therapies in cell-based medicine.

A Drug-Tunable Gene Therapy for Broad-Spectrum Protection against Retinal Degeneration.

Retinal degenerations are a large cluster of diseases characterized by the irreversible loss of light-sensitive photoreceptors that impairs the vision of 9.1 million people in the US. An attractive treatment option is to use gene therapy to deliver broad-spectrum neuroprotective factors. However, this approach has had limited clinical translation because of the inability to control transgene expression. To address this problem, we generated an adeno-associated virus vector named RPF2 that was engineered to express domains of leukemia inhibitory factor fused to the destabilization domain of bacterial dihydrofolate reductase. Fusion proteins containing the destabilization domain are degraded in mammalian cells but can be stabilized with the binding of the drug trimethoprim. Our data show that expression levels of RPF2 are tightly regulated by the dose of trimethoprim and can be reversed by trimethoprim withdrawal. We further show that stabilized RPF2 can protect photoreceptors and prevent blindness in treated mice.

An inhibitor screen identifies histone-modifying enzymes as mediators of polymer-mediated transgene expression from plasmid DNA.

Effective transgene expression in mammalian cells relies on successful delivery, cytoplasmic trafficking, and nuclear translocation of the delivered vector, but delivery is impeded by several formidable physicochemical barriers on the surface of and within the target cell. Although methods to overcome cellular exclusion and endosomal entrapment have been studied extensively, strategies to overcome inefficient nuclear entry and subsequent intranuclear barriers to effective transient gene expression have only been sparsely explored. In particular, the role of nuclear packaging of DNA with histone proteins, which governs endogenous gene expression, has not been extensively elucidated in the case of exogenously delivered plasmids. In this work, a parallel screen of small molecule inhibitors of chromatin-modifying enzymes resulted in the identification of class I/II HDACs, sirtuins, LSD1, HATs, and the methyltransferases EZH2 and MLL as targets whose inhibition led to the enhancement of transgene expression following polymer-mediated delivery of plasmid DNA. Quantitative PCR studies revealed that HDAC inhibition enhances the amount of plasmid DNA delivered to the nucleus in UMUC3 human bladder cancer cells. Native chromatin immunoprecipitation (N-ChIP)-qPCR experiments in CHO-K1 cells indicated that plasmids indeed interact with intracellular core Histone H3, and inhibitors of HDAC and LSD1 proteins are able to modulate this interaction. Pair-wise treatments of effective inhibitors led to synergistic enhancement of transgene expression to varying extents in both cell types. Our results demonstrate that the ability to modulate enzymes that play a role in epigenetic processes can enhance the efficacy of non-viral gene delivery, resulting in significant implications for gene therapy and industrial biotechnology.

Autochthonous tumors driven by Rb1 loss have an ongoing requirement for the RBP2 histone demethylase.

Inactivation of the retinoblastoma gene (RB1) product, pRB, is common in many human cancers. Targeting downstream effectors of pRB that are central to tumorigenesis is a promising strategy to block the growth of tumors harboring loss-of-function RB1 mutations. One such effector is retinoblastoma-binding protein 2 (RBP2, also called JARID1A or KDM5A), which encodes an H3K4 demethylase. Binding of pRB to RBP2 has been linked to the ability of pRB to promote senescence and differentiation. Importantly, genetic ablation of RBP2 is sufficient to phenocopy pRB's ability to induce these cellular changes in cell culture experiments. Moreover, germline Rbp2 deletion significantly impedes tumorigenesis in Rb1+/- mice. The value of RBP2 as a therapeutic target in cancer, however, hinges on whether loss of RBP2 could block the growth of established tumors as opposed to simply delaying their onset. Here we show that conditional, systemic ablation of RBP2 in tumor-bearing Rb1+/- mice is sufficient to slow tumor growth and significantly extend survival without causing obvious toxicity to the host. These findings show that established Rb1-null tumors require RBP2 for growth and further credential RBP2 as a therapeutic target in human cancers driven by RB1 inactivation.

Protective efficacy of a plasmid DNA vaccine against transgene-specific tumors by Th1 cellular immune responses after intradermal injection.

