Generation of a Beta-Cell Transplant Animal Model of Diabetes Using CRISPR Technology.

Since insulin deficiency results from pancreatic beta-cell destruction, all type 1 and most type 2 diabetes patients eventually require life-long insulin injections. Insulin gene synthesis could also be impaired due to insulin gene mutations as observed in diabetic patients with MODY 10. At this point, insulin gene therapy could be very effective to recompense insulin deficiency under these circumstances. For this reason, an HIV-based lentiviral vector carrying the insulin gene under the control of insulin promoter (LentiINS) was generated, and its therapeutic efficacy was tested in a beta-cell transplant model lacking insulin produced by CRISPR/Cas9-mediated genetically engineered pancreatic beta cells. To generate an insulin knockout beta-cell transplant animal model of diabetes, a dual gene knockout plasmid system involving CRISPR/Cas9 was transfected into a mouse pancreatic beta cell line (Min6). Fluorescence microscopy and antibiotic selection were utilized to select the insulin gene knockout clones. Transplantation of the genetically engineered pancreatic beta cells under the kidney capsule of STZ-induced diabetic rats revealed LentiINS- but not LentiLacZ-infected Ins2KO cells transiently reduced hyperglycemia similar to that of MIN6 in diabetic animals. These results suggest LentiINS has the potential to functionally restore insulin production in an insulin knockout beta-cell transplant animal model of diabetes.

Genome engineering and disease modeling via programmable nucleases for insulin gene therapy; promises of CRISPR/Cas9 technology.

Targeted genome editing is a continually evolving technology employing programmable nucleases to specifically change, insert, or remove a genomic sequence of interest. These advanced molecular tools include meganucleases, zinc finger nucleases, transcription activator-like effector nucleases and RNA-guided engineered nucleases (RGENs), which create double-strand breaks at specific target sites in the genome, and repair DNA either by homologous recombination in the presence of donor DNA or via the error-prone non-homologous end-joining mechanism. A recently discovered group of RGENs known as CRISPR/Cas9 gene-editing systems allowed precise genome manipulation revealing a causal association between disease genotype and phenotype, without the need for the reengineering of the specific enzyme when targeting different sequences. CRISPR/Cas9 has been successfully employed as an ex vivo gene-editing tool in embryonic stem cells and patient-derived stem cells to understand pancreatic beta-cell development and function. RNA-guided nucleases also open the way for the generation of novel animal models for diabetes and allow testing the efficiency of various therapeutic approaches in diabetes, as summarized and exemplified in this manuscript.

Current Update on Severe Acute Respiratory Syndrome Coronavirus 2 Vaccine Development with a Special Emphasis on Gene Therapy Viral Vector Design and Construction for Vaccination.

Severe acute respiratory syndrome (SARS) is a newly emerging infectious disease (COVID-19) caused by the novel coronavirus SARS-coronavirus 2 (CoV-2). To combat the devastating spread of SARS-CoV-2, extraordinary efforts from numerous laboratories have focused on the development of effective and safe vaccines. Traditional live-attenuated or inactivated viral vaccines are not recommended for immunocompromised patients as the attenuated virus can still cause disease via phenotypic or genotypic reversion. Subunit vaccines require repeated dosing and adjuvant use to be effective, and DNA vaccines exhibit lower immune responses. mRNA vaccines can be highly unstable under physiological conditions. On the contrary, naturally antigenic viral vectors with well-characterized structure and safety profile serve as among the most effective gene carriers to provoke immune response via heterologous gene transfer. Viral vector-based vaccines induce both an effective cellular immune response and a humoral immune response owing to their natural adjuvant properties via transduction of immune cells. Consequently, viral vectored vaccines carrying the SARS-CoV-2 spike protein have recently been generated and successfully used to activate cytotoxic T cells and develop a neutralizing antibody response. Recent progress in SARS-CoV-2 vaccines, with an emphasis on gene therapy viral vector-based vaccine development, is discussed in this review.

Lentiviral gene therapy vectors encoding VIP suppressed diabetes-related inflammation and augmented pancreatic beta-cell proliferation.

