CA2637137A1 - A novel method of protecting islet cells from apoptosis during the donor harvesting process - Google Patents
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Abstract
The present invention relates to methods for improving the viability and recovery of islets that are separated from a donor organ for subsequent transplantation and more particularly relates to the use of eIF5A siRNAs to enhance the viability of islets.
Description
A NOVEL METHOD OF PROTECTING ISLET CELLS FROM APOPTOSIS DURING
THE DONOR HARVESTING PROCESS
RELATED APPLICATIONS
This application claims priority to U.S. Application 60/783,414 filed March 20, 2007, the entire contents of which are incorporated herein.
BACKGROUND OF THE INVENTION
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.
Separation or isolation of the islets from the connective matrix and remaining exocrine tissue is advantageous and beneficial for laboratory experimentation and transplantation purposes. 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, Apri12001, 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. Following the digestion step, there are several successive steps for separating the islets from other cells in the pancreas.
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.
Altogether, the steps of the Edmonton Protocol create a vigorous process that compromises the viability of islets, which have a fragile, three-dimensional structure and require large amounts of oxygen for growth and viability. During the process, islets may be damaged or destroyed due to non-optimal conditions of oxygen delivery, affecting the yield of healthy islets that are retrieved from a given donor pancreas. Furthermore, islet transplantation is severely limited by donor availability; frequently, two pancreata are required to obtain insulin independence in just one patient.
Islet transplantation, together with steroid-free, nondiabetogenic immunosuppressive therapy, has been used to treat patients with type 1 diabetes. However, such treatments can lead to increased risk of hyperlipidemia and hypertension, and long-term studies demonstrate that islet viability is impaired.
As a result, there is a need for a method of protecting islet cells from apoptosis during the harvesting process. The present invention provides this need.
SUMMARY OF THE INVENTION
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 eIF5A 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 eIF5A. A
preferred siRNA comprises the nucleotide sequence AGUCGACCUUCAGUAAGGCdTdT.
THE DONOR HARVESTING PROCESS
RELATED APPLICATIONS
This application claims priority to U.S. Application 60/783,414 filed March 20, 2007, the entire contents of which are incorporated herein.
BACKGROUND OF THE INVENTION
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.
Separation or isolation of the islets from the connective matrix and remaining exocrine tissue is advantageous and beneficial for laboratory experimentation and transplantation purposes. 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, Apri12001, 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. Following the digestion step, there are several successive steps for separating the islets from other cells in the pancreas.
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.
Altogether, the steps of the Edmonton Protocol create a vigorous process that compromises the viability of islets, which have a fragile, three-dimensional structure and require large amounts of oxygen for growth and viability. During the process, islets may be damaged or destroyed due to non-optimal conditions of oxygen delivery, affecting the yield of healthy islets that are retrieved from a given donor pancreas. Furthermore, islet transplantation is severely limited by donor availability; frequently, two pancreata are required to obtain insulin independence in just one patient.
Islet transplantation, together with steroid-free, nondiabetogenic immunosuppressive therapy, has been used to treat patients with type 1 diabetes. However, such treatments can lead to increased risk of hyperlipidemia and hypertension, and long-term studies demonstrate that islet viability is impaired.
As a result, there is a need for a method of protecting islet cells from apoptosis during the harvesting process. The present invention provides this need.
SUMMARY OF THE INVENTION
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 eIF5A 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 eIF5A. 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 eIF5A 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 eIF5A in the islet cells and wherein the inhibition of eIF5A
expression inhibits apoptosis.
The present invention also provides a composition for inhibiting apoptosis in islet cells, comprising eIF5A siRNA, wherein the siRNA inhibits expression of eIF5A and thereby inhibits apoptosis in the islet cells. A preferred composition comprises eIF5A siRNA
comprisubg the nucleotide sequence AGUCGACCUUCAGUAAGGCdTdT.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 provides results of RT-PCR performed for 0-actin, mAAT and eIF5A
after perfusion through the portal vein with eIF-5A siRNA. This figure shows that eIF5A expression is measurable and was thus incorporated into islets.
