NZ711827B2 - MicroRNA-BASED APPROACH TO TREATING MALIGNANT PLEURAL MESOTHELIOMA - Google Patents

Abstract

The invention relates to microRNA mimics, corresponding to the miR-15/107 family, and to methodology for using microRNA mimics to treat malignant pleural mesothelioma (MPM) by restoring regulation of the expression of target genes of the miR-15/107 family in MPM tumor cells.

Description

[Annotation] KJM None set by KJM [Annotation] KJM MigrationNone set by KJM [Annotation] KJM ed set by KJM [Annotation] KJM None set by KJM [Annotation] KJM MigrationNone set by KJM [Annotation] KJM Unmarked set by KJM NA-BASED APPROACH TO NG MALIGNANT PLEURALMESOTHELIOMA CROSS-REFERENCE TO RELATED ATIONS This ation claims the benefit under 35 U.S.C. § 120 to U.S. application serial No. 13/801,010, filed March 13, 2013, the contents of which are incorporated here by nce in their entirety.

OUND OF THE INVENTION Technical Field The invention relates generally to the field of molecular biology and cancer. More specifically, the invention relates to microRNA mimics corresponding to the miR-15/107 family and related methods of using microRNA mimics to treat malignant pleural mesothelioma (MPM) by restoring the regulation of expression of target genes of the miR- /107 family in MPM tumor cells.

Background Malignant pleural mesothelioma is an almost invariably fatal cancer for which few ents are available. New therapies are urgently , and dysregulated microRNA expression provides a source of novel therapeutic targets.

NAs are transcribed by RNA polymerase II (pol II) or RNA polymerase III (pol III). See Qi et al. (2006) Cellular & Molecular Immunology, Vol. 3: 411-19. They arise from initial transcripts, termed primary microRNA transcripts (pri-microRNAs), which generally are several thousand bases long. Pri-microRNAs are processed in the nucleus by the RNase Drosha into about 70-to about 100-nucleotide hairpin-shaped precursors (premicroRNAs ). Following transport, to the cytoplasm, the hairpin pre-microRNA is further processed by Dicer to produce a double-stranded mature NA. The mature microRNA strand is then incorporated into the RNA-induced silencing complex (RISC), where it associates with its target mRNAs by base-pair complementarity. In the relatively rare cases in which a microRNA base pairs perfectly with an mRNA target, it promotes mRNA [Annotation] KJM None set by KJM [Annotation] KJM ionNone set by KJM [Annotation] KJM Unmarked set by KJM [Annotation] KJM None set by KJM [Annotation] KJM MigrationNone set by KJM [Annotation] KJM ed set by KJM degradation. More commonly, microRNAs form imperfect heterduplexes with target mRNAs, either ing mRNA stability or inhibiting mRNA translation.

Multiple studies have d mRNA gene expression in MPM to identify potential s, and more recently, NA expression profiles have been generated, initially for diagnostic purposes using s derived from normal and tumor cell lines, MPM tumors and pooled normal pericardium, or MPM and lung cancer. They also have been generated for prognostic purposes, i.e., within MPM tumors of different classification. See, e.g., Busacca et al., Am. J. Respir. Cell. Mol. Biol. 42: 312—19 (2010). However, none have made an extensive comparison between MPM tumors and normal pleural tissue.

Further, while relatively easy to use in vitro, microRNA mimics typically suffer, in terms of in viva eff1cacy, due to two problems: (1) poor activity (including low RISC incorporation and off-target effects) and (2) inefficient delivery (related to stability and specific/selective distribution to the site of action).

As mentioned, multiple studies have profiled gene expression in MPM. This has been with the aim of understanding the disease process as well as to identify potential targets.

These studies have characterized a general upregulation of lic and cell cycle genes in MPM, with onal changes in apoptotic genes associated with an altered tic response linked to resistance to chemotherapy. To date, these studies have yet to reveal the overarching mechanism of genetic control of the phenotypes common to MPM tumors.

However, as microRNAs are considered global modulators of gene expression, downregulation of expression of microRNAs represent a potential explanation for the upregulation of families of genes (i.e., loss of microRNA expression causes target gene upregulation).

SUMMARY OF THE INVENTION The invention is based in part on the identification of a family of NAs that are down-regulated in MPM tumor samples as compared with normal pleural tissue from cted individuals. In particular, the inventors ered a marked down-regulation in expression of the miR-15/ 107 family of microRNAs in MPM tissue.

D 2 [Annotation] KJM None set by KJM [Annotation] KJM MigrationNone set by KJM [Annotation] KJM ed set by KJM [Annotation] KJM None set by KJM [Annotation] KJM MigrationNone set by KJM [Annotation] KJM Unmarked set by KJM [0008a] Accordingly, in one aspect the present invention relates to a double-stranded microRNA mimic comprising: (a) a mature sequence corresponding to a /107 family member; and (b) a passenger strand, wherein the mature sequence contains AGCAGC at positions 2-7 or 1-6 at the 5’ end and wherein the mature sequence comprises a sequence selected from the group consisting of SEQ ID NOS: 11-14. [0008b] In another aspect, the present invention relates to the use of a double-stranded microRNA mimic according to the invention in the manufacture of a medicament for treating malignant pleural mesothelioma (MPM). [0008c] In another aspect, the present invention relates to the use of a microRNA mimic according to the ion and an adjunct anti-cancer y in the manufacture of a medicament for treating malignant l mesothelioma (MPM). [0008d] In another , the present invention relates to the use of a double-stranded microRNA mimic according to the invention in the manufacture of a medicament for increasing sensitivity of a malignant pleural elioma (MPM) cancer cell to an anti-cancer therapy.

In another aspect the present invention relates to a double-stranded microRNA mimic that is useful for the treatment of MPM. Such a miRNA mimic of the invention comprises: (1) a mature sequence corresponding to a miR-15/107 family member and that contains AGCAGC at positions 2-7 or 1-6 at the 5’ end; and (2) a ger , which can be inactivated by chemical modification. The mature sequence can comprise a ce selected from the group consisting of SEQ ID NOS: 11-14. The passenger strand can comprise a sequence ed from the group consisting of SEQ ID NOS: 15-18. 3 followed by 3A [Annotation] KJM None set by KJM [Annotation] KJM MigrationNone set by KJM ation] KJM Unmarked set by KJM [Annotation] KJM None set by KJM ation] KJM MigrationNone set by KJM [Annotation] KJM Unmarked set by KJM In accordance with another aspect, the invention provides a method for treating MPM in a subject suffering from the disease. The method comprises administering an effective amount of a double-stranded miRNA mimic, as described above, where such administration mimics endogenous expression of the miR-15/107 family, thereby restoring regulation of the expression of target genes of the miR-15/107 family in the subject. The administration preferably is effected using an intact, bacterially derived minicell for delivery of the miRNA mimic. Pursuant to the inventive methodology, the step of administering microRNA mimic comprises simultaneous or serial co-administration of an adjunct ancer therapy to the subject.

In another aspect of the invention, a method is provided for increasing sensitivity of a MPM cancer cell to the cytotoxic effects of an anti-cancer therapy. The method comprises administering to the cell at least one miRNA mimic as described above, such that the sensitivity of the MPM cancer cell is increased.

These and other aspects of this invention are r described below.

BRIEF DESCRIPTION OF THE GS So that the matter in which the recited features, advantages and objects of the invention, as well as others which will become clear, are attained and can be tood in detail, more particular descriptions and certain embodiments of the invention briefly summarized above are illustrated in the appended figures. These figures form a part of the 3A followed by 4 ation] KJM None set by KJM [Annotation] KJM MigrationNone set by KJM [Annotation] KJM Unmarked set by KJM [Annotation] KJM None set by KJM [Annotation] KJM MigrationNone set by KJM [Annotation] KJM Unmarked set by KJM specification. They illustrate particular embodiments of the invention and, hence, are not limiting of it.

