Note: Descriptions are shown in the official language in which they were submitted.
CA 02637137 2008-07-14
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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
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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.
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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.
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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
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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
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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.
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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.
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