FORM 2
THE PATENTS ACT, 1970
(39 of 1970)
&
THE PATENT RULES, 2003
PROVISIONAL SPECIFICATION
(See Section 10; rule 13)
"RNA INTERFERENCE MEDIATED
INHIBITION OF
NUCLEAR MITOTIC APPARATUS PROTEIN (NUMA )
AND IT'S COMBINATIONS
AS ANTICANCER THERAPY."
RELIANCE LIFE SCIENCES PVT. LTD.
an Indian Company having its Registered Office at Dhirubhai Ambani Life Sciences Centre, R-282, TTC Area of MIDC, Thane Belapur Road, Rabale, Navi Mumbai - 400 701 Maharashtra India.
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FIELD OF THE INVENTION:
The present invention relates to the field of short interfering nucleic acid (SIRNA) molecules responsible for modulating gene expression. The invention in particular relates to compounds, compositions and uses of short interfering nucleic acid molecules of 21, 23, 27 nucleotides in modulation of Nuclear mitotic apparatus protein (NuMA) gene expression. The compounds of the present invention have applications in cancer therapy whether conventional or alternative, either alone or in conjunction with other therapies.
BACKGROUND OF THE INVENTION
Mitosis is the process by which a cell duplicates its genetic information (DNA), in order to generate two, identical, daughter cells. The spindle apparatus is a structure of the eukaryotic cytoskeleton involved in mitosis and meiosis, often referred to as the mitotic spindle during mitosis and the meiotic spindle during meiosis. Its function is to segregate chromosomes during cell division (either mitosis or meiosis) to the daughter cells . It consists of a bundle of microtubules joined at the ends but spread out in the middle, vaguely ellipsoid in shape. In the wide middle portion, known as the spindle midzone, antiparallel microtubules are bundled by kinesins. At the pointed ends, known as spindle poles, microtubules are nucleated by the centrosomes.
During spindle assembly in prometaphase, some of the spindle's microtubules attach to the kinetochores that assemble on the centromere portion of the chromosomes. The chromosomes are pulled into alignment along the spindle midzone to form the metaphase spindle. Once all the chromosomes are aligned with sister chromatids pointing to opposite ends of the spindle, the cell enters anaphase, in which the chromatids separate and move toward their respective poles. Since the center of the spindle specifies the plane along which the cell will divide during cytokinesis, this ensures that each daughter cell will receive one of each chromatid.
The nuclear mitotic apparatus protein (NuMA) was first discovered by Lyderson and Pettijohn in 1980 as a non-histone protein that leaves the nucleus at mitosis and became associated with the poles of the mitotic spindle. They described NuMA as a
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predominantly nuclear protein that is present in the interphase nucleus and is concentrated in the spindle pole of mitotic cells.
Recently several nuclear proteins similar to that of NuMA have then been identified. These proteins are also known as centrophilin ( Toussen et al 1991), SPN ( Kallajoki et al 1991) SP-H ( Maekawa et al 1991) 1H1/1F1 ( Compton et al 1991) and Wl ( Tang et al 1993b). These proteins have been demonstrated by various monoclonal antibodies or autoimmune sera. However, the sequence obtained for NuMA, SPN, SP-H, 1H1/1F1 and Wl have different names for the same protein.
There have been many studies based on microinjections and cell cycle dependent localization experiments, which have led to the determination of the role of NuMA in maintaining nuclear structure possibly as a structural component of the nuclear matrix.
Nuclear mitotic apparatus protein (NuMA) is a large 236 KDa coiled-coil protein with globular head and tail. NuMA converge microtubules at minus ends, a function that is essential for spindle organization. In dividing cells, upon phosphorylation, NuMA disperses into the cytoplasm, associates with cytoplasmic dynein/dynactin to form a complex, and translocates along microtubules to the spindle poles where it organizes and tethers microtubules to spindle poles. NuMA becomes dephosphorylated, loses its association with dynein/dynactin, and releases from spindle poles after anaphase onset to allow spindle disassembly and reformation of interphase daughter nuclei. The cell-cycle-dependent phosphorylation of NuMA is regulated by the balanced activities of protein kinases and phosphatases. It has been shown that phosphorylation of NuMA by cyclin B/cdc2 kinase allows NuMA to release from the nucleus and to associate with centrosomes and/or microtubules at the spindle poles, while NuMA's dephosphorylation due to the cyclin B degradation allows NuMA to dissociate from the spindle poles after anaphase onset. Overexpression of NuMA interferes with spindle-associated dynein localization and promotes multipolar spindle formation and cancer. On the other hand, NuMA is absent in many kinds of non-proliferating cells and highly differentiated cells. NuMA also functions during meiotic spindle organization in male and female germ cells.
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Degradation of NuMA results in the breakdown of normal nuclear structure, and has been used as a marker of cell apoptosis.
Any discrepancy in the function of NuMA leads to disruption of microtubule focusing at spindle poles leading to splaying of microtubule ends. NuMA resides in the nucleus during interphase and becomes transiently associated with mitotic centrosomes after multiple steps of phosphorylations. NuMA does respond to external signals such as hormones that induce cell divisions or heat shock that induce apoptosis. At prophase NuMA disperses in the cytoplasm and associates with microtubules. During meta or anaphase NuMA gets associated with chromatin and finally to the reconstituted nucleus. NuMA is a cell cycle-related protein essential for normal mitosis that gets degraded in early apoptosis.
Studies conducted by Comptom and Cleveland (1993) have suggested that NuMA is required for the proper terminal phases of chromosome separation and /or nuclear reassembly during mitosis. Microinjection of anti- NuMA antibodies into early mitotic or metaphase cells was found by Yang and Snyder 1992 (Yang C H, Lambie E J, Snyder M. NuMA: an unusually long coiled-coil related protein in the mammalian nucleus. J Cell Biol. 1992 116(6): 1303-1317) that it prevents the formation or causes the collapse of the mitotic spindle apparatus thus suggesting that NuMA may play an important role during mitosis.
Variations in the NuMA gene are likely to be responsible for the observed increase in familial breast cancer risk. NuMA forms a complex with cytoplasmic dynein and dynactin. The depletion of the complex lead to failure in normal assembly of mitotic spindles. NuMA gets PARsylated by tankyrase-1 during mitosis.
NuMA, a nuclear mitotic apparatus protein, is released from cells undergoing apoptosis (Miller et al., Biotechniques, 15:1042, 1993). NuMA has been detected in the serum of patients with a wide range of cancers (Miller et al., Cancer Res., 52:422, 1992), and
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specifically in the urine of patients with bladder cancer (Stampfer et al., J. Urol., 159:394, 1998).
WO/2005/014846 provides methods for identifying risk of breast cancer in a subject and/or a subject at risk of breast cancer, reagents and kits for carrying out the methods, methods for identifying candidate therapeutics for treating breast cancer, and therapeutic methods for treating breast cancer in a subject. This patent application apart from various targets has focused on NuMA also.
