Modification of a Constitutive to Glucose-Responsive Liver-Specific Promoter Resulted in Increased Efficacy of Adeno-Associated Virus Serotype 8-Insulin Gene Therapy of Diabetic Mice.

We have previously used a hepatotropic adeno-associated viral (AAV) vector with a modified human insulin gene to treat diabetic mice. The HLP (hybrid liver-specific promoter) used was constitutively active and non-responsive to glucose. In this study, we examined the effects of addition of glucose responsive elements (R3G) and incorporation of a 3' albumin enhancer (3'iALB) on insulin expression. In comparison with the original promoter, glucose responsiveness was only observed in the modified promoters in vitro with a 36 h lag time before the peak expression. A 50% decrease in the number of viral particles at 5 × 109 vector genome (vg)/mouse was required by AAV8-R3GHLP-hINSco to reduce the blood sugar level to near normoglycemia when compared to the original AAV8-HLP-hINSco that needed 1 × 1010 vg/mouse. The further inclusion of an 860 base-pairs 3'iALB enhancer component in the 3' untranslated region increased the in vitro gene expression significantly but this increase was not observed when the packaged virus was systemically injected in vivo. The addition of R3G to the HLP promoter in the AAV8-human insulin vector increased the insulin expression and secretion, thereby lowering the required dosage for basal insulin treatment. This in turn reduces the risk of liver toxicity and cost of vector production.

Characterization of Insulin-Secreting Porcine Bone Marrow Stromal Cells Ex Vivo and Autologous Cell Therapy In Vivo.

Cell therapy could potentially meet the need for pancreas and islet transplantations in diabetes mellitus that far exceeds the number of available donors. Bone marrow stromal cells are widely used in clinical trials mainly for their immunomodulatory effects with a record of safety. However, less focus has been paid to developing these cells for insulin secretion by transfection. Although murine models of diabetes have been extensively used in gene and cell therapy research, few studies have shown efficacy in large preclinical animal models. Here we report optimized conditions for ex vivo expansion and characterization of porcine bone marrow stromal cells and their permissive expression of a transfected insulin gene. Our data show that these cells resemble human bone marrow stromal cells in surface antigen expression, are homogeneous, and can be reproducibly isolated from outbred Yorkshire-Landrace pigs. Porcine bone marrow stromal cells were efficiently expanded in vitro to >10(10) cells from 20 ml of bone marrow and remained karyotypically normal during expansion. These cells were electroporated with an insulin expression plasmid vector with high efficiency and viability, and secreted human insulin and C-peptide indicating appropriate processing of proinsulin. We showed that autologous insulin-secreting bone marrow stromal cells implanted and engrafted in the liver of a streptozotocin-diabetic pig that modeled type 1 diabetes resulted in partial, but significant, improvement in hyperglycemia that could not be ascribed to regeneration of endogenous ß-cells. Glucose-stimulated insulin secretion in vivo from implanted cells in the treated pig was documented by a rise in serum human C-peptide levels during intravenous glucose tolerance tests. Compared to a sham-treated control pig, this resulted in significantly reduced fasting hyperglycemia, a slower rise in serum fructosamine, and prevented weight loss. Taken together, this study suggests that bone marrow stromal cells merit further development as autologous cell therapy for diabetes.

Insulin-producing cells from adult human bone marrow mesenchymal stem cells control streptozotocin-induced diabetes in nude mice.

Harvesting, expansion, and directed differentiation of human bone marrow-derived mesenchymal stem cells (BM-MSCs) could provide an autologous source of surrogate ß-cells that would alleviate the limitations of availability and/or allogenic rejection following pancreatic or islet transplantation. Bone marrow cells were obtained from three adult type 2 diabetic volunteers and three nondiabetic donors. After 3 days in culture, adherent MSCs were expanded for two passages. At passage 3, differentiation was carried out in a three-staged procedure. Cells were cultured in a glucose-rich medium containing several activation and growth factors. Cells were evaluated in vitro by flow cytometry, immunolabeling, RT-PCR, and human insulin and c-peptide release in responses to increasing glucose concentrations. One thousand cell clusters were inserted under the renal capsule of diabetic nude mice followed by monitoring of their diabetic status. At the end of differentiation, ∼5-10% of cells were immunofluorescent for insulin, c-peptide or glucagon; insulin, and c-peptide were coexpressed. Nanogold immunolabeling for electron microscopy demonstrated the presence of c-peptide in the rough endoplasmic reticulum. Insulin-producing cells (IPCs) expressed transcription factors and genes of pancreatic hormones similar to those expressed by pancreatic islets. There was a stepwise increase in human insulin and c-peptide release by IPCs in response to increasing glucose concentrations. Transplantation of IPCs into nude diabetic mice resulted in control of their diabetic status for 3 months. The sera of IPC-transplanted mice contained human insulin and c-peptide but negligible levels of mouse insulin. When the IPC-bearing kidneys were removed, rapid return of diabetic state was noted. BM-MSCs from diabetic and nondiabetic human subjects could be differentiated without genetic manipulation to form IPCs that, when transplanted, could maintain euglycemia in diabetic mice for 3 months. Optimization of the culture conditions are required to improve the yield of IPCs and their functional performance.

