Diabetes is a disease of epidemic proportions and is

We have previously demonstrated that adenoviral gene transfer of the NF-B inhibitor I B to human islets results in protection from interleukin (IL)-1␤-mediated dysfunction and apoptosis. Here we report that human and mouse islets can be efficiently transduced by a cationic peptide transduction domain (PTD-5) without impairment of islet function. PTD mediated delivery of a peptide inhibitor of the IL-1␤-induced I B kinase (IKK), derived from IKK␤ (NBD; Nemo-binding domain), and completely blocked the detrimental effects of IL-1␤ on islet function and NF-B activity, in a similar manner to Ad-I B. We also demonstrate that mouse islets can be transduced in situ by infusion of the transduction peptide through the bile duct prior to isolation, resulting in 40% peptide transduction of the ␤-cells. Delivery of the IKK inhibitor transduction fusion peptide (PTD-5-NBD) in situ to mouse islets resulted in improved islet function and viability after isolation. These results demonstrate the feasibility of using PTD-mediated delivery to transiently modify islets in situ to improve their viability and function during isolation, prior to transplantation.

ObjectivesThe CHARM-01 study characterized the safety, acceptability, pharmacokinetics (PK), and pharmacodynamics (PD) of three tenofovir (TFV) gels for rectal application. The vaginal formulation (VF) gel was previously used in the CAPRISA 004 and VOICE vaginal microbicide Phase 2B trials and the RMP-02/MTN-006 Phase 1 rectal safety study. The reduced glycerin VF (RGVF) gel was used in the MTN-007 Phase 1 rectal microbicide trial and is currently being evaluated in the MTN-017 Phase 2 rectal microbicide trial. A third rectal specific formulation (RF) gel was also evaluated in the CHARM-01 study.MethodsParticipants received 4 mL of the three TFV gels in a blinded, crossover design: seven daily doses of RGVF, seven daily doses of RF, and six daily doses of placebo followed by one dose of VF, in a randomized sequence. Safety, acceptability, compartmental PK, and explant PD were monitored throughout the trial.ResultsAll three gels were found to be safe and acceptable. RF and RGVF PK were not significantly different. Median mucosal mononuclear cell (MMC) TFV-DP trended toward higher values for RF compared to RGVF (1136 and 320 fmol/106 cells respectively). Use of each gel in vivo was associated with significant inhibition of ex vivo colorectal tissue HIV infection. There was also a significant negative correlation between the tissue levels of TFV, tissue TFV-DP, MMC TFV-DP, rectal fluid TFV, and explant HIV-1 infection.ConclusionsAll three formulations were found to be safe and acceptable. However, the safety profile of the VF gel was only based on exposure to one dose whereas participants received seven doses of the RGVF and RF gels. There was a trend towards higher tissue MMC levels of TFV-DP associated with use of the RF gel. Use of all gels was associated with significant inhibition of ex vivo tissue HIV infection.Trial RegistrationClinicalTrials.gov NCT01575405

Transplantation of allogeneic pancreatic islets is an effective approach to treat type 1 diabetes. To bypass the need for systemic administration of immunosuppression drugs following transplantation, approaches to genetically modify allogeneic islets to express anti-inflammatory, immunosuppressive, or antiapoptotic proteins prior to transplantation are being developed. Adeno-associated viral (AAV) based vectors have been used for gene transfer to islets, but the efficiency of functional transduction is low. Recently, doublestranded (ds) or double-copy (dc) based AAV vectors have been developed that allow for more rapid and efficient AAVmediated transgene expression following transduction. Here we demonstrate that intact human and murine islets can be transduced with dsAAV2-eGFP efficiently compared to single-stranded AAV2-eGFP. Furthermore, our results demonstrate that murine islets transduced with dsAAV2-eGFP have normal islet glucose responsiveness, viability, and islet insulin content. Transplantation of the dsAAV2-eGFP transduced islet restored normal glycemia in diabetic mice without eliciting an immune response. Significant dsAAV2-mediated eGFP expression was observed in the islet grafts for at least 6 months post-transplant. Finally, we demonstrated that dsAAV serotypes 2, 6, and 8 infect human islets efficiently. Taken together, these results suggest that dsAAV based vectors are highly appropriate for gene transfer to islets to facilitate transplantation.

