Wai Har Ng

Biosafety assessment of site-directed transgene integration in human umbilical cord-lining cells.

Biosafety and efficacy considerations that impede clinical application of gene therapy could be addressed by nonviral ex vivo cell therapy, utilizing transgenic cells that have been comprehensively pre-evaluated for genotoxic potential and transgene expression. We evaluated the genotoxic potential of phiC31 bacteriophage integrase-mediated transgene integration in cord-lining epithelial cells (CLECs) readily cultured from the outer membrane of human umbilical cords, by sequencing and mapping integration sites, spectral karyotyping, high-resolution genome copy number, transcriptome, and transgene copy number analyses and in vivo tumorigenicity. Of 44 independent integration events, <5% were exonic and 85% of modified cells had integrated <or=2 transgene(s). Expression of 95.6% of genes was unaltered in modified cells. Only three small regions showed genome copy number changes that did not correlate with altered gene expression or integration sites. Spectral karyotyping revealed rare nonrecurrent occurrence of three different translocations. Integrase-modified cells were not tumorigenic in immunocompromised mice for at least 4 months. Stable integration of a human factor VIII (FVIII) construct conferred durable FVIII secretion in vitro. Xenoimplantation of FVIII-secreting CLECs in immunocompetent hemophilic mice achieved significant phenotypic correction. Pre-evaluated clonal populations of phiC31 integrase-modified CLECs could be useful as bioimplants for monogenic diseases such as hemophilia.

The distinctive gastric fluid proteome in gastric cancer reveals a multi-biomarker diagnostic profile

BackgroundOverall gastric cancer survival remains poor mainly because there are no reliable methods for identifying highly curable early stage disease. Multi-protein profiling of gastric fluids, obtained from the anatomic site of pathology, could reveal diagnostic proteomic fingerprints.MethodsProtein profiles were generated from gastric fluid samples of 19 gastric cancer and 36 benign gastritides patients undergoing elective, clinically-indicated gastroscopy using surface-enhanced laser desorption/ionization time-of-flight mass spectrometry on multiple ProteinChip arrays. Proteomic features were compared by significance analysis of microarray algorithm and two-way hierarchical clustering. A second blinded sample set (24 gastric cancers and 29 clinically benign gastritides) was used for validation.ResultsBy significance analysyis of microarray, 60 proteomic features were up-regulated and 46 were down-regulated in gastric cancer samples (p < 0.01). Multimarker clustering showed two distinctive proteomic profiles independent of age and ethnicity. Eighteen of 19 cancer samples clustered together (sensitivity 95%) while 27/36 of non-cancer samples clustered in a second group. Nine non-cancer samples that clustered with cancer samples included 5 pre-malignant lesions (1 adenomatous polyp and 4 intestinal metaplasia). Validation using a second sample set showed the sensitivity and specificity to be 88% and 93%, respectively. Positive predictive value of the combined data was 0.80. Selected peptide sequencing identified pepsinogen C and pepsin A activation peptide as significantly down-regulated and alpha-defensin as significantly up-regulated.ConclusionThis simple and reproducible multimarker proteomic assay could supplement clinical gastroscopic evaluation of symptomatic patients to enhance diagnostic accuracy for gastric cancer and pre-malignant lesions.

Global molecular dysfunctions in gastric cancer revealed by an integrated analysis of the phosphoproteome and transcriptome

We integrated LC-MS/MS-based and protein antibody array-based proteomics with genomics approaches to investigate the phosphoproteome and transcriptome of gastric cancer cell lines and endoscopic gastric biopsies from normal subjects and patients with benign gastritis or gastric cancer. More than 3,000 non-redundant phosphorylation sites in over 1,200 proteins were identified in gastric cancer cells. We correlated phosphoproteome data with transcriptome data sets and reported the expression of 41 protein kinases, 5 phosphatases and 65 phosphorylated mitochondrial proteins in gastric cancer cells. Transcriptional expression levels of 190 phosphorylated proteins were >2-fold higher in gastric cancer cells compared to normal stomach tissue. Pathway analysis demonstrated over-presentation of DNA damage response pathway and underscored critical roles of phosphorylated p53 in gastric cancer. This is the first study to comprehensively report the gastric cancer phosphoproteome. Integrative analysis of the phosphoproteome and transcriptome provided an expansive view of molecular signaling pathways in gastric cancer.

