Patent Application: US-201314443360-A

Abstract:
a method of treating cancer in a subject is provided . the method includes administering a splice modulating oligonucleotide to the subject in an amount effective to provide a therapeutic benefit to the subject . in the method , the oligonucleotide modifies splicing of a pre - mrna encoding a polypeptide , and either : reduces expression of the polypeptide if the polypeptide promotes cancer cell survival , proliferation , and / or metastasis , or promotes angiogenesis , or a combination thereof ; or increases expression of the polypeptide if the polypeptide inhibits cancer cell survival , proliferation and / or metastasis , or inhibits angiogenesis , or a combination thereof . similar methods of treating diseases involving prolactin or the prolactin receptor are also provided .

Description:
the following applications are incorporated by reference herein : provisional patent application no . 61 / 727 , 030 , filed on nov . 15 , 2012 . alternative splicing of pre - mrna can lead to the production of mature mrnas that code for different forms of a protein , including wild - type , constitutively active , or dominant negative forms , by either inclusion or exclusion of particular exons in mature mrnas . a variety of mammalian conditions and diseases , including certain types of cancer , arise and / or progress from mis - expression of wild - type , constitutively - active , or dominant negative forms of proteins ( e . g ., ligands , receptors , kinases , and transcription factors ). techniques and compositions useful for selectively modulating the production , from pre - mrna , of mature mrna or the translation thereof that codes for wild - type , constitutively active , and / or dominant negative forms of protein would have application in , e . g ., inhibiting incidence or progression of mammalian disease , such as certain types of cancer . certain embodiments of the invention provide methods of modulating the splicing of a pre - mrna present in a cell of a mammal . in some embodiments , such methods involve bringing the cell into contact with an effective amount of a splice modulating oligonucleotide ( smo ). in some embodiments , the smo is characterized by having a single - stranded sequence , at least eight nucleotides in length , that facilitates smo hybridization to a target sequence in the pre - mrna that comprises or overlaps at least one of an intron - exon junction , a 5 ′ splice site , a 3 ′ splice site , a branch site , an intronic splice enhancer , and an exonic splice enhancer . and smo hybridization to the target sequence inhibits splicing an exon , in the pre - mrna , into a mature mrna , produced from the pre - mrna , that codes for a protein , expression of which in the cell of the mammal inhibits an incidence or a progression of a cancer in the mammal . certain embodiments of the invention provide methods of modulating the splicing of a pre - mrna present in a cell of a mammal . in some embodiments , such methods involve bringing the cell into contact with an effective amount of a smo . in some embodiments , the smo is characterized by having a single - stranded sequence , at least eight nucleotides in length , that facilitates smo hybridization to a target sequence in the pre - mrna that comprises or overlaps at least one of an intronic splice silencer and an exonic splice silencer . and smo hybridization to the target sequence promotes splicing of an exon , which resides in the pre - mrna , into a first mature mrna that codes for a protein , expression of which in the cell inhibits an incidence or a progression of a cancer in the mammal . certain embodiments of the invention provide smos for modulating splicing of a pre - mrna present in a cell of a mammal . in such embodiments , smos can be characterized by having a single - stranded sequence , at least eight nucleotides in length , configured to facilitate smo hybridization to a target sequence in the pre - mrna that comprises or overlaps at least one of an intron - exon junction , a 5 ′ splice site , a 3 ′ splice site , a branch site , an intronic splice enhancer , and an exonic splice enhancer . and smo hybridization to the target sequence inhibits splicing an exon , in the pre - mrna , into a mature mrna , produced from the pre - mrna , that codes for a protein , expression of which in the cell of the mammal inhibits an incidence or a progression of a cancer in the mammal certain embodiments of the invention provide smos for modulating splicing of a pre - mrna present in a cell of a mammal . in such embodiments , smos can be characterized by having a single - stranded sequence , at least eight nucleotides in length , configured to facilitate smo hybridization to a target sequence in the pre - mrna that comprises or