We have demonstrated the ability to deliver and express genes specifically in beta-cells for at least 6 months, using a murine insulin promoter (mIP) in a double-stranded, self-complementary AAV vector (dsAAV8-mIP). In this study, we evaluated the effects of dsAAV8-mIP-mediated delivery of interleukin 4 (mIL-4) to endogenous beta-cells in nonobese diabetic (NOD) mice. In 4-week-old NOD mice, the extent of gene transfer and expression in endogenous beta-cells after ip delivery of dsAAV8-mIP-enhanced green fluorescent protein (eGFP) was comparable to normal BALB/C mice. Further, after IP delivery of dsAAV8-mIP-IL4, expression of mIL-4 was detected in islets isolated from the treated mice and cultured. AAV8-mIP-mediated gene expression of mIL-4 in endogenous beta- cells of 4- and 8-week-old NOD mice prevented the onset of hyperglycemia in NOD mice and reduced the severity of insulitis. Moreover, expression of mIL-4 also maintained the level of CD4(+)CD25(+)FoxP3(+) cells, and adoptive transfer of splenocytes from nondiabetic dsAAV8-mIP-IL-4 mice to NODscid mice was able to block the diabetes induced by splenocytes co-adoptively transferred from nondiabetic dsAAV-mIP-eGFP mice. Taken together, these results demonstrate that local expression of mIL-4 in islets prevents islet destruction and blocks autoimmunity, partly through regulation of T-cell function.

A major concern in using porcine organs for transplantation is the potential of transmission of porcine endogenous retrovirus (PERV). To investigate the long-term effects of PERV infection on human cells, human embryonic kidney cell line HEK-293 infected with PERV PK-15 was maintained for up to 72 passages and samples were harvested at intervals for use in morphological, growth, and genomic analyses. Morphology, DNA content/cell, and doubling time of uninfected and infected cells were similar. Restriction fragment length polymorphism (RFLP) analysis of PCR-amplified nearly full-length PERV genome showed no alterations in band pattern. RFLP analysis of the long terminal repeats (LTR) showed some changes in band pattern, but not in length. Southern blot analysis of genomic DNA of infected cells indicated random integration of PERV without structural alterations in proviral genome. Semi-quantitative PCR demonstrated a gradual increase of proviral load in the infected cells. Sequence analysis of the LTR region of PERV from infected cells indicated a relatively low rate (6.0 x 10(-4)/bp or about 2 x 10(-6)/bp/generation) of mutation. There were also indications of recombination of PERV strains A and B. Finally, PERV infection had no effect on transcription of human endogenous retrovirus-K (HERV-K) genes. Together, no significant effect attributable to PERV infection was evident on chronically PERV-infected HEK-293 cells.

These results demonstrate that local inhibition of the CD40-CD40L pathway by adenoviral gene transfer of sCD40-Ig to the islets prior to transplant significantly prolonged islet allograft acceptance. This approach could be used clinically to facilitate islet transplantation.

The ontogeny of gamma-glutamyl transferase (GGTase; E.C.2.3.2.2) and tyrosine aminotransferase (TAT; E.C.2.6.1.5) activities in 14 to 36 weeks gestational and neonatal hepatocytes during development of human fetal liver was studied. Subsequently, 20-24 weeks gestational hepatocytes were cultured in media supplemented with epidermal growth factor (EGF) and insulin with or without glucagon and dexamethasone to investigate the proliferation and differentiation of fetal hepatocyte in vitro using GGTase and TAT as biochemical markers. During the development of the liver, the activity of GGTase increased continuously from the first trimester through the third trimester and decreased (p < 0.001) in neonates. A low basal level of TAT activity was seen only during the third trimester, which then increased significantly (p < 0.001) in neonates. Fetal hepatocytes, in the presence of EGF and insulin, undergo proliferation from the fourth to 10th day with an increase in cell number (p < 0.001) and concomitant increase (p < 0.001) in GGTase activity. As the cells attain confluence, enzyme activity decreased significantly (p < 0.001) from the 10th to 16th day. Maximal TAT activity (p < 0.001) was observed at 48 h of culture, which decreased, but not significantly, during cell proliferation and the enzyme activity was regained as the cultures attained confluence. Furthermore, TAT activity was induced synergistically (p<0.001) in the presence of glucagon and dexamethasone, while GGTase was inhibited (p<0.001). These results indicate that GGTase increases with proliferation, whereas TAT, once it has been expressed, is not suppressed during cell proliferation. In conclusion, human fetal hepatocytes undergo enzymic differentiation by 48 h of culture, and proliferate with an increase in GGTase in the presence of growth factors with maintenance of differentiated status up to the studied 16 days of culture.