Note: Descriptions are shown in the official language in which they were submitted.
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USE OF A TRUNCATED EIF-5A1 POLYNUCLEOTIDE
TO INDUCE APOPTOSIS IN CANCER CELLS
RELATED APPLICATIONS
This application claims priority to U.S. provisional application 61/093,749,
which was filed on September 3, 2008, and U.S. application 12/400,742 filed on
March 19, 2009, which are herein incorporated by reference in its entirety.
BACKGROUND OF THE INVENTION
Relatively recently, researchers observed that double stranded RNA
("dsRNA") could be used to inhibit protein expression. This ability to silence
a gene
has broad potential for treating human diseases, and many researchers and
commercial entities are currently investing considerable resources in
developing
therapies based on this technology.
Double stranded RNA induced gene silencing can occur on at least three
different levels: (i) transcription inactivation, which refers to RNA guided
DNA or
histone methylation; (ii) siRNA induced mRNA degradation; and (iii) mRNA
induced
transcriptional attenuation.
It is generally considered that the major mechanism of RNA induced silencing
(RNA interference, or RNAi) in mammalian cells is mRNA degradation. Initial
attempts to use RNAi in mammalian cells focused on the use of long strands of
dsRNA. However, these attempts to induce RNAi met with limited success, due in
part to the induction of the interferon response, which results in a general,
as opposed
to a target-specific, inhibition of protein synthesis. Thus, long dsRNA is not
a viable
option for RNAi in mammalian systems.
More recently it has been shown that when short (18-30 bp) RNA duplexes are
introduced into mammalian cells in culture, sequence-specific inhibition of
target
mRNA can be realized without inducing an interferon response. Certain of these
short
dsRNAs, referred to as small inhibitory RNAs ("siRNAs"), can act catalytically
at
sub-molar concentrations to cleave greater than 95% of the target mRNA in the
cell.
A description of the mechanisms for siRNA activity, as well as some of its
applications are described in Provost et al. (2002) Ribonuclease Activity and
RNA
Binding of Recombinant Human Dicer, EMBO J. 21(21): 5864-5874; Tabara et al.
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(2002) The dsRNA Binding Protein RDE-4 Interacts with RDE-1, DCR-1 and a
DexH-box Helicase to Direct RNAi in C. elegans, Cell 109(7):861-71; Ketting et
al.
(2002) Dicer Functions in RNA Interference and in Synthesis of Small RNA
Involved
in Developmental Timing in C. elegans; Martinez et al., Single-Stranded
Antisense
siRNAs Guide Target RNA Cleavage in RNAi, Cell 110(5):563; Hutvagner &
Zamore (2002) A microRNA in a multiple-turnover RNAi enzyme complex, Science
297:2056.
From a mechanistic perspective, introduction of long double stranded RNA
into plants and invertebrate cells is broken down into siRNA by a Type III
endonuclease known as Dicer. Sharp, RNA interference--2001, Genes Dev. 2001,
15:485. Dicer, a ribonuclease-III-like enzyme, processes the dsRNA into 19-23
base
pair short interfering RNAs with characteristic two base 3' overhangs.
Bernstein,
Caudy, Hammond, & Hannon (2001) Role for a bidentate ribonuclease in the
initiation step of RNA interference, Nature 409:363. The siRNAs are then
incorporated into an RNA-induced silencing complex (RISC) where one or more
helicases unwind the siRNA duplex, enabling the complementary antisense strand
to
guide target recognition. Nykanen, Haley, & Zamore (2001), ATP requirements
and
small interfering RNA structure in the RNA interference pathway, Cell 107:309.
Upon binding to the appropriate target mRNA, one or more endonucleases within
the
RISC cleaves the target to induce silencing. Elbashir, Lendeckel, & Tuschl
(2001)
RNA interference is mediated by 21- and 22-nucleotide RNAs, Genes Dev. 15:188,
FIG. 1.
The interference effect can be long lasting and may be detectable after many
cell divisions. Moreover, RNAi exhibits sequence specificity. Kisielow, M. et
al.
(2002), Isoform-specific knockdown and expression of adaptor protein ShcA
using
small interfering RNA, J. Biochem. 363:1-5. Thus, the RNAi machinery can
specifically knock down one type of transcript, while not affecting closely
related
mRNA. These properties make siRNA a potentially valuable tool for inhibiting
gene
expression and studying gene function and drug target validation. Moreover,
siRNAs
are potentially useful as therapeutic agents against: (1) diseases that are
caused by
over-expression or misexpression of genes; and (2) diseases brought about by
expression of genes that contain mutations.
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Successful siRNA-dependent gene silencing depends on a number of factors.
One of the most contentious issues in RNAi is the question of the necessity of
siRNA
design, i.e., considering the sequence of the siRNA used. Early work in C.
elegans
and plants circumvented the issue of design by introducing long dsRNA (see,
for
instance, Fire, A. et al. (1998) Nature 391:806-811). In this primitive
organism, long
dsRNA molecules are cleaved into siRNA by Dicer, thus generating a diverse
population of duplexes that can potentially cover the entire transcript. While
some
fraction of these molecules are non-functional (i.e., induce little or no
silencing) one
or more have the potential to be highly functional, thereby silencing the gene
of
interest and alleviating the need for siRNA design. Unfortunately, due to the
interferon response, this same approach is unavailable for mammalian systems.
While this effect can be circumvented by bypassing the Dicer cleavage step and
directly introducing siRNA, this tactic carries with it the risk that the
chosen siRNA
sequence may be non-functional or semi-functional.
A number of researches have expressed the view that siRNA design is not a
crucial element of RNAi. On the other hand, others in the field have begun to
explore
the possibility that RNAi can be made more efficient by paying attention to
the design
of the siRNA.
To treat various diseases or disorders, the upregulation of certain proteins
is
desirable but this may not be all that is needed. For example, the
combinatorial use of
siRNA to knock down or knock out expression of an endogenous protein or a
different protein may be needed. The present invention fulfills this need and
provides
methods of treating cancer, especially multiple myeloma.
Cancer, including multiple myeloma are diseases which would benefit from
the ability to induce apoptosis. Conventional therapies for of multiple
myeloma
include chemotherapy, stem cell transplantation, high-dose chemotherapy with
stem
cell transplantation, and salvage therapy. Chemotherapies include treatment
with
Thalomid (thalidomide), bortezomib, Aredia (pamidronate), steroids, and
Zometa (zoledronic acid). However many chemotherapy drugs are toxic to
actively
dividing non-cancerous cells, such as of the bone marrow, the lining of the
stomach
and intestines, and the hair follicles. Therefore, chemotherapy may result in
a
decrease in blood cell counts, nausea, vomiting, diarrhea, and loss of hair.
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Conventional chemotherapy, or standard-dose chemotherapy, is typically the
primary or initial treatment for patients with of multiple myeloma. Patients
also may
receive chemotherapy in preparation for high-dose chemotherapy and stem cell
transplant. Induction therapy (conventional chemotherapy prior to a stem cell
transplant) can be used to reduce the tumor burden prior to transplant.
Certain
chemotherapy drugs are more suitable for induction therapy than others,
because they
are less toxic to bone marrow cells and result in a greater yield of stem
cells from the
bone marrow. Examples of chemotherapy drugs suitable for induction therapy
include dexamethasone, thalidomide/dexamethasone, VAD (vincristine, Adriamycin
(doxorubicin), and dexamethasone in combination), and DVd (pegylated liposomal
doxorubicin (Doxil , Caelyx ), vincristine, and reduced schedule dexamethasone
in
combination).
The standard treatment for of multiple myeloma is melphalan in combination
with prednisone (a corticosteroid drug), achieving a response rate of 50%.
Unfortunately, melphalan is an alkylating agent and is less suitable for
induction
therapy. Corticosteroids (especially dexamethasone) are sometimes used alone
for
multiple myeloma therapy, especially in older patients and those who cannot
tolerate
chemotherapy. Dexamethasone is also used in induction therapy, alone or in
combination with other agents. VAD is the most commonly used induction
therapy,
but DVd has recently been shown to be effective in induction therapy.
Bortezomib has
been approved recently for the treatment of multiple myeloma, but it is very
toxic.
However, none of the existing therapies offer a significant potential for a
cure. Thus,
there still remains a need to find a suitable treatment for cancer and
multiple
myeloma. The present invention fulfills this need.
SUMMARY OF INVENTION
The present invention provides an isolated polynucelotide encoding a
truncated form of eIF-5Al as well as a truncated eIF-Al polypeptide. The
truncated
eIF-5A l polynucleotide is useful in inducing apoptosis and killing cancer
cells in a
subject The truncated polnucleotide may be used within an expression vector
which
is then administered to a subject. The truncated eIF-5A form is expressed
within the
mammal and kills cancer cells. The truncated eIF-5Al protein is about 16 kDA
as
opposed to the full length elf-5Al protein, which is about 17 kDa. A
polynucleotide
encoding a truncated eIF5A1 protein may be used to make a medicament used to
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induce apoptosis in a cancer cell or a tumor in a subject. The polynucleotide
encoding
a truncated eIF5Al protein may be used in conjunctions with other embodiments
described herein (for example in combination with a polynucleotide encoding
full
length eIF5Al, with a polynucleotide encoding full length mutant eIF5Al,
and/or
with an siRNA targeted against the 3'UTR of eIF5A).
The present invention also relates to the combinatorial use of an siRNA
targeted against an endogenous gene to knock out or knock down expression of
the
endogenous gene in a host and a delivery of a polynucleotide encoding the gene
in a
delivery vehicle/expression vector to the host to provide expression in the
host of the
protein encoded by the polynucleotide. A polynucleotide encoding a normal (non
faulty) protein (or the protein itself) is administered to the host and is
expressed (in
the case of the polynucleotide) so that the normal protein can perform its
necessary
function. The siRNA is preferably designed to target a region of the gene so
it either
knocks down or knocks out endogenous expression of the faulty protein but at
the
same time will not effect exogenous expression of the administered
polynucleotide
encoding the normal protein.
The invention provides a composition comprising a complex of an eIF5Al
siRNA targeted against the 3' end of eIF5Al, an expression vector comprising a
polynucleotide encoding a mutant eIF5Al wherein the mutant eIF5Al is unable to
be
hypusinated, and wherein the siRNA and the expression vector are complexed to
polyethylenimine to form a complex.
The invention provides a composition comprising an siRNA targeted against a
target gene to suppress endogenous expression of the target gene is a subject,
and a
polynucleotide encoding a target protein capable of being expressed in the
subject. In
certain embodiments the polynucleotide is in RNAi resistant plasmid (will not
be
suppressed by the siRNA). The siRNA and the plasmid are preferably complexed
to
polyethylenimine to form a complex.
In certain embodiments the siRNA has the sequence shown in figure 25 and
wherein the polynucleotide encoding the mutant eIF5Al is eIF5Alx5ox The
expression vector comprises a polynucleotide encoding a mutant eIF5Al and a
promoter operably linked to provide expression of the polynucleotide in a
subject.
The promoter preferably is either tissue specific or systemic. For example, if
the
composition is used to treat cancer, then preferably the promoter is tissue
specific for
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the tissue in which the cancer resides. For example, for treating multiple
myeloma, it
is preferable to use a B cell specific promoter, such as B29. In certain
embodiments,
the expression vector comprises a pCpG plasmid.
In certain embodiments, the eIF5Al siRNA and the expression vector
comprising the mutant eIF5Al polynucleotide are independently complexed to
polyethylenimine, such as in vivo JetPEITM. In other embodiments, the eIF5Al
siRNA and the expression vector comprising the mutant eIF5Al polynucleotide
are
complexed together to polyethylenimine.
The present invention further provides a composition comprising an eIF5Al
siRNA targeted against the 3' end of eIF5Al and an expression vector
comprising a
polynucleotide encoding a mutant eIF5Al wherein the mutant eIF5Al is unable to
be
hypusinated, and wherein the siRNA and the expression vector are delivered to
a
subject to treat cancer. The cancer may be any cancer including multiple
myeloma.
The present invention further provides a method of treating cancer comprising
administering composition of the present invention to a subject (including but
not
limited to mammals and humans).
The composition may be administered any acceptable route, such as, but not
limited to intravenously, intra peritoneally, subcutaneously or intra
tumorally. The
siRNA and the expression vector may be administered at different times and via
different routes or may be administered together at the same time and via the
same
route. For example, but not limited to, the siRNA may be delivered naked or
complexed to a carrier such as in vivo jetPEI via IV and the expression vector
may be
administered intra tumorally, or both the siRNA and the expression vector may
be
administered IV or intratumorally, etc.
The present invention provides a method of inhibiting cancer cell growth
and/or killing cancer cells. The present invention also provides a method of
inhibiting
or slowing down the ability of a cancer cell to metastasize. Inhibiting cancer
growth
includes a reduction in the size of a tumor, a decrease in the growth of the
tumor, and
can also encompass a complete remission of the tumor. The cancer can be any
cancer
or tumor, including but not limited to colon cancer, colorectal
adenocarcinoma,
bladder carcinoma, cervical adenocarcinoma, and lung carcinoma. Preferably the
cancer is multiple myeloma.
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In preferred embodiments, the eIF-5A is a mutant that is unable to be
hypusinated. Exemplary mutants are described herein.
In addition to providing eIF-5A or a polynucleotide encoding eIF-5A to a
subject (to provide expression of the eIF-5A), siRNA is provided to knock out
or
knock down endogenous expression of eIF-5A.
The present invention also provides the use of eIF5A, polynucleotides
encoding eIF5Al and siRNA against eIF5Al to make a medicament to treat cancer
kill multiple myeloma cells in a subject having multiple myeloma. Preferably
the
polynucleotides encoding a mutant eIF-5A are unable to be hypusinated.
The present invention also provides a method of treating sickle cell anemia. A
polynucleotide encoding a healthy hemoglobin gene (such as HBB) is
administered to
a patient suffering from sickle cell anemia. In conjunction, the patient is
also
administered siRNA that targets the gene encoding the faulty hemoglobin gene
(such
as the gene encoding the mutant HbS) to knock down or knock out expression of
the
faulty protein. The treatment may further comprise administration of other
known
medicines or treatments commonly used in treating sickle cell anemia.
BRIEF DESCRIPTION OF THE FIGURES
Figure 1 provides the amino acid sequence of human eIF-5Al and shows
various important sites.
Figure 2 shows that mutation of eIF-5Al at K50 and K67 increases
accumulation of transfected protein. See example 1.
Figure 3 shows that mutation of eIF5Al at K47, K50 and K67 increases
accumulation of transfected protein. See example 2.
Figure 4 shows that mutation of eIF5Al at K50 and K67 results in induction
of apoptosis when transfected into KAS cells. See example 3.
Figure 5 shows that mutation of eIF5Al at K50 and K67 results in induction
of apoptosis when transfected into KAS cells. See example 4.
Figure 6 shows that mutation of eIF5Al at K50 and K67 results in induction
of apoptosis when transfected into KAS cells. See example 5.
Figure 7A shows transfection with siRNA and treating with an adenovirus that
is modified to express eIF-5Al results in apoptosis in KAS cells. See example
6A.
Figure 7B shows that pre-treatment with eIF5Al siRNA (against target #1 (SEQ
ID
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NO: 1))(the sequence of the siRNA construct shown in figure 25 reduced
expression
of endogenous eIF5Al but allows accumulation of RNAi-resistant eIF5Alk5oA
expressed by adenovirus. See example 6B. Figure 7C shows that pre-treatment
with
eIF5Al siRNA against target #1 prior to adenovirus infection reduces
expression of
phosphorylated NF-KB in human multiple myeloma cells. See example 6C. Figure
7D shows that pre-treatment with eIF5Al siRNA against target #1 prior to
Adenovirus infection reduces expression of phosphorylated NF-kB and ICAM-1 in
human multiple myeloma cells. See example 6D. Figure 7E shows that siRNA-
mediated suppression of eIF5A in human multiple myeloma cells inhibits LPS-
mediated induction of NFkB DNA-Binding Activity. The inhibition of NFkB
activity
by eIF5A siRNA could explain it's ability to increase apoptosis induction when
combined with over-expression of eIF5Ax5oR since NF-kB regulates many pro-
survival and anti-apoptosis pathways. Figure 7F shows that pretreatment of KAS
cells with siRNA increases apoptosis by eIF5Alk50R gene delivery in the
presence of
IL-6. See example 6E.
Figure 8 shows that co-administration of eIF5Al plasmid and eIF5Al siRNA
delays growth of multiple myeloma subcutaneous tumors. The data shown is the
tumor volume for all the mice in each group. See example 7.
Figure 9 shows that co-administration of eIF5Al plasmid and eIF5Al siRNA
delays growth of multiple myeloma subcutaneous tumors. The data shown is the
average tumor volume per group +/- standard error. See example 7.
Figure 10 shows that co-administration of eIF5Al plasmid and eIF5Al siRNA
reduces weight of multiple myeloma subcutaneous tumors. See example 7.
Figure 11 shows that co-administration of eIF5Al plasmid and eIF5Al siRNA
delays growth of multiple myeloma subcutaneous tumors and results in tumor
shrinkage. See Example 8.
Figure 12 shows that administration of eIF5Al siRNA intra-venously (i.v.)
and PEI/eIF5A1K50R plasmid complexes intra-tumorally (i.t.) results in tumor
shrinkage of multiple myeloma subcutaneous tumors. See example 9.
Figure 13A shows that treatment with eIF5Al plasmid and eIF5Al siRNA
delays growth of multiple myeloma subcutaneous tumors and results in tumor
shrinkage. Figure 13B shows that co-administration of eIF5Al plasmid and
eIF5Al
siRNA results in tumor shrinkage. Figure 13C shows that administration of
eIF5Al
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siRNA intra-venously (i.v.) and PEI/eIF5Alx50R plasmid complexes intra-
tumorally
(i.t.) results in tumor shrinkage of multiple myeloma subcutaneous tumors.
Figure 14 shows that intra-venous co-administration of eIF5Al plasmid and
eIF5Al siRNA delays growth of multiple myeloma subcutaneous tumors. See
example 10.
Figure 15 shows that administration of eIF5Al siRNA intra-venously (i.v.)
and PEI/eIF5A1K50R plasmid complexes intra-venously (i.v.) or intra-peritoneal
(i.p.) delays growth of multiple myeloma subcutaneous tumors. See example 11.
Figure 16 shows that treatment with eIF5Al plasmid and eIF5Al siRNA
delays growth of multiple myeloma subcutaneous tumors.
Figure 17 shows that co-administration of eIF5Al plasmid and eIF5Al siRNA
delays growth of multiple myeloma subcutaneous tumors and results in tumor
shrinkage. See example 12.
Figure 18 shows that administration of eIF5Al siRNA intra-venously (i.v.)
and PEI/eIF5A1K50R plasmid complexes intra-tumorally (i.t.) results in tumor
shrinkage of multiple myeloma subcutaneous tumors. See example 13.
Figure 19 shows co-administration of eIF5Alx50R plasmid, driven by either the
EF1 or B29 promoter, and eIF5Al siRNA delays growth of multiple myeloma
subcutaneous tumors and results in tumor shrinkage (KAS-SQ-S). See example 14.
Figure 20 shows co-administration of eIF5Al siRNA increases anti-tumor
effect of eIF5Alx50R plasmid, driven by either the EF1 or B29 promoter, on
multiple
myeloma subcutaneous tumors and results in reduced tumor burden (KAS-SQ-S).
See
example 15.
Figure 21 shows eIF5Al siRNA synergistically increases apoptosis resulting
from infection with Ad-eIFSA in lung adenocarcinoma cells. See example 16.
Figure 22 shows the map of pExp5A, the construction of which is
described in Example 17.
Figure 23 shows the predicted sequence of pExp5A (3371 bp). See
example 17.
Figure 24 shows the rexpression of eIF5A1K5O in various cell lines. See
example 18.
Figure 25 shows the target sequence and the sequence of a preferred eIF5Al
siRNA.
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Figure 26 provides the results of efficacy studies in multiple myeloma. See
example 21.
Figure 27 provides the results of efficacy studies in multiple myeloma. See
example 21.
Figure 28 provides the sequence of eIF-5Alk5 ' cDNA.
Figure 29 provides the alignment of human eIF-5A l against human
eIF5Alk5ox Figure 30 shows the effect of DNA:siRNA ratio on HA-eIF5AK5ox
expression. See example 23.
Figure 31 shows the effect of DNA:siRNA ratio on apoptosis induced by
nanoparticle transfection. See example 24.
Figure 32 shows administration of PEI complexes (N/P = 6 or 8) containing
eIF5A1K5OR plasmid and eIF5Al siRNA (siSTABLE or non-siSTABLE) inhibits
growth of multiple myeloma subcutaneous tumors and results in tumor shrinkage.
See example 25.
Figure 33 shows that the JET PEITM nanoparticles are being effectively taken
up by tumor tissue and that nanoparticles are delivering plasmid and siRNA to
the
same cell. See example 26.
Figure 34 shows that KAS cells undergo apoptosis in response to Actinomycin
D. KAS cells were either left untreated or were treated with 0.5 g/ml
Actinomycin
D. Twenty-four hours after the intiation of treatment, the cells were
harvested,
washed, and apoptotic cells were labeled using Annexin/PI (BD Bioscience) and
analyzed by flow cytometry.
Figure 35 shows that a truncated form of eIF-5Al accumulates in response to
actinomycin D treatment. KAS cells were treated with 0.5 g/ml Actinomycin D
for
time periods ranging from 0 hours to 30 hours. Cell lysate was harvested and
analyzed by Western blot using an antibody against eIF5Al. Accumulation of a
smaller molecular weight (cleaved) form of eIF5Al was observed beginning at
approximately 8 hours after Actinomycin D treatment and was present throughout
the
remainder of the treatment. Equal loading is demonstrated by Western blot of
the
same membrane using an antibody against actin.
Figure 36 shows that the truncated form of eIF-5Al has a higher isolectric
point than the full length elf-5Al . KAS cells were either left untreated or
were
treated with 0.5 g/ml Actinomycin D. Seventeen hours after the initiation of
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treatment, the cell lysates were harvested and separated by 2-D gel
electrophoresis
followed by Western blot analysis using an antibody against eIF5Al. A large
amount
of the full-length eIF5Al can be observed in the untreated sample while
accumulation
of a cleaved form of eIF5Al with a higher pI can be observed in the
Actinomycin D-
treated sample.