overlaps at least one of an intronic splice silencer and an exonic splice silencer . and smo hybridization to the target sequence promotes splicing of an exon , in the pre - mrna , into a first mature mrna that codes for a protein , expression of which in the cell inhibits an incidence or a progression of a cancer in the mammal . certain embodiments of the invention provide methods of decreasing an amount of a protein present in a cell of a mammal , comprising exposing the cell to an effective amount of a smo , in which the smo comprises a single - stranded sequence , at least eight nucleotides in length , configured to facilitate smo hybridization to one or more target nucleotide sequence ( s ) in the pre - mrna that comprise ( s ) or overlap ( s ) a first nucleotide sequence or second and third nucleotide sequences ; the first nucleotide sequence resides in an exon of the pre - mrna ; the second and third nucleotide sequences reside in two different exons of the pre - mrna ; smo hybridization to the one or more target sequence ( s ): ( i ) inhibits translation of a mature mrna produced from the pre - mrna , ( ii ) promotes degradation of the pre - mrna and thereby inhibits production of the mature mrna ; or ( iii ) both ( i ) and ( ii ); and the mature mrna codes for a protein , expression of which in the cell is associated with an incidence or a progression of a cancer in the mammal . certain embodiments of the invention provide a smo , for increasing a degradation rate of a pre - mrna present in a cell of a mammal , that comprises a single - stranded sequence , at least eight nucleotides in length , configured to facilitate smo hybridization to one or more target sequence ( s ) in the pre - mrna that comprise ( s ) or overlap ( s ) a first nucleotide sequence or second and third nucleotide sequences , in which the first nucleotide sequence resides in an exon of the pre - mrna ; the second and third nucleotide sequences reside in two different exons of the pre - mrna ; and smo hybridization to the one or more target sequence ( s ) promotes degradation of the pre - mrna and inhibits production of a mature mrna from the pre - mrna that codes for a protein , expression of which in the cell is associated with an incidence or a progression of a cancer in the mammal . as used herein , the term , “ splice modulating oligonucleotide ” refers to an oligonucleotide , 7 - 100 nucleotides in lengths , characterized by having a nucleotide sequence that is complementary to a target nucleotide sequence in a pre - mrna present in a mammalian cell and renders the oligonucleotide operative , when contacted with the mammalian cell in therapeutically effective amounts , to modulate ( e . g ., up - regulate or down - regulate ) an amount of a mrna splice variant produced from the pre - mrna in the cell or to increase a degradation rate of the pre - mrna in the cell and / or decrease a translation rate of a mrna . in embodiments in which smos modulate splicing of a pre - mrna , such smos may comprise a nucleotide sequence complementary to a target sequence in the pre - mrna that comprises or overlaps : an intron - exon junction ; an intronic splice silencer ; and / or an intronic splice enhancer . in embodiments in which smos increase a pre - mrna degradation rate or decrease a mrna translation rate , such smos may comprise a nucleotide sequence complementary to one or more target sequence ( s ) in the pre - mrna that comprise ( s ) or overlap ( s ) a nucleotide sequence from a single exon or two or more nucleotide sequences from two or more exons . the nucleotides of smos can be modified or unmodified dna nucleotides , rna nucleotides , or combinations thereof . in some embodiments , an smo composition comprises at least one of a dna oligonucleotide , an rna oligonucleotide , a morpholino , or a “ vivo morpholino ”. in particular , the oligonucleotide can include modified oligonucleotide backbones such as , but not limited to , phosphorothioates , chiral phosphorothioates , phosphorodithioates , phosphotriesters , aminoalkylphosphotriesters , methyl and other alkyl phosphonates 3 ′- alkylene phosphonates and chiral phosphonates ), phosphinates , phosphoramidates 3 ′- amino phosphoramidate and aminoalkylphosphoramidates ), thionophosphoramidates , thionoalkylphosphonates , thionoalkyl phosphotriesters , and boranophosphates having normal 3 ′- 5 ′ linkages , as well as 2 ′- 5 ′ linked analogs of these , and those having inverted polarity wherein the adjacent pairs of nucleoside units are linked 3 ′- 5 ′ to 5 ′- 3 ′ or 2 ′- 5 ′ to various salts , mixed salts and free acid forms are also included . references that teach the preparation of such modified backbone oligonucleotides are provided , for example , in u . s . pat . nos . 