US patent number 6,287,790 provides a method for distinguishing malignant and proliferating non-malignant cells by cell immunostaining using a NuMA specific antibody, and microscopic analysis of NuMA distribution within each nucleus.
US patent number 6,864,238 provides polypeptides, and polynucleotides encoding such polypeptides, that are useful for destabilizing microtubules. Since microtubules play an essential role in cell division, which occurs more frequently in tumor cells, the polypeptides and polynucleotides can be useful in preparing a composition for inhibiting cell proliferation for treating a tumor .
US patent number 6,855,515 provides a method of producing autoantigens, compositions comprising autoantigenic fragments and methods of using autoantigenic fragments in the treatment of a condition associated with an autoimmune response. In particular embodiments, autoantigenic fragments of DNA Pk.sub.cs, NuMA or PARP are generated by the action of granzyme B.
US 20030044352 provides a method to evaluate the response of animals and humans to administration of immune stimulatory and/or apoptosis-inducing compositions comprising bacterial DNA (B-DNA) administered with a pharmaceutically acceptable carrier, or B-DNA complexed to bacterial cell walls (BCC) and administered with a pharmaceutically acceptable carrier. These biological markers include without limitation, interleukins (IL) such as IL-12 and IL-18, the protein soluble Fas ligand (sFasL) and
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nuclear mitotic apparatus protein (NuMA). Any change in one or more biological molecules derived from the animal or human receiving the compositions indicates a response to the compositions.
US 20030125290 provides a composition comprising useful triethyleneglycol cholesteryl oligonucleotides for induction of response in a cell, including but not limited to inhibition of cellular proliferation, induction of cell cycle arrest, induction of caspase activation, cleavage of poly(ADP-ribose) polymerase, induction of apoptosis or modulation of extracellular matrix-cell interactions, or combinations thereof, in cancer cells or synovial cells, and methods of using this composition for treating disease. The release of nuclear mitotic apparatus protein (NuMA) was used as a measure of apoptosis.
US patent publication 20040043950 provides methods for inhibiting the growth of breast cancer cells and methods for treating breast cancers expressing Wilms' Tumor 1 (WTl) gene product using a WTl antisense oligonucleotide. It further provides methods of predicting breast cancer progression and methods for the screening of candidate substances for activity against breast cancer, wherein western blot analysis of WTl expresses in nuclear extracts of breast cancer cells. Nuclear mitotic apparatus protein (NUMA) was used as an internal control.
WO9640917A provides methods and compositions for identifying proteins which interact non-covalently with the Nuclear Mitotic Apparatus protein (NuMA) in a cell, novel proteins identified by the method, and methods and compositions for interfering with this interaction in vivo
W09845433 Alprovides new nucleic acid encoding human highly expressed in cancer nuclear protein - used for diagnosis and for modulation of the cell cycle to control malignant and other cell growth abnormalities
Till date past research has more focused on NuMA as diagnostic marker for risk assessment of an individual prone to breast cancer and as a biological marker for understanding prognosis of tumor.. There have been no reports on modulation of the
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NuMA gene expression through short interfering nucleic acids (siRNA) molecule.. The present invention focuses on modulation of the NuMA gene expression through short interfering nucleic acids (siRNA) with the aim of offering therapeutic intervention..
OBJECT OF THE INVENTION
It is the principle object of the present invention to provide modulation of the NuMA gene expression through short interfering nucleic acids (siRNA) molecule.
It is the object of the present invention to provide 21, 23 and 27 mer short nucleic acid molecules for modulation of NuMA gene expression.
It is the object of the present invention to provide compounds having 21, 23 or 27 mer short nucleic acid molecules for treatment of different types of cancers more particularly breast, lung, prostate, colorectal, cervical, epidermoid and oral cancers.
It is the object of the present invention to provide 21, 23 and 27 mer short nucleic acid molecules site directed against the target.
It is the object of the present invention to provide SNP specific siRNA molecules so as to offer personalized treatment to patients with NuMA indisposition.
It is the object of the present invention to provide 21, 23 and 27 mer short nucleic acid molecules, which can be used alone or in combination with other therapies for effective management of cancer treatment.
It is the object of the present invention to provide 21, 23 and 27 mer short interfering nucleic acid molecules, which can be combined with conjugates not limiting to lipids, polymers and monoclonal antibodies.
It is the object of the present invention to determine the quantity of NuMA that gets co-localized at the site of its action in comparison with the mock treated controls.
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SUMMARY OF THE INVENTION
The present invention is directed to modulation of the NuMA gene expression through short interfering nucleic acids (siRNA) molecule. In particular the present invention relates to compounds, compositions and uses of 21, 23 or 27 mer short interfering nucleic acid (SiRNA) molecules directed against NuMA in modulation of its expression. The compounds of the present invention are useful in therapy of cancer either alone or in combination with other treatments or therapies.
In one embodiment, the short nucleic acid molecules of the present invention is also featured as short interfering nucleic acid (SIRNA), short interfering RNA (SiRNA), double stranded RNA (dsRNA), micro RNA (mRNA), deoxyribose nucleic acid intereference (DNAi) and short hairpin RNA (shRNA) molecules. The short nucleic acid molecules can be unmodified or modified chemically. In the preferred embodiments the present invention relates to 21, 23 or 27 mer short interfering RNA. In the present invention the efficiency of SIRNA is determined by the ability to reduce the quantity of the target protein so that the functional properties associated with that protein gets impaired.
In another embodiment SIRNA of 21, 23 or 27 mers can be synthesized either chemically or enzymatically or expressed from a vector In preferred embodiments, present invention relates to the chemically synthesized SIRNA of 21, 23 or 27 mers in length to reduce expression levels of NuMA either alone or in combination with other SIRNA directed against genes that are responsible in regulating various cancers.
In the preferred embodiment, the present invention provides short nucleic acid molecules for treatment of various types of cancers which include breast, lung, prostate, colorectal, cervical, epidermoid, oral cancers, glioma and leukemia.
In one embodiment, the present invention provides techniques used to validate the efficacy of siRNA of 21, 23 or 27 mers with biomarkers of cancer. The present invention
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provides the efficacy testing with specific biomarkers of cancer such as PCNA, KI-67, and BCL-2 antigen expression.
In one embodiment, the present invention provides combination of SIRNA targeting NuMA and combinations there of, for the treatment of various types of cancers which include breast, lung, prostate, colorectal, cervical, epidermoid, oral cancers, glioma and leukemia.
In yet another embodiment, the present invention provides SIRNA molecules which can be used alone or in combination with other SIRNA or small molecules which are related to the regulation and expression of genes of NuMA and such proteins which are associated with cancer or any other conditions or disease that respond to the levels of NuMA in a cell or tissue. The preferred embodiment is the use of SIRNA of the present invention in any therapy of genes encoding sequence of NuMA shown in table I. Genbank Accession Number NM006185.