Figure 37 provides a photograph of the truncated eIF-5Al 2-D gel. KAS cells
were treated with 0.5 g/ml Actinomycin D. Seventeen hours after the
initiation of
treatment, the cell lysate was harvested and separated by 2-D gel
electrophoresis
followed by Coomassie Blue staining. The position of the cleaved form of
eIF5Al
that was collected for mass spectrometry analysis is indicated.
Figure 38 provides the results of sequencing by mass spectrometry of the
truncated eIF-SAl. (A) The full-length amino acid sequence of eIF5Al is shown
in
black. Sequenced peptides identified by Mass spectrometry (B) that aligned
with the
full-length sequence of eIF5Al are shown in underlined (A). Note that the
first six
amino acids of eIF5Al are missing from the sequenced peptides.
Figure 39 shows that caspase 3, 8 and 9 inhibitors inhibit formation of the
truncated form of eIF-5Al. Human multiple myeloma KAS cells were incubated
with
different caspase inhibitors 8 hours before being co-treated with 0.5 g/ml
Actinomycin D (ActD) for another 16 hours. Cell lysates were collected.
Proteins
were isolated by 2D-PAGE and subjected to western analysis using antibody
against
eIF5A. The caspase inhibitors used were Z-VAD-FMK (general caspase inhibitor),
Z-LEHD-FMK (caspase 9 inhibitor), Z-DEVD-FMK (caspase 3/7 inhibitor), Z-IETD-
FMK (caspase 6/8 inhibitor), and Z-YVAD-FMK (caspase 1 inhibitor). Data
indicated that the general caspase inhibitor and inhibitors for caspase 3, 8
and 9 all
strongly prevent the formation of the cleavage form of eIF5A that accumulates
during
ActD-induced apoptosis in KAS cells. Caspase 1 inhibitor also reduces, but not
complete blocks, the formation of the cleavage product. These results indicate
that
eIF5Al is cleaved by caspases following genotoxic stress (induced by
Actinomycin
D); cleavage of eIF5Al could alter its activity ie increase pro-apoptotic
activity.
Figure 40 shows that a deacetylase inhibitor (nicotinamide) promotes
formation of the truncated form of eIF-5Al. eIF5A which can be acetylated on
Lysine47 is a substrate of a Sir2-related deacetylase Hst2 (Shirai et al.,
2008). Human
cervical carcinoma Hela S3 cells were treated with 0.5 g/ml Actinomycin D
(ActD)
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and/or 20 M nicotinamide, a potent inhibitor of Sir2, for indicated lengths
of time.
Cell lysates were collected. Proteins were isolated by 2D-PAGE and subjected
to
western analysis using antibody against eIF5A. Treatment of Hela S3 cells with
ActD
or nicotinamide alone does not induce the accumulation of the cleavage form of
eIF5A. However, in HeLa cells treated with both Actinomycin D and
nicotinamide,
the cleavage form of eIF5A is up-regulated at least 4 hours after being
exposed to
ActD, suggesting that acetylation may promote the cleavage of eIF5A during
apoptosis.
Figure 41 provides the DNA sequence of truncated human eIF5Al.
Figure 42 provides the protein sequence of full length eIF5Al, truncated
eIF5As and the cleavage cite.
Figure 43: Infection of Hela cells with adenovirus constructs results in
accumulation of eIF5A transgene. Hela S3 cells were infected with different
adenovirus constructs at 500 infectious units per cell. Cell lysates were
collected at 48
and 72 hours after infection and subjected to SDS-PAGE and western blotting
analysis using antibodies against eIF5A and actin. Accumulation of truncated
eIF5A
can be observed.
Figure 44: Induction of apoptosis through treatment with Actinomycin D,
sodium nitroprusside, or withdrawal of IL-6 results in the accumulation of a
truncated
form of eIF5Al (eIF5A1A1-6) in human myeloma cells. KAS-6/1 cells were treated
as indicated and then subjected to 2D-PAGE and western blotting analysis using
antibody against eIF5A. Induction of apoptosis in KAS-6/1 cells via with
Actinomycin D, the NO donor, sodium nitroprusside, or IL-6 starvation all
induced
the accumulation of truncated eIF5Al. Over-expression of truncated eIF5Al
using
either Ad-Al A(2-6) or Ad-Al A(2-6)/K50R induced the accumulation of eIF5Al
proteins have a smaller pI than the truncated eIF5A1(eIF5A1A1-6) produced
during
apoptosis, indicating that other post-translational modifications are present
on
eIF5A1A1-6).
Figure 45: eIF5AID6E and eIF5A I D6E/K5 OR are resistant to Actinomycin
D-induced cleavage during apoptosis. Human multiple myeloma KAS-6/1 cells were
treated as indicated and then subjected to 2D-PAGE and western blot analysis
using
antibody against eIF5A. Over-expressed eIF5Al (Ad-Al) is subjected to cleavage
during treatment with Actinomycin D. However, eIF5AID6E or eIF5A I D6E/K5 OR
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are resistant to cleavage following treatment with Actinomycin D. These
results are
consistent with the hypothesis that amino acids 3 to 6 of eIF5Al constitute a
caspase
cleavage site.
Figure 46: eIF5Al is cleaved in vitro by recombinant caspases. Human
eIF5Al (Al) and the mutants eIF5AlD6E (D6E) and eIF5A1D6E/K50R (D6E/K5OR)
were subcloned into the pHM6 vector (Roche) which results in expression of an
HA-
tagged protein. pHM6-HA-A 1, pHM6-HA-A1 (D6E), and pHM6-HA-A1 (D6E/K50R)
were in vitro transcribed and translated (TNT T7 Quick Coupled
Transcription/Translation Systems; Promega) and labelled with Transcend Non-
Radioactive Translation Detection Systems (Promega) following the
manufacturer's
instructions. Three microliters of each the in vitro translation products were
incubated with one microliter of recombinant caspase (caspases 1, 2, 3, 6, 7,
8, 9, or
10; Calbiochem) in caspase reaction buffer (50 mM Hepes, 0.1% CHAPS, 10%
sucrose, 100 mM NaCl, 10 mM DTT, 1 mM EDTA) at 37 C for 2 hours. Final
products were subjected to SDS-PAGE, and the biotinylated proteins were
visualized
by incubation with Streptavidin-HRP, followed by chemiluminescent detection.
The
wild-type eIF5Al is cleaved by caspases 2, 3, 6, 7, 8, 9, and 10 but not by
caspase 1.
HA-tagged eIF5AlD6E and eIF5AlD6E/K50R are effectively cleaved in vitro by
caspases 3, 7 and 8 but are not efficiently cleaved by caspases 9 and 10.
However,
this cleavage may be due to the highly abundant caspase activity in the in
vitro system
as well as the high structural similarity between glutamic acid and aspartic
acid,
thereby allowing cleavage of eIF5AlD6E, although at a reduced efficiency
(particularly since eIF5AID6E and eIF5A1D6E/K50R were resistant to cleavage in
an in vivo assay (Figure 45) .
Figure 47: Infection of Hela cells with Ad-eIF5A1A(2-6) or Ad-
eIF5A1K50RA(2-6) results in increased apoptosis compared to infection with
wild-
type Ad-eIF5Al or Ad-eIF5A1K50R. Human cervical cancer Hela S3 cells were
infected with different adenovirus constructs at 500 infectious units per
cell. At 48 or
72 hours after infection, cells were collected and subjected to Annexin V/PI
staining.
Cells were then sorted by flow cytometry. Percentages of cells at early stage
of
apoptosis (Annexin V+/PI-), as well as cells at late stage of apoptosis
(Annexin
V+/PI+) are shown. Truncation of amino acids 2 to 6 increases the apoptotic
activity
of eIF5Al or eIF5A1K50R. Conversely, mutation of the predicted cleavage site
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reduces, but does not eliminate, the apoptotic activity of eIF5Al or
eIF5A1K50R.
These results indicate that caspase-mediated cleavage of eIF5Al results in a
truncated
form of eIF5Al with increased apoptotic activity.
Figure 48 provides a model of the contribution of caspase-mediated truncation
of eIF-5Al in apoptosis.
Figure 49 shows that cleaved eIF5A accumulates in the nucleus following
actinomycin D treatment in KAS human myeloma cells.
Figure 50 shows that cleaved eIF5A accumulates in the nucleus following
actinomycin D treatment in KAS human myeloma cells. Key: UN= untreated; actD=
treatment with actinomycin D; C=cytoplasmic fraction ; N=nuclear fraction, a-
tubilin
and a-PCNA are controls. This figure also shows that the truncated form
accumulates
predominantly in the nucleus, while the full length eIF5A is evenly
distributed
between the cytoplasm and nucleus.
Figure 51 shows that the truncated form of eIF5A accumulates in response to
different apoptotic stimuli in multiple cell lines and cell types indicating
that caspase-
mediated cleavage of eIF5A is a common phenomena during apoptosis. This figure
also shows that the truncated form accumulates predominantly in the nucleus,
while
the full length eIF5A is evenly distributed between the cytoplasm and nucleus.
Figure 52 provides results of a study that shows truncated eIF5A is produced
in different cell lines in response to different apoptotic stimuli -- KAS
human multiple
myeloma cells following treatment with IL-6 starvation or IL-6 and FBS
starvation
and in UACC 1598 (human ovarian cancer cell line) following treatment with
actinomycin D.
Figure 53 provides the nucleotide sequence of human eIF5Al and eIF5A2.
DETAILED DESCRIPTION OF THE INVENTION
The present invention provides an isolated polynucelotide encoding a
truncated form of eIF-5Al as well as a truncated eIF-Al polypeptide. The
truncated
eIF-5A l polynucleotide is useful in inducing apoptosis and killing cancer
cells. The
truncated polnucleotide may be used within an expression vector which is then
administered to a mammal. The truncated eIF-5A form is expressed within the
mammal and kills cancer cells. The truncated eIF-5Al protein is about 16 kDA
as
opposed to the full length elf-5Al protein, which is about 17 kDa.
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Formation of a smaller molecular weight form of eIF5A (- 16 kDa) has been
observed in cells undergoing apoptosis. This has been observed in beta islet
cells
treated with a cytokine cocktail and in human myeloma cells (KAS cells)
treated with
the cytotoxic drug, Actinomycin D. See Figure 35. The smaller molecular weight
form of eIF5A has also been found to accumulate in HeLa cervical cancer cells
undergoing apoptosis following infection with Ad-eIF5Al, suggesting that the
smaller
molecular weight form of eIF5A results from cleavage by proteases rather than
being
the result of alternative splicing. The observation that the accumulation of
the smaller
molecular weight form of eIF5A is accompanied by a dramatic reduction in the
amount of full-length hypusinated eIF5A further supports that the smaller
molecular
weight form of eIF5A is due to cleavage rather than alternative splicing. See
figure
36.
The present invention provides a composition comprising an eIF5Al
polynucleotide that encodes a truncated eIF5Al protein. This composition is
useful to
make a medicament used to induce apoptosis in a cancer cell or a tumor in a
subject.
The eIF5Al polynucleotide encodes a truncated eIF5Al protein preferally
comprising
the amino acid sequence set forth in SEQ ID NO:37 shown in figure 38. In
certain
embodiments, the eIF5Al polynucleotide encodes a truncated eIF5Al protein that
is
about 16 kDA.
The composition and or the medicament can be administered to mammals,
including humans. As used herein, "subject" includes mammals and humans.
Inducing apoptosis can have the following effects in a cancer cell or tumor:
slows cancer cell or tumor growth, arrests cancer cell or tumor cell growth,
or kills the
cancer cell or reduces the tumor size and any combination of the above. Any
cancer
can be treated and in certain embodiments, the cancer is multiple myeloma.
In certain embodiments the eIF5Al polynucleotide comprises the sequence set
forth in SEQ ID NO:38 (as shown in figure 41). In certain embodiments the
eIF5Al
polynucleotide is comprised within a plasmid or expression vector. Plasmids
and
expression vectors are described herein below in more detail. In certain
embodiments
the expression vector is an adenovirus expression vector or is pHM6. In
certain
embodiments the expression vector comprise a tissue specific promoter, such as
a B
cell specific promoter (i.e. B29) when the composition or medicament is used
to treat
multiple myeloma. The expression vector may comprise a pCpG plasmid. As
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discussed in more detail hereinbelow, the expression vector may be complexed
to
polyethylenimine.
The composition or medicament is preferably administered intratumorally,
intravenously or subcutaneously.
The present invention also provides an isolated polynucleotide encoding a
truncated eIF5Al protein wherein the polynucleotide comprises the sequence set
forth
in SEQ ID NO:38 (as shown in Figure 41).
The invention also provides an isolated polynucleotide encoding a truncated
eIF5Al protein wherein the truncated protein comprises the amino acid sequence
set
forth in SEQ ID NO:37.
As discussed hereinbelow, the eIF5Al is formed by caspase-mediated
cleavage. Accordinlgy the present invention further provides an isolated
truncated
eIF5Al polypeptide formed by caspase mediated cleavage of eIF5Al.
The present invention further provides a composition or the use of the
composition to make a medicament comprsing an eIF5Al polynucleotide that
encodes
a truncated eIF5Al protein in combination with a full length eIF5Al
polynucleotide.
This composition is useful to make a medicament to induce apoptosis in a
cancer cell
or tumor in a subject. The eIF5Al polynucleotide encodind a truncated eIF5A
protein
is as desribed above. The full length eIF5Al polynucleotide encodes a protein
comprising the amino acid sequence set forth in SEQ ID NO:35. The full length
eIF5Al polynucleotide is described in more detail below. In certain
embodiments,
the eIF5Al polynucleotide comprises the sequence set forth in SEQ ID NO:38 and
the
full length eIF5Al polynucleotide comprises sequence set forth SEQ ID NO:43
(as
shown in figure 53).
The full length and truncated polynucleotides may be present in expression
vectors as described herein. Additionally the vectors may comprise the
promoters
described herein as well as any other useful promoter.
In certain embodiments, the full length eIF5A polynucleotide encodes a
mutant eIF5Al, wherein the mutation prevents or inhibits hypusination by
deoxyhypusine synthase and/or wherein the mutation is present at the
ubiquinization
site and/or the acetylation site. The mutants are described in more detail
below. In
certain embodiments, the mutant is selected from the group consisting of K50A,
K50R, K67A, K47R, K67R, K50A/K67A, K50A/K47R, K50A/K67R, K50R/K67A,
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K50R /K47R, K50R/K67R, and K47A/K67A.
The present invention also provides a composition comprising a
polynucleotide encoding a truncated eIF5A, a nucleotide encoding a mutant
eIF5A
and an siRNA targeted against the 3'UTR of eIF5A. The combination/dual use of
a
nucleotide encoding a mutant eIF5A and an siRNA targeted against the 3'UTR of
eIF5A is described more fully below. The composition is useful to make a
medicament to administer to a subject to induce apoptosis in cancer cells or
tumors.
In certain preferred embodiments, the siRNA targets the sequence in eIF5Al
of 5'-GCT GGA CTC CTC CTA CAC A-3'. siRNA is dsRNA and one strand of the
dsRNA comprises the sequence of 5'-GCU GGA CUC CUC CUA CAC A-3'. In
certain embodiments the siRNA is stabilized to prevent degradation in serum.
The full length eIF5Al polynucleotide and/or the eIF5A polynucleotide
encoding the truncated eIF5Al protein may be present in an expression vector.
In
certain embodiments, the full length eIF5Al polynucleotide and/or the eIF5A
polynucleotide encoding the truncated eIF5Al protein and/or the siRNA are
complexed to polyethylenimine. The full length eIF5Al polynucleotide and/or
the
eIF5A polynucleotide encoding the truncated eIF5Al protein and/or the siRNA
are
independently complexed to polyethylenimine.
The present invention also provides a method of inducing apoptosis is a
mammalian cancer cell or mammalian tumor by providing to the mammal a
composition or medicament described herein, for example comprising a
nucleotide
encoding a truncated eIF5Al, optionally comprising a nucleotide encoding a
full
length eIF5A or a a nucleotide encoding a full length mutant eIF5A and
optionally
comprising an siRNA targeted against the 3'UTR of eIF5A. In certain
embodiments
the cancer is multiple myeloma and the composition/medicament is administered
intravenously, intra peritoneally or intra tumorally.
The present invention aslso relates to the combinatorial use of an siRNA
targeted against an endogenous gene to knock out or knock down expression of
the
endogenous gene in a subject and a of a polynucleotide encoding the gene in a
delivery vehicle/expression vector provided to the subject to provide
expression in the
host of the protein encoded by the polynucleotide.
This combination is useful in treating a subject with a disease or condition
caused by the existence of a faulty or mutant protein, i.e. where the protein
produced
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in the subject is unable to perform it necessary function or alternatively,
fowls up a
metabolic pathway or biomolecule interaction because of its faulty structure.
The
siRNA is designed to target the gene encoding the faulty protein, and knock
down or
knock out expression of that faulty protein. A polynucleotide encoding a
normal (non
faulty) protein is administered to the subject and is expressed in the subject
so that the
normal protein is available to perform its necessary function.
In another embodiment, instead of administering the polynucleotide encoding
the desired protein, the protein is administered to the subject. The terms
protein,
peptide and polypeptide are used herein interchangeably.
The siRNA is preferably designed to target a certain region of the gene so it
either knocks down or knocks out endogenous expression of the faulty protein
but at
the same time will not effect exogenous expression of the administered
polynucleotide encoding the normal protein. For example, the siRNA may target
the
3'UTR so it does not effect exogenous expression of the administered sense
construct
(the polynucleotide encoding the protein). By knocking down or knocking out
endogenous expression of the faulty gene, there will be less or none of the
faulty
protein to compete with the normal protein expressed from the exogenous
polynucleotide.
One example of a disease state where this application would be useful
concerns sickle cell anemia. Sickle cell anemia is a blood disorder that
affects
hemoglobin, the protein found in red blood cells (RBCs) that helps carry
oxygen
throughout the body.
Sickle cell anemia occurs when a person inherits two abnormal genes (one
from each parent) that results in expression of a mutant hemoglobin (Hbs). The
mutant hemoglobin causes the RBCs to change shape. Red blood cells with normal
hemoglobin (hemoglobin A, or HbA) move easily through the bloodstream,
delivering
oxygen to all of the cells of the body. They can easily "squeeze" through even
very
small blood vessels. Sickle cell anemia occurs because the abnormal form of
hemoglobin (HbS) tends to clump together, making red blood cells sticky,
stiff, and
more fragile, and causing them to form into a curved, sickle shape.
Although several hundred HBB gene variants are known, sickle cell anemia is
most commonly caused by the hemoglobin variant HbS. In this variant, the
hydrophobic amino acid valine takes the place of hydrophilic glutamic acid at
the
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sixth amino acid position of the HBB polypeptide chain. This substitution
creates a
hydrophobic spot on the outside of the protein structure that sticks to the
hydrophobic
region of an adjacent hemoglobin molecule's beta chain. This clumping together
(polymerization) of HbS molecules into rigid fibers causes the "sickling" of
red blood
cells.
Polymerization occurs only after red blood cells have released the oxygen
molecules that they carry to various tissues throughout the body. Once red
blood cells
return to the lungs where hemoglobin can bind oxygen, the long fibers of HbS
molecules depolymerize or break apart into single molecules. Cycling between
polymerization and depolymerization causes red blood cell membranes to become
rigid. The rigidity of these red blood cells and their distorted shape when
they are not
carrying oxygen can result in blockage of small blood vessels. This blockage
can
cause episodes of pain and can damage organs.
Sickle cell anemia is an autosomal recessive genetic disorder. For the disease
to be expressed, a person must inherit either two copies of HbS variant or one
copy of
HbS and one copy of another variant. Carriers, who have one copy of the normal
HBB gene (HbA) and one copy of HbS, are described as having sickle cell trait
and
do not express disease symptoms.
Thus, one embodiment of the present invention provides a method of treating
subjects with sickle cell anemia. siRNA targeted to the HBB gene is
administered to
the patient. The siRNA is designed to knock down and preferably knock out the
expression of the Hbs variant of hemoglobin. A polynucleotide encoding a
normal
hemoglobin is provided to the subject so the subject expresses a normal
hemoglobin.
The siRNA is also designed so that it will not interfere with expression of
the
exogenous polynucleotides encoding the normal hemoglobin. Thus, the subject no
longer makes the variant hemoglobin (or makes substantially less) and instead
makes
normal healthy hemoglobin, resulting in more normal red blood cells, which
function
normally.
The present invention is also useful in situations where a post translational
modification occurs to the protein, which causes or leads to a disease state.
siRNA is
used to knock down expression of the endogenous protein so none or less is
available
for the post translational modification. Then, a polynucleotide encoding a
protein is
provided to the patient for exogenous expression. The protein is modified so
that it is
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unable to be post translationally modified. This protein is then available to
the body
for its appropriate use, but will not lead to the disease state because it is
not able to be
post translationally modified. One skilled in the art would understand
different post
translational modifications. For example, after translation, the
posttranslational
modification of amino acids extends the range of functions of the protein by
attaching
to it other biochemical functional groups such as acetate, phosphate, various
lipids
and carbohydrates, by changing the chemical nature of an amino acid (e.g.
citrullination) or by making structural changes, like the formation of
disulfide bridges.
Also, enzymes may remove amino acids from the amino end of the protein, or cut
the
peptide chain in the middle. For instance, the peptide hormone insulin is cut
twice
after disulfide bonds are formed, and a propeptide is removed from the middle
of the
chain; the resulting protein consists of two polypeptide chains connected by
disulfide
bonds. Also, most nascent polypeptides start with the amino acid methionine
because
the "start" codon on mRNA also codes for this amino acid. This amino acid is
usually
taken off during post-translational modification. Other modifications, like
phosphorylation, are part of common mechanisms for controlling the behavior of
a
protein, for instance activating or inactivating an enzyme. Another post
translational
modification includes the hypusination of eukarotic initiation factor 5A
(eIF5A) by
deoxyhypusine synthase (DHS).
Thus, the invention provides a method of altering expression of a gene in a
subject, wherein a polynucleotide encoding a protein is provided to a patient
and is
expressed in the patient. The protein may be a normal/wild type protein or a
mutated
protein. Expression of the corresponding endogenous gene is suppressed with
the
siRNA that is administered to the subject.
The method further comprises providing a construct comprising a
polynucleotide encoding the target protein wherein the polynucleotide is
expressed in
the subject to produce the target protein. In certain embodiments where the
endogenous gene expresses a faulty protein, the polynucleotide is designed to
encode
either a normal/healthy protein. The siRNA is administered to suppress
expression of
the faulty endogenous protein. In certain embodiments where the endogenous
gene
expresses a normal healthy protein, the polynucleotide is designed to encode a
mutant
protein that can not be postranslationally modified as would occur with a
normal/healthy or non mutant protein. The siRNA is administered to suppress
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expression of the endogenous protein so there is less of this protein to be
available for
posttranslational modification.