Type 1 diabetes (T1DM) is an autoimmune condition in which the immune system attacks and destroys insulin-producing beta cells in the pancreas leading to hyperglycemia. Vasoactive intestinal peptide (VIP) manifests insulinotropic and anti-inflammatory properties, which are useful for the treatment of diabetes. Because of its limited half-life due to DPP-4-mediated degradation, constant infusions or multiple injections are needed to observe any therapeutic benefit. Since gene therapy has the potential to treat genetic diseases, an HIV-based lentiviral vector carrying VIP gene (LentiVIP) was generated to provide a stable VIP gene expression in vivo. The therapeutic efficacy of LentiVIP was tested in a multiple low-dose STZ-induced animal model of T1DM. LentiVIP delivery into diabetic animals reduced hyperglycemia, improved glucose tolerance, and prevented weight loss. Also, a decrease in serum CRP levels, and serum oxidant capacity, but an increase in antioxidant capacity were observed in LentiVIP-treated animals. Restoration of islet cell mass was correlated with an increase in pancreatic beta-cell proliferation. These beneficial results suggest the therapeutic effect of LentiVIP is due to the repression of diabetes-induced inflammation, its insulinotropic properties, and VIP-induced beta-cell proliferation.

High-Grade Purification of Third-Generation HIV-Based Lentiviral Vectors by Anion Exchange Chromatography for Experimental Gene and Stem Cell Therapy Applications.

Lentiviral vectors (LVs) have been increasingly used in clinical gene therapy applications particularly due to their efficient gene transfer ability, lack of interference from preexisting viral immunity, and long-term gene expression they provide. Purity of LVs is essential in in vivo applications, for a high therapeutic benefit with minimum toxicity. Accordingly, laboratory scale production of LVs frequently involves transient cotransfection of 293T cells with packaging and transfer plasmids in the presence of CaPO4. After clearance of the cellular debris by low-speed centrifugation and filtration, lentivectors are usually concentrated by high-speed ultracentrifugation in sucrose cushion. Concentrated viral samples are then purified by anion exchange chromatography (AEX) after benzonase treatment to remove the residual cellular DNA. Here, we describe an improved practical method for LV purification using AEX, useful for experimental studies concerning gene and stem cell therapy.

HIV-based lentivirus-mediated vasoactive intestinal peptide gene delivery protects against DIO animal model of Type 2 diabetes.

Type 2 diabetes mellitus (T2DM) is characterised by insulin resistance, glucose intolerance and beta cell loss leading to hyperglycemia. Vasoactive intestinal peptide (VIP) has been regarded as a novel therapeutic agent for the treatment of T2DM because of its insulinotropic and anti-inflammatory properties. Despite these beneficial properties, VIP is extremely sensitive to peptidases (DPP-4) requiring constant infusion or multiple injections to observe any therapeutic benefit. Thus, we constructed an HIV-based lentiviral vector encoding human VIP (LentiVIP) to test the therapeutic efficacy of VIP peptide in a diet-induced obesity (DIO) animal model of T2DM. VIP gene expression was shown by immunocytochemistry (ICC) and VIP peptide secretion was confirmed by ELISA both in HepG2 liver and MIN6 pancreatic beta cell lines. Functional properties of VIP were demonstrated by cAMP production assay and glucose-stimulated insulin secretion test (GSIS). Intraperitoneal (IP) delivery of LentiVIP vectors into mice significantly increased serum VIP concentrations compared to control mice. Most importantly, LentiVIP delivery in DIO animal model of T2DM resulted in improved insulin sensitivity, glucose tolerance and protection against STZ-induced diabetes in addition to reduction in serum triglyceride/cholesterol levels. Collectively, these data suggest LentiVIP delivery should be evaluated as an experimental therapeutic approach for the treatment of T2DM.

Clinical utility of insulin and insulin analogs.

