Anti-inflammatory properties of exenatide in human pancreatic islets.

Exenatide is an analog of the incretin hormone glucagon-like peptide (GLP-1) that is used for the treatment of T2D for their metabolic effects. In addition to its insulinotropic effects, exenatide increases functional islet mass and improves their survival. Improved outcomes have been reported in recent clinical islet transplantation trials for the treatment of type 1 diabetes. The purpose of this study was to investigate whether exenatide has anti-inflammatory properties in human islets. Exenatide treatment improved islet function, significantly reduced content of inflammation-related molecules (tissue factor, IFN-γ, IL-17, IL-1ß, and IL-2) and caspase 3 activation, whereas increased phosphorylation of ERK1/2, STAT3, and Akt in vitro. Immunostaining showed expression of GLP-1R in ß-cells but not in α-cells. IL-1ß colocalized with GLP-1R in ß-cells. Induction of serine proteinase inhibitor 9 (PI-9) was detected after exposure of human islets to exenatide in vitro and after transplantation into immunodeficient mice. GLP-1 induced PI-9 expression in vitro but to a lower extent than exenatide. This effect was partially blocked by the antagonist exendin-9 in vitro. As assessed by immunostaining PI-9 is mostly expressed in ß-cells but not in α-cells. In conclusion, we describe anti-inflammatory and cytoprotective properties of exenatide in human islets. Exenatide-mediated PI-9 expression, the only known granzyme B inhibitor, unveils potential immunoregulatory properties.

Efficient ex vivo transduction of pancreatic islet cells with recombinant adeno-associated virus vectors.

The ability to transfer immunoregulatory, cytoprotective, or antiapoptotic genes into pancreatic islet cells may allow enhanced posttransplantation survival of islet allografts and inhibition of recurrent autoimmune destruction of these cells in type 1 diabetes. However, transient transgene expression and the tendency to induce host inflammatory responses have limited previous gene delivery studies using viral transfer vectors. We demonstrate here that recombinant adeno-associated virus (rAAV) serotype 2, a vector that can overcome these limitations, effectively transduces both human and murine pancreatic islet cells with reporter genes as well as potentially important immunoregulatory cytokine genes (interleukin-4, interleukin-10), although a very high multiplicity of infection (10,000 infectious units/islet equivalent) was required. This requirement was alleviated by switching to rAAV serotype 5, which efficiently transduced islets at a multiplicity of infection of 100. Although adenovirus (Ad) coinfection was required for efficient ex vivo expression at early time points, islets transduced without Ad expressed efficiently when they were transplanted under the renal capsule and allowed to survive in vivo. The rAAV-delivered transgenes did not interfere with islet cell insulin production and were expressed in both beta- and non-beta-cells. We believe rAAV will provide a useful tool to deliver therapeutic genes for modulating immune responses against islet cells and markedly enhance longterm graft survival.

Keratinocyte gene therapy for adenosine deaminase deficiency: a model approach for inherited metabolic disorders.

Disorders in which there is toxic buildup of circulating substrate may be treated by furnishing an enzyme reservoir capable of metabolically processing the excess substrate. The epidermal keratinocyte is a potential site for such a reservoir. In this study, we explore the capacity of genetically modified keratinocytes to metabolize extracellular substrate in a culture model that resembles in vivo epidermal architecture. Keratinocytes from adenosine deaminase (ADA)-deficient patients were transduced with a retroviral vector encoding the human ADA gene and the capacity of this tissue to deaminate deoxyadenosine (dAdo) in vitro was measured. The results show that at a substrate concentration of 10 microM, ADA-corrected keratinocytes deaminate dAdo at a rate of 0.38 nmol/min.10(6) cells. These results indicate that keratinocytes process extracellular substrate at rates that suggest complete substrate conversion in a single pass. This study provides a strong indication that the epidermis, the largest and most accessible tissue of the body, is a valuable site for designing clinically relevant gene therapies.

Loss of expression of a retrovirus-transduced gene in human keratinocytes.

