Preclinical evaluation of ex vivo expanded/activated gammadelta T cells for immunotherapy of glioblastoma multiforme.

J Neurooncol. 2010 Jun 10;

Authors: Bryant NL, Gillespie GY, Lopez RD, Markert JM, Cloud GA, Langford CP, Arnouk H, Su Y, Haines HL, Suarez-Cuervo C, Lamb LS

We have previously shown that expanded/activated gammadelta T cells from healthy donors are cytotoxic to GBM cell lines and primary GBM explants. In this report, we examined the therapeutic effect of intracranial infusion of expanded/activated gammadelta T cells on human minimal and established U251 tumor xenografts in athymic nude mice. Immunohistochemistry was used to determine the presence of NKG2D ligands on cell lines and tumors, and blocking studies were used to determine the effect of these ligands on gammadelta T cell recognition. Expanded/activated gammadelta T cells were prepared by 18-day culture in RPMI, human serum (HS), anti-CD2, IL-12, IFN-gamma, and OKT-3. Anti-GBM activity of the cell product was assessed using in vitro cytotoxicity assays against the GBM cell line U251MG in suspension and in adherent culture. Ex vivo expanded/activated gammadelta T cells were of the effector/memory phenotype, expressed Th1 cytokines, and effectively killed U251 cells in vitro. Xenografts were prepared using a U251 cell line following transfection with a firefly luciferase gene to monitor tumor progression. Mice treated with gammadelta T cells showed slower progression of both new and established GBM xenografts versus mice that received vehicle only as determined by photon emission over time. Median survival was improved in all gammadelta T cell treated groups between 32 and 50 days by Kaplan-Meier analysis. U251 cells expressed ULBP-2 and ULBP-3, although blocking of these reduced in vitro cytotoxicity of gammadelta T cells to U251MG by only 33 and 25%, respectively. These studies show that expanded/activated gammadelta T cells can mediate killing of new or established GBM xenografts, reduce tumor progression, and constitute a potentially effective novel immunotherapeutic strategy against GBM.

PMID: 20532954 [PubMed - as supplied by publisher]

Bioengineering a Unique Deimmunized Bispecific Targeted Toxin That Simultaneously Recognizes Human CD22 and CD19 Receptors in a Mouse Model of B-Cell Metastases.

Mol Cancer Ther. 2010 Jun 8;

Authors: Vallera DA, Oh S, Chen H, Shu Y, Frankel AE

A drug of high potency and reduced immunogenicity is needed to develop a targeted biological drug that when injected systemically can penetrate to malignant B cells. Therefore, a novel deimmunized bispecific ligand-directed toxin targeted by dual high-affinity single-chain Fvs (scFv) spliced to PE38 with a KDEL COOH-terminus was genetically engineered. The aims were to reduce toxin immunogenicity using mutagenesis, measure the ability of mutated drug to elicit antitoxin antibody responses, and show that mutated drug was effective against systemic B-cell lymphoma in vivo. Both human anti-CD22 scFv and anti-CD19 scFv were cloned onto the same single-chain molecule with truncated pseudomonas exotoxin (PE38) to create the drug. Site-specific mutagenesis was used to mutate amino acids in seven key epitopic toxin regions that dictate B-cell generation of neutralizing antitoxin antibodies. Bioassays were used to determine whether mutation reduced potency, and ELISAs were done to determine whether antitoxin antibodies were reduced. Finally, a powerful genetically altered luciferase xenograft model was used that could be imaged in real time to determine the effect on systemic malignant human B-cell lymphoma, Raji-luc. Patient B-lineage acute lymphoblastic leukemia, B-cell chronic lymphocytic leukemia, and B lymphoma were high in CD22 and CD19 expression. 2219KDEL7mut was significantly effective against systemic Raji-luc in mice and prevented metastatic spread. Mutagenesis reduced neutralizing antitoxin antibodies by approximately 80% with no apparent loss in in vitro or in vivo activity. Because 2219KDEL7mut immunogenicity was significantly reduced and the drug was highly effective in vivo, we can now give multiple drug treatments with targeted toxins in future clinical trials. Mol Cancer Ther; 9(6); 1872-83. (c)2010 AACR.

PMID: 20530709 [PubMed - as supplied by publisher]

A bioluminescent mouse model of pancreatic {beta}-cell carcinogenesis.