Gene and stem cell therapy for diabetes.

Gene and stem cell therapy has been on the scientific agenda in many laboratories for more than 20 years. The literature is enormous, but practical applications have been few. Recently advances in stem cell biology and gene therapy are clarifying some of the issues. I have made a few observations concerning our own studies on bone marrow mesenchymal stem cells cultured to produce a small percentage of insulin-producing cells and human insulin gene engineered into Lenti and AA viruses. The aim of clinical application would still seem to be several years away, if all goes well. The first step will be to produce enough insulin-secreting cells to be of potential value to patients. The next crucial question will be how to persuade the cells to respond to blood glucose levels swiftly and appropriately. With both stem cell and gene therapy, another important factor will be to ensure that any positive results will continue long enough to be preferable to insulin injections.

Organ transplantation has come of age.

Organ transplantation started in the mid-1950s with a kidney transplant between identical twins, demonstrating the surgical technique could provide successful therapy. The immunological barrier to be overcome, however proved to be far more difficult to deal with. The introduction of immunosuppressive agents produced some success but it was not until Cyclosporin became available in the 1980s that results became sufficiently good for widespread acceptance and rapid development of organ grafting. Now with more powerful and selective agents, although there is still much room for improvement in immunosuppression, one of the main problems in organ transplantation is a result of its success, namely a shortage of organ donors. In this review, I summarise these matters.

Stem cell and gene therapies for diabetes mellitus.

In this Perspectives article, we comment on the progress in experimental stem cell and gene therapies that might one day become a clinical reality for the treatment of patients with diabetes mellitus. Research on the ability of human embryonic stem cells to differentiate into islet cells has defined the developmental stages and transcription factors involved in this process. However, the clinical applications of human embryonic stem cells are limited by ethical concerns, as well as the potential for teratoma formation. As a consequence, alternative forms of stem cell therapies, such as induced pluripotent stem cells and bone marrow-derived mesenchymal stem cells, have become an area of intense study. Finally, gene therapy shows some promise for the generation of insulin-producing cells. Here, we discuss two of the most frequently used approaches: in vitro gene delivery into cells which are then transplanted into the recipient and direct delivery of genes in vivo.

The role of research in transplantation.

In the past 50 years, organ transplantation has developed from an improbable laboratory exercise to a major therapeutic success. The surgical problems of organ grafting have, for the most part, been solved. Rejection of grafts is now partially understood and usually controllable by powerful immunosuppressive drugs. A steady improvement in patient outcome, especially following the introduction of cyclosporin as an immunosuppressive agent has resulted in a worldwide shortage of organs for transplantation. This has provoked serious ethical dilemmas in every country. These matters are summarised in the following text.

Alemtuzumab (CAMPATH-1H) for the treatment of acute rejection in kidney transplant recipients: long-term follow-up.

BACKGROUND: Alemtuzumab (MabCampath, Campath-1H) is a lymphocyte-depleting monoclonal antibody increasingly used in renal transplantation. This article reports the long-term follow-up data from the first series of patients treated with alemtuzumab for biopsy-proven acute rejection (BPAR). METHODS: Fifteen patients were identified who had received alemtuzumab for BPAR between November 1991 and June 1994. Patient and allograft survival were compared with a control group consisting of 25 patients with BPAR from the same era treated with intravenous methyl prednisolone, and with a contemporaneous UK renal transplant cohort. RESULTS: All rejection episodes responded to treatment with alemtuzumab but there was an excess of early infection-associated death in this group. Long-term transplant survival was similar in both groups as was allograft function (mean creatinine concentration at 10 years was 143 micromol/L in alemtuzumab cohort and 183 micromol/L in control cohort, P=0.06). There was no excess incidence of malignancy or cytomegalovirus infection in this prolonged follow-up period.

Transplantation: current developments and future directions.

