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  • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
  • C12N5/06—Animal cells or tissues; Human cells or tissues
  • C12N5/0602—Vertebrate cells
  • C12N5/0676—Pancreatic cells
• • C—CHEMISTRY; METALLURGY
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  • C12N2310/00—Structure or type of the nucleic acid
  • C12N2310/10—Type of nucleic acid
  • C12N2310/14—Type of nucleic acid interfering nucleic acids [NA]

Definitions

• the islets of Langerhans is a multi-cellular entity containing cells that produce insulin within the pancreas.
• the average person has about a million islets, and they contain approximately two to three percent of the total number of cells in the pancreas.
• the pancreas contains the islets of Langerhans, which house beta cells that produce insulin.
• the beta cells monitor glucose levels in the blood and release finely measured amounts of insulin to counterbalance glucose peaks. Type I and II diabetes develop when more than 90 percent of these beta cells are damaged.
• Islet transplantation is a most promising and minimally physiologically invasive procedure for treatment of type I diabetes mellitus. Transplanting islets rather than complete pancreatic tissue has the distinct advantages of ease of transplantation, and the elimination of the pancreatic exocrine function of the donor tissue involving secretion of digestive enzymes. Liberating islets from pancreatic exocrine tissue is the initial and crucial step that influences islet transplantations. The important objective in islet isolations is to provide sufficient numbers of viable functional and potent islets for transplantation. The "Edmonton Protocol" transplants healthy islets into diabetic patients.
• Islet transplantation using the Edmonton Protocol is described in Shapiro, Ryan, and Lakey, Clinical Islet Transplantation—State of the Art, Transplantation Proceedings, 33, pp. 3502-3503 (2001); Ryan et al., Clinical Outcomes and Insulin Secretion After Islet Transplantation With the Edmonton Protocol, Diabetes, Vol. 50, April 2001, pp. 710-719; and Ryan et al., Continued Insulin Reserve Provides Long-Term Glycemic Control, Diabetes, Vol. 51, July 2002, pp. 2148- 2157. Once in the liver, the cells develop a blood supply and begin producing insulin.
• the Edmonton Protocol may include 7-10 steps depending on the method employed.
• the first step involves the delivery of a specific enzyme (liberase) to a donor pancreas, which digests the pancreas tissue, but does not digest the islets.
• a specific enzyme liberase
• the separated islets are transplanted into the main vessel of the liver, known as the portal vein.
• the liver is able to regenerate itself when damaged, building new blood vessels and supporting tissue. Therefore, when islets are transplanted into the liver, it is believed that new blood vessels form to support the islets.
• the insulin that the cells produce is absorbed into the blood stream through these surrounding vessels and distributed through the body to control glucose levels in the blood.
• Islet transplantation together with steroid-free, nondiabetogenic immunosuppressive therapy, has been used to treat patients with type 1 diabetes.
• Such treatments can lead to increased risk of hyperlipidemia and hypertension, and long-term studies demonstrate that islet viability is impaired.
• the present invention provides a method for inhibiting islet cells from undergoing apoptosis during a donor harvesting process comprising administering eIF5A siRNA to the islet cells of an islet cell donor prior to islet isolation, wherein the eIF5 A siRNA inhibits expression of eIF5A in the islet cells and thereby inhibits apoptosis in the islet cells.
• eIF5 A siRNA inhibits expression of eIF5A in the islet cells and thereby inhibits apoptosis in the islet cells.
• Any siRNA or antisense construct can be used, as long as such construct inhibits expression of eIF5 A.
• a preferred siRNA comprises the nucleotide sequence AGUCGACCUUCAGUAAGGCdTdT.
• Administration of siRNA may be by any suitable route. Exemplary administration methods include perfusion through the portal vein of the islet cell donor and hydrodynamic perfusion through the portal vein of the islet cell donor.
• the present invention also provides a method for inhibiting expression of eIF5 A in islet cells comprising administering eIF5A siRNA to the islet cells, wherein the eIF5A siRNA inhibits expression of eIF5A in the islet cells.