Figure 1 depicts the expression of the miR-15/107 family which is downregulated in MPM cell lines and tumors.

Figure 2 illustrates that continuous tile exposure reduces miR-15/ 107 family microRNA expression in MeT-SA immortalized mesothelial cells Figure 3 depicts that restoring expression of miR-15a, miR-15b or miR-16 leads to growth tion of MPM cells in vitro.

Figure 4 shows that mimics comprising a sequence corresponding to the miR- /107 consensus are more effective than native miR—16 in ting MPM cell proliferation.

Figure 5 illustrates that transfection with miR-16 downregulates target genes.

Figure 6 depicts the effects of miR—16 on gemcitabine and pemetrexed toxicity in MPM cells.

Figure 7 depicts the effects of miR—16 replacement in MPM in vivo, delivered as EGFRminicellsmiR_16 (bispecific antibody targeted, ll-packaged miR-16 mimic where the tumor cell targeting sequence in the bispecif1c antibody is ed to EGFR).

Figure 8 shows the effect on tumor growth in vivo of con15/107.2, a mimic derived from the consensus.

DETAILED DESCRIPTION OF THE INVENTION As noted, a key aspect of the invention is the identification of a family of microRNAs that are down-regulated in MPM tumor samples as compared with normal pleural tissue from unaffected individuals. Thus, a marked down-regulation of the expression of the miR-15/107 family of microRNAs occurs in MPM tissue.

In exemplary embodiments, therefore, the present invention relates to the , synthesis, construction, composition, terization and use of eutic microRNAs D 4 [Annotation] KJM None set by KJM [Annotation] KJM MigrationNone set by KJM [Annotation] KJM Unmarked set by KJM [Annotation] KJM None set by KJM [Annotation] KJM MigrationNone set by KJM [Annotation] KJM Unmarked set by KJM corresponding to the miR-15/107 family for treating MPM. More specifically, the invention is directed to microRNA mimics that act to restore expression of the miR-15/ 107 family in MPM tumor cells by mimicking the activity of the endogenous members of the miR—15/107 family, thereby re-establishing l of MPM cell growth. The microRNA mimics can therefore be used in a replacement therapy approach to e expression of the miR-15/ 107 family in MPM cells.

In further exemplary embodiments, the microRNA mimics are modified to e stability, reduce off-target effects and increase activity. Additional embodiments relate to the use of a minicell to r the microRNA mimics. In another aspect of the ion, microRNA mimics e to enhance the efficacy of other clinically used drugs for the treatment of MPM.

The miR-15/107 Family The miR—15/107 family was identified previously and is characterized as a microRNA super family in which each member contains the sequence AGCAGC starting at either the first or second nucleotide from the 5’ end of the mature microRNA strand. It is believed that the miR-15/107 family is involved with the regulation of us cell activities that represent intervention points for cancer therapy and for therapy of other diseases and disorders. See Finnerty et al. (2010) J. M01. Biol, Vol. 402: 491—509, the contents of which are incorporated here by reference in its entirety. For ce, the miR- /107 family is believed to be involved in regulating gene expression relating to cell division, metabolism, stress response and angiogenesis in vertebrate species, as well as human s, cardiovascular disease and neurodegenerative diseases.

The search for novel approaches to developing cancer therapies often begins with attempts to identify differences between normal and tumor tissue to enable specific or selective targeting of tumor cells. For MPM this has proved difficult as many changes in gene expression, while specific to the tumor cells, are either unable to be targeted (i.e., non— druggable) or provide a eutic window that is too narrow (i.e., normal cells are affected).

Furthermore, target expression is not always correlated with treatment s. While previous s have indicated that a number of microRNAs have tumor—suppressor or oncogenic functions in MPM, these have not been observed to occur frequently in a large D 5 [Annotation] KJM None set by KJM [Annotation] KJM MigrationNone set by KJM [Annotation] KJM Unmarked set by KJM [Annotation] KJM None set by KJM [Annotation] KJM MigrationNone set by KJM [Annotation] KJM Unmarked set by KJM tion of tumors or cell lines. In this context, the discovery that the entire miR-15/107 family is downregulated in all MPM cell lines and tumors analyzed (Figure 1) provides a strong rationale for targeting these changes as a treatment ch. Together with data showing asbestos—induced downregulation of the /107 family of NAs (Figure 2), this evidences an important causative role of these changes in MPM biology. In this regard, the identification of downregulation of the miR-15/ 107 family is significant for at least two reasons: first, it is highly specific for MPM (change is present in all samples, Figure 1); second, increasing levels of miR-15/107 family affects MPM cell growth but has no effect on normal mesothelial cells (Figure 3). In addition, using a microRNA as a therapeutic entity for MPM provides the ability to correct, with a single agent, multiple aberrantly expressed genes (Figure 5), thereby decreasing proliferation (Figure 3) and sing drug sensitiviy (Figure 6). To formulate microRNAs that are capable of restoring expression of the miR—15/107 family, the inventors considered characteristics such as ce similarity in order to e an effective microRNA therapeutic approach.

The miR—15/ 107 family shares a common seed sequence, and thus the mRNA targets of each member overlap significantly. As used , the term RNA fan'lily” refers to a group of niiRNA species that share identity across at least 6 consecutive nucleotides, also referred to as the “seed sequence,” as bed in Brenneeke, J. et 3.1., Piaf} bio] 3 (3):pe'85 (2005). As used in this description, “seed ce” denotes nucleotides at positions l—e, l—7, 23.7, or 23?» of a mature miRNA sequence. The microRNA seed sequence typically is located at the 5" end of the miRNA, “Mature sequence” refers to the strand of a fully processed NA that enters RISC. {8&8} Accordingly, for the purposes of the present invention the miR-15/107 family is comprised of ten sequences as follows: miR-15a-5p (SEQ ID NO:1 uagcagcacauaaugguuugug, MIMAT0000068); -Sp (SEQ ID NO:2 uagcagcacaucaugguuuaca, MIMAT0000417); miR5p (SEQ ID NO:3 uagcagcacguaaauauuggcg, MIMAT0000069); miR-l 95 -5p (SEQ ID NO:4 uagcagcacagaaauauuggc, MIMAT0000461); D 6 [Annotation] KJM None set by KJM [Annotation] KJM ionNone set by KJM [Annotation] KJM Unmarked set by KJM [Annotation] KJM None set by KJM ation] KJM MigrationNone set by KJM [Annotation] KJM Unmarked set by KJM miR5p (SEQ ID I\O:5 cagcagcaauucauguuuugaa, MIMAT0001341); miR5p (SEQ ID 1\O:6 cagcagcacacugugguuugu, 02820); miR5p (SEQ ID I\O:7 uagcagcgggaacaguucugcag, MIMAT0002874); miR5p (SEQ ID 1\O:8 aagcagcugccucugaggc, MIMAT0003316); 3a-3p (SEQ ID \IO:9 agcagcauuguacagggcuauga, MIMAT0000101); and miR-107 (SEQ ID NO:10 auuguacagggcuauca, MIMAT0000104).

To control all targets of the /107 family effectively, in theory all ten members would need to be reintroduced to MPM cells using NA mimics specific to each sequence listed above. Yet, this is not an efficient ch to clinical treatment.

Instead, the invention provides a microRNA mimic approach in which a consensus sequence of the entire miR-15/107 family has been designed that operates as a mimic to perform the functions of the endogenous miR-15/107 family, thereby restoring expression of the miR- /107 family in MPM cells (Figure 3). As used in this description, the phrase “consensus sequence” refers to a nucleotide sequence that shares high sequence, structural and/or functional identity among a group of sequences. In this regard, a microRNA mimic comprising a consensus sequence is capable of mimicking the functions of the entire miR— /107 family. The design of a consensus sequence of the entire /107 family therefore affords the advantage of maximizing the number of desired targets (i.e., the ten miR—15/107 family members) for which endogenous expression can be mimicked, while at the same time limiting the number of mimics to a single sequence entity.

