Efficient AAV1-AAV2 hybrid vector for gene therapy of hemophilia.

Adeno-associated virus (AAV) serotype 1 (AAV1) has been shown to be more effective than the well-studied AAV serotype 2 (AAV2) in muscle gene transfer. Replacement of amino acids 350 to 430 of AAV2 VP1 with the corresponding amino acids from VP1 of AAV1 resulted in a hybrid vector, termed AAV-221-IV, which behaved similarly to AAV1 in vitro and in vivo in muscle. Intramuscular injection of 1x10(11) vector particles per mouse of hybrid vector carrying a human FIX transgene in CD4 knockout mice resulted in an average level of human FIX in the plasma of 450 ng/ml, 4- to 10-fold higher than in mice injected with an AAV2 vector carrying the same transgene, and 80% of the transgene levels in animals treated with the same dose of AAV1. DNA analysis of injected muscle showed a 10-fold higher copy number after gene delivery by the hybrid vector compared with AAV2. A comparison of total DNA versus DNA from intact virus particles suggests a higher stability of hybrid virus particles. These results suggest that changes in the AAV capsid have an effect on virus-cell receptor interaction, and also influence trafficking and processing of the virus particle in the cell. This "hybrid vector" retains the heparin-binding sites of AAV2 and, therefore, can be purified by passage through a heparin-Sepharose column with the same efficiency as AAV2. When tested in vivo, either in CD4 knockout mice or in a hemophilic mouse model, the heparin-purified hybrid vector showed >10-fold higher activity than similarly purified AAV2. This demonstrates the utility of this hybrid vector in the performance of large-scale heparin column purification to generate a vector with a high expression profile for muscle-directed gene delivery. Initiation of clinical studies with this hybrid vector may be facilitated because it differs from AAV2 by only nine amino acids.

FTY720: a promising agent for treatment of metastatic hepatocellular carcinoma.

PURPOSE: Recurrence after resection and metastasis are common in hepatocellular carcinoma and are associated with poor prognosis. Therefore, effective treatment is urgently needed for improvement of patients' survival. Previously, we reported that FTY720 has an antimetastatic effect on hepatocellular carcinoma cell line through down-regulation of Rac signaling pathway. This study aims to investigate the in vivo antimetastatic potential of FTY720 in an orthotopic nude mice model using metastatic human hepatocellular carcinoma cell lines MHCC-97L (lower metastatic potential) and MHCC-97H (higher metastatic potential). EXPERIMENTAL DESIGN: The nude mice bearing liver tumors were randomized into a treatment group and a control group, each with 12 mice. FTY720 was administered at a dosage of 5 or 10 mg/kg via i.p. injection after 7 days of tumor inoculation. Thirty-five days later, the mice were sacrificed for record of intrahepatic and pulmonary metastases. RESULTS: After 35 days of FTY720 treatment at the dosages of 5 and 10 mg/kg, all 12 mice in the treatment group were alive and well. FTY720 at the dosages of 5 and 10 mg/kg significantly suppressed the tumor volume and intrahepatic and pulmonary metastases in the metastatic nude mice model. FTY720 suppressed intrahepatic and pulmonary metastases by inhibition of Rac expression, which at least in part down-regulated the vascular endothelial growth factor expression and CD34 staining in a dose-dependent manner. CONCLUSION: FTY720 is a promising novel therapeutic drug for treatment of hepatocellular carcinoma metastasis.

Magnetic resonance spectroscopic imaging of tumor metabolic markers for cancer diagnosis, metabolic phenotyping, and characterization of tumor microenvironment.

Cancer cells display heterogeneous genetic characteristics, depending on the tumor dynamic microenvironment. Abnormal tumor vasculature and poor tissue oxygenation generate a fraction of hypoxic tumor cells that have selective advantages in metastasis and invasion and often resist chemo- and radiation therapies. The genetic alterations acquired by tumors modify their biochemical pathways, which results in abnormal tumor metabolism. An elevation in glycolysis known as the "Warburg effect" and changes in lipid synthesis and oxidation occur. Magnetic resonance spectroscopy (MRS) has been used to study tumor metabolism in preclinical animal models and in clinical research on human breast, brain, and prostate cancers. This technique can identify specific genetic and metabolic changes that occur in malignant tumors. Therefore, the metabolic markers, detectable by MRS, not only provide information on biochemical changes but also define different metabolic tumor phenotypes. When combined with the contrast-enhanced Magnetic Resonance Imaging (MRI), which has a high sensitivity for cancer diagnosis, in vivo magnetic resonance spectroscopic imaging (MRSI) improves the diagnostic specificity of malignant human cancers and is becoming an important clinical tool for cancer management and care. This article reviews the MRSI techniques as molecular imaging methods to detect and quantify metabolic changes in various tumor tissue types, especially in extracranial tumor tissues that contain high concentrations of fat. MRI/MRSI methods have been used to characterize tumor microenvironments in terms of blood volume and vessel permeability. Measurements of tissue oxygenation and glycolytic rates by MRS also are described to illustrate the capability of the MR technology in probing molecular information non-invasively in tumor tissues and its important potential for studying molecular mechanisms of human cancers in physiological conditions.

Complete correction of hemophilia A with adeno-associated viral vectors containing a full-size expression cassette.