Carcinogenesis. 2010 Jun 8;

Authors: Zumsteg A, Strittmatter K, Klewe-Nebenius D, Antoniadis H, Christofori G

The Rip1Tag2 transgenic mouse model of pancreatic beta-cell carcinogenesis has been instrumental in identifying several hallmarks of cancer, including tumor cell evasion from apoptosis, tumor angiogenesis, and tumor invasion. Moreover, Rip1Tag2 mice have been helpful in the development and testing of innovative cancer therapies and tumor imaging protocols. However, based on tumor localization in the mouse, primary tumor growth and metastatic dissemination cannot be easily monitored in a longitudinal axis by non-invasive and low-technology approaches. Here, we report the generation of a new transgenic mouse line as a versatile tool to study beta-cell carcinogenesis. Transgenic expression of a bicistronic mRNA encoding simian virus large T antigen and firefly luciferase in pancreatic beta-cells recapitulates insulinoma development in a reproducible multistage process. In the mouse line called RTL1 (RipTag-IRES-Luciferase line 1), the beta-cell-specific expression of luciferase allows the non-invasive monitoring of primary tumor growth over time in vivo and the detection and quantification of disseminated tumor cells and micro-metastases in distant organs ex vivo. When crossed to mouse lines in which the expression of cancer "modifier" genes has been manipulated, tumor initiation and tumor progression are similarly affected as previously reported for Rip1Tag2 mice, indicating a robust tumor progression pathway shared between the two different transgenic mouse lines. Together, the data indicate that the RTL1 mouse line will be of great value to study anti-tumoral therapeutic approaches as well as to define the functional roles of cancer- and metastasis-related genes when crossed to appropriate transgenic or gene-targeted mouse lines.

PMID: 20530553 [PubMed - as supplied by publisher]

Elevated phosphate activates N-ras and promotes cell transformation and skin tumorigenesis.

Cancer Prev Res (Phila Pa). 2010 Mar;3(3):359-70

Authors: Camalier CE, Young MR, Bobe G, Perella CM, Colburn NH, Beck GR

Recent results suggest a paradigm shift from viewing inorganic phosphate as a passive requirement for basic cell functions to an active regulator of cell behavior. We have previously shown that elevated concentrations of phosphate increased cell proliferation and expression of protumorigenic genes such as Fra-1 and osteopontin in a preosteoblast cell line. Therefore, we hypothesized that elevated phosphate concentrations would promote cell transformation in vitro and tumorigenesis in vivo. Supplementation of medium with phosphate increased anchorage-independent transformation and proliferation of BALB/c mouse JB6 epidermal cells, activation of N-ras, ERK1/2, and activator protein-1, and increased gene expression of Fra-1, COX-2, and osteopontin in a dose-dependent manner. These in vitro results led to the hypothesis that varying the levels of dietary inorganic phosphate would alter tumorigenesis in the mouse model of skin carcinogenesis. Female FVB/N mice were treated with 7,12-dimethylbenz(a)anthracene/12-O-tetradecanoylphorbol-13-acetate and fed high- or low-phosphate diets (1.2% versus 0.2% of the diet) for 19 weeks. The high-phosphate diet increased skin papilloma number by approximately 50% without changing feed intake and body weights. High dietary phosphate increased serum concentrations of phosphate, parathyroid hormone, and osteopontin and decreased serum concentrations of calcium. Thus, we conclude that elevated phosphate promotes cell transformation and skin tumorigenesis partly by increasing the availability of phosphate for activation of N-ras and its downstream targets, which defines reducing dietary phosphate as a novel target for chemoprevention.

PMID: 20145188 [PubMed - indexed for MEDLINE]

Survivin knockdown by short hairpin RNA abrogates the growth of human hepatocellular carcinoma xenografts in nude mice.

Cancer Gene Ther. 2010 Apr;17(4):275-88

Authors: Zhang R, Ma L, Zheng M, Ren J, Wang T, Meng Y, Zhao J, Jia L, Yao L, Han H, Li K, Yang A

Abnormal high activation of survivin is involved in carcinogenesis of various types of cancer. Survivin has been shown to promote cell proliferation in human hepatocellular carcinoma (HCC). Survivin-targeting approaches have become a promising strategy for treating HCC. Here, we used a reporter system to screen effective survivin siRNA sequences. The effect of vector-based survivin short hairpin RNA (shRNA) on the malignant phenotype of HCC cells in vitro and in vivo was determined, and an adenovirus-mediated shRNA expression vector was developed to decrease survivin expression of the established HCC tumor in nude mice. In vitro study showed that stable survivin knockdown inhibited cancer cell proliferation, enhanced apoptotic susceptibility, arrested cell cycle in the G1 phase and resulted in apparent mitotic catastrophe. Moreover, cells stably expressing survivin shRNA showed decreased tumorigenicity in nude mice. An additional in vivo study showed that intratumoral injection of adenovirus-delivered survivin shRNA suppressed tumor growth by spontaneous apoptosis of cancer cells and significantly prolonged animal survival. In conclusion, we proved the therapeutic potential of survivin shRNA for the treatment of HCC. And our results indicated that adenovirus-delivered shRNA may serve as a novel therapeutic for HCC.