Although the present invention is related to regulate NuMA gene, the embodiments includes all homolog genes, and transcript variants of NuMA and other genes involved in NuMA regulatory pathway and polymorphism (eg: single nucleotide polymorphism) associated with NuMA.
In one embodiment the nucleic acid molecule of the present invention comprises between 19,20,21,22,23,24,25,26,27,28,29 &30 base pairs.
In another embodiments the nucleic acid molecule of the present invention comprises 19,20,21,22,23,24,25,26,27,28,29 & 30 base pairs complementary to RNA having an NuMA nucleic acid sequence.
In one embodiment, a siRNA molecule of the present invention comprises a double stranded RNA, wherein one strand of the RNA is complimentary to the RNA of NuMA. In another embodiment, a siRNA molecule of the present invention comprises a double
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stranded RNA, wherein one strand of the RNA comprises a portion of a sequence of RNA having NuMA sequence.
In one embodiment, the invention features a mammalian cell, for example a human cell, including a nucleic acid molecule of the invention.
The present invention features method of down-regulating NuMA activity in a cell, comprising contacting the cell with an enzymatic nucleic acid molecule or antisense nucleic acid molecule, or other nucleic acid molecule of the invention, under conditions suitable for down-regulating of NuMA activity.
The present invention also features method of treatment of a subject having a condition associated with the level of NuMA, comprising contacting cells of the subject with an enzymatic nucleic acid molecule or antisense nucleic acid molecule or other nucleic acid molecule of the present invention under conditions suitable for such treatment.
In one embodiment, the present invention also features method of treatment of a subject having a condition associated with the level of NuMA, comprising contacting cells of the subject with the enzymatic nucleic acid molecule or antisense nucleic acid molecule or other nucleic acid molecule of the present invention, under conditions suitable for treatment.
In one embodiment, a method of treatment of the invention comprises the use of one or more drug therapies under conditions suitable for said treatment.
In another embodiment, other drug therapies contemplated by the invention include monoclonal antibodies, chemotherapy, or radiation therapy or a combination thereof.
In another embodiment, an expression vector of the invention further comprises an antisense nucleic acid molecule complementary to RNA of a subunit of NuMA. In yet another embodiment, an expression vector of the invention comprises a nucleic
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acid sequence encoding two or more enzymatic nucleic acid molecules, which can be the same or different.
The present invention also features a method for treatment of different types of cancers, not limited to breast cancer, lung cancer, prostate cancer, colorectal cancer, brain cancer, esophageal cancer, stomach cancer, bladder cancer, pancreatic cancer, cervical cancer, head and neck cancer, ovarian cancer, melanoma, lymphoma, glioma, or multidrug resistant cancer, comprising administering to a subject an enzymatic nucleic acid molecule or antisense nucleic acid molecule or other nucleic acid molecule of the invention under conditions suitable for said treatment.
The present invention features compositions comprising the enzymatic nucleic acid and/or antisense nucleic acid molecules of the invention in a pharmaceutically acceptable carrier.
The invention also features a method of administering to a cell, such as mammalian cell (e.g. human cell), where the cell can be in culture or in a mammal, such as a human, an enzymatic nucleic acid molecule or antisense molecule of the instant invention, comprising contacting the cell with the enzymatic nucleic acid molecule or antisense molecule or other nucleic acid molecule of the invention under conditions suitable for such administration. The method of administration can be in the presence of a delivery reagent, for example a lipid, cationic lipid, phospholipids or liposome.
BRIEF DESCRIPTION OF THE DRAWINGS
The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present disclosure, the inventions of which can be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.
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Figure 1A Western blot showing knock down of NuMA protein after 72 h of siRNA transfection where 236 KDa represents NuMA protein. Endogenous control tubulin was re presented by 51 KDa protein band. Lane M represents molecular weight markers. Lane 1-4 represents lung cancer cell line A549 transfected with siRNA 1, 2, 3 & 4 respectively. A very faint band of NuMA is present in all siRNA transfected cells in comparison over mock treated cells.
Lane 5-8 represents normal fibroblasts cell line HFF-2 transfected with siRNA 1, 2, 3 & 4 respectively. A very faint band of NuMA is present in all siRNA transfected cells in comparison over mock treated cells.
Figure IB Western blot showing knock down of NuMA protein after 72 h of siRNA transfection where 236 KDa represents NuMA protein. Endogenous control tubulin was re presented by 51 KDa protein band. Lane M represents molecular weight markers. Lane 1 - 4 represents epidermoid cancer cell line A431 transfected with siRNA 1, 2, 3 & 4 respectively. A very faint band of NuMA is present in all siRNA transfected cells in comparison over mock treated cells.
Lane 5 & 4 represents prostate cancer cell line PC3 transfected with siRNA 1, 2, 3 & 4 respectively. A very faint band of NuMA is present in all siRNA transfected cells in comparison over mock treated cells.
Figure 1C Western blot showing knock down of NuMA protein after 72 h of siRNA transfection where 236 KDa represents NuMA protein. Endogenous control tubulin was re presented by 51 KDa protein band. Lane M represents molecular weight markers. Lane 1 - 4 represents cervical cancer cell line HeLa transfected with siRNA 1, 2, 3 & 4 respectively. A very faint band of NuMA is present in all siRNA transfected cells in comparison over mock treated cells.
Figure 2: Colony forming efficiency of siRNA tested by seeding 300 cells per well of a 6-well plate in triplicate each. At the end of 10 days of incubation the number of colonies were counted after crystal violet staining. The mean average percent of number of colonies has been determined with respective to mock treated controls.
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DETAILED DESCRIPTION OF THE INVENTION
Definitions:
The term " Short interfering nucleic acid", "SiNA" or SINA" molecules, "short interfering RNA", "SIRNA", "short interfering nucleic acid molecule", "short interfering oligonucleotide molecule", as used herein refers to any nucleic acid molecule capable of inhibiting or down regulating gene expression.
The term " RNA" as used herein means a molecule comprising atleast one ribonucleotide residue which includes double stranded RNA, single stranded RNA, isolated RNA, partially purified, pure or synthetic RNA, recombinantly produced RNA, as well as altered RNA such as analogs or analogs of naturally occurring RNA.
The term "modulate" as used herein means that the expression of the gene or level of RNA molecule or equivalent RNA molecules encoding one or more protein or protein subunits, or activity of one or more protein subunits is up regulated or down regulated such that the expression, level or activity is greater than or less than that observed in the absence of the modulator. The term" modulate" also can mean "inhibit" but the use of the terms is not limited in this definition.
The term "gene" as used herein means a nucleic acid that encodes a RNA sequence including but not limited to structural genes encoding a polypeptide.