Figure 2 shows slows retrograde portal vein perfusion. Bile duct (clear) and portal vein (red) ready for preparatory knot (dark suture). The needle enters below the knot (direction indicated by arrow), cross under the knot and releases siRNA into vessels that reach pancreas, spleen, intestine and a third of distal colon.
Figure 3 shows that perfusion of eIF5A siRNA into islets causes a reduction of expression of eIF5A (shown is reduction in mRNA levels of eIF5A).
Figure 4 shows a reduction of apoptosis of islets cells having been treated with eIF5 siRNA as compared to control and saline treated islets (here n=2 per group).
Figure 5 shows a reduction of apoptosis of islets cells having been treated with eIF5 siRNA as compared to control and saline treated islets (here n=3 per group).
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.
The present invention also provides a method for inhibiting expression of eIF5A 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 eIF5A in the islet cells and wherein the inhibition of eIF5A
expression inhibits apoptosis.
The present invention also provides a composition for inhibiting apoptosis in islet cells, comprising eIF5A siRNA, wherein the siRNA inhibits expression of eIF5A and thereby inhibits apoptosis in the islet cells. A preferred composition comprises eIF5A siRNA
comprisubg the nucleotide sequence AGUCGACCUUCAGUAAGGCdTdT.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 provides results of RT-PCR performed for 0-actin, mAAT and eIF5A
after perfusion through the portal vein with eIF-5A siRNA. This figure shows that eIF5A expression is measurable and was thus incorporated into islets.
Figure 2 shows slows retrograde portal vein perfusion. Bile duct (clear) and portal vein (red) ready for preparatory knot (dark suture). The needle enters below the knot (direction indicated by arrow), cross under the knot and releases siRNA into vessels that reach pancreas, spleen, intestine and a third of distal colon.
Figure 3 shows that perfusion of eIF5A siRNA into islets causes a reduction of expression of eIF5A (shown is reduction in mRNA levels of eIF5A).
Figure 4 shows a reduction of apoptosis of islets cells having been treated with eIF5 siRNA as compared to control and saline treated islets (here n=2 per group).
Figure 5 shows a reduction of apoptosis of islets cells having been treated with eIF5 siRNA as compared to control and saline treated islets (here n=3 per group).
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 l0A and B provide the nucleotide sequence of human eIF5A with exemplary siRNAs.
Figure 11 provides the nucleotide sequence of human eIF5A with exemplary siRNAs.
DETAILED DESCRIPTION OF THE INVENTION
It has been previously shown that 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 Ju12029, 2002 and McCaffrey AP, et al., Nature 418:38-39, 2002).
The present invention provides a method for inhibiting expression of eIF5A in islet cells comprising administering eIF5A siRNA to the islet cells, wherein the eIF5A
siRNA inhibits expression of eIF5A 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 eIF5A siRNA treated islet cells do indeed express less eIF5A siRNA. By inhibiting eIF5A
expression, apoptosis is also inhibited. Figures 4 and 5 shows that treating islets cells with eIF5A
siRNA prior to isolation, inhibited these cells from apoptosis (as demonstrated by a reduction of the number of cells in the sub-Gl phase). Accordingly, 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.
Any eIF5A siRNA that inhibits expression of eIF5A may be used. The term "inhibits"
also means reduce. One exemplary eIF5A siRNA comprises the sequence:
AGUCGACCUUCAGUAAGGCdTdT. Co-pending application 11/293,391, which was filed on November 28, 2005 (which is herein incorporated by reference in its entirety) provides additional exemplary eIF5A siRNAs and other antisense constructs that have been used to inhibit expression of eIF5A in other cell types and were also shown to inhibit apoptosis. One skilled in the art could design other eIF5A siRNAs given the eIF5A sequence and can easily test for the siRNAs ability to inhibit expression without undue experimentation. Figures 6-11 provide sequences of eIF5A, exemplary eIF5A siRNAs and antisense constructs. In another embodiment of the invention, antisense constructs of eIF5A may be used to inhibit expression of eIF5A and thus inhibit apoptosis of the islet cells.
In preferred embodiments the eIF5A siRNA comprises the nucleotide sequence AGUC GAC CUUCAGUAAG G C dT dT .
