EP2386640A2

Delivering functional nucleic acids to mammalian cells via bacterially-derived, intact minicells

Abstract

Intact bacterially derived minicells containing functional nucleic acids or plasmids encoding functional nucleic acids can reduce, in targeted mammalian cells, drug resistance, apoptosis resistance, and neoplasticity, respectively. Methodology that employs minicells to deliver functional nucleic acids, targeting the transcripts of proteins that contribute to drug resistance or apoptosis resistance, inter alia, can be combined with chemotherapy to increase the effectiveness of the chemotherapy.

EP2386640A2, drawing sheet 1 — Sheet 1 of 7

Term

Term ended

Projected expiry passed 25 August 2025, 1.1 years ago.

15 claims: 11 independent, 4 dependent

1
A composition comprising (a) an intact, bacterially derived minicell that contains a functional nucleic acid molecule or a plasmid comprising a segment that encodes a functional nucleic acid molecule and (b) a pharmaceutically acceptable carrier therefor, wherein said functional nucleic acid molecule targets the gene or the transcript of a protein that contributes to resistance to a chemotherapeutic drug.
3
A intact, bacterially derived minicell containing a functional nucleic acid molecule or a plasmid comprising a segment that encodes a functional nucleic acid molecule, where the functional nucleic acid molecule targets a gene or transcript of a protein that promotes resistance to a chemotherapeutic drug, for use as a medicament.
4
An intact, bacterially derived minicell containing a functional nucleic acid molecule or a plasmid comprising a segment that encodes a functional nucleic acid molecule, where the functional nucleic acid molecule targets the gene or transcript of a protein that promotes resistance to a chemotherapeutic drug, for use in the treatment of a malignancy resistant to said chemotherapeutic drug, wherein said intact minicell is administered simultaneously or consecutively with said chemotherapeutic drug to allow the functional nucleic acid to diminish resistance to said chemotherapeutic drug.
5
A chemotherapeutic drug for use in the treatment of a drug resistant malignancy, wherein an intact, bacterially derived minicell containing a functional nucleic acid molecule or a plasmid comprising a segment that encodes a functional nucleic acid molecule, where the functional nucleic acid molecule targets the gene or transcript of a protein that promotes resistance to said chemotherapeutic drug, is administered simultaneously or consecutively with said chemotherapeutic drug to allow the functional nucleic acid to diminish resistance to said chemotherapeutic drug.
6
A product containing a chemotherapeutic drug and an intact, bacterially derived minicell, said minicell containing a functional nucleic acid molecule or a plasmid comprising a segment that encodes a functional nucleic acid molecule, where the functional nucleic acid molecule targets the gene or transcript of a protein that promotes resistance against said drug, for use in the treatment of a malignancy resistant to said chemotherapeutic drug, wherein said drug and intact minicell is administrated simultaneously or consecutively.
10
The intact, bacterially derived minicell, the chemotherapeutic drug or the product according to any of the preceding claims, wherein said malignancy resistant to said chemotherapeutic drug is selected from the list consisting of cancer, AIDS and tuberculosis.
11
The intact, bacterially derived minicell, the chemotherapeutic drug or the product according to any of the preceding claims, wherein said minicell further comprises a bispecific ligand.
12
The intact, bacterially derived minicell, the chemotherapeutic drug or the product according to any of the preceding claims, wherein said bispecific ligand is selected from the list consisting of receptors, enzymes, binding peptides, fusion/chimeric proteins and small molecules.
13
The intact, bacterially derived minicell, the chemotherapeutic drug or the product according to any of the preceding claims, wherein said bispecific ligand has specificity for both a surface component on the minicell and a surface component on a non-phagocytic mammalian cell.
14
The intact bacterially derived minicell, the chemotherapeutic drug or the product according to any of the preceding claims, wherein the target of said minicell is a phagocysis- or endocytosis-competent mammalian cell.
15
The intact, bacterially derived minicell, the chemotherapeutic drug or the product according to any of the preceding claims for administration at least once a week over several weeks to several months.

Independent claims11

245 paragraphs in 5 sections, as filed

BACKGROUND OF THE INVENTION

0001The present invention relates to ongoing efforts to achieve effective delivery of functional nucleic acids to mammalian cells. More specifically, the invention relates to using bacterial minicell vectors to deliver functional nucleic acids to mammalian cells. The invention has particular utility for eliminating drug resistance, especially in the context of cancer and AIDS therapy, for promoting apoptosis and for countering neoplasticity in targeted cells.

0002Recent advances have highlighted a variety of techniques for introducing functional nucleic acids into cells. For example, liposome-based transfection methods can deliver exogenously produced nucleic acids. Such an exogenous approach has the drawback, however, of effecting only transient inhibition of a target. Additionally, liposomes are unstable in vivo. As an alternative to delivery of exogenously produced nucleic acids, vectors can deliver plasmids that encode functional nucleic acids, which are produced endogenously. The viral vectors currently useful for this purpose, however, poses serious safety concerns. Illustrative problems include recombination with wild-type viruses, insertional and oncogenic potential, virus-induced immunosuppression, limited capacity of the viral vectors to carry large segments of DNA, reversion to virulence of attenuated viruses, difficulties in recombinant virus manufacture and distribution, low stability, and adverse reactions, such as an inflammatory response, caused by existing immunity. An approach that obviated these problems would offer significant benefit in making delivery of functional nucleic acids safer and more effective.

0003An effective method of delivering functional nucleic acids would be particularly beneficial for reversing drug resistance. Mammalian cells employ a variety of biological processes to resist drugs, which poses a major obstacle to the successful treatment of cancer. Similarly, drug resistance limits the efficacy of HIV treatment, particularly to highly active antiretroviral therapy (HAART), which is based on a combination of nucleoside reverse transcriptase inhibitors (NRTIs) and protease inhibitors (PIs) or a non-nucleoside reverse transcriptase inhibitor (NNRTI).

0004Clinical tumor resistance to chemotherapy can be intrinsic or acquired. Intrinsic resistance exists at the time of diagnosis in tumors that fail to respond to first-line chemotherapy. Acquired resistance occurs in tumors that may respond well to initial treatment, but exhibit a resistant phenotype upon recurrence. Such tumors gain resistance both to previously used drugs and to new drugs, including drugs with different structures and mechanisms of action. The term MDR (multidrug resistance) describes this phenomenon in which tumor cells become cross-resistant to several structurally unrelated drugs after exposure to a single drug.

0005The mechanisms for multi-drug resistance are complex and multifactorial, owing largely to the high level of genomic instability and mutations in cancer cells. Exemplary mechanisms are drug inactivation, extrusion of drug by cell membrane pumps, decreased drug influx, mutations of drug targets and failure to initiate apoptosis (Bredel, 2001; Chen et al., 2001; White and McCubrey, 2001; Sun et al., 2001).

0006Drug extrusion is particularly common, and can result from over-expression of membrane-associated proteins that pump drugs from the intracellular to the extracellular environment. Such pumps often are members of the ATP-binding cassette (ABC) transporter superfamily (Doige et al., 1993). P-glycoprotein (Pgp) is one such example, and is a major contributor to MDR in a variety of cancer cells (Endicott et al., 1989; Litman et al., 2001). Other examples include the MDR-associated protein (MRP; Cole et al., 1992), breast cancer resistance protein (BCRP; Litman et al., 2000), and lung resistance-related protein (LRP; a major vault protein; Scheffer et al., 2000). Other multidrug transporter proteins also have been identified in cancer cells (Gottesman et al., 2002) and in pathogenic microorganisms (Van Bambeke et al., 2000).

0007Resistance to apoptosis (programmed cell death) of tumor cells induced by cytotoxic agents and radiation (Sellers and Fisher, 1999) is another common mechanism. This mechanism frequently involves over-expression of anti-apoptotic proteins, such as B-cell leukemia protein 2 (Bcl-2), Bcl-XL, Bcl-W, A1/Bf11, Mcl-1 and mutations in the p53 protein. Although a precise understanding of how proteins like Bcl-2 exerts their anti-apoptotic effects remains elusive, the proteins are over-expressed in many cancers including colorectal, prostate, and breast cancers (Hanada, et al., 1995; Bakhshi et al., 1985; Wang et al., 1996). Increased expression of the transcription factor nuclear factor kappa B (NF-κB) also is a major mechanism for tumor cells to acquire chemotherapy resistance (Wang et al., 1999).

0008Drugs to counter MDR have been identified, such as drugs that block the action of P-glycoprotein (List et al., 1993; Miller et al., 1991; Wishart et al., 1992). Many such drugs were ineffective in clinical trials, however, because they bound to the plasma of patients, could not reach their destination (Ayesh et al., 1996a; Broxterman et al., 1987; Lehnert et al., 1996) and were toxic to normal cells. The use of functional nucleic acids to counter MDR also has been attempted. Yet, as noted above, existing vectors for this purpose are unstable or toxic, or they pose other serious safety issues, which hamper their use in humans (Sioud, 2004).

0009Accordingly, a continuing need exists for tools and methods for delivering functional nucleic acids that reduce drug resistance, promote apoptosis, and counter neoplasticity in target cells.

SUMMARY OF THE INVENTION

0010To address these and other needs, the present invention provides, in one aspect, a method of delivering a functional nucleic acid, comprising (a) providing an intact minicell that contains a functional nucleic acid molecule or contains a plasmid comprising a segment that encodes a functional nucleic acid molecule, then (b) bringing the minicell into contact with a target mammalian cell, such that the mammalian cell engulfs the minicell. Following engulfment of the minicell, the functional nucleic acid molecule is released into the cytoplasm, transported to the nucleus and expressed by the target cell. The aforementioned plasmid also may contain a regulatory element, such as a promoter, a terminator, an enhancer or a signal sequence that is operably linked to the segment that encodes a functional nucleic acid molecule. It is particularly advantageous for the plasmid to comprise a promoter that is dependent on either RNA polymerase (pol) II or pol III, such as the RNA III polymerase promoters human 7SK, H1 and U6. Further, the plasmid may contain a reporter element, such as a nucleic acid segment coding for green fluorescent protein. Contact between the minicell and the mammalian cell may be in vitro or in vivo.

0011In relation to this invention, the category of "functional nucleic acids" encompasses: siRNA molecules, including shRNA molecules; miRNA molecules, antisense molecules; and ribozyme molecules. Preferably, the functional nucleic acid molecule targets the gene or transcript of a protein that promotes drug resistance, inhibits apoptosis, or contributes to a neoplastic phenotype. Particularly useful targets that contribute to drug resistance include ATP binding cassette transporters such as P-glycoprotein, MDR-2, MDR-3, BCRP, APT11a and LRP. Particularly useful targets that contribute to apoptosis resistance include Bcl-2 (B cell leukemia/lymphoma), Bcl-XL, A1/Bfl 1, focal adhesion kinase and p53 mutant protein. Other useful targets are oncogenic proteins and mutant tumor suppressor proteins.