With DNA vaccines, it is important to monitor the movement of transfectants and to overcome immune deviations. We used a pCMV-LacZ plasmid (expressing ß-galactosidase) and a pcDNA-hNIS plasmid (expressing the human sodium/iodide symporter [hNIS] gene) as non-secreted visual-imaging markers. Transfectants carrying the hNIS or LacZ gene migrated to peripheral lymphoid tissues. hNIS-expressing cells were observed specifically in the LNs and spleen. Anti-ß-galactosidase was detected in LacZ DNA immunized mice after boosting twice, suggestive of Th2 humoral immune responses. Antibody isotyping defined the humoral immune response. A dominant IgG2a type occurred in hNIS-immunized mice in ELISAs. IgG2a/IgG1 ratios increased after hNIS DNA vaccination. High levels of INF-γ-secreting cells were identified in ELISpot and increased IFN-γ levels were found in cytokine ELISAs. Tumor growth decreased in hNIS DNA-immunized mice. In conclusion, humoral immune responses switched to the Th1 cellular immune response, even though we administered plasmid DNA by intra dermal injection.

A new adenovector system for implementing thymulin gene therapy for inflammatory disorders.

Thymulin is a thymic peptide possessing anti-inflammatory effects. In order to manipulate thymulin expression in gene therapy studies, we built a bidirectional regulatable two-vector Tet-Off system and the corresponding control system. The experimental two-vector system, ETV, consists of a recombinant adenovector (RAd) harboring an expression cassette centered on a Tet-Off bidirectional promoter flanked by a synthetic gene for thymulin and the gene for humanized Green Fluorescent Protein (hGFP). The second adenovector of this system, RAd-tTA, constitutively expresses the regulatory protein tTA. When cells are co-transduced by the two adenovector components, tTA activates the bidirectional promoter and both transgenes are expressed. In the presence of the antibiotic doxycycline (DOX) transgene expression is deactivated. The control two-vector system, termed CTV, is similar to ETV but only expresses hGFP. In CHO-K1, BHK, and C2C12 cells, ETV and CTV induced a dose-dependent hGFP expression. In CHO-K1 cells, transgene expression was almost completely inhibited by DOX (1mg/ml). After intracerebroventricular injection of ETV in rats, thymulin levels increased significantly in the cerebrospinal fluid and there was high hGFP expression in the ependymal cell layer. When injected intramuscularly the ETV system induced a progressive increase in serum thymulin levels, which were inhibited when DOX was added to the drinking water. We conclude that our regulatable two-adenovector system is an effective molecular tool for implementing short and long-term anti-inflammatory thymulin gene therapy in animal models of acute or chronic inflammation.

High-fidelity Glucagon-CreER mouse line generated by CRISPR-Cas9 assisted gene targeting.

OBJECTIVE: α-cells are the second most prominent cell type in pancreatic islets and are responsible for producing glucagon to increase plasma glucose levels in times of fasting. α-cell dysfunction and inappropriate glucagon secretion occur in both type 1 and type 2 diabetes. Thus, there is growing interest in studying both normal function and pathophysiology of α-cells. However, tools to target gene ablation or activation specifically of α-cells have been limited, compared to those available for ß-cells. Previous Glucagon-Cre and Glucagon-CreER transgenic mouse lines have suffered from transgene silencing, and the only available Glucagon-CreER "knock-in" mouse line results in glucagon haploinsufficiency, which can confound the interpretation of gene deletion analyses. Therefore, we sought to develop a Glucagon-CreERT2 mouse line that would maintain normal glucagon expression and would be less susceptible to transgene silencing. METHODS: We utilized CRISPR-Cas9 technology to insert an IRES-CreERT2 sequence into the 3' UTR of the Glucagon (Gcg) locus in mouse embryonic stem cells (ESCs). Targeted ESC clones were then injected into mouse blastocysts to obtain Gcg-CreERT2 mice. Recombination efficiency in GCG+ pancreatic α-cells and glucagon-like peptide 1 positive (GLP1+) enteroendocrine L-cells was measured in Gcg-CreERT2 ;Rosa26-LSL-YFP mice injected with tamoxifen during fetal development and adulthood. RESULTS: Tamoxifen injection of Gcg-CreERT2 ;Rosa26-LSL-YFP mice induced high recombination efficiency of the Rosa26-LSL-YFP locus in perinatal and adult α-cells (88% and 95%, respectively), as well as in first-wave fetal α-cells (36%) and adult enteroendocrine L-cells (33%). Mice homozygous for the Gcg-CreERT2 allele were phenotypically normal. CONCLUSIONS: We successfully derived a Gcg-CreERT2 mouse line that expresses CreERT2 in pancreatic α-cells and enteroendocrine L-cells without disrupting preproglucagon gene expression. These mice will be a useful tool for performing temporally controlled genetic manipulation specifically in these cell types.