In certain embodiments, the siRNA is chosen or designed to target regions of
the gene so as to not effect expression of the exogenous polynucleotide. For
example,
the siRNA may target the 3' UTR or 3' end.
The siRNA may be delivered to the patient as either naked siRNA or naked
siRNA stabilized for serum. The siRNA may by either injected systemically,
i.e. IP
or IV. Alternatively, the siRNA may be injected or delivered locally to the
desired
area of the body. In certain embodiments, the siRNA may be administered in a
delivery vehicle such as but not limited to dendrimers, liposomes, or
polymers.
The polynucleotides encoding the desired protein may be administered
through any delivery means that provide or allow expression of the nucleotide.
The
term polynucleotide and nucleotide are used herein interchangeably. Delivery
may be
through any viral or non-viral mechanism, such as but not limited to plasmids,
expression vectors, viral constructs, adenovirus constructs, dendrimers,
liposomes, or
polymers.
In certain embodiments an expression plasmid having reduced CpG
dinucleotides is used to express the polynucleotides. Any promoter capable of
promoting expression of the polynucleotide may be used, which may be chosen
based
on the application desired for the therapy. For example, for killing multiple
myeloma,
a promoter specific for B cells may be desirable, such as human B29
promoter/enhancer. In other embodimetns, the promoter may be another tissue
specific promoter, or may be a system promoter. The polynucleotides encoding
the
target protein may be delivered through IV or subcutaneous injection or any
other
biologically suitable delivery mechanism. Alternatively, the polynucleotides
may
be delivered in liposomes or any other suitable "carrier" or "vehicle" that
provides for
delivery of the DNA (or plasmid or expression vector) to the target tumor or
cancer
cells. See for example, Luo, Dan, et al., Nature Biotechnology, Vol. 18,
January
2000, pp. 33-37 for a review of synthetic DNA delivery systems. Thus, it may
be
preferable to deliver the nucleotides/plasmid/expression vector via a vehicle
of
nanometer size such as liposomes, dendrimers or a similar non-toxic nano-
particle.
The vehicle preferably protects the nucleotides/plasmid/expression vector from
premature clearance or from causing an immune response while delivering an
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effective amount of the nucleotides/plasmid/expression vector to the subject,
tumor or
cancer cells. Exemplary vehicles may range from a simple nano-particle
associated
with the nucleotides/plasmid/expression vector to a more complex pegylated
vehicle
such as a pegylated liposome having a ligand attached to its surface to target
a
specific cell receptor.
Liposomes and pegylated liposomes are known in the art. In conventional
liposomes, the molecules to be delivered (i.e. small drugs, proteins,
nucleotides or
plasmids) are contained within the central cavity of the liposome. One skilled
in the
art would appreciate that there are also "stealth," targeted, and cationic
liposomes
useful for molecule delivery. See for example, Hortobagyi, Gabriel N., et al.,
J.
Clinical Oncology, Vol. 19, Issue 14 (July) 2001:3422-3433 and Yu, Wei, et
al.,
Nucleic Acids Research. 2004, 32(5);e48. Liposomes can be injected
intravenously
and can be modified to render their surface more hydrophilic (by adding
polyethylene
glycol ("pegylated") to the bilayer, which increases their circulation time in
the
bloodstream. These are known as "stealth" liposomes and are especially useful
as
carriers for hydrophilic (water soluble) anticancer drugs such as doxorubicin
and
mitoxantrone. To further the specific binding properties of a drug carrying
liposome
to a target cell, such as a tumor cell, specific molecules such as antibodies,
proteins,
peptides, etc. may be attached on the liposome surface. For example,
antibodies to
receptors present on cancer cells maybe used to target the liposome to the
cancer cell.
In the case of targeting multiple myeloma, folate, 11-6 or transferrin for
example, may
be used to target the liposomes to multiple myeloma cells.
Dendrimers are also known in the art and provide a preferable delivery
vehicle. See for example Marjoros, Istvan, J., et al, "PAMAM Dendrimer-Based
Multifunctional Conjugate for Cancer Therapy: Synthesis, Characterization, and
Functionality," Biomacromolecules, Vol. 7, No. 2, 2006; 572-579, and Majoros,
Istvan J., et al., J. Med. Chem, 2005. 48, 5892-5899 for a discussion of
dendrimers.
In a preferred embodiment, the delivery vehicle comprises a polyethylenimine
nanoparticle. An exemplary polyethylenimine nanoparticle is the in vivo
jetPEITM,
currently produced by Polyplus Transfection, Inc. In vivo-jetPEITM is cationic
polymer transfection agent useful as a DNA and siRNA delivery agent. In vivo-
j etPEITM from Polyplus Transfection is a linear polyethylenimine reagent that
provides reliable nucleic acid delivery in animals. It is used for gene
therapy (Ohana
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et al., 2004. Gene Ther Mol Bio 8:181-192; Vernejoul et al., 2002. Cancer
Research
62:6124-3 1), RNA interference, (Urbain-Klein et al., 2004. Gene therapy 23:1-
6;
Grezelinski et al., 2006. Human Gene Therapy 17:751-66), and genetic
vaccination
(Garzon et al., 2005. Vaccine 23:1384-92). In vivo JET-PEI is currently in use
in
human clinical trials as a delivery vector for cancer gene therapy (Lemkine et
al.,
2002. Mol. Cell. Neurosci. 19:165-174).
In vivo-jetPEITM condenses nucleic acids into roughly 50 nm nanoparticles,
which are stable for several hours. As a result of this unique protection
mechanism,
aggregation of blood cells following injection is reduced compared to other
reagents
thereby preventing restricted diffusion within a tissue, erythrocyte
aggregation and
microembolia. These nanoparticles are sufficiently small to diffuse into the
tissues
and enter the cells by endocytosis. In vivo-jetPEITM favors nucleic acids
release from
the endosome and transfer across of the nuclear membrane.
In a preferred embodiment, both the siRNA and a vector/plasmid comprising
the polynuncleotide are administered to the subject via an in vivo j etPEITM
complex.
The siRNA and the vector/plasmid comprising the polynucleotide maybe complexed
together via a polymer complex such as polyethylenimine or the in vivo
jetPEITM
complex or may separately complexed to a polymer. For instance, where the
siRNA
and the vector/plasmid comprising the polynucleotide are to be administered
separately to the subject (separately in the meaning of time and/or delivery
site) it is
preferable to have the siRNA and the polynucleotide complexed to a different
carrier.
Where the administration will occur at the same time and at the same site, it
may be
preferable to complex the siRNA and the polynucleotide together.
In another embodiment, instead of a plasmid or vector being administered to
deliver a polynucleotide that will be expressed in the subject, the protein
per se is
delivered to the subject. The protein may be either isolated or may be
synthetic.
One embodiment of the present invention provides a method of treating cancer
in a subject, including mammals and humans. Treating cancer includes, but is
not
limited to inducing apoptosis in cancer cells, killing cancer cells, reducing
the number
of cancer cells and reducing tumor volume/weight. The method comprises
administering a composition comprising eIF5Al siRNA and a polynucleotide
encoding a mtuant eIF5Al. The composition and eIF5Al siRNA and a
polynucleotide encoding a mtuant eIF5Al are discussed herein below.
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All cells produce eukaryotic initiation factor 5A ("eIF-5A")(or also referred
to
herein as "factor 5A"). Mammalian cells produce two isoforms of eIF-5Al (eIF-
5Al
and eIF-5A2). eIF-5Al has been referred to as apoptosis-specific eIF-5A, as it
is
upregulated in cells undergoing apoptosis. Human eIF-5Al has the accession
number
NM 001970 and is shown in figure 1. It is believed that eIF-5A l is
responsible for
shuttling out of the nucleus subsets of mRNAs encoding proteins necessary for
apoptosis. eIF-5A2 has been referred to as proliferation eIF-5A as it is
believed to be
responsible for shuttling out of the nucleus subsets of mRNAs encoding
proteins
necessary for cellular proliferation. See Liu & Tartakoff (1997) Supplement to
Molecular Biology of the Cell, 8, 426a. Abstract No. 2476, 37th American
Society for
Cell Biology Annual Meeting, and Rosorius et al. (1999) J. Cell Science, 112,
2369-
2380.
Both factor 5As are post translationally modified by deoxyhypusine synthase
("DHS"). DHS hypusinates the eIF-5As. Hypusine, a unique amino acid, is found
in
all examined eukaryotes and archaebacteria, but not in eubacteria, and eIF-5A
is the
only known hypusine-containing protein. Park (1988) J. Biol. Chem., 263, 7447-
7449;
Schumann & Klink (1989) System. Appl. Microbiol., 11, 103-107; Bartig et al.
(1990)
System. Appl. Microbiol., 13, 112-116; Gordon et al. (1987a) J. Biol. Chem.,
262,
16585-16589. Hypusinated eIF-5A is formed in two post-translational steps: the
first
step is the formation of a deoxyhypusine residue by the transfer of the 4-
aminobutyl
moiety of spermidine to the a-amino group of a specific lysine of the
precursor eIF-
5A catalyzed by deoxyhypusine synthase. The second step involves the
hydroxylation of this 4-aminobutyl moiety by deoxyhypusine hydroxylase to form
hypusine.
The amino acid sequence of eIF-5A is well conserved between species, and
there is strict conservation of the amino acid sequence surrounding the
hypusine
residue in eIF-5A, which suggests that this modification may be important for
survival. Park et al. (1993) Biofactors, 4, 95-104. This assumption is further
supported by the observation that inactivation of both isoforms of eIF-5A
found to
date in yeast, or inactivation of the DHS gene, which catalyzes the first step
in their
activation, blocks cell division. Schnier et al. (1991) Mol. Cell. Biol., 11,
3105-3114;
Sasaki et al. (1996) FEBS Lett., 384, 151-154; Park et al. (1998) J. Biol.
Chem., 273,
1677-1683. However, depletion of eIF-5A protein in yeast resulted in only a
small
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decrease in total protein synthesis suggesting that eIF-5A may be required for
the
translation of specific subsets of mRNA's rather than for protein global
synthesis.
Kang et al. (1993), "Effect of initiation factor eIF-5A depletion on cell
proliferation
and protein synthesis," in Tuite, M. (ed.), Protein Synthesis and Targeting in
Yeast,
NATO Series H. The recent finding that ligands binding eIF-5A share highly
conserved motifs also supports the importance of eIF-5A. Xu & Chen (2001) J.
Biol.
Chem., 276, 2555-2561. In addition, the hypusine residue of modified eIF-5A
was
found to be essential for sequence-specific binding to RNA, and binding did
not
provide protection from ribonucleases.
The present inventors have shown that when polynucleotides encoding eIF-5A
are administered to cells, there is an increase in apoptosis those cells. They
have
shown that they have been able to push cancer cells into apoptosis by
administering
eIF-5Al polynucleotides that are then expressed in the cancer cells. See co-
pending
applications 10/200,148; 11/287,460; 11/293,391 and 11/637,835, all of which
are
incorporated by reference in their entireties.
The present inventors have additionally determined that when cells have a
build up of the hypusinated form of factor 5A, the cells enter into a survival
mode and
do not undergo apoptosis as they normally would over time. Notably, in cancer
cells,
there is a significant amount of hyspusinated factor 5A and thus, the cells do
not enter
into apoptosis (and do not die). Thus, to treat cancer by killing the cancer
cells (push
the cancer cells to enter into the apoptosis pathway), a polynucleotide
encoding eIF-
5Al is administered to the subject or to the cancer cells or tumor to provide
increased
expression of eIF-SAl, which in turn causes apoptosis in the cancer cells and
ultimately cell death and tumor shrinkage. However, if one were to only
provide
polynucleotides encoding the eIF-5Al protein to up regulate gene expression of
eIF-
5Al and not also use siRNA to knock down endogenous expression of eIF-SAl,
there
is a tug of war: the eIF-5Al expression directing the cells towards the
apoptosis
pathway competes with the presence of the hypusinated factor 5A directing the
cells
towards the cell survival pathway. The present invention eliminates this tug
of war
and represents an improvement over only increasing expression of eIF-5Al. The
polynucleotides administered to the subject or cell are mutated so that the
resulting
expressed protein can not be hypusinated. In addition endogenous expression of
factor 5A is knocked out/down with siRNA targeted against eIF-5A so there is
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none/less endogenous eIF-5A l around to by hypusinated. Thus, since there is
no (or
substantially less) hypusinated eIF-5A in the cells, they are not pushed into
survival
mode.
The polynucleotide encoding a mutated eIF-5A l is preferably mutated so that
it can not be hypusinated and thus will not be available to drive the cell
into survival
mode. For example, in one embodiment, the polynucleotide encoding eIF-5A is
mutated to so that the lysine (K) at position 50, which is normally
hypusinated by
DHS, is changed to an alanine (A) (which can not be hypusinated). This mutant
is
denoted as K50A.
In another embodiment, the lysine at position 67 is changed to an arginine
(R).
This mutant is denoted as (K67R). In another embodiment the lysine (K) at
position
67 is changed to an alanine (A) and is denoted as (K67A). In another
embodiment,
the lysine (K) at position 50 is changed to an arginine (K50R) and another
embodiment provides a mutant where the lysine (K) at position 47 is changed to
an
arginine (K47R).
In other embodiments, a double mutant is used. One double mutant is where
the lysine (K) at position 50 is changed to an arginine (R) and the lysine (K)
at
position 67 is changed to a arginine (R). This double mutant is referred to as
K50R/K67R. This double mutant is similarly unable to be hypusinated but the
changes in the amino acids do not alter the 3-D structure of eIF-5Al as much
as the
single mutation (K50A). The double mutation thus provides a protein that is
very
similar in 3-D shape and folding as the wild type and thus is more stable than
the
single mutant. Being more stable, it exists longer in the body to provide
longer
therapeutic benefit. Thus, the body will have the factor 5A it needs for
normal cell
function but it will not be able to hypusinated so the cells do not get locked
into the
cell survival mode and escape apoptosis.
Another double mutant is where the lysine (K) at position 47 is changed to an
arginine (R) and the lysine at position 50 is changed to an arginine (R). This
mutant
is denoted as (K47R/K50R). The invention provides another double mutant where
the
lysine (L) at position 50 is changes to an alanine (A) and the lysine at
position 67 is
changes to an alanine (A). This mutant is denoted as (K50A/K67A).
Because the body needs factor 5A for normal cell survival and healthy cell
proliferation, it is preferable not to shut off expression completely in the
subject with
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the siRNA, if the siRNA is delivered systemically. Control of eIF-5A
expression can
be achieved by either using an siRNA that is not as good at shutting off
expression
(i.e. shuts down or reduces expression but does not completely shut off
expression) or
alternatively, or utilizing a dosing and/or treatment regimen to balance
expression
levels to allow normal growth and functioning of healthy cells but also to
push
cancerous cells to apoptosis.
Alternatively, one may utilize local delivery of siRNA. If the siRNA is
delivered locally to the cancer cell or tumor, then the expression is
preferably knocked
out. By knocking out expression, there is no factor 5A around that can be
hypusinated and thus there is no hypusinated eIF-5A to lock the cells into
survival
mode. Since the siRNA is delivered locally to the cancer or tumor, there is no
need to
have eIF-5A available for regular cell growth.
In certain embodiments, the endogenous gene is eIF5Al. siRNA targeted
against eIF5Al is administered to the subject to suppress expression of the
endogenous eIF-SAl. In certain embodiments the siRNA comprises SEQ ID NO:1 or
SEQ ID NO:2 or is any siRNA targeted against eIF5Al that will suppress
expression
of endogenous eIF-SAl. In certain embodiments, the eIF5Al is human eIF-5Al
(shown in figure 1) and the subject is a human. Other siRNAs targeted against
human
eIF-5Al are known and disclosed in co-pending applications 11/134,445;
11/287,460;
11/184,982; 11/293,391; 11/725,520; 11/725,470; 11/637,835. In other
embodiments,
the subject is a mammal and the eIF5Al is specific to the mammal. For example,
the
subject is a dog and the eIF5Al is canine eIF5Al. In certain embodiments, the
siRNA consists essentially of the siRNA construct shown in figure 25. For
example,
the siRNA contains nucleic acids targeted against the eIF5Al but also contains
overhangs such as U or T nucleic acids or also contains tags, such as a his
tag (often
referred to as HA tag -which is often used in in vitro studies). Molecules or
additional nucleic acids attached at either the 5' or 3' end (or even within
the
consecutive string of nucleic acids shown in figure 25, for example) may be
included
and fall within the "consisting essentially of' as long as the siRNA construct
is able to
reduce expression of the target gene. Preferably the siRNA targets regions of
the
eIF5Al gene so as to not effect expression of the exogenous polynucleotide.
For
example the eIF5Al siRNA targets the 3' UTR or the 3' end. The siRNA shown in
figure 25 an exemplary eIF5Al siRNA.
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The polynucleotide encodes eIF5Al wherein the polynucleotide is mutated to
encode an eIF5Al variant. The mutated eIF5Al is designed so that the variant
eIF5Al can not be post translationally modified (can not be hypusinated).
Exemplary
mutants are discussed herein above.
In the case of cancer involving solid tumors, it may be desirable to deliver
the
siRNA directly to the tumor. The siRNA maybe administered separately with
respect
to time as well as the delivery site from the polynucleotide or may
administered
together at the same time and/or at the same delivery site. One skilled in the
art
would understand that the timing of administration of the siRNA may be
necessarily
administered when the endogenous protein is being translated and not after it
is
already made.
Although the present inventors have earlier shown that eIF5Al is non toxic to
normal tissue (see pending application Ser. No. 11/293,39 1, filed Nov. 28,
2005,
which is incorporated herein by reference in its entirety), a delivery complex
(as
compared to direct administration of the eIF5A
polynucleotides/plasmid/expression
vector) may be preferred. A preferred delivery system provides an effective
amount
of eIF5Al to the subject, tumor or group of cancer cells, as well as
preferably
provides a targeted delivery to the tumor or group of cancer cells. Thus, in
certain
embodiments, it is preferable to deliver the eIF5Al
nucleotides/plasmid/expression
vector via a vehicle of nanometer size such as liposomes, dendrimers or a
similar non-
toxic nano-particle such as a polyethylenimine polymer (such as an in vivo
JetPEITM
complex).
The eIF5Al protein may also be delivered directly to the site of the tumor.
One skilled in the art would be able to determine the dose and length of
treatment
regimen for delivery of eIF5Al protein.
The molecular basis for the induction of apoptosis by eIF5Al is discussed
below.
Death Receptor Signaling
Treatment of cancer cells with Ad-eIF5Al (adenovirus with a wild type
eIF5Al) or Ad-eIF5A1(K50A) induces activation of caspase 8, which is initiated
by death receptor - ligand binding, and caspase 3, the executioner caspase.
These
are likely to be indirect effects of eIF5Al, and the fact that caspase 8 and
caspase 3
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are also activated following treatment with eIF5A1(K50A), which cannot be
hypusinated, indicates that the effect is attributable to lysine5o eIF5Al.
Treatment
with Ad-eIFSAl also appears to result in up-regulation of death receptors as
shown
previously with upregulation of TNFR1.
Mitochondrial Pathway
Direct or indirect involvement of lysine5o eIFSAl in the mitochondrial
pathway for apoptosis is supported by a number of observations including the
finding that caspase 9 is activated by treatment of cancer cells with either
eIFSAl
or eIFSAl (K50A). As well, p53, which plays a role in activation of the
mitochondrial apoptotic pathway, appears to be regulated by eIFSAl. For
example, treatment of cancer cells with Actinomycin D up-regulates p53, and
this
up-regulation of p53 is inhibited by eIFSAl siRNA. Consistent with this,
treatment of cancer cells with Ad-eIF5Al up-regulates p53 mRNA. Treatment of
cancer cells with eIF5Al also induces migration of Bax from the cytosol to
mitochondria, ensuing loss of mitochondrial membrane potential and release of
cytochrome C from the intra-mitochondrial space into the cytosol. In addition,
this
treatment results in up-regulation of cleaved Bc12, Bim and spliced Bim, which
are
all pro-apoptotic.
MAPK Signaling
In addition, the present inventors have obtained evidence for the
involvement of eIF5Al in MAPK signaling related to apoptosis. For example,
treatment of cancer cells with Ad-eIF5Al up-regulated P-JNK, which in turn
inhibits anti-apoptotic Bc12. In addition Ad-eIF5Al and Ad-eIF5A1(KSOA) both
induce the formation of P-p38, which can in turn initiate apoptosis by
impacting a
variety of pro-apoptotic agents including TNFR1 & TNF; FAS & FASL; caspase
8; Bid; Cytochrome C and Capase 3.
NF-KB Signaling
There is evidence that NF-KB signaling supports myeloma growth. For
example, myeloma cell adhesion to bone marrow stromal cells induces NF-KB -
dependent transcriptional up-regulation of IL-6, which is both a growth and
anti-
apoptotic factor in multiple myeoloma [Chauhan et al. (1996) Blood 87, 1104.]
In
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addition, TNF-a secreted by myeloma cells activates NF-KB in bone marrow
stromal cells, thereby up-regulating IL-6 transcription and secretion. TNF-a
also
activates NF-KB in myeloma cells resulting in up-regulation of the
intracellular
adhesion molecule-1 (ICAM-1; CD54) and the vascular cell adhesion molecule-1
(VCAM-1; CD 106) on both myeloma cells and bone marrow stromal cells
[Hideshima et al. (2001) Oncogene 20,4519]. This in turn enhances the
association
of myeloma cells with bone marrow stromal cells [Hideshima et al. (2001)
Oncogene 20,4519]. Conversely, these effects are inhibited by blocking TNFa-
induced NF-KB activation [Hideshima et al. (2001) Oncogene 20,4519]. Indeed,
it
seems likely that NF-KB mediates protection against TNFa-induced apoptosis in
myeloma cells [Hideshima et al. (2002) JBC 277, 16639]. These and other
observations have prompted the view that NF-kB signaling may be an attractive
target for multiple myeloma therapies.
The inventors have shown that eIF5Al siRNA inhibits both the activation of
NF-KB and the formation of ICAM-1 in human myeloma cells. These observations
indicate that eIF5Al plays a role in NF-KB activation, and inasmuch as the
ensuing
effects of NF-KB activation are pro-survival in nature, we predict that this
activation is
mediated, directly or indirectly, by hypusinated eIF5Al.