PMID: 19876077 [PubMed - indexed for MEDLINE]

Estrogen-Like Effects of Cadmium <i>in vivo</i> do not Appear to be Mediated via the Classical Estrogen Receptor Transcriptional Pathway.

Environ Health Perspect. 2010 Jun 4;

Authors: Ali I, Penttinen-Damdimopoulou PE, Mäkelä SI, Berglund M, Stenius U, Akesson A, Håkansson H, Halldin K

Background: Cadmium (Cd), a ubiquitous food contaminant of health concern, is proposed to be an endocrine disruptor by inducing estrogenic responses in vivo. Several in vitro studies suggest that these effects are mediated via estrogen receptors (ERs). The present study was performed to clarify whether Cd induced effects in vivo are mediated via classical ER-signaling through ERE-regulated genes or if other signaling pathways are involved. Methods: We investigated the estrogenic effects after CdCl2 exposure in vivo by applying the Organisation for Economic Co-operation and Development (OECD) uterotrophic bioassay in rodents to transgenic ERE-luciferase reporter mice. Immature female mice were injected subcutaneously with CdCl2 at 5, 50 or 500 microg/kg b.w. or with 17alpha-ethinylestradiol (EE2) on three consecutive days. Uterus weight and histology, vaginal opening, body and organ weights, Cd tissue retention, activation of MAPK pathways and ERE dependent luciferase expression were investigated. Results: CdCl2 increased the height of the uterine luminal epithelium in a dose dependent manner without increasing the uterine wet weight, altering the timing of vaginal opening, or affecting the luciferase activity in either reproductive or non-reproductive organs. However, changes in the phosphorylation of Mdm2 and Erk1/2 were observed in the liver after CdCl2 exposure. As expected, EE2 advanced vaginal opening and increased the height of the uterine epithelium, uterine wet weight, and luciferase activity in various tissues.. Conclusion: Our data suggest that Cd exposure induces a limited spectrum of estrogenic responses in vivo and that, in certain targets, effects of Cd might not be mediated via classical ER-signaling through ERE-regulated genes.

PMID: 20525538 [PubMed - as supplied by publisher]

Micro-RNA-31 controls hair cycle-associated changes in gene expression programs of the skin and hair follicle.

FASEB J. 2010 Jun 3;

Authors: Mardaryev AN, Ahmed MI, Vlahov NV, Fessing MY, Gill JH, Sharov AA, Botchkareva NV

The hair follicle is a cyclic biological system that progresses through stages of growth, regression, and quiescence, which involves dynamic changes in a program of gene regulation. Micro-RNAs (miRNAs) are critically important for the control of gene expression and silencing. Here, we show that global miRNA expression in the skin markedly changes during distinct stages of the hair cycle in mice. Furthermore, we show that expression of miR-31 markedly increases during anagen and decreases during catagen and telogen. Administration of antisense miR-31 inhibitor into mouse skin during the early- and midanagen phases of the hair cycle results in accelerated anagen development, and altered differentiation of hair matrix keratinocytes and hair shaft formation. Microarray, qRT-PCR and Western blot analyses revealed that miR-31 negatively regulates expression of Fgf10, the components of Wnt and BMP signaling pathways Sclerostin and BAMBI, and Dlx3 transcription factor, as well as selected keratin genes, both in vitro and in vivo. Using luciferase reporter assay, we show that Krt16, Krt17, Dlx3, and Fgf10 serve as direct miR-31 targets. Thus, by targeting a number of growth regulatory molecules and cytoskeletal proteins, miR-31 is involved in establishing an optimal balance of gene expression in the hair follicle required for its proper growth and hair fiber formation.-Mardaryev, A. N., Ahmed, M. I., Vlahov, N. V., Fessing, M. Y., Gill, J. H., Sharov, A. A., and Botchkareva, N. V. Micro-RNA-31 controls hair cycle-associated changes in gene expression programs of the skin and hair follicle.

PMID: 20522784 [PubMed - as supplied by publisher]

Optimisation of bioluminescent reporters for use with mycobacteria.

