Dynamic distribution and expression in vivo of human endostatin gene delivered by adenoviral vector

Endostatin, a 20-kDa carboxyl-terminal fragment of collagen XVIII, is a potent inhibitor of endothelial cell proliferation and tumor angiogenesis. We have constructed replication-deficient recombinant adenovirus (Ad-rhE), which encoded secreted human endostatin, and our previous studies showed that Ad-rhE had a potent suppression of tumor growth in vivo. In the present study, we investigated the dynamic distribution and expression of human endostatin gene in vivo using fluorogenic real-time quantitative PCR and enzyme-linked immunosorbent assay(ELISA), respectively, with an injection of 2.0 x10(9)pfu of Ad-rhE. After injection, the Ad-rhE DNAs decreased sharply, but lasted a relative long-term at low concentration (10,000--20,000 copies/mg tissues). Whereas the expressed endostatin rose up rapidly, and reached to the top on day 5 after injection of Ad-rhE, and then decreased sharply, but endostatin in tumors sustained to over 9 days at a certain level. Both Ad-rhE DNAs and endostatin mainly enriched in tumors in vivo, and then in livers. These results suggest that endostatin gene delivered by adenoviral vector can generate a high expression in vivo, and both the metabolism pathways of Ad-rhE DNAs and endostatin in vivo are through the systems of livers.     

Quantitative bioluminescence imaging of transgene expression in vivo

Bioluminescent imaging (BLI) is a widely used in vivo method to determine the location and relative intensity of luciferase expression in mice. Luciferase expression is observed following an i.p. dose of d-luciferin, resulting in bioluminescence that is detected in anesthetized mice by a charge-coupled device camera. To establish whether BLI could be used as a quantitative measurement of non-viral-mediated luciferase expression, precise quantities of plasmid DNA encoding the luciferase gene were hydrodynamically dosed in mice. The results established a linear correlation between the DNA dose and the BLI response measured in liver which spanned five orders of magnitude. The level of luciferase expression was found to be a direct function of d-luciferin dose. The time course of luciferase expression and the influence of multidosing of substrate were measured by BLI. The recovery of luciferase from the liver of hydrodynamically dosed mice allowed calibration of the BLI measurements. The results establish BLI's limit-of-detection at 20 pg of luciferase per liver following a hydrodynamic dose of 100 pg of plasmid DNA. These results demonstrate that BLI is both sensitive and linear and should allow for the direct comparison of the efficiency of gene transfer vectors that target the liver.     

Transposition from a gutless adeno-transposon vector stabilizes transgene expression in vivo

A major limitation of adenovirus-mediated gene therapy for inherited diseases is the instability of transgene expression in vivo, which originates at least in part from the loss of the linear, extrachromosomal vector genomes. Herein we describe the production of a gene-deleted adenovirus-transposon vector that stably maintains virus-encoded transgenes in vivo through integration into host cell chromosomes. This system utilizes a donor transposon vector that undergoes Flp-mediated recombination and excision of its therapeutic payload in the presence of the Flp and Sleeping Beauty recombinases. Systemic in vivo delivery of this system resulted in efficient generation of transposon circles and stable transposase-mediated integration in mouse liver. Somatic integration was sufficient to maintain therapeutic levels of human coagulation Factor IX for more than six months in mice undergoing extensive liver proliferation. These vectors combine the versatility of adenoviral vectors with the integration capabilities of a eukaryotic DNA transposon and should prove useful in the treatment of genetic diseases.     