IL-1
Over-production of the pro-inflammatory cytokine, IL-1, by myeloma cells
is a characteristic feature of multiple myeloma that leads to deterioration of
bone
tissue. eIF5Al siRNA has been shown to dramatically reduce the overproduction
of IL-1 induced by an LPS challenge in mice.
One embodiment of the present invention provides a method of treating
multiple myeloma. Multiple myeloma ("MM") is a progressive and fatal disease
characterized by the expansion of malignant plasma cells in the bone marrow
and
by the presence of osteolytic lesions. Multiple myeloma is an incurable but
treatable cancer of the plasma cell. Plasma cells are an important part of the
immune system, producing immunoglobulins (antibodies) that help fight
infection
and disease. Multiple myeloma is characterized by excessive numbers of
abnormal
plasma cells in the bone marrow and overproduction of intact monoclonal
immunoglobulins (IgG, IgA, IgD, or IgE; "M-proteins") or Bence-Jones protein
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(free monoclonal light chains). Hypocalcaemia, anemia, renal damage, increased
susceptibility to bacterial infection, and impaired production of normal
immunoglobulin are common clinical manifestations of multiple myeloma.
Multiple myeloma is often also characterized by diffuse osteoporosis, usually
in
the pelvis, spine, ribs, and skull.
The present invention seems to be well suited to treat multiple myeloma
because of the stimulation feedback loop found in multiple myeloma. For
instance,
multiple myeloma produces I1-1 in low concentrations in bone marrow. The I1-1
in
turn stimulates stromal cells to produce IL-6, which then goes onto stimulate
growth
of the multiple myeloma. The inventors have previously shown (see pending
application 11/725,539 and 11/184,982) that siRNA directed against eIF-5Al was
able to inhibit expression of proinflammatory cytokines, such as I1-1; TNF-a,
and I1-
8). Thus, the siRNA would not only knock down expression of eIF-5A so less is
available for hypusination, it would also cut off or decrease the I1-1/11-6
feedback
loop.
An siRNA targeting human eIF5A was used to suppress levels of endogenous
hypusinated eIF5A in tumors, while an RNAi-resistant plasmid expressing a
mutant
of eIF5A (eIF5Ax5ox), that is incapable of being hypusinated, was used to
raise the
levels of unmodified eIF5A in vivo. Intra-tumoral injection of PEI
nanocomplexes
containing eIF5A siRNA inhibited MM tumor growth by more than 80 % (* * * p =
0.0003) versus complexes containing a control siRNA, indicating that
suppressing
levels of hypusinated eIF5A has an anti-tumoral effect. PEI complexes
containing an
eIF5AK50R expression plasmid had a similar effect and inhibited tumor growth
by
more than 70 % (** = p 0.001) versus complexes containing a control plasmid.
Thus,
MM tumor growth can be inhibited either by suppression of the growth-promoting
hypusinated eIF5A or by increasing levels of the pro-apoptotic unhypusinated
form of
eIF5A. Intra-tumoral delivery of complexes containing both eIF5A siRNA and
RNAi-resistant eIF5AK50R plasmid had a synergistic effect on tumor growth and
resulted in significant tumor shrinkage, inhibiting tumor growth by 94 % (* *
* p =
0.0002). Intra-venous delivery of eIF5A siRNA/eIF5AK50R PEI complexes also
efficiently reduced tumor growth by 95 % (* * p = 0.002) indicating systemic
delivery
of the therapeutic is feasible.
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Both local and systemic delivery of eIF5A siRNA/eIF5Ax5ox pDNA PEI
complexes resulted in a significant anti-tumoral response in multiple myeloma.
The present invention further provides a composition useful in the
treatment of cancer, including multiple myeloma. In a preferred embodiment,
the
composition is a complex of a plasmid DNA encoding point-mutated eIF5Al that
cannot be hypusinated and eIF5Al siRNA that selectively suppresses endogenous
human eIF5Al but has no effect on the point-mutated eIF5Al encoded by the
plasmid. eIF5Al siRNAs and polynucleotides encoding mutant eIF5Al are
discussed above. The plasmid DNA and the siRNA are both preferably complexed
to PEI (polyethylenimine) nanoparticles. They may be complexed separately and
administered seperately or together or they may be complexed together. The DNA
and the RNA bind to positively charged amino groups on the PEI and are
released
when the nanoparticles are taken up into cells. It has been demonstrated that
PEI-
nucleic acid complexes are effectively taken up into both dividing and non-
dividing cells .
The plasmid DNA preferably encodes eIF5A1(K5 OR) which, like
eIF5A1(K50A), cannot be hypusinated and, accordingly, is strongly apoptogenic.
The expression of eIF5Al (KS OR) is preferably regulated by a B-cell-specific
promoter.
The eIF5Al siRNA is preferably specific to the 3'-end of endogenous
human eIF5Al and has no effect on expression of the trans eIF5A1(KSOR). An
exemplary preferred eIF5Al siRNA comprises, consists essentially of or
consists
of the siRNA shown in figure 25. The rationale for including the eIF5Al siRNA
is: (1) to deplete endogenous eIF5Al, which is almost all hypusinated and
hence
in the pro-survival form; (2) to inhibit activation of NF-KB, and thereby
reduce the
production of IL-6 and the formation of intracellular adhesion molecules; and
(3)
to inhibit the formation of IL-1. That eIF5Al siRNA acts synergistically with
eIF5A1(KSOR) to induce apoptosis in myeloma cells. Inasmuch as (2) and (3)
above are pro-survival events, they are likely mediated by hypusinated eIF5Al,
and hence not affected by eIF5A1(KSOR) which cannot by hypusinated. This
approach results in a larger pool of unhypusinated eIF5A leading to apoptosis
of
cancer cells, including multiple myeloma cells, with little effect on healthy
cells.
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A preferred composition is referred to herein as SNSO1. SNSOI is a complex
containing both, an RNAi-resistant plasmid DNA encoding eIF5AK50R driven by a
promoter that restricts expression to cells of B-cell origin (including
myeloma cells)
for enhanced safety, and an siRNA targeting human eIF5A with dTdT 3' overhangs
for enhanced nuclease resistance and which the siRNA and the plasmid are
complexed to in vivo JetPEITM.
EXAMPLES
Example 1: Transfection of HeLaS3 cells with wild type and variants of eIF-5Al
HeLa S3 cells were transfected using Lipofectamine 2000 with plasmids
expressing HA-tagged eIF5Al variants including wild-type eIF5Al (WT),
eIF5A1K50R (K50R), eIF5A1K67R (K67R), eIF5A1K67A (K67A),
eIF5A1K47R/K50R (K4750R), eIF5A1K50R/K67R (K5067R), or
eIF5A1K50A/K67A (K5067A). A plasmid expressing
LacZ was used as a control. At 24 and 48 hours (A) or 28 and 52 hours (B)
after
transfection, the cell lysate was harvested and fractionated by SDS-PAGE.
Expression
levels of transfected eIF5Al was detected using an antibody against HA.
Result:
Mutation of eIF5Al at a lysine in the putative ubiquination site (K67R)
increased the
accumulation of the eIF5Al transgene above wild-type (A). Mutation of eIF5Al
at
the lysine required for hypusination (K50R) also increased accumulation of
eIF5Al
transgene above wild-type eIF5Al (B). A double mutant form of eIF5Al
(K50A/K67A) was expressed particularly well when compared to the unmutated
wild-
type eIF5Al transgene (A + B). See Figure 2.
Example 2: Transfection of KAS cells with wild type and variants of eIF-5Al
KAS cells were transfected using PAMAM dendrimer (FMD44) with
plasmids expressing HA-tagged eIF5Al variants including wild-type eIF5Al
(5Al),
eIF5A1K67A (K67A), eIF5A1K50A/K67A (K50A K67A), eIF5A1K50R (K50R),
eIF5A1K47R (K47R), eIF5A1K67R (K67R), eIF5A1K47R/K50R (K47R K50R),or
eIF5A1K50R/K67R (K50R K67R). A plasmid expressing LacZ was used as a control.
48 hours (after transfection, the cell lysate was harvested and fractionated
by SDS-
PAGE. Expression levels of transfected eIF5Al was detected using an antibody
against HA. Equal loading was verified using an antibody against actin.
Result:
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Mutation of eIF5Al at a lysine in the putative ubiquination site (K67A or
K67R)
increased the accumulation of the eIF5Al transgene above wild-type. Mutation
of
eIF5Al at the lysine required for hypusination (K50R) or at an acetylation
site
(K47R) also increased accumulation of eIF5Al transgene above wild-type eIF5A.
A
double mutant form of eIF5Al (K50A/K67A) was also expressed at higher levels
when compared to the unmutated wild-type eIF5Al transgene. See Figure 3.
Example 3: Transfection of KAS cells using PAMAM dendrimer
KAS cells were transfected using PAMAM dendrimer (FMD45-2) with
plasmids expressing HA-tagged eIF5Al variants including eIF5A1K50R (K50R),
eIF5A1K50A/K67A (K50A/K67A), or eIF5A1K50R/K67R (K50R K67R). A plasmid
expressing LacZ was used as a control. Seventy-two hours after transfection,
the cells
were stained with Annexin/PI and analyzed by FACS. Cells that stained
positively for
Annexin V and negatively for PI (propidium iodide) were considered to be in
the
early stages of apoptosis (Ann+/PI-) and cells that stained positively for
both Annexin
V and PI were considered to be in the late stages of apoptosis (Ann+/PI+).
Result:
Mutation of eIF5Al at a lysine in the hypusination site (K50R) or in the
putative
ubiquination site
(K67R), as well as the double mutant (K50A/K67A) resulted in apoptosis of KAS
cells significantly above the levels of the LacZ control. See figure 4.
Example 4: Transfection of KAS cells with plasmids expressing eIF-5A1 and eIF-
5Al variants
KAS cells were transfected using Lipofectamine 2000 with plasmids
expressing HA-tagged eIF5Al variants including eIF5A1K50A (K50A),
eIF5A1K50R (K50R), eIF5A1K67R (K67R), eIF5A1K50A/K67A (K50A/K67A), or
eIF5A1K50R/K67R (K50R K67R). A plasmid expressing LacZ was used as a control.
Seventy-two hours after transfection, the cells were stained with Annexin/PI
and
analyzed by FACS. Cells that stained positively for Annexin V and negatively
for PI
(propidium iodide) were considered to be in the early stages of apoptosis
(Ann+/PI-)
and cells that stained positively for both Annexin V and PI were considered to
be in
the late stages of apoptosis (Ann+/PI+). Result: Mutation of eIF5Al at a
lysine in the
hypusination site (K50R) or In the putative ubiquination site (K67R), as well
as the
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double mutant (K50A/K67A) resulted in apoptosis of KAS cells significantly
above
the levels of the LacZ control. See figure 5.
Example 5: The use of Mutated eIF-5Al to treat KAS cells results in apoptosis
KAS cells were transfected using Lipofectamine 2000 with plasmids
expressing HA-tagged eIF5Al variants eIF5A1K50R (K50R) or eIF5A1K50A/K67A
(K50A/K67A. A plasmid expressing LacZ was used as a control. Seventy-two hours
aftertransfection, the cells were stained with Annexin/PI and analyzed by
FACS. Cells
that stained positively for Annexin V and negatively for PI (propidium iodide)
were
considered to be in the early stages of apoptosis (Ann+/PI-) and cells that
stained
positively for both Annexin V and PI were considered to be in the late stages
of
apoptosis (Ann+/PI+). Result: Mutation of eIF5Al at a lysine in the
hypusination site
(K50R) or mutation of eIF5Al at both the hypusination site and in the putative
ubiquination site (K50A/K67A) resulted in apoptosis of KAS cells significantly
above
the levels of the LacZ control. See figure 6.
Example 6A: siRNA/Adenovirus-mediated killing of multiple myeloma cells
KAS (human multiple myeloma) cells were maintained in S 10 media [RPMI
1640 with 4 ng/ml IL-6, 10 % fetal bovine serum (FBS), and
penicillin/streptomycin
(P/S)]. KAS cells were transfected with 58.7 pmoles of siRNA using
Lipofectamine
2000 (Invitrogen). Mock transfected cells were treated with Lipofectamine 2000
in
the absence of siRNA. Transfection was conducted in the antibiotic-free S 10
media.
a) siRNAs targeting human eIF5Al :
eIF5Al siRNA target #1 (the siRNA targets this region of human
eIF5Al : 5'-AAGCTGGACTCCTCCTACACA-3' (SEQ ID NO: ). The siRNA
sequence is shown in figure 25 and is often referred to herein as hSAl.
eIF5Al siRNA target #2 eIF5Al (this siRNA targets this region of
human eIF5A1 : 5'-AAAGGAATGACTTCCAGCTGA-3' (SEQ ID NO: ). (The
siRNA sequence is often referred to herein ashSAl-ALT)
b) control siRNA : The control siRNA had the following sequence: sense
strand, 5 -ACACA L C ='1_ CC: ~ CAGCa1_ C _ dTd"C-3 `; and anti sense strand,
3'-
dTdTUGUGUEGG AGGAGUCCAGC-5'".
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Other controls that have been used inclhide non-targeting validated siRNAs
frog Dharr aeon since they have been micro-array tested to limit unwanted off-
targeting effects. For example, for in vitro work studyign NFkB, the control
used was
Dharmacon's non-targeting siRNA's (sequence D-001%00-01) and for in vivo work,
the control used was Dharmacon's (sequence D-001810--01).
Four hours after transfection, the cells were pelleted and resuspended in 1 ml
of S10 media. Seventy-two hours after the initial siRNA transfection, the
transfected
KAS cells were counted and seeded at 300,000 cells/well in a 24-well plate and
transfected with the same siRNA a second time.
Four hours after transfection, the cells were pelleted and resuspended in 1 ml
of S10 media (without IL-6) containing 3000 ifu of either Ad-LacZ (Adenovirus
expressing -galactosidase) or Ad-5AiM (Adenovirus expressing human
eIF5AiK5oA)
Seventy-two hours later the cells were harvested and analyzed for apoptosis by
staining with Annexin V-FITC and PI (BD Bioscience) followed by FACS analysis.
a) early apoptosis was defined as cells that were positively stained with
Annexin- FITC and negative for PI-staining (Ann +/PI -)
b) total apoptosis was defined as the total of cells either in early apoptosis
(Ann +/PI -) or late apoptosis/necrosis (Ann +/PI +)
The 5A1 siRNA targeting #1 targets the 3'UTR of human eIF5Al and
therefore will not affect expression of eIF5Al from adenovirus. 5A1 siRNA
targeting
#2 targets within the open reading frame of human eIF5Al and so it could
potentially
interfere with expression of eIF5Al from the adenovirus.
Results: Cells treated with siRNA and infected with adenovirus expressing
the eIF-5A1 K50A variant undergo apoptosis in greater numbers than non-treated
cells and cells treated only with siRNA. See Figure 7.
Example 6B: Pre-treatment with eIF5Al siRNA against eIF5Al target #1 (shown in
figure 25), reduced expression of endogenous eIF5Al but allows accumulation of
RNAi-resistant eIF5Alk50A expressed by adenovirus.
KAS cells were transfected using Lipofectamine 2000 with either a control
siRNA (C) or one of two siRNAs targeting eIF5Al (#1 and #2). The eIF5Al siRNA
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#1 targets the 3'UTR of eIF5Al and therefore does not interfere with
expression of
eIF5Al from adenovirus since it contains only the open reading frame of
eIF5A1.
The sequence of the siRNA is shown in figure 25. The eIF5A1 #2 siRNA targets
the
open reading frame of eIF5Al and will therefore affect expression of both
endogenous and exogenously-expressed eIF5Al. Seventy-two hours after the
initial
transfection hours the cells were transfected with the same siRNA a second
time. Four
hours later the transfection complexes were removed from the cells and
replaced with
growth media (-) IL6 containing either Ad-LacZ (L) or Ad-eIF5A1K5oA (5M).
Seventy-two hours later the cell lysate was harvested and analyzed by Western
blot
using antibodies against eIFSA and actin. See Figure 7B. Accumulation of
virally
expressed eIF5Al can be observed (lane 1 vs lane 2) and reduction of eIFSA
expression by eIF5Al siRNAs targeting # 1 and # 2 can be clearly seen (lanes 5
and 7
vs lane 3). As expected, the eIF5Al siRNA # 1 does not affect accumulation of
the
virally expressed eIF5A1K5oA (lane 6 vs lane 4) while the eIF5Al siRNA # 2
only
moderately affects expression of the virally-expressed transgene (lane 8 vs
lane 4).
Example 6C: pre-treatment with eIF5Al siRNA against target #1 prior to
adenovirus
infection reduces expression of phosphorylated NF-KB in human multiple myeloma
cells.
KAS cells were transfected using Lipofectamine 2000 with either a control
siRNA (hC) or an siRNA targeting eIF5Al (#1). The eIF5Al siRNA #1 targets the
3'UTR of eIF5Al and will therefore not interfere with expression of eIF5Al
from
adenovirus since it contains only the open reading frame of eIF5Al. Seventy-
two
hours after the initial transfection hours the cells were transfected with the
same
siRNA a second time. Four hours later the transfection complexes were removed
from
the cells and replaced with growth media (+) IL6 containing either Ad-LacZ (L)
or
Ad-eIF5A1K5oA (5M). Twenty-four hours later the cell lysate was harvested and
analyzed by Western blot using antibodies against phospho-NF-kB p65 (Ser 536)
and
eIFSA. As expected, the eIF5Al siRNA # 1 does not affect accumulation of the
virally expressed eIF5A1KIIA Phosphorylation of NF-kB p65 at serine 536
regulates
activation, nuclear localization, protein-protein interactions, and
transcriptional
activity. See Figure 7C.
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Example 6D: Pre-treatment with eIF5Al siRNA #1 prior to Adenovirus infection
reduces expression of phosphorylated NF-kB and ICAM-1 in human multiple
myeloma cells.
KAS cells were transfected using Lipofectamine 2000 with either a control
siRNA (C) or one of two siRNAs targeting eIF5Al (#1 and #2). Seventy-two hours
after the initial transfection hours the cells were transfected with the same
siRNA a
second time. Four hours later the transfection complexes were removed from the
cells
and replaced with growth media (+) IL6. Twenty-four hours after the second
transfection, the cells were stimulated with 40 ng/ml TNF-a and cell lysate
was
harvested at 0, 4, or 24 hours and analyzed by Western blot using antibodies
against
phospho-NF-kB p65 (Ser 536), ICAM-1 and actin. A reduction in TNF-a induced
NF-kB p65 phosphorylation (ser 536) and ICAM-1 expression was observed
following transfection with both eIF5Al-specific siRNAs. Phosphorylation of NF-
kB
p65 at serine 536 regulates activation, nuclear localization, protein-protein
interactions, and transcriptional activity. ICAM-1 is an inter-cellular
adhesion surface
glycoprotein that is believed to be involved in the pathogenesis of multiple
myeloma.
See figure 7D.
Example 6E: pretreatment of KAS cells with siRNA increases apoptosis by
eIF5Alk5ox gene delivery in the presence of IL-6.
KAS cells were transfected with either control siRNA (hcon) or human
eIF5Al siRNA (h5Al) using Lipofectamine 2000. Seventy-two hours later the
cells
were re-transfected with siRNA. PEI complexes of empty vector (mcs) or
eIF5Alk5ox
(K50R) plasmids were added to the cells four hours later following removal of
siRNA
transfection medium. The growth medium used throughout the study contained IL-
6.
Apoptosis was measured seventy-two hours later by staining the cells with
Annexin/PI and FACS analysis. See figure 7F.
Example 7: Co-administration of eIF-5Al plasmid and eIF-5Al siRNA delays
growth of multiple myeloma subcutaneous tumors (figures 8-10).
SCID mice were injected subcutaneously with KAS cells. Treatment was
initiated when palpable tumors were observed. Six 3-5 week old SCID/CB17 mice
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were injected with 10 million KAS-6/1 myeloma cells in 200 gL PBS in their
right
flank and treatment was initiated when the tumors reached a minimum size of 4
mm3.
Control mice were injected intra-tumorally 2 times per week with PEI
complexes containing pCpG-mcs (empty vector) and control siRNA (control group
was made up of 3 mice: C-1, C-2, and C-3). Treated mice were injected intra-
tumorally 2 times per week with PEI complexes containing the RNAi-resistant
plasmid pCpG-eIF5A1k50R and eIF5A1 siRNA (treated group was made up of 3
mice: 5A-1, 5A-2, and 5A-3). Injections were given at multiple sites within
the
tumor to prevent reflux and a slow rate of injection was used to increase
uptake. The
data in figure 8 shows the tumor volume for all the mice in the group. The
data
shown in figure 9 is the average tumor volume per group +/- standard error.
Asterix
denote statistical significance (* = p < 0.025; n =3).
Figure 10 shows that co-administration of eIF-5Al plasmid and eIF-5Al
siRNA reduces the weight of multiple myeloma subcutaneous tumors. The data
shown is the average tumor weight per group +/- standard error. Asterix denote
statistical significance (* = p < 0.05; n =3).
JET-PEI (PolyPlus) at 2 x 0.1 ml was used for the in vivo tests. The N/P ratio
was 8. The PEI/DNA/siRNA complexes were formed in a total volume of 0.1 ml in
5% glucose. The protocol for forming complexes was as follows.
1. Bring components to room temperature. Keep sterile.
2. Dilute 20 gg of plasmid DNA (- 10 gl at 2 mg/ml) and 10 gg siRNA (10 gl at
1
mg/ml) into a total volume of 25 l. Use sterile water to make up difference.
3. Adjust the volume of DNA solution to 50 gl 5 % glucose by adding 25 gl of
10 %
glucose. Vortex gently and centrifuge briefly.
4. Dilute 4.8 gl of in vivo JETPEI into a total volume of 25 gl of 10 %
glucose.
Adjust volume to 50 gl with sterile water to end up with a final concentration
of 5
% glucose. Vortex gently and centrifuge briefly.
5. Immediately add 50 gl of diluted PEI to the 50 gl of diluted DNA (do not
reverse
the order). Vortex briefly and immediately spin down.
6. Incubate for 15 minutes prior to injection. Complexes are stable for 6
hours.