Nonvirally Modified Autologous Primary Hepatocytes Correct Diabetes and Prevent Target Organ Injury in a Large Preclinical Model

Background Current gene- and cell-based therapies have significant limitations which impede widespread clinical application. Taking diabetes mellitus as a paradigm, we have sought to overcome these limitations by ex vivo electrotransfer of a nonviral insulin expression vector into primary hepatocytes followed by immediate autologous reimplantation in a preclinical model of diabetes. Methods and Results In a single 3-hour procedure, hepatocytes were isolated from a surgically resected liver wedge, electroporated with an insulin expression plasmid ex vivo and reimplanted intraparenchymally under ultrasonic guidance into the liver in each of 10 streptozotocin-induced diabetic Yorkshire pigs. The vector was comprised of a bifunctional, glucose-responsive promoter linked to human insulin cDNA. Ambient glucose concentrations appropriately altered human insulin mRNA expression and C-peptide secretion within minutes in vitro and in vivo. Treated swine showed correction of hyperglycemia, glucose intolerance, dyslipidemia and other metabolic abnormalities for ≥47 weeks. Metabolic correction correlated significantly with the number of hepatocytes implanted. Importantly, we observed no hypoglycemia even under fasting conditions. Direct intrahepatic implantation of hepatocytes did not alter biochemical indices of liver function or induce abnormal hepatic lobular architecture. About 70% of implanted hepatocytes functionally engrafted, appeared histologically normal, retained vector DNA and expressed human insulin for ≥47 weeks. Based on structural tissue analyses and transcriptome data, we showed that early correction of diabetes attenuated and even prevented pathological changes in the eye, kidney, liver and aorta. Conclusions We demonstrate that autologous hepatocytes can be efficiently, simply and safely modified by electroporation of a nonviral vector to express, process and secrete insulin durably. This strategy, which achieved significant and sustained therapeutic efficacy in a large preclinical model without adverse effects, warrants consideration for clinical development especially as it could have broader future applications for the treatment of other acquired and inherited diseases for which systemic reconstitution of a specific protein deficiency is critical.

Multidimensional Identification of Tissue Biomarkers of Gastric Cancer

Gastric cancer remains highly fatal due to a dearth of diagnostic biomarkers for early stage disease and molecular targets for therapy. Plasma membrane proteins, including cluster of differentiation (CD) proteins and receptor tyrosine kinases (RTKs), are a rich reservoir of biomarkers. Recognizing that interrogating plasma membrane proteins individually overlooks extensive interactions among them, we have systematically investigated the membrane proteomes and transcriptomes of six gastric cancer cell lines. Our data revealed aberrantly high expression of proteins whose functions accurately reflect the clinical phenotype of gastric cancer, and prioritized critical RTKs and CD proteins in gastric cancer. Expression of selected surface proteins was confirmed by flow cytometry and immunostaining of clinical gastric cancer tissues. Close to 90% of the gastric cancer tissues in a cohort showed up-regulation of at least one of four proteins, that is, MET, EPHA2, FGFR2, and CD104/ITGB4. All intestinal type gastric cancer tumors in this cohort overexpressed at least one of a panel of three proteins, MET, FGFR2, and EPHA2. This study reports the first quantitative global landscape of the surface proteome of gastric cancer cells and provides a shortlist of gastric cancer biomarkers.

Unique Triphenylphosphonium Derivatives for Enhanced Mitochondrial Uptake and Photodynamic Therapy

In this study, unique methyl-functionalized derivatives (T*PP+) of the drug carrier triphenylphosphonium (TPP+) that exhibit significant enhancement of the accumulation of both the cation and its conjugated cargo in cell mitochondria are designed. We show that the presence of methyl group(s) at key positions within the phenyl ring results in an increase in the hydrophobicity and solvent accessible surface area of T*PP+. In particular, when the para position of the phenyl ring in T*PP+ is functionalized with a methyl group, the cation is most exposed to the surrounding environment, leading to a large decrease in water entropy and an increase in the level of van der Waals interaction with and partition into a nonpolar solvent. Therefore, stronger binding between the hydrophobic T*PP+ and mitochondrial membrane occurs. This is exemplified in a (hexachloro-fluorescein)-TPP+ conjugate system, where an ∼12 times increase in the rate of mitochondrial uptake and a 2 times increase in photodynamic therapy (PDT) efficacy against HeLa and FU97 cancer cells are achieved when TPP+ is replaced with T*PP+. Importantly, nearly all the FU97 cells treated with the (hexachloro-fluorescein)-T*PP+ conjugate are killed as compared to only half the population of cells in the case of the (hexachloro-fluorescein)-TPP+ conjugate at a similar PDT light dosage. This study thus forms a platform for the healthcare community to explore alternative TPP+ derivatives that can act as optimal drug transporters for enhanced mitochondrially targeted therapies.

Multidimensional Genome-wide Analyses Show Accurate FVIII Integration by ZFN in Primary Human Cells

Costly coagulation factor VIII (FVIII) replacement therapy is a barrier to optimal clinical management of hemophilia A. Therapy using FVIII-secreting autologous primary cells is potentially efficacious and more affordable. Zinc finger nucleases (ZFN) mediate transgene integration into the AAVS1 locus but comprehensive evaluation of off-target genome effects is currently lacking. In light of serious adverse effects in clinical trials which employed genome-integrating viral vectors, this study evaluated potential genotoxicity of ZFN-mediated transgenesis using different techniques. We employed deep sequencing of predicted off-target sites, copy number analysis, whole-genome sequencing, and RNA-seq in primary human umbilical cord-lining epithelial cells (CLECs) with AAVS1 ZFN-mediated FVIII transgene integration. We combined molecular features to enhance the accuracy and activity of ZFN-mediated transgenesis. Our data showed a low frequency of ZFN-associated indels, no detectable off-target transgene integrations or chromosomal rearrangements. ZFN-modified CLECs had very few dysregulated transcripts and no evidence of activated oncogenic pathways. We also showed AAVS1 ZFN activity and durable FVIII transgene secretion in primary human dermal fibroblasts, bone marrow- and adipose tissue-derived stromal cells. Our study suggests that, with close attention to the molecular design of genome-modifying constructs, AAVS1 ZFN-mediated FVIII integration in several primary human cell types may be safe and efficacious.