0012In another aspect, the invention provides a method of overcoming drug resistance or apoptosis resistance and treating a malignancy in a subject. The method comprises (a) providing an intact minicell that contains a functional nucleic acid molecule or a plasmid comprising a segment that encodes a functional nucleic acid molecule, where the functional nucleic acid molecule targets the transcript of a protein that promotes drug resistance (b) bringing the minicell into contact with a target mammalian cell, such that the mammalian cell engulfs the minicell, and (c) delivering a chemotherapeutic drug to the target mammalian cell. Preferably, step (c) is performed after steps (a) and (b), to allow the functional nucleic acid to diminish resistance to the drug prior to the drug's administration. The drug may be delivered by any conventional means, but it preferably is delivered via an intact minicell.

0013In certain embodiments of the invention, the minicell is brought into contact with the target mammalian cell via a bispecific ligand. The bispecific ligand has specificity for both a surface component on the minicell and a surface component on the mammalian cell, such as a receptor. As a result, the ligand causes the minicell to bind to the mammalian cell, the minicell is engulfed by the mammalian cell, and the minicell payload is released into the cytoplasm of the mammalian cell. In other embodiments of the invention, the minicell is brought into contact with a target mammalian cell that is phagocytosis- or endocytosis-competent. The use of bispecific ligands is optional when a target cell is phagocytosis-competent.

0014In another aspect, the invention provides a composition comprising (i), intact minicells and (ii) a pharmaceutically acceptable carrier therefor, where the minicells contain a functional nucleic acid molecule or a plasmid that encodes a functional nucleic acid molecule. The functional nucleic acid molecule may be an shRNA or miRNA or other siRNA molecule, an antisense molecule, or a ribozyme molecule. Preferably, the functional nucleic acid molecule targets the gene or transcript of a protein that promotes drug resistance, inhibits apoptosis, or contributes to a neoplastic phenotype. Particularly useful targets that contribute to drug resistance include ATP binding cassette transporters such as P-glycoprotein, MDR-2 and MDR-3. Particularly useful targets that contribute to apoptosis resistance include Bcl-2 (B cell leukemia/lymphoma), Bcl-XL, Al/Bfl 1, focal adhesion kinase and p53 mutant protein. Other useful targets are oncogenic proteins and mutant tumor suppressor proteins. The plasmid may contain a regulatory element, such as a promoter, a terminator, an enhancer or a signal sequence that is operably linked to the segment that encodes a functional nucleic acid molecule. Further, the plasmid may contain a reporter element. The functional nucleic acid molecule may comprise multiple RNA interference sequences as miRNA or shRNA and these may be co-cistronic or expressed from separate promoters to enable simultaneous knockdown of multiple targets associated with drug resistance. In preferred embodiments, the composition contains fewer than one contaminating parent cell per 107, 108, 109, 1010, 1011 or 1012 minicells.

0015In still another aspect, the invention provides for a use of intact minicells in the preparation of a medicament for use in a method of overcoming drug resistance or promoting apoptosis by administration of the medicament to a cell, tissue or organ. In the medicament, minicells contain a functional nucleic acid molecule or a plasmid encoding a functional nucleic acid molecule, where the functional nucleic acid molecule targets the transcript of a protein that promotes drug resistance or inhibits apoptosis. The disease treated in this context may be a cancer, for example, or an acquired disease, such as AIDS and tuberculosis.

0016The invention affords significant improvements over conventional methods and formulations for delivering functional nucleic acids in the context of cancer and HIV by (i) providing safe and stable vehicles for delivering functional nucleic acids, (ii) countering the principal mechanisms of drug resistance in diseased cells, (iii) reducing toxic side-effects associated with overcoming drug resistance, and (iv) providing targeted and drug-packaged vehicles to treat the disease.

BRIEF DESCRIPTION OF THE DRAWINGS

0017<figref idref="f0001">Figure 1</figref> shows the effect of various treatments on the viability of human colon cancer cell line Caco-2. The treatments are shown on the x-axis (minicells are designated by "M") and percent cell viability is shown by the bars. Each bar is a mean of six independent measurements and standard deviation of the mean is shown.

0018<figref idref="f0002">Figure 2</figref> shows regression of human colon cancer (Caco-2) xenografts in nude mice (11 mice per group) following dual treatment with (1) targeted recombinant minicells carrying shRNA encoding plasmids (anti-bcl2 or anti-Mdr1) and (2) targeted minicells packaged with the chemotherapeutic drug Irinotecan. The bispecific antibody used to target the colon cancer cells carried specificity against S. typhimurium O-antigen on one arm and human epidermal growth factor receptor (EGFR) on the other arm. The targeted recombinant minicells were injected intravenously on days 9 and 23, and the targeted Irinotecan packaged minicells were given intravenously on days 15, 18, 29 and 32. Other control treatments administered intravenously include: Group 1 - tumor only, Group 2 - free irinotecan, Group 3 - EGFRminicellsIrino, Group 4 - EGFRminicellsshRNA-MDR-1, Group 5 - EGFRminicellsshRNA-bcl-2 and Group 6 - EGFRminicellsshRNA-MDR-1 followed by free Irino. Tumor volume is shown on the y-axis. SEM is shown for each measurement.

0019<figref idref="f0003">Figure 3</figref> shows regression of human colon cancer (Caco-2) xenografts in nude mice (11 mice per group) following dual treatment with (1) targeted recombinant minicells carrying shRNA encoding plasmids (anti-bcl2 or anti-Mdr1) and (2) targeted minicells packaged with the chemotherapeutic drug 5-FU. The bispecific antibody used to target the colon cancer cells carried specificity against S. typhimurium O-antigen on one arm and human epidermal growth factor receptor (EGFR) on the other arm. The targeted recombinant minicells were injected intravenously on days 9 and 23, and the targeted 5-FU packaged minicells were given intravenously on days 15, 18, 29 and 32. Other control treatments administered intravenously include: G1 - tumor only, G2 (control), free 5-FU (5 x 104 ng/gm of mouse body weight ~ 1 x 106 ng per mouse), G3 (control),EGFRminicells5-FU. G4 (control),EGFRminicellsshRNA-MDR-1, G5 (control),EGFRminicellsshRNA-bcl-2, G6 (control),EGFRminicellsshRNA-MDR-1 followed by CMVminicells5-FU, G7 (control),EGFRminicellsshRNA-nonsense followed by EGFRminicells5-FU, G8 (control),EGFRminicellsshRNA-MDR-1 followed by free 5-FU, G9 (expt),EGFRminicellsshRNA-MDR-1 followed by EGFRminicells5-FU, and G10 (expt),EGFRminicellsshRNA-bcl-2 followed by EGFR minicells5-FU. Tumor volume is shown on the y-axis. SEM is shown for each measurement.

0020<figref idref="f0004">Figure 4</figref> shows regression of human breast cancer (MDA-MB-468) xenografts in nude mice (11 mice per group) following dual treatment with (1) targeted recombinant minicells carrying shRNA encoding plasmid (anti-MDR-1) and (2) targeted minicells packaged with chemotherapeutic drug doxorubicin. The bispecific antibody used to target the breast cancer cells carried specificity against S. typhimurium O-antigen on one arm and human EGFR on the other arm. The targeted recombinant minicells were injected intravenously on day 21 and the targeted Dox-packaged minicells were given intravenously on days 27, 34 and 41. Treatments administered intravenously include: G1 - tumor only, G2 (control),EGFRminicellsDox, and G3 (expt), EGFRminicellsshRNA-MDR-1 followed by EGFRminicellsDox. Tumor volume is shown on the y-axis. SEM is shown for each measurement.

0021<figref idref="f0005">Figure 5</figref> shows the effect of dosing schedules on reversal of drug-resistance and therapeutic effect. Human colon cancer (Caco-2) xenografts were established in nude mice and the following intravenous treatments were administered: G1 - tumor only, G2 (control), free irinotecan, G3 (expt),EGFRminicellsshRNA-MDR-1 followed 96 hrs later by EGFRminicellsIrino, G4 (expt),EGFRminicellsshRNA-MDR-1 followed 120 hrs later by EGFRminicellsIrino, and G5 (expt),EGFRminicellsshRNA-MDR-1 followed 144 hrs later by EGFRminicellsIrino. The bispecific antibody used to target the breast cancer cells carried specificity against S. typhimurium O-antigen on one arm and human EGFR on the other arm. Tumor volume is shown on the y-axis. SEM is shown for each measurement.

0022<figref idref="f0006">Figure 6</figref> shows the effect of dosing schedules on reversal of drug-resistance and therapeutic effect. Human colon cancer (Caco-2) xenografts were established in nude mice and the following intravenous treatments were administered: G1 - tumor only, G2 (control), free 5-FU, G3 (expt),EGFRminicellsshRNA-MDR-1 followed 96 hrs later by EGFRminicells5-FU, G4 (expt),EGFRminicellsshRNA-MDR-1 followed 120 hrs later by EGFRminicells5-FU, and G5 (expt),EGFRminicellsshRNA-MDR-1 followed 144 hrs later by EGFRminicells5-FU. The bispecific antibody used to target the breast cancer cells carried specificity against S. typhimurium O-antigen on one arm and human EGFR on the other arm. Tumor volume is shown on the y-axis. SEM is shown for each measurement.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

0023The inventors have discovered that intact bacterially derived minicells can safely and effectively introduce into target mammalian cells any of a range of functional nucleic acids, such as an siRNA molecule, a miRNA molecule, a ribozyme, or an antisense nucleic acid. In the particular context of cancer and HIV infection, the inventors have found that the introduction of functional nucleic acids into target cells, via intact minicells, can diminish drug resistance or apoptosis resistance in the target cells.