CpG-free Cloning Vectors and pCpG Plasmids were obtained from
InvivoGen. These plamids are completely devoid of CpG dinucleotides, named
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pCpG. These plasmids yield high levels of transgene expression both in vitro
and in
vivo, and in contrast to CMV-based plasmids allow sustained expression in
vivo.
pCpG plasmids contain elements that either naturally lack CpG dinucleotides,
were
modified to remove all CpGs, or entirely synthesized such as genes encoding
selectable markers or reporters. Synthesis of these new alleles was made
possible by
the fact that among the sixteen dinucleotides that form the genetic code, CG
is the
only dinucleotide that is non-essential and can be replaced. Eight codons
contain a
CG encoding for five different amino acids. All eight codons can be
substituted by at
least a choice of two codons that code for the same amino acid to create new
alleles
that code for proteins having amino acid sequences that remain identical to
the wild
type and thus are as active as their wild-type counterparts. These new alleles
are
available individually in a plasmid named pMOD from which they can be easily
excised.
pCpG plamids allow long lasting expression in vivo, and represent valuable
tools to study the effects of CpGs on gene expression in vivo and in vitro,
using cell
lines expressing TLR9, as well as their effects on the innate and acquired
immune
systems. The empty vector, pCpG-mcs (Invivogen) is a vector with no
expressed gene product, only a multiple cloning site, and was used as the
control
vector. An HA-tagged eIF5Al"50R cDNA was subcloned into the Ncol and Nhel
sites
of a pCpG-LacZ vector (Invivogen), from which the LacZ gene had been removed,
to
give rise to the treatment vector pCpG-eIF5Al (K50R). The DNA was prepared
using
Endo-Free Qiagen kit. Endotoxin levels measured and are < 0.03 EU/ ug; DNA
should be at 2 mg/ml in water.
The control siRNA used in the experiments was a micro-array validated non-
targeting control siRNA from Dharmacon (D-001810-01). The siRNA was obtained
with a modification (siSTABLE) to prevent degradation in serum.
The eIF5Al siRNA used in the experiments was designed against the 3'UTR
of human eIF5Al. There is no similarity between the eIF5Al siRNA and mouse
eIF5Al and the siRNA should therefore only suppress human (but not mouse)
eIF5Al. The siRNA also has no similarity to eIF5A2 (either human or mouse).
The
siRNA was obtained with a modification (siSTABLE) to prevent degradation in
serum. The eIF5Al siRNA has the following target sequence :
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5' GCU GGA CUC CUC CUA CAC A (UU) 3
The siSTABLE siRNA was dissolved at 1 mg/ml in water (stored in aliquots
at -20C).
Tumor dimensions of length (1) and width (w) were measured 2-3 times per
week using digital calipers. Tumor volume was calculated according to the
following
equation:
1= length; smallest dimension
w = width; largest dimension
tumor volume (mm) = 12 * w * 0.5
Statistical analyses
Student's t-test was used for statistical analysis. Significance was deemed to
be a confidence level above 95 % (p < 0.05).
Example 8: Co-administration of eIF5Al plasmid and eIF5Al siRNA delays growth
of multiple myeloma subcutaneous tumors and results in tumor shrinkage.
In another study, again SCID mice were injected subcutaneously with KAS
cells. Treatment was initiated when palpable tumors were observed. Control
mice
were injected intra-tumorally 2 times per week with PEI complexes containing
pCpG-
mcs (empty vector) and control siRNA (control group G-1, G-2 and G-3). Treated
mice were injected intra-tumorally 2 times per week with PEI complexes
containing
the RNAi-resistant plasmid pCpG-eIF5A1k50R (20 gg of plasmid DNA) and eIF5Al
siRNA (10 gg of siRNA)(treated group G-4, G-5 and G-6). The data shown in
figure
11 is the average tumor volume per group +/- standard error. Asterix denote
statistical significance (* = p < 0.025; n =3). Six injections over a period
of 21 days
were given (red arrows).
Example 9: Administration of eIF5Al siRNA intra-venously (i.v.) and
PEI/eIF5A1K50R plasmid complexes intra-tumorally (i.t.) results in tumor
shrinkage
of multiple myeloma subcutaneous tumors (Group 2B).
SCID mice were injected subcutaneously with KAS cells. When palpable
tumors were observed treatment was initiated with an initial tail injection of
50
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micrograms of either control siRNA (control group) or human eIF5Al siRNA
(treated
group). Control Mice were subsequently treated by intra-tumoral injections 2
times
per week with PEI complexes containing pCpG-mcs (empty vector; control group;
G-
1, G-2, and G-3). Treated mice were subsequently treated by intra-tumoral
injections
2 times per week with PEI complexes containing the RNAi-resistant plasmid pCpG-
eIF5A1k5OR (20 gg plasmid DNA)(treated group; G-4, G-5, and G-6). Control mice
continued to receive control siRNA (control group R-1, R-2, and R-3) by i.v.
injection
once per week. Treated mice continued to receive human eIF5Al siRNA (20
gg)(treated group R-4, R-5, and R-6) by i.v. injection once per week. The data
shown
in figure 12 is the tumor volume for all the mice in each group. Six
intramural
injections of PEI/DNA (red arrows) and four i.v. injections of siRNA (blue
arrows)
were given over a period of 21 days.
Figure 13 provides an overlay of the results from Example 8 and 9. SCID
mice were injected subcutaneously with KAS cells. The data shown in figure 13
is
the average tumor volume for the mice in each group +/- standard error.
Asterix
denote statistical significance between treated and control groups (* * = p <
0.01;
= p < 0.001; n =3).
Protocol for forming PEI complexes:
1. Bring components to room temperature. Keep sterile.
2. Dilute plasmid DNA or plasmid DNA + siRNA into a total volume of 25 l. Use
sterile water to adjust the volume.
a) For plasmid DNA only complexes:
Dilute 20 gg of plasmid DNA (10 gl at 2 mg/ml) into a total volume of 25 l.
Use
sterile water to make up difference.
b) For plasmid DNA + siRNA complexes:
Dilute 20 gg of plasmid DNA (- 10 gl at 2 mg/ml) and 10 gg of siRNA (10 gl at
1
mg/ml) into a total volume of 25 l. Use sterile water to make up difference.
3. Adjust the volume of DNA solution to 50 p15 % glucose by adding 25 gl of 10
%
glucose (provided with PEI kit). Vortex gently and centrifuge briefly.
4. Dilute in vivo JETPEI into a total volume of 25 gl of 10 % glucose.
a) For plasmid DNA only complexes:
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Dilute 3.2 gl of in vivo JETPEI into a total volume of 25 gl of 10 % glucose.
Adjust volume to 50 gl with sterile water to end up with a final concentration
of 5
% glucose. Vortex gently and centrifuge briefly.
b) For plasmid DNA + siRNA complexes:
Dilute 4.8 gl of in vivo JETPEI into a total volume of 25 gl of 10 % glucose.
Adjust
volume to 50 gl with sterile water to end up with a final concentration of 5 %
glucose. Vortex gently and centrifuge briefly.
5. Immediately add 50 gl of diluted PEI to the 50 gl of diluted DNA (do not
reverse
the order!). Vortex briefly and immediately spin down.
6. Incubate for 15 minutes prior to injection. Complexes are stable for 6
hours.
Regarding the tail-vein injection of siRNA, the initial siRNA injection was 50
micrograms. siRNA was diluted to 0.4 mg/ml in PBS. 125 gl per mouse (50 g)
was
injected into the tail vein. Subsequent injections of serum-stabilised siRNA
were
given two times per week at 20 gg per mouse. siRNA was diluted to 0.4 mg/ml in
PBS. 50 gl per mouse (20 g) was injected into the tail vein.
Figure 13B shows that co-administration of eIF5Al plasmid and eIF5Al
siRNA results in tumor shrinkage. SCID mice were injected subcutaneously with
KAS cells. Treatment was initiated when palpable tumors were observed. Mice
were
injected intra-tumorally 2 times per week with PEI complexes containing the
RNAi-
resistant plasmid pCpG-eIF5A1k50R and eIF5Al siRNA (treated group; G-4, G-5,
and G-6). Six injections over a period of 21 days were given. Forty-two days
after the
initiation of treatment the mice were sacrificed and the skin under the tumor
site was
opened and examined for evidence of tumor growth. No tumor growth was observed
in any of the group 2A treated mice.
Figure 13C shows that administration of eIF5Al siRNA intra-venously (i.v.)
and PEI/eIF5A1K50R plasmid complexes intra-tumorally (i.t.) results in tumor
shrinkage of multiple myeloma subcutaneous tumors. SCID mice were injected
subcutaneously with KAS cells. When palpable tumors were observed treatment
was
initiated with an initial injection of 50 micrograms of human eIF5Al siRNA
(treated
group). Mice were subsequently treated by intra-tumoral injections 2 times per
week
with PEI complexes containing the RNAi-resistant plasmid pCpG-eIF5A1k50R
(treated group; R-4, R-5, and R-6). Mice continued to receive human eIF5Al
siRNA
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by i.v. injection once per week. Treatment ended 21 days after initiation of
treatment.
Forty-two days after the initiation of treatment the mice were sacrificed and
the skin
under the tumor site was opened and examined for evidence of tumor growth.
There
was no evidence of tumor growth in one mouse of the treatment group (group
2B).
Example 10: Intra-venous co-administration of eIF5Al plasmid and eIF5Al siRNA
delays growth of multiple myeloma subcutaneous tumors.
SCID mice were injected subcutaneously with KAS cells. When palpable
tumors were observed treatment was initiated with an initial injection of 50
micrograms of either control siRNA (control group) or human eIF5Al siRNA
(treated
group). Mice were subsequently treated by intra-venous (red arrows) or intra-
peritoneal injections (green arrow) - twice per week with either PEI complexes
containing pCpG-mcs (empty vector; control group Al, A2, and A3) or PEI
complexes containing the RNAi-resistant plasmid pCpG-eIF5A1k5OR (treated
group;
A4, A5, and A6). Mice continued to receive either control siRNA (control group
Al,
A2, and A3) or human eIF5Al siRNA (treated group A4, AS, and A6) by i.v.
injection (blue arrows) once per week. The data shown is the tumor volume for
all
the mice in each group. The data shown in Figure 14 is the tumor volume for
all the
mice in each group.
Example 11: Administration of eIF5Al siRNA intra-venously (i.v.) and
PEI/eIF5A1K50R plasmid complexes intra-venously (i.v.) or intra-peritoneal
(i.p.)
delays growth of multiple myeloma subcutaneous tumors.
SCID mice were injected subcutaneously with KAS cells. When palpable
tumors were observed treatment was initiated with an initial injection of 50
micrograms of either control siRNA (control group) or human eIF5Al siRNA
(treated
group). Control mice were subsequently treated by intra-venous or intra-
peritoneal
injections - once per week with PEI complexes containing pCpG-mcs (empty
vector;
control group was three mice: Bl, B2,and B3). Treated mice were subsequently
treated by intra-venous or intra-peritoneal injections - once per week with
PEI
complexes containing the RNAi-resistant plasmid pCpG-eIF5Alx50R (treated
group;
B4, B5, and B6). Mice continued to receive either control siRNA (control group
Bl,
B2, and B3) or human eIF5Al siRNA (treated group was three mice: B4, B5, and
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B6) by i.v. injection once per week. The experiment began with initial siRNA
injection of 50 gg (day-2 on graph in Figure 15). Subsequent injections used
20
micrograms of siRNA once weekly. The siRNA was given naked, i.e. no delivery
vehicle. PEI complexes contained 20 gg of plasmid DNA. The initial PEI
injection
was given i.p. and subsequent injections were given i.v. The data shown in
Figure 15
is the tumor volume for all the mice in each group.
Figure 16 provides an overlay of Example 10 and 11. SCID mice were
injected subcutaneously with KAS cells. Treatment was initiated when palpable
tumors were observed. One set of mice received i.v. injections of either
control
siRNA (control; Group A) or eIF5Al siRNA (treated; Group A) once per week and
either i.v. or i.p. of either PEI complexes containing pCpG-mcs (control;
Group A) or
PEI complexes containing the RNAi-resistant plasmid pCpG-eIF5Alx5ox (treated;
Group A). A second set of mice were given i.v. or i.p. injections - 2 times
per week
with either PEI complexes containing pCpG-mcs (empty vector) and control siRNA
(control; Group B.) or PEI complexes containing the RNAi-resistant plasmid
pCpG-
eIF5A1k50R and eIF5Al siRNA (treated; Group B). The data shown is the average
tumor volume for the mice in each group +/- standard error. Asterix denote
statistical
significance between treated and control groups (* = p < 0.05; * * * = p <
0.001; n =3).
The protocols for preparing the PEI complexes and the siRNA are as described
in previous examples.
Example 12: Co-administration of eIF5Al plasmid and eIF5Al siRNA delays growth
of multiple myeloma subcutaneous tumors and results in tumor shrinkage.
SCID mice were injected subcutaneously with KAS cells. Treatment was
initiated when palpable tumors were observed. Control mice were injected intra-
tumorally 2 times per week with PEI complexes containing pCpG-mcs (empty
vector)
and control siRNA (control group had 3 mice: control 1, control 2, and control
3).
Treated mice were injected intra-tumorally 2 times per week with PEI complexes
containing the RNAi-resistant plasmid pCpG-eIF5Alx5ox and eIF5Al siRNA
(treated
group contained 4 mice: 5A-1, 5A-2, 5A-3, and 5A-4). The intra-tumoral
rejections
of PEI complexes contained both 20 gg of plasmid DNA and 10 gg of siRNA. The
data shown in Figure 17 is the tumor volume for all the mice in each group.
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Example 13: Administration of eIF5Al siRNA intra-venously (i.v.) and
PEI/eIF5Alx5ox plasmid complexes intra-tumorally (i.t.) results in tumor
shrinkage of
multiple myeloma subcutaneous tumors.
SCID mice were injected subcutaneously with KAS cells. When palpable
tumors were observed treatment was initiated with an initial injection of 50
micrograms of either control siRNA (control group had three mice: control 1,
control
2 and control 3) or human eIF5Al siRNA (treated group had 3 mice: 5A-1, 5A-2,
5A-3). Control mice were subsequently treated by intra-tumoral injections 2
times
per week with PEI complexes containing pCpG-mcs (20 g) (control group 1-3).
Treated mice were subsequently treated by intra-tumoral injections 2 times per
week
with PEI complexes containing the RNAi-resistant plasmid pCpG-eIF5Alx5ox (20
g)
(5A-1, 5A-2, 5A-3). Control mice continued to receive either control siRNA (20
g)
by tail vein i.v. injection twice per week. Treated mice continued to receive
human
eIF5Al siRNA (20 g) by tail vein i.v. injection twice per week. The
injections were
given 48 hours prior to the intra-tumoral injections. The siRNA was given as
naked
siRNA, i.e. no delivery vehicle. The data shown in Figure 18 is the tumor
volume for
all the mice in each group.
Example 14: co-administration of eIF5Alx50R plasmid, driven by either the EF1
or
B29 promoter, and eIF5Al siRNA delays growth of multiple myeloma subcutaneous
tumors and results in tumor shrinkage.
SCID mice were injected subcutaneously with KAS cells. Treatment was
initiated when palpable tumors were observed. Mice were injected intra-
tumorally 2
times per week with PEI complexes containing either control vector (G1 and G2)
or
an eIF5Al plasmid driven by either the B29 promoter (G3 and G4) or the EF1
promoter (G5 and G6) and either control siRNA (G1, G3, G5) or h5Al siRNA (G2,
G4, G6). The data shown is the average tumor volume +/- standard error for
each
group. Note: the B29 promoter was intended as a B-cell-specific promoter.
However,
although the B29 promoter/mCMV enhancer used in this study was found to drive
high expression of HA-eIF5Alx50R in KAS cells in vitro, it does not appear to
be 13-
cell-specific (likely due to CMV enhancer). See figure 19.
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Example 15: Co-administration of eIF5Al siRNA increases anti-tumor effect of
eIF5Alx5ox plasmid, driven by either the EF1 or B29 promoter, on multiple
myeloma
subcutaneous tumors and results in reduced tumor burden.
SCID mice were injected subcutaneously with KAS cells. Treatment was
initiated when palpable tumors were observed. Mice were injected intra-
tumorally 2
times per week with PEI complexes containing either control vector (G1 and G2)
or
an eIF5Al plasmid driven by either the B29 promoter (G3 and G4) or the EF1
promoter (G5 and G6) and either control siRNA (G1, G3, G5) or h5Al siRNA (G2,
G4, G6). The mice were sacrificed 24 days after the initiation of treatment
and the
subcutaneous tumor was removed and weighed. The data shown is the average
tumor
weight +/- stadard error for all groups. See figure 20.
Example 16: eIF5Al siRNA Synergistically Increases Apoptosis Induction
Resulting
from Infection with Ad-eIF5A in Lung Adenocarcinoma Cells.
A549 cells were infected with either Ad-LacZ or Ad-eIF5A. Cells were
transfected with either a control siRNA or an siRNA targeting human eIF5Al
(h5A1)
by adding the transfection media to cells immediately following addition of
the virus.
Four hours after transfection with the siRNA and infection with virus, the
media was
replaced with fresh media and the cells were incubated for 72 hours prior to
labelling
with Annexin/PI to detect apoptotic cells. Note: over-expression of eIF5A in
this cell
line results in the accumulation of unhypusinated eIF5A due to limiting
amounts of
DHS and DOHH and therefore results in same pro-apoptotic effect as over-
expression
of eIF5Ax5ox These data indicate that the synergistic effect in apoptosis
caused by
simultaneous suppression of hypusinated eIF5A and over-expression of
unhypusinated eIF5A is observed in non-myeloma tumor-types as well. See figure
21.
Example 17: Construction of plamsid pExp5A.
pExp5A is an expression plasmid with reduced CpG dinucleotides designed
to drive expression of human eIF5Alx50R predominantly in cells of B cell
lineage.
The vector is derived from pCpG-LacZ, a plasmid completely devoid of CpG
dinucleotides (Invivogen). All the elements required for replication and
selection in
E. coli are free of CpG dinucleotides. The original CMV enhancer/promoter and
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LacZ gene from the CpG-LacZ vector have been replaced with a human minimal B
cell specific promoter (B29/CD79b; Invivogen) and human eIF5Alx50R,
respectively,
in order to drive B-cell specific expression of eIF5Alx50x The B29 DHS4.4 3'
enhancer has been introduced into the plasmid downstream of the eIF5A1
expression
cassette in order to enhance activity of the B29 promoter and reduce
expression in
non-B cells (Malone et at. 2006. B29 gene silencing in pituitary cells is
regulated by
its 3' enhancer. J. Mol. Biol. 362: 173-183). Incorporation of the B29 minimal
promoter, eIF5Alx50R, and the B29 DHS4.4 3' enhancer has introduced 32 CpG
dinucleotides into the vector.
Elements for expression in E. coli
Origin of replication: E. coli R6K gamma on.
* Due to the presence of the R6K gamma origin of replication, pCpG plasmids
can
only be amplified in E. coli mutant strain expressing a pir mutant gene. They
will not
replicate in standard E. coli strains. Therefore, pCpG plasmids are provided
with the
E. coli GT 115 strain, a pir mutant also deficient in Dcm methylation
(Invivogen).
Bacterial promoter: EM2K, a CpG-free version of the bacterial EM7 promoter.
Selectable marker: ZeocinTM resistance gene; a synthetic allele with no CpGs.
Elements for expression in mammalian cells
Mammalian promoter: the human -167bp minimal B29 (CD79b) promoter for tissue-
specific expression in B cells. A synthetic intron (1140) is also present in
the 5'UTR.
Polyadenylation signal: a CpG dinucleotide-free version of the late SV40
polyadenylation signal.
3' Enhancer: the human B29 DHS4.4 3' enhancer.
MAR: Two CpG-free Matrix attached regions (MAR) are present between the
bacterial and mammalian transcription units. One MAR is derived from the 5'
region
of the human IFN-(3 gene and one from the 5' region of the (3-globin gene.
The predicted Sequence of pExp5A (3371 bp is provided at figure 23.
Amino Acid Sequence of eIF5A1K50R
* K50R mutation is underlined
48
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MADDLDFETGDAGASATFPMQCSALRKNGFVVLKGRPCKIVEMSTSKTGRH
GHAKVHLVGIDIFTGKKYEDICPSTHNMDVPNIKRNDFQLIGIQDGYLSLLQD
SGEVREDLRLPEGDLGKEIEQKYDCGEEILITVLSAMTEEAAVAIKAMAK
Construction of pExp5A --Outline of Construction:
Step 1: Cloning of B29 DHS4.4 3' enhancer and subcloning into pGEM T easy
(Promega) -- creates pGEM-4.4enh #8.
Step 2: Subcloning of minimal B29 promoter into pCpG-LacZ (Invivogen) --
creates
B29-5 #3.
Step 3: Subcloning of HA-eIF5A1K50R into B29-5# 3 vector -- creates B29-5#3-
eIF5A1 K50R
Step 4: Creation of new multiple cloning site in pCpG-mcs (Invivogen) --
creates
pCpG-Linker4.
Step 5: Subcloning of B29 DS4.4 3' enhancer into pCpG-Linker4
-- creates pCpG-DHS4.4.
Step 6: Subcloning of B29 promoter + HA-eIF5A1K50R + SV40 pA expression
cassette into pCpG-DHS4.4 creates pExp-5.
Step 7: Replacement of HA-eIF5A1K50R in pExp-5 with non-HA eIF5A1K50R
creates final vector, pExp5A.
Construction in Detail:
Step 1: Cloning of B29 DHS4.4 3' enhancer and subcloning into pGEM T easy
(Promega) -- creates pGEM-4.4enh #8.
The B29 DHS4.4 3' enhancer was cloned by PCR from genomic DNA
isolated from KAS cells (human multiple myeloma cell line) using the following
primers:
forward 5'-CAGCAAGGGAGCACCTATG-3' and reverse 5'-
GTTGCAGTGAGCGGAGATG-3'. The primers were designed using the sequence of
the human CD79B/GH-1 Intergenic region (Accession AB062674). The resulting 608
bp PCR fragment was subcloned into the pGEM T easy cloning vector (Promega)
and sequenced. Komatsu et al. 2002. Novel regulatory regions found downstream
of
the rat B29/Ig-b gene. Eur. J. Biochem. 269 : 1227-1236.