Accordingly, in specific embodiments, the microRNA mimics are used as a form of replacement therapy to treat MPM cells, wherein the microRNA mimics are capable of performing the functions of the miR-15/107 family, thereby ing expression of the miR- /107 family. As such, in the t of this disclosure, the term “restoring expression” refers to the restoration of expression of the miR-15/107 family through the use of microRNA mimics that are capable of mimicking the functions of the nous miR—15/ 107 family.

D 7 [Annotation] KJM None set by KJM [Annotation] KJM MigrationNone set by KJM [Annotation] KJM Unmarked set by KJM [Annotation] KJM None set by KJM [Annotation] KJM MigrationNone set by KJM [Annotation] KJM ed set by KJM microRNA Mimics As used herein, the term “microRNA mimic” refers to synthetic non-coding RNAs that are capable of entering the RNAi pathway and regulating gene expression. As used herein, “synthetic microRNA” refers to any type of RNA sequence, other than endogenous microRNA. microRNA mimics imitate the function of endogeneous microRNAs and can be designed as , double—stranded les or mimic sors (e.g., pri— or pre— microRNAs). MicroRNA mimics can be comprised of modified or unmodified RNA, DNA, RNA-DNA hybrids or alternative nucleic acid chemistries.

As disclosed above, the / 107 family comprises ten microRNAs that share the sequence 5’—AGCAGC—3’ at the 5’—terminal end of the active (guide) strand. The fact that all members of the miR-15/107 family have the same seed sequence provides an opportunity to correct global regulation of gene expression with a single microRNA mimic. Accordingly, the invention provides microRNA mimics corresponding to the miR—15/107 family which comprise a sus sequence, n the microRNA mimics are capable of mimicking the nous activity of the entire miR—15/ 107 family. Therefore, restoration of microRNA expression is achieved through the use of these NA mimics.

Exemplary consensus sequences of the invention are as follows: con15/ 107.1 (SEQ ID NO:11 UAGCAGCACAUAAUGGUUUGCG); con15/107.2 (SEQ ID NO: 12 UAGCAGCACAUAAUGGUUUGCGGA; con15/ 107.3 (SEQ ID NO:13 UAGCAGCACAUAAUGGUUUGCU); and con15/107.4 (SEQ ID NO: 14 UAGCAGCACAGUAUGGUUUGCG). con15/107 . 1, complement A (SEQ ID NO: 15 CCAULAUGUGCUGCUA); con15/ 107.2, complement A (SEQ ID NO: 16 UCCGCAAACCAUUAUGUGCUGCUA); con15/ 107.3, complement A (SEQ ID NO: 17 AGCAAACCAUUAUGUGCUGCUA); con15/ 107.4, complement A (SEQ ID NO: 18 CGCAAACCAUACUGUGCUGCUA); D 8 [Annotation] KJM None set by KJM [Annotation] KJM MigrationNone set by KJM [Annotation] KJM Unmarked set by KJM [Annotation] KJM None set by KJM [Annotation] KJM MigrationNone set by KJM [Annotation] KJM Unmarked set by KJM con15/107. l, ment B (SEQ ID \0: 19 CGCAAACCAUUAUGUGCUGCUU); con15/ 107.2, complement B (SEQ ID \O:20 UCCGCAAACCAUUAUGUGCUGCUU); con15/ 107.3, complement B (SEQ ID \O:21 AGCAAACCAUUAUGUGCUGCUU); con15/ 107.4, complement B (SEQ ID \O:22 CGCAAACCAUACUGUGCUGCUU); con15/107. l, complement C (SEQ ID \O:23 CGCAAACCAUUAUGUGCUGCUUUA); con15/ 107.2, ment C (SEQ ID \O:24 AACCAUUAUGUGCUGCUUUA); con15/ 107.3, ment C (SEQ ID \O:25 AGCAAACCAUUAUGUGCUGCUUUA); con15/ 107.4, complement C (SEQ ID \O:26 CGCAAACCAUACUGUGCUGCUUUA); con15/107. l, complement D (SEQ ID \O:27 CGCAAACCAUUAUUGUGCUGCUUUA); con15/ 107.2, complement D (SEQ ID \O:28 UCCGCAAACCAUUAUUGUGCUGCUUUA); con15/ 107.3, complement D (SEQ ID \O:29 AGCAAACCAUUAUUGUGCUGCUUUA); and con15/107 .4, complement D (SEQ ID \O:30 CGCAAACCAUACUUGUGCUGCUUUA).

A preferred embodiment of the invention comprises a synthetic consensus microRNA (“guide”) sequence in full or in part (SEQ IDs NO: 11-14), together with the complementary ce as a passenger strand (SEQ IDs NO: 15—18). A double—stranded RNA mimic in which terminal ches, overhangs and/or internal bulges are incorporated between the guide and passenger strand, which are introduced to increase RISC loading, is also plated by the invention (see SEQ ID NOs: 21-30). Other variations of the sequence corresponding to the sus sequence of all family members, where the seed sequence AGCAGC is present within the first 7 nucleotides of the guide strand, i.e., positions 1—6 or positions 2-7, are also contemplated. Those skilled in the art will tand that other variations can promote RISC loading to increase activity. By way of example, these include a one or two nucleotide overhang at the 3’ end of the guide strand; a DNA nucleotide (or other chemical modification) at the 3’ end of the passenger strand.

D 9 [Annotation] KJM None set by KJM [Annotation] KJM MigrationNone set by KJM [Annotation] KJM Unmarked set by KJM [Annotation] KJM None set by KJM [Annotation] KJM MigrationNone set by KJM [Annotation] KJM Unmarked set by KJM Chemical Modifications Generally, microRNA mimics have been found to be inefficient in ive use. In this , to improve ncy the t invention employs a microRNA mimic comprising a structurally and chemically modified double-stranded RNA. In exemplary embodiments, in order to overcome the limitations of microRNA mimics, non-toxic chemical modifications to the mimic sequence have been uced to improve stability, reduce off— target effects and increase activity.

In one embodiment, the microRNA mimic includes an RNA duplex sing the mature microRNA sequence and a passenger strand. In one , the passenger strand is structurally and chemically modified to enable the retention of activity of the duplex mimic while inactivating the passenger , thereby reducing off-target effects. In a further aspect, chemical modification inhibits nuclease activity, thereby sing stability.

In particular embodiments, the microRNA mimics of the invention contemplate the use of nucleotides that are modified to enhance their activities. Such tides include those that are at the 5' or 3' terminus of the RNA as well as those that are internal within the molecule. Modified nucleotides used in the complementary strands of microRNAs either block the 5'OH or phosphate of the RNA or introduce internal sugar modifications that t uptake and activity of the inactive strand of the microRNA. ations for the microRNA inhibitors include internal sugar modifications that enhance ization as well as stabilize the molecules in cells and terminal modifications that further stabilize the nucleic acids in cells. Further contemplated are modifications that can be detected by microscopy or other methods to identify cells that contain the microRNAs.

In other aspects, modifications may be made to the sequence of a microRNA or a pre-microRNA without disrupting microRNA activity. As used herein, the term ional variant” of a microRNA sequence refers to an oligonucleotide sequence that varies from the natural microRNA sequence, but retains one or more functional characteristics of the NA (e.g., enhancement of cancer cell susceptibility to chemotherapeutic agents, cancer cell proliferation inhibition, induction of cancer cell sis, specific microRNA target tion). In some embodiments, a functional variant of a microRNA sequence retains all of the functional characteristics of the microRNA. In certain embodiments, a D 10 ation] KJM None set by KJM [Annotation] KJM MigrationNone set by KJM [Annotation] KJM Unmarked set by KJM [Annotation] KJM None set by KJM [Annotation] KJM MigrationNone set by KJM [Annotation] KJM Unmarked set by KJM functional variant of a microRNA has a nucleobase sequence that is a least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the microRNA or sor thereof over a region of about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, , 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 or more nucleobases, or that the functional variant hybridizes to the complement of the microRNA or precursor thereof under stringent hybridization conditions. Accordingly, in certain embodiments the nucleobase sequence of a functional variant may be capable of hybridizing to one or more target sequences of the microRNA.