4 , 469 , 863 and 5 , 750 , 666 , all incorporated by reference herein [ 29 , 30 ]. the design and synthesis of antisense oligonucleotides is well known in the art [ 31 ]. computer programs for the design of antisense oligonucleotide sequences are also available [ 32 ]. in some embodiments , at least seven , eight , 10 , 15 , 20 , 25 , 30 , 40 , or 50 contiguous nucleotides in a smo are complementary to the target nucleotide sequence in the selected pre - mrna that comprises , or overlaps , a sequence motif selected from the group consisting of a 5 ′ splice site , a 3 ′ splice site , a branch site , an intronic splice enhancer , an intronic splice silencer , an exonic splice enhancer , and an exonic splice silencer . in some embodiments , at 8 - 12 , 8 - 15 , 8 - 20 , 10 - 25 , 15 - 30 , 20 - 40 , or 30 - 50 contiguous nucleotides in a smo are complementary to the target nucleotide sequence in the selected pre - mrna that comprises , or overlaps , a sequence motif selected from the group consisting of a 5 ′ splice site , a 3 ′ splice site , a branch site , an intronic splice enhancer , an intronic splice silencer , an exonic splice enhancer , and an exonic splice silencer . in some embodiments , an smo is 7 - 50 nucleotides in length , 7 - 25 nucleotides in length , 7 - 15 nucleotides in length , 8 - 12 nucleotides in length , 8 - 17 nucleotides in length , 8 - 25 nucleotides in length , 8 - 50 nucleotides in length , 9 - 12 nucleotides in length , 9 - 25 nucleotides in length , 10 - 17 nucleotides in length , 10 - 25 nucleotides in length , 20 - 30 nucleotides in length , or 25 nucleotides in length . in embodiments where an smo comprises at least 75 % rna nucleotides , the smo can be less than 21 nucleotides in length . in some embodiments , an smo is entirely single - stranded . in some embodiments , an smo is partially single - stranded . in some embodiments , an smo is capable of reversibly or permanently forming stem loop structures , hairpin structures , and the like . as used herein , the term “ complementary ” is used to in reference oligonucleotide sequences capable of base pairing to form a double - stranded nucleic acid molecule , under conditions selected from physiologic , high stringency , medium stringency , and low stringency . as used herein , the terms “ hybridize ” and “ hybridization ” refer to single stranded nucleic acid molecules base pairing with one another to form a double - stranded helix molecule , the nucleic acid molecules characterized by being complementary , either exactly or to a high percentage , such as 70 %, 75 %, 80 %, 85 %, 90 %, 95 %, 96 %, 97 %, 98 %, 99 %, and 100 %. hybridization stringency is a function of probe length , probe composition ( g + c content ), salt concentration , organic solvent concentration , and temperature of hybridization or wash conditions . stringency is typically compared by the parameter t m , which is the temperature at which 50 % of the complementary molecules in the hybridization are hybridized , in terms of a temperature differential from t m . high stringency conditions are those providing a condition of t m - 5 ° c . to t m - 10 ° c . medium or moderate stringency conditions are those providing t m - 20 ° c . to t m - 29 ° c . low stringency conditions are those providing a condition of t m - 40 ° c . to t m - 48 ° c . the relationship of hybridization conditions to tm ( in ° c .) is expressed in the mathematical equations : t m = 81 . 5 − 16 . 6 ( log 10 [ na +])+ 0 . 41 (% g + c )−( 600 / n ) ( equ . 1 ), where n is the length of the probe . this equation works well for probes 14 to 70 nucleotides in length that are identical to the target sequence . the equation below for t m of dna - dna hybrids is useful for probes in the range of 50 to greater than 500 nucleotides , and for conditions that include an organic solvent ( formamide ). tm = 81 . 5 + 16 . 6 log ([ na +]/( 1 + 0 . 7 [ na +])+ 0 . 41 (% g + c )− 500 / l 0 . 63 (% formamide ) ( equ . 2 ), where l is the length of the probe in the hybrid [ 14 ]. the t m of equation ( 2 ) is affected by the nature of the hybrid ; for dna - rna hybrids t m is 10 - 15 ° c . higher than calculated , for rna - rna hybrids tm is 20 - 25 ° c . higher . because the tm decreases about 1 ° c . for each 1 % decrease in homology when a long probe is used ( see , bonner et al ., j . mol . biol . 