49
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Sequence of B29 DHS4.4 3' enhancer PCR fragment _ t (297 bp) in pGEM-4.4enh #8
ACCACCCTGGGCCAGGCTGGGCCAAGCCAGGCGGCCCCTGTGTTTTCCCC
AGTCTCTGGGCTGCTGGAGGGAACCAGGTTGTTTTGGCATCAGCCTCTACT
GAGCCGGAGCCCTTCCTTTCCTGCTGCTTTGCATAGTGGCACTAATTCCG
TCCTCCTACCTCCACCAGGGACCTAGGCAGCCGGGTAGATGGTGGGAGGA
GGCTTCACTTCTCCCCCAAGCAGGGTCTCCACCTGCTTGAGGCTGCCCTGG
GTTGGGGGAGGCCTTGGCTTTACCTAAAGACTTTTTAACACCTCT
o +4.4 regions contains several transcription factor binding sites
^ SRY GTTGTTT
^ GATA CATCAGC
^ OCT-X GCTGCTTTGCATAG
^ NF-KB GAGGCTGCCC
Alignment of B29 DHS4.4 3' enhancer PCR fragment (297 bp) in pGEM-4.4enh #8
with sequence of the human CD79B/GH-1 Intergenic __ig on (Accession AB062674).
1 10 20 30 40 50 60 70 80 90 100 110 120 130
g,"6
GGGRRCRGCTGCCfl6CTGGGRGflCCfl8GTGCfl8TCRRCCTGCRCGTGCflflBGCCTCCCTCCCRRGCCflGGCT
GTGCTCCflCTTCCTGTTGncCCTGGflGGGRRTCCTTCGRGGCCCCTCTGCTflTTCCTG
PCR
Consensus
...............................................................................
...................................................
131 140 150 160 170 180 190 200 210 220 230 240 250 260
g,"6
CTCTGflflTTCCBGCflflGGGflGCflCCTBTGCTGTGGGflGCTGCCflGTTTflflCTGGGGflflTCflfl6fl
CCBGCBCBGGGGflflCTflGTGflGflflCflGTGCCflflTTTTCflCCflGflTTCCCTCTGGflflTTCCflGGT
GG
PCR
Consensus
...............................................................................
...................................................
261 270 280 290 300 310 320 330 340 350 360 370 380 390
gen6
66cnGGTGGGTflflGGCCCCCBCGCCTGCflGTTTCflGGTflflflTCTCTCCEECCflCE:C2iCE:F3i(iCTa3
f&CF:REE&3;C-flEia3:&GF:CE:CTf EEiET37F:CE:C33f
EE:E3:31iFi6E:33iCEEiu3E&GFiREEC3;R6EiE:&T
PCR EEtcPoCF.'CT&G&C_A.GGCT&
&c'CRE&cCIZFs&c&GCCE:CTGFFsET3lCCE:C33GFF.'Ei3'G&GC3GCFFs&3E&G&R Etc FIGFs T&
Consensus
................................................3ECC.PoCf';:T&6&CCY.C;&CT&
&CCtZ3EC CNF,&c&6CCE:CTGFF,ET3lCCE:C33GFf'EC3'G&6E:3 C;CFFI 3E&6&tZEECC.y6F,
ET&7
391 400 410 420 430 440 450 460 470 400 490 500 510 520
--------"----------------------------------------------------------------------
------------------*---------*-------------------I
gen6 F7YfFC:TFf.R6F.'7C7FICT&f7GCF.of:AfE'.C:?TC-CTFTCCT6::?fC-
TTFf:C(1TRi~?fGCftCTA(1TF::i'fPCCFf.C7FIC::?f.C-flF.'f:&G6R
i'TR6F~Cf:&CCe&TR6ftFf:&L6e ~Rf6flF ~f.YLE=R YTC.
PCR F7?fi&C:T?cflGF.CTC78CT&flGCF.^&f:FicCC::?TC-CTFTCCT6::?fC-TTFf:C8T8F~?f
GCElCTF78TF::CfTCF.Ff.C78C::?f.'C-
flF.Cf:&6&BCCTFEGGCf:&CC6:c&TBGEIFf:&T&6:iFf&flG"af.?TF-3CFTC
Consensus ET373GCEE33;RGE:CTClFICT&33&CE:&3EflGF:CE:3l1:0 E7CC3G?:3331:F1
E3EC13TRii333GCEEC7fl13T F?:C333 CE:E3:C'lFICE:33;C-
flE:C3E&6FiRE:CTRGEEC3E&CF:6Ei&T&GEE E3E&T&6Ei833&flEi&3:3 TC2E37C
521 530 540 550 560 570 500 590 600 610 620 630 640 650
gen6
TCfCCCflf:6C863CTCTCLCflCCT6fFT6fl61fT6CCCFf66TFf6f66:Z&fCCTFf6CTFTPfC.T@fEf6fE
CFTFTTfl@,3:CCTCT6flflCflflCBCfl6TTT000TGflGACTTT6flfl6CTCTTGTTTTBTTTfl
PCR TCfti-Cfflf:6C86:'s6TCTF.Cf:CCT6:.?TC.flfiaf.?GCC:.?fl:6TFf:6GG6:T&fl-
4TFf:&CTFiFfl-TBflf.&BCF7YTTNBCf.CCTC7
Consensus TCf.C-CCBf:&f86Fo&TC-TF.Cf:CCT6::?T[iflFo-
Sf.YfiE'.C::?f[i6TFf:&fi66:T&fC-CTFf:&CTFTFf.C-TF1Rf:&(1CFT?TPflFI'f:CCTCT
.............................................
651 660 670 680 690 700 710 720 730 740 750 760 770 780
gen6
TTTflTTTBTTTRTTTflTTTflCTTRTTTflTTTflTTTGCflGflCRGflGTCTCflCTCTGTTGCCCflGflCTGG
RGTGCflGTGGCflCCflTCTCCGCTCRCTGCflflCCTCCGTCTCCTGflGTTCBflGCflflTTCTCCT
PCR
Consensus
...............................................................................
...................................................
781 790 800
gen6 GCCTCRGCCTCCRRRGTOCC
PCR
Consensus ....................
Step 2: Subcloning of minimal B29 promoter into pCpG-LacZ (Invivogen) --
creates
B29-5 #3.
CA 02735823 2011-03-02
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The minimal -167 human B29 promoter was amplified from a commercial
plasmid bearing the full length human B29 promoter (pDrive-hB29; Invivogen)
using
the following primers : forward 5'-CCAACTAGTGCGACCGCCAAACCTTAGC-
3'; reverse : 5'-CAAAAGCTTGACAACGTCCGAGGCTCCTTGG-3'. The
resulting PCR fragment was digested with Spel and Hindlll and ligated into the
Spel
and Hindlll sites of the pCpG-LacZ vector (Invivogen) to create B29-5 #3.
Sequence of B29 minimal promoter PCR fragment (188 bp) in B29-5 #3
GCGACCGCCAAACCTTAGCGGCCCAGCTGACAAAAGCCTGCCCTCCCCCA
GGGTCCCCGGAGAGCTGGTGCCTCCCCTGGGTCCCAATTTGCATGGCAGG
AAGGGGCCTGGTGAGGAAGAGGCGGGGAGGGGACAGGCTGCAGCCGGTG
CAGTTACACGTTTTCCTCCAAGGAGCCTCGGACGTTGTC
Alignment of B29 minimal promoter PCR fragment (B29 min) in B29-5 #3 with full
length human B29 promoter from pDrive-hB29
1 10 20 30 40 50 60 70 80 90 100 110 120 130
E------------------------------------------------------------------------------
-------------------------------------------------- E
B29_pron
CCTGCflGGGCCCflciRGTflflflCGGflGGGTTGTGflGGflGflGTGflGflGGTGGflCfl6flG6GCflCCGf
lCGflTTTflGCflTCTCTTCCTCTCCTGGGGGTCGHGGHTGHGflGflCflflflflflfl6flfl6CT6CCBGGflf
lflC
B29_nin
Consensus
...............................................................................
...................................................
131 140 150 160 170 180 190 200 210 220 230 240 250 260
E-------- *---------- ---------------------------------------------------------
--------------------------------------------------- E
B29_pron
flTHHnHTTCBGHGGGCTURGUTGCBGGGEIGH66TciGcnHGCHTGUTGiGincnUTTGTGCBTGTTGTG000TGERC
HHGGGCHTCICTGflHGGGGCTGCHciGGnECCHGGGGCHGGGGCGCHHHGGT
629_",n
Consensus
...............................................................................
...................................................
261 270 280 290 300 310 320 330 340 350 360 370 380 390
E-------- *---------- ---------------------------------------------------------
--------------------------------------------------- E
B29_pron
GBGTTTBTflTCflGTTCCTGflGCBCTGTGGCTCCBTCCflGCflCTCTGflGGflCBGGCflGGflTHURGCTGGfl
GGflCCTGflGGGCTCCCCCflCflCCflGCTCCTGTTCCCTGCCCflBGflCCCCCTGGflCCTGCBG
629_nin
Consensus
...............................................................................
...................................................
391 400 410 420 430 440 450 460 470 480 490 500 510 520
E-------- *---------- ---------------------------------------------------------
--------------------------------------------------- E
B29_pron
flCHHCHHTTCflncGuflcicntiHGICCUHCHGTTHHGnHCTCCCTGflfl6flfl6CCCCCflGTGGUTGCGTGGT
GGHTTITCGCflnHGUTGICTUCHUCTHCHTCERCECTGTI166URG000UTnURTnCTCT
629_nin
Consensus
...............................................................................
...................................................
521 530 540 550 560 570 580 590 600 610 620 630 640 650
E-------- *---------- ---------------------------------------------------------
--------------------------------------------------- E
B29pron
TTCBCflGCflTGflGGflBGGGflGGCCTCTCflCCflBGflCCTGGflCTGflflTCTTCTCCCflGTGGCTGCCfl
CflCCTGflCCTGCTCTT6CTCCfl6flflCCTCTGTGGCT000flTCCTCCflCflGGGTCflflCTTCCflflC
629_nin
Consensus
...............................................................................
...................................................
651 660 670 680 690 700 710 720 730 740 750 760 770 780
E-------- *---------- ---------------------------------------------------------
--------------------------------------------------- E
B29pron
flTGGCTGCCTGCflCTCCflGCCflflGflGGCTCTGCTCTGGGCCCCTCCfl6niGCCTGflCCTGGGTCTGTGGCT
GCCCTGTCCTTCTTCBGTGCTCCTCTTCCCGCTGGGTGflGGflflTflGTTCBGGBCflGflGG
629_nin
Consensus
...............................................................................
...................................................
781 790 800 810 820 830 840 850 860 870 880 890 900 910
E-------- *---------- ---------------------------------------------------------
--------------------------------------------------- E
B29pron
flGCTnHGTTCnuGiicnITCHTntiGnuflGGTGCCTRITTcGCTCflCGGCCCntiGnHTntiRGHCTTGCCGGGCT
CGGCCUTiuGGGGnUTTGGCHGHcGGcnGHGGGGHGGUTGGCTG6000BGGGGBTGH
629_nin
Consensus
...............................................................................
...................................................
911 920 930 940 950 960 970 980 990 1000 1010 1020 1030 1040
E-------- *---------- ---------------------------------------------------------
--------------------------------------------------- E
B29_pron
CCRCCGGTGGGGTflfl6CflCflGflCflGflGGGGRGCRCRGGCTTCCCCCfl6flflGRCTGflGflGGLCCLCCH
GRGGCRTCCRCAGRGGRCCCCRGCTGTGLTGLCCRRGLTGG6fFiRLC9LfftRf.CLTFI5LF6f.
629_nin GfGR::CfCfIRICCTTI8f.GRE'.
Consensus
...............................................................................
.............................ExfER.-f;E-EIRILfTFI5fG6f.
1041 1050 1060 1070 1080 1090 1100 1110 1120 1130 1140 1150 1160 1170
E-------'*---------*--------'*---------*'--------*---------*'--------*---------
*---------*--------'*---------*---------*---------
B29_pron L-LFELL.3GFEL3Ef7Hf7GL'LTGC~'LGI.L-LL'ftGGGTLi:LLf-GE4's3EGCT6i3
Uf_Y.TLCCCT6iL"fCLL:~3E 3"f7'GftTGGf'f1ieGHf7GL'LL~CCTLie3 Uf7GL'ft3EGHG6:LUf-
GE4'sGGGNLFEL!,f_"FL'L3EGLL-613!,CH6'
B29_nin CLRfcCTGRL3Ef7E3RGELYSCCL?GCCLEBSSGFLCLCG6RsFGCT&iTGCCFLCCCTfiiL-
TiCCI$TTTG.BYGGCft~eSE3RGGfsSfCFB&TGR6G8FG65&f LGGfifEsSGGNLRL-
GCTGLRSCCfi1TGCBG
Consensus R.Rf CTGRLRflFiRGff.Tl;CLf.FfCfF.Lf!&l;GFLCf.CGGR:'sFfti-
TfiaT6fLF:.CLti-
TG=:f:FfLLIRTTTG.flYGGLft~rfRRG6is6LCFEsST[RG6RFfN5Gff:6GGR:'s&l;I:RLRf:6fT6:.F
f;C-fG=iT6ffl6
1171 1180 1190 1200 1210 1220 1230 1237
E------------------- -------'*-------------------*---------*'-----
B29_pronfTRi:flI:GTTETCCTCi:
fl33GGEEfifCFE:GEeRI.GFTfiTCflLGGGTTTGGGGTCGGGGflCflGflGLGGTGflL
B29_nin TTR9:flffiT T?fCCZIIIIi.S11TC.f:l.fiT78fC
Consensus TT3i:f71'FiFT?TCCTLCf7HGGEEfsLGTf6&51.6'FTf.ff
...............................
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Step 3: Subcloning of HA-eIF5A1K50R into B29-5# 3 vector -- creates pB29-
eIF5A1K50R_7.
HA-eIF5A1K5oR was amplified by PCR using the pHM6-eIF5A1K50R as a
DNA template and the following primers : forward 5'-
CGCCATGGACATGTACCCTTACGACGTCCCAGACTACGCTGCAGATGATTT
GGACTTCGAG-3' and reverse 5'-CGCGCTAGCCAGTTATTTTGCCATCGCC-3'.
The resulting PCR fragment was digested with Ncol and Nhel and subcloned into
the
Ncol and Nhel sites of B29-5 # 3 to replace LacZ.
Sequence of HA-eIF5A1K50R PCR fragment (497 bp) in pB29-eIF5A1K50R7
ACATGTACCCTTACGACGTCCCAGACTACGCTGCAGATGATTTGGACTTCG
AGACAGGAGATGCAGGGGCCTCAGCCACCTTCCCAATGCAGTGCTCAGCA
TTACGTAAGAATGGTTTTGTGGTGCTCAAGGGCCGGCCATGTAAGATCGT
CGAGATGTCTACTTCGAAGACTGGCAGGCATGGCCATGCCAAGGTCCATC
TGGTTGGCATTGATATTTTTACTGGGAAGAAATATGAAGATATCTGCCCGT
CGACTCATAACATGGATGTCCCCAACATCAAAAGGAATGATTTCCAGCTG
ATTGGCATCCAGGATGGGTACCTATCCCTGCTCCAGGACAGTGGGGAGGT
ACGAGAGGACCTTCGTCTGCCTGAGGGAGACCTTGGCAAGGAGATTGAGC
AGAAGTATGACTGTGGAGAAGAGATCCTGATCACAGTGCTGTCCGCCATG
ACAGAGGAGGCAGCTGTTGCAATCAAGGCGATGGCAAAATAACTG
Translation of HA-eIF5A1K50R PCR fragment in pB29-eIF5A1K50R7
HA epitope
eIF5AI K50R
K50R
MDMYPYDVPDYAADDLDFETGDAGASATFPMQCSALRKNGFVVLKGRPCKI
VEMSTSKTGRHGHAKVHLVGIDIFTGKKYEDICPSTHNMDVPNIKRNDFQLIG
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IQDGYLSLLQDSGEVREDLRLPEGDLGKEIEQKYDCGEEILITVLSAMTEEAAV
AIKAMAK
Alignment of HA-eIF5Alx50x PCR fragment in pB29-eIF5AlK50R7 with human
eIF5A1 (Accession # NP_001961)
1 10 20 30 40 50 60 70 80 90 100 110 120 130
-------------- ----------------------------------------------------------------
-------------------------------------------------
eIF5fl Hfff;f3E IIfFTFiF3:}4rHCNIFaY=ft~SRf_dtY=3dl:FV;13
YCRPCY.i+~Ft1.iF53:Y6KFi:"sklflK;'FEf_u6ZpFFFf
Y=KYFElFf.FSTFi:Fiff7+:PE7FFKl;FE1f_1CZ6lfJE'sY3 CI EgElSf:F;'FIL kEPF69L
Hfl-5H _K50R IIHIIYPYHYPHYflflE~{3E.IIfF7EiF~
Frf7CHTFaYSdC.SRf_EtY3dC.FV;~3.Y,liRPF.Y.1+~FtIaT53:T6RFi:'sklfiK;'FEf_u&ZPTFFf
Y,b:YFE7Ff.FSTFi:Fiff1+~PE7TKRKC7Fl~f_1&ZL?{7FsY c3_E.gE75f:F;'RFIi kEPFe i
Consensus
...........aIdORE.ERFIEiRlSfdSHIF?Y=GC.it1P_EtKtd[iFV'd3.KGEiPEKiS[ki-
iTSKY6rNiohf7KYFEP_u6ZDIFFf KP:YFE1Ff.F.iTNEFRf16PF7FKFtdOFIP_?6ZQ{J&Y3
SI_E.4E1Sf:F'7RFl4 RQPE64
131 140 150 160 165
1--------=---------=---------=----E
eIF511 6KE1EQKYE: EL1LId 4LSFR31 ELFIHYRIKtdRHK
HR-5F1 K50.1 Vi
Consensus tiKt:-~E~7KYF3:,Et1LI ~+iE5NttFfFflfl4EZIK33RflK
Step 4: Creation of new multiple cloning site in pCpG-mcs (Invivogen) --
creates
pCpG-Linker4.
The pCpG cloning vector, pCpG-mcs G2 (Invivogen), was digested with
EcoRl to remove the mammalian expression cassette contaning the mCMV enhancer,
the hEFI promoter, the synthetic intron, the multiple cloning site, and the
SV40
polyadenylation signal. The EcoRl-digested pCpG-mcs G2 vector was then ligated
to
a synthetic linker with EcoRl sticky ends to create a promoterless vector with
a new
multiple cloning site (pCpG-Linker4).
E00 M
slay W-8d
.. ... .... . .. ..... .......... . ...................
Xho I GI I Not I MIu I
as~ W.4
Synthetic nk r Linker with two E co R1
!
Sequence of region surrounding new multiple cloning site in pCpG-Linker4
1 GGCATGTGAACTGGCTGTCTTGGTTTTCATCTGTACTTCATCTGCTACCT 50
51 CTGTGACCTGAAACATATTTATAATTCCATTAAGCTGTGCATATGATAGA 100
101 TTTATCATATGTATTTTCCTTAAAGGATTTTTGTAAGAACTAATTGAATT 150
151 GATACCTGTAAAGTCTTTATCACACTACCCAATAAATAATAAATCTCTTT 200
201 GTTCAGCTCTCTGTTTCTATAAATATGTACAAGTTTTATTGTTTTTAGTG 250
53
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251 GTAGTGATTTTATTCTCTTTCTATATATATACACACACATGTGTGCATTC 300
301 ATAAATATATACAATTTTTATGAATAAAAAATTATTAGCAATCAATATTG 350
351 AAAACCACTGATTTTTGTTTATGTGAGCAAACAGCAGATTAAAAGGAATT 400
401 CTCGAGTCATCGATAAGCGGCCGCAGACGCGTAATTCAGTCAATATGTTC 450
451 ACCCCAAAAAAGCTGTTTGTTAACTTGCCAACCTCATTCTAAAATGTATA 500
501 TAGAAGCCCAAAAGACAATAACAAAAATATTCTTGTAGAACAAAATGGGA 550
551 AAGAATGTTCCACTAAATATCAAGATTTAGAGCAAAGCATGAGATGTGTG 600
601 GGGATAGACAGTGAGGCTGATAAAATAGAGTAGAGCTCAGAAACAGACCC 650
651 ATTGATATATGTAAGTGACCTATGAAAAAAATATGGCATTTTACAATGGG 700
701 AAAATGATGATCTTTTTCTTTTTTAGAAAAACAGGGAAATATATTTATAT 750
751 GTAAAAAATAAAAGGGAACCCATATGTCATACCATACACACAAAAAAAAT 800
801 TCCAGTGAATTATAAGTCTAAATGGAGAAGGCAAAACTTTAAATCTTTTA 850
851 GAAAATAATATAGAAGCATGCCATCAAGACTTCAGTGTAGAGAAAAATTT 900
901 CTTATGACTCAAAGTCCTAACCACAAAGAAAAGATTGTTAATTAGATTGC 950
951 ATGAATATTAAGACTTATTTTTAAAATTAAAAAACCATTAAGAAAAGTCA 1000
1001 GGCCATAGAATGACAGAAAATATTTGCAAC 1030
The numbering of the sequence above is for ease of reference to the following
features: 13Glo MAR (nucleotides 1-380); EcoRl recognition sequence
(nucleotides
396-401); Xhol recognition sequence (nucleotides 401-406); Clal recognition
sequence (nucleotides 409-414); Nod recognition sequence (nucleotides 417-
424);
Mlul recognition sequence (nucleotides 427-432);IFNB S/MAR (nucleotides 438-
1,030).
Step 5: Subcloning of B29 DS4.4 3' enhancer into pCpG-Linker4 -- creates pCpG-
DHS4.4.
The B29 DHS4.4 3' enhancer was amplified by PCR using pGEM-4.4enh #8
as a template and the following primers : forward 5'-
GAAGCGGCCGCACCACCCTGGGCCAGGCTGG-3'; and reverse 5'-
CCACGCGTAGAGGTGTTAAAAAGTCTTTAGGTAAAG-3'. The resulting PCR
fragment was digested with Nod and Mlul and ligated into the Nod and Mlul
sites in
the new multiple cloning site of pCpG-Linker4 to create pCpG-DHS4.4.
