Plasmid-based gene therapy of diabetes mellitus

Abstract

Type I diabetes mellitus (T1D) is due to a loss of immune tolerance to islet antigen and thus, there is intense interest in developing therapies that can re-establish it. Tolerance is maintained by complex mechanisms that include inhibitory molecules and several types of regulatory T cells (Tr). A major historical question is whether gene therapy can be employed to generate Tr cells. This review shows that gene transfer of immunoregulatory molecules can prevent T1D and other autoimmune diseases. In our studies, non-viral gene transfer is enhanced by in vivo electroporation (EP). This technique can be used to perform DNA vaccination against islet cell antigens and when combined with appropriate immune ligands results in the generation of Tr cells and protection against T1D. In vivo EP can also be applied for non-immune therapy of diabetes. It can be used to deliver protein drugs such as glucagon-like peptide 1 (GLP-1), leptin or transforming growth factor beta (TGF-b). These act in T1D or type II diabetes (T2D) by restoring glucose homeostasis, promoting islet cell survival and growth or improving wound healing and other complications. Furthermore, we show that in large animals EP can deliver peptide hormones, such as growth hormone releasing hormone (GHRH). We conclude that the non-viral gene therapy and EP represent a safe and efficacious approach with clinical potential.

Prototype development and preclinical immunogenicity analysis of a novel minimally invasive electroporation device

“…In this study, we targeted the induction of protective humoral responses against influenza A/H1N1 strains, including the novel influenza A/Mexico/09/H1N1 (2009). Having failed to achieve appreciable expression of plasmid through IM/ID injection alone (without EP) at these doses (GFP data above; and previous immunogenicity studies with DNA alone, [17][18][19][20][21][22] we focused our experiments instead on determining the level of humoral immune responses induced by MID delivery. Animals were vaccinated with a combination of plasmids targeting the H1N1 strains (100 mg per plasmid) containing pGX2005 (SynCon vaccine construct that encodes a consensus sequence of hemagglutinin (HA) from H1N1 viruses) 27 and pGX2009 (SynCon vaccine construct that encodes a consensus HA sequence derived from the swine origin influenza A/H1N1 (2009) strains in a volume of 50 ml diluted in 1Âphosphate-buffered saline (PBS).…”

“…A visual estimation of enhancement of expression is in excess of 100-1000-fold based on number of GFP positive cells relative to DNA alone and is consistent with previous reports of enhancement of expression achieved with other EP devices. [18][19][20] Minimally invasive EP results in a positive transfection in a wide spectrum of species To ensure that the robust transfection was not a guinea pig-specific phenomenon, we carried out GFP localization in a wide range of species. Skin sites on mice, rabbits, rats and pigs were injected with 50 ml (30 ml in mice) of 1 mg ml À1 GFP plasmid and immediately pulsed using MID.…”

“…As such, EP increases the uptake, as well as the extent to which drugs and DNA are delivered to their target tissue. [17][18][19][20][21] Although the precise mechanism by which EP functions has not been elucidated, a proposed theoretical model involves a poration event because of the destabilization of the membrane, followed by the electrophoretic movement of charged molecules into the cell. For EP to occur the formation of pores requires that a threshold energy be achieved and the movement produced by the electrophoretic effect depends on both the electric field and the pulse length.…”

Combined effects of IL-12 and electroporation enhances the potency of DNA vaccination in macaques

“…Electroporation (EP), a method traditionally used for enhancing the transfection efficiency of cells in vitro, has been studied to enhance the uptake and expression of both therapeutic and vaccine plasmids [20,21] and increase immune responses in tumor animal models [22]. More recently, EP has been studied as a method to enhance the potency of DNA vaccines in primates with encouraging results [23,24].…”

Transfection of Rat Pancreatic Islet Tissue by Polymeric Gene Vectors

“…It was hypothesized that such genetic modification would improve the viability and functionality of transplanted islets. [9][10][11][12][13] Islet transfection has mostly been conducted using viral or liposomal gene carriers. The use of viral vectors raises several safety concerns (e.g., toxicity, mutagenesis, carcinogenesis, and antiviral responses) [14][15][16][17] and presents serious technical challenges (e.g., limited loading capacity, limited gene size, and difficulties in production=purification).…”