In some embodiments, the complementary strand is modified so that a chemical group other than a phosphate or yl is at its 5' terminus. The presence of the 5' modification apparently eliminates uptake of the complementary strand and subsequently favors uptake of the active strand by the microRNA protein complex. The 5' modification can be any of a variety of les known in the art, including NHZ, 3, and biotin.

In another ment, the uptake of the complementary strand by the microRNA pathway is reduced by incorporating nucleotides with sugar modifications in the first 2-6 nucleotides of the complementary strand. It should be noted that such sugar modifications can be combined with the 5' terminal modifications described above to further enhance microRNA activity. Sugar modifications plated in microRNA mimics include, but are not limited to, a sugar substitute group selected from: F; O—, S—, or N—alkyl; O—, S—, or N— alkenyl; O-, S- or N—alkynyl; or l-O-alkyl, wherein the alkyl, alkenyl and alkynyl may be substituted or tituted C1 to C10 alkyl or C2 to C10 alkenyl and alkynyl. In some embodiments, these groups may be chosen from: O(CH2)XOCH3, O((CH2)XO)yCH3, O(CH2)XNH2, O(CH2)XCH3, O(CH2)XONH2 and O(CH2)XON((CH2)XCH3)2, where x and y are from 1 to 10. d base moieties or altered sugar moieties also include other modifications consistent with the purpose of a microRNA mimic. Such oligomeric nds are best described as being structurally distinguishable from, yet functionally interchangeable with, naturally occurring or synthetic fied ucleotides. As such, all such oligomeric compounds are contemplated to be encompassed by this invention so long as they function D 11 [Annotation] KJM None set by KJM ation] KJM ionNone set by KJM [Annotation] KJM Unmarked set by KJM [Annotation] KJM None set by KJM [Annotation] KJM MigrationNone set by KJM [Annotation] KJM Unmarked set by KJM effectively to mimic the structure or function of a desired RNA oligonucleotide strand corresponding to the miR-15/ 107 family.

In some embodiments, the complementary strand is ed so that nucleotides in the 3' end of the complementary strand are not complementary to the active strand. This results in double-stranded hybrid RNAs that are stable at the 3' end of the active strand but relatively unstable at the 5' end of the active strand. This difference in stability enhances the uptake of the active strand by the microRNA pathway, while reducing uptake of the complementary strand, thereby enhancing microRNA activity.

Concerning other modifications, contemplated for use in the practice of the invention, see also the disclosure in US Patent Pub. No. 2012/0259001 that addition of 2'-O— methyl modifications to the sense strand of miRNA mimics, inter alia, can have negative effects on that molecule. Ameliorating these negative s is a miRNA mimic that comprises (i) a sense strand that ranges in size from about 16 to about 31 nucleotides in which about 40% to about 90% of the nucleotides of the sense strand are chemically modified; (ii) an antisense strand that ranges in size from about 16 to about 31 nucleotides in which about 40% to about 90% of the nucleotides of the nse strand are chemically modified tides; and (iii) at least one of a mismatch between nucleotide l on the antisense strand and the opposite nucleotide on the sense ; and a mismatch between nucleotide 7 on the antisense strand and the opposite nucleotide on the sense strand. Also advantageous in this context, as disclosed, is the attachment to the sense strand of the miRNA mimic, via a linker molecule that is from about 3 to about 9 atoms in length, of a conjugate moiety selected from the group consisting of terol, cholestanol, stigmasterol, cholanic acid, and ergosterol. The linker molecule can be 5 to 8 atoms in length, for example, and the linker molecule can attach the conjugate moiety to the 3' end of the sense strand.

In some embodiments, microRNA sequences of the invention may be ated with a second RNA sequence that may be d on the same RNA molecule or on a separate RNA molecule as the microRNA sequence. In such cases, the microRNA sequence may be referred to as the active strand, while the encoded RNA sequence, which is at least partially complementary to the NA sequence, may be referred to as the complementary strand. The active and complementary s may be ized to generate D 12 [Annotation] KJM None set by KJM [Annotation] KJM MigrationNone set by KJM [Annotation] KJM Unmarked set by KJM [Annotation] KJM None set by KJM [Annotation] KJM ionNone set by KJM [Annotation] KJM Unmarked set by KJM a double-stranded RNA that is similar to a naturally occurring microRNA precursor. The activity of a microRNA may be optimized by maximizing uptake of the active strand and minimizing uptake of the complementary strand by the microRNA protein complex that tes gene translation. This can be done through cation and/or design of the complementary strand, for instance.

A nucleic acid may be made by any technique known to one of ordinary skill in the art, such as for example, chemical synthesis, enzymatic production or biological production.

In some embodiments, microRNA compositions of the invention are chemically synthesized.

A red embodiment has a particular set of such ations. These are chemical modification of 2 to 6 nucleotides at each end of the ger strand with 2’0- methyl—modified sugars, and include any combination of 2, 3, 4, 5 or 6 modified nucleotides at the 5’end of the passenger strand with 2, 3, 4, 5 or 6 modified nucleotides at the 3’end of the passenger strand. ative chemical modification strategies imparting similar onality will be apparent to those skilled in the art.

Method of stration NA mimics can be administered to a subject by any means suitable for delivering these compounds to cancer cells of the subject. For example, the microRNA mimics can be administered by methods suitable to transfect cells of the subject with the mimics, or with nucleic acids sing sequences ng these compounds. In one embodiment, the cells are transfected with a plasmid or viral vector comprising sequences encoding a microRNA mimic.

In one particular embodiment of the invention, to circumvent the problems associated with cient delivery in viva, a mimic according to the invention preferably is delivered via the “EnGeneIC Delivery Vehicle” system developed by EnGeneIC Molecular Delivery Pty Ltd (Sydney), which is based on the use of intact, bacterially derived minicells.

The EDVTM system is described, for example, in published international applications and , the respective contents of which are incorporated here by reference.

D 13 ation] KJM None set by KJM [Annotation] KJM MigrationNone set by KJM [Annotation] KJM Unmarked set by KJM [Annotation] KJM None set by KJM [Annotation] KJM MigrationNone set by KJM [Annotation] KJM Unmarked set by KJM In an exemplary embodiment, ore, the NA mimics described here are delivered using intact, bacterially derived minicells. These minicells are delivered specifically to target s, using if1c antibodies. One arm of such an dy has specificity for the target tissue, while the other has specificity for the minicell. The antibody brings minicells to the target cell surface, and then the minicells are brought into the cell by endocytosis. After uptake into the tumor cell there is a release of the minicell contents, i.e., the microRNA mimic(s). For an antibody in this regard, specificity t any cell surface marker for MPM could be used in accordance with the ion. Thus, illustrative of such specificity suitable for a if1c antibody in the present context could be a specificity to human mesothelin, expressed on 100% of epithelioid mesotheliomas, for which therapeutic antibodies are in development (see Kelly et al., M01. Cancer Ther. 11: 517—22 (2012)), or to intelectin-l, which is expressed specifically in MPM and gastrointestinal goblet cells (see Washimi et al., PLoS One 7: e39889 (2012)).

Other methods of administering nucleic acids are well known in the art. In particular, the routes of administration y in use for nucleic acid therapeutics, along with formulations in current use, provide preferred routes of administration and formulation for the nucleic acids. Nucleic acid compositions can be administered by a number of routes including, but not limited to: oral, intravenous, intrapleural, intraperitoneal, intramuscular, transdermal, subcutaneous, topical, sublingual, or rectal means.

