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      Combined Small Interfering RNA Therapy and In Vivo Magnetic Resonance Imaging in Islet Transplantation

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          Abstract

          OBJECTIVE

          Recent advances in human islet transplantation are hampered by significant graft loss shortly after transplantation and inability to follow islet fate directly. Both issues were addressed by utilizing a dual-purpose therapy/imaging small interfering RNA (siRNA)-nanoparticle probe targeting apoptotic-related gene caspase-3. We expect that treatment with the probe would result in significantly better survival of transplanted islets, which could be monitored by in vivo magnetic resonance imaging (MRI).

          RESEARCH DESIGN AND METHODS

          We synthesized a probe consisting of therapeutic (siRNA to human caspase-3) and imaging (magnetic iron oxide nanoparticles, MN) moieties. In vitro testing of the probe included serum starvation of the islets followed by treatment with the probe. Caspase-3 gene silencing and protein expression were determined by RT-PCR and Western blot, respectively. In vivo studies included serial MRI of NOD-SCID mice transplanted with MN-small interfering (si)Caspase-3–labeled human islets under the left kidney capsule and MN-treated islets under the right kidney capsule.

          RESULTS

          Treatment with MN-siCaspase-3 probe resulted in decrease of mRNA and protein expression in serum-starved islets compared with controls. In vivo MRI showed that there were significant differences in the relative volume change between MN-siCaspase-3–treated grafts and MN-labeled grafts. Histology revealed decreased caspase-3 expression and cell apoptosis in MN-siCaspase-3–treated grafts compared with the control side.

          CONCLUSIONS

          Our data show the feasibility of combining siRNA therapy and in vivo monitoring of transplanted islets in mice. We observed a protective effect of MN-siCaspase-3 in treated islets both in vitro and in vivo. This study could potentially aid in increasing the success of clinical islet transplantation.

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          Most cited references17

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          Five-year follow-up after clinical islet transplantation.

          Islet transplantation can restore endogenous beta-cell function to subjects with type 1 diabetes. Sixty-five patients received an islet transplant in Edmonton as of 1 November 2004. Their mean age was 42.9 +/- 1.2 years, their mean duration of diabetes was 27.1 +/- 1.3 years, and 57% were women. The main indication was problematic hypoglycemia. Forty-four patients completed the islet transplant as defined by insulin independence, and three further patients received >16,000 islet equivalents (IE)/kg but remained on insulin and are deemed complete. Those who became insulin independent received a total of 799,912 +/- 30,220 IE (11,910 +/- 469 IE/kg). Five subjects became insulin independent after one transplant. Fifty-two patients had two transplants, and 11 subjects had three transplants. In the completed patients, 5-year follow-up reveals that the majority ( approximately 80%) have C-peptide present post-islet transplant, but only a minority ( approximately 10%) maintain insulin independence. The median duration of insulin independence was 15 months (interquartile range 6.2-25.5). The HbA(1c) (A1C) level was well controlled in those off insulin (6.4% [6.1-6.7]) and in those back on insulin but C-peptide positive (6.7% [5.9-7.5]) and higher in those who lost all graft function (9.0% [6.7-9.3]) (P < 0.05). Those who resumed insulin therapy did not appear more insulin resistant compared with those off insulin and required half their pretransplant daily dose of insulin but had a lower increment of C-peptide to a standard meal challenge (0.44 +/- 0.06 vs. 0.76 +/- 0.06 nmol/l, P < 0.001). The Hypoglycemic score and lability index both improved significantly posttransplant. In the 128 procedures performed, bleeding occurred in 15 and branch portal vein thrombosis in 5 subjects. Complications of immunosuppressive therapy included mouth ulcers, diarrhea, anemia, and ovarian cysts. Of the 47 completed patients, 4 required retinal laser photocoagulation or vitrectomy and 5 patients with microalbuminuria developed macroproteinuria. The need for multiple antihypertensive medications increased from 6% pretransplant to 42% posttransplant, while the use of statin therapy increased from 23 to 83% posttransplant. There was no change in the neurothesiometer scores pre- versus posttransplant. In conclusion, islet transplantation can relieve glucose instability and problems with hypoglycemia. C-peptide secretion was maintained in the majority of subjects for up to 5 years, although most reverted to using some insulin. The results, though promising, still point to the need for further progress in the availability of transplantable islets, improving islet engraftment, preserving islet function, and reducing toxic immunosuppression.
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            siRNA-mediated off-target gene silencing triggered by a 7 nt complementation