The present invention also provides a method for inhibiting islet cells from undergoing apoptosis during a donor harvesting process. As discussed above, many islets cells undergo apoptosis when they are harvested. The present inventors have shown that providing eIF5A
siRNA to the islet cells prior to harvesting, offers a protective benefit against apoptosis. The eIF5A siRNA is administered to the islet cells of an islet cell donor prior to islet isolation. The donor (and hence islet cells) may be any animal, including human islet cells.
Any method of administration may be used. For example, 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. In a mouse model, 1 ml saline or siRNA (5 g) is released slowly, the needle is removed and the knot is closed behind the needle to prevent fluid escape. At this point the mouse is turned around and the bile duct accessed for pancreas digestion.
The pancreas may be held longer with siRNA. Alternatively, it can be removed but kept cold with collagenase longer. Regular islet isolation methods are followed and the islets (50) may be incubated in for 16 hours.
The present invention also provides a composition for inhibiting apoptosis in islet cells, comprising eIF5A siRNA, wherein the siRNA inhibits expression of eIF5A 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 AGUC GAC CUUCAGUAAG G C dT dT .
EXAMPLES
Mouse islets express eIF5A.
Total RNA was extracted from isolated mouse islets and RT-PCR was performed for ((3-actin and for eIFSA (fig. 1). Resting non-stimulated islets exhibited positive levels of eIFSA-mRNA.
eIF5A-mRNA levels diminished after e1F5A-siRNA delivery: portal vein slow pe~fusion.
Mice were introduced 1 ml of siRNA (CT (control) sequence or eIF5A, 5 g) or saline, n = 2 per group, by slow retrograde portal vein perfusion (fig. 2). 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. Total RNA was then extracted and RT-PCR
was performed for 0-actin and for eIF5A (fig. 3). Ratio of mRNA for eIF5A/0-actin was 5.24 (CT-siRNA) and 3.01 (eIF5A-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 e1F5A-siRNA
delivery: portal vein hydrodynamic perfusion.
Mice were introduced 1 ml of siRNA (CT or eIF5A, 5 g) or saline, n = 2 per group, by hydrodynamic retrograde portal vein perfusion, which was completed within 5 seconds.
Figure 9 provides the nucleotide sequence of human eIF5A with exemplary antisense oligonucleotides.
Figures l0A and B provide the nucleotide sequence of human eIF5A with exemplary siRNAs.
Figure 11 provides the nucleotide sequence of human eIF5A with exemplary siRNAs.
DETAILED DESCRIPTION OF THE INVENTION
It has been previously shown that 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 Ju12029, 2002 and McCaffrey AP, et al., Nature 418:38-39, 2002).
The present invention provides a method for inhibiting expression of eIF5A in islet cells comprising administering eIF5A siRNA to the islet cells, wherein the eIF5A
siRNA inhibits expression of eIF5A 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 eIF5A siRNA treated islet cells do indeed express less eIF5A siRNA. By inhibiting eIF5A
expression, apoptosis is also inhibited. Figures 4 and 5 shows that treating islets cells with eIF5A
siRNA prior to isolation, inhibited these cells from apoptosis (as demonstrated by a reduction of the number of cells in the sub-Gl phase). Accordingly, 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.
Any eIF5A siRNA that inhibits expression of eIF5A may be used. The term "inhibits"
also means reduce. One exemplary eIF5A siRNA comprises the sequence:
AGUCGACCUUCAGUAAGGCdTdT. Co-pending application 11/293,391, which was filed on November 28, 2005 (which is herein incorporated by reference in its entirety) provides additional exemplary eIF5A siRNAs and other antisense constructs that have been used to inhibit expression of eIF5A in other cell types and were also shown to inhibit apoptosis. One skilled in the art could design other eIF5A siRNAs given the eIF5A sequence and can easily test for the siRNAs ability to inhibit expression without undue experimentation. Figures 6-11 provide sequences of eIF5A, exemplary eIF5A siRNAs and antisense constructs. In another embodiment of the invention, antisense constructs of eIF5A may be used to inhibit expression of eIF5A and thus inhibit apoptosis of the islet cells.