Nucleic acids can also be stered via liposomes or rticles. Such administration routes and appropriate formulations are generally known to those of skill in the art. Administration of the formulations described herein may be accomplished by any acceptable method that allows the microRNA or nucleic acid encoding the NA to reach its . The particular mode selected will depend of course, upon exemplary factors such as the particular formulation, the severity of the state of the subject being treated, and the dosage required for therapeutic efficacy. As generally used herein, an “effective amount” of a nucleic acid is the amount that is able to treat one or more symptoms of cancer or related disease, reverse the progression of one or more symptoms of cancer or related disease, halt the progression of one or more symptoms of cancer or related disease, or prevent the occurrence of one or more symptoms of cancer or related disease in a subject to whom the D 14 [Annotation] KJM None set by KJM [Annotation] KJM MigrationNone set by KJM [Annotation] KJM Unmarked set by KJM [Annotation] KJM None set by KJM [Annotation] KJM MigrationNone set by KJM [Annotation] KJM Unmarked set by KJM formulation is administered, as compared to a matched subject not receiving the compound or therapeutic agent. The actual ive amounts of drug can vary according to the specific drug or combination thereof being utilized, the ular composition formulated, the mode of administration, and the age, weight, condition of the t, and severity of the symptoms or condition being treated.

Other delivery systems le include but are not limited to time—release, delayed release, sustained release, or controlled release delivery systems. Such systems may avoid repeated administrations in many cases, increasing convenience to the subject and the physician. Many types of e delivery systems are available and known to those of ordinary skill in the art. They include, for e, polymer—based systems such as polylactic and/or polyglycolic acids, polyanhydrides, polycaprolactones, copolyoxalates, polyesteramides, polyorthoesters, polyhydroxybutyric acid, and/or combinations of these.

Pharmaceutical compositions of the invention containing microRNA mimics can also comprise conventional pharmaceutical excipients and/or additives. Suitable pharmaceutical excipients include stabilizers, antioxidants, osmolality adjusting agents, buffers, and pH ing agents. Suitable ves include, e.g., physiologically biocompatible buffers (e. g., tromethamine hydrochloride), additions of chelants (e. g., DTPA or DTPA-bisamide) or calcium chelate complexes (e.g., calcium DTPA, CaNaDTPA- bisamide), or, optionally, additions of calcium or sodium salts (e.g., calcium chloride, calcium ascorbate, calcium ate or calcium e).

Dosing As the relationship n loss of microRNA expression and MPM is consistent across the samples, one aspect of the invention is directed to microRNA replacement in MPM tumor cells. In this aspect, based on l animal experiments, it is contemplated that the ssion of MPM will be halted by treatment with the miR-containing miRs of the invention. In further aspects, it is contemplated that le doses over a continued time frame can be administered to the subject. In some aspects, although one dose per week may have a suitably efficacious , some subjects may trate more markedly efficacious effects in response to increasing frequency and/or increasing dosage.

D 15 [Annotation] KJM None set by KJM [Annotation] KJM MigrationNone set by KJM ation] KJM Unmarked set by KJM [Annotation] KJM None set by KJM [Annotation] KJM MigrationNone set by KJM [Annotation] KJM Unmarked set by KJM In another ment of the invention, chronic dosing will be able to achieve MPM disease control. In yet other ments, it is contemplated that MPM tumor cells receiving microRNA mimics will enter permanent growth arrest. In this regard, permanent growth arrest can be attributed to the mechanism of action of the microRNA mimics (downregulation of cell cycle- and metabolism-promoting genes, anti-apoptotic pro-survival genes, and drug ance genes) and phenotypic consequences (cell cycle arrest). Continued ent thus is contemplated, in accordance with one aspect of the invention, to induce disease control or maintenance (i.e., stable disease). Pursuant to aspect of the invention, tumor regression (i.e., a partial/complete response) is contemplated where the MPM tumors enter ent cell growth arrest.

Furthermore, as discussed in further detail below, the microRNA mimics of the invention further function to sensitize cells to conventional cancer therapies such as chemotherapy and radiation. In this aspect, it is contemplated that combining the mimics with chemotherapy will provide onal therapeutic effects.

With all current treatments for MPM, response to therapy is invariably followed by relapse. Thus, in yet another aspect of the invention it is contemplated that tumors will retain this expression profile upon relapse in view of the relationship between microRNA expression and MPM. In this regard, a relapse as described will allow re-treatment with the same microRNA mimics disclosed herein. In this embodiment, long-term tumor suppression s the nature of disease and potentially changes MPM into a form of chronic e.

In other s, dosages for a particular subject can be determined by one of ordinary skill in the art using conventional considerations, (e. g., by means of an appropriate, conventional pharmacological protocol). A physician may, for example, prescribe a relatively low dose at first, subsequently sing the dose until an appropriate response is ed. The dose administered to a subject is sufficient to effect a beneficial therapeutic response in the subject over time, or, e.g., to reduce ms, or other appropriate ty, depending on the ation. The dose is determined by the efficacy of the particular formulation, and the activity, stability or serum half-life of the microRNA employed and the condition of the subject, as well as the body weight or surface area of the subject to be treated. The size of the dose is also determined by the existence, nature, and extent of any D 16 ation] KJM None set by KJM [Annotation] KJM MigrationNone set by KJM [Annotation] KJM Unmarked set by KJM [Annotation] KJM None set by KJM [Annotation] KJM MigrationNone set by KJM [Annotation] KJM Unmarked set by KJM adverse side—effects that accompany the administration of a particular vector, and ation, in a particular subject.

Combination Therapy The microRNA mimics bed herein can supplement treatment conditions by any known conventional therapy, including, but not limited to, antibody administration, vaccine administration, administration of cytotoxic agents, natural amino acid polypeptides, nucleic acids, nucleotide analogues, and biologic response modifiers. Two or more combined compounds may be used together or sequentially. For e, the microRNA mimics can also be administered in therapeutically effective amounts as a portion of an anticancer cocktail. An ancer cocktail is a mixture of the microRNA mimic(s) with one or more anti-cancer drugs in addition to a pharmaceutically able carrier for delivery. The use of anti-cancer cocktails as a cancer ent is routine.

In one embodiment, it is oned to use a microRNA mimic in combination with other therapeutic ties. Thus, in addition to the microRNA therapies described above, one may also provide to the subject more “standard” therapies such as, but not limited to, conventional cancer therapeutic agents.

Anti-cancer drugs that are well known in the art and can be used as a treatment in combination with the nucleic acids described herein include, but are not limited to: actinomycin D, aminoglutethimide, asparaginase, bleomycin, busulfan, carboplatin, tine, chlorambucil, cisplatin (cis-DDP), cyclophosphamide, cytarabine HCl (cytosine arabinoside), dacarbazine, factinomycin, daunorubicin HCl, doxorubicin HCl, Estramustine phosphate sodium, etoposide (VP 16—213), floxuridine, 5—fluorouracil (5—FU), flutamide, hydroxyurea (hydroxycarbamide), Ifosfamide, Interferon apha-2a, interferon alpha-2b, leuprolide acetate (LHRH-releasing factor analog), lomustine, mechlorethamine HCl (nitrogen d), melphalan, mercaptopurine, mesna, methotrexate (MTX), mitomycin, mitoxantrone hcl, octreotide, plicamycin, procarbazine hcl, ozocin, tamoxifen citrate, thioguanine, thiotepa, vinblastine sulfate, vincristine e, ine, azacitidine, hexamethylmelamine, interleukin-2, mitoguazone, pentostatin, semustine, teniposide, and vindesine sulfate.