Providing sustained transgene induction through affinity-based drug delivery.

Small molecule drug activators of gene expression have been used in applications ranging from gene therapy, to tissue engineering and regenerative medicine. One concern is that for sustained gene expression, a long-term, controlled delivery system is needed. Insoluble polymers containing a high proportion of cyclodextrin (CD) affinity groups have been shown to prolong drug delivery far beyond that capable of polymers relying on diffusion alone. In this study we evaluate the capacity of such polymers to deliver the transgene inducer doxycycline. Our results show that initial drug loading is proportional to affinity, with ∼8% loading in high-affinity γ-CD polymers; ∼7% loading in moderate-affinity ß-CD polymers; and only ∼4.5% loading in the non-affinity control polymer made from linear dextran. When release aliquots from these polymers were incubated with cells genetically modified for inducible transgene expression we observed activation of transgene expression for up to three weeks from samples released by affinity-based polymers. We showed that drug stability is maintained over the course of the study using a bacterial zone of inhibition assay where again affinity-based polymers show sustained availability of drug, weeks longer than non-affinity controls. Lastly we provide theoretical calculations of strength of binding interactions between cyclodextrins and many additional transgene inducers demonstrating the broad utility of this delivery platform.

Lentiviral vector system for coordinated constitutive and drug controlled tetracycline-regulated gene co-expression.

Constitutive co-expression of cooperating transgenes using retroviral integrating vectors is frequently used for genetic modification of different cell types to establish therapeutic or cancer models. However, such approaches are unable to dissect the influence of dose, order and reversibility of transgene expression on the fate of newly developed therapeutic/malignant phenotypes. We present a modular lentiviral vector system, which provides expression of constitutive and inducible components. To demonstrate its functionality, we constitutively expressed the well-described transcription factor Meis1 followed by inducible co-expression of collaborating partner Hoxa9 under the control of tetracycline responsive promoters in murine fibroblasts and primary hematopoietic progenitor cells (HPCs). Fluorescent markers to track transgene co-expression revealed tightly controlled, efficiently inducible and reversible but cell type dependent gene transfer over time. We demonstrated dose-dependent blockade of myeloid differentiation when both Meis1/Hoxa9 were concomitantly overexpressed in primary HPCs in vitro, but the absence of the transformed phenotype in non-induced samples or when Hoxa9 expression was down-regulated. This system combines the advantages of lentiviral gene transfer and the opportunity for drug-controlled co-expression of multiple transgenes to dissect, among others, gene networks governing complex cell behavior, such as proto-oncogene dose-dependent leukemogenic pathways or collaborating mechanisms of genes enhancing competitive fitness of hematopoietic cells.

RN-1, a potent and selective lysine-specific demethylase 1 inhibitor, increases γ-globin expression, F reticulocytes, and F cells in a sickle cell disease mouse model.

Increased levels of fetal hemoglobin are associated with decreased symptoms and increased lifespan in patients with sickle cell disease (SCD). Hydroxyurea, the only drug currently approved for SCD, is not effective in a large fraction of patients, and therefore, new agents are urgently needed. Recently it was found that lysine demethylase 1, an enzyme that removes monomethyl and dimethyl residues from the lysine 4 residue of histone H3, is a repressor of γ-globin gene expression. In this article, we have compared the ability of tranylcypromine (TCP) and a more potent TCP derivative, RN-1, to increase γ-globin expression in cultured baboon erythroid progenitor cells and in the SCD mouse model. The results indicate that the ability of RN-1 to induce F cells and γ-globin mRNA in SCD mice is similar to that of decitabine, the most powerful fetal hemoglobin-inducing drug known, and greater than that of either TCP or hydroxyurea. We conclude that RN-1 and other lysine demethylase 1 inhibitors may be promising new γ-globin-inducing agents for the treatment of SCD that warrant further studies in other preclinical models, such as nonhuman primates.

DNA methylation and histone modifications are the molecular lock in lentivirally transduced hematopoietic progenitor cells.