Hemophilia A is caused by a deficiency in the factor VIII (FVIII) gene. Constrained by limited packaging capacity, even the 4.3-kb B domain-deleted FVIII remained a challenge for delivery by a single adeno-associated viral (AAV) vector. Studies have shown that up to a 6.6-kb vector sequence may be packaged into AAV virions, which suggested an alternative strategy for hemophilia A gene therapy. To explore the usefulness of AAV vectors carrying an oversized FVIII gene, we constructed the AAV-FVIII vector under the control of a beta-actin promoter with a cytomegalovirus enhancer (CB) and a bovine growth hormone (bGH) poly(A) sequence. The CB promoter plus bGH signal was shown to be 3- to 5-fold more potent than the mini-transthyretin (TTR) promoter with a synthetic poly(A) sequence for directing FVIII expression in the liver. Despite the 5.75-kb genome size of pAAV-CB-FVIII, sufficient AAV vectors were produced for in vivo testing. Approximately 3- to 5-fold more FVIII secretion was observed in animals receiving AAV-CB-FVIII vectors than in those receiving standard-sized AAV-TTR-FVIII vectors. Both the activated partial thromboplastin time assay and the whole blood thromboelastographic analysis confirmed that AAV-FVIII vectors fully corrected the bleeding phenotype of hemophilia mice. These results suggest that AAV vectors with an oversized genome should be useful for not only hemophilia A gene therapy but also other diseases with large cDNA such as muscular dystrophy and cystic fibrosis.

Significance of the Rac signaling pathway in HCC cell motility: implications for a new therapeutic target.

Recurrence and metastasis are commonly associated with poor prognosis of hepatocellular carcinoma (HCC). Therefore, a better understanding of molecular mechanisms involved in HCC metastasis may lead to more effective treatment for HCC patients. Rac plays important roles in cytoskeletal reorganization leading to cell motility in renal and breast carcinomas. However, the role of Rac is controversial in tumors and has not been studied in HCC. The aim of this study was to investigate the importance of the Rac signaling pathway in HCC cell motility and the anti-metastatic potential of FTY720. Recently a pair of HCC cell lines from a primary tumor (H2P) and its matched metastasis (H2M) was established. These two cell lines provide a useful tool for the study of HCC metastasis. The results show that the Rac signaling pathway is activated in the metastatic HCC cell line (H2M) compared with the primary HCC cell line (H2P). FTY720 specifically suppressed H2M cell motility by down-regulation of the Rac-GTP level through inhibition of phosphoinositide 3-kinase activity. To conclude, this study is the first to demonstrate an essential role of Rac signaling pathway activation in HCC metastasis and suppression of cell motility by FTY720 through blocking of the Rac pathway.

Glycyl C(alpha) chemical shielding in tripeptides: measurement by solid-state NMR and correlation with X-ray structure and theory.

We report here (13)C(alpha) chemical shielding parameters for central Gly residues in tripeptides adopting alpha-helix, beta-strand, polyglycine II, and fully extended 2 degrees structures. To assess experimental uncertainties in the shielding parameters and the effects of (14)N-(13)C(alpha) or (15)N-(13)C(alpha) dipolar coupling, stationary and magic angle spinning (MAS) spectra with and without (15)N decoupling were obtained from natural abundance and double-labeled samples containing [2-(13)C, (15)N]Gly. We find that accurate (<1 ppm uncertainty) shielding parameters are measured with good sensitivity and resolution in (15)N decoupled 1D or 2D MAS spectra of double-labeled samples. Compared to variations of isotropic shifts with peptide angles, those of (13)C(alpha) shielding anisotropy and asymmetry are greater. Trends relating shielding parameters to the 2 degrees structure are apparent, and the correlation of the experimental values with unscaled ab initio shielding calculations has an rms error of 3 ppm. Using the experimental data and the ab initio shielding values, the empirical trends relating the 2 degrees structure to shielding are extended to the larger range of torsion angles found in proteins.

FTY720 induces apoptosis of human hepatoma cell lines through PI3-K-mediated Akt dephosphorylation.

Our aim was to study the anticancer effect of the novel immunomodulator FTY720 in vitro and in vivo by investigation of cell cycle entry, cell cycle regulation, cell survival and apoptosis pathways. Three hepatoma cell lines with different p53 statuses (HepG2, Huh-7 and Hep3B) and one non-tumorigenic immortalized liver cell line (MIHA) were used for an in vitro study. The in vivo effects of FTY720 were evaluated in a nude mouse tumor model. Cell cycle distribution and cell cycle regulator proteins p27(Kip1) and cyclin D1, together with the PI3-K/Akt pathway, mitogen-activated protein kinases and cleaved caspase-3 and caspase-9, were evaluated. FTY720 selectively induced cell apoptosis in hepatoma cell lines with overexpression of cleaved caspase-3 and caspase-9, but the same phenomena were not found in MIHA cells. FTY720 induced Akt dephosphorylation at Ser473 mediated by phosphoinositide 3-kinase (PI3-K) inhibition. Dephosphorylation led to down-regulation of p42/p44 and dephosphorylation of Forkhead transcription factor and GSK-3beta and, subsequently, up-regulation of p27(Kip1) and down-regulation of cyclin D1. In our in vivo model FTY720 induced apoptosis of tumor cells by down-regulation of the Akt pathway. FTY720 suppressed tumor growth without notable side-effects in normal liver. In conclusion, FTY720 is a novel anticancer agent that induces apoptosis of hepatoma cell lines both in vitro and in vivo through PI3-K-mediated Akt dephosphorylation in a p53-independent manner.