Recent Gene Therapy Research & Selected Publications

Summary

BACKGROUND: To increase the success rate in xenogeneic islet transplantation, proper assessment of graft mass is required following transplantation. For this reason, we aimed to develop a suitable fluorescence imaging system to monitor islet xenograft survival in diabetic mice.

METHODS: Adenovirus vector encoding enhanced green fluorescent protein-transduced rat pancreatic islets were transplanted under the renal capsule of streptozotocin-induced diabetic mice and the fluorescence signal was quantified over time using a cooled charge-coupled device. Non-fasting blood glucose levels were recorded during the same period. Insulin release from transduced and control islets was detected via enzyme-linked immunosorbent assay.

RESULTS: Adenovirus vector encoding enhanced green fluorescent protein infection did not alter the function or survival of pancreatic islets post transduction. A direct correlation was found between the number of islets (250-750) transplanted under the kidney capsule and the blood glucose recovery.

CONCLUSIONS: Fluorescence imaging appears to be a useful tool for quantitative assessment of islet cell viability post transplantation and could permit earlier detection of graft rejection.

Summary

PURPOSE: We tested a new light detection cooled charge-coupled device (CCD) for in vivo assessment of noninvasive, whole-body fluorescence optical imaging of adenovirus directed enhanced green fluorescent protein (AdEGFP) expression.

PROCEDURES:
AdEGFP was injected i.v. into BALB/c mice via tail vein. Whole-body fluorescence optical imaging of AdEGFP expression was performed using a Kodak 2000MM Image Station before and after vector administration.

RESULTS:
EGFP expression was exclusively detected around the abdominal cavity, and the fluorescent signal peaked at day 4 and then remained detectable for at least 30 days. Ex vivo fluorescence imaging confirmed that EGFP expression was restricted to the liver, and transgene expression was homogeneously diffused into all four lobes.

CONCLUSIONS:
These findings demonstrate that in vivo fluorescence imaging provides functional data indicating the approximate location, magnitude, and duration of AdEGFP expression.

Summary

Type 1 diabetes (T1D), characterized by permanent destruction of insulin-producing beta cells, is lethal unless conventional exogenous insulin therapy or whole-organ transplantation is employed. Although pancreatic islet transplantation is a safer and less invasive method compared with whole-organ transplant surgery, its treatment efficacy has been limited by islet graft malfunction and graft failure. Thus, ex vivo genetic engineering of beta cells is necessary to prolong islet graft survival. For this reason, a novel gene therapy approach involving adenovirus-mediated TRAIL gene delivery into pancreatic islets was tested to determine whether this approach would defy autoreactive T cell assault in streptozotocin (STZ)-induced diabetic rats. To test this, genetically modified rat pancreatic islets were transplanted under the kidney capsule of STZ-induced diabetic rats, and diabetic status (blood sugar and body weight) was monitored after islet transplantation. STZ-induced diabetic rats carrying Ad5hTRAIL-infected islets experienced prolonged normoglycemia compared with animals grafted with mock-infected or AdCMVLacZ-infected islets. In addition, severe insulitis was detected in animals trans- planted with mock-infected or AdCMVLacZ-infected islets, whereas the severity of insulitis was reduced in animals engrafted with Ad5hTRAIL-infected islets. Thus, TRAIL overexpression in pancreatic islets extends allograft survival and function, leading to a therapeutic benefit in STZ-induced diabetic rats.

Summary

High levels of decoy receptor 2 (DcR2; TRAIL-R4) expression are correlated with TRAIL resistance in prostate cancer cells. In addition, upregulation of TRAIL death receptor (DR4 and DR5) expression, either by ionizing radiation or chemotherapy, can sensitize cancer cells to TRAIL. Considering more than half of human cancers are TRAIL resistant, modulation of surface TRAIL receptor expression appears to be an attractive treatment modality to counteract TRAIL resistance. In this study, three siRNA duplexes targeting DcR2 receptor were tested. Ad5hTRAIL infections were performed to overexpress human full-length TRAIL to induce cell death, and the in vitro tumorigenic potential of prostate cancer cells was assessed using colony-forming assays on soft agar. The DU145 and LNCaP prostate cancer cell lines, which express high levels of DcR2, were resistant to Ad5hTRAIL-induced death. Downregulation of surface DcR2 expression by siRNA sensitized these prostate cancer cell lines to Ad5hTRAIL. In addition, DcR2 siRNA-mediated knockdown of DcR2, followed by Ad5hTRAIL infection, dramatically reduced the in vitro tumorigenic potential of prostate cancer cells. Collectively, our results suggest the potential for combining receptor-specific siRNA with TRAIL in the treatment of certain cancers.

Summary

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) selectively induces apoptosis in cancer cells but not in normal cells. However, studies have indicated that more than half of human tumors exhibit TRAIL resistance. Although the mechanism of TRAIL resistance is not understood, it represents a barrier to any TRAIL-mediated gene therapy approach. In addition, no correlation between TRAIL receptor (TRAIL-R) expression profile and TRAIL resistance has been demonstrated in cancer cells. In this study, three different lung cancer cell lines and three different primary cell cultures established from patients with lung cancer (two patients with squamous cell lung carcinoma and one with adenocarcinoma) were screened for sensitivity to adenoviral delivery of TRAIL. Whereas TRAIL-resistant primary lung cell cultures and the A549 lung cancer cell line exhibited high levels of surface decoy receptor-2 (DcR2/TRAIL-R4) expression, TRAIL-sensitive lung cancer cell lines (HBE and H411) failed to express it. A DcR2 short interfering RNA (siRNA) approach involving three different siRNA constructs in combination downregulated DcR2/TRAIL-R4 expression and sensitized lung cancer cells to TRAIL-induced apoptosis. Immunohistochemical staining of samples from 10 patients with lung carcinoma suggested that high-level DcR2/TRAIL-R4 expression is a common phenotype observed in patients with non-small cell lung carcinoma.

Summary

Despite the fact that tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) can selectively induce apoptosis in cancer cells, TRAIL resistance in cancer cells has challenged the use of TRAIL as a therapeutic agent. First, prostate carcinoma cell lines (DU145, LNCaP and PC3) were screened for sensitivity to adenovirus delivery of TRAIL (Ad5hTRAIL). As amplified Ikappa B kinase (IKK) activity is responsible for the constitutive nuclear factor-B (NF-B) activation leading to uncontrolled cell growth and metastasis, a dual vector approach using both an adenovirus vector (Ad) expressing the dominant-negative mutant of IKK (AdIKKKA) and Ad5hTRAIL was employed to determine if prostate cancer cells were sensitized to TRAIL in the setting of IKK inhibition. Inhibition of the NF-B pathway through IKK blockade sensitized all three prostate cancer cell lines to TRAIL, regardless of NF-B activation or decoy receptor gene expression. Moreover, a novel quantitative real-time RT-PCR assay and conventional flow cytometry analysis indicated that TRAIL-resistant DU145 and LNCaP cells, but not TRAIL-sensitive PC3 cells, expressed substantial amounts of TRAIL Decoy Receptor 4. In conclusion, TRAIL decoy receptor expression appeared to be the chief determinant of TRAIL resistance encountered in prostate carcinoma cell lines.