>pCpG-DHS4.4 full-length sequences (2,282 bp)
1 TTAATTAAAATTATCTCTAAGGCATGTGAACTGGCTGTCTTGGTTTTCAT 50
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51 CTGTACTTCATCTGCTACCTCTGTGACCTGAAACATATTTATAATTCCAT 100
101 TAAGCTGTGCATATGATAGATTTATCATATGTATTTTCCTTAAAGGATTT 150
151 TTGTAAGAACTAATTGAATTGATACCTGTAAAGTCTTTATCACACTACCC 200
201 AATAAATAATAAATCTCTTTGTTCAGCTCTCTGTTTCTATAAATATGTAC 250
251 AAGTTTTATTGTTTTTAGTGGTAGTGATTTTATTCTCTTTCTATATATAT 300
301 ACACACACATGTGTGCATTCATAAATATATACAATTTTTATGAATAAAAA 350
351 ATTATTAGCAATCAATATTGAAAACCACTGATTTTTGTTTATGTGAGCAA 400
401 ACAGCAGATTAAAAGGAATTCTCGAGTCATCGATAAGCGGCCGCACCACC 450
451 CTGGGCCAGGCTGGGCCAAGCCAGGCGGCCCCTGTGTTTTCCCCAGTCTC 500
501 TGGGCTGCTGGAGGGAACCAGGTTGTTTTGGCATCAGCCTCTACTGAGCC 550
551 GGAGCCCTTCCTTTCCTGCTGCTTTGCATAGTGGCACTAATTCCGTCCTC 600
601 CTACCTCCACCAGGGACCTAGGCAGCCGGGTAGATGGTGGGAGGAGGCTT 650
651 CACTTCTCCCCCAAGCAGGGTCTCCACCTGCTTGAGGCTGCCCTGGGTTG 700
701 GGGGAGGCCTTGGCTTTACCTAAAGACTTTTTAACACCTCTACGCGTAAT 750
751 TCAGTCAATATGTTCACCCCAAAAAAGCTGTTTGTTAACTTGCCAACCTC 800
801 ATTCTAAAATGTATATAGAAGCCCAAAAGACAATAACAAAAATATTCTTG 850
851 TAGAACAAAATGGGAAAGAATGTTCCACTAAATATCAAGATTTAGAGCAA 900
901 AGCATGAGATGTGTGGGGATAGACAGTGAGGCTGATAAAATAGAGTAGAG 950
951 CTCAGAAACAGACCCATTGATATATGTAAGTGACCTATGAAAAAAATATG 1000
1001 GCATTTTACAATGGGAAAATGATGATCTTTTTCTTTTTTAGAAAAACAGG 1050
1051 GAAATATATTTATATGTAAAAAATAAAAGGGAACCCATATGTCATACCAT 1100
1101 ACACACAAAAAAATTCCAGTGAATTATAAGTCTAAATGGAGAAGGCAAAA 1150
1151 CTTTAAATCTTTTAGAAAATAATATAGAAGCATGCCATCAAGACTTCAGT 1200
1201 GTAGAGAAAAATTTCTTATGACTCAAAGTCCTAACCACAAAGAAAAGATT 1250
1251 GTTAATTAGATTGCATGAATATTAAGACTTATTTTTAAAATTAAAAAACC 1300
1301 ATTAAGAAAAGTCAGGCCATAGAATGACAGAAAATATTTGCAACACCCCA 1350
1351 GTAAAGAGAATTGTAATATGCAGATTATAAAAAGAAGTCTTACAAATCAG 1400
1401 TAAAAAATAAAACTAGACAAAAATTTGAACAGATGAAAGAGAAACTCTAA 1450
1451 ATAATCATTACACATGAGAAACTCAATCTCAGAAATCAGAGAACTATCAT 1500
1501 TGCATATACACTAAATTAGAGAAATATTAAAAGGCTAAGTAACATCTGTG 1550
1551 GCTTAATTAAAACAGGTAGTTGACAATTAAACATTGGCATAGTATATCTG 1600
1601 CATAGTATAATACAACTCACTATAGGAGGGCCATCATGGCCAAGTTGACC 1650
1651 AGTGCTGTCCCAGTGCTCACAGCCAGGGATGTGGCTGGAGCTGTTGAGTT 1700
1701 CTGGACTGACAGGTTGGGGTTCTCCAGAGATTTTGTGGAGGATGACTTTG 1750
1751 CAGGTGTGGTCAGAGATGATGTCACCCTGTTCATCTCAGCAGTCCAGGAC 1800
1801 CAGGTGGTGCCTGACAACACCCTGGCTTGGGTGTGGGTGAGAGGACTGGA 1850
1851 TGAGCTGTATGCTGAGTGGAGTGAGGTGGTCTCCACCAACTTCAGGGATG 1900
1901 CCAGTGGCCCTGCCATGACAGAGATTGGAGAGCAGCCCTGGGGGAGAGAG 1950
1951 TTTGCCCTGAGAGACCCAGCAGGCAACTGTGTGCACTTTGTGGCAGAGGA 2000
2001 GCAGGACTGAGGATAACCTAGGAAACCTTAAAACCTTTAAAAGCCTTATA 2050
2051 TATTCTTTTTTTTCTTATAAAACTTAAAACCTTAGAGGCTATTTAAGTTG 2100
2101 CTGATTTATATTAATTTTATTGTTCAAACATGAGAGCTTAGTACATGAAA 2150
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2151 CATGAGAGCTTAGTACATTAGCCATGAGAGCTTAGTACATTAGCCATGAG 2200
2201 GGTTTAGTTCATTAAACATGAGAGCTTAGTACATTAAACATGAGAGCTTA 2250
2251 GTACATACTATCAACAGGTTGAACTGCTGATC 2282
The numbering of the sequence above is for ease of reference to the following
features: 13Glo MAR (nucleotides 1-400); EcoRl recognition sequence
(nucleotides
416-421); Xhol recognition sequence (nucleotides 421-426); Clal recognition
sequence (nucleotides 429-434); Nod recognition sequence (nucleotides 437-
444);
DHS4.4 (nucleotides 445-741); Mlul recognition sequence (nucleotides 742-
747);IFNB S/MAR (nucleotides 753-1,569).
Step 6: Subcloning of B29 promoter + HA-eIF5Al x5ox + SV40 pA expression
cassette into pCpG-DHS4.4 -- creates pExp-5.
The B29-eIF5Al expression cassette containing the minimal B29 promoter,
the synthetic intron, HA-eIF5Al x5ox, and the SV40 pA, was amplified from pB29-
eIF5A1K5OR7 (Step 3) by PCR using the following primers : forward 5'-
GTTATCGATACTAGTGCGACCGCCAAACC-3'; and reverse 5'-
CAAGCGGCCGCCATACCACATTTGTAGAGGTTTTAC-3'. The resulting PCR
fragment was digested with Clal and Nod and subloned into the Clal and Nod
sites in
the multiple cloning site of pCpG-DHS4.4 to create pExp-5.
Step 7: Replacement of HA-eIF5A1x5ox in pExp-5 with non-HA eIF5Alx5ox
creates final vector, pExp5A.
The pExp-5 plasmid was digested with Ncol and Nhel to remove HA-
eIF5Alx5ox A non-HA-tagged eIF5Alx50R PCR fragment was amplified from pHM6-
eIF5Alx50x by PCR using the following primers : forward 5'-
CACCA'I'GGCAGATGA'1"1"I'GGACT'1'C--3'; and reverse 5'-
CGCGCTAGCCAGTTATTTTGCCATCGCC-3'. The resulting PCR product was
digested with Ncol and Nhel and ligated into the Ncol and Nhel sites of B29-5
#3 to
generate B29-K50R. B29-K5OR was digested with Ncol and Nhel and the 470 bp
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eIF5Alx50R fragment was gel purified and ligated to Ncol/Nhel-digested pExp-5
to
generate the final expression vector, pExp5A.
Example 18: Testing of pExp5A
Various cell lines were transfected with plasmids using Lipofectamine 2000
and expression of HA-eIF5A1K50R was determined 24 hours following transfection
by Western blotting with an anti-HA antibody (Roche). Different cells lines
tested
were P3X63Ag8.653 (mouse B lymphoblast - myeloma), KAS (human myeloma),
HepG2 (huma liver hepatocellular carcinoma), T24 (human bladder carcinoma); HT-
29 (human colorectal adenocarcinoma), HEK-293 (human embryonic kidney cells),
PC3 (human prostrate adenocarcinoma); HeLa (human cervical adenocarcinoma),
and
A549 (lung carcinoma). See Figure 24.
pExp-5 plasmid expresses HA-eIF5Alx50R in both human and mouse
myeloma cell lines at comparable levels to a plasmid with the constitutive EF1
promoter (CpG-eIF5Alx50x) However expression of HA-eIF5Alx50R driven by
pExp-5 is limited in non-B cell lines compared to expression by a constitutive
promoter. The one exception was in HEK-293 cells, a human embryonic kidney
cell
line where high levels of HA-eIF5Alx50R expression was observed following pExp-
5
transfection - this may be due to the embryonic nature of the cell line; at
this time we
do not know if pExp-5 expresses in adult kidney cells. The final plasmid for
use in
toxicity studies and clinical trial will be a version of pExp-5 in which HA-
eIF5Al K50R
has been replaced by non-HA tagged eIF5Alx50R (pExp5A). pExp-5 contains HA-
tagged eIF5Al K50R under the control of the minimal human B29
promoter/enhancer;
expression of HA-eIF5Alx50R was compared to that driven by plasmids with
constitutive expression as well as to a plasmid containing the full-length B29
promoter
Example 19: formation of in vivo JETPEITM nanoparticle
This example given is for formation of the in vivo JETPEITM nanoparticle
complex for injection into 20 g mouse for a dose of 1.5 mg/kg (0.1 mL) --1.5
mg/kg =
1.0 mg pExp5A/kg + 0.5 mg h5Al/kg -- DNA: siRNA ratio = 2:1.
Dilute plasmid DNA and siRNA into a total volume of 25 ml. Use sterile
water to adjust the volume. * Dilute 20 mg of plasmid DNA (10 ml at 2 mg/ml)
and
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mg of siRNA (10 ml at 1 mg/ml) into a total volume of 25 ml. Use sterile water
to
make up difference. Adjust the volume of DNA solution to 50 ml 5 % glucose by
adding 25 ml of 10 % glucose (provided with PEI kit). Vortex gently and
centrifuge
briefly. Dilute in vivo JETPEITM into a total volume of 25 ml of water. *
Dilute 3.6
5 ml of in vivo JETPEITM into a total volume of 25 ml of water. Adjust final
volume to
50 ml with 10 % glucose to end up with a final concentration of 5 % glucose.
Vortex
gently and centrifuge briefly. Immediately add 50 ml of diluted PEI to the 50
ml of
diluted DNA (do not reverse the order!). Vortex briefly and immediately spin
down.
After formation the complex is stable for 8 to 10 hours. The N/P ratio of the
10 complex should be 6. The N/P ratio is the ratio of the number of positively
charged
nitrogen residues of in vivo-jetPEI to the number of negatively charged
phosphate
residues of DNA and siRNA. DNA and siRNA contain the same number of phosphate
groups per gram. The N/P ratio is therefore a measure of the ionic balance
within the
complex. Increasing the N/P ratio of the complex can increase the toxicity of
the
complex. In vivo JET-PEI is provided as a 150 mM solution (expressed as
nitrogen
residues) while DNA contains 3 nmoles of anionic phosphate in 1 mg. The final
concentration of DNA in the final volume should not exceed 0.5 mg/ml. The DNA
should be of high quality and prepared in water. In vivo-jetPEI and 10%
glucose
should be brought to room temperature prior to use.
Example20: Dose Range-Finding and Repeat Dose Studies with Intra-Venous SNSO1
and SNS-EF1/UU in Mice.
SNSO1 is one embodiment of the present invention - it is a cancer therapy
biologic targeted to the treatment of multiple myeloma. SNSO1 is comprised of
three
components: a DNA vector expressing a pro-apoptotic mutant of eIF5A (see
figure
22); an siRNA that targets the native eIF5A that promotes growth/anti-
apoptosis of
cancer cells (see the sequence in figure 25); and a synthetic polymer called
polyethylenimine (In vivo-jetPEI; Polyplus Transfection Inc.) that acts as a
delivery
vehicle.
The purpose of the studies was to determine the maximum tolerated dose and
the feasibility of long-term administration of therapeutic doses of intra-
venous SNSO1
into mice. Two separate studies were performed. The maximum tolerated dose
(Study
ID : MTD) study was an 8-day study in which mice received two intra-venous
doses
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of increasing amounts of SNSO1 (from 2.2 mg/kg to 3.7 mg/kg) and toxicity was
assessed by monitoring clinical signs, body weight, organ weight and liver
enzymes.
The 9-week repeated dose study (Study ID : EX6) was a study designed to assess
toxicity following long-term administration of twice-weekly therapeutic doses
(1.5
mg/kg) of SNS-EF1/UU and as well as it's various components. SNS-EF1/UU is a
preclinical version of SNSO1 and differs mainly in that expression of
eIF5AK50R is
driven by a constitutive promoter (one that expresses in all tissues at all
times) rather
than a B-cell-specific promoter as in the SNSO1 complex. The use of the B cell-
specific B29 promoter in SNSO1 was designed to enhance the safety of the
therapeutic
by limiting expression of the pro-apoptotic eIF5A mutant to cells of B-cell
origin,
including myeloma cells. The EX6 study also included a group of mice that were
dosed with a mouse-specific eIF5A siRNA to determine whether there were any
toxic
effects of suppressing eIF5A in mouse tissues. Toxicity in the repeated dose
study
was assessed by monitoring clinical signs, body weight, hematology, liver
enzymes,
as well as histopathology.
Study ID Animal Model Injection Duration of Test Article
Schedule Treatment
MTD CD-1 (female) Twice weekly 8-Days SNSO1
Intra-venous (2 injections)
EX6 Balb/c Twice weekly 9-Weeks -SNS-EF1/UU
(female) Intra-venous
-SNS-EF 1 /UU
components
tested
individually
-Mouse eIF5A
siRNA
Test Article Plasmid DNA siRNA Vehicle Material
Grade
SNSOla pExp5A eIF5A siRNA In vivo- GLP-grade
j etPEITM materials
(eIF5AK50R
expression (dTdT
driven by B29 overhangs)
B-cell-specific
promoter) human-specific
eIF5A siRNA
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SNS-EF1/UU pCpG-HA- h5Al In vivo- Research-
eIF5AK5OR (UU overhang) jetPEITM grade
materials
(HA- human-specific
eIF5AK5OR eIF5A siRNA
expression
driven by
ubiquitous EF1
promoter)
a- SNSO1 contains GLP-grade materials and is being developed for the clinic
b- SNS-EF1/UU is a test article used in preclinical research that led to the
development of SNSO1
c- the pExp5A plasmid is RNAi-resistant due to the fact that the plasmid
contains
only the open reading frame of eIF5A while the eIF5A siRNA (h5Al) targets the
3'
UTR of eIF5A
d - the sequence of the SNSO1 eIF5A siRNA and the h5Al siRNA are identical
except for the presencee of a dTdT overhang rather than a UU overhang on the
3'
terminal ends of the siRNA; the dTdT overhang does not affect the target
selectivity
or efficacy of the siRNA but has been proposed to enhance stability
Preclinical experiments have indicated that SNSO1 is therapeutic at doses of
0.75 mg/kg to 1.5 mg/kg (Study EX9). In the 8-day dose range-finding study
(Study
ID : MTD) significantly higher doses than the therapeutic range was tested to
determine the upper limit of the dose range. Twice-weekly intra-venous doses
of the
test article was well tolerated at the lower dose levels of 2.2 mg/kg and 2.9
mg/kg
although one mouse reached morbidity at the 2.9 mg/kg and was euthanized.
Doses at
3.3 mg/kg or above resulted in survival rates of approximately 20-25 %.
Therefore,
the maximum tolerated dose is between 2.2 mg/kg and 2.9 mg/kg and is well
above
the therapeutic range of 0.75 mg/kg to 1.5 mg/kg.
In the 9-week repeated dose study (Study ID : EX6) the mice received twice
weekly tail vein injections of therapeutic doses (1.5 mg/kg) of SNS-EF1/UU and
no
test article-related toxic effects were observed over the period of the study.
The DNA
and siRNA were also tested separately in this study and both were well
tolerated by
the mice. Since the human eIF5A siRNA is not active in mice, a mouse eIF5A-
specific siRNA was also included in this study. No toxic effects related to
chronic
administration of the mouse eIF5A siRNA were observed over the 9-week period.
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These results indicate that therapeutic doses of SNSO1 and SNS-EF1/UU are non-
toxic to mice even when administered over long periods.
Test Article and Vehicle
Test Article Manufacturer Manufacturer Manufacturer Formation of
of Plasmid of siRNA of PEI Test Article
DNA component component
component
SNSO1 VGXI Avecia Polyplus Components
Lot # Transfection were
pExp5A.08L007 Inc combined
GLP-Grade with 10%
glucose to
form nano -
complexes;
complexes
were injected
within 2-4
hours
SNS- Qiagen Thermo Scientific Polyplus Components
EF1/UU EndoFree Dharmacon Transfection were
Plasmid Mega RNAi Inc. combined
Kit Technologies Research- with 10%
< 0.1 EU/ g Grade glucose to
DNA form nano -
complexes;
complexes
were injected
within 2-4
hours
Storage -20 0 C -200 4 Room
Conditions (> 1 year) (> 1 year) (> 1 year) Temperature
(Stability) (> 6 hours)
Test Systems and Study Designs
All aspects of this study were conducted in accordance with the guidelines set
out by
the University of Waterloo Animal Care Committee (Waterloo, Ontario, Canada)
as
established by the Canadian Council on Animal Care and the Province of Ontario
Animals for Research Act.
The CD-1 and BALB/c mice were obtained from Charles River Laboratories
(Quebec,
Canada). Mice from both studies received the test article twice-weekly via
tail vein
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intra-venous injections. Slow injections (- 2-3 minutes) were used to deliver
volumes
greater than 0.2 ml.
The mice for the 8-day study were approximately 6-9 weeks old at the start of
study.
The mice for the 9-week repeated dose study were approximately 5-6 weeks old
at the
initiation of the study.
STUDY ID : MTD
Group No. Test N/P Dose Injection Total #
Animals Article ratio' level Volume injections
(female) (mg/kg)
MTD-C 5 5 % - - 0.33 mL 2
Glucose
MTD-PA 5 SNSO1 6 2.2 0.20 mL 2
mg/kg
MTD-PB 5 SNSO1 6 2.9 0.27 mL 2
mg/kg
MTD-PC 4 SNSO1 6 3.3 0.30 mL 1
mg/kg
MTD-PD 5 SNSO1 6 3.7 0.33 mL 1
mg/kg
a- N/P ratio = ratio of positively-charged nitrogens on PEI to the negatively-
charged
phosphate groups of the nucleic acids
b - due to toxicity the surviving mice were not given a second injection
STUDY ID : EX6
Group No. Test N/P Dose level Injection Total #
Animals Article ratio (mg/kg) Volume injections
(female)
Ex6-G1 4 5% - - 0.10 mL 20
Glucose
Ex6-G2 5 Vehicle 8 1.5 mg/kg 0.10 mL 20
Control'
Ex6-G3 5 siRNA 8 1.5 mg/kg 0.10 mL 20
Ex6-G4 6 DNA 8 1.5 mg/kg 0.10 mL 20
Ex6-G5 4 SNS- 8 1.5 mg/kg 0.10 mL 20
EF1/UU
Ex6-G6 6 Mouse 8 1.5 mg/kg 0.10 mL 20
siRNAd
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a - PEI complex containing a non-expressing plasmid (same vector background as
pExp5A) and a non-targeting siRNA
b - PEI complex containing a non-expressing plasmid (same vector background as
pExp5A) and the h5Al siRNA
c - PEI complex containing pCpG-HA-eIF5AK50R plasmid and a non-targeting
siRNA
d - PEI complex containing a non-expressing plasmid (same vector background as
pExp5A) and a mouse-specific eIF5A siRNA (the human eIF5A siRNA is not active
in mouse)
8-Day Maximum Tolerated Dose Study (MTD)
The two-dose 8-day study was a dose range-finding study designed to
determine the maximum tolerated dose of SNSO1. The dose range was 2.2 mg/kg to
3.7 mg/kg and is well above the therapeutic dose range of 0.75 mg/kg to 1.5
mg/kg.
At the lowest dose (2.2 mg/kg) of SNSO1 no clinical signs of toxicity were
observed
except for one mouse that exhibited slightly ruffled fur and decreased
activity that
resolved within one hour. No clinical signs of toxicity were observed
following the
2"d injection of 2.2 mg/kg of SNSO1. All the mice maintained their weight
throughout
the study. No macroscopic changes in the organs were observed. The organ
weight
to body weight ratios were unchanged from the control group except for a
modest
increase in the ratio of the liver weight : body weight ratio. However, since
an
increase in this ratio was not observed in any of the higher dose level groups
it is
unlikely to be related to the test article.
Four out of five mice tolerated 2.9 mg/kg of SNSO1 with no clinical signs of
toxicity. However, one mouse experienced convulsions and mild respiratory
distress
within 1 hour of injection and had to be humanely euthanized. No clinical
signs of
toxicity were observed following the 2nd injection of SNSO1 in the remaining
mice.
The mice maintained their weight throughout the study and no macroscopic
changes
in the organs or changes in the organ weight to body weight ratios were
observed.
There was a slight increase in the serum levels of ALT following two doses of
2.9
mg/kg SNSO1.
As expected, doses of SNSO1 at or above 3.3 mg/kg were not well tolerated
and in both groups all mice but one had to be humanely euthanized. In all
cases
where mice were humanely euthanized due to morbidity the clinical signs
appeared
within 1 hour of injection and were consistent with other reported studies
using high
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doses of PEI. The surviving mice of the 3.3 mg/kg and 3.7 mg/kg recovered
completely within 4 hours after the injection and maintained their weight
throughout
the study, although they did not receive a 2"d dose. The maximum tolerated
dose of
SNSO1 therefore appears to be between 2.2 mg/kg and 2.9 mg/kg.
9-Week Repeated Dose Study (EX6)
The purpose of the 9-week repeated dose study was to assess the safety of
chronic administration of therapeutic doses (1.5 mg/kg) of SNS-EF1/UU, a
complex
that was used for preclinical studies during development of SNSO1. SNS-EF1/UU
does not differ significantly from SNSO1, the major difference being that the
materials
are research-grade and that eIF5Ax5ox expression is driven by the constitutive
human
EF1 promoter that is active in all cell types. Although SNSO1 uses a B-cell-
specific
promoter to drive eIF5AK50R expression, the use of a constitutive promoter in
this
safety study allows for the assessment of toxicity resulting from the
accumulation of
the mutant eIF5AK50R protein in non-B-cell tissues. Another aspect of the 9-
week
repeated dose study was the inclusion of groups to test the safety of the
individual
components of SNS-EF1/UU. The DNA group (Ex6-G3) was dosed with a complex
containing the eIF5A plasmid and a non-targeting control siRNA while the siRNA
group (Ex6-G4) was dosed with a complex containing the human eIF5A (h5Al)
siRNA and a non-expressing plasmid. Since the test article SNS-EF1/UU contains
a
human eIF5A siRNA that will not affect expression of the endogenous mouse
eIF5A,
another feature of this study was the inclusion of a group (Ex6-G6) that was
dosed
with PEI complexes containing a non-expressing plasmid and an siRNA that
efficiently targets mouse eIF5A. This group allowed assessment of the safety
of
chronic administration of an active eIF5A siRNA.