D 17 [Annotation] KJM None set by KJM [Annotation] KJM MigrationNone set by KJM [Annotation] KJM Unmarked set by KJM [Annotation] KJM None set by KJM [Annotation] KJM MigrationNone set by KJM [Annotation] KJM Unmarked set by KJM Chemotherapeutic agents, for example, can be agents that directly cross-link DNA, agents that intercalate into DNA, and agents that lead to chromosomal and c aberrations by affecting nucleic acid synthesis. Agents that directly link nucleic acids, specifically DNA, are envisaged and are shown herein, to eventuate DNA damage leading to a synergistic antineoplastic combination. Agents such as tin, and other DNA alkylating agents may be used. Agents that damage DNA also e compounds that ere with DNA replication, mitosis, and somal segregation. Examples of these compounds include adriamycin (also known as doxorubicin), VP-l6, also known as etoposide, verapamil, podophyllotoxin, and the like. Exemplary chemotherapeutics include at least I) antibiotics, such as doxorubicin, daunorubicin, mitomycin, Actinomycin D; 2) platinum-based agents, such as cisplatin; 3) plant alkaloids, such as taxol and vincristine, stine; 4) alkylating agents, such as carmustine, melphalan, cyclophosphamide, chlorambucil, busulfan, and lomustine.

In exemplary embodiments of the invention, the microRNA mimic(s) can be administered as a single agent but can also be used in combination with other drugs, e.g., pemetrexed, cisplatin (or latin), and gemcitabine, etc.

Combination therapies may be achieved by contacting MPM tumor cells with a single composition or a pharmacological formulation that includes one or more microRNA mimics and a second cancer eutic agent, or by contacting the tumor cell with two distinct compositions or ations, at the same time, wherein one composition includes one or more microRNA mimics and the other includes the second cancer therapeutic agent.

Alternatively, administration of one or more microRNA mimics may precede or follow administration of the other cancer therapeutic agent by intervals ranging from s to weeks. In embodiments where the other cancer therapeutic agent and one or more microRNA mimics are applied separately to the subject, one would generally ensure that a significant period of time did not expire between the time of each delivery, such that the cancer therapeutic agent and the one or more microRNA mimics would still be able to exert an advantageously combined effect on the tumor cell.

Further cological therapeutic agents and methods of administration, dosages, etc., are well known to those of skill in the art (see, for example, the “Physicians Desk D 18 [Annotation] KJM None set by KJM [Annotation] KJM MigrationNone set by KJM [Annotation] KJM Unmarked set by KJM [Annotation] KJM None set by KJM [Annotation] KJM MigrationNone set by KJM [Annotation] KJM Unmarked set by KJM Reference,’ 3 Klaasen’s “The Pharmacological Basis of Therapeutics,’ 3 “Remington’s Pharmaceutical Sciences,” and “The Merck Index, Eleventh Edition,” incorporated herein by reference in relevant parts), and may be ed with the invention in light of the disclosures herein. Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, ine the appropriate dose for the dual subject, and such individual determinations are within the skill of those of ordinary skill in the art.

EXAMPLES The following examples are given for the purpose of illustrating various embodiments of the invention. They are not meant to limit the invention in any fashion. One skilled in the art will appreciate that the invention is well adapted to carry out the s and obtain the ends and advantages mentioned, as well any objects, ends and advantages inherent herein. The present es (along with the methods described herein) are tly representative of preferred embodiments. They are exemplary, and are not intended as limitations on the scope of the invention. Variations and other uses which are encompassed within the spirit of the invention as defined by the scope of the claims will occur to those skilled in the art.

Example 1 ery of the Reduced Expression of the miR-15/107 Family in MPM MicroRNAs have important roles in cancer development and their expression is ulated in tumors, including MPM. The inventors identified changes in the miR-l6 expression in MPM cell lines and a small set of tumor samples, and further assessed expression of the entire miR-15/ 107 family of d microRNAs in MPM cell lines and a larger set of tumor ens (Figure l).

A panel of 7 MPM cell lines was compared with a mesothelial cell line .

Cells were obtained from ATCC (H28, H2052, H2452, H226 and MSTO-2llH) and collaborators (MM05 and Ren) and cultured in the recommended medium at 37°C with 5% C02. Total RNA was isolated from cells (grown to 80% confluence) using TriZOL® (Life D 19 [Annotation] KJM None set by KJM [Annotation] KJM MigrationNone set by KJM [Annotation] KJM Unmarked set by KJM [Annotation] KJM None set by KJM [Annotation] KJM MigrationNone set by KJM ation] KJM Unmarked set by KJM Technologies). Sixty tumor specimens ted of formalin—fixed, paraffin—embedded blocks. Tumor microRNA expression was compared with 23 formalin-fixed samples of normal l . To isolate RNA from tumor specimens, an experienced pathologist marked tumor content on H&E d slides from each block which were used as a guide for capture microdissection to enrich tumor content. Briefly, samples were mounted onto membrane slides (Zeiss), and LCM was carried out using the PALM system (Zeiss). ed tissue was collected in adhesive collection tubes, and deparaff1nisation was performed in xylene. RNA was extracted from the captured tumor specimens and the normal tissues using the FFPE RNeasy Mini kit (Qiagen) according to the manufacturer’s instructions. RNA from cell line and tissue samples was quantified using a Nanophotometer with readings at 260 and 280 nm. For both cell line and tumor samples, reverse transcription (RT) used microRNA—speciflc stem—loop primers (Life Technologies). 100 ng total RNA was used as template in the RT reaction, also including 4 pl RT primer mix (with up to 10 microRNA-specif1c RT primers multiplexed in an equimolar mix), with the reaction carried out following the cturer’s ctions in a volume of 10 pl. The ant complementary DNA (cDNA) was diluted by the addition of 57.8 pl water, and from this on, 2.25 pl cDNA was added as template to the qPCR reaction. The qPCR further contained microRNA-specif1c TaqMan primers/probes and TaqMan GeneExpression Mastermix (both Life logies) with a total reaction volume of 10 pl. The reactions were set up manually and run in duplicate on a Mx3000P real-time PCR machine (Stratagene) with 10 min enzyme activation at 95° C followed by 40 cycles of 15 sec denaturing at 95° C and 60 sec annealing/elongation at 60° C. Values for Cq (quantification cycle) were determined applying adaptive—baseline and background—based threshold algorithms using the MxPro software. Analysis of the qPCR results was performed using the Z'AACq method (as described by Livak et al), and included normalization to RNU6B expression levels. Expression in each sample was calculated relative to the e expression of controls.

Of the ten microRNAs in the miR-15/107 family, 6 (miR—l6, a, miR-15b, miR—l95, miR-103 and miR—lO7) were detected in all samples, and 4 (miR-424, miR-497, miR—503 and miR—646) were undetected. In cell lines, an average 2— to 5-fold downregulation of all six detectable microRNAs was observed as compared to expression in D 20 ation] KJM None set by KJM [Annotation] KJM MigrationNone set by KJM [Annotation] KJM Unmarked set by KJM [Annotation] KJM None set by KJM [Annotation] KJM MigrationNone set by KJM [Annotation] KJM Unmarked set by KJM immortalized normal mesothelial cells (see Figure 1). In tumors, miR-l6, miR-15a, miR-le and miR—l95 were downregulated by on average 8— to 25—fold, whereas miR-103 and miR— 107 were downregulated by 4— to 6—fold, when compared with levels of these microRNAs in normal pleura (id).

Example 2 Reduction of miR—15/107 Family sion Upon Exposure of MPM Cells to Chrysotile Asbestos Fibres Asbestos is the etiological agent in MPM, but effects on microRNA expression are unknown. s observed following asbestos exposure would suggest a causative role in MPM y. Previous work in the field has focused on the effects on cells of acute exposure to cytotoxic concentrations of asbestos fibres. While s in gene sion are observed in these cases, the predominant result of such treatment is a combination apoptosis and necrosis leading to extensive cytotoxicity and cell death.