Diabetes is a pandemic disease characterized by autoimmune, genetic and metabolic abnormalities. While insulin deficiency manifested as hyperglycemia is a common sequel of both Type-1 and Type-2 diabetes (T1DM and T2DM), it does not result from a single genetic defect--rather insulin deficiency results from the functional loss of pancreatic ß cells due to multifactorial mechanisms. Since pancreatic ß cells of patients with T1DM are destroyed by autoimmune reaction, these patients require daily insulin injections. Insulin resistance followed by ß cell dysfunction and ß cell loss is the characteristics of T2DM. Therefore, most patients with T2DM will require insulin treatment due to eventual loss of insulin secretion. Despite the evidence of early insulin treatment lowering macrovascular (coronary artery disease, peripheral arterial disease and stroke) and microvascular (diabetic nephropathy, neuropathy and retinopathy) complications of T2DM, controversy exists among physicians on how to initiate and intensify insulin therapy. The slow acting nature of regular human insulin makes its use ineffective in counteracting postprandial hyperglycemia. Instead, recombinant insulin analogs have been generated with a variable degree of specificity and action. Due to the metabolic variability among individuals, optimum blood glucose management is a formidable task to accomplish despite the presence of novel insulin analogs. In this article, we present a recent update on insulin analog structure and function with an overview of the evidence on the various insulin regimens clinically used to treat diabetes.

Insulin gene therapy from design to beta cell generation.

Despite the fact that insulin injection can protect diabetic patients from developing diabetes-related complications, recent meta-analyses indicate that rapid and long-acting insulin analogues only provide a limited benefit compared with conventional insulin regarding glycemic control. As insulin deficiency is the main sequel of type-1 diabetes (T1D), transfer of the insulin gene-by-gene therapy is becoming an attractive treatment modality even though T1D is not caused by a single genetic defect. In contrast to human insulin and insulin analogues, insulin gene therapy targets to supplement patients not only with insulin but also with C-peptide. So far, insulin gene therapy has had limited success because of delayed and/or transient gene expression. Sustained insulin gene expression is now feasible using current gene-therapy vectors providing patients with basal insulin coverage, but management of postprandial hyperglycaemia is still difficult to accomplish because of the inability to properly control insulin secretion. Enteroendocrine cells of the gastrointestinal track (K cells and L cells) may be ideal targets for insulin gene therapy, but cell-targeting difficulties have limited practical implementation of insulin gene therapy for diabetes treatment. Therefore, recent gene transfer technologies developed to generate authentic beta cells through transdifferentiation are also highlighted in this review.

Therapeutic potential of VIP vs PACAP in diabetes.

Type 2 diabetes (T2D) is characterized by chronic insulin resistance and a progressive decline in beta-cell function. Although rigorous glucose control can reduce morbidity and mortality associated with diabetes, achieving optimal long-term glycemic control remains to be accomplished in many diabetic patients. As beta-cell mass and function inevitably decline in T2D, exogenous insulin administration is almost unavoidable as a final outcome despite the use of oral antihyperglycemic agents in many diabetic patients. Pancreatic islet cell death, but not the defect in new islet formation or beta-cell replication, has been blamed for the decrease in beta-cell mass observed in T2D patients. Thus, therapeutic approaches designed to protect islet cells from apoptosis could significantly improve the management of T2D, because of its potential to reverse diabetes not just ameliorate glycemia. Therefore, an ideal beta-cell-preserving agent is expected to protect beta cells from apoptosis and stimulate postprandial insulin secretion along with increasing beta-cell replication and/or islet neogenesis. One such potential agent, the islet endocrine neuropeptide vasoactive intestinal peptide (VIP) strongly stimulates postprandial insulin secretion. Because of its broad spectrum of biological functions such as acting as a potent anti-inflammatory factor through suppression of Th1 immune response, and induction of immune tolerance via regulatory T cells, VIP has emerged as a promising therapeutic agent for the treatment of many autoimmune diseases including diabetes.

Tracing of islet graft survival by way of in vivo fluorescence imaging.

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.

NF-κB targeting by way of IKK inhibition sensitizes lung cancer cells to adenovirus delivery of TRAIL.