The term " Nuclear associated mitotic protein" , "NuMA" as used herein refers to any NuMA protein, peptide, , or polypeptide having NuMA or Centrophilin activity such as encoded by genbank accession number NM006185: It also refers to nucleic acid sequence encoding NuMA protein, peptide, or polypeptide having isoforms, mutant genes, splice variants and polymorphisms.
The term" target nucleic acid" as used herein means any nucleic acid sequence whose expression or activity is to be modulated. The target nucleic acid can be DNA or RNA.
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The term "sense region" as used herein means a nucleotide sequence of a SINA molecule having complementary to a target nucleic acid sequence. In addition, the sense region of a SIRNA molecule can comprise a nucleic acid sequence having homology with a target nucleic acid sequence.
The term "antisense region" as used herein means a nucleotide sequence of a SIRNA molecule having a complementarity to a target nucleic acid sequence. It can also comprise a nucleic acid sequence having complementarity to a sense region of the SIRNA molecule.
The term "complementarity" as used herein means a nucleic acid can form hydrogen bonds with another nucleic acid molecule.
The term "cancer" or "proliferative diseases" as used herein means any disease, condition, trait, genotype or phenotype characterized by unregulated cell growth or replication as is known in the art. It can include all types of cancer, tumors, lymphomas, carcinomas that can respond to the modulation of disease related NuMA gene expression in a cell or tissue alone or in combination with other therapies.
The present invention provides for the compounds of short interfering nucleotides and their uses in modulation of NuMA gene expression. The main features of the studies conducted are as follows:
1. Design of SIRNA.
2. Preparation of SIRNA
3. Efficacy testing of the compounds
4. Comparative data of 21, 23, and 27 mer
5. Potency evaluation in animal models
The design of SIRNA involves the design of the SIRNA with 21, 23, and 27 nucleotides for modulation of NuMA either with or without any chemical modification. Since the design of SIRNA is the main key step in gene knockdown experiments, the general
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requirements were followed as provided by the literature available. For all the SIRNA
irrespective of their length following general requirements were considered while
designing the molecules as stated below
i. No runs of 4 or more A, T, G, or U allowed
ii. The following sequences were avoided which are responsible for interferon response. A)
5'-UGUGU-3' and B) 5'-GUCCUUCAA-3'.
iii. Each SIRNA duplex was checked in-silico to avoid silencing of off-target effects made
on BLAST search considering the following parameters:
A) Low complexity filtering was removed to avoid insignificance by BLAST resulting in limited or no query sequencer.
B) The word size was set to 7 letters, the minimal value algorithm.
C) Expect value threshold was set at 1000 to avoid probability of short sequence occurrence. Further the target gene NuMA was screened for accessible sites and the SIRNA was synthesized considering the ORF sequences.
The synthesis of SIRNA was done by commercially available methods. Most preferably these could be synthesized by standard chemicals techniques provided by Qiagen. The chemical methods involve the addition of chemically protected monomeric units called phospharmidites sequentially to generate the desired oligonucleotide sequence. The synthesis involves mainly four steps such as coupling, capping, oxidation and 5'-deprotection. The purification of the SIRNA molecules was done either by PAGE, desalting or by IE-HPLC. The quality of each SIRNA was analyzed by MALDI-TOF and the yields were determined by integrated spectrophotometer.
The efficacy testing of the siRNA molecule was done in different cell lines. The cell lines related to breast cancer such as MCF-7, SKBR-3, prostate cancer such as PC3,lung cancer such as A549, skin cancer such as A431, cervical cancer line HeLa, were obtained from ATCC and were cultured as per the recommendation of ATCC. The cell lines were transfected with SIRNAs and incubated.
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The transfection efficiencies were obtained for each cell line by counting number of cells showing Cy3 labeled SIRNA after 16 hours of transfection.
Apart from the percent transfection, the morphological features of the cell lines were also observed in comparison with the untreated cell lines.
The potencies of the different lengths of SIRNAs were checked by their efficiency in inhibiting proliferation of cancer cell lines. After transfection of SIRNA for 72 hours the cells were incubated with BrdU as per the protocol of Calbiochem. This test determines the ability to incorporate Brdu into DNA of actively proliferating cells. The quantity of BrdU incorporated was estimated by the absorbance values and was compared with the mock treated cells. It is a known fact that incorporation of BrdU occurs only when there is DNA synthesis. During the S-phase of mitosis synthesis of DNA occurs resulting in doubling of chromosomes. In cancer cells, the number of cells that undergo the process of DNA synthesis indicates the growing potential of cells resulting in growth of tumor. Thus the amount of BrdU incorporated into the cells is directly proportionate to the growing potential of tumor cells.
The cells transfected with the SIRNA were also analyzed for specific mRNA knockdown effects using Real time quantitative PCR analysis. The present invention has studied the relative mRNA quantities of NuMA in cells transfected either with siRNA specific for NuMA or scrambled siRNA The fold change in mRNA levels was determined by the protocol of Kenneth JL and Thomas DS (2001).
The proliferative and metastasis potential of cancer cell lines treated with the siRNA molecules was obtained by measuring the levels of PCNA (proliferaive cell nuclear antigen) or Ki-67 antigen.
The protein levels of NuMA were analyzed by western blot analysis. Since proteins are the functional units that are responsible to affect any biological function in the cell, the estimation of protein quantity helps to understand the metabolic status of the cell.
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Although transfections of SIRNA results in successful knockdown of their respective mRNA levels, the cells have various mechanisms by which they can cope up with the loss of mRNA levels by enhancing the gene expression activity so as to meet the required protein demand of the cell. Hence the efficacy of SIRNA in the present invention is determined by the ability to reduce the quantity of the target protein so that the functional properties associated with that protein gets impaired.
As stated earlier, the inhibition in the protein levels of NuMA has various effects on metabolic activity of the cells, which leads to functional impairment of cells. The present invention has measured by colony forming assays, which basically identifies the ability of the single cancer cell to initiate cell cycle process resulting in development of tumor if they get metastasized.
The cytotoxicity effect of the transfected cell lines with SIRNA was studied by analyzing the amount of LDH released into the medium due to compromise on membrane integrity.. As described earlier, the knockdown of gene NuMA results in failure of cells to divide as a result of inability of spindle pole to organize properly. This results in activation of mitotic check points resulting in arrest of cell cycle. These cells may under go membrane integrity compromise resulting in release of cytosolic LDH.
NuMA is found to be localized at the spindle poles and is responsible for focus of minus ends of microtubules at the spindle poles. NuMA in order to be functional during cell division process needs to get co-localized at appropriate quantities. Absence of co-localization of NuMA leads to defective spindle assembly. Even though the methods such as mRNA quantification and protein quantification are available, these methods do not however indicate the minimum threshold levels of NuMA required for normal cell function. Hence the present invention aims to determine the quantity of NuMA that gets co-localized at the site of its action in comparison with the mock treated controls.