0024The inventors also have discovered that minicells can sequentially transfect the same target mammalian cells, particularly in vivo, and that minicells can sequentially deliver a range of different payloads to the same target mammalian cells. These discoveries are the first for any macroparticulate delivery vehicle and provide, for the first time, a method to treat complex multifactorial diseases like cancer and HIV where different therapeutic payloads need to be delivered to the same cell before a therapeutic effect is achieved. Similarly, the inventors have discovered that the complex problem of drug resistance associated with multiple mutations in different genes can be addressed with minicells that introduce multiple RNAi sequences into a host cell to counteract the multitude of genetic defects, and that following minicell-mediated RNAi delivery and allowing sufficient time for knockdown of target drug resistance-mediating proteins, cancer cells formerly resistant to specific chemotherapeutic drugs can effectively be treated with minicells packaged with the same drugs. This is the first in vivo demonstration of effectively treating cancer that is refractory to all other methods of treatment. The concentration of chemotherapeutic drugs, delivered via minicells, required to treat drug-resistant cancer cells effectively is discovered to be over 1000-fold less than free drug treatment. This is a surprising discovery because all previous methods to reverse drug resistance using RNAi or inhibitors of drug resistance-mediating proteins still required drug concentrations that can cause severe toxicity to a mammalian subject. Thus, methods of the invention, i.e., minicell-mediated delivery of RNAi followed by minicell-mediated chemotherapeutic drug, has the potential to treat cancer effectively without severe toxicity.

0025Additionally, the inventors have discovered that the serotype of minicells can be adapted to overcome a host immune response against minicells.

0026The following description outlines the invention related to these discoveries, without limiting the invention to the particular embodiments, methodology, protocols, or reagents described. Likewise, the terminology used here describes particular embodiments only and does not limit the scope of the invention.

Definitions

0027Unless defined otherwise, all technical and scientific terms used in this description have the same meaning as commonly understood by those skilled in the relevant art.

0028For convenience, the meaning of certain terms and phrases employed in the specification, examples, and appended claims are provided below. Other terms and phrases are defined throughout the specification.

0029The singular forms "a," "an," and "the" include plural reference unless the context clearly dictates otherwise.

0030"Antisense oligonucleotide" refers to a nucleic acid molecule complementary to a portion of a particular gene transcript that can hybridize to the transcript and block its translation. An antisense oligonucleotide may comprise RNA or DNA.

0031"Biomolecular sequence" or "sequence" refers to all or a portion of a polynucleotide or polypeptide sequence.

0032"Cancer," "neoplasm," "tumor," "malignancy" and "carcinoma," used interchangeably herein, refer to cells or tissues that exhibit an aberrant growth phenotype characterized by a significant loss of control of cell proliferation. The methods and compositions of this invention particularly apply to precancerous, malignant, pre-metastatic, metastatic, and non-metastatic cells.

0033"Complementary" refers to the topological compatibility or matching together of the interacting surfaces of two molecules, such as a functional nucleic acid molecule and its target. The molecules can be described as complementary, and furthermore, the contact surface characteristics are complementary to each other.

0034"Corresponds to" or "represents" when used in the context of, for example, a polynucleotide or sequence that "corresponds to" or "represents" a gene means that a sequence of the polynucleotide is present in the gene or in the nucleic acid gene product, e.g., mRNA. The polynucleotide may be wholly present within an exon of a genomic sequence of the gene, or different portions of the sequence of the polynucleotide may be present in different exons, e.g., such that the contiguous polynucleotide sequence is present in an mRNA, either pre- or post-splicing, that is an expression product of the gene.

0035"Cytokine" is a generic term for proteins released by one cell population that act on another cell population as intercellular mediators.

0036"Drug" refers to any physiologically or pharmacologically active substance that produces a local or systemic effect in animals, particularly mammals and humans.

0037"Expression" generally refers to the process by which a polynucleotide sequence undergoes successful transcription and translation such that detectable levels of the amino acid sequence or protein are expressed. In certain contexts herein, expression refers to the production of mRNA. In other contexts, expression refers to the production of protein.

0038"Functional nucleic acid" refers to a nucleic acid molecule that, upon introduction into a host cell, specifically interferes with expression of a protein. In general, functional nucleic acid molecules have the capacity to reduce expression of a protein by directly interacting with a transcript that encodes the protein. Ribozymes, antisense nucleic acids and siRNA molecules, including shRNA molecules, short RNAs (typically less than 400 bases in length), micro-RNAs (miRNAs) constitute exemplary functional nucleic acids.

0039"Gene" refers to a polynucleotide sequence that comprises control and coding sequences necessary for the production of a polypeptide or precursor. The polypeptide can be encoded by a full length coding sequence or by any portion of the coding sequence. A gene may constitute an uninterrupted coding sequence or it may include one or more introns, bound by the appropriate splice junctions. Moreover, a gene may contain one or more modifications in either the coding or the untranslated regions that could affect the biological activity or the chemical structure of the expression product, the rate of expression, or the manner of expression control. Such modifications include, but are not limited to, mutations, insertions, deletions, and substitutions of one or more nucleotides. In this regard, such modified genes may be referred to as "variants" of the "native" gene.

0040"Host cell" refers to a cell that may be, or has been, used as a recipient for a recombinant vector or other transfer of polynucleotides, and includes the progeny of the original cell that has been transfected. The progeny of a single cell may not necessarily be completely identical in morphology or in genomic or total DNA complement as the original parent due to natural, accidental, or deliberate mutation.

0041"Hybridization" refers to any process by which a polynucleotide sequence binds to a complementary sequence through base pairing.

0042"Individual," "subject," "host," and "patient," used interchangeably herein, refer to any mammalian subject for whom diagnosis, treatment, or therapy is desired. In one preferred embodiment, the individual, subject, host, or patient is a human. Other subjects may include, but are not limited to, cattle, horses, dogs, cats, guinea pigs, rabbits, rats, primates, and mice.

0043"Label" refers to agents that are capable of providing a detectable signal, either directly or through interaction with one or more additional members of a signal producing system. Labels that are directly detectable and may find use in the invention include fluorescent labels. Specific fluorophores include fluorescein, rhodamine, BODIPY, cyanine dyes and the like. The invention also contemplates the use of radioactive isotopes, such as 35S, 32P, 3H, and the like as labels. Colorimetric labels such as colloidal gold or colored glass or plastic (e.g., polystyrene, polypropylene, latex) beads may also be utilized. For instance, see <patcit id="pcit0001" dnum="US4366241A"><text>U.S. patents No. 4,366,241</text></patcit>, No. <patcit id="pcit0002" dnum="US4277437A"><text>4,277,437</text></patcit>, No. <patcit id="pcit0003" dnum="US4275149A"><text>4,275,149</text></patcit>, No. <patcit id="pcit0004" dnum="US3996345A"><text>3,996,345</text></patcit>, No. <patcit id="pcit0005" dnum="US3939350A"><text>3,939,350</text></patcit>, No. <patcit id="pcit0006" dnum="US3850752A"><text>3,850,752</text></patcit>, and No. <patcit id="pcit0007" dnum="US3817837A"><text>3,817,837</text></patcit>.

0044"Oligonucleotide" refers to a polynucleotide comprising, for example, from about 10 nucleotides (nt) to about 1000 nt. Oligonucleotides for use in the invention are preferably from about 10 nt to about 150 nt. The oligonucleotide may be a naturally occurring oligonucleotide or a synthetic oligonucleotide. Oligonucleotides may be modified.

0045"Minicell" refers to anucleate forms of bacterial cells, engendered by a disturbance in the coordination, during binary fission, of cell division with DNA segregation. Minicells are distinct from other small vesicles that are generated and released spontaneously in certain situations and, in contrast to minicells, are not due to specific genetic rearrangements or episomal gene expression. For practicing the present invention, it is desirable for minicells to have intact cell walls ("intact minicells").

0046"Modified oligonucleotide" and "Modified polynucleotide" refer to oligonucleotides or polynucleotides with one or more chemical modifications at the molecular level of the natural molecular structures of all or any of the bases, sugar moieties, internucleoside phosphate linkages, as well as to molecules having added substitutions or a combination of modifications at these sites. The internucleoside phosphate linkages may be phosphodiester, phosphotriester, phosphoramidate, siloxane, carbonate, carboxymethylester, acetamidate, carbamate, thioether, bridged phosphoramidate, bridged methylene phosphonate, phosphorothioate, methylphosphonate, phosphorodithioate, bridged phosphorothioate or sulfone internucleotide linkages, or 3'-3', 5'-3', or 5'-5' linkages, and combinations of such similar linkages. The phosphodiester linkage may be replaced with a substitute linkage, such as phosphorothioate, methylamino, methylphosphonate, phosphoramidate, and guanidine, and the ribose subunit of the polynucleotides may also be substituted (e.g., hexose phosphodiester; peptide nucleic acids). The modifications may be internal (single or repeated) or at the end(s) of the oligonucleotide molecule, and may include additions to the molecule of the internucleoside phosphate linkages, such as deoxyribose and phosphate modifications which cleave or crosslink to the opposite chains or to associated enzymes or other proteins. The terms "modified oligonucleotides" and "modified polynucleotides" also include oligonucleotides or polynucleotides comprising modifications to the sugar moieties (e.g., 3'-substituted ribonucleotides or deoxyribonucleotide monomers), any of which are bound together via 5' to 3' linkages.

0047The phrase "nucleic acid molecules" and the term "polynucleotides" denote polymeric forms of nucleotides of any length, either ribonucleotides or deoxynucleotides. They include single-, double-, or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or a polymer comprising purine and pyrimidine bases or other natural, chemically or biochemically modified, non-natural, or derivatized nucleotide bases. The backbone of a polynucleotide can comprise sugars and phosphate groups (as may typically be found in RNA or DNA), or modified or substituted sugar or phosphate groups. Alternatively, the backbone of the polynucleotide can comprise a polymer of synthetic subunits such as phosphoramidites and thus can be an oligodeoxynucleoside phosphoramidate or a mixed phosphoramidate-phosphodiester oligomer. A polynucleotide may comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs, uracyl, other sugars, and linking groups such as fluororibose and thioate, and nucleotide branches. A polynucleotide may be further modified, such as by conjugation with a labeling component. Other types of modifications include caps, substitution of one or more of the naturally occurring nucleotides with an analog, and introduction of means for attaching the polynucleotide to proteins, metal ions, labeling components, other polynucleotides, or a solid support.

0048"Pharmaceutically acceptable" refers to physiological compatibility. A pharmaceutically acceptable carrier or excipient does not abrogate biological activity of the composition being administered, is chemically inert and is not toxic to the organism in which it is administered.

0049"Polypeptide" and "protein," used interchangeably herein, refer to a polymeric form of amino acids of any length, which may include translated, untranslated, chemically modified, biochemically modified, and derivatized amino acids. A polypeptide or protein may be naturally occurring, recombinant, or synthetic, or any combination of these. Moreover, a polypeptide or protein may comprise a fragment of a naturally occurring protein or peptide. A polypeptide or protein may be a single molecule or may be a multi-molecular complex. In addition, such polypeptides or proteins may have modified peptide backbones. The terms include fusion proteins, including fusion proteins with a heterologous amino acid sequence, fusions with heterologous and homologous leader sequences, with or without N-terminal methionine residues, immunologically tagged proteins, and the like.