BACKGROUND: Lung cancer causes the highest rate of cancer-related deaths both in men and women. As many current treatment modalities are inadequate in increasing patient survival, new therapeutic strategies are required. TNF-related apoptosis-inducing ligand (TRAIL) selectively induces apoptosis in tumor cells but not in normal cells, prompting its current evaluation in a number of clinical trials. The successful therapeutic employment of TRAIL is restricted by the fact that many tumor cells are resistant to TRAIL. The goal of the present study was to test a novel combinatorial gene therapy modality involving adenoviral delivery of TRAIL (Ad5hTRAIL) and IKK inhibition (AdIKKßKA) to overcome TRAIL resistance in lung cancer cells. METHODS: Fluorescent microscopy and flow cytometry were used to detect optimum doses of adenovirus vectors to transduce lung cancer cells. Cell viability was assessed via a live/dead cell viability assay. Luciferase assays were employed to monitor cellular NF-κB activity. Apoptosis was confirmed using Annexin V binding. RESULTS: Neither Ad5hTRAIL nor AdIKKßKA infection alone induced apoptosis in A549 lung cancer cells, but the combined use of Ad5hTRAIL and AdIKKßKA significantly increased the amount of A549 apoptosis. Luciferase assays demonstrated that both endogenous and TRAIL-induced NF-κB activity was down-regulated by AdIKKßKA expression. CONCLUSIONS: Combination treatment with Ad5hTRAIL and AdIKKßKA induced significant apoptosis of TRAIL-resistant A549 cells, suggesting that dual gene therapy strategy involving exogenous TRAIL gene expression with concurrent IKK inhibition may be a promising novel gene therapy modality to treat lung cancer.

Tumor necrosis factor-related apoptosis inducing ligand-R4 decoy receptor expression is correlated with high Gleason scores, prostate-specific antigen recurrence, and decreased survival in patients with prostate carcinoma.

OBJECTIVE: Tumor necrosis factor-related apoptosis inducing ligand (TRAIL) has recently been investigated because of its ability to selectively kill cancer cells. Despite recent publications mainly focusing on TRAIL resistance in cancer cells, little is known about how TRAIL contributes to the carcinogenesis process. Because the expression patterns of TRAIL and its receptors in patients with prostate carcinoma have recently been reported, this study investigated the significance of TRAIL and TRAIL receptor expression in connection to serum prostate-specific antigen (PSA) and Gleason scoring. MATERIALS AND METHODS: A total of 98 patients were included in the study. Gleason scores, PSA, TRAIL, and TRAIL receptor expressions were used for the comparison purposes. The Spearman rho correlation test was administered to reveal the correlations among the variants. The Kruskal Wallis-Mann Whitney U or Friedman-Wilcoxon signed ranks test determined the statistical significance between the pairs. Multinomial and/or multiple binary logistic regression analyses were deployed to test whether TRAIL markers were independent variables to predict the prognosis of prostate cancer. Kaplan-Meier and log-rank tests were used to determine the survival rates. RESULTS: High-serum PSA levels were correlated with higher levels of TRAIL and TRAIL receptor expressions. Patients with high Gleason scores had higher levels of TRAIL-R4 decoy receptor expression but lower levels of TRAIL death ligand expression. CONCLUSIONS: TRAIL-R4 decoy receptor expression is strongly correlated with PSA recurrence, which is suggestive of poor prognosis. High levels of TRAIL-R4 expression but low levels of TRAIL death ligand expression are connected to decreased survival.

TRAIL death receptor-4 expression positively correlates with the tumor grade in breast cancer patients with invasive ductal carcinoma.

PURPOSE: Tumor necrosis factor-related apoptosis inducing ligand (TRAIL) selectively induces apoptosis in cancer cells but not in normal cells, and a number of clinical trials have recently been initiated to test the safety and antitumoral potential of TRAIL in cancer patients. Four different receptors have been identified to interact with TRAIL: two are death-inducing receptors (TRAIL-R1 [DR4] and TRAIL-R2 [DR5]), whereas the other two (TRAIL-R3 [DcR1] and TRAIL-R4 [DcR2]) do not induce death upon ligation and are believed to counteract TRAIL-induced cytotoxicity. Because high levels of DcR2 expression have recently been correlated with carcinogenesis in the prostate and lung, this study investigated the importance of TRAIL and TRAIL receptor expression in breast cancer patients with invasive ductal carcinoma, taking various prognostic markers into consideration. METHODS AND MATERIALS: Immunohistochemical analyses were performed on 90 breast cancer patients with invasive ductal carcinoma using TRAIL and TRAIL receptor-specific antibodies. Age, menopausal status, tumor size, lymph node status, tumor grade, lymphovascular invasion, perineural invasion, extracapsular tumor extension, presence of an extensive intraductal component, multicentricity, estrogen and progesterone receptor status, and CerbB2 expression levels were analyzed with respect to TRAIL/TRAIL receptor expression patterns. RESULTS: The highest TRAIL receptor expressed in patients with invasive ductal carcinoma was DR4. Although progesterone receptor-positive patients exhibited lower DR5 expression, CerbB2-positive tissues displayed higher levels of both DR5 and TRAIL expressions. CONCLUSIONS: DR4 expression positively correlates with the tumor grade in breast cancer patients with invasive ductal carcinoma.