All animals survived to the scheduled sacrifice date. No clinical signs of
toxicity were observed in any of the groups over the course of the 9-week
study and
the mice in all groups continued to gain weight during the study period. Red
and
white blood cell counts were measured three and six weeks after the initiation
of
treatment and were normal for all dosing groups. Serum liver enzyme levels
were
measured following sacrifice and were within the normal range for all mice. No
changes in the macroscopic appearance of the organs were observed in any of
the
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groups. Histopathological analysis of the major organs was conducted by an
independent pathologist (and revealed no toxicity attributable to the test
article.
Chronic administration of therapeutic doses of SNS-EF1/UU is well tolerated
by mice and no adverse effects were observed. In addition, chronic
administration of
a mouse-specific eIF5A siRNA revealed no toxic effects, indicating that the
administration of PEI complexes containing a human eIF5A siRNA should be safe
for
humans.
Example 21: Therapeutic Efficacy Studies with Intra-Venous SNS-B29/UU and
SNSO1 in Multiple Myeloma Tumor-Bearing Mice.
SNSO1 is as described above. The test article SNS-B29/UU is a preclinical
version of SNSO1. SNS-B29/UU differs very little from SNSO1, the chief
difference
being that the components are of research-grade rather than GLP-grade. The
purpose
of the study reported here was to determine the minimum effective dose of SNS-
B29/UU and to confirm that the GLP-grade materials that comprise SNSO1 perform
as well as the research-grade materials that were used for the preclinical
studies. The
repeated dose tumor study (Study ID : EX9) was a 5-week study in which the
ability
of increasingly smaller twice-weekly doses of SNS-B29/UU to inhibit
subcutaneous
tumor growth in mice was assessed in order to determine the optimal
therapeutic dose
of SNSO1. The treated animals were also assessed for signs of toxicity by
monitoring
clinical signs, body weight and organ weight.
Study ID Animal Model Injection Duration of Test Article
Schedule Treatment
Ex9 C.B17 (SCID) Twice weekly 35 Days SNS-B29/UU
(female) mice Intra-venous
bearing
subcutaneous Twice weekly 25 Days SNSO1
human multiple Intra-venous
myeloma
tumors
(KAS-6/1)
Test Article Plasmid DNA siRNA Vehicle Material
Grade
SNSOla pExp5A eIF5A siRNA In vivo- GLP-grade
j etPEITM materials
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(eIF5AK50R (dTdT
expression overhang')
driven by B29
B-cell-specific human-specific
promoter) eIF5A siRNA
SNS-B29/UU pExp5A h5Al In vivo- Research-
(UU overhang ) jetPEITM grade
materials
human-specific
eIF5A siRNA
a- SNSO1 contains GLP-grade materials and is being developed for the clinic
b- SNS-B29/UU is a test article used in preclinical research that led to the
development of SNSO1
c- the pExp5A plasmid is RNAi-resistant due to the fact that the plasmid
contains
only the open reading frame of eIF5A while the eIF5A siRNA (h5Al) targets the
3'
UTR of eIF5A
d - the sequence of the eIF5A siRNA and the h5Al siRNAs are identical except
for
the presencee of a dTdT overhang rather than a UU overhang on the 3' terminal
ends
of the siRNA; the dTdT overhang does not affect the target selectivity or
efficacy of
the siRNA but has been proposed to enhance stability
The therapeutic range of SNS-B29/UU was determined in SCID mice bearing
subcutaneous human multiple myeloma tumors. Doses of SNS-B29/UU between 0.15
mg/kg and 1.5 mg/kg were tested. The anti-tumoral efficacy of the test article
was
determined by twice-weekly tumor volume measurements and by excising and
weighing tumor tissue following sacrifice. SNS-B29/UU doses of 0.75 mg/kg and
1.5
mg/kg resulted in significant tumor shrinkage indicating that the therapeutic
range of
SNS-B29/UU lies between 0.75 mg/kg and 1.5 mg/kg. Effective inhibition of
growth
of subcutaneous tumors was also observed at 0.38 mg/kg SNS-B29/UU although no
tumor shrinkage was observed. Some inhibition of tumor growth was even
observed
at doses as low as 0.15 mg/kg SNS-B29/UU indicating a broad therapeutic range.
See
Figures 26 and 27.
The efficacy of SNSO1 made using GLP-grade components was compared to
SNS-B29/UU and was found have a comparable efficacy in the inhibition of tumor
growth. Treatment of tumor-bearing SCID mice with SNSO1 and SNS-B29/UU was
well tolerated and the mice continued to gain weight throughout the study.
Test Article and Vehicle
Test Article Manufacturer Manufacturer Manufacturer Formation of
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of Plasmid of siRNA of PEI Test Article
DNA component component
component
SNSO1 VGXI Avecia Polyplus Components
Lot # Transfection were
pExp5A.08L007 Inc combined
GLP-Grade with 10%
glucose to
form nano -
complexes;
complexes
were injected
within 2-4
hours
SNS- Qiagen Thermo Scientific Polyplus Components
B29/UU EndoFree Dharmacon Transfection were
Plasmid Mega RNAi Inc. combined
Kit Technologies Research- with 10%
< 0.1 EU/ g Grade glucose to
DNA form nano -
complexes;
complexes
were injected
within 2-4
hours
Storage -20 0 C -200 4 Room
Conditions (> 1 year) (> 1 year) (> 1 year) Temperature
(Stability) (> 6 hours)
Test Systems and Study Design
The female C.B.17/IcrHsd-Prkdc (SCID) mice were obtained from Harlan
(Indianapolis, IN, USA). Subcutaneous tumors were established by injecting
1Ox106
viable KAS-6/1 (human multiple myeloma) cells into the right flank of 5 to 6
week-
old mice. Treatment with SNS-B29/UU began when the tumors reached an
approximate size of 20 to 40 mm3 (approximately 4 weeks after tumor cell
injection).
Treatment with SNSO1 began when the tumors reached an approximate size of 130
mm3 (approximately 6 weeks after tumor cell injection). Mice received the test
article
twice-weekly via tail vein intra-venous injection.
STUDY ID : EX9
Group No. Test N/P Dose level Injection Total #
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Animals Article ratio (mg/kg) Volume injections
(female)
Ex9-G1 3 Controla 6 1.5 mg/kg 0.1 mL 11
Ex9-G2 4 SNS- 6 1.5 mg/kg 0.1 mL 11
B29/UU
Ex9-G3 4 SNS- 6 0.75 0.05 mL 11
B29/UU mg/kg
Ex9-G4 4 SNS- 6 0.38 0.025 mL 11
B29/UU mg/kg
Ex9-G5 3 SNS- 6 0.15 0.01 mL 11
B29/UU mg/kg
Ex9-G9 3 SNSO1 6 1.5 mg/kg 0.1 mL 7
a - PEI complex containing a non-expressing plasmid (same vector background as
pExp5A) and a non-targeting siRNA
Repeated Dose Tumor Study
The repeated dose tumor study was designed to determine the minimum
effective therapeutic dose of SNS-B29/UU and to confirm that the GLP-grade
SNSO1
test article retained the tumor inhibition activity demonstrated by the
research-grade
test article SNS-B29/UU. A secondary objective was to assess any toxic effects
of the
treatment by monitoring the treated mice for clinical signs, body weight, and
organ
weight. Test article therapeutic anti-tumoral activity was monitored by twice-
weekly
tumor volume measurements using digital calipers. Upon sacrifice the tumors
were
excised and weighed.
All the mice survived to the scheduled sacrifice date. Control mice that were
treated with PEI nanocomplexes containing a non-expressing plasmid and a non-
targeting siRNA had an average tumor volume of 284 mm3 at the time of
sacrifice
while mice treated with 1.5 mg/kg SNS-B29/UU had an average tumor volume of
only 13 mm3, a 95 % (*p = 0.026) reduction in tumor growth. However, when it
was
attempted to excise the tumors from mice that had been treated with 1.5 mg/kg
SNS-
B29/UU, no evidence of a tumor ws found in any of the mice. Decreasing the
dose of
SNS-B29/UU by half to 0.75 mg/kg still resulted in a 91 % (*p = 0.03) and 87 %
(*p
= 0.04) decrease in tumor volume and weight, respectively, and in one mouse
the
tumor had completely disappeared. Therefore, the optimum therapeutic dose for
twice-weekly injections of SNS-B29/UU appears to be between 0.75 mg/kg and 1.5
mg/kg. Twice-weekly doses of SNS-B29/UU doses as low as 0.15 mg/kg still
resulted in a 60 % reduction in the final tumor volume indicating that SNS-
B29/UU
has potent anti-tumoral activity over a wide dose range.
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In addition to inhibiting tumor growth, treatment with SNS-B29/UU and
SNSO1 at 0.75 mg/kg and 1.5 mg/kg resulted in significant reduction in tumor
volume
indicating that this treatment is capable of inducing tumor regression, likely
through
the induction of apoptosis in the tumor. The percent change in tumor volume in
tumor-bearing mice treated with SNS-B29/UU, at dose levels of 0.75 mg/kg and
1.5
mg/kg, was -244 % and - 245 %, respectively. The tumors of control mice
increased
in size by more than 2000 % during the same time period. Twice-weekly
injections of
SNSO1 also significantly shrunk multiple myeloma tumors. The percent change in
tumor volume for mice treated with 1.5 mg/kg SNSO1 was -349 %, indicating that
SNSO1 is just as effective as SNS-B29/UU. The use of GLP-grade materials may
in
fact have increased the biological activity since treatment with SNSO1
achieved a 349
% decrease in tumor volume following only 25 days of treatment while SNS-
B29/UU
achieved a 245 % reduction in tumor volume following 35 days of treatment. In
addition, the tumors treated with SNSO1 were quite large (- 130 mm) indicating
that
treatment with SNSO1 is effective against well-established tumors.
The treatment was well tolerated by all mice and no clinical signs of toxicity
were observed. The mice in all groups continued to gain weight throughout the
study.
No changes in the macroscopic appearance of the organs was observed at
necropsy
and no significant changes in the organ weight to body weight ratios occurred.
Therefore SNSO1 (and its preclinical version SNS-B29/UU) is well tolerated
by SCID mice and is extremely effective in treating subcutaneous human
multiple
myeloma tumors when delivered by intra-venous injection twice per week. All
doses
of SNS-B29/UU that were tested were effective at inhibiting tumor growth but
the
highest dose of 1.5 mg/kg successfully eliminated tumors in all mice receiving
treatment.
Example 22: Biodistribution of plasmid DNA and siRNA polyethylenimine (JetPEI)
complexes
Green fluorescent protein ("GFP")GFP-expression constructs were used to
determine localization of plasmid DNA delivered by PEI complexes. Two
promoters
were used to drive GFP expression: EF1: ubiquitous promoter (EF1::GFP) or B29:
13-
cell specific promoter (B29::GFP). PEI complexes containing 20 micrograms of
GFP
plasmid DNA and 10 micrograms of a fluorescently-labelled (DY547) h5Al siRNA
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were prepared at an N/P ratio of 6. Ba1B/C mice were injected intra-venously
with
either 5% glucose or PEI complexes on two consecutive days. Seventy-two hours
following the firsts injection the mice were euthanized and their organs were
harvested and analyzed for GFP expression and DY547-siRNA by confocal
microscopy.
Bone Marrow: In most cases there was evidence of DY547-siRNA but no GFP
expression. Timing of organ harvest may not coincide with peak expression of
GFP;
and there may be quenching of GFP signal or GFP may not be expressed. However,
GFP and DY547 that colocalized to the same bone marrow cells in some instances
was observed. Therefore, this provides evidence that PEI nanoparticles can
transfect
bone marrow cells in a live animal when given by intra-venous injection
Lung: In most cases there was evidence of DY547-siRNA but no GFP expression.
Timing of organ harvest may not coincide with peak expression of GFP or there
may
be quenching of GFP signal or GFP may not be expressed.
Spleen: Evidence of GFP expression (when driven by EFl promoter) colocalizing
in
cells also positive for the presence of DY547-siRNA was seen. Expression of
GFP
was much lower in spleen cells when driven by the B29 promoter. This shows
that
PEI nanoparticles appear to transfect cells of the spleen.
Kidney: No GFP or DY547 was observed indicating nanoparticles may not enter
kidney.
Liver: In most cases there was evidence of DY547-siRNA but no GFP expression.
This provides evidence that PEI nanoparticles are transfecting cells of the
liver.
Heart: Colocalization of EFl ::GFP and DY547-siRNA in tissue of heart was
seen,
thus indicating that PEI nanoparticles may be transfecting this organ. No GFP
was
observed with B29 promoter.
Example 23: Effect of DNA:siRNA ratio on HA-eIF5AK50R Expression.
KAS cells were transfected with nanoparticles containing B29-HA-eIF5AK50R
(plasmid driven by B-cell-specific promoter) and h5Al siRNA. JET PEITM
nanoparticles containing different ratios of pExp5A and h5Al siRNA were made
and
incubated for 4 hours at room temperature prior to addition to KAS cells. Four
hours
after transfection, the nanoparticle-containing media was replaced with fresh
media.
Twenty-four hours later the cell lysate was harvested and used for western
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analysis with an antibody against HA. The ratio of DNA:siRNA was varied from
the
standard ratio of 2:1. The accumulation of HA-eIF5Ax50x peaked at ratios of
1:0, 3:1,
and 2:1. See figure 30.
Example 24: Effect of DNA:siRNA ratio on apoptosis induced by nanoparticle
transfection.
Nanoparticles containing different ratios of pExp5A and h5A1 siRNA were
made and incubated for 4 hours at room temperature prior to addition to KAS
cells.
Four hours after transfection, the nanoparticle-containing media was replaced
with
fresh media. Forty-eight hours later the cells were harvested, labelled with
Annexin
V/PI and analyzed by FACS. The induction of apoptosis was highest in cells
transfected with nanoparticles with the standard DNA:siRNA ratio of 2:1. See
figure
31.
Example 25: Administration of PEI complexes (N/P = 6 or 8) containing
eIF5A1K50R plasmid and eIF5A1 siRNA (siSTABLE or non-siSTABLE) inhibits
growth of multiple myeloma subcutaneous tumors and results in tumor shrinkage.
SCID mice were injected subcutaneously with KAS cells. Treatment was
initiated when palpable tumors were observed. Mice were injected intra-
venously 2
times per week with either: (G1) PEI complexes containing 20 mg of pCpG-mcs
(empty vector) and 10 mg of control siRNA at N/P = 8 (medium dose); (G5) PEI
complexes containing 20 mg of the RNAi-resistant plasmid pCpG-eIF5A1k50R and
10 mg of siSTABLE h5Al siRNA at N/P = 8 (lmedium dose, siSTABLE); (G8) PEI
complexes containing 20 mg of the RNAi-resistant plasmid pCpG-eIF5A1k50R and
10 mg of h5Al siRNA at N/P = 6 (medium dose, N/P = 6). The data shown is the
individual tumor volume for the mice in each group. The final injection was
given at
day 40 after initiation of treatment. See figure 32.
Example 26: JET PEITM nanoparticles are being effectively taken up by tumor
tissue
and that nanoparticles are delivering plasmid and siRNA to the same cell.
Tumor section 48 hours after injection with nanoparticles containing pExp-
GFP (GFP under control of B-cell-specific promoter) and DY547-siRNA
(fluorescently-labelled siRNA). Co-localized expression of GFP and DY547 is
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observed in tumor section following confocal microscopy indicating that the
nanoparticles are being effectively taken up by tumor tissue and that
nanoparticles are
delivering plasmid and siRNA to the same cell. See figure 33.
Example 27: Adenovirus Constructs created for truncation study.
Adenovirus (serotype 5, E3-deleted)
1) Ad-eIF5A1O(2-6) [A(2-6)] - this is a human eIF5Al lacking amino acids 2 to
6.
2) Ad-eIF5A1K50RA(2-6) [A(2-6)/K50R] this is human eIF5Al lacking amino
acids 2 to 6. Additionally, the "K50R" means that the Lysine (K) at position
50 has been mutated to arginine (R) and is hence believed unable to by
hypusinated by DHS.
3) Ad-eIF5A1D6E [D6E] - this is human eIF5Al in which the predicted
cleavage site has been mutated (D6 to E).
4) Ad-eIF5A1D6E/K50R [D6E/K50R] - this is human eIF5A1K50R in which
predicted cleavage site has been mutated (D6 to E). Additionally, the "K50R"
means that the Lysine (K) at position 50 has been mutated to arginine (R) and
is hence believed unable to by hypusinated by DHS.
Example 28: Caspase mediated cleavage of eIF5A
To identify the site of cleavage, protein lysate isolated from KAS cells
treated
with Actinomycin D was separated by 2-D gel electrophoresis (Figure 37) and
the
spot corresponding to the smaller molecular weight eIF5A cleavage product was
cut
out of the gel and sequenced by mass spectroscopy (Figure 38B). Although a
full-
length sequence of the cleavage product was not obtained, it was determined
that the
cleavage occurred following the 6th amino acid from the N-terminal end of the
protein
(Figure 38A). The sequence immediately preceding the suspected cleavage site
is
`DDLD', a sequence that has been identified as a caspase cleavage site (Chat'
et al.,
2002).
To determine whether production of the cleavage fragment is caspase-
dependent, the ability of caspase inhibitors to block the production of the
cleavage
fragment produced during Actinomycin D-induced apoptosis in human myeloma
cells
was tested (Figure 41). A general caspase inhibitor and specific inhibitors of
caspase
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3, 8 and 9 all strongly prevent the formation of the cleavage form of eIF5A
that
accumulates during ActD-induced apoptosis in KAS cells. A caspase 1 inhibitor
also
reduced, but did not completely block, the formation of the cleavage product.
Incubation with the caspase inhibitors not only prevented the accumulation of
the
cleavage product, but also inhibited loss of the hypusinated form of eIF5A
(Figure
41), indicating that loss of hypusinated eIF5A during Actinomycin D-induced
apoptosis is a result of cleavage.
Caspase cleavage of proteins during apoptosis (reviewed by Fisher et at.,
2003) can have a myriad of purposes: 1) to inactivate pro-survival or anti-
apoptotic
proteins - for example: the eukaryotic translation initiation factor 4G 1,
which binds to
the 5' cap structure of mRNAs and facilitates binding of capped mRNA to 40S
ribosomal subunits, is inactivated by caspase cleavage, thereby inhibiting
translation;
2) to create a dominant negative form of the protein (for example, NF-kappa B
p65 is
cleaved by caspases during apoptosis to create a dominant-negative fragment
that
binds DNA but has no trans-activating activity and therefore acts as a
dominant-
negative inhibitor of NF-kB); 3) gain-of-function cleavage resulting in the
activation
of a pro-apoptotic protein by removal of an inhibitory or regulatory domain
thereby
resulting in formation of a fragment with a new activity or increased activity
(for
example, BRCA- 1, a breast cancer suppressor protein that mediates cell cycle
arrest
and apoptosis, is activated by caspase cleavage which releases a pro-apoptotic
cleavage fragment. DAPS, a member of the eIF4G family is activated following
caspase cleavage; the cleavage product stimulates translation of apoptosis-
related
proteins); 4) conversion of an anti-apoptotic protein to a pro-apoptotic
protein (for
example, apoptotic inhibitors, such as Bcl-xL and c-IAP1, can be turned into
pro-
apoptotic proteins through cleavage by caspase); and 5) cellular
redistribution (for
example, upon cleavage by caspase 8, the pro-apopototic p15 fragment of Bid
undergoes myristoylation at a glycine residue exposed by cleavage which
subsequently enables Bid to translocate to the mitochondria where it enhances
cytochrome c release).
To determine whether caspase-mediated cleavage of eIF5A during
Actinomycin D-induced apoptosis is a general phenomena or specific to myeloma
cells, HeLa cells treated with Actinomycin D were examined for the presence of
cleaved eIF5A (Figure 42). No accumulation of the eIF5A cleavage fragment was
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observed in Actinomycin D-treated HeLa cells. In an effort to determine
whether
post-translation modification (such as phosphorylation or acetylation) of
eIF5A may
be a pre-requisite for cleavage by caspases, the effect of a deacetylase
inhibitor,
nicotinamide, on the formation of the cleavage fragment was examined.
Acetylation
of eIF5A has been observed by mass spectroscopy analysis of eIF5A by our lab
and
others (Kim et at., 2006). Furthermore, yeast eIF5A has been identified as a
substrate
of the Sir2-related deacetylase Hst2 (Shirai et at., 2008) and the authors
observed that
acetylation of eIF5A was only observed in yeast lacking Hst2 or in yeast in
which
eIF5A was over-expressed, indicating that eIF5A is generally present in the
unacetylated form. Incubation of Actinomycin D-treated HeLa cells with the
Sir2
deacetylase inhibitor nicotinamide, resulted in the accumulation of the eIF5A
cleavage product indicating that acetylation of eIF5A is required for caspase-
mediated
cleavage to occur. These data suggest that eIF5A may normally be protected
from
caspase activity until an apoptotic signal triggers acetylation of eIF5A and
allows
caspase-mediated cleavage.
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Chay KO, Park SS, Mushinski JF (2002). Linkage of caspase-mediated degradation
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paxillin to apoptosis in Ba/F3 murine pro-B lymphocytes. J Biol Chem. 277,
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Fischer U, Janicke RU, Schulze-Osthoff K (2003). Many cuts to ruin: a
comprehensive update of caspase substrates. Cell Death and Differentiation 10,
76-
100.
Kim, S. C., Sprung, R., Chen, Y., Xu, Y., Ball, H., Pei, J., Cheng, T., Kho,
Y., Xiao,
H., Xiao, L., Grishin, N. V., White, M., Yang, X. J., and Zhao, Y. (2006)
Substrate
and Functional Diversity of Lysine Acetylation Revealed by a Proteomics
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Shirai et al. (2008). Global analysis of gel mobility of proteins and its use
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