0050"Purified" refers to a compound that is removed from its natural environment and is at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 99.99% free from other components with which it is naturally associated.

0051"Ribozyme" refers to an RNA molecule having an enzymatic activity that can repeatedly cleave other RNA molecules in a nucleotide base sequence-specific manner.

0052"RNA interference" (RNAi) refers to sequence-specific or gene specific suppression of gene expression (protein synthesis) that is mediated by short interfering RNA (siRNA), short-haripin RNA, short RNA or micro-RNA.

0053"Sequence Identity" refers to a degree of similarity or complementarity. There may be partial identity or complete identity. A partially complementary sequence is one that at least partially inhibits an identical sequence from hybridizing to a target polynucleotide; it is referred to using the functional term "substantially identical." The inhibition of hybridization of the completely complementary sequence to the target sequence may be examined using a hybridization assay (Southern or Northern blot, solution hybridization, and the like) under conditions of low stringency. A substantially identical sequence or probe will compete for and inhibit the binding (i.e., the hybridization) of a completely identical sequence or probe to the target sequence under conditions of low stringency. This is not to say that conditions of low stringency are such that non-specific binding is permitted; low stringency conditions require that the binding of two sequences to one another be a specific (i.e., selective) interaction. The absence of non-specific binding may be tested by the use of a second target sequence which lacks even a partial degree of complementarity (e.g., less than about 30% identity); in the absence of non-specific binding, the probe will not hybridize to the second non-complementary target sequence.

0054Another way of viewing sequence identity, in the context to two nucleic acid or polypeptide sequences, entails referencing residues in the two sequences that are the same when aligned for maximum correspondence over a specified region. As used here, "percentage of sequence identity" means the value determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid base occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity.

0055"Short interfering RNA" (siRNA) refers to double-stranded RNA molecules, generally, from about 10 to about 30 nucleotides long that are capable of mediating RNA interference (RNAi). As used herein, the term siRNA includes short hairpin RNAs, also known as shRNAs.

0056The terms "treatment," "treating," "treat," and the like refer to obtaining a desired pharmacological and/or physiologic effect. The effect may be prophylactic in terms of completely or partially preventing a disease or symptom thereof and/or may be therapeutic in terms of a partial or complete stabilization or cure for a disease and/or adverse effect attributable to the disease. "Treatment" covers any treatment of a disease in a mammal, particularly a human, and includes: (a) preventing the disease or symptom from occurring in a subject which may be predisposed to the disease or symptom but has not yet been diagnosed as having it; (b) inhibiting the disease symptom, i.e., arresting its development; or (c) relieving the disease symptom, i.e., causing regression of the disease or symptom.

II.

Delivery of Functional Nucleic Acids via Minicells

0057In one aspect, the invention provides a method of delivering a functional nucleic acid to a target cell, comprising (a) providing an intact minicell that contains a functional nucleic acid molecule or a plasmid comprising a segment that encodes a functional nucleic acid molecule, then, (b) bringing the minicell into contact with a target mammalian cell, such that the mammalian cell engulfs the minicell. Following engulfment of the minicell, the functional nucleic acid molecule is released into the cytoplasm of the target cell or expressed by the target cell. Minicells may be brought into contact with the target mammalian cells via bispecific ligands, as described in <patcit id="pcit0008" dnum="WO2005056749A"><text>WO 2005/056749</text></patcit>. Contact between the minicell and the target mammalian cell may be in vitro or in vivo.

Minicells

0058Minicells of the invention are anucleate forms of E. coli or other bacterial cells, engendered by a disturbance in the coordination, during binary fission, of cell division with DNA segregation. Prokaryotic chromosomal replication is linked to normal binary fission, which involves mid-cell septum formation. In E. coli, for example, mutation of min genes, such as minCD, can remove the inhibition of septum formation at the cell poles during cell division, resulting in production of a normal daughter cell and an anucleate minicell. See de Boer et al., 1992; Raskin & de Boer, 1999; Hu & Lutkenhaus, 1999; Harry, 2001. Minicells are distinct from other small vesicles that are generated and released spontaneously in certain situations and, in contrast to minicells, are not due to specific genetic rearrangements or episomal gene expression. For practicing the present invention, it is desirable for minicells to have intact cell walls ("intact minicells").

0059In addition to min operon mutations, anucleate minicells also are generated following a range of other genetic rearrangements or mutations that affect septum formation, for example in the divIVB1 in B. subtilis. See Reeve and Cornett, 1975; Levin et al., 1992. Minicells also can be formed following a perturbation in the levels of gene expression of proteins involved in cell division/chromosome segregation. For example, overexpression of minE leads to polar division and production of minicells. Similarly, chromosome-less minicells may result from defects in chromosome segregation for example the smc mutation in Bacillus subtilis (Britton et al., 1998), spoOJ deletion in B. subtilis (Ireton et al., 1994), mukB mutation in E. coli (Hiraga et al., 1989), and parC mutation in E. coli (Stewart and D'Ari, 1992). Gene products may be supplied in trans. When over-expressed from a high-copy number plasmid, for example, CafA may enhance the rate of cell division and/or inhibit chromosome partitioning after replication (Okada et al., 1994), resulting in formation of chained cells and anucleate minicells (Wachi et al., 1989; Okada et al., 1993). Minicells can be prepared from any bacterial cell of Gram-positive or Gram-negative origin.

0060In accordance with the invention, minicells contain a functional nucleic acid or a plasmid that encodes a functional nucleic acid for which delivery is desired. "Functional" nucleic acid molecules of the invention have the capacity to reduce expression of a protein by directly interacting with a transcript that encodes the protein. siRNA molecules, ribozymes, and antisense nucleic acids constitute exemplary functional nucleic acids.

siRNA Molecules

0061Short interfering RNA (siRNA) molecules are useful for performing RNA interference (RNAi), a post-transcriptional gene silencing mechanism. siRNA generally refers to double-stranded RNA molecules from about 10 to about 30 nucleotides long that are named for their ability specifically to interfere with protein expression. Preferably, siRNA molecules are 12-28 nucleotides long, more preferably 15-25 nucleotides long, still more preferably 19-23 nucleotides long and most preferably 21-23 nucleotides long. Therefore, preferred siRNA molecules are 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27 28 or 29 nucleotides in length.

0062The length of one strand designates the length of an siRNA molecule. For instance, an siRNA that is described as 21 ribonucleotides long (a 21-mer) could comprise two opposite strands of RNA that anneal together for 19 contiguous base pairings. The two remaining ribonucleotides on each strand would form an "overhang." When an siRNA contains two strands of different lengths, the longer of the strands designates the length of the siRNA. For instance, a dsRNA containing one strand that is 21 nucleotides long and a second strand that is 20 nucleotides long, constitutes a 21-mer.

0063siRNAs that comprise an overhang are desirable. The overhang may be at the 5' or the 3' end of a strand. Preferably, it is at the 3' end of the RNA strand. The length of an overhang may vary, but preferably is about 1 to about 5 bases, and more preferably is about 2 nucleotides long. Preferably, the siRNA of the present invention will comprise a 3' overhang of about 2 to 4 bases. More preferably, the 3' overhang is 2 ribonucleotides long. Even more preferably, the 2 ribonucleotides comprising the 3' overhang are uridine (U).

0064According to the invention, the term siRNA includes short hairpin RNAs (shRNAs). shRNAs comprise a single strand of RNA that forms a stem-loop structure, where the stem consists of the complementary sense and antisense strands that comprise a double-stranded siRNA, and the loop is a linker of varying size. The stem structure of shRNAs generally is from about 10 to about 30 nucleotides long. Preferably, the stem of shRNA molecules are 12-28 nucleotides long, more preferably 15-25 nucleotides long, still more preferably 19-23 nucleotides long and most preferably 21-23 nucleotides long. Therefore, preferred shRNA molecules comprise stems that are 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27 28 or 29 nucleotides in length.

0065siRNAs of the invention are designed to interact with a target ribonucleotide sequence, meaning they complement a target sequence sufficiently to hybridize to the target sequence. In one embodiment, the invention provides an siRNA molecule comprising a ribonucleotide sequence at least 70%, 75%, 80%, 85% or 90% identical to a target ribonucleotide sequence or the complement of a target ribonucleotide sequence. Preferably, the siRNA molecule is at least 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the target ribonucleotide sequence or the complement of the target ribonucleotide sequence. Most preferably, an siRNA will be 100% identical to the target nucleotide sequence or the complement of the ribonucleotide sequence. However, siRNA molecules with insertions, deletions or single point mutations relative to a target may also be effective.

0066Tools to assist siRNA design are readily available to the public. For example, a computer-based siRNA design tool is available on the internet at www.dharmacon.com.

Ribozymes

0067Ribozymes are RNA molecules having an enzymatic activity that can repeatedly cleave other RNA molecules in a nucleotide base sequence-specific manner. Such enzymatic RNA molecules may be targeted to virtually any RNA transcript, and efficient cleavage achieved in vitro.

0068Six basic varieties of naturally-occurring enzymatic RNAs are known presently. Each can catalyze the hydrolysis of RNA phosphodiester bonds in trans (and thus can cleave other RNA molecules) under physiological conditions. In general, enzymatic polynucleotides act by first binding to a target RNA. Such binding occurs through the target binding portion of a enzymatic polynucleotide which is held in close proximity to an enzymatic portion of the molecule that acts to cleave the target RNA. Thus, the enzymatic polynucleotide first recognizes and then binds a target RNA through complementary base-pairing, and once bound to the correct site, acts enzymatically to cut the target RNA. Strategic cleavage of such a target RNA will destroy its ability to direct synthesis of an encoded protein. After an enzymatic polynucleotide has bound and cleaved its RNA target, it is released from that RNA to search for another target and can repeatedly bind and cleave new targets.

0069The enzymatic nature of a ribozyme is advantageous. Because a single ribozyme molecule is able to cleave many molecules of target RNA, effective concentrations of ribozyme can be quite low.

0070Useful ribozymes may comprise one of several motifs, including hammerhead (Rossi et al. (1992)), hairpin (Hampel and Tritz, (1989), Hampel et al. (1990)), hepatitis delta virus motif (Perrotta and Been (1992), group I intron (<patcit id="pcit0009" dnum="US4987071A"><text>U.S. Patent No. 4,987,071</text></patcit>), RNaseP RNA in association with an RNA guide sequence (Guerrier-Takada et al. (1983)), and Neurospora VS RNA (Saville & Collins (1990); Saville & Collins (1991); Collins & Olive (1993)). These specific motifs are not limiting, as all that is important in a ribozyme of this invention is that it has a specific substrate binding site that is complementary to one or more target RNA regions, and that it have nucleotide sequences within or surrounding that substrate binding site which impart an RNA cleaving activity to the molecule.