In preferred embodiments the eIF5A siRNA comprises the nucleotide sequence AGUC GAC CUUCAGUAAG G C dT dT .
The present invention also provides a method for inhibiting islet cells from undergoing apoptosis during a donor harvesting process. As discussed above, many islets cells undergo apoptosis when they are harvested. The present inventors have shown that providing eIF5A
siRNA to the islet cells prior to harvesting, offers a protective benefit against apoptosis. The eIF5A siRNA is administered to the islet cells of an islet cell donor prior to islet isolation. The donor (and hence islet cells) may be any animal, including human islet cells.
Any method of administration may be used. For example, 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. In a mouse model, 1 ml saline or siRNA (5 g) is released slowly, the needle is removed and the knot is closed behind the needle to prevent fluid escape. At this point the mouse is turned around and the bile duct accessed for pancreas digestion.
The pancreas may be held longer with siRNA. Alternatively, it can be removed but kept cold with collagenase longer. Regular islet isolation methods are followed and the islets (50) may be incubated in for 16 hours.
The present invention also provides a composition for inhibiting apoptosis in islet cells, comprising eIF5A siRNA, wherein the siRNA inhibits expression of eIF5A 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 AGUC GAC CUUCAGUAAG G C dT dT .
EXAMPLES
Mouse islets express eIF5A.
Total RNA was extracted from isolated mouse islets and RT-PCR was performed for ((3-actin and for eIFSA (fig. 1). Resting non-stimulated islets exhibited positive levels of eIFSA-mRNA.
eIF5A-mRNA levels diminished after e1F5A-siRNA delivery: portal vein slow pe~fusion.
Mice were introduced 1 ml of siRNA (CT (control) sequence or eIF5A, 5 g) or saline, n = 2 per group, by slow retrograde portal vein perfusion (fig. 2). 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. Total RNA was then extracted and RT-PCR
was performed for 0-actin and for eIF5A (fig. 3). Ratio of mRNA for eIF5A/0-actin was 5.24 (CT-siRNA) and 3.01 (eIF5A-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 e1F5A-siRNA
delivery: portal vein hydrodynamic perfusion.
Mice were introduced 1 ml of siRNA (CT or eIF5A, 5 g) or saline, n = 2 per group, by hydrodynamic retrograde portal vein perfusion, which was completed within 5 seconds.
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). Levels of mRNA for eIF5A/0-actin were again higher in CT-siRNA group than in eIFSA-siRNA group. Apoptosis rate was reduced by 28.1 %
(fig. 4). This experiment was repeated with n = 3, apoptosis rate again diminished (fig. 5).
Islets perfusion with biotinylated-siRNA.
Biotinylated-siRNA (50 g) was perfused into islets as described above (slow perfusion, n = 1). Pancreas was fixed in formalin for staining.
siRNA.
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.
RT-PCR.
Total RNA was extracted from cells using Qiagen RNeasy kit. eIF5A Primers:
Forward 5'-GAC AGT GGG GAG GTA CGA GA-3'; Reverse 5'-GGG GTG AGG AAA ACC AAA AT-3'.
Propidium iodide (PI) apoptosis stain.
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.
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). Levels of mRNA for eIF5A/0-actin were again higher in CT-siRNA group than in eIFSA-siRNA group. Apoptosis rate was reduced by 28.1 %
(fig. 4). This experiment was repeated with n = 3, apoptosis rate again diminished (fig. 5).
Islets perfusion with biotinylated-siRNA.
Biotinylated-siRNA (50 g) was perfused into islets as described above (slow perfusion, n = 1). Pancreas was fixed in formalin for staining.
siRNA.
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.
RT-PCR.
Total RNA was extracted from cells using Qiagen RNeasy kit. eIF5A Primers:
Forward 5'-GAC AGT GGG GAG GTA CGA GA-3'; Reverse 5'-GGG GTG AGG AAA ACC AAA AT-3'.
Propidium iodide (PI) apoptosis stain.
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.
Claims (8)
1. 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 eIF5A siRNA inhibits expression of eIF5A in the islet cells and thereby inhibits apoptosis in the islet cells.