Stable introduction of a functional gene in hematopoietic progenitor cells (HPCs) has appeared to be an alternative approach to correct genetically linked blood diseases. However, it is still unclear whether lentiviral vector (LV) is subjected to gene silencing in HPCs. Here, we show that LV carrying green fluorescent protein (GFP) reporter gene driven by cytomegalovirus (CMV) promoter was subjected to transgene silencing after transduction into HPCs. This phenomenon was not due to the deletion of proviral copy number. Study using DNA demethylating agent and histone deacetylase (HDAC) inhibitor showed that the drugs could either prevent or reverse the silencing effect. Using sodium bisulfite sequencing and chromatin immunoprecipitation (ChIP) assay, we demonstrated that DNA methylation occurred soon after LV transduction. At the highest level of gene expression, CMV promoter was acetylated and was in a euchromatin state, while GFP reporter gene was acetylated but was strangely in a heterochromatin state. When the expression declined, CMV promoter underwent transition from acetylated and euchromatic state to a heterochromatic state, while the GFP reporter gene was in deacetylated and heterochromatic state. With these, we verify that DNA methylation and dynamic histone modifications lead to transgene silencing in HPCs transduced with LV.

A novel adenoviral vector carrying an all-in-one Tet-On system with an autoregulatory loop for tight, inducible transgene expression.

BACKGROUND: One of the most commonly used vectors for gene therapy is the adenoviral vector; its ability to tightly regulate transgene expression is critical for optimizing therapeutic outcomes. The tetracycline-regulated system (especially the Tet-On system) for gene expression is one of the most valuable tools for controlling gene expression. The major problem of an adenoviral vector carrying a Tet-On system is suboptimal regulation of transgene expression. RESULTS: We constructed a single adenoviral vector carrying in its E1 region a novel "all-in-one" Tet-On system with an autoregulatory loop. This system had improved Dox-inducible gene expression in terms of low basal expression, high induced expression and high responsiveness to Dox. To our knowledge, this is the first reported adenovirus-based, all-in-one Tet-On system with an autoregulatory loop inserted into a single region of adenoviral genome. This system was further tested by inducible expression of soluble tumor necrosis factor-related apoptosis-inducing ligand (sTRAIL). The adenovirus that expressed soluble TRAIL under the control of this novel Tet-On system showed tumor-derived cells inhibitory activity in SW480 cells only under induced conditions. CONCLUSIONS: Our novel, single adenoviral vector carrying in its E1 region an all-in-one Tet-On system with an autoregulatory loop displayed tight regulation of transgene expression in vitro. This system has great potential for a variety of applications, including gene therapy and the study of gene function.

Pristimerin enhances recombinant adeno-associated virus vector-mediated transgene expression in human cell lines in vitro and murine hepatocytes in vivo.

OBJECTIVE: In the present study, we systemically evaluated the ability of two bioactive compounds from traditional Chinese medicine, celastrol and pristimerin, to enhance recombinant adeno-associated virus (rAAV) serotype vector-mediated transgene expression both in human cell lines in vitro, and in murine hepatocytes in vivo. METHODS: Human cell lines were infected with rAAV vectors with either mock treatment or treatment with celastrol or pristimerin. The transgene expression, percentage of nuclear translocated viral genomes and the ubiquitination of intracellular proteins were investigated post-treatment. In addition, nonobese diabetic/severe combined immunodeficient gamma (NSG) mice were tail vain-injected with rAAV vectors and co-administered with either dimethyl sulfoxide, celastrol, pristimerin or a positive control, bortezomib. The transgene expression in liver was detected and compared over time. RESULTS: We observed that treatment with pristimerin, at as low as 1 µmol/L concentration, significantly enhanced rAAV2 vector-mediated transgene expression in vitro, and intraperitoneal co-administration with pristimerin at 4 mg/(kg·d) for 3 d dramatically facilitated viral transduction in murine hepatocytes in vivo. The transduction efficiency of the tyrosine-mutant rAAV2 vectors as well as that of rAAV8 vectors carrying oversized transgene cassette was also augmented significantly by pristimerin. The underlying molecular mechanisms by which pristimerin mediated the observed increase in the transduction efficiency of rAAV vectors include both inhibition of proteasomal degradation of the intracellular proteins and enhanced nuclear translocation of the vector genomes. CONCLUSION: These studies suggest the potential beneficial use of pristimerin and pristimerin-containing herb extract in future liver-targeted gene therapy with rAAV vectors.

Effectiveness and preclinical safety profile of doxycycline to be used "off-label" to induce therapeutic transgene expression in a phase I clinical trial for glioma.