Decoy receptor-2 small interfering RNA (siRNA) strategy employing three different siRNA constructs in combination defeats adenovirus-transferred tumor necrosis factor-related apoptosis-inducing ligand resistance in lung cancer cells.

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.

Surface TRAIL decoy receptor-4 expression is correlated with TRAIL resistance in MCF7 breast cancer cells.

BACKGROUND: Tumor Necrosis Factor (TNF)-Related Apoptosis-Inducing Ligand (TRAIL) selectively induces apoptosis in cancer cells but not in normal cells. Despite this promising feature, TRAIL resistance observed in cancer cells seriously challenged the use of TRAIL as a death ligand in gene therapy. The current dispute concerns whether or not TRAIL receptor expression pattern is the primary determinant of TRAIL sensitivity in cancer cells. This study investigates TRAIL receptor expression pattern and its connection to TRAIL resistance in breast cancer cells. In addition, a DcR2 siRNA approach and a complementary gene therapy modality involving IKK inhibition (AdIKKbetaKA) were also tested to verify if these approaches could sensitize MCF7 breast cancer cells to adenovirus delivery of TRAIL (Ad5hTRAIL). METHODS: TRAIL sensitivity assays were conducted using Molecular Probe's Live/Dead Cellular Viability/Cytotoxicity Kit following the infection of breast cancer cells with Ad5hTRAIL. The molecular mechanism of TRAIL induced cell death under the setting of IKK inhibition was revealed by Annexin V binding. Novel quantitative Real Time RT-PCR and flow cytometry analysis were performed to disclose TRAIL receptor composition in breast cancer cells. RESULTS: MCF7 but not MDA-MB-231 breast cancer cells displayed strong resistance to adenovirus delivery of TRAIL. Only the combinatorial use of Ad5hTRAIL and AdIKKbetaKA infection sensitized MCF7 breast cancer cells to TRAIL induced cell death. Moreover, novel quantitative Real Time RT-PCR assays suggested that while the level of TRAIL Decoy Receptor-4 (TRAIL-R4) expression was the highest in MCF7 cells, it was the lowest TRAIL receptor expressed in MDA-MB-231 cells. In addition, conventional flow cytometry analysis demonstrated that TRAIL resistant MCF7 cells exhibited substantial levels of TRAIL-R4 expression but not TRAIL decoy receptor-3 (TRAIL-R3) on surface. On the contrary, TRAIL sensitive MDA-MB-231 cells displayed very low levels of surface TRAIL-R4 expression. Furthermore, a DcR2 siRNA approach lowered TRAIL-R4 expression on surface and this sensitized MCF7 cells to TRAIL. CONCLUSION: The expression of TRAIL-R4 decoy receptor appeared to be well correlated with TRAIL resistance encountered in breast cancer cells. Both adenovirus mediated IKKbetaKA expression and a DcR2 siRNA approach sensitized MCF7 breast cancer cells to TRAIL.

The assessment of PTEN tumor suppressor gene in combination with Gleason scoring and serum PSA to evaluate progression of prostate carcinoma.