• Another embodiment of the invention provides a method for inhibiting apoptosis in harvested islet cells comprising administering eIF5A siRNA to the islet cells, wherein the eIF5A siRNA inhibits expression of eIF5 A in the islet cells and wherein the inhibition of eIF5 A expression inhibits apoptosis.
• the present invention also provides a composition for inhibiting apoptosis in islet cells, comprising eIF5 A siRNA, wherein the siRNA inhibits expression of eIF5 A and thereby inhibits apoptosis in the islet cells.
• a preferred composition comprises eIF5A siRNA comprisubg the nucleotide sequence AGUCGACCUUCAGUAAGGCdTdT.
• Figure 1 provides results of RT-PCR performed for ⁇ -actin, mAAT and eIF5 A after perfusion through the portal vein with eIF-5 A siRNA. This figure shows that eIF5 A expression is measurable and was thus incorporated into islets.
• Figure 2 shows slows retrograde portal vein perfusion. Bile duct (clear) and portal vein
• Figure 3 shows that perfusion of eIF5 A siRNA into islets causes a reduction of expression of eIF5 A (shown is reduction in mRNA levels of eIF5 A).
• Figure 6 provides the nucleotide sequence of human eIF5Al aligned against eIF5A2.
• Figure 7 provides the amino acid sequence of human eIF5Al aligned against eIF5A2.
• Figure 8 provides the nucleotide sequence of human eIF5A with exemplary antisense oligonucleotides.
• Figure 9 provides the nucleotide sequence of human eIF5A with exemplary antisense oligonucleotides .
• Figures 1OA and B provide the nucleotide sequence of human eIF5A with exemplary siRNAs.
• Figure 11 provides the nucleotide sequence of human eIF5A with exemplary siRNAs.
• siRNA incorporation into islets can be achieved by pancreatic perfusion via retrograde portal vein inoculation. See Bradley, et al., Transplantation Proceedings, 37, 233-236, 2005. Briefly, Cy-3 labeled Luciferase (Luc) siRNA GL2 duplex was used either packaged with Lipofectamine 2000 or unpackaged, and injected either through tail vein (in vivo, 50 ⁇ g per mouse) or directly into the pancreas by retrograde portal vein inoculation (in situ, 2 ⁇ g per mouse).
• Pancreata were procured and stored at 4°C for 24 hours after in situ delivery, or 4 hours after in vivo delivery, and islets were isolated and cultured an extra 16 hours before examination. To visualize siRNA distribution, pancreata were stained for insulin and examined under a fluorescent microscope. Isolated islets were directly examined under a fluorescent microscope. Unpackaged siRNA reached islets to a similar extent as observed using liposomal-packaged siRNA, agreeing with reports of so-called "naked"-siRNA delivery in vivo. Lewis et al., Nat. Genet. 32:107-108, Epub 2002 JuI 2029, 2002 and McCaffrey AP, et al., Nature 418:38-39, 2002).
• the present invention provides a method for inhibiting expression of eIF5 A in islet cells comprising administering eIF5 A siRNA to the islet cells, wherein the eIF5 A siRNA inhibits expression of eIF5 A in the islet cells.
• Figure 1 shows that perfusion to the islet cells provides a suitable delivery mechanism to the islet cells and
• Figure 3 shows that the eIF5 A siRNA treated islet cells do indeed express less eIF5A siRNA.
• apoptosis is also inhibited.
• Figures 4 and 5 shows that treating islets cells with eIF5 A siRNA prior to isolation, inhibited these cells from apoptosis (as demonstrated by a reduction of the number of cells in the sub-Gl phase).
• the present invention also provides a method for inhibiting apoptosis in harvested islet cells comprising administering eIF5A siRNA to the islet cells, wherein the eIF5A siRNA inhibits expression of eIF5A in the islet cells and wherein the inhibition of eIF5A expression inhibits apoptosis.
• eIF5 A siRNA that inhibits expression of eIF5 A may be used.
• the term “inhibits” also means reduce.