PLoS One. 2010;5(5):e10777

Authors: Andreu N, Zelmer A, Fletcher T, Elkington PT, Ward TH, Ripoll J, Parish T, Bancroft GJ, Schaible U, Robertson BD, Wiles S

BACKGROUND: Mycobacterium tuberculosis, the causative agent of tuberculosis, still represents a major public health threat in many countries. Bioluminescence, the production of light by luciferase-catalyzed reactions, is a versatile reporter technology with multiple applications both in vitro and in vivo. In vivo bioluminescence imaging (BLI) represents one of its most outstanding uses by allowing the non-invasive localization of luciferase-expressing cells within a live animal. Despite the extensive use of luminescent reporters in mycobacteria, the resultant luminescent strains have not been fully applied to BLI. METHODOLOGY/PRINCIPAL FINDINGS: One of the main obstacles to the use of bioluminescence for in vivo imaging is the achievement of reporter protein expression levels high enough to obtain a signal that can be detected externally. Therefore, as a first step in the application of this technology to the study of mycobacterial infection in vivo, we have optimised the use of firefly, Gaussia and bacterial luciferases in mycobacteria using a combination of vectors, promoters, and codon-optimised genes. We report for the first time the functional expression of the whole bacterial lux operon in Mycobacterium tuberculosis and M. smegmatis thus allowing the development of auto-luminescent mycobacteria. We demonstrate that the Gaussia luciferase is secreted from bacterial cells and that this secretion does not require a signal sequence. Finally we prove that the signal produced by recombinant mycobacteria expressing either the firefly or bacterial luciferases can be non-invasively detected in the lungs of infected mice by bioluminescence imaging. CONCLUSIONS/SIGNIFICANCE: While much work remains to be done, the finding that both firefly and bacterial luciferases can be detected non-invasively in live mice is an important first step to using these reporters to study the pathogenesis of M. tuberculosis and other mycobacterial species in vivo. Furthermore, the development of auto-luminescent mycobacteria has enormous ramifications for high throughput mycobacterial drug screening assays which are currently carried out either in a destructive manner using LuxAB or the firefly luciferase.

PMID: 20520722 [PubMed - in process]

Rosiglitazone Attenuates Endothelin-1-Induced Vasoconstriction by Upregulating Endothelial Expression of Endothelin B Receptor.

Hypertension. 2010 Jun 1;

Authors: Tian J, Wong WT, Tian XY, Zhang P, Huang Y, Wang N

Thiazolidinediones improve insulin resistance and endothelial dysfunction. However, the mechanisms underlying the vasoprotective effects of thiazolidinediones remain to be fully elucidated. The present study aimed to examine the molecular mechanism for the anti-vasoconstrictive effects of rosiglitazone in response to endothelin (ET) 1. Mouse aortas were treated with rosiglitazone for 24 hours, and ET-1-induced vasoconstriction was assessed by wire myography. The results showed that rosiglitazone attenuated ET-1-induced contraction in mouse aortas; this effect was abolished by ET-B receptor (ETBR) antagonist, NO synthase inhibitor, and by the removal of endothelium. By using Northern blotting, real-time RT-PCR, Western blotting, and immunohistochemical techniques, we found that rosiglitazone upregulated expression of ETBR at both mRNA and protein levels in mouse aortas and human vascular endothelial cells. The induction of ETBR was prevented by peroxisome proliferator-activated receptor-gamma antagonism. Luciferase reporter assay showed that rosiglitazone enhanced ETBR gene promoter activity. Furthermore, chromatin immunoprecipitation assays demonstrated that peroxisome proliferator-activated receptor-gamma can directly bind to ETBR gene promoter. Furthermore, in vivo treatment with rosiglitazone also attenuated the ET-1-induced vasoconstrictions and increased the ETBR expression in mouse aortas and mesenteric arteries. In conclusion, these results demonstrate that rosiglitazone attenuated ET-1-induced vasoconstriction through the upregulation of endothelial ETBR, which is a peroxisome proliferator-activated receptor-gamma direct target.

PMID: 20516393 [PubMed - as supplied by publisher]

Inhibition of Dengue Virus by an Ester Prodrug of an Adenosine Analog.

Antimicrob Agents Chemother. 2010 Jun 1;

Authors: Chen YL, Yin Z, Lakshminarayana SB, Qing M, Schul W, Duraiswamy J, Kondreddi RR, Goh A, Xu HY, Yip A, Liu B, Weaver M, Dartois V, Keller TH, Shi PY