In order to identify the physiologically relevant effects of asbestos exposure on microRNA expression in mesothelial cells, MeT—SA cells were continuously exposed to chrysotile asbestos fibres. MeT—SA cells were grown in recommended culture conditions in the presence of 0, 0.1 or 1 pg chrysotile asbestos fibres continuously for 3 months. At the ted time points, cells were harvested and RNA isolated for microRNA expression measurements. RNA isolation and RT-qPCR was carried as described in Example 1. Levels of microRNA were normalized to U6 expression and are sed relative to the normalized expression in untreated cells. Figure 2 demonstrates that uous exposure to asbestos at 1 uM leads to decreased expression of miR—l6, miR-15b, miR-l95, miR—103 and miR—107 at all time points, and ses in miR—15a expression from 70 days on. These results are the first to link asbestos re to changes in microRNA sion in general, and the first to analyze changes in gene expression related to erm asbestos exposure. They provide a direct link between asbestos exposure and miR—15/107 family expression, and thus suggest that these may be early and important changes in MPM progression.

D 21 [Annotation] KJM None set by KJM [Annotation] KJM ionNone set by KJM [Annotation] KJM Unmarked set by KJM [Annotation] KJM None set by KJM [Annotation] KJM MigrationNone set by KJM [Annotation] KJM Unmarked set by KJM Example 3 Replacing miR-15/107 Family Leads to Growth Inhibition of MPM cells in vitro In order to determine the effects of restoring miR—15/ 107 family members on MPM cell growth, MPM cell lines were transfected with microRNA mimics and the effect on cell growth measured. Mimics consisted of double—stranded RNAs corresponding to the sequence of mature miR—16, miR—15a or miR—15, or a synthetic sequence corresponding to the sus sequence of the miR—15/107 , and were provided as HPLC—purified lyophilized es hai GenePharma). Mimics were pended in water at a tration of 20 uM. These were then reverse transfected into cells using Lipofectamine RNAiMAX (‘LRM’, Life Technologies) at the indicated concentrations. First, lipoplexes were generated by mixing the appropriate concentration of mimic in serum-free medium with an equal volume of a 1% solution of LRM in serum-free medium, and incubating for 20 to 120 s at room temperature. Lipoplexes were distributed to replicate multiwell plates and cells in suspension (medium containing 10% FCS) were added to the lipoplex mix in each well such that the final density of cells was 7500/cm2. Transfection was allowed to proceed for 24 hours after which medium was replaced with fresh medium containing 10 % FCS and cells were further incubated at 37°C until harvest. Thereafter, replicate plates were harvested at 48, 72, 96 and 120 hours post-transfection, which involved ng medium and freezing plates at -80 °C. At the conclusion of the experiment, plates were thawed and 150 pl lysis buffer containing 0.01% SYBR Green added to each well to measure DNA content. After incubation overnight in the dark, DNA content was quantified by measuring fluorescence in a Fluostar Optima meter, set at excitation of 485 nm and on 535 nm. Total fluorescence (i.e., DNA per well) in this assay displays a linear relationship with cell number, allowing it to determine proliferation.

Figure 3 shows that, following transfection with microRNA mimics corresponding in sequence to miR—15a, miR—15b or miR—16, there was a dose— and time—dependent decrease in proliferation in 4 MPM cell lines (H28, MM05, H2052 and MSTO). There was no effect of these treatments on the normal mesothelial cell line MeT-SA. Therefore, restoring microRNA expression and thus control of target gene expression resulted specifically in the inhibition of proliferation of MPM cells.

D 22 [Annotation] KJM None set by KJM [Annotation] KJM MigrationNone set by KJM ation] KJM Unmarked set by KJM [Annotation] KJM None set by KJM ation] KJM MigrationNone set by KJM [Annotation] KJM Unmarked set by KJM To investigate further the effects of the miR—15/ 107 family on MPM cells, mimics were designed that correspond to the consensus sequence of the family. The consensus mimics appear below.

Table 1 Mimics Sense (passenger) nse ) con 1 5/ 107.1 mCmeCmAAACCAUUAUGUGCUmeCmUmA UAGCAGCACAUAAUGGUUUGCG con15/107.2 mGCAAACCAUUAUGUGCUmeCmUmA UAGCAGCACAUAAUGGUUUGCGGA con 1 5/ 107.3 AAACCAUUAUGUGCUmeCmUmA UAGCAGCACAUAAUGGUUUGCU con 1 5/ 107.4 AAACCAUACUGUGCUmeCmUmA UAGCAGCACAGUAUGGUUUGCG These consensus sequences varied depending on the number of microRNAs included in the alignment, varying from the miR-15 family only / 107.1) to the entire /107 family (con15/107.2 to 4). The consensus length in con15/107.3 was increased to account for the longer mature sequence of some microRNAs. These four consensus microRNAs then were transfected into MSTO or H28 cells at varying concentrations and the effect on cell growth assessed as above. Figure 4 shows that all of the mimics based on the miR—15/107 consensus sequence were more growth inhibitory than the native miR—16 sequence. This indicates that the consensus sequences are more promising therapeutic candidates than the natural miR-16.

Example 4 Transfection with miR-16 Downregulates Target Genes MicroRNAs are responsible for post—transcriptional gene regulation. While the primary mechanism of action of microRNAs is via inhibition of translation of mRNA into protein, this frequently leads to destabilization of target mRNA. Therefore, the ability of a microRNA to regulate gene expression of predicted targets can be measured by analyzing target mRNA levels following modulation of the microRNA of interest. Targets regulated in D 23 [Annotation] KJM None set by KJM [Annotation] KJM MigrationNone set by KJM [Annotation] KJM Unmarked set by KJM [Annotation] KJM None set by KJM [Annotation] KJM ionNone set by KJM [Annotation] KJM Unmarked set by KJM this way are then candidates for genes involved in the phenotypic effects observed following microRNA mimic transfection. Those genes also downregulated at the protein level are considered more likely to be bona fide targets of the microRNA.

To link effects of mimic transfection on cell y of MPM cells, expression of 24 candidate miR-l6 target genes was measured in MPM cells treated with miR-l6 mimic e 5). H28 and MSTO cells were reverse transfected in 6-well , with miR-l6 mimic or control (each 5 nM) as per the method described for Example 3. After 48 h transfection, RNA was isolated using TriZOL and quantified using a nanophotometer (Implen, Munich, Germany). From replicate wells, protein was isolated and fied by Pierce BCA Protein assay (Thermo Fisher Scientific). Synthesis of cDNA used 250 ng RNA as template, with a mix of random oligos and oligodT as s. This cDNA was then used as template in 10 pl qPCR reactions with primers specific for the predicted targets of miR-l6, using Brilliant II SYBR green chemistry (Agilent Technologies) mix as per manufacturer’s instructions, and the reactions were run on an MX3000P real time PCR machine (Agilent Technologies). The qPCR results were analyzed by the AACt method, y target gene expression was normalized to expression of 18S, and results from mimic-transfected cells expressed relative to the normalized expression of targets in control-transfected cells. These results demonstrated a egulation in 24 targets ranging from 1.2 to 4-fold. On the n level, expression of CCNDland Bcl-2 were analyzed by western blot. n (20 pg) was separated on a 10% precast polyacrylamide gel (Mini Protein TGX Precast Gels, Biorad) and transferred to PVDF membranes using the Biorad Trans-Blot Turbo Transfer System d, NSW, Australia). Membranes were blocked using milk powder then probed with target specific antibodies (Bel-2; CCNDl), followed by detection with a rabbit or mouse specific secondary antibody (all antibodies from Cell ing Inc). uminesence (Supersignal West Femto Maximum Sensitivity substrate kit, Thermo Fisher Scientific) was used to detect the presence of the protein and was measured using a Kodak Geologic 2200 imaging system. Expression of beta-actin in) was included to control for equal protein loading. Protein expression of both CCNDl and Bcl-2 were significantly down-regulated in miR—l6 treated cells compared with controls (see Figure 5). Together, these changes in mRNA and protein expression show that the observed phenotypic effects of miR-l6 mimic transfection are d to genes involved in proliferation and altered apoptotic response.