2. The method of claim 1 wherein the eIF5A siRNA comprises the nucleotide sequence AGUCGACCUUCAGUAAGGCdTdT.
3. The method of claim 1 wherein the siRNA is administered via perfusion through the portal vein of the islet cell donor.
4. The method of claim 1 wherein the siRNA is administered via hydrodynamic perfusion through the portal vein of the islet cell donor.
5. A method for inhibiting expression of eIF5A in islet cells comprising administering eIF5A
siRNA to the islet cells, wherein the eIF5A siRNA inhibits expression of eIF5A
in the islet cells.
siRNA to the islet cells, wherein the eIF5A siRNA inhibits expression of eIF5A
in the islet cells.
6. 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.
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.
7. A composition for inhibiting apoptosis in islet cells, comprising eIF5A
siRNA, wherein the siRNA inhibits expression of eIF5A and thereby inhibits apoptosis in the islet cells.
siRNA, wherein the siRNA inhibits expression of eIF5A and thereby inhibits apoptosis in the islet cells.
8. The composition of claim 7 wherein the siRNA comprises the nucleotide sequence AGUCGACCUUCAGUAAGGCdTdT.
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US78341406P | 2006-03-20 | 2006-03-20 | |
| US60/783,414 | 2006-03-20 | ||
| PCT/US2007/064424 WO2007109677A2 (en) | 2006-03-20 | 2007-03-20 | A novel method of protecting islet cells from apoptosis during the donor harvesting process |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2637137A1 true CA2637137A1 (en) | 2007-09-27 |
Family
ID=39680975
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|---|---|---|---|
| CA002637137A Abandoned CA2637137A1 (en) | 2006-03-20 | 2007-03-20 | A novel method of protecting islet cells from apoptosis during the donor harvesting process |
Country Status (7)
| Country | Link |
|---|---|
| EP (1) | EP1996707A2 (en) |
| JP (1) | JP5543774B2 (en) |
| AR (1) | AR060785A1 (en) |
| AU (1) | AU2007226878A1 (en) |
| CA (1) | CA2637137A1 (en) |
| IL (1) | IL192388A0 (en) |
| WO (1) | WO2007109677A2 (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20090093434A1 (en) * | 2007-08-20 | 2009-04-09 | Senesco Technologies, Inc. | Use of eif-5a1 sirna to protect islets cells from apoptosis and to preserve their functionality |
Family Cites Families (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US7166467B2 (en) * | 2001-07-23 | 2007-01-23 | Senesco Technologies, Inc. | Nucleic acids, polypeptides, compositions, and methods for modulating apoptosis |
| JP2006520611A (en) * | 2003-03-05 | 2006-09-14 | セネスコ テクノロジーズ，インコーポレイティド | Use of antisense oligonucleotides or siRNA to suppress the expression of eIF-5A1 |
| WO2005007853A2 (en) * | 2003-06-06 | 2005-01-27 | Senesco Technologies, Inc. | Inhibition of apoptosis-specific elf-5a (“eif-5a1”) with antisense oligonucleotides and sirnas as anti-inflammatory therapeutics |
-
2007
- 2007-03-20 CA CA002637137A patent/CA2637137A1/en not_active Abandoned
- 2007-03-20 WO PCT/US2007/064424 patent/WO2007109677A2/en active Application Filing
- 2007-03-20 AU AU2007226878A patent/AU2007226878A1/en not_active Abandoned
- 2007-03-20 JP JP2009501701A patent/JP5543774B2/en not_active Expired - Fee Related
- 2007-03-20 EP EP07758926A patent/EP1996707A2/en not_active Withdrawn
- 2007-05-07 AR ARP070101971A patent/AR060785A1/en not_active Application Discontinuation
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2008
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Also Published As
| Publication number | Publication date |
|---|---|
| JP2009530418A (en) | 2009-08-27 |
| AU2007226878A1 (en) | 2007-09-27 |
| AR060785A1 (en) | 2008-07-10 |
| JP5543774B2 (en) | 2014-07-09 |
| IL192388A0 (en) | 2008-12-29 |
| WO2007109677A3 (en) | 2008-04-24 |
| EP1996707A2 (en) | 2008-12-03 |
| WO2007109677A2 (en) | 2007-09-27 |
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