0071Ribozymes of the invention may comprise modified oligonucleotides (e.g., for improved stability, targeting, etc.). Nucleic acid sequences encoding the ribozymes may be under the control of a strong constitutive promoter, such as, for example, RNA Polymerase II or RNA Polymerase III promoter, so that transfected cells will produce sufficient quantities of the ribozyme to destroy target endogenous messages and inhibit translation.

Antisense Oligonucleotides

0072Antisense oligonucleotides of the invention specifically hybridize with a nucleic acid encoding a protein, and interfere with transcription or translation of the protein. In one embodiment, an antisense oligonucleotide targets DNA and interferes with its replication and/or transcription. In another embodiment, an antisense oligonucleotide specifically hybridizes with RNA, including pre-mRNA and mRNA. Such antisense oligonucleotides may affect, for example, translocation of the RNA to the site of protein translation, translation of protein from the RNA, splicing of the RNA to yield one or more mRNA species, and catalytic activity that may be engaged in or facilitated by the RNA. The overall effect of such interference is to modulate, decrease, or inhibit target protein expression.

0073There are several sites within a gene that may be utilized in designing an antisense oligonucleotide. For example, an antisense oligonucleotide may bind the region encompassing the translation initiation codon, also known as the start codon, of the open reading frame. In this regard, "start codon and "translation initiation codon" generally refer to the portion of such mRNA or gene that encompasses from at least about 25 to at least about 50 contiguous nucleotides in either direction (i.e., 5' or 3') from a translation initiation codon.

0074Another site for antisense interaction to occur is the termination codon of the open reading frame. The terms "stop codon region" and "translation termination codon region" refer generally to a portion of such a mRNA or gene that encompasses from at least about 25 to at least about 50 contiguous nucleotides in either direction form a translation termination codon.

0075The open reading frame or coding region also may be targeted effectively. The open reading frame is generally understood to refer to the region between the translation initiation codon and the translation termination codon. Another target region is the 5' untranslated region, which is the portion of a mRNA in the 5' direction from the translation initiation codon. It includes the nucleotides between the 5' cap site and the translation initiation codon of a mRNA or corresponding nucleotides on the gene.

0076Similarly, the 3' untranslated region may be used as a target for antisense oligonucleotides. The 3' untranslated region is that portion of the mRNA in the 3' direction from the translation termination codon, and thus includes the nucleotides between the translation termination codon and the 3' end of a mRNA or corresponding nucleotides of the gene.

0077An antisense oligonucleotide may also target the 5' cap region of an mRNA. The 5' cap comprises an N7-methylated guanosine residue joined to the 5'-most residue of the mRNA via 5'-5' triphosphate linkage. The 5' cap region is considered to include the 5' cap structure itself as well as the first 50 nucleotides adjacent to the cap.

0078Although some eukaryotic mRNA transcripts are directly translated, many contain one or more intron regions, which are excised from a transcript before it is translated. The remaining (and therefore translated) exon regions are spliced together to form a continuous mRNA sequence. mRNA splice sites, i.e., intron-exon junctions, represent possible target regions, and are particularly useful in situations where aberrant splicing is implicated in disease, or where an overproduction of a particular mRNA splice product is implicated in disease. Moreover, aberrant fusion junctions due to rearrangements or deletions are also possible targets for antisense oligonucleotides.

0079With these various target sites in mind, antisense oligonucleotides that are sufficiently complementary to the target polynucleotides must be chosen. There must be a sufficient degree of complementarity or precise pairing such that stable and specific binding occurs between the oligonucleotide and the polynucleotide target. Importantly, the sequence of an antisense oligonucleotide need not be 100% complementary to that of its target polynucleotide to be specifically hybridizable. An antisense oligonucleotide is specifically hybridizable when binding of the antisense oligonucleotide to the target polynucleotide interferes with the normal function of the target polynucleotide to cause a loss of utility, and there is a sufficient degree of complementarity to avoid non-specific binding of the antisense oligonucleotide to non-target sequences under conditions in which specific binding is desired, i.e., under physiological conditions in the case of in vivo assays or therapeutic treatment, and in the case of in vitro assays, under conditions in which the assays are performed.

0080The antisense oligonucleotides may be at least about 8 nt to at least about 50 nt in length. In one embodiment, the antisense oligonucleotides may be about 12 to about 30 nt in length.

0081The antisense oligonucleotides used in accordance with this invention may be conveniently and routinely made through the well-known technique of solid phase synthesis. Equipment for such synthesis is sold by several vendors including, for example, Applied Biosystems (Foster City, CA). Any other means for such synthesis known in the art may additionally or alternatively be employed. It is well known to use similar techniques to prepare oligonucleotides such as the phosphorothioates and alkylated derivatives.

Nucleic Acids Encoding Functional Nucleic Acids

0082In preferred embodiments of the invention, minicells comprise nucleic acids that encode functional nucleic acids. For example, a plasmid may encode a functional nucleic acid that is expressed inside of mammalian target cells. This makes possible endogenous delivery of functional nucleic acids, which has advantages over the transient nature of exogenous delivery.

0083Thus, recombinant intact minicells may carry plasmid DNA encoding one or more siRNA sequences aimed at silencing drug resistance or apoptosis resistance genes. Using minicells that encode multiple functional nucleic acids, it is possible to treat cells that express multiple drug resistance mechanisms. Different siRNA sequences can be expressed individually from different promoters. For example, siRNA targeting Pgp mRNA can be expressed from the U6 promoter and siRNA targeting Bcl-2 mRNA can be expressed from the H1 promoter. These multiple expression cassettes preferably are carried on a single plasmid, but may also be on different plasmids. Different siRNA sequences also can be expressed from a single promoter, where the recombinant plasmid carries an expression cassette comprised of multiple siRNA-encoding sequences, which are linked together via noncoding polynucleotide sequences. A single gene transcription terminator can be placed downstream of the complete expression cassette.

0084In one strategy, a plasmid encodes the sense and antisense strands of an siRNA as two independent transcripts that, after expression within a target cell, hybridize to form functional siRNA duplexes. In a second preferred strategy, a plasmid encodes one or more siRNAs that each are expressed as a single transcript that forms a short hairpin RNA stem-loop structure. The hairpin structure may be processed by a Dicer enzyme into functional siRNA.

Reporter Elements

0085A nucleic acid molecule to be introduced via the approach of the present invention can include a reporter element. A reporter element confers on its recombinant host a readily detectable phenotype or characteristic, typically by encoding a polypeptide, not otherwise produced by the host, that can be detected, upon expression, by histological or in situ analysis, such as by in vivo imaging techniques. For example, a reporter element delivered by an intact minicell, according to the present invention, could code for a protein that produces, in the engulfing host cell, a colorimetric or fluorometric change that is detectable by in situ analysis and that is a quantitative or semi-quantitative function of transcriptional activation. Illustrative of these proteins are esterases, phosphatases, proteases and other enzymes, the activity of which generates a detectable chromophore or fluorophore.

0086Preferred examples are E. coli β-galactosidase, which effects a color change via cleavage of an indigogenic substrate, indolyl-β-D-galactoside, and a luciferase, which oxidizes a long-chain aldehyde (bacterial luciferase) or a heterocyclic carboxylic acid (luciferin), with the concomitant release of light. Also useful in this context is a reporter element that encodes the green fluorescent protein (GFP) of the jellyfish, Aequorea victoria, as described by Prasher et al. (1995). The field of GFP-related technology is illustrated by two published <patcit id="pcit0010" dnum="WO09521191A"><text>PCT applications, WO 095/21191</text></patcit> (discloses a polynucleotide sequence encoding a 238 amino-acid GFP apoprotein, containing a chromophore formed from amino acids 65 through 67) and <patcit id="pcit0011" dnum="WO09521191A"><text>WO 095/21191</text></patcit> (discloses a modification of the cDNA for the apopeptide of A. victoria GFP, providing a peptide having altered fluorescent properties), and by a report of Heim et al. (1994) of a mutant GFP, characterized by a 4-to-6-fold improvement in excitation amplitude.

0087Another type of a reporter element is associated with an expression product that renders the recombinant minicell resistant to a toxin. For instance, the neo gene protects a host against toxic levels of the antibiotic G418, while a gene encoding dihydrofolate reductase confers resistance to methotrexate, and the chloramphenicol acetyltransferase (CAT) gene bestows resistance to chloramphenicol.

0088Other genes for use as a reporter element include those that can transform a host minicell to express distinguishing cell-surface antigens, e.g., viral envelope proteins such as HIV gp120 or herpes gD, which are readily detectable by immunoassays.

Regulatory Elements

0089A nucleic acid molecule to be introduced via the approach of the present invention also can have a desired encoding segment linked operatively to a regulatory element, such as a promoter, a terminator, an enhancer and/or a signal sequence. A suitable promoter can be tissue-specific or even tumor-specific, as the therapeutic context dictates.

0090A promoter is "tissue-specific" when it is activated preferentially in a given tissue and, hence, is effective in driving expression, in the target tissue, of an operably linked structural sequence. The category of tissue-specific promoters includes, for example: the hepatocyte-specific promoter for albumin and a1― antitrypsin, respectively; the elastase I gene control region, which is active in pancreatic acinar cells; the insulin gene control region, active in pancreatic beta cells; the mouse mammary tumor virus control region, which is active in testicular, breast, lymphoid and mast cells; the myelin basic protein gene control region, active in oligodendrocyte cells in the brain; and the gonadotropic releasing hormone gene control region, which is active in cells of the hypothalamus. See Frain et al. (1990), Ciliberto et al. (1985), Pinkert et al., (1987), Kelsey et al. (1987), Swift et al. (1984), MacDonald (1987), Hanahan, (1985), Leder et al. (1986), Readhead et al. (1987), and Mason et al. (1986).

0091There also are promoters that are expressed preferentially in certain tumor cells or in tumor cells per se, and that are useful for treating different cancers in accordance with the present invention. The class of promoters that are specific for cancer cells is illustrated by: the tyrosinase promoter, to target melanomas; the MUC1/Df3 promoter, to target breast carcinoma; the hybrid myoD enhancer/SV40 promoter, which targets expression to rhabdomyosarcoma (RMS); the carcinoembryonic antigen (CEA) promoter, which is specific for CEA-expressing cells such as colon cancer cells, and the hexokinase type II gene promoter, to target non-small cell lung carcinomas. See Hart (1996), Morton & Potter (1998), Kurane et al. (1998) and Katabi et al. (1999).