Glioblastoma multiforme (GBM) is the most common malignant primary brain cancer in adults; it carries a dismal prognosis despite improvements in standard of care. We developed a combined gene therapy strategy using (1) herpes simplex type 1-thymidine kinase in conjunction with the cytotoxic prodrug ganciclovir to kill actively proliferating tumor cells and (2) doxycycline (DOX)-inducible Fms-like tyrosine kinase 3 ligand (Flt3L), an immune stimulatory molecule that induces anti-GBM immunity. As a prelude to a phase I clinical trial, we examined the efficacy and safety of this approach (Muhammad et al., 2010, 2012). In the present article, we investigated the efficacy and safety of the "off-label" use of the antibiotic DOX to turn on the high-capacity adenoviral vector (HC-Ad) encoding therapeutic Flt3L expression. DOX-inducible Flt3L expression in male Lewis rats was assessed using DOX doses of 30.8 mg/kg/day (low-DOX) or 46.2 mg/kg/day (high-DOX), which are allometrically equivalent (Voisin et al., 1990) to the human doses that are recommended for the treatment of infections: 200 or 300 mg/day. Naïve rats were intracranially injected with 1×10(9) viral particles of HC-Ad-TetOn-Flt3L, and expression of the therapeutic transgene, that is, Flt3L, was assessed using immunohistochemistry in brain sections after 2 weeks of DOX administration via oral gavage. The results show robust expression of Flt3L in the rat brain parenchyma in areas near the injection site in both the low-DOX and the high-DOX groups, suggesting that Flt3L will be expressed in human glioma patients at a DOX dose of 200 or 300 mg/day. These doses have been approved by the U.S. Food and Drug Administration to treat infections in humans and would thus be considered safe for an off-label use to treat GBM patients undergoing HC-Ad-mediated gene therapy in a phase I clinical trial.

Airway-specific inducible transgene expression using aerosolized doxycycline.

Tissue-specific transgene expression using tetracycline (tet)-regulated promoter/operator elements has been used to revolutionize our understanding of cellular and molecular processes. However, because most tet-regulated mouse strains use promoters of genes expressed in multiple tissues, to achieve exclusive expression in an organ of interest is often impossible. Indeed, in the extreme case, unwanted transgene expression in other organ systems causes lethality and precludes the study of the transgene in the actual organ of interest. Here, we describe a novel approach to activating tet-inducible transgene expression solely in the airway by administering aerosolized doxycycline. By optimizing the dose and duration of aerosolized doxycycline exposure in mice possessing a ubiquitously expressed Rosa26 promoter-driven reverse tet-controlled transcriptional activator (rtTA) element, we induce transgene expression exclusively in the airways. We detect no changes in the cellular composition or proliferative behavior of airway cells. We used this newly developed method to achieve airway basal stem cell-specific transgene expression using a cytokeratin 5 (also known as keratin 5)-driven rtTA driver line to induce Notch pathway activation. We observed a more robust mucous metaplasia phenotype than in mice receiving doxycycline systemically. In addition, unwanted phenotypes outside of the lung that were evident when doxycycline was received systemically were now absent. Thus, our approach allows for rapid and efficient airway-specific transgene expression. After the careful strain by strain titration of the dose and timing of doxycycline inhalation, a suite of preexisting transgenic mice can now be used to study airway biology specifically in cases where transient transgene expression is sufficient to induce a phenotype.

Transient JMJD2B-mediated reduction of H3K9me3 levels improves reprogramming of embryonic stem cells into cloned embryos.

Correct reprogramming of epigenetic marks in the donor nuclei is crucial for successful cloning by nuclear transfer. Specific epigenetic modifications, such as repressive histone lysine methylation marks, are known to be very stable and difficult to reprogram. The discovery of histone lysine demethylases has opened up opportunities to study the effects of removing repressive histone lysine methylation marks in donor cells prior to nuclear transfer. In this study, we generated mouse embryonic stem (ES) cells for the inducible expression of JMJD2B (also known as KDM4B), a demethylase that primarily removes the histone-3 lysine-9 trimethylation (H3K9me3) mark. Induction of jmjd2b in the ES cells decreased total levels of H3K9me3 by 63%. When these cells were used for nuclear transfer, H3K9me3 levels were normalized within minutes following fusion with an enucleated oocyte. This transient reduction of H3K9me3 levels improved in vitro development into cloned embryos by 30%.

Enhancement of gene transfer efficiency in the Bcap-37 cell line by dimethyl sulphoxide and menthol.