81 : 123 ( 1973 )), stringency conditions can be adjusted to favor detection of identical genes or related family members . equation ( 2 ) is derived assuming equilibrium and therefore , hybridizations according to the present invention are most preferably performed under conditions of probe excess and for sufficient time to achieve equilibrium . the time required to reach equilibrium can be shortened by inclusion of a hybridization accelerator such as dextran sulfate or another high volume polymer in the hybridization buffer . stringency can be controlled during the hybridization reaction or after hybridization has occurred by altering the salt and temperature conditions of the wash solutions used . the formulas shown above are equally valid when used to compute the stringency of a wash solution . preferred wash solution stringencies lie within the ranges stated above ; high stringency is 5 - 8 ° c . below tm , medium or moderate stringency is 26 - 29 ° c . below tm and low stringency is 45 - 48 ° c . below tm . as used herein , the term “ intron - exon junction ” refers to sequences of nucleotides in a pre - mrna that reside at the junction of an intron and an exon in the pre - mrna . an intron - exon junction may extend up to 4 nucleotides , 5 nucleotides , 6 nucleotides , 7 nucleotides , 8 nucleotides , 9 nucleotides , 10 nucleotides , 15 nucleotides , 20 nucleotides , 25 nucleotides , 30 nucleotides , 35 nucleotides , 40 nucleotides , 45 nucleotides , and 50 nucleotides in both 5 ‘- ward and 3 ’- ward directions from the intron - exon border . methods for introducing smos of the invention into the body of a mammal ( e . g ., a human or mouse ) in need thereof include any route of administration effective to enable the smos to perform their intended function , non - limiting examples of which are orally , intranasally , parenterally ( intravenously , intramuscularly , intraperitoneally , or subcutaneously ), intratumorally ( including by one or more intratumoral injections ), rectally , via an implant , an implanted pump , and topically . amounts of smos of the invention administered to a mammal ( e . g ., a human ) include any amount effective to enable the smos to perform their intended function . the amount of active compound to be administered to the patient varies according to the weight of the patient , the mode of administration , the indication and the severity of the disease . the clinician will be well suited to make such decisions based on the efficacy and toxicity ( if any ) of the therapeutic formulations . implantable drug delivery devices , also referred to as implanted pumps or drug delivery pumps , can be used to administer therapeutic agents to various locations within the body . some drug delivery devices have variable volumetric flow rates , adjustable through an external programmer device . other implantable drug delivery devices have fixed volumetric flow rates , but can be activated and deactivated externally . still other drug delivery devices have fixed volumetric flow rates and are not adapted to be controlled from outside of the body . for example , implantable , electromechanical drug delivery devices can include , within a fluid impermeable and sealed casing , a watch - type drive mechanism that drives a circular wheel . microchip based drug delivery devices are also available , and can include a plurality of drug reservoirs etched into a substrate to produce , for example , a single microchip . embodiments including a pharmaceutical composition will typically contain a pharmaceutically acceptable carrier . depending on the intended mode of administration , the pharmaceutical composition may be in the form of solid , semi - solid or liquid dosage forms , such as , for example , tablets , suppositories , pills , capsules , powders , liquids , suspensions , ointments or lotions . a carrier can be any and all solvents , dispersion media , vehicles , coatings , diluents , antibacterial and antifungal agents , isotonic and absorption delaying agents , buffers , carrier solutions , suspensions , colloids , and the like . the use of such media and agents for pharmaceutical active substances is well known in the art . except insofar as any conventional media or agent is incompatible with the active ingredient , its use in the therapeutic compositions is contemplated . supplementary active ingredients can also be incorporated into the compositions . preferably in unit dosage form suitable for single administration of a precise dosage . one skilled in the art may formulate the compound in an appropriate manner , and in accordance with accepted practices , such as those disclosed in remington &# 39 ; s pharmaceutical sciences , gennaro , ed ., mack publishing co ., easton , pa . 