Organ transplantation has emerged from a few sporadic failed attempts to one of the most successful branches of surgery in the course of 50 years since the first identical twin transplant was performed in Boston. In this article I will attempt to portray the historical background and the recent shift of attitude regarding immunosuppression for solid organ transplants. Previously a culture of increasing immunosuppression and incorporating new and powerful agents into an already effective regimen has resulted in over-immunosuppression and more sepsis without an improvement in long-term graft survival. Over-immunosuppression is probably detrimental in preventing the natural control and "switching off" of the immune response as a vital function of the immune system and as a consequence any attempts to produce immunological tolerance are likely to be impaired by excessive immunosuppressive regimens. I will therefore,, explain and advocate a minimalistic approach to immunosuppression, a background on tissue typing and a summary of clinical results. Now that the procedure is perceived worldwide as an excellent therapy for previously doomed patients, there is an increasing mismatch between the number of donor organs available and patients in need of a graft. This has produced ethical dilemmas previously unknown in the medical profession. These are extremely important considerations as they can undermine the Hippocratic tradition and the high ethical standing previously enjoyed by our profession.

Alotransplante de ilhotas de Langerhans no fígado de ratos submetidos a manipulação tímica com células não-parenquimatosas / Allogenic islet transplantation on the rat liver after allogenic nonparenchymal cells injection in the thymus

RACIONAL: A maior indicação do transplante de pâncreas ou de ilhotas de Langerhans é o diabetes mellitus do tipo I. O processo deve suprir as necessidades de insulina, mantendo os níveis glicêmicos dentro da normalidade OBJETIVOS: Estudar o alotransplante de ilhotas de Langerhans no fígado de ratos Lewis (RT1¹), tendo como doadores de ilhotas ratos Wistar (RT1u). No grupo controle (n = 8) injetava-se, no timo, solução de Hanks e no grupo de estudo(n = 8), células não-parenquimatosas hepáticas MATERIAL E MÉTODOS: No grupo controle com o método de separação e purificação das ilhotas de Langerhans obteve-se 3.637 ± 783,3 ilhotas com pureza de 85 ± 3,52 por cento. No grupo de estudo obteve-se 3.270 ± 770 ilhotas de Langerhans com pureza de 84,25 ± 2,76 por cento e com o método de isolamento e purificação das células não-parenquimatosas hepáticas obteve-se 2 x 10(6) células RESULTADOS: No grupo controle, o transplante de 3.637 ± 783,3 ilhotas de Langerhans no fígado, quase normalizou a glicemia que chegou a 17,95 ± 5,33 mmol/L no 2° dia do pós-operatório (diferença significante com relação ao pré-operatório). Do pós-operatório imediato até o 8° dia do pós-operatório a glicemia não se elevou significativamente, porém a partir do 10° dia do pós-operatório houve aumento significativo deste parâmetro, o que pode ser compatível com rejeição aguda do enxerto. No grupo de estudo, o transplante de 3.270 ± 770 ilhotas de Langerhans no fígado, quase normalizou a glicemia que chegou a 17,95 ± 5,33 mmol/L no 2° dias do pós-operatório (diferença significante com relação ao pré-operatório). Do 4° ao 10° pós-operatório a glicemia elevou-se significativamente, o que pode ser compatível com quadro de rejeição aguda do enxerto e certamente precoce CONCLUSÃO: A inoculação de células alogênicas apresentadoras de antígenos (células não-parenquimatosas hepáticas) no timo de ratos imunossuprimidos e diabéticos, antes...

BACKGROUD: The major indication for pancreas or islet transplantation is diabetes mellitus type I. This process has to supply the insulin necessity keeping glucose under control AIM: We studied allogenic islet transplantation on the rat liver, Wistar (RT1u) to Lewis (RT1¹) as a recipient. Control group (n = 8) and nonparenchymal cell group (n = 8) respectively with injection of Hanks solution and nonparenchymal cells in the thymus before islet transplantation. MATERIAL AND METHODS: With the method of isolation and purification of the islets we obtained both in the control group 3.637 ± 783,3 islets with purity of 85 ± 3,52 percent and nonparenchymal cell group 3.270 ± 770 islets with purity of 84,25 ± 2,76 percent. The nonparenchymal cells were retrieved from the liver and we obtained 2 x 106 cells. Diabetes was induced by i.v. streptozotocin RESULTS: Control group the transplantation of 3.637 ± 783,3 islets in the rat liver normalized glucose test, 7,21 ± 0,57 mmol/L in the 2nd postoperative day. Acute rejection came in the 6th postoperative day with significantly increase of glucose test in nonparenchymal cell group, the transplantation of 3.270 ± 770 islets in the rat liver, almost normalized the glucose test was 17,95 ± 5,33 mmol/L in the 2nd postoperative day. From the 4th postoperative day to 10th postoperative day. The glucose test increase significantly showing an early acute rejection CONCLUSION: The injection of nonparenchymal cells in the thymus before allogenic islet transplantation in the rat liver lead to an early acute rejection.