Retroviral-mediated transfer of new genetic information into keratinocytes is a key step in epidermal gene therapy. An obstacle to the use of retroviruses for gene therapy is that although high levels of expression of the transduced gene can be maintained in tissue culture, expression is often lost when the cells are transplanted to an animal host. To examine some of the factors involved in this instability of expression, we transduced keratinocytes with a retrovirus encoding the gene for human factor IX and monitored secretion of the transduced gene. We observed continued secretion of factor IX through five passages in culture. When, however, sheets of these cells were grafted to athymic mice, factor IX expression was reduced or lost within 6 wk. We show that the reduction of factor IX expression in grafted keratinocytes did not result from a loss of grafted cells, nor was there a block to systemic delivery of a secreted endogenous product.

Keratinocyte gene transfer and gene therapy.

Gene therapy has moved beyond the pre-clinical stage to the treatment of a variety of inherited and acquired diseases. For such therapy to be successful, genes must be efficiently delivered to target cells and gene products must be expressed for prolonged periods of time without toxic effects to the host. This may be achieved by means of an in vivo strategy where genes are transferred directly into a host cell, or by means of an ex vivo approach through which cells are removed, cultured, targeted for gene delivery, and grafted back to the host. Several obstacles continue to delay safe and effective clinical application of gene therapy in a variety of target cells. The limited survival of transplanted cells, transient expression of transferred genes, and difficulties in targeting stem cells are technical issues requiring further investigation. Epidermal and oral keratinocytes are potential vehicles for gene therapy. Several features of these tissues can be utilized to achieve delivery of therapeutic gene products for local or systemic delivery. These qualities include: (1) the presence of stem cells; (2) the cell-, strata-, and site-specific regulation of keratinocyte gene expression; (3) tissue accessibility; and (4) secretory capacity. Such features can be exploited by the use of gene therapy strategies to facilitate: (1) identification, enrichment, and targeting of stem cells to ensure the continued presence of the transferred gene; (2) high-level and persistent transgene expression using keratinocyte-specific promoters; (3) tissue access needed for culture and grafting for ex vivo therapy and direct in vivo gene transfer; (4) secretion of transgene product for local or systemic delivery; and (5) monitoring of genetically modified tissue and removal if treatment termination is required. Optimal gene therapy strategies are being tested in a variety of tissues to treat dominant and recessive genetic disorders as well as acquired diseases such as neoplasia and infectious disease. This experience provides a basis for the application of such clinical studies to a spectrum of diseases effecting epidermal and oral keratinocytes. Gene therapy is in an early stage yet holds great promise for its ultimate clinical application.

Approaches to gene transfer in keratinocytes.

The introduction and expression of exogenous genetic material in cultured cells has provided a powerful tool for studying gene function and regulation. Immortalized cell lines have been useful for establishing gene transfer methodologies that are generally inefficient. For investigators of epidermal and mucosal biology, wishing to make use of the tissue architecture produced by primary keratinocytes in vitro, the limited life span of these cells presents a host of unique problems. Primary cells require the use of gene transfer methods that are highly efficient and will not significantly alter the cell's normal differentiation pathway. The purpose of this review is to evaluate gene transfer technology as it applies to keratinocytes.

Systemic delivery of secreted protein by grafts of epidermal keratinocytes: prospects for keratinocyte gene therapy.

Grafts of autologous keratinocytes genetically altered to secrete a new gene product are a potential vehicle for gene therapy. To consider the feasibility of such an approach, we have examined the ability of keratinocytes to secrete and deliver apolipoprotein E (apoE) to the circulation of mice bearing grafts of human keratinocytes. The grafted keratinocytes secreted two forms of apoE, an endogenous apoE encoded in the genome and a recombinant apoE encoded in a transfected gene construct. In vitro studies showed that endogenous apoE was secreted from basal keratinocytes whereas recombinant apoE was secreted from basal as well as suprabasal cells. On the basis of amounts of recombinant apoE present in the serum of grafted mice, we estimate that a graft occupying 2% of the surface area of an adult human would deliver 6.5-8.3 mg of recombinant apoE protein per day.

Secretion of apolipoprotein E by basal cells in cultures of epidermal keratinocytes.

