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.

Lentivirus Mediated Pancreatic Beta-Cell-Specific Insulin Gene Therapy for STZ-Induced Diabetes.

Autoimmune destruction of pancreatic beta cells is the characteristic feature of type 1 diabetes mellitus. Consequently, both short- and intermediate-acting insulin analogs are under development to compensate for the lack of endogenous insulin gene expression. Basal insulin is continuously released at low levels in response to hepatic glucose output, while post-prandial insulin is secreted in response to hyperglycemia following a meal. As an alternative to multiple daily injections of insulin, glucose-regulated insulin gene expression by gene therapy is under development to better endure postprandial glucose excursions. Controlled transcription and translation of proinsulin, presence of glucose-sensing machinery, prohormone convertase expression, and a regulated secretory pathway are the key features unique to pancreatic beta cells. To take advantage of these hallmarks, we generated a new lentiviral vector (LentiINS) with an insulin promoter driving expression of the proinsulin encoding cDNA to sustain pancreatic beta-cell-specific insulin gene expression. Intraperitoneal delivery of HIV-based LentiINS resulted in the lowering of fasting plasma glucose, improved glucose tolerance and prevented weight loss in streptozoticin (STZ)-induced diabetic Wistar rats. However, the combinatorial use of LentiINS and anti-inflammatory lentiviral vector (LentiVIP) gene therapy was required to increase serum insulin to a level sufficient to suppress non-fasting plasma glucose and diabetes-related inflammation.

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.