Genetic engineering for haemophilia A.

At first sight, haemophilia A would appear to be an ideal candidate for treatment by gene therapy. There is a single gene defect; cells in different parts of the body, but especially the liver, produce Factor VIII, and only 5% of normal levels of Factor VIII are necessary to prevent the serious symptoms of bleeding. This review attempts to outline the status of gene therapy at present and efforts that have been made to overcome the difficulties and remaining problems that require solving. Undoubtedly, success will be achieved, but it is likely that considerably more work will be necessary before experimental models can be introduced into the clinic with any likelihood of success. The most successful results in animals that may have clinical application were from introducing the Factor VIII gene to newborn animals before antibodies are produced, presumably inducing a state of tolerance.

[Allogenic islet transplantation on the rat liver after allogenic nonparenchymal cells injection in the thymus]. / Alotransplante de ilhotas de Langerhans no fígado de ratos submetidos a manipulação tímica com células não-parenquimatosas.

BACKGROUND: [corrected] The major indication for pancreas or islet transplantation is diabetes mellitus type I. This process has to supply the insulin necessity keeping glucose under control AIM: We studied allogenic islet transplantation on the rat liver, Wistar (RT1u) to Lewis (RT1(1)) as a recipient. Control group (n = 8) and nonparenchymal cell group (n = 8) respectively with injection of Hanks solution and nonparenchymal cells in the thymus before islet transplantation. MATERIAL AND METHODS: With the method of isolation and purification of the islets we obtained both in the control group 3.637 +/-783,3 islets with purity of 85 +/- 3,52% and nonparenchymal cell group 3.270 +/- 770 islets with purity of 84,25 +/- 2,76%. The nonparenchymal cells were retrieved from the liver and we obtained 2 x 106 cells. Diabetes was induced by i.v. streptozotocin RESULTS: Control group the transplantation of 3.637 +/- 783,3 islets in the rat liver normalized glucose test, 7,21 +/- 0,57 mmol/L in the 2nd postoperative day. Acute rejection came in the 6th postoperative day with significantly increase of glucose test in nonparenchymal cell group, the transplantation of 3.270 +/- 770 islets in the rat liver, almost normalized the glucose test was 17,95 +/- 5,33 mmol/L in the 2nd postoperative day. From the 4th postoperative day to 10th postoperative day. The glucose test increase significantly showing an early acute rejection CONCLUSION: The injection of nonparenchymal cells in the thymus before allogenic islet transplantation in the rat liver lead to an early acute rejection.

Gene therapy for hemophilia A.

Hemophilia A is an X-linked bleeding disorder caused by defective coagulation Factor VIII (FVIII). Although the efficacies of existing treatment using purified or recombinant FVIII are good, there remain shortcomings in using this particular form of treatment. A few FVIII gene therapy clinical trials have been initiated with modest improvements recorded, but these are no longer being continued due to insufficient efficacy. However, with the progress in the development of gene delivery vectors and the availability of mouse and canine hemophilia A models, gene therapy of hemophilia A remains an area of hot pursuit.

[Alogenic islet transplantation on the rat liver after alogenic dendritic cells injection in the thymus]. / Alotransplante de ilhotas de Langerhans no fígado de ratos submetidos a manipulação tímica com células dendríticas.

BACKGROUND: The major indication for pancreas or islet transplantation is diabetes mellitus type I. This process has to supply the insulin necessity keeping glucose under control. AIM: We studied alogenic islet transplantation on the rat liver, Wistar (RT1u) to Lewis (RT1(1)) as a recipient. Control group (n = 8) and dendritic cell group (n = 9) respectively with injection of Hanks solution and dendritic cells in the thymus before islet transplantation. MATERIAL AND METHODS: With the method of isolation and purification of the islets we obtained both in the control group 3637 +/- 783,3 islets with purity of 85 +/- 3,52% and dendritic cell group 3268 +/- 378 islets with purity of 87 +/- 4,47%. The dendritic cells were retrieved from the spleen and we obtained 3,34 x 105+/-1,16 cells. Diabetes was induced by i.v. streptozotocin. RESULTS: Control group the transplantation of 3637 +/- 783,3 islets in the rat liver normalized glucose test, 7,21 +/- 0,57 mmol/L in the second post-operative day. Acute rejection came in the 10 postoperative day with significantly increase of glucose test. Dendritic cell group, the transplantation of 3258 +/- 378 islets in the rat liver, normalized the glucose test was 9,3 +/- 2,85 mmoL/L in the second postoperative day. From the 4th postoperative day to 10th postoperative day the glucose test increase significantly showing an early acute rejection. CONCLUSION: The injection of dendritic cells in the thymus before alogenic islet transplantation in the rat liver lead to an early acute rejection.