Left ventricular targeting of reporter gene expression in vivo by human BNP promoter in an adenoviral vector

To selectively introduce genes into the mouse myocardium, we used a recombinant adenovirus encoding a transgene composed of a cardiac-specific promoter [the proximal human brain natriuretic peptide (hBNP) promoter] coupled to a luciferase reporter gene (Ad.hBNPLuc). Activity in vitro and in vivo was compared with Ad.CMVLuc, which contained the cytomegalovirus (CMV) enhancer/promoter. We tested cell-specific and inducible regulation of Ad.hBNPLuc in vitro. Expression was higher in neonatal cardiac myocytes than in a fibroblast cell line and was induced by interleukin-1beta, phenylephrine, and isoproterenol in myocytes. For in vivo experiments, Ad.hBNPLuc, Ad.CMVLuc, or vehicle was injected into the left ventricular (LV) free wall of the mouse heart. In Ad.hBNPLuc-injected mice, luciferase activity was only detected in the heart. In contrast, Ad.CMVLuc-injected mice had detectable luciferase activity in all tissues examined. Our studies indicate that 1) the cardiac-specific hBNP promoter and direct cardiac injection limit expression of the transgene to the LV free wall; and 2) transgene expression in vitro is inducible and cardiac myocyte specific. Thus the use of the proximal hBNP promoter in recombinant adenoviral vectors may allow cardiac-specific and inducible expression of therapeutic genes in vivo and prevent some of the side effects of systemic adenovirus administration.     

Prolonged liver-specific transgene expression by a non-primate lentiviral vector

Liver-directed gene therapy has the potential for treatment of numerous inherited diseases affecting metabolic functions. The aim of this study was to evaluate gene expression in hepatocytes using feline immunodeficiency virus-based lentiviral vectors, which may be potentially safer than those based on human immunodeficiency virus. In vitro studies revealed that gene expression was stable for up to 24 days post-transduction and integration into the host cell genome was suggested by Alu PCR and Southern blot analyses. Systemic in vivo administration of viral particles by the hydrodynamics method resulted in high levels of gene expression exclusively in the liver for over 7 months whereas injection of plasmid DNA by the same method led to transient expression levels. Our studies suggest that feline immunodeficiency-based lentiviral vectors specifically transduce liver cells and may be used as a novel vehicle of gene delivery for treatment of metabolic disease.     

A lentiviral vector with novel multiple cloning sites: stable transgene expression in vitro and in vivo

Gene delivery has become an important tool for biological research and gene therapy trials. Lentiviral vector (LV) mediated gene transfer is a preferred approach for stable, sustained transgenic expression. We report here step-wise development of an Indian human immunodeficiency virus type 2 (HIV-2) isolate derived third generation lentiviral vector with a novel, versatile multiple cloning site (MCS) that can also facilitate single step sub-cloning of a PCR amplified transgene cassette by T/A cloning strategy apart from useful cohesive/blunt end cloning. Efficiency of the vector systems was functionally demonstrated by development of a transgenic enhanced green fluorescence protein (GFP) expressing cell line. Further, a GFP down regulated cell line was derived from the said cell line through LV mediated shRNA expression by cloning the GFP-shRNA cassette using the T/A cloning strategy. Subsequently long term expression of GFP transgene in nude mouse spleen/liver was also documented till 30 days. This LV platform with the enhanced user friendly cloning options will be an important advancement in gene transfer technology.     

Mucosally delivered E1-deleted adenoviral vaccine carriers induce transgene product-specific antibody responses in neonatal mice

E1-deleted adenoviral vectors of the human serotype 5 (AdHu5) and the chimpanzee serotype 68 (AdC68) expressing the rabies virus glycoprotein (rab.gp) were tested for induction of transgene product-specific Abs upon intranasal or oral immunization of newborn mice. Both vectors induced Abs to rabies virus that could be detected in serum and from mucosal secretions. Serum rabies virus neutralizing Ab titers sufficed to protect neonatally vaccinated mice against a subsequent challenge with rabies virus. The efficacy of the AdHu5rab.gp vector given orally to newborn mice born to AdHu5 virus-immune dams was not impaired by maternally transferred Abs to the vaccine carrier.     

Noninvasive monitoring of placenta-specific transgene expression by bioluminescence imaging

Background

Placental dysfunction underlies numerous complications of pregnancy. A major obstacle to understanding the roles of potential mediators of placental pathology has been the absence of suitable methods for tissue-specific gene manipulation and sensitive assays for studying gene functions in the placentas of intact animals. We describe a sensitive and noninvasive method of repetitively tracking placenta-specific gene expression throughout pregnancy using lentivirus-mediated transduction of optical reporter genes in mouse blastocysts.