Further the present invention also provides cholesterol conjugated SIRNAs of NuMA. The serum stability of these molecules is also studied.
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The effects of these conjugated SIRNAs will be evaluated for their interferon response. The present invention provides the preclinical evaluation studies in animals wherein the efficacy of SIRNA is checked for causing tumor regression. The knockdown affect of NuMA on solid tumors and metastatic tumors will be tested by induction of xenograft tumors in 6-8 weeks old Nude mice by injecting cell lines (SKBR-3, MCF-7, HCC-38, SCC-4, A549 & PC-3) at a density of lx 106 cells ml/1 in 100 mL of volume either subcutaneously or intravenously. Once Xenograft tumors reaches size of 50-100 mm in volume (mm3) the tumors will be treted with siRNA against NuMA. The preclinical studies comprises following groups of animals to test the efficacy of siRNA in causing tumor regression.
Each group consists of 10 Nude mouse including placebo treated controls-
1. Group A - Mice treated with NuMA siRNA.
2. Group B - Mice treated with NuMA and any other siRNA.
3. Group C - Mice treated with placebo.
SiRNA conjugated with cholesterol and may or may not be complexed with Polyethylene imine, Chemically modified siRNA, siRNA conjugated with cholesterol as well as linked to monoclonal antibodies will be injected directly at the site of tumor origin or by intravenous injection. Following two doses (at a concentration of 5mg of SIRNA) at an interval of 24 h each the tumor regression will be monitored by in-vivo imaging system and X-ray radio graphy. Depending on the tumor regression dosage will be enhanced or reduced. The longevity studies will be performed with periodic dosage application of siRNA to record any enhancement in life span of tumor mice.
The following examples are included to demonstrate preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the
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present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.
EXAMPLE 1; Design of 21,23, and 27mer SIRNA for modulation of NuMA
Identification of target sites:
Based on the literature of Henshel et.al (2004), Ui-Tei et. al.(2004), Sui et. al (2002),
Elbashir et. A; (2002), Duxburg and Whang (2004), Kim et. al (2005), Horunung et. al
(2005) & Judge et. al (2005). 21, 23 or 27 mers were designed. The following basic
requirment were met when designing si RNAs:
For designing 21mer siRNAs:
1.A11 SIRNA has GC content between 30-50 %
2.3'- of each siRNA has a over hang of dTdT
For designing 23mer siRNAs:
1.A11 siRNAs start at 5'- either with G/C
2.3'- of each siRNA strand has a over hang of dTdT
3.GC content of duplex is between 40 -50 %
For designing 27mer siRNAs:
1. GC content of duplex is between 40 -55 %.
2. Sense strand is of 25 nucleotides where as antisense strand are always 27 nucleotides resulting in over hand at 3'- of antisense strand.
3. Last 2 nucleotides of 3'-sense strand contains deoxy sugar instead of ribosugar back bone.
4. 5'- of sense strand contains overhang while 3'- is blunt ended.
TARGET SITES:
The sequence of NuMA was screened for accessible sites which could meet above mentioned criteria using various online available algorithms as well as manually. Based on these criteria the following sites were taken into consideration for designing siRNA to NuMA, a 236 kDa coiled-coil protein.
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Table 1: Target ORF sequences of NuMA for which siRNA were synthesized

SIRNA Gene ID Target Sequence in ORF Start site End site
1 (21mer) NM_006185 5 '-GAGGTACGATTCCGGAGAA-3' 20 40
2 (23mer) NM_006185 5'-GACCATGAGGACGGGCTA AAC-3' 578 598
3 (27mer) NM_006185 5'-CGAGAAGGATGCACAGATAGC CATG-3' 905 929
EXAMPLE 2: PREPARATION OF RNAi MOLECULES
The RNAi molecules were synthesized by chemicals means employing commercially available machinery from various companies such as Applied Biosystems, Beckmen etc. These could be synthesized by any of the following standard chemical methods or procured from Qiagen. The chemical methods were classified based on the type of protecting group incorporated at 2'-carbon position of the ribose sugar -
1. 21-t-butyldimethylsilyl (TBDMS)
2. 2'-0-triisopropylsilyloxymethyl (TOM)
3. 2'-acetoxyethoxy chemistry (ACE)
The cycle begins with the 3'-most nucleoside attached to solid support material or bead. The second nucleotide is coupled to the 5-hydroxyl of the first nucleoside. Capping prevents effectively the propagation of failed or short nucleosides. The internucleotidic phosphate bond is then oxidized to the final P (V) state. Finally, the 5'-protecting group on the new nucleotide is removed and the growing oligonucleotide is ready for addition of the next nucleotide. Once nucleic acid molecule reaches desired length it is further de-protected, cleaved from the solid support and analyzed for purity and yield.
Purification:
The SIRNAs were purified by PAGE, desalting, PAGE (Polyacrylamidegel electrophoresis) or by IE-HPLC (Ion Exchange - High Performance Liquid Chromatography) and quality of each RNA strand was analyzed MALDI-TOF and yields were determined by integrated spectrophotometer absorbance at 30nm. During quality control by MALDI-TOF a difference of 4 atomic mass units is the maximum allowed difference than the predicted difference. And also after obtaining comparable yields for
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each strand as determined by absorbance at 30 nm sense and antisense strands were annealed, vacuum lyophilized. At the time of experiment the lyophilized powders are suspended in RNA suspension buffer consisting of 100 mM KC1, 30 mM HEPES buffer (pH 7.5), and 1 mM MgCl2 . and heated for 1 min at 90 C and incubated at 37 C for 1 h to dissolve the lyophilized powder well. By following these manufacturing protocols the following siRNA having different 3'-end modifications and lengths were synthesized (Table2).
Table2: siRNA synthesized and their end modifications for NuMA.

siRNA Duplex sequence with overhangs Yield
1 SENSE ANTISENSE 5' r(GAG GUA CGA UUC CGG AGA A)dTdT3' 5'r(UUC UCC GGA AUC GUA CCU C)dTdT 3' 296mgmL"1
2 SENSE ANTISENSE 5' r(GAC CAU GAG GAC GGG CUA AAC)dTdT 3' 5' r(GUU UAG CCC GUC CUC AUG GUC)dTdT 3' 325mmL"
3 SENSEANTSENSE 5' r(CGA GAA GGA UGC ACA GAU AGC CA)dTdG 3' 5' r(CAU GGC UAU CUG UGC AUC CUU CUC GGU) 3' 297mgmL'1
4 SENSE ANTISENSE 5' r(GAG GAG GAA GCG CCC AAU AUC)dTdT 3' 5' r(GAU AUU GGG CGC UUC CUC CUC)dTdT 3' 325mgmL_1
siRNA 4 is scramble siRNA for NuMA. This means that these siRNA does not target any gene of interest. These were used as negative controls and always referred in experiments as mock treated.