0092Promoters that are dependent on either RNA polymerase (pol) II or pol II are preferred promoters. Highly preferred promoters are the RNA III polymerase promoters H1 and U6.

0093A signal sequence can be used, according to the present invention, to effect secretion of an expression product or localization of an expression product to a particular cellular compartment. Thus, a therapeutic polynucleotide molecule that is delivered via intact minicells may include a signal sequence, in proper reading frame, such that the expression product of interest is secreted by an engulfing cell or its progeny, thereby to influence surrounding cells, in keeping with the chosen treatment paradigm. Illustrative signal sequences include the haemolysin C-terminal secretion sequence, described in <patcit id="pcit0012" dnum="US5143830A"><text>U.S. patent No. 5,143,830</text></patcit>, the BAR1 secretion sequence, disclosed in <patcit id="pcit0013" dnum="US5037743A"><text>U.S. patent No. 5,037,743</text></patcit>, and the signal sequence portion of the zsig32 polypeptide, described in <patcit id="pcit0014" dnum="US6025197A"><text>U.S. patent No. 6,025,197</text></patcit>.

Targets of Functional Nucleic Acids

0094Functional nucleic acids of the invention target the gene or transcript of a protein that promotes drug resistance, inhibits apoptosis or promotes a neoplastic phenotype. Successful application of functional nucleic acid strategies in these contexts have been achieved in the art, but without the benefits of minicell vectors. See, e.g., Sioud (2004), Caplen (2003), Wu et al. (2003), Nieth et al. (2003), Caplen and Mousses (2003), Duxbury et al. (2004), Yague et al. (2004), Duan et al. (2004),

0095Proteins that contribute to drug resistance constitute preferred targets of functional nucleic acids. The proteins may contribute to acquired drug resistance or intrinsic drug resistance. When diseased cells, such as tumor cells, initially respond to drugs, but become refractory on subsequent treatment cycles, the resistant phenotype is acquired. Useful targets involved in acquired drug resistance include ATP binding cassette transporters such as P-glycoprotein (P-gp, P-170, PGY1, MDR1, ABCB1, MDR-associated protein, Multidrug resistance protein 1), MDR-2 and MDR-3. MRP2 (multi-drug resistance associated protein), BCR-ABL (breakpoint cluster region ― Abelson protooncogene), a STI-571 resistance-associated protein, lung resistance-related protein, cyclooxygenase-2, nuclear factor kappa, XRCC1 (X-ray cross-complementing group 1), ERCC1 (Excision cross-complementing gene), GSTP1 (Glutathione S-transferase), mutant β-tubulin, and growth factors such as IL-6 are additional targets involved in acquired drug resistance. When previously untreated cells fail to respond to one or more drugs, the resistant phenotype is intrinsic. An example of a protein contributing to intrinsic resistance is LRP (lung resistance-related protein).

0096Useful targets also include proteins that contribute to apoptosis resistance. These include Bcl-2 (B cell leukemia/lymphoma), Bcl-XL, A1/Bfl 1, focal adhesion kinase and p53 mutant protein.

0097Useful targets further include oncogenic and mutant tumor suppressor proteins. Examples include β-Catenin, PKC-α (protein kinase C), C-RAF, K-Ras (V12), DP97 Dead box RNA helicase, DNMT1 (DNA methyltransferase 1), FLIP (Flice-like inhibitory protein), C-Sfc, 53BPI, Polycomb group protein EZH2 (Enhancer of zeste homologue), ErbB1, HPV-16 E5 and E7 (human papillomavirus early 5 and early 7), Fortilin & MCI1P (Myeloid cell leukemia 1 protein), DIP13α (DDC interacting protein 13a), MBD2 (Methyl CpG binding domain), p21, KLF4 (Kruppel-like factor 4), tpt/TCTP (Translational controlled tumor protein), SPK1 & SPK2 (Sphingosine kinase), P300, PLK1 (Polo-like kinase-1), Trp53, Ras, ErbB1, VEGF (Vascular endothelial growth factor), and BAG-1 (BCL2-associated athanogene 1).

0098With regard to HIV infection, targets include HIV-Tat, HIV-Rev, HIV-Vif, HIV-Nef, HIV-Gag, HIV-Env, LTR, CD4, CXCR4 (chemokine receptor) and CCR5 (chemokine receptor).

0099Because of tumor cell heterogeneity, a number of different drug resistance or apoptosis resistance pathways may be operational in target cells. Therefore, the functional nucleic acids used in methods of the invention may require change over time. For instance, if biopsy samples reveal new mutations that result in acquired drug resistance, specific siRNAs can be designed and encoded on a suitable expression plasmid, which is transformed into a minicell-producing bacterial strain, which is used to produce recombinant minicells that are administered to address the acquired drug resistance.

III.

Method of Overcoming Drug Resistance and Treating Disease

0100In another aspect, the invention provides a method of overcoming drug resistance and treating a disease, such as cancer or AIDS, in a subject. The method comprises (a) providing an intact minicell that contains a functional nucleic acid molecule or a plasmid comprising a segment that encodes a functional nucleic acid molecule, where the functional nucleic acid molecule targets the gene or transcript of a protein that promotes drug resistance, (b) bringing the minicell into contact with a target mammalian cell, such that the mammalian cell engulfs the minicell, and (c) delivering a drug to the target mammalian cell. Preferably, step (c) is performed after steps (a) and (b), to allow the functional nucleic acid to diminish resistance to the drug prior to the drug's administration. Delivery of the drug and introduction of the functional nucleic acid can occur consecutively, in any order, or simultaneously.

0101According to the invention, drugs may be delivered by any conventional means. For example, drugs may be delivered orally, parenterally (including subcutaneously, intravenously, intramuscularly, intraperitoneally, and by infusion), topically, transdermally or by inhalation. The appropriate mode of delivery and dosage of each drug is easily ascertainable by those skilled in the medical arts.

Drug Delivery via Minicells

0102Although drug delivery may occur via conventional means, delivery via minicells is preferred. In this regard, the inventors have discovered that the same mammalian cells can be successfully re-transfected by targeted intact minicells that are packaged with different payloads. For example, siRNA-encoding plasmid-packaged minicells can transfect a mammalian cell, after which drug-packaged minicells can deliver drug to the same mammalian cell. This discovery was a surprise, and indicates that the intracellular processes associated with minicell breakdown, endosomal release of a payload and escape of the payload to intracellular targets remains fully functional after the first round of transfection and payload delivery.

0103The drug may be packaged in a separate minicell from the functional nucleic acid or plasmid encoding the functional nucleic acid. Alternatively, the drug may be packaged in the same minicell as the functional nucleic acid molecule or plasmid encoding the functional nucleic acid molecule. Certain drugs may interact with nucleic acids and preclude co-packaging of drug and nucleic acid in the same minicell. For example, Doxorubicin is known to interact with DNA.

0104Preferably, minicells of the invention contain a sufficient quantity of drug to exert the drug's physiological or pharmacological effect on a target cell. Also preferably, drugs contained within the minicells are heterologous, or foreign, to the minicells, meaning that the minicells' parent bacterial cells do not normally produce the drug.

0105Both hydrophilic and hydrophobic drugs can be packaged in minicells by creating a concentration gradient of the drug between en extracellular medium containing minicells and the minicell cytoplasm. When the extracellular medium contains a higher drug concentration than the minicell cytoplasm, the drug naturally moves down this concentration gradient, into the minicell cytoplasm. When the concentration gradient is reversed, however, the drug does not move out of the minicells.

0106To load minicells with drugs that normally are not water soluble, the drugs initially can be dissolved in an appropriate solvent. For example, Paclitaxel can be dissolved in a 1:1 blend of ethanol and cremophore EL (polyethoxylated castor oil), followed by a dilution in PBS to achieve a solution of Paclitaxel that is partly diluted in aqueous media and carries minimal amounts of the organic solvent to ensure that the drug remains in solution. Minicells can be incubated in this final medium for drug loading. Thus, the inventors discovered that even hydrophobic drugs can diffuse into the cytoplasm of minicells to achieve a high and therapeutically significant cytoplasmic drug load. This is unexpected because the minicell membrane is composed of a hydrophobic phospholipid bilayer, which would be expected to prevent diffusion of hydrophobic molecules into the cytoplasm.

0107Another method of loading minicells with a drug involves culturing a recombinant parent bacterial cell under conditions wherein the parent bacterial cell transcribes and translates a nucleic acid encoding the drug, such that the drug is released into the cytoplasm of the parent bacterial cell. For example, a gene cluster encoding the cellular biosynthetic pathway for a desired drug can be cloned and transferred into a parent bacterial strain that is capable of producing minicells. Genetic transcription and translation of the gene cluster results in biosynthesis of the drug within the cytoplasm of the parent bacterial cells, filling the bacterial cytoplasm with the drug. When the parent bacterial cell divides and forms progeny minicells, the minicells also contain the drug in their cytoplasm. The pre-packaged minicells can be purified by any of the minicell purification processes known in the art and described above.

0108Similarly, another method of loading minicells with a drug involves culturing a recombinant minicell that contains an expression plasmid encoding the drug under conditions such that the gene encoding the drug is transcribed and translated within the minicell.

Drugs

0109Drugs useful in the invention may be any physiologically or pharmacologically active substance that produces a desired local or systemic effect in animals, particularly mammals and humans. Drugs may be inorganic or organic compounds, without limitation, including peptides, proteins, nucleic acids, and small molecules, any of which may be characterized or uncharacterized. They may be in various forms, such as unchanged molecules, molecular complexes, pharmacologically acceptable salts, such as hydrochloride, hydrobromide, sulfate, laurate, palmitate, phosphate, nitrite, nitrate, borate, acetate, maleate, tartrate, oleate, salicylate, and the like. For acidic drugs, salts of metals, amines or organic cations, for example, quaternary ammonium, can be used. Derivatives of drugs, such as bases, esters and amides also can be used. A drug that is water insoluble can be used in a form that is a water soluble derivative thereof, or as a base derivative thereof, which in either instance, or by its delivery, is converted by enzymes, hydrolyzed by the body pH, or by other metabolic processes to the original therapeutically active form.

0110Useful drugs include chemotherapeutic agents, immunosuppressive agents, cytokines, cytotoxic agents, nucleolytic compounds, radioactive isotopes, receptors, and pro-drug activating enzymes, which may be naturally occurring or produced by recombinant methods.