Characterization of Insulin-Secreting Porcine Bone Marrow Stromal Cells Ex Vivo and Autologous Cell Therapy In Vivo.

Cell therapy could potentially meet the need for pancreas and islet transplantations in diabetes mellitus that far exceeds the number of available donors. Bone marrow stromal cells are widely used in clinical trials mainly for their immunomodulatory effects with a record of safety. However, less focus has been paid to developing these cells for insulin secretion by transfection. Although murine models of diabetes have been extensively used in gene and cell therapy research, few studies have shown efficacy in large preclinical animal models. Here we report optimized conditions for ex vivo expansion and characterization of porcine bone marrow stromal cells and their permissive expression of a transfected insulin gene. Our data show that these cells resemble human bone marrow stromal cells in surface antigen expression, are homogeneous, and can be reproducibly isolated from outbred Yorkshire–Landrace pigs. Porcine bone marrow stromal cells were efficiently expanded in vitro to >1010 cells from 20 ml of bone marrow and remained karyotypically normal during expansion. These cells were electroporated with an insulin expression plasmid vector with high efficiency and viability, and secreted human insulin and C-peptide indicating appropriate processing of proinsulin. We showed that autologous insulin-secreting bone marrow stromal cells implanted and engrafted in the liver of a streptozotocin-diabetic pig that modeled type 1 diabetes resulted in partial, but significant, improvement in hyperglycemia that could not be ascribed to regeneration of endogenous β-cells. Glucose-stimulated insulin secretion in vivo from implanted cells in the treated pig was documented by a rise in serum human C-peptide levels during intravenous glucose tolerance tests. Compared to a sham-treated control pig, this resulted in significantly reduced fasting hyperglycemia, a slower rise in serum fructosamine, and prevented weight loss. Taken together, this study suggests that bone marrow stromal cells merit further development as autologous cell therapy for diabetes.

Mitochondrial-Targeting MET Kinase Inhibitor Kills Erlotinib-Resistant Lung Cancer Cells

Lung cancer cells harboring activating EGFR mutations acquire resistance to EGFR tyrosine kinase inhibitors (TKIs) by activating several bypass mechanisms, including MET amplification and overexpression. We show that a significant proportion of activated MET protein in EGFR TKI-resistant HCC827 lung cancer cells resides within the mitochondria. Targeting the total complement of MET in the plasma membrane and mitochondria should render these cells more susceptible to cell death and hence provide a means of circumventing drug resistance. Herein, the mitochondrial targeting triphenylphosphonium (TPP) moiety was introduced to the selective MET kinase inhibitor PHA665752. The resulting TPP analogue rapidly localized to the mitochondria of MET-overexpressing erlotinib-resistant HCC827 cells, partially suppressed the phosphorylation (Y1234/Y1235) of MET in the mitochondrial inner membrane and was as cytotoxic and apoptogenic as the parent compound. These findings provide support for the targeting of mitochondrial MET with a TPP-TKI conjugate as a means of restoring responsiveness to chemotherapy.

Durable engraftment of genetically modified FVIII-secreting autologous bone marrow stromal cells in the intramedullary microenvironment

Genetically modified FVIII‐expressing autologous bone marrow‐derived mesenchymal stromal cells (BMSCs) could cure haemophilia A. However, culture‐expanded BMSCs engraft poorly in extramedullary sites. Here, we compared the intramedullary cavity, skeletal muscle, subcutaneous tissue and systemic circulation as tissue microenvironments that could support durable engraftment of FVIII‐secreting BMSC in vivo. A zinc finger nuclease integrated human FVIII transgene into PPP1R12C (intron 1) of culture‐expanded primary canine BMSCs. FVIII‐secretory capacity of implanted BMSCs in each dog was expressed as an individualized therapy index (number of viable BMSCs implanted × FVIII activity secreted/million BMSCs/24 hours). Plasma samples before and after implantation were assayed for transgenic FVIII protein using an anti‐human FVIII antibody having negligible cross‐reactivity with canine FVIII. Plasma transgenic FVIII persisted for at least 48 weeks after implantation in the intramedullary cavity. Transgenic FVIII protein levels were low after intramuscular implantation and undetectable after both intravenous infusion and subcutaneous implantation. All plasma samples were negative for anti‐human FVIII antibodies. Plasma concentrations and durability of transgenic FVIII secretion showed no correlation with the therapy index. Thus, the implantation site microenvironment is crucial. The intramedullary microenvironment, but not extramedullary tissues, supported durable engraftment of genetically modified autologous FVIII‐secreting BMSCs.