High-Titer Production of HIV-Based Lentiviral Vectors in Roller Bottles for Gene and Cell Therapy.

Lentiviral vectors are becoming preferred vectors of choice for clinical gene therapy trials due to their safety, efficacy, and the long-term gene expression they provide. Although the efficacy of lentiviral vectors is mainly predetermined by the therapeutic genes they carry, they must be produced at high titers to exert therapeutic benefit for in vivo applications. Thus, there is need for practical, robust, and scalable viral vector production methods applicable to any laboratory setting. Here, we describe a practical lentiviral production technique in roller bottles yielding high-titer third-generation lentiviral vectors useful for in vivo gene transfer applications. CaPO4-mediated transient transfection protocol involving the use of a transfer vector and three different packaging plasmids is employed to generate lentivectors in roller bottles. Following clearance of cellular debris via low-speed centrifugation and filtration, virus is concentrated by high-speed ultracentrifugation over sucrose cushion.

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.

Therapeutic Potential of Lentivirus-Mediated Glucagon-Like Peptide-1 Gene Therapy for Diabetes.

Postprandial glucose-induced insulin secretion from the islets of Langerhans is facilitated by glucagon-like peptide-1 (GLP-1)-a metabolic hormone with insulinotropic properties. Among the variety of effects it mediates, GLP-1 induces delta cell secretion of somatostatin, inhibits alpha cell release of glucagon, reduces gastric emptying, and slows food intake. These events collectively contribute to weight loss over time. During type 2 diabetes (T2DM), however, the incretin response to glucose is reduced and accompanied by a moderate reduction in GLP-1 secretion. To compensate for the reduced incretin effect, a human immunodeficiency virus-based lentiviral vector was generated to deliver DNA encoding human GLP-1 (LentiGLP-1), and the anti-diabetic efficacy of LentiGLP-1 was tested in a high-fat diet/streptozotocin-induced model of T2DM. Therapeutic administration of LentiGLP-1 reduced blood glucose levels in obese diabetic Sprague Dawley rats, along with improving insulin sensitivity and glucose tolerance. Normoglycemia was correlated with increased blood GLP-1 and pancreatic beta cell regeneration in LentiGLP-1-treated rats. Plasma triglyceride levels were also normalized after LentiGLP-1 injection. Collectively, these data suggest the clinical potential of GLP-1 gene transfer therapy for the treatment of T2DM.

Incretins: their physiology and application in the treatment of diabetes mellitus.

Therapies targeting the action of incretin hormones have been under close scrutiny in recent years. The incretin effect has been defined as postprandial enhancement of insulin secretion by gut-derived factors. Likewise, incretin mimetics and incretin effect amplifiers are the two different incretin-based treatment strategies developed for the treatment of diabetes. Although, incretin mimetics produce effects very similar to those of natural incretin hormones, incretin effect amplifiers act by inhibiting dipeptidyl peptidase-4 (DPP-4) enzyme to increase plasma concentration of incretins and their biologic effects. Because glucagon-like peptide-1 (GLP-1) is an incretin hormone with various anti-diabetic actions including stimulation of glucose-induced insulin secretion, inhibition of glucagon secretion, hepatic glucose production and gastric emptying, it has been evaluated as a novel therapeutic agent for the treatment of type 2 diabetes mellitus (T2DM). GLP-1 also manifests trophic effects on pancreas such as pancreatic beta cell growth and differentiation. Because DPP-4 is the enzyme responsible for the inactivation of GLP-1, DPP-4 inhibition represents another potential strategy to increase plasma concentration of GLP-1 to enhance the incretin effect. Thus, anti-diabetic properties of these two classes of drugs have stimulated substantial clinical interest in the potential of incretin-based therapeutic agents as a means to control glucose homeostasis in T2DM patients. Despite this fact, clinical use of GLP-1 mimetics and DPP-4 inhibitors have raised substantial concerns owing to possible side effects of the treatments involving increased risk for pancreatitis, and C-cell adenoma/carcinoma. Thus, controversial issues in incretin-based therapies under development are reviewed and discussed in this manuscript.