EXAMPLE 3: TESTING OF EFFICACY
A) Oligonucleotide Transfections/siRNA transfections:
The cell lines A549, MCF-7, SKBR-3, A431 and HFF-2 cell lines were obtained from ATCC and cell lines were maintained at 70-80 % confluence with change of medium prior to 24 h of transfection in T-25 flasks (cat #156367, Nunc). Cell lines were used for all transfections of siRNA before reaching passage number ten unless otherwise mentioned. At the time of transfection cells were trypsinized and reseeded into either 24-well plate (cat # 143982, Nunc) or any other standard tissue culture disposable plastic
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ware at appropriate cell density. For most of the experiments unless otherwise stated all transfections were carried out in a 24-well plate with varying cell densities depending on cell lines used for a given experiment. Each well of 24-well plate is seeded with appropriate cell densities one hour prior to transfections with growth medium not exceeding 400mL and incubated in 37 C incubator with 5 % C02. To this medium diluted siRNA were added to a final concentration of lOnM (In 97mL of Opti-MEM I added 0.3mL of siRNA from a 20mM stock. To the diluted siRNA 3 mL of Hiperfect transfection agent (Qiagen) was added and mixed by vortexing before incubating at room temperature for 10 min. In all experiments mock or negative control were used where siRNA 4 was scrambled siRNAs against the gene NuMA. Henceforth mock denotes use of scrambled siRNA 4 pertaining to NuMA gene.
All siRNA and transfection mixes were performed as master mixes from which appropriate volumes were added to the wells seeded with cells. At the end of incubation siRNA-liposome complexes, were mixed thoroughly and added drop wise gently to each well. The wells of 24-well plate were mixed to uniformity and gently by rocking the plate back and forth as well as sideways. The plates were incubated till appropriate incubation times in 37 ° C CO2 incubators for further analysis of cells. Transfection efficiencies are obtained for each cell line by counting number of cells showing Cy3 labeled siRNA after 16h of transfection. Transfected cells were trypsinised and washed once in PBS and suspended in PBS. Cells were observed with inverted fluorescent microscope and counted number of fluorescent labeled cells and total number of cells in 15 different fields of microscope each field having a minimum of 3 cells each. The percentage of cells that were labeled with Cy3 siRNA was determined and thus transfection efficiency was derived. Of all the cell lines tested SKBR-3 & HFF-2 were showing 97 % of transfection efficiency where as MCF-7 was showing only 63 % efficiency as shown in the following Table 3.
Table3. Percent of transfection efficiencies as determined by Cy3 labeled SIRNA for different cell lines.

Cell line % of Transfection
A549 67
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MCF-7 63
SKBR-3 97
A431 89
HFF-2 97
A) Inhibition of NuMA by siRNA knockdown results in proliferation Inhibition of different cancer cell lines:
PC3 (Prostate cancer), A549 (Lung cancer), HeLa (Cervical cancer) and A431 (Epidermoid cancer) cell lines were transfected with different lengths of siRNA (siRNA 1,2 , 3 & 4). Twenty four hours after transfection, the cells were plated at a density of 2000 cells per well in a 96-well plate in triplicate for each. At the end of 72h, cells were incubated for three hours with the addition of Brdu following protocol of Brdu cell proliferation assay kit (cat # QIA58, Calbiochem). This enables incorporation of Brdu into DNA of actively proliferating cells. At the end of incubation time reaction was stopped by the addition of fixation reagent and permeabilized for anti Brdu antibody binding. The quantity of Brdu incorporated into actively proliferating cells was estimated from absorbance values at 450 nm with a reference filter at 540 nm. From the absorbance values the percent of cells that were in S-phase of cell cycle was obtained with reference to the mock treated cells. All the experiments were performed in triplicate and their mean percentages were obtained (Table 4).
Table4: Percent of cells that were S-phase of cell cycle as determined by Brdu incorporation after 72 h of siRNA transfection.

Cell line (Cancer) siRNA 1 siRNA2 siRNA3 siRNA4 (Mock)
PC3 (Prostate) 75 73 80 100
A549 (Lung) 88 N.D* N.D 100
HeLa (Cervical) 60 76 74 100
A431 (Epidermoid) 65 57 57 100
*N.D - Not determined
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Of different cancer cell lines tested siRNA 2 & 3 were showing maximum proliferation inhibition in case of A431 cells which was 43 % in comparison to that of mock treated cells as shown in Table 4. These results suggest that siRNA 1, 2 and 3 are potent and have ability to inhibit cell proliferation even when applied at similar concentrations and for time periods than other siRNA.
C) Real Time Quantitative PCR analysis
Transfection of cells with siRNA results in activation of RNAi pathway. During this mRNA complementary to the siRNA is degraded resulting in production of reduced quantities of protein being encoded by the gene for which siRNA are designed. By measuring the quantity of mRNA leftover after activation of RNAi pathway gives the potency of each siRNA being tested. This can be done by quantitative real time PCR which provides the relative quantity of mRNA in siRNA against NuMA vs mock siRNA transfected cells. The preparation of first strand cDNA for real time quantitative PCR analysis was carried-out using Qiagen Fast lane cell cDNA kit (Cat.no. 215011) with minor modifications. Briefly 20,000 cells were pelleted and washed once with buffer FCW. Cells were lysed for 15 min at room temperature using buffer FCP. Genomic DNA contamination was eliminated by the addition of gDNA wipeout buffer by incubating at 42.5 C for 30min. First strand cDNA was synthesized by the addition of Quantiscript reverse transcriptase at 42.5 C for 45 min followed by incubation at 95 C for 3 min. The first strand cDNA prepared was either used immediately for quantitative real time PCR or stored till further use at -20 C.
Here in this invention real time quantitative PCR is the preferred method as this can be accomplished following standard protocols and using commercially available machines such as ABI 7800, 7400 or 7900. In this study cells transfected with antisense RNAs were analyzed either at 24 h, 48 h, 72 h, and 96 h or after 10 days from the date of transfection. The first strand cDNA was prepared as described above from the experimental cells following protocol of Fast lane cell cDNA kit (Qiagen). First strand cDNA from antisense, mock and untreated samples were used as template and quantified the levels of mRNA by normalizing against the internal control (b-actin. The mRNA
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levels were knocked down to different folds depending on the cell line and siRNA as shown in the following Table 5.
Results obtained shows that siRNA 1 has showed 18 folds decrease in breast cancer cell line SKBR -3 while siRNA 3 was showing only 13 folds decrease in mRNA levels of NuMA after 72 h of transfection.