0111Drugs that are affected by classical multidrug resistance have particular utility in the invention, such as vinca alkaloids (e.g., vinblastine and vincristine), the anthracyclines (e.g., doxorubicin and daunorubicin), RNA transcription inhibitors (e.g., actinomycin-D) and microtubule stabilizing drugs (e.g., paclitaxel). (Ambudkar et al., 1999)

0112In general, cancer chemotherapy agents are preferred drugs. Useful cancer chemotherapy drugs include nitrogen mustards, nitrosorueas, ethyleneimine, alkane sulfonates, tetrazine, platinum compounds, pyrimidine analogs, purine analogs, antimetabolites, folate analogs, anthracyclines, taxanes, vinca alkaloids, topoisomerase inhibitors and hormonal agents. Exemplary chemotherapy drugs are Actinomycin-D, Alkeran, Ara-C, Anastrozole, Asparaginase, BiCNU, Bicalutamide, Bleomycin, Busulfan, Capecitabine, Carboplatin, Carboplatinum, Carmustine, CCNU, Chlorambucil, Cisplatin, Cladribine, CPT-11, Cyclophosphamide, Cytarabine, Cytosine arabinoside, Cytoxan, Dacarbazine, Dactinomycin, Daunorubicin, Dexrazoxane, Docetaxel, Doxorubicin, DTIC, Epirubicin, Ethyleneimine, Etoposide, Floxuridine, Fludarabine, Fluorouracil, Flutamide, Fotemustine, Gemcitabine, Herceptin, Hexamethylamine, Hydroxyurea, Idarubicin, Ifosfamide, Irinotecan, Lomustine, Mechlorethamine, Melphalan, Mercaptopurine, Methotrexate, Mitomycin, Mitotane, Mitoxantrone, Oxaliplatin, Paclitaxel, Pamidronate, Pentostatin, Plicamycin, Procarbazine, Rituximab, Steroids, Streptozocin, STI-571, Streptozocin, Tamoxifen, Temozolomide, Teniposide, Tetrazine, Thioguanine, Thiotepa, Tomudex, Topotecan, Treosulphan, Trimetrexate, Vinblastine, Vincristine, Vindesine, Vinorelbine, VP-16, and Xeloda.

0113Useful cancer chemotherapy drugs also include alkylating agents such as Thiotepa and cyclosphosphamide; alkyl sulfonates such as Busulfan, Improsulfan and Piposulfan; aziridines such as Benzodopa, Carboquone, Meturedopa, and Uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, trietylenephosphoramide, triethylenethiophosphaoramide and trimethylolomelamine; nitrogen mustards such as Chlorambucil, Chlornaphazine, Cholophosphamide, Estramustine, Ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, Melphalan, Novembiehin, Phenesterine, Prednimustine, Trofosfamide, uracil mustard; nitroureas such as Cannustine, Chlorozotocin, Fotemustine, Lomustine, Nimustine, and Ranimustine; antibiotics such as Aclacinomysins, Actinomycin, Authramycin, Azaserine, Bleomycins, Cactinomycin, Calicheamicin, Carabicin, Carminomycin, Carzinophilin, Chromoinycins, Dactinomycin, Daunorubicin, Detorubicin, 6-diazo-5-oxo-L-norleucine, Doxorubicin, Epirubicin, Esorubicin, Idambicin, Marcellomycin, Mitomycins, mycophenolic acid, Nogalamycin, Olivomycins, Peplomycin, Potfiromycin, Puromycin, Quelamycin, Rodorubicin, Streptonigrin, Streptozocin, Tubercidin, Ubenimex, Zinostatin, and Zorubicin; anti-metabolites such as Methotrexate and 5-fluorouracil (5-FU); folic acid analogues such as Denopterin, Methotrexate, Pteropterin, and Trimetrexate; purine analogs such as Fludarabine, 6-mercaptopurine, Thiamiprine, and Thioguanine; pyrimidine analogs such as Ancitabine, Azacitidine, 6-azauridine, Carmofur, Cytarabine, Dideoxyuridine, Doxifluridine, Enocitabine, Floxuridine, and 5-FU; androgens such as Calusterone, Dromostanolone Propionate, Epitiostanol, Rnepitiostane, and Testolactone; anti-adrenals such as aminoglutethimide, Mitotane, and Trilostane; folic acid replenisher such as frolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; Amsacrine; Bestrabucil; Bisantrene; Edatraxate; Defofamine; Demecolcine; Diaziquone; Elfornithine; elliptinium acetate; Etoglucid; gallium nitrate; hydroxyurea; Lentinan; Lonidamine; Mitoguazone; Mitoxantrone; Mopidamol; Nitracrine; Pentostatin; Phenamet; Pirarubicin; podophyllinic acid; 2-ethylhydrazide; Procarbazine; PSK®; Razoxane; Sizofrran; Spirogermanium; tenuazonic acid; triaziquone; 2, 2',2"-trichlorotriethylamine; Urethan; Vindesine; Dacarbazine; Mannomustine; Mitobronitol; Mitolactol; Pipobroman; Gacytosine; Arabinoside ("Ara-C"); cyclophosphamide; thiotEPa; taxoids, e.g., Paclitaxel (TAXOL®, Bristol-Myers Squibb Oncology, Princeton, NJ) and Doxetaxel (TAXOTERE®, Rhone-Poulenc Rorer, Antony, France); Chlorambucil; Gemcitabine; 6-thioguanine; Mercaptopurine; Methotrexate; platinum analogs such as Cisplatin And Carboplatin; Vinblastine; platinum; etoposide (VP-16); Ifosfamide; Mitomycin C; Mitoxantrone; Vincristine; Vinorelbine; Navelbine; Novantrone; Teniposide; Daunomycin; Aminopterin; Xeloda; Ibandronate; CPT-11; topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO); retinoic acid; Esperamicins; Capecitabine; and pharmaceutically acceptable salts, acids or derivatives of any of the above. Also included are anti-hormonal agents that act to regulate or inhibit hormone action on tumors such as anti-estrogens including for example Tamoxifen, Raloxifene, aromatase inhibiting 4(5)-imidazoles, 4 Hydroxytamoxifen, Trioxifene, Keoxifene, Onapristone, And Toremifene (Fareston); and anti-androgens such as Flutamide, Nilutamide, Bicalutamide, Leuprolide, and Goserelin; and pharmaceutically acceptable salts, acids or derivatives of any of the above.

0114Useful drugs also include cytokines. Examples of such cytokines are lymphokines, monokines, and traditional polypeptide hormones. Included among the cytokines are growth hormones such as human growth hormone, N-methionyl human growth hormone, and bovine growth hormone; parathyroid hormone; thyroxine; insulin; proinsulin; relaxin; prorelaxin; glycoprotein hormones such as follicle stimulating hormone (FSH), thyroid stimulating hormone (TSH), and luteinizing hormone (LH); hepatic growth factor; fibroblast growth factor; prolactin; placental lactogen; tumor necrosis factor-α and -β; mullerian-inhibiting substance; mouse gonadotropin-associated peptide; inhibin; activin; vascular endothelial growth factor; integrin; thrombopoietin (TPO); nerve growth factors such as NGF-β; platelet growth factor; transforming growth factors (TGFs) such as TGF-α and TGF-β; insulin-like growth factor-I and -II; erythropoietin (EPO); osteoinductive factors; interferons such as interferon-α, -β and -y; colony stimulating factors (CSFs) such as macrophage-CSF (M-CSF); granulocyte-macrophage-CSF (GM-CSF); and granulocyte-CSF (GCSF); interleukins (ILs) such as IL-1, IL-la, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-11, IL-12, IL-15; a tumor necrosis factor such as TNF-α or TNF-β; and other polypeptide factors including LIF and kit ligand (KL). As used herein, the tern cytokine includes proteins from natural sources or from recombinant cell culture and biologically active equivalents of the native sequence cytokines.

0115The drugs may be prodrugs, subsequently activated by a prodrug-activating enzyme that converts a prodrug like a peptidyl chemotherapeutic agent to an active anti-cancer drug. See, e.g.,<patcit id="pcit0015" dnum="WO8807378A"><text>WO 88/07378</text></patcit>; <patcit id="pcit0016" dnum="WO8101145A"><text>WO 81/01145</text></patcit>; <patcit id="pcit0017" dnum="US4975278A"><text>U.S. Patent No. 4,975,278</text></patcit>. In general, the enzyme component includes any enzyme capable of acting on a prodrug in such a way so as to covert it into its more active, cytotoxic form.

IV.

Directing Minicells to Specific Mammalian Cells

0116In one aspect of the invention, a minicell is directed to a target mammalian cell via a bispecific ligand, as described in <patcit id="pcit0018" dnum="WO2005056749A"><text>WO 2005/056749</text></patcit>. The bispecific ligand, having specificity for both minicell and mammalian cell components, causes the minicell to bind to the mammalian cell, such that the minicell is engulfed by the mammalian cell, and the mammalian cell produces the functional nucleic acid molecule. This targeted delivery method may be performed in vivo or in vitro, or both in vivo and in vitro.

0117Contact between bispecific ligand, minicell and mammalian cell may occur in a number of different ways. For in vivo delivery, it is preferable to administer a minicell that already has the bispecific ligand attached to it. Thus, minicell, bispecific ligand and target cell all are brought into contact when the bispecific ligand-targeted minicell reaches the target cell in vivo. Alternatively, bispecific ligand and minicell can be separately administered in vivo.

0118Contact between the bispecific ligands, minicells and mammalian cells also may occur during one or more incubations in vitro. In one embodiment, the three elements are incubated together all at once. Alternatively, step-wise incubations may be performed. In one example of a step-wise approach, minicells and bi-specific ligands are first incubated together to form bispecific ligand-targeted minicells, which are then incubated with target cells. In another example, bispecific ligands are first incubated with target cells, followed by an incubation with minicells. A combination of one or more in vitro incubations and in vivo administrations also may bring bispecific ligands, minicells and mammalian target cells into contact.

0119The inventors found that the targeted delivery approach is broadly applicable to a range of mammalian cells, including cells that normally are refractory to specific adhesion and endocytosis of minicells. For example, bispecific antibody ligands with anti-O-polysaccharide specificity on one arm and anti-HER2 receptor, anti-EGF receptor or anti-androgen receptor specificity on the other arm efficiently bind minicells to the respective receptors on a range of target non-phagocytic cells. These cells include lung, ovarian, brain, breast, prostate and skin cancer cells. Moreover, the efficient binding precedes rapid endocytosis of the minicells by each of the non-phagocytic cells.

0120Target cells of the invention include any cell into which a functional nucleic acid is to be introduced. Desirable target cells are characterized by expression of a cell surface receptor that, upon binding of a ligand, facilitates endocytosis. Preferred target cells are non-phagocytic, meaning that the cells are not professional phagocytes, such as macrophages, dendritic cells and Natural Killer (NK) cells. Preferred target cells also are mammalian.