            A growing body of evidence suggests that siRNA could generate off-target effects through different mechanisms. However, the full impact of off-target gene regulation on phenotypic induction and accordingly on data interpretation in the context of large-scale siRNA library screen has not been reported. Here we report on off-target gene silencing effects observed in a large-scale knockdown experiment designed to identify novel regulators of the HIF-1 pathway. All of the three ‘top hits’ from our screen have been demonstrated to result from off-target gene silencing. Two of the three ‘siRNA hits’ were found to directly trigger down-regulation of hif-1α mRNA through a 7 nt motif, AGGCAGT, that is present in both the hif-1α mRNA and the siRNAs. Further analysis revealed that the generation of off-target gene silencing via this 7 nt motif depends on the characteristics of the target mRNA, including the sequence context surrounding the complementary region, the position of the complementary region in the mRNA and the copy number of the complementary region. Interestingly, the off-target siRNA against hif-1α was also shown to trigger mRNA degradation with high probability of other genes that possess multiple copies of the AGGCAGT motif in the 3′-untranslated region. Lessons learned from this study will be a valuable asset to aid in designing siRNAs with more stringent target selectivity and improving ‘hits-follow-up’ strategies for future large-scale knockdown experiments.
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              Inflammation-mediated dysfunction and apoptosis in pancreatic islet transplantation: implications for intrahepatic grafts.

              Recent advances in clinical protocols have improved the outcomes of pancreatic islet transplantation (PIT), yet PIT recipients typically require pancreatic islet grafts derived from multiple donors to achieve insulin independence. This along with experimental models of syngeneic PIT, showing that up to 60% of pancreatic islet tissue undergoes apoptosis within the first several days post-transplantation, strongly suggest the involvement of nonalloantigen-specific, inflammatory events in partial destruction of the graft following PIT. Interleukin-1beta appears to be among the most important inflammatory mediators, causing pancreatic islet dysfunction and apoptosis through the up-regulation of inducible nitric oxide (NO) synthase and cyclooxygenase-2. Kupffer cells secrete many molecules, including cytokines, NO, and free radicals, which are known to be directly toxic to the pancreatic islets, and depletion or inhibition of Kupffer cells improves outcomes following experimental PIT. Immediately after transplantation, the pancreatic islets are perfused only by portal vein blood until the process of angiogenesis restores arterial blood flow some 7-10 days later. This delayed vascularization may have implications for the expression of leukocyte adhesion molecules, the effects of free radicals, and the role of ischemia-reperfusion injury. Finally, in the immediate post-transplant period, hepatocytes may contribute to pancreatic islet injury through the production of NO. This paper reviews literature regarding the inflammatory events that follow PIT as well as the pathogenesis of diabetes and the pathophysiology of hepatic ischemia-reperfusion and their relation to the survival and function of intrahepatic pancreatic islet grafts.
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                Author and article information

                Journal
                Diabetes
                diabetes
                diabetes
                Diabetes
                Diabetes
                American Diabetes Association
                0012-1797
                1939-327X
                February 2011
                21 January 2011
                : 60
                : 2
                : 565-571
                Affiliations
                [1] 1Molecular Imaging Laboratory, MGH/MIT/HMS Athinoula A. Martinos Center for Biomedical Imaging, Department of Radiology, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts
                [2] 2The Transplantation Biology Research Center, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts
                Author notes
                Corresponding author: Anna Moore, amoore@ 123456helix.mgh.harvard.edu .
                Article
                1400
                10.2337/db10-1400
                3028356
                21270267
                0126ecb7-abf3-457a-8a4c-95bc8328f073
                © 2011 by the American Diabetes Association.

                Readers may use this article as long as the work is properly cited, the use is educational and not for profit, and the work is not altered. See http://creativecommons.org/licenses/by-nc-nd/3.0/ for details.

                History
                : 1 October 2010
                : 17 November 2010
                Categories
                Immunology and Transplantation

                Endocrinology & Diabetes
                Endocrinology & Diabetes

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