• One exemplary eIF5A siRNA comprises the sequence:
• antisense constructs of eIF5A may be used to inhibit expression of eIF5A and thus inhibit apoptosis of the islet cells.
• the eIF5A siRNA comprises the nucleotide sequence
• the present invention also provides a method for inhibiting islet cells from undergoing apoptosis during a donor harvesting process.
• many islets cells undergo apoptosis when they are harvested.
• the present inventors have shown that providing eIF5 A siRNA to the islet cells prior to harvesting, offers a protective benefit against apoptosis.
• the eIF5 A siRNA is administered to the islet cells of an islet cell donor prior to islet isolation.
• the donor and hence islet cells
• the donor may be any animal, including human islet cells. Any method of administration may be used.
• the siRNA may be administered via perfusion through the portal vein of the islet cell donor or via hydrodynamic perfusion through the portal vein of the islet cell donor.
• Perfusion through portal vein is similar to canulation of the bile duct, but the needle points the opposite way.
• the portal vein is exposed by retraction of liver and shifting of visceral organs to the mouse's left.
• a preparative knot is made around it and includes the bile duct. After puncturing the vessel a blunted needle is advanced toward the pancreas and the knot is tightened around it.
• 1 ml saline or siRNA 5 ⁇ g
• the needle is removed and the knot is closed behind the needle to prevent fluid escape.
• the pancreas may be held longer with siRNA.
• the present invention also provides a composition for inhibiting apoptosis in islet cells, comprising eIF5 A siRNA, wherein the siRNA inhibits expression of eIF5 A and thereby inhibits apoptosis in the islet cells.
• the composition may comprise other or additional eIF5A siRNAs as discussed above.
• a preferred siRNA comprises the nucleotide sequence AGUCGACCUUCAGUAAGGCdTdT.
• Mouse islets express eIF5A.
• eIF5A-mRNA levels diminished after elF5A-siRNA delivery: portal vein slow perfusion.
• Pancreata were digested by collagenase irrigation of pancreatic duct and islets were isolated as described by Lewis et al., Proc. Natl. Acad. Sci. USA, 102:12153-12158 Epub 12005 Aug. 12110, 2005. Islets (50 per mouse) were incubated for 16 hours.
• RNA was then extracted and RT-PCR was performed for ⁇ -actin and for eIF5A (fig. 3). Ratio of mRNA for eIF5A/ ⁇ -actin was 5.24 (CT-siRNA) and 3.01 (eIF5 A-siRNA). Figure 3 shows that mRNA levels of eIF5A were reduced in those cells treated with siRNA. This experiment was repeated with n 3 mice and islets were incubated for RNA extraction in triplicates; results were consistent with initial observation.
• eIF5A-mRNA levels diminished and islet apoptosis rate reduced after elF5 A-siRNA delivery: portal vein hydrodynamic perfusion.
• Pancreata were digested by collagenase irrigation of pancreatic duct and islets were isolated. Islets were incubated for 16 hours and then divided: one group was stained with propidium iodide for evaluation of apoptosis (50 islets per mouse) and the other group was processed for RT-PCR (25 islets per mouse).
• siRNA molecules were synthesized by Dharmacon, Lafayette, CO. The sequence of the eIF5A and control siRNA were: 5' CGGAAUGACUUCCAGCUGAdTdT 3' and 5' AGUCGACCUUCAGUAAGGCdTdT 3', respectively.
• eIF5A Primers Forward 5'-GAC AGT GGG GAG GTA CGA GA-3 1 ; Reverse 5'-GGG GTG AGG AAA ACC AAA AT- 3'.
• PI Propidium iodide
• Single cell suspension of islets was achieved by gentle trypsinization.
• Cells were washed with PBS and added saponin-PI mixture containing 0.3 % Saponin, EDTA 1 mM, Rnase, 1 % Azide, 1 % FCS and 50 ⁇ g/ml PI in PBS.
• Cells were thoroughly vortexed and incubated at 4°C in the dark for 6 hours before analyzed for sub-GI population by FACS.

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