Dengue virus (DENV) is the most prevalent mosquito-borne viral pathogen that infects humans. Neither vaccine nor antiviral therapy is currently available for DENV. Here we report an adenosine nucleoside prodrug that potently inhibits DENV replication both in cell culture and in a DENV mouse model. NITD449 (2'-C-acetylene-7-deaza-7-carbamoyladenosine) was initially identified as a parental compound that inhibits all four serotypes of DENV with low cytotoxicity. However, in vivo pharmacokinetic studies indicated that NITD449 had a low exposure in plasma when dosed orally. To increase the oral bioavailability, we covalently linked isobutyric acids to the 3'-, and 5'-hydroxyl groups of ribose via ester linkage to NITD449, leading to prodrug NITD203 (3',5'-O-diisobutyryl-2'-C-acetylene-7-deaza-7-carbamoyl-adenosin). Pharmacokinetic analysis showed that upon oral dosing of the prodrug, NITD203 was readily converted to NITD449, resulting in improved exposure of the parental compound in plasma in both mouse and rat. In DENV-infected AG129 mice, oral dosing of the prodrug at 25 mg/kg reduced peak viremia by 30 folds. Antiviral spectrum analysis showed that NITD203 inhibited various flaviviruses (DENV, Yellow Fever virus, and West Nile virus) and hepatitis C virus, but not Chikungunya virus (an alphavirus). Mode-of-action analysis, using a luciferase reporting replicon, indicated that NITD203 inhibited DENV RNA synthesis. Although NITD203 exhibited potent in vitro and in vivo efficacies, the compound could not reach satisfactory NOAEL (no observable adverse effect level) in a two-week in vivo toxicity study. Nevertheless, our results demonstrate that a prodrug approach of nucleoside analog could potentially be developed for flavivirus antiviral therapy.

PMID: 20516277 [PubMed - as supplied by publisher]

Netrin-1 dampens pulmonary inflammation during acute lung injury.

Am J Respir Crit Care Med. 2010 Apr 15;181(8):815-24

Authors: Mirakaj V, Thix CA, Laucher S, Mielke C, Morote-Garcia JC, Schmit MA, Henes J, Unertl KE, Köhler D, Rosenberger P

RATIONALE: Acute lung injury (ALI) is an inflammatory disorder characterized by hypoxemia and diffuse infiltration of neutrophils into the alveolar space. The migration and extravasation of neutrophils is guided through positive guidance cues, such as chemokines. Recent work has identified the neuronal guidance protein netrin-1 to be a negative guidance cue for leukocyte migration and to hold antiinflammatory potential. OBJECTIVES: To test the role of pulmonary netrin-1 during ALI. METHODS: Pulmonary netrin-1 expression was evaluated during acute inflammation in vitro and in vivo; the netrin-1 promoter was studied using pGL4 luciferase reporter. ALI was induced through LPS inhalation and mechanical ventilation in wild-type, Ntn1(+/-), and A2BAR(-/-) animals. Exogenous netrin-1 was used to evaluate its impact on pulmonary inflammation. MEASUREMENTS AND MAIN RESULTS: Wild-type animals demonstrated repression of pulmonary netrin-1 after LPS inhalation. In vitro studies confirmed the repression of netrin-1. Studies in the putative netrin-1 promoter identified a nuclear factor-kappaB-dependent mechanism to be involved in this repression. Ntn1(+/-) animals demonstrated increased inflammatory changes after LPS inhalation compared with Ntn1(+/+) animals. Reconstitution with netrin-1 dampened the infiltration of neutrophils and cytokine production in the alveolar space. This effect was dependent on the adenosine 2b receptor. The importance of netrin-1 for the control of pulmonary inflammation could be corroborated in a model of ventilator-induced lung injury. CONCLUSIONS: Pulmonary netrin-1 levels are repressed during ALI. This results in pronounced pulmonary damage, an increased infiltration of neutrophils, and increased pulmonary inflammation. Exogenous netrin-1 significantly dampens the extent of ALI through the adenosine 2B receptor.

PMID: 20075388 [PubMed - indexed for MEDLINE]

Ultrasound-assisted non-viral gene transfer to the salivary glands.

Gene Ther. 2010 May 27;

Authors: Passineau MJ, Zourelias L, Machen L, Edwards PC, Benza RL

We report a non-viral gene transfer method using ultrasound induced microbubble destruction to allow the uptake of plasmid gene transfer vectors to the cells of the mouse salivary gland. The Luciferase (Luc) reporter gene, driven by a cytomegalovirus (CMV) promoter, was delivered unilaterally to the submandibular salivary gland via retroductal cannulation and Luc expression was monitored with in vivo imaging. The CMV-Luc plasmid was delivered to the salivary gland in a carrier solution containing microbubbles composed of lipid-encased perfluoropropane gas, with two different concentrations of microbubbles used (100 and 15% volume/volume). An Adenoviral (Ad) vector using an identical CMV-Luc expression cassette was used as a positive control at two different dosages. Whereas ultrasound-assisted gene transfer (UAGT) with 100% microbubbles was weak and rapidly extinguished, UAGT with the 15% microbubble solution was robust and stable for 28 days. UAGT seems to be a practicable and promising method for non-viral gene delivery to the salivary glands.Gene Therapy advance online publication, 27 May 2010; doi:10.1038/gt.2010.86.