1990 . to determine the effects on splicing in vivo , pumps can be implanted into mice that administered thereto 1 μl / hr of one of the following four solutions . a solution that contains 100 μm of a smo that targets mouse prlr — long form ( high ), a solution that contains 10 μm of the smo ( mid ), a solution that contained 1 μm of the smo ( low ), or a control pbs solution that contains no smo ( pbs ). five days after pump implantation , expression levels of mouse prlr — long form mrna , mouse prlr — short form s2 mrna , and gapdh mrna can be assayed , for example , in liver tissue , ovary and mammary gland tissue . expression levels of mouse prlr — long form mrna and mouse prlr — short form s2 mrna can be normalized to gapdh in corresponding tissue samples , and reported in arbitrary units on the y axes of graphs . in other assays , a solution can contain , for example , 100 μm of a smo that targets mouse prlr — long form , a solution that contains 100 μm control oligomer , or a control pbs solution that contains no smo ( pbs ). five days after pump implantation , expression levels of mouse prlr — long form mrna , mouse prlr — short form s2 mrna , and gapdh mrna can be assayed in liver tissue , ovary tissue or mammary gland tissue . expression levels of mouse prlr — long form mrna and mouse prlr — short form s2 mrna can be normalized to gapdh in corresponding tissue samples , and reported in arbitrary units on the y axes of graphs . the terms “ treatment ” and “ to treat ” refer to providing therapeutic benefit including anything that promotes or enhances the well - being of the subject with respect to the medical treatment of his condition . a list of nonexhaustive examples of this therapeutic benefit includes extension of the subject &# 39 ; s life by any period of time , a decrease in pain to the subject that can be attributed to the subject &# 39 ; s condition , a decrease in the severity of the disease , an increase in the therapeutic effect of a therapeutic agent , an improvement in the prognosis of the condition or disease , a decrease in the amount or frequency of administration of a therapeutic agent , an alteration in the treatment regimen of the subject that reduces invasiveness of treatment , a decrease in the number of normal ( non - cancerous ) cells undergoing apoptosis so as to reduce injury to a tissue , an increase in the number of cells undergoing apoptosis when hyperproliferation is at least partially responsible for a condition or disease , and a decrease in the severity or frequency of side effects from a therapeutic agent . with respect to the treatment of cancer , therapeutic benefits also include a decrease or delay in the neoplastic development of the disease , decrease in hyperproliferation , reduction in tumor growth , delay or reduction of metastases , reduction of cancer stem cell number , and reduction in cancer cell or tumor cell proliferation rate . pertinent to certain embodiments of the invention , a wild - type form of human estrogen receptor α ( erα ) protein is encoded by mrna comprised of exons 1 - 8 of erα pre - mrna ( fig1 a ) spliced together . and splice variants produced from the erα pre - mrna code for constitutively active and dominant negative forms of erα protein . a δ5erα splice variant : comprises exons 1 - 4 and 6 - 8 of the erα pre - mrna ( fig1 a ) spliced together , and codes for a constitutively active form of erα protein . a δ7erα splice variant : comprises exons 1 - 6 and 8 of the erα pre - mrna ( fig1 a ) spliced together ; and codes for another dominant negative form of erα protein . in some embodiments of the invention , δ5erα mrna can be advantageously modulated in a mammalian ( e . g ., a human or mouse ) cell using smos . in some embodiments of the invention , δ7erα mrna can be advantageously modulated in a mammalian ( e . g ., a human or mouse ) cell using smos . pertinent to certain embodiments of the invention , a wild - type form of human estrogen receptor β ( erβ ) protein is encoded by mrna comprised of exons 1 - 9 of erβ pre - mrna ( fig2 a ) spliced together . and a splice variant produced from erβ pre - mrna codes for a dominant negative form of erβ protein , δ5erβ , which comprises exons 1 - 4 and 6 - 9 of the erα pre - mrna ( fig2 a ) spliced together . in some embodiments of the invention , δ5erβ mrna can be advantageously modulated in a mammalian ( e . g ., a human or mouse ) cell using smos . pertinent to certain embodiments of the invention , a wild - type form of human signal transducer and activator of transcription 5a ( stat5a ) protein is encoded by mrna comprised of exons 1 - 20 of stat5a pre - mrna ( fig3 a ) spliced together . a δ5stat5a splice variant that comprises exons 1 - 4 and 6 - 20 is expressed in certain cancers . in some embodiments of the invention , the expression of δ5stat5a splice variant mrna can be advantageously modulated in a mammalian ( e . g ., a mouse or human ) cell using smos . pertinent to certain embodiments of the invention , a wild - type form of human growth hormone receptor ( ghr ) protein is encoded by mrna comprised of exons 1 - 10 of ghr pre - mrna ( fig4 a ) spliced together . and a pδ9ghr splice variant : ( i ) comprises exons 1 - 8 and part of exon 9 of the ghr pre - mrna ( fig4 a ) spliced together , and ( ii ) codes