Methodology/Principal Findings

Zona-free blastocysts were incubated with lentivirus expressing firefly luciferase (Fluc) and Tomato fluorescent fusion protein for trophectoderm-specific infection and transplanted into day 3 pseudopregnant recipients (GD3). Animals were examined for Fluc expression by live bioluminescence imaging (BLI) at different points during pregnancy, and the placentas were examined for tomato expression in different cell types on GD18. In another set of experiments, blastocysts with maximum photon fluxes in the range of 2.0E+4 to 6.0E+4 p/s/cm²/sr were transferred. Fluc expression was detectable in all surrogate dams by day 5 of pregnancy by live imaging, and the signal increased dramatically thereafter each day until GD12, reaching a peak at GD16 and maintaining that level through GD18. All of the placentas, but none of the fetuses, analyzed on GD18 by BLI showed different degrees of Fluc expression. However, only placentas of dams transferred with selected blastocysts showed uniform photon distribution with no significant variability of photon intensity among placentas of the same litter. Tomato expression in the placentas was limited to only trophoblast cell lineages.

Conclusions/Significance

These results, for the first time, demonstrate the feasibility of selecting lentivirally-transduced blastocysts for uniform gene expression in all placentas of the same litter and early detection and quantitative analysis of gene expression throughout pregnancy by live BLI. This method may be useful for a wide range of applications involving trophoblast-specific gene manipulations in utero.     

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.     

Fish transgene expression by direct injection into fish muscle.

The ability of a promoter sequence to drive expression of a reporter gene can be determined by direct injection of copies of the cloned sequence into fish muscle, followed by biopsy of muscle from the site of injection. We describe a set of experiments in which copies of the constructs FV1 and FV2, both comprising a carp beta-actin promoter sequence spliced to the bacterial reporter gene CAT, were injected into the muscle of tilapia fish )Oreochromis niloticus) of between 5 and 8 cm body length. The site of injection was carefully determined so that biopsy samples could be recovered from the injection site 24 hours, 48 hours, and 7 days after injection. Biopsy samples of muscle were homogenized and used for CAT assays. CAT activity was successfully detected in many of the muscle samples.     

Bystander macrophages silence transgene expression driven by the retroviral long terminal repeat

The Moloney murine leukemia virus (MLV)-based retroviral vector has been widely used for transfer of exogenous genes to various organs and tissues. Although the long terminal repeat (LTR) of MLV allows for transgene expression in a wide range of cell type, its activity is often silenced in vivo. In reporter macrophages transduced with a MLV-based retroviral vector, activity of the LTR was transiently and reversibly suppressed following stimulation by lipopolysaccharide (LPS). When unstimulated reporter macrophages were co-cultured with LPS-stimulated, untransduced macrophages, the LTR activity was similarly depressed. Activity of the LTR in retrovirus-transduced, mesangial cells was also down-regulated when co-cultured with activated macrophages. This suppressive effect was reproduced by cross-feeding with culture media conditioned by activated macrophages. LPS-stimulated macrophages abundantly expressed cytokines including IL-1beta, tumor necrosis factor-alpha (TNF-alpha) and transforming growth factor-beta1 (TGF-beta1). When externally added, TNF-alpha and/or TGF-beta1, but not IL-1beta, depressed activity of the LTR in reporter macrophages and reporter mesangial cells. These results raise a possibility that expression of transgenes driven by the MLV-LTR may be silenced in vivo when the retrovirally-transduced cells are co-localized with activated macrophages.     