PMID: 20508599 [PubMed - as supplied by publisher]

Identification of MicroRNA-93 as a novel regulator of vascular endothelial growth factor (VEGF) In hyperglycemic conditions.

J Biol Chem. 2010 May 25;

Authors: Long J, Wang Y, Wang W, Chang BH, Danesh FR

Vascular endothelial growth factor (VEGF) is a dimeric glycoprotein which plays a crucial role in microvascular complications of diabetes, including diabetic nephropathy. However, the precise regulatory mechanisms governing VEGF expression in the diabetic milieu are still poorly understood. Here we provide evidence that microRNA-93 (miR-93) regulates VEGF expression in experimental models of diabetes both in vitro and in vivo. Comparative miRNA expression profile arrays identified miR-93 as a signature miRNA in hyperglycemic conditions. We identified VEGF-A as a putative target of miR-93 in the kidney with a perfect complementarity between miR-93 and the 3'-UTR of VEGF in several species. When cotransfected with a luciferase reporter construct containing the mouse VEGF 3'-UTR, expression of miR-93 markedly decreased the luciferase activity. We showed that forced expression of miR-93 in cells abrogated VEGF protein secretion. Conversely, anti-miR-93 inhibitors increased VEGF release. Transfection of miR-93 also prevented the effect of high glucose on VEGF downstream targets. Using transgenic mice containing VEGF/LacZ bicistronic transcripts, we found that inhibition of glomerular miR-93 by peptide-conjugated morpholino oligomers elicited increased expression of VEGF. Our findings also indicate that high glucose decreases miR-93 expression by downregulating the promoter of the host MCM7 gene. Taken together, our findings provide new insights into the role of miR-93 in VEGF signaling pathway, and offer a potentially novel target in preventing the progression of diabetic nephropathy.

PMID: 20501654 [PubMed - as supplied by publisher]

Targeting of mesenchymal stem cells to ovarian tumors via an artificial receptor.

J Ovarian Res. 2010 May 25;3(1):12

Authors: Komarova S, Roth J, Alvarez R, Curiel DT, Pereboeva L

ABSTRACT: Background Mesenchymal Progenitor/Stem Cells (MSC) respond to homing cues providing an important mechanism to deliver therapeutics to sites of injury and tumors. This property has been confirmed by many investigators, however, the efficiency of tumor homing needs to be improved for effective therapeutic delivery. We investigated the feasibility of enhancing MSC tumor targeting by expressing an artificial tumor-binding receptor on the MSC surface. Methods Human MSC expressing an artificial receptor that binds to erbB2, a tumor cell marker, were obtained by transduction with genetically modified adenoviral vectors encoding an artificial receptor (MSC-AR). MSC-AR properties were tested in vitro in cell binding assays and in vivo using two model systems: transient transgenic mice that express human erbB2 in the lungs and ovarian xenograft tumor model. The levels of luciferase-labeled MSCs in erbB2-expressing targeted sites were evaluated by measuring luciferase activity using luciferase assay and imaging. RESULTS: The expression of AR enhanced binding of MSC-AR to erbB2-expressing cells in vitro, compared to unmodified MSCs. Furthermore, we have tested the properties of erbB2-targeted MSCs in vivo and demonstrated an increased retention of MSC-AR in lungs expressing erbB2. We have also confirmed increased numbers of erbB2-targeted MSCs in ovarian tumors, compared to unmodified MSC. The kinetic of tumor targeting by ip injected MSC was also investigated. CONCLUSION: These data demonstrate that targeting abilities of MSCs can be enhanced via introduction of artificial receptors. The application of this strategy for tumor cell-based delivery could increase a number of cell carriers in tumors and enhance efficacy of cell-based therapy.

PMID: 20500878 [PubMed - as supplied by publisher]

Sonoporation mediated immunogene therapy of solid tumors.