D 24 ation] KJM None set by KJM [Annotation] KJM MigrationNone set by KJM [Annotation] KJM Unmarked set by KJM [Annotation] KJM None set by KJM [Annotation] KJM MigrationNone set by KJM [Annotation] KJM Unmarked set by KJM Example 5 s of miR-16 on Gemcitabine and Pemetrexed Toxicity in MPM Cells MPM is considered resistant to herapy, and this resistance is believed to relate to s in apoptotic responses of the tumor cells. As many of the predicted targets of the miR-15/107 family are genes related to these processes, one might predict that microRNA mimics would sensitize MPM cells to chemotherapy drugs. This was tested for the combination of miR-l6 and the drugs gemcitabine and pemetrexed (Figure 6).

To test the effect of restoring miR-l6 expression on drug toxicity, the normal mesothelial cell line MeT-5A (A, D), and two MPM lines — MM05 (B, E) and l 1H (C, F) - were transfected with 1 or 5 nM miR—16 (closed ls) or control mimic (closed symbols) in 96—well plates as described in Example 3. Thereafter, medium was replaced after 24 hours with medium ning a serial dilution of pemetrexed (1.95 to 500 nM) or gemcitabine (0.625 to 160 nM) with each concentration assayed in triplicate. After 72 hours, plates were harvested and DNA content measured as bed in Example 3. The growth of cells exposed to drug was normalized to untreated cells, and the concentration inhibiting growth by 50 % (leo value) was determined. The effects of miR—16 on drug sensitivity was determined by ing IC50 values in mimic and control transfected cells. This is demonstrated in Figure 6, which shows a dose-dependent 2— to 5—fold sensitization of MM05 cells (B, E) and MSTO-leH cells (C, F) to both drugs, but no effect on normal MeT-5A cells (A, D).

Example 6 Effects of miR-16 Replacement in MPM in vivo, Delivered as VectEDVmiR-l6 The growth (and other) inhibitory effects observed upon restoration of lost expression of tumor suppressor microRNAs in cancer suggests that they represent novel therapeutic targets. In order to investigate this ility, this must be tested in pre-clinical mouse models. In order to ate these in vitro effects in the in viva situation, however, the microRNA mimics must overcome hurdles limiting delivery to the cells within the tumor where they have their therapeutic effect. Here the delivery of miR—16 or consensus mimics D 25 [Annotation] KJM None set by KJM [Annotation] KJM MigrationNone set by KJM [Annotation] KJM Unmarked set by KJM [Annotation] KJM None set by KJM [Annotation] KJM MigrationNone set by KJM ation] KJM Unmarked set by KJM used a targeted minicell approach. Minicells are nanoparticles derived from asynchronous division of bacteria, as disclosed above in reference to US. Patent Pub. No. 201 1/011 1041.

In vivo efficacy of miR—16 restoration was evaluated in a subcutaneous human xenograft model of MPM in nude mice (Figures 7 and 8). Athymic (nu/nu) mice (4—6 weeks old) were purchased from the Animal ces Centre (Perth Western Australia) and all animal experiments were approved by the Sydney Local Health cts Animal Ethics Committee, Concord and RPAH. MSTO cells were cultured and 1.5 1:: 106 cells in 50 l-Ll serum-free media together with 50 I-Ll growth factor reduced el (BD Biosciences) and injected aneously between the shoulder blades. Tumor volume (mm3) was determined by measuring length (l) and width (w) and calculating volume (V = lw2/2) as described on the ted days. Experimental and control treatments were carried out once the tumor s were on average 100 mm3, at which time the tumor mass was y palpable and vascularized, as determined following excision and histological examination of tumors. Mice were randomized to different groups before starting the various treatments. All tumor volume—measurements were performed by an investigator blinded to the ents administered.

Three experiments were carried out. In the first experiment, mice were treated on the indicated days with indicated dose of 1x109 miR or control-containing minicells 1, 2 or 4 times per week (Figure 7). The tumors in the control mice (treated with saline or empty minicells) sed from 100 to 400 mm3 in the course of the experiment. Tumors in those mice receiving miR—16 mimic grew more , and effects were dependent on number of treatments. Tumor-bearing mice receiving 1 dose per week had tumors that grew more slowly than those in control-treated mice, and this inhibition of growth was maintained until day 30, at which point the size of these tumors was similar to that of controls. Mice receiving 2 doses per week had tumors that increased in size to approximately 200 mm3 by the end of the experiment on day 33, and were considerably smaller than those in mice receiving control minicells throughout. Mice treated 4 times per week had tumors that did not increase in size for the first week following the initial administration of the miR-16 mimic. By the end of the experiment, these tumors sed in size to only 170 mm3, corresponding to a 75% inhibition of growth when compared with controls.

D 26 ation] KJM None set by KJM [Annotation] KJM MigrationNone set by KJM [Annotation] KJM Unmarked set by KJM [Annotation] KJM None set by KJM [Annotation] KJM ionNone set by KJM [Annotation] KJM Unmarked set by KJM In the second experiment, mice were d 4 times per week with a dose of 2x109 miR-l6 or control-loaded minicells (Figure 7). In this experiment, tumors in the mice treated with l minicells grew at a comparable rate to those in the first experiment that received half the dose. In mice receiving the increased dose of miR-l6, there was a marked increase in umor effect of the miR-l6 mimic. In the initial phase following treatment, the volume of these tumors decreased from the 100 mm3 starting point. From there, tumor volume ed around 80 mm3 and, despite a slight increase following cessation of treatment on day 26 and the end of the experiment on day 29, remained below 100 mm3.

This corresponds to a complete inhibition of tumor growth in these -treated animals, compared with saline and control—treated groups.

In the third experiment, mice were treated 4 times per week with a dose of 1x109 con15/ 107.2 mimic or control-loaded minicells, with treatments beginning once tumors d an average of 100 mm3 (Figure 8). In this experiment, tumors in the mice treated with control minicells grew at a rate similar to the first ment, with a slight reduction in growth as compared to the tumors in the saline-treated mice. In mice receiving the con15/ 107.2 mimic, there was a clear anti-tumor effect. For the first 10 days of the ment, corresponding to 8 treatments, the volume of the tumors was below the initial size of 100 mm3. Thereafter, tumor volume increased gradually to around 150 mm3 by the end of the experiment, compared with 250 and 225 mm3 in the saline and control-treated mice, respectively.

The inhibition of growth of MSTO—2llH-derived xenograft tumors observed in mice following administration of VCCtEDVmiR46 or V6“EDVc0n15/107_2 is exceptionally strong and s the inhibition observed in vitro in cultures of the same (and other) MPM cells (compare Figure 3 with Figures 7 and 8). This is remarkable considering the fact that in vitro >95% of cells are transfected with the miR mimic, whereas in vivo the number of tumor cells receiving the mimic is likely to be 510%.

The observed effect is believed to be caused by the inhibitory effects of miR-l6 (and other family members) on endothelial cells, thereby effectively targeting both tumor cells and stromal cells involved in angiogenesis, which is required for tumor growth. The growth D 27 inhibitory effects of the mimic treatment in the subcutaneous xenograft model are greater than effects reported for other systemic treatments in the same model.

********************** The present invention is well adapted to attain the ends and advantages mentioned as well as those that are inherent n. The particular embodiments disclosed above are illustrative only, as the present invention may be modified and practiced in different but equivalent manners nt to those skilled in the art having the benefit of the teachings herein. Furthermore, no limitations are ed to the details of uction or design herein shown, other than as described in the claims below. It is therefore evident that the particular illustrative embodiments disclosed above may be altered or modified and all such variations are considered within the scope and spirit of the present invention.

Claims (1)

1. A double-stranded microRNA mimic comprising: (a) a mature sequence corresponding to a miR-