Isolation and live imaging of enteric progenitors based on Sox10-Histone2BVenus transgene expression

To facilitate dynamic imaging of neural crest (NC) lineages and discrimination of individual cells in the enteric nervous system (ENS) where close juxtaposition often complicates viewing, we generated a mouse BAC transgenic line that drives a Histone2BVenus (H2BVenus) reporter from Sox10 regulatory regions. This strategy does not alter the endogenous Sox10 locus and thus facilitates analysis of normal NC development. Our Sox10-H2BVenus BAC transgene exhibits temporal, spatial, and cell-type specific expression that reflects endogenous Sox10 patterns. Individual cells exhibiting nuclear-localized fluorescence of the H2BVenus reporter are readily visualized in both fixed and living tissue and are amenable to isolation by fluorescence activated cell sorting (FACS). FACS-isolated H2BVenus+ enteric NC-derived progenitors (ENPs) exhibit multipotency, readily form neurospheres, self-renew in vitro and express a variety of stem cell genes. Dynamic live imaging as H2BVenus+ ENPs migrate down the fetal gut reveals cell fragmentation suggesting that apoptosis occurs at a low frequency during normal development of the ENS. Confocal imaging both during population of the fetal intestine and in postnatal gut muscle strips revealed differential expression between individual cells consistent with down-regulation of the transgene as progression towards non-glial fates occurs. The expression of the Sox10-H2BVenus transgene in multiple regions of the peripheral nervous system will facilitate future studies of NC lineage segregation as this tool is expressed in early NC progenitors and maintained in enteric glia.     

Efficient liver-specific deletion of a floxed human angiotensinogen transgene by adenoviral delivery of Cre recombinase in vivo.

Tissue-specific ablation of gene function is possible in vivo by the Cre-loxP recombinase system. We generated transgenic mice containing a human angiotensinogen gene flanked by loxP sites (hAGT(flox)). To examine the physiologic consequences of tissue-specific loss of angiotensinogen gene function in vivo, we constructed an adenovirus expressing Cre recombinase. Studies were performed in several independent lines of hAGT(flox) mice before and after intravenous administration of either Adcre or AdbetaGal as a control. Systemic administration of Adcre caused a significant decrease in circulating human angiotensinogen and markedly blunted the pressor response to administration of purified recombinant human renin. Southern blot analysis of genomic DNA from various organs revealed that the Cre-mediated deletion was liver-specific. Further analysis revealed the absence of full-length human angiotensinogen mRNA and protein in the liver but not the kidney of Adcre mice, consistent with the liver being the target for adenoviruses administered intravenously. These studies demonstrate that extra-hepatic sources of angiotensinogen do not contribute significantly to the circulating pool of angiotensinogen and provide proof-of-principle that the Cre-loxP system can be used effectively to examine the contribution of the systemic and tissue renin-angiotensin system to normal and pathological regulation of blood pressure.     

A new transgene reporter for in vivo magnetic resonance imaging

We report a new platform technology for visualizing transgene expression in living subjects using magnetic resonance imaging (MRI). Using a vector, we introduced an MRI reporter, a metalloprotein from the ferritin family, into specific host tissues. The reporter is made superparamagnetic as the cell sequesters endogenous iron from the organism. In this new approach, the cells construct the MRI contrast agent in situ using genetic instructions introduced by the vector. No exogenous metal-complexed contrast agent is required, thereby simplifying intracellular delivery. We used a replication-defective adenovirus vector to deliver the ferritin transgenes. Following focal inoculation of the vector into the mouse brain, we monitored the reporter activity using in vivo time-lapse MRI. We observed robust contrast in virus-transduced neurons and glia for several weeks. This technology is adaptable to monitor transgene expression in vivo in many tissue types and has numerous biomedical applications, such as visualizing preclinical therapeutic gene delivery.     

腺病毒载体疫苗的临床研究进展

腺病毒载体是目前最有应用前景的疫苗载体之一,具有高表达、可同时诱导特异性体液免疫和细胞免疫反应、安全性好、易于制备、无需添加佐剂等优点,被广泛应用于各种预防性或治疗性疫苗的研发。本文就进入临床试验的重要腺病毒载体疫苗作一综述。