Ultrasound Med Biol. 2010 Mar;36(3):430-40

Authors: Casey G, Cashman JP, Morrissey D, Whelan MC, Larkin JO, Soden DM, Tangney M, O'Sullivan GC

Development of gene-based therapies for the treatment of inherited and acquired diseases, including cancer, has seen renewed interest in the use of nonviral vectors coupled to physical delivery modalities. Low-frequency ultrasound (US), with a well-established record in a clinical setting, has the potential to deliver DNA efficiently, accurately and safely. Optimal in vivo parameters for US-mediated delivery of naked plasmid DNA were established using the firefly luciferase reporter gene construct. Optimized parameters were used to administer a therapeutic gene construct, coding for granulocyte-macrophage colony-stimulating factor (GM-CSF) and B7-1 costimulatory molecule, to growing murine fibrosarcoma tumors. Tumor progression and animal survival was monitored throughout the study and the efficacy of the US-mediated gene therapy determined and compared with an electroporation-based approach. Optimal parameters for US-mediated delivery of plasmid DNA to tumors were deduced to be 1.0 W/cm(2) at 20% duty cycle for 5 min (60 J/cm(2)). In vivo US-mediated gene therapy resulted in a 55% cure rate in tumor-bearing animals. The immunological response invoked was cell mediated, conferring resistance against re-challenge and resistance to tumor challenge after transfer of splenocytes to naïve animals. US treatment was noninjurious to treated tissue, whereas therapeutic efficacy was comparable to an electroporation-based approach. US-mediated delivery of an immune-gene construct to growing tumors was therapeutically effective. Sonoporation has the potential to be a major factor in the development of nonviral gene delivery approaches.

PMID: 20133039 [PubMed - indexed for MEDLINE]

AAV6 capsid tyrosine to phenylalanine mutations improve gene transfer to skeletal muscle.

Hum Gene Ther. 2010 May 24;

Authors: Qiao C, Zhang W, Yuan Z, Shin JH, Li J, Jayandharan GR, Zhong L, Srivastava A, Xiao X, Duan D

Adeno-associated virus (AAV) vectors are the most efficient in vivo gene transfer tools for gene therapy applications. In recent years, efforts have been made in translating encouraging results from small animal models to human patients. However the large quantities of vector needed for clinical application remains a great challenge. Developing novel AAV vectors with enhanced infectivity may reduce high vector dose requirement for many applications such as gene therapy for muscular dystrophy. Selective mutation of AAV capsid surface-exposed tyrosine (Y) is a novel strategy to improve transduction efficiency. AAV6 has been considered as one of the most robust muscle gene delivery vehicles. Here, we hypothesize that AAV6 transduction efficiency can be further enhanced by mutating surface Y to F. We found that mutants AAV6-Y445F and AAV6-Y731F, especially the former, achieved more efficient gene transfer than the original AAV6 after intramuscular administration in mice. Expression of both firefly luciferase and alkaline phosphatase reporter genes increased up to 8-fold and DNA copy numbers in the muscle increased up to 6-fold. Our results suggest that tyrosine-mutant AAV6 vectors may represent powerful tools for testing muscle gene therapy in animal models and potentially in humans.

PMID: 20497037 [PubMed - as supplied by publisher]

The positive feedback system provides efficient and persistent transgene expression.

Mol Pharm. 2010 May 24;

Authors: Ochiai H, Harashima H, Kamiya H

The two-step transcriptional amplification (TSTA) system, using artificial transcription factors, effectively enhances transgene expression. In this study, a TSTA system-based positive feedback system was developed to achieve efficient and persistent transgene expression. A fusion protein of the sequence-specific DNA binding domain of yeast GAL4 and the transcriptional activation domain of herpes simplex virus VP16 (GAL4-VP16) was used as an "activator" to amplify the expression of the luciferase "reporter" gene. It was found that the introduction of five tandem copies of the GAL4 recognition sequence (G5) into both the upstream and downstream regions of the expression cassette synergistically enhanced the transgene expression. The upstream and downstream G5 sequences were introduced into the expression cassette of the activator itself, and into that of the reporter, to form the positive feedback loop that enabled continuous activator expression. This positive feedback system maintained the expression levels of the reporter for 4 days in HeLa cells and for a week in mouse liver, while those from the usual plasmids decreased by 30- and 50-fold, respectively. These results constitute the first evidence that the positive feedback system is a useful method for long-term transgene expression in cultured cells and in vivo. This system would be applicable to gene therapy, in vivo imaging, and biotechnology.

PMID: 20496887 [PubMed - as supplied by publisher]

Conditional Bicistronic Cre Reporter Line Expressing Both Firefly Luciferase and beta-galactosidase.