for a dominant negative form of ghr protein . in some embodiments of the invention , pδ9ghr mrna can be advantageously modulated in a mammalian ( e . g ., a mouse or human ) cell using smos . pertinent to certain embodiments of the invention , a wild - type form of erbb4 protein is encoded by mrna comprised of exons 1 - 28 of erbb4 pre - mrna ( fig5 a ) spliced together . and a δ26erbb4 splice variant : ( i ) comprises exons 1 - 25 and 27 - 28 of erbb4 pre - mrna ( fig5 a ) spliced together ; and ( ii ) codes for a dominant negative form of erbb4 protein . in some embodiments of the invention , δ26erbb4 mrna can be advantageously modulated in a mammalian ( e . g ., a mouse or human ) cell using smos . pertinent to certain embodiments of the invention , a wild - type form of a human insulin receptor ( ir ) protein is encoded by mrna comprised of exons 1 - 22 of ir pre - mrna ( fig6 a ) spliced together . and a δ11ir splice variant : ( i ) comprises exons 1 - 10 and 12 - 22 of ir pre - mrna ( fig6 a ) spliced together ; and ( ii ) codes for a form of ir protein found in cancer . in some embodiments of the invention , the expression δ11ir mrna can be advantageously modulated in a mammalian ( e . g ., a mouse or human ) cell using smos in a variety of contexts . pertinent to certain embodiments of the invention , a wild - type form of her2 protein is encoded by mrna comprised of exons 1 - 27 of her2 pre - mrna ( fig7 a ) spliced together . and a δ16her2 splice variant : comprises exons 1 - 15 and 17 - 27 of her2 pre - mrna ( fig7 a ) spliced together , and codes for a constitutively active form of her2 protein . in some embodiments of the invention , the expression of δ16her2 mrna can be advantageously down - regulated in a mammalian ( e . g ., a mouse or human ) cell using smos . for example , overexpression of wild - type her2 is found in aggressive breast and gastric cancers characterized by poor prognosis for time to progression and survival . the δ16her2 mrna codes for a protein that has a small deletion in the extracellular domain of wild - type her2 protein . the deletion removes some extracellular cysteine residues , leaving some remaining extracellular cysteine residues unpaired and available for intermolecular disulfide bonding ( i . e ., putative formation of a constitutively active , δ16her2 homodimer ). δ16her2 transcripts have been detected in a majority of breast tumors and reported to comprise 4 - 9 % of total her2 transcripts . the δ16her2 protein has been associated with trastuzumab ( herceptin ®) resistance . and oncogenic transformation associated with wild - type her2 overexpression may very well be a function of the associated increase in absolute levels of the δ16her2 protein to a critical threshold required for constitutive activation of the her2 pathway by δ16her2 homodimer activity . pertinent to certain embodiments of the invention , a long form of a human prolactin receptor ( alternatively called “ prlr1f ” or “ lf prlr ”) protein is encoded by mrna comprised of exons 1 - 10 of prlr pre - mrna ( fig8 a ) spliced together . and a short form human prlr splice variant comprises exons 3 - 9 and 11 of human prolactin receptor short form 1 b pre - mrna ( fig9 a ) spliced together , and codes for a dominant negative form of human prlr ( see refseq database , accession number nm — 000949 , incorporated by reference herein ). in some embodiments of the invention , prlrlf mrna can be advantageously modulated in a mammalian ( e . g ., a mouse or human ) cell using smos . antimaia is the name we have given to our splice - modulating oligomer that inhibits expression of the lf prolactin receptor and by so doing increases the relative expression of the short form prolactin receptors . human and mouse forms of antimaia have the following sequences : gcccttctattaaaacacagacaca ( human ); gcccttctattgaaacacagataca ( mouse ). the oligonucleotide is derivatized by adding morpholino groups to enhance half life and by octaguanidine groups to effect cell penetration . the present invention may be better understood by referring to the accompanying examples , which are intended for illustration purposes only and should not in any sense be construed as limiting the scope of the invention . pumps containing antimaia were implanted into mice , and antimaia was used at 1 , 10 and 100 pmoles / hour / mouse for 5 days . as shown in fig9 a - 9c , antimaia reduced production of the long form of the prolactin receptor in mammary gland , liver and ovary . a shown in fig1 a - 10d , antimaia had no effect on the short prolactin receptor forms ( called s1 - 3 in mouse ) or a related receptor , the growth hormone receptor ( ghr ). potential cancer drugs are typically tested by analyzing their ability to shrink a primary tumor . while shrinkage of primary