Table 5: Fold decrease in mRNA levels of NuMA over untreated controls after 72h of siRNA transfection for different cell lines
Cell line siRNA 1 siRNA 2 siRNA3
Non-small cell lung cancer ( A549) 4.31 2.86 4.35
Breast cancer Cell line (MCF-7) 2.36 1.02 13.17
Breast cancer cell line (SKBR-3) 18.18 1.66 5.04
Epidermoid cancer cell line (A431) N.D 3.83 3.23
Normal diploid fibroblasts (HHF-2) 1.24 1.21 1.06
D) Analysis of protein levels of NuMA: Since proteins are the functional units that are responsible to affect any biological function in the cell estimating the quantity of a protein will help us to understand the metabolic status of a cell and possible functions that may get impaired due to down regulation of the protein of our interest. Hence in this study cells transfected with siRNA 1, 2, 3 & 4 independently to perform western blot analysis. The cells (A549, MCF-7, A431, PC3 & HeLa) transfected with siRNA were subjected to total protein extraction after 72 h of transfection. The protein extraction was performed following the protocol of MPER (Cat # 78501, Peirce Biosciences) with the inclusion of protease inhibitor cocktail from Roche. Total protein was estimated by Bradford method and a 7.5% SDS PAGE was run with 50mg of protein per well at a voltage of 200 for 90 min on Biorads mini protean gel system.
The proteins resolved over the SDS-PAGE were subjected to western blot transfer at 110 V for 70 min. onto a pre wet nitrocellulose membrane along with pre-stained rainbow molecular weight markers (Cat # RPN756, Amersham Biosciences). The transfer of
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proteins by electro blotting was confirmed by Ponceau S staining (Sigma). The blot was incubated in blocking solution (5 % skim milk powder) for lh at room temperature on an orbital rocking platform. Before incubating with mouse anti NuMA anti body (Cat # Calbiochem NA 09L) and mouse alpha tubulin antibody (Cat # T6199, Sigma) as internal control the blot was washed over an orbital shaker for 5 min each with change of TBST (Tris buffered saline containing 0.1% Tween 20). The blot was incubated overnight at 4 C before washing with TBST as above to remove any non-specific bound primary antibodies. After washing with TBST the blots were incubated with secondary antibodies conjugated with alkaline phospohotase for two hours at room temperature over an orbital shaker. These secondary antibodies include goat anti mouse antibody conjugated with alkaline phosphotase (Cat # A3438, Sigma) to detect tubulin while goat anti rabbit antibody (Cat # 111-055-003, Jackson) to detect NuMA anti body. Blot was washed three times with TBST for 10 min each before being developed with BCIP/NBT substrate solution (Cat # B6404, Sigma).
Results obtained from western blots have identified 90 % protein being getting knockdown in case of NuMA siRNA treated cell lines (Figure 1A - 1C) in comparison with that of mock treated samples. This confirms our previous results where there was remarkable decrease in levels of mRNA in NuMA treated cancer cell lines by quantitative real time PCR analysis. Thus quantification of these protein after SIRNA transfections has revealed that indeed knockdown of their mRNAs has resulted in reduced levels of their proteins which are functional units of any given cell.
E) Determination of Proliferation & Metastatic Potential of Cancer cell lines treated with Antisense RNA
1) Biomarker analysis for cell proliferation efficiency: Prognosis of cancer is being understood by various biomarkers. These biomarkers include estimation of PCNA levels and Ki-67 antigen expression. Cell lines (A549, MCF-7, SKBR-3, A431, SCC-4 & HFF-
2) will be treated with siRNA. At the end of 72 h of incubation period cells will be subjected to analysis of Ki-67 antigen, PCNA (Proliferative Cell Nuclear Antigen) by
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ELISA and quantitative real time PCR to measure the levels of proliferation markers in comparison with that of the untreated cells.
2) Metabolic status of different cancer cells treated with siRNA against NuMA: siRNA 1,2 & 3 have successfully knocked down NuMA mRNA as well as protein levels relative to their mock treated samples to various extents as detailed in earlier sections. However to understand the affect of knockdown of expression levels of NuMA one needs to under stand the metabolic status of SIRNA treated cells. Metabolically active cells have ability to convert NAD or NADP to NADH or NADPH by dehydrogenase enzymes. The present invention has exploited this phenomenon of cells and measured the number of cells that were metabolically active which in turn determines the proliferation status of the cancer cells. For this purpose the cell titer 96 aqueous non-radioactive assay kit from Promega will be used. Because any sort of discrepancy in functional state of a cells get reflected on the metabolic status of the cell. After 24h of SIRNA transfection, cell lines (A549, MCF-7, SKBR-3, A431, HFF-2, HCC-38, PC3 & HeLa) will be trypsinized, and replated at a concentration of 1000 cells per well in a 96-well plate in having each well 200mL of growth medium. Cells were incubated for another 48h before measuring cell metabolic status following protocol of Promega Cell titer aqueous 96 kit. Briefly l00 mL of growth medium was withdrawn from 96 well plates and added 20mL of Cell titer aqueous 96 solution, incubated for 4h at 37°C in CO2 incubator. At the end of incubation plates were read at 490 run with the inclusion of reference filter 630 nm. Standard deviation of the absorbance value from triplicate will be determined and percentage of metabolically active cells or cell proliferation will be estimated with respective to the mock treated cells.
F) Colony forming assay:
The present invention studied the ability of SIRNA treated cells to initiate and develop a tumor when gets metastasized was assessed by employing colony forming assays. After 24 h of transfection of different cell lines (A549, PC-3 & HeLa) with siRNA cells were trypsinized and seeded at a concentration of 300 cells per each 6-well plate in triplicate along with mock treated cell lines with 3.0 mL of growth medium. After 10 days of
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incubation wells of 6-well plates were washed with PBS once and added 300 mL of 0.1 % crystal violet. The plates were incubated for 5 min before rinsing with PBS three times as above. The plates were reversed and grid lines were drawn to count the number of colonies in each well. Wells with a minimum of 10 colonies and each colony with a minimum of 50 cells were taken into consideration while counting under a microscope as shown in Figure 2 and Table 6.
Table 6: Percent of colony forming units (CFU) as determined by crystal violet staining after 10 days of siRNA transfection over mock treated controls.

Cell line SiRNA l siRNA2 siRNA3
A549 33.00 35.00 54.00
PC3 40.00 60.00 92.00
HeLa 24.00 03.00 27.00
The mean percent of colony forming units was derived from average of triplicates for each treatment. The percentage of colony forming efficiency/survivability was obtained with respective to the mock treated cells. Treatment of different cancer cell lines showed inhibition of colony forming ability from 8-97 % in cell lines as well as siRNA dependent manner as shown in Table 6.