0121Ligands useful in the targeted delivery methods of this invention include any agent that binds to a surface component on a target cell and to a surface component on a minicell. Preferably, the surface component on a target cell is a receptor, especially a receptor capable of mediating endocytosis. The ligands may comprise a polypeptide and/or carbohydrate component. Antibodies are preferred ligands. For example, a bispecific antibody that carries dual specificities for a surface component on bacterially derived intact minicells and for a surface component on target mammalian cells, can be used efficiently to target the minicells to the target mammalian cells in vitro and in vivo. Useful ligands also include receptors, enzymes, binding peptides, fusion/chimeric proteins and small molecules.

0122The selection of a particular ligand is made on two primary criteria: (i) specific binding to one or more domains on the surface of intact minicells and (ii) specific binding to one or more domains on the surface of the target cells. Thus, ligands preferably have a first arm that carries specificity for a bacterially derived intact minicell surface structure and a second arm that carries specificity for a mammalian cell surface structure. Each of the first and second arms may be multivalent. Preferably, each arm is monospecific, even if multivalent.

0123For binding to bacterially derived minicells, it is desirable for one arm of the ligand to be specific for the O-polysaccharide component of a lipopolysaccharide found on the parent bacterial cell. Other minicell surface structures that can be exploited for ligand binding include cell surface-exposed polypeptides and carbohydrates on outer membranes, such as pilli, fimbrae and flagella cell surface exposed peptide segments.

0124For binding to target cells, one arm of the ligand is specific for a surface component of a mammalian cell. Such components include cell surface proteins, peptides and carbohydrates, whether characterized or uncharacterized. Cell surface receptors, especially those capable of activating receptor-mediated endocytosis, are desirable cell surface components for targeting. Such receptors, if over-expressed on the target cell surface, confer additional selectivity for targeting the cells to be treated, thereby reducing the possibility for delivery to non-target cells.

0125By way of example, one may target tumor cells, metastatic cells, vasculature cells, such as endothelial cells and smooth muscle cells, lung cells, kidney cells, blood cells, bone marrow cells, brain cells, liver cells, and so forth, or precursors of any selected cell by selecting a ligand that specifically binds a cell surface receptor motif on the desired cells. Examples of cell surface receptors include carcinoembryonic antigen (CEA), which is overexpressed in most colon, rectum, breast, lung, pancreas and gastrointestinal tract carcinomas (Marshall, 2003); heregulin receptors (HER-2, neu or c-erbB-2), which is frequently overexpressed in breast, ovarian, colon, lung, prostate and cervical cancers (Hung et al., 2000); epidermal growth factor receptor (EGFR), which is highly expressed in a range of solid tumors including those of the breast, head and neck, non-small cell lung and prostate (Salomon et al., 1995); asialoglycoprotein receptor (Stockert, 1995); transferrin receptor (Singh, 1999); serpin enzyme complex receptor, which is expressed on hepatocytes (Ziady et al., 1997); fibroblast growth factor receptor (FGFR), which is overexpressed on pancreatic ductal adenocarcinoma cells (Kleeff et al., 2002); vascular endothelial growth factor receptor (VEGFR), for anti-angiogenesis gene therapy (Becker et al., 2002 and Hoshida et al., 2002); folate receptor, which is selectively overexpressed in 90% of nonmucinous ovarian carcinomas (Gosselin and Lee, 2002); cell surface glycocalyx (Batra et al., 1994); carbohydrate receptors (Thurnher et al., 1994); and polymeric immunoglobulin receptor, which is useful for gene delivery to respiratory epithelial cells and attractive for treatment of lung diseases such as Cystic Fibrosis (Kaetzel et al., 1997).

0126Preferred ligands comprise antibodies and/or antibody derivatives. As used herein, the term "antibody" encompasses an immunoglobulin molecule obtained by in vitro or in vivo generation of an immunogenic response. The term "antibody" includes polyclonal, monospecific and monoclonal antibodies, as well as antibody derivatives, such as single-chain antibody fragments (scFv). Antibodies and antibody derivatives useful in the present invention also may be obtained by recombinant DNA techniques.

0127Wild type antibodies have four polypeptide chains, two identical heavy chains and two identical light chains. Both types of polypeptide chains have constant regions, which do not vary or vary minimally among antibodies of the same class, and variable regions. Variable regions are unique to a particular antibody and comprise an antigen binding domain that recognizes a specific epitope. The regions of the antigen binding domain that are most directly involved in antibody binding are "complementarity-determining regions" (CDRs).

0128The term "antibody" also encompasses derivatives of antibodies, such as antibody fragments that retain the ability to specifically bind to antigens. Such antibody fragments include Fab fragments (a fragment that contains the antigen-binding domain and comprises a light chain and part of a heavy chain bridged by a disulfide bond), Fab' (an antibody fragment containing a single antigen-binding domain comprising a Fab and an additional portion of the heavy chain through the hinge region, F(ab')2 (two Fab' molecules joined by interchain disulfide bonds in the hinge regions of the heavy chains), a bispecific Fab (a Fab molecule having two antigen binding domains, each of which may be directed to a different epitope), and an scFv (the variable, antigen-binding determinative region of a single light and heavy chain of an antibody linked together by a chain of amino acids.)

0129When antibodies, including antibody fragments, constitute part or all of the ligands, they preferably are of human origin or are modified to be suitable for use in humans. So-called "humanized antibodies" are well known in the art. See, e.g., Osbourn et al., 2003. They have been modified by genetic manipulation and/or in vitro treatment to reduce their antigenicity in a human. Methods for humanizing antibodies are described, e.g., in <patcit id="pcit0019" dnum="US6639055B"><text>U.S. patents No. 6,639,055</text></patcit>, <patcit id="pcit0020" dnum="US5585089A"><text>No. 5,585,089</text></patcit>, and <patcit id="pcit0021" dnum="US5530101A"><text>No. 5,530,101</text></patcit>. In the simplest case, humanized antibodies are formed by grafting the antigen-binding loops, known as complementarity-determining regions (CDRs), from a mouse mAb into a human IgG. See Jones et al., 1986; Riechmann et al., 1988; and Verhoeyen et al., 1988. The generation of high-affinity humanized antibodies, however, generally requires the transfer of one or more additional residues from the so-called framework regions (FRs) of the mouse parent mAb. Several variants of the humanization technology also have been developed. See Vaughan et al., 1998.

0130Human antibodies, rather than "humanized antibodies," also may be employed in the invention. They have high affinity for their respective antigens and are routinely obtained from very large, single-chain variable fragments (scFvs) or Fab phage display libraries. See Griffiths et al., 1994; Vaughan et al., 1996; Sheets et al., 1998; de Haard et al., 1999; and Knappik et al., 2000.

0131Useful ligands also include bispecific single chain antibodies, which typically are recombinant polypeptides consisting of a variable light chain portion covalently attached through a linker molecule to a corresponding variable heavy chain portion. See <patcit id="pcit0022" dnum="US5455030A"><text>U.S. patents No. 5,455,030</text></patcit>, <patcit id="pcit0023" dnum="US5260203A"><text>No. 5,260,203</text></patcit>, and <patcit id="pcit0024" dnum="US4496778A"><text>No. 4,496,778</text></patcit>. Bispecific antibodies also can be made by other methods. For example, chemical heteroconjugates can be created by chemically linking intact antibodies or antibody fragments of different specificities. See Karpovsky et al., 1984. However, such heteroconjugates are difficult to make in a reproducible manner and are at least twice as large as normal monoclonal antibodies. Bispecific antibodies also can be created by disulfide exchange, which involves enzymatic cleavage and reassociation of the antibody fragments. See Glennie et al., 1987.

0132Because Fab and scFv fragments are monovalent they often have low affinity for target structures. Therefore, preferred ligands made from these components are engineered into dimeric, trimeric or tetrameric conjugates to increase functional affinity. See Tomlinson and Holliger, 2000; Carter, 2001; Hudson and Souriau, 2001; and Todorovska et al., 2001. Such conjugate structures may be created by chemical and/or genetic cross-links.

0133Bispecific ligands of the invention preferably are monospecific at each end, i.e., specific for a single component on minicells at one end and specific for a single component on target cells at the other end. The ligands may be multivalent at one or both ends, for example, in the form of so-called diabodies, triabodies and tetrabodies. See Hudson and Souriau, 2003. A diabody is a bivalent dimer formed by a non-covalent association of two scFvs, which yields two Fv binding sites. Likewise, a triabody results from the formation of a trivalent trimer of three scFvs, yielding three binding sites, and a tetrabody results from the formation of a tetravalent tetramer of four scFvs, yielding four binding sites.

0134Several humanized, human, and mouse monoclonal antibodies and fragments thereof that have specificity for receptors on mammalian cells have been approved for human therapeutic use, and the list is growing rapidly. See Hudson and Souriau, 2003. An example of such an antibody that can be used to form one arm of a bispecific ligand has specificity for HER2: Herceptin™; Trastuzumab.

0135Antibody variable regions also can be fused to a broad range of protein domains. Fusion to human immunoglobulin domains such as IgG1 CH3 both adds mass and promotes dimerization. See Hu et al., 1996. Fusion to human Ig hinge-Fc regions can add effector functions. Also, fusion to heterologous protein domains from multimeric proteins promotes multimerization. For example, fusion of a short scFv to short amphipathic helices has been used to produce miniantibodies. See Pack and Pluckthun, 1992. Domains from proteins that form heterodimers, such as fos/jun, can be used to produce bispecific molecules (Kostelny et al., 1992) and, alternately, homodimerization domains can be engineered to form heterodimers by engineering strategies such as "knobs into holes" (Ridgway et al., 1996). Finally, fusion protein partners can be selected that provide both multimerization as well as an additional function, e.g. streptavidin. See Dubel et al., 1995.

Delivery to phagocytosis- or endocytosis-competent cells

0136The invention further provides for delivery by means of bringing bacterially derived minicells into contact with mammalian cells that are phagocytosis-or endocytosis-competent. Such mammalian cells, which are capable of engulfing parent bacterial cells in the manner of intracellular bacterial pathogens, likewise engulf the minicells, which release their payload into the cytoplasm of the mammalian cells. This delivery approach can be effected without the use of targeting ligands.

0137A variety of mechanisms may be involved in the engulfing of minicells by a given type of cell, and the present invention is not dependent on any particular mechanism in this regard. For example, phagocytosis is a well-documented process in which macrophages and other phagocyte cells, such as neutrophils, ingest particles by extending pseudopodia over the particle surface until the particle is totally enveloped. Although described as "non-specific" phagocytosis, the involvement of specific receptors in the process has been demonstrated. See Wright & Jong (1986); Speert et al. (1988).