Recently it has been shown that apolipoprotein E (apoE) secreted by keratinocytes in transplanted epidermal grafts reaches the systemic circulation. In this study we ask which cells in cultures of epidermal keratinocytes, basal or suprabasal, are the source of apoE. By fractionating disaggregated cultures in gradients of Ficoll400, the small nondifferentiated cells derived from the basal compartment were shown to be the source of apoE. The larger more differentiated cells derived from suprabasal layers could not be shown to contain or secrete apoE, although they did contain the apoE mRNA. Basal cells are the primary site for replication. However, analysis during growth in culture indicated that secretion did not correlate with cell replication but appeared to be linked to specific changes in metabolic activity of the basal cell compartment. Localization of apoE secretion to the basal compartment may provide a mechanism for lipid uptake and redistribution within the epidermis and may be viewed within the larger context of keratinocyte differentiation.

Retrovirus-mediated transduction of cultured epidermal keratinocytes.

Retrovirus-mediated gene transfer is an efficient means of introducing and expressing exogenous gene(s) in many cell types including keratinocytes. However, parameters of transduction and gene expression have not been systematically analyzed for keratinocytes. To carry out such a study we have transduced cultures of newborn foreskin cells with retroviral vectors that encode the genes for neomycin resistance (neor) and for beta-galactosidase (B-gal). The neor gene is a dominant selectable marker and the B-gal gene encodes a histochemically detectable product. Our key findings are the following: 1) all keratinocytes that form colonies can be successfully transduced at a viral titer greater than 5 x 10(6) colony-forming units/ml; 2) transduction is effected by integration of a single copy of retroviral DNA; 3) transduced cells are not at a growth disadvantage and, in fact, single clones of transduced keratinocytes can be expanded to yield over 10(9) cells, suggesting that stem cells are transduced; 4) whereas most transduced colonies exhibit B-gal staining in a high percentage of constituent cells, some colonies had a mosaic or sectored staining pattern; 5) expression of the non-selectable B-gal gene was somewhat greater in differentiated cells of the culture as compared to nondifferentiated precursors. The ability to transduce stem cells at a high efficiency and to follow expression of transduced genes in clonal progeny will allow lineage mapping in stratified epithelial tissues.

Systemic distribution of apolipoprotein E secreted by grafts of epidermal keratinocytes: implications for epidermal function and gene therapy.

In the present study, human apolipoprotein E (apoE) was monitored in the circulation of athymic mice and rats bearing human epidermal grafts. Human apoE was detected in the systemic circulation of graft-bearing animals as long as the graft remained on the animal. Within 24 hr of graft removal, human apoE was not detectable in plasma, indicating that apoE resulted from continuous production of the protein by grafted keratinocytes. These results show that proteins as large as apoE (299 amino acids) traverse the epidermal-dermal barrier and achieve systemic distribution where they may produce effects on distal tissues. The feasibility of using grafts of genetically-altered keratinocytes for the delivery of secreted proteins is clearly reinforced by the demonstration that an epidermally derived protein exhibits systemic distribution. Finally, by virtue of its systemic distribution, apoE produced in a peripheral tissue such as skin, may function in the reverse transport of cholesterol from peripheral tissues to the liver.

Synthesis and secretion of apolipoprotein E by cultured human keratinocytes.

Non-polar lipids are synthesized by keratinocytes in the epidermis and transported to the extracellular space where they contribute to formation of a permeability barrier. Transport of non-polar lipids in other organs and tissues usually occurs with the lipid complexed to an apolipoprotein. In this study we set out to learn if apolipoprotein E is produced by human epidermal keratinocytes in culture. Analysis of total cellular RNA from cultured keratinocytes showed the presence of human apolipoprotein E mRNA at concentrations ranging from 2.5 to 35 molecules/cell. The cells secrete a protein identified as apo E on the basis of molecular weight, isoform pattern, and immunoreactivity. Enzyme linked immunosorbent assay of media from keratinocyte cultures indicated that apolipoprotein E is secreted at a rate of 0.92 ng/h/10(6) cells.