Alotransplante de ilhotas de Langerhans no fígado de ratos submetidos a manipulação tímica com células dendríticas / Alogenic islet transplantation on the rat liver after alogenic dendritic cells injection in the thymus

RACIONAL: A maior indicação do transplante de pâncreas ou de ilhotas de Langerhans é o diabetes mellitus do tipo I. O processo deve suprir as necessidades de insulina, mantendo os níveis glicêmicos dentro da normalidade. OBJETIVOS: Estudou-se o alotransplante de ilhotas de Langerhans no fígado de ratos Lewis, tendo como doadores de ilhotas ratos Wistar. No grupo controle (n = 8) injetava-se, no timo, solução de Hanks e no grupo de estudo (n = 9), células dendríticas. MATERIAL E MÉTODOS: No grupo controle com o método de separação e purificação das ilhotas de Langerhans obteve-se 3637 ± 783,3 ilhotas com pureza de 85 por cento ± 3,52 por cento. No grupo de estudo obteve-se 3268 ± 378 ilhotas de Langerhans com pureza de 87 por cento ± 4,47 por cento e com o método de isolamento e purificação das células dendríticas do baço obteve-se 3,34 x 105 ± 1,16 células. RESULTADOS: No grupo controle, o transplante de 3637 ± 783,3 ilhotas de Langerhans no fígado, normalizou a glicemia que chegou a 7,21 ± 0,57 mmol/L no segundo pós-operatório (diferença significativa com relação ao pré-operatório). Do pós-operatório imediato até o 8° pós-operatório a glicemia não se elevou significativamente, porém a partir do 10° pós-operatório houve aumento significativo deste parâmetro, o que pode ser compatível com rejeição aguda do enxerto. No grupo de estudo, o transplante de 3258 ± 378 ilhotas de Langerhans no fígado, normalizou a glicemia, que chegou a 9,3 ± 2,85 mmol/L no segundo pós-operatório (diferença significativa com relação ao pré-operatório). Do 4° ao 10° pós-operatório, a glicemia elevou-se significativamente, o que pode ser compatível com quadro de rejeição aguda do enxerto e certamente precoce. CONCLUSÃO: A inoculação de células alogênicas apresentadoras de antígenos (células dendríticas) no timo de ratos imunossuprimidos e diabéticos, antes do alotransplante de ilhotas de Langerhans no fígado, ao contrário de inibir a reação do receptor contra o enxerto, prolongando a sobrevida média das ilhotas e, possivelmente, levando ao estado de tolerância imunológica, induziu ao processo de rejeição aguda precoce.

Insulin-secreting cells derived from stem cells: clinical perspectives, hypes and hopes.

Diabetes is a degenerative disease that results from the selective destruction of pancreatic beta-cells. These cells are responsible for insulin production and secretion in response to increases in circulating concentrations of nutrients, such as glucose, fatty acids and amino acids. This degenerative disease can be treated by the transplantation of differentiated islets obtained from cadaveric donors, according to a new surgical intervention developed as Edmonton protocol. Compared to the classical double transplant kidney-pancreas, this new protocol presents several advantages, concerning to the nature of the implant, immunosuppressive drug regime and the surgical procedure itself. However, the main problem to face in any islet transplantation program is the scarcity of donor pancreases and the low yield of islets isolated (very often around 50%) from each pancreas. Nevertheless, transplanted patients presented no adverse effects and no progression of diabetic complications. In the search of new cell sources for replacement trials, stem cells from embryonic and adult origins represent a key alternative. In order to become a realistic clinical issue transplantation of insulin-producing cells derived from stem cells, it needs to overcome multiple experimental obstacles. The first one is to develop a protocol that may allow obtaining a pure population of functional insulin-secreting cells as close as possible to the pancreatic beta-cell. The second problem should concern to the transplantation itself, considering issues related to immune rejection, tumour formation, site for implant, implant survival, and biosafety mechanisms. Although transplantation of bioengineered cells is still far in time, experience accumulated in islet transplantation protocols and in experiments with appropriate animal models will give more likely the clues to address this question in the future.