Mol Imaging Biol. 2010 May 22;

Authors: Ishikawa TO, Herschman HR

PURPOSE: The Cre-loxP system has become an important strategy for conditional gene deletion and conditional gene expression in genetically engineered mice. To evaluate Cre recombinase expression, we generated reporter mice that permit both noninvasive imaging in living animals and either ex vivo histochemical/immunohistochemical tissue transgene expression analysis or quantitative enzyme analysis in the same animal. PROCEDURES: Transgenic reporter mice were generated in which a loxP-flanked enhanced green fluorescent protein (EGFP) reporter gene and STOP sequence are placed after the nearly ubiquitously expressed CAG promoter, but before a bicistronic transcriptional unit containing luciferase and beta-galactosidase reporter gene coding sequences. RESULTS: After global deletion of the floxed STOP sequence by germ line Cre deletion, the reporter mouse expresses luciferase and beta-galactosidase in all tissues examined. Tissue-specific expression of both reporter genes occurs in reporter mouse strains expressing Cre in skin (K14 keratin Cre), heart (myosin light chair Cre), or colon (Villin Cre). CONCLUSION: The luc-gal(Tg) reporter mouse allows noninvasive imaging of target Cre activation both in living animals and in tissues and cells following necropsy, using loss of EGFP expression, gain of luciferase expression, and gain of beta-galactosidase expression as alternatives within the same animal for qualitative analysis of Cre expression.

PMID: 20495880 [PubMed - as supplied by publisher]

Bioluminescent imaging study: FAK inhibitor, PF-562,271, preclinical study in PC3M-luc-C6 local implant and metastasis xenograft models.

Cancer Biol Ther. 2010 Jul 9;10(1)

Authors: Sun H, Pisle S, Gardner ER, Figg WD

Focal adhesion kinase (FAK) is essential in regulating integrin signaling pathways responsible for cell survival and proliferation, as well as motility, making FAK a distinctive target in the field of anticancer drug development, especially with regards to metastatic disease.(1) Our objective was to demonstrate tumor growth inhibition by PF-562,271, a selective inhibitor of FAK and FAK2, or Pyk2,(2) in mouse xenograft models, both subcutaneous and metastatic, employing the human prostate cancer cell line PC3M-luc-C6, a modified PC3M cell line that expresses luciferase. After two weeks of treatment with PF-562,271, 25 mg/kg PO BID 5x/wk, the subcutaneous model showed a 62% tumor growth inhibition compared to control based on tumor measurements (p < 0.05), with a 88% vs. a 490% increase in bioluminescent signal for treatment and control respectively (p < 0.05). In the metastasis model, the percent change from baseline, after 18 days of treatment, of the treatment group was 2,854% vs. 14,190% for the vehicle (p < 0.01). These results show that PF-562,271 has a potent effect on metastatic prostate cancer growth in vivo.

PMID: 20495381 [PubMed - as supplied by publisher]

Delivery of Na/I Symporter Gene into Skeletal Muscle Using Nanobubbles and Ultrasound: Visualization of Gene Expression by PET.

J Nucl Med. 2010 May 19;

Authors: Watanabe Y, Horie S, Funaki Y, Kikuchi Y, Yamazaki H, Ishii K, Mori S, Vassaux G, Kodama T

The development of nonviral gene delivery systems is essential in gene therapy, and the use of a minimally invasive imaging methodology can provide important clinical endpoints. In the current study, we present a new methodology for gene therapy-a delivery system using nanobubbles and ultrasound as a nonviral gene delivery method. We assessed whether the gene transfer allowed by this methodology was detectable by PET and bioluminescence imaging. METHODS: Two kinds of reported vectors (luciferase and human Na/I symporter [hNIS]) were transfected or cotransfected into the skeletal muscles of normal mice (BALB/c) using the ultrasound-nanobubbles method. The kinetics of luciferase gene expression were analyzed in vivo using bioluminescence imaging. At the peak of gene transfer, PET of hNIS expression was performed using our recently developed PET scanner, after (124)I injection. The imaging data were confirmed using reverse-transcriptase polymerase chain reaction amplification, biodistribution, and a blocking study. The imaging potential of the 2 methodologies was evaluated in 2 mouse models of human pathology (McH/lpr-RA1 mice showing vascular disease and C57BL/10-mdx Jic mice showing muscular dystrophy). RESULTS: Peak luciferase gene activity was observed in the skeletal muscle 4 d after transfection. On day 2 after hNIS and luciferase cotransfection, the expression of these genes was confirmed by reverse-transcriptase polymerase chain reaction on a muscle biopsy. PET of the hNIS gene, biodistribution, the blocking study, and autoradiography were performed on day 4 after transfection, and it was indicated that hNIS expression was restricted to the site of plasmid administration (skeletal muscle). Similar localized PET and (124)I accumulation were successfully obtained in the disease-model mice. CONCLUSION: The hNIS gene was delivered into the skeletal muscle of healthy and disease-model mice by the ultrasound-nanobubbles method, and gene expression was successfully visualized with PET. The combination of ultrasound-nanobubble gene transfer and PET may be applied to gene therapy clinical protocols.

PMID: 20484436 [PubMed - as supplied by publisher]