OBJECTIVE: The purpose of the study was to determine if the tumor suppressor gene phosphate and tensin homolog (PTEN) (mutated in multiple advanced cancers 1) in combination with Gleason scoring and serum prostate specific antigen (PSA) could be employed to better predict the progression of prostate carcinoma. MATERIALS AND METHODS: The study group consisted of 43 patients with benign prostate hyperplasia (BPH), 15 with organ confined prostate carcinoma (OCPCa), and 18 with advanced prostate carcinoma (APCa). Prostate tissue samples were obtained from radical prostatectomy, transurethral resection, and TRUS guided trans-rectal needle biopsy and then evaluated for biomarker expression. The clinical stage was assessed according to tumor node metastasis classification and grade according to Gleason system. Serum PSA was measured by conventional techniques and Western blotting analysis was used to determine PTEN expression in the primary tissue. Multivariate analysis was performed to analyze whether these markers could individually predict the progression of prostate carcinoma. RESULTS: APCa patients displayed higher Gleason scores and serum PSA levels. But much lower PTEN expression was detected in prostate of APCa patients compared to patients with BPH or OCPCa. Hormone refractory (HR) and hormone sensitive (HS) APCa cases did not yield any significant differences in terms of Gleason scoring, serum PSA and PTEN expression. PSA levels were significantly higher in patients with OCPCa or APCa compared to patients with BPH. CONCLUSION: Our results suggested that both PTEN and serum PSA appeared to be useful as independent markers to depict the nature of tumor behavior as benign or malign. In addition, PTEN also appeared to be useful as an independent marker to predict the progression of prostate carcinoma.

Novel approaches to augment adeno-associated virus type-2 endocytosis and transduction.

Recombinant adeno-associated virus (rAAV) receptor binding, endocytosis, nuclear trafficking and second strand gene conversion have been described as potential rate-limiting steps in rAAV type-2 (rAAV-2) transduction. Several strategies have been developed to enhance rAAV-2 intracellular trafficking and gene conversion in an attempt to increase the efficiency of this virus as a gene therapy vector. To this end, the current study has investigated novel methods for augmenting rAAV transduction by enhancing endocytosis of rAAV-2. A selective trypsinization assay demonstrated that the abundance of internalized rAAV ssDNA was increased only in cells treated with both pyrrolidinedithiocarbonate (PDTC) and a genotoxic agent. Treating cells with each of these agents alone had no effect on rAAV endocytosis in comparison to controls. To investigate the mechanisms of this synergistic effect on rAAV transduction, the involvement of Rac1 protein was evaluated. Inhibition of the Rac1 pathway by expression of a dominant negative mutant of Rac1 (N17Rac1) decreased rAAV transduction. In contrast, expression of a dominant active form of Rac1 (V12Rac1) alone mimicked the up-regulated response seen in the presence of PDTC and genotoxic agents. These studies provide potential insights into the importance of the Rac1 pathway to enhance uptake of rAAV-2.

Fundamental principals of tumor necrosis factor-alpha gene therapy approach and implications for patients with lung carcinoma.

Apoptosis, known as programmed cell death, is defined as a cell's preferred form of death under hectic conditions through genetically conserved and complex pathways. There is a decisive balance between stimulatory and inhibitory signaling pathways to maintain homeostasis in cells. In order to shift the balance towards apoptosis, the modulation of both apoptotic and anti-apoptotic pathways represents an attractive target for cancer therapeutics. Currently, chemotherapy and radiotherapy are among the most commonly used treatment modalities against lung cancer. Tumor suppressor gene, p53, is required in order for both of these treatment methods to work as anti-tumor agents. As a result, tumors lacking p53 display resistance to both chemotherapy and radiotherapy. However, death ligands induce apoptosis regardless of p53 status of cells. Thus, these methods constitute a complementary therapeutic approach to currently employed conventional treatment modalities. At present, death ligands are being evaluated as potential cancer therapeutic agents. Since resistance to tumor necrosis factor (TNF)-alpha-mediated apoptosis represented an obstacle for the treatment of patients with lung carcinoma in the earlier attempts, an extensive research was recently initiated to understand molecular mechanism of TNF-alpha signaling. NF-kappaB transcription factors have been demonstrated to modulate the apoptotic program, mostly as blockers of apoptosis in different cell types. In this review, we concentrate on the current progress in the understanding of TNF-alpha-mediated apoptosis for lung carcinoma. Representative models of NF-kappaB-inhibiting gene therapy strategies from various labs including ours are also provided as examples of up-to-date approaches to defeat TNF resistance. In order to give the reader better understanding and appreciation of such approaches, previously unpublished in vivo assays are also incorporated into this review. Current progress in clinical trials using adenovirus-mediated delivery of TNF-alpha is also discussed.