tumors can be crucial for inoperable tumors , the vast majority of primary tumors are surgically excised . moreover , patient death results from cells that escaped the primary tumor , becoming metastases . also , sometimes cancer recurs years after an apparent cure , suggesting that some cancer stem cells were dormant and then re - activated . to determine the ability of smos to inhibit metastatic spread and kill cancer stem cells , two highly aggressive and metastatic models were used . in the first model , mouse breast cancer cells are used in normal mice . this allows an assessment of the efficacy of treatment in a situation where there is a normal hormone and immune environment , both of which would be present in a patient . a solution of the antimaia oligonucleotide was placed in a pump that delivered a constant dose , and mice were checked for metastases at various time points . primary tumors were formed in the breast , and natural metastases emanating from the primary tumor were followed . 4t1 cells are a syngeneic balb / c mouse breast cancer cell line . after suspension in matrigel , cells were injected into the mammary fat pads . numbers of cells injected and time after injection before analysis varied with the experiment . metastatic spread was quantified on the basis of tumor cell resistance to 6 - thioguanine normal cells are not resistant . lung tissue was dissociated and the cells were plated in medium containing 6 - thioguanine only the tumor cells survive and form colonies . the number of colonies produced by cells derived from control mice and mice treated with antimaia can then be counted . results showed that antimaia markedly reduced metastases to the lung at a time point ( post 40 days ) when metastatic spread was so bad that it killed two of the animals in the control group ( fig1 ). antimaia also decreased metastases in the liver and increased the immune response to tumor cells in the liver . single cell suspensions were made from dissected livers and 1 - 2 × 10 6 cells in rpmi - 1640 ( plus 10 % fbs + penstrep ) were incubated with 4t1 tumor cell lysate in the presence of purified mouse anti - cd28 antibody for an hour at 37 ° c ./ 5 % co 2 humidified incubator , after which golgi stop ( monensin ) was added and incubated for 5 hr . cells were then washed and first stained with fluorochrome - conjugated rat anti - mouse cd4 antibody for t helper cells . after washing and permeabilization with fixation and permeabilization buffer , cells were stained for intracellular ifn - g and il - 2 with appropriate fluorochrome - conjugated rat anti - mouse ifn - g and anti - il - 2 antibodies . after staining , paraformaldehyde fixed cells were acquired in a bd facsaria flow cytometer and analyzed by flowjo software . livers were also fixed in paraformaldehyde and stained with hematoxylin and eosin for routine histopathological analysis . as shown in fig1 a - 12 b , liver sections from control animals displayed metastases and poor tissue architecture ( 12 a ), while liver sections from antimaia treated animals showed immune cell infiltration without signs of inflammatory destruction ( 12 b ). antimaia also increased the number of immune cells that are capable of killing tumor cells in the liver . single cell suspensions were made from dissected livers and 1 - 2 × 10 6 cells in rpmi - 1640 ( plus 10 % fbs + penstrep ) were incubated with 4t1 tumor cell lysate in presence of purified mouse anti - cd28 antibody for an hour at 37 ° c ./ 5 % co 2 humidified incubator , after which golgi stop monensin was added and incubated for 5 hr . cells were then washed and first stained with fluorochrome conjugated rat anti - mouse cd8 antibody for effector cytotoxic t cells . after washing and permeabilization with fixation and permeabilization buffer , cells were stained for intracellular ifn - g and il - 2 with appropriate fluorochrome conjugated rat anti - mouse ifn - g and anti - il - 2 antibodies . after staining , paraformaldehyde fixed cells were acquired in bd facsaria flow cytometer and analyzed by flowjo software . as shown in table 1 , antimaia treatment increased the percentage of il - 2 positive cd8 + t cells in liver . antimaia also reduced the number of cancer stem cells . under experimental conditions , only stem cells survive and give rise to colonies greater than 50 μm in size . 4000 4t1 cells were cultured in serum free dmem / f12 medium supplemented with 2 % b27 and 20 ng / ml hegf and plated in ultra - low attachment plates with either 1 um control smo or antimaia or without treatment for 7 days . after 7 days , numbers of formed mammaosphperes bigger than 50 uμm were counted . results are shown in fig1 . a second assay for stem cells gave similar results . 