G) Cancer cell invasibility assay:
Metastatic ability of tumor cells is one of major causes of morbidity in patients with cancer. Metastasis of tumor cells is a complex phenomenon where there is nt any satisfactory explanation so far. However studies have identified that cancer cells have ability to secrete matrix metalloproteinase's which aid in dissolution of various matrix proteins and thus are capable of either entering blood stream or spreading to near by organs. Metastatic ability of cancer cells is aided by linker cell-cell linker protein like E-cadeherin. Hence in the present study a wound or scratch assay will be performed in a mono layer of SIRNA transfected cells as well as mock treated cells. Cell lines will be plated in a collagen IV coated 24-well plates at a density of 3x 104 cells/per well
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immediately after transfection in triplicate. Twenty Four hours after plating in 24-well plate a scratch will be made using pipette tip across the well where a monolayer of cells have been established. The number of cells that have migrated into the scratched area will be counted with respective to different time intervals (8h and 24 h after scratch inflicted. The number of cells that have migrated into the scratch area with respective to the time of incubation will be determined from the microscope observations as well as image analysis with respective to the mock treated controls. Their standard deviation will be obtained from triplicate studies.
FDEffect of siRNA on cancer cell lines in inducing apoptosis:
The siRNA treated cell lines (SKBR-3, MCF-7, HCC-38, SCC-4, A549 & PC-3) will be subjected to apoptosis assay at different time intervals (include 24h, 48h and 72h) following protocol of Annexin V-PE apoptosis detection kit (Cat # CBA060, Cal biochem). The protocol involves suspension of siRNA treated cells at a density of lx 105 cells mL"1 in IX binding buffer followed by addition of 5mL of annexin V-PE and incubation at room temperature in dark for five minutes. At the end of incubation cell suspension will be transferred on to the slide glass and covered with coverslip. Number of cells that were showing phosphatidylserine - annexin complex over their cell membrane will be counted at 10 different microscopic fields and thus the percentage of cells undergoing apoptosis will be obtained from the total number of cells counted. All experiments for each transfection will be performed in triplicate and for each atleast 300 cells will be counted to derive statistical significance. Identification or quantification of cells that will be undergoing apoptosis will indicate the fate of metabolically inactive cells and possible cause of inhibition of cell proliferation.
I) Efficacy of siRNA transfection over distribution/localization NuMA by Confocal Immunofluorescence Microscopy:
siRNA transfections were carried out in triplicate as explained earlier (SKBR-3, MCF-7, HCC-38, SCC-4, A549 & PC-3) with a single modification where cover slips were laid at the bottom of 24-well plates of before adding cells to the well. At the end of 72 h of incubation spent medium will be withdrawn and washed three times with PBS to remove
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dead and unadhered cells from the cover slips. Using ice cold methanol cells will be fixed for 15 min to permeabilize their membranes. Anti NuMA anti body will be added to each well and will be incubated for 1 h at 37 C. At the end of incubation the cover slips will be washed three times for 10 min each over an orbital shaker in 24-well plates and will be incubated for additional lh with secondary antibody conjugated with FITC against anti NuMA antibody. At the end of incubation time cells will be washed as described elsewhere. Cover slips will be withdrawn from the 24-well plate and will be mounted in a mounting medium containing DAPI over a slide glass and sealed with nail polish. Slides will be observed over a spinning disc laser confocal microscope and images will be captured in z-series. The fluorescence intensity and localization patterns will be recorded. NuMA is expected to be localized within the nucleus during interphase and to the minus ends of microtubules during prop or metaphase. NuMA plays its function role during cell division process. The functional protein needs to get co-localized at appropriate quantities failure of which will lead to defectives in focusing of spindle poles. However the methods such as mRNA quantification and protein quantification do not yield the minimum thresh hold levels of NuMA required for a cell to perform its functions. This can be affectively determined by measuring quantity of NuMA that gets co-localized at the site of its action in comparison with that of mock treated control.
J) Efficacy of siRNA conjugated with cholesterol and Monoclonal antibodies:
Cholesterol conjugated NuMA siRNA will be linked to thiolated F(ab)2 fragments of monoclonal antibodies of anti EGFR1. Monoclonal antibody linked cholesterol conjugated siRNA (MABCC siRNA) will be transfected in different cancer cell lines as described above and their knock down efficiencies determined by real time quantitative PCR as well as western blotting.
K) Induction of Interferon response by siRNA transfection of various cancer cell lines:
NuMA siRNAs (cholesterol conjugated siRNA, CCPC, siRNA+PEI complexed, cholesterol conjugated siRNA+MAB linked) will be transfected into different cancer cell lines (SKBR-3, MCF-7, HCC-38, SCC-4, A549 & PC-3) as described elsewhere and
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incubated for 72 h. At the end of 72 h 200mL of supernatants will be incubated overnight at 4 C in poly-L-Lysine coated round bottom ELISA plate to coat interferon that had released due to siRNA mediated stimulation of interferon pathway. At the end of incubation time wells will be briefly washed with PBST (Phosphated Buffered Saline containing 0.1% Tween 20) to remove unbound antigen. ELISA plate wells incubated with 5 % skim milk powder for 30 min at room temperature. At the end of incubation wells will be washed 5 min each for three times with PBST before incubating with anti rabbit anti interferon alpha antibodies for lh at room temperature. At the end of incubation wells will be washed as before three times and incubated with HRP conjugated goat anti rabbit antibodies for additional lh at room temperature. At the end of incubation time HRP substrate TMB/H202 will be added. Absorbance values will be noted at 450 nm with a reference of 560 run. Based on the absorbance values with respective to the mock treated samples the interferon response obtained will be documented.
References
1. Merdes A and Cleveland D W (1998). The role of NuMA in the interphase nucleus. J Cell Sci. (Ill): 71-79.
2. Taiman P and Kallajoki M (2003). NuMA and nuclear lamins behave differently in Fas-mediated apoptosis. J Cell Sci. (116): 571-583.
3. Hsin-Ling Hsu and Ning-Hsing yeh (1996). Dynamic changes of NuMA during cell cycle and possible apperarence of a truncated form of NuMA during apoptosis.J Cell Sci. (109): 277-288.
4. Saredi A, Howard L and Compton D A (1997). Phosphorylation regulates the assembly of NuMA in a mammalian mitotic extract. J Cell Sci. (110) 1287-1297.
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All of the compositions and methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the compositions and/or methods and in the steps or in the sequence of steps of the methods described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents that are chemically or physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention.

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ABSTRACT
This invention relates to the use of short interfering nucleic acid molecules (SIRNA) in modulation of Nuclear Mitotic Apparatus Protein (NuMA) gene expression. The invention in particular related to the compounds, compositions and methods useful for modulation of expression and activity of other genes involved in pathways of NuMA gene. In particular the present invention provides small nucleic acid molecules of 21, 23 and 27 nucleotides in length which can be used in treating, preventing, inhibiting cancer and for any other disease, traits or conditions in which is related to or will respond to levels of NuMA in a cell or tissue. The present invention provides short interfering nucleic acid molecules, which can be used alone or in combination with other treatments or therapies.
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