Simple and efficient gene transfer into the nucleus would facilitate non-viral gene delivery. One promising method of non-viral gene delivery is to apply penetration enhancers. Chemicals, such as dimethyl sulfoxide (DMSO) and menthol, may have promise as non-toxic vehicles in improving gene transfer efficiency. In this study, the cytotoxic effects of DMSO and menthol were evaluated using MTT assays. Gene delivery efficiency in a human breast cancer cell line (Bcap-37) was investigated by quantitative PCR, fluorescence microscopy and flow cytometry. Non-toxic concentrations of DMSO (2%) and menthol (12.5 µM) enhanced the efficiency of liposome-mediated gene delivery in Bcap-37 cells. Quantitative PCR results showed that growth hormone (GH) mRNA expression in the post-menthol and pre-DMSO treatment groups was 10-fold higher compared to that in the liposome group, while in the pre-menthol and post-DMSO treatment groups, a 30-fold increase in GH mRNA expression was observed. Both DMSO and menthol treatments increased green fluorescent protein (GFP) expression efficiency as shown by fluorescence microscopy experiments. Compared to the liposome group, the number of positive cells in the pre-menthol and post-DMSO treatment groups was significantly increased by 15%. Furthermore, cell cycle analysis demonstrated that there were significant differences among the DMSO-treated group, the menthol-treated group and the normal group, which implied different effects of DMSO and menthol treatments. In conclusion, both non-toxic and harmless DMSO (2%) and menthol (12.5 µM) treatments improve gene transfer efficiency, while post-DMSO treatment may be the most effective protocol in increasing transgene expression efficiency.

PAMAM dendrimer-baculovirus nanocomplex for microencapsulated adipose stem cell-gene therapy: in vitro and in vivo functional assessment.

The present study aims to develop a new stem cell based gene delivery system consisting of human adipose tissue derived stem cells (hASCs) genetically modified with self-assembled nanocomplex of recombinant baculovirus and PAMAM dendrimer (Bac-PAMAM) to overexpress the vascular endothelial growth factor (VEGF). Cells were enveloped into branched PEG surface functionalized polymeric microcapsules for efficient transplantation. In vitro analysis confirmed efficient transduction of hASCs expressing 7.65 ± 0.86 ng functionally active VEGF per 10(6) microencapsulated hASCs (ASC-VEGF). To determine the potential of the developed system, chronically infarcted rat hearts were treated with either empty microcapsules (MC), microencapsulated hASCs expressing MGFP reporter protein (MC+ASC-MGFP), or MC+ASC-VEGF, and analyzed for 10 weeks. Post-transplantation data confirmed higher myocardial VEGF expressions with significantly enhanced neovasculature in the MC+ASC-VEGF group. In addition, the cardiac performance, as measured by percentage ejection fraction, also improved significantly in the MC+ASC-VEGF group (48.6 ± 6.1%) compared to that in MC+ASC-MGFP (38.8 ± 5.3%) and MC groups (31.5 ± 3.3%). Collectively, these data demonstrate the feasibility of this system for improved stem cell therapy applications.

Roles for ß-catenin and doxycycline in the regulation of respiratory epithelial cell frequency and function.

The expression of ß-catenin-dependent genes can be increased through the Cre recombinase (Cre)-mediated elimination of the exon 3-encoded sequence. This mutant ß-catenin is termed DE3, and promotes the expression of ß-catenin-dependent genes. Our previous study used the DE3 model to demonstrate that persistent ß-catenin activity inhibited bronchiolar Clara-to-ciliated cell differentiation. The present study was designed to evaluate the roles of ß-catenin in regulating the tracheal progenitor cell hierarchy. However, initial experiments demonstrated that the tetracycline-responsive element-Cre transgene (TRE-Cre) was active in the absence of a reverse tetracycline transactivator driver or inducer, doxycycline (Dox). This spurious TRE-Cre transgene activity was not detected using the ROSA26-floxed STOP-LacZ reporter. To determine if the phenotype was a consequence of genotype or treatment with Dox, tracheal and lung specimens were evaluated using quantitative histomorphometric techniques. Analyses of uninduced mice demonstrated a significant effect of genotype on tracheal epithelial cell mass, involving basal, Clara-like cell types. The bronchial and bronchiolar Clara cell mass was also decreased. Paradoxically, an effect on ciliated cell mass was not detected. Activation of the ß-catenin reporter transgene TOPGal demonstrated that ß-catenin-dependent gene expression led to the genotype-dependent tracheal and bronchiolar phenotype. Comparative analyses of wild-type or keratin 14-rtTA(+/0)/TRE-cre(+/0)/DE3(+/+) mice receiving standard or Dox chow demonstrated an effect of treatment with Dox on basal, Clara-like, and Clara cell masses. We discuss these results in terms of cautionary notes and with regard to alterations of progenitor cell hierarchies in response to low-level injury.