5 × 10 5 4t1 cells were seeded on plates and treated with 800 pmoles of control smo or antimaia per 8 hours or were without any treatment . medium was changed every 8 hours for the entire 15 - day experiment . the 15 day treatment was begun on day1 , the 9 day treatment started on day7 , the 5 day treatment started on day 11 and the 2 day treatment started on day14 so that all samples were collected at the same time for flow cytometric analysis . to analyze the population of cancer stem cells , cells were stained with antibodies to lin , cd24 , cd29 and aldh and the stem cells marked as lin −/ cd24 +/ cd29 +/ aldh + were gated . results showed longer treatment with antimaia decreased the population of cancer stem cells . results from the 15 day treatment are shown in fig1 . in the second model , human breast cancer cells were used in a mouse that would not reject the cancer because it has an impaired immune system . one million human bt474 cells were injected orthotopically into the mouse fat pad . the mice were treated with control smo or human antimaia for 25 days ( 100 pmoles / h / mouse ) after which time the lungs were fixed for histopathological analysis of hematoxylin and eosin - stained sections . morphometric analysis quantified the area of sections occupied by metastatic densities . as shown in fig1 , lungs of human - antimaia treated animals showed markedly reduced metastatis . antimaia also increased apoptosis and necrosis in the primary tumors . three hours before sacrifice , tumor - bearing mice were injected with edu ( 5 - ethynyl - 2 ′- deoxyuridine ) that incorporates into dna of dividing cells . tumors formed by 4t1 cells in the mammary fat pad were fixed and processed for histopathology . sections were stained for the presence of edu and the percentage of positive dividing cells quantified by morphometric analysis . sections were also stained to detect apoptosis using reagents that recognize cut ends of dna produced by apoptosis . results are shown in fig1 a - 16b . analysis of liver enzymes in the blood , differential blood counts , and histological analysis of multiple tissues indicated that antimaia showed no evidence of toxicity . liver enzymes were analyzed using commercial clinical kits scaled down to accommodate sample size . differential blood counts were performed on blood smears taken at the time of sacrifice . histopathological analysis of routinely fixed and prepared tissues was performed on lung , heart , liver , and gastrointestinal tract / results for liver enzymes are shown in fig1 a - 17b ( alt , alanine transaminase ; ast , aspartate amino transferase ). antimaia is predicted ( based on charge considerations ) not to cross the blood brain barrier and so would not be expected to affect cognition . in addition , antimaia : has a major inhibitory effect on metastatic spread to the lungs and liver in both models ; kills tumor cells by inducing apoptosis ; in the normal mouse model , also stimulates an immune response to tumor cells ; increased central primary tumor death ; reduced the number of tumor stem cells , suggesting the possibility that it could eradicate all tumor cells ; showed no toxic effects within the experimental time frames used . the inventors hypothesized that an observed reduction in tumor size and increased central death of tumors in the antimaia - treated group had an immunologic component and resulted , at least in part , from anti - tumor immunity by t cells . to test the hypothesis , isolated cells were isolated from liver ( an organ with different grades of metastases ) of the two groups of mice , and in ex vivo experiments , incubated the cells with lysed tumor cell ( 4t1 ) antigen ( ag ) s , and detected any intracellular production of ifn - γ or il - 2 by t cells at the single cell level by multicolor flow cytometry . cd4 + t cells in three out of 6 in the antimaia group ( 3 / 6 ), but none ( 0 / 6 ) in the control group secreted ifn - γ in response to the tumor ag with a percentage (%) range varying between ( 0 . 42 %- 1 . 02 %, mean 0 . 69 +/− sd 0 . 3 )— shown in fig1 . similarly , four out of 6 ( 4 / 6 ) in the antimaia group secreted il - 2 in response to tumor ag (% range : 0 . 22 %- 0 . 94 %, mean 0 . 61 +/− sd 0 . 3 ) as compared to 1 in 6 ( 1 / 6 ) in the control group . similarly , antigen - specific cd8 + il - 2 + cells were present in three out of 6 ( 3 / 6 ) mice in the antimaia group (% range : 0 . 89 %- 1 . 1 %, mean 0 . 98 +/− sd 0 . 11 ) as compared to 1 in 6 mice ( 1 / 6 ) in the control group . this analysis was conducted in a blinded fashion and then combined with the effect on tumor growth . at this point , failure in the delivery system for antimaia in two non - responding animals is suspected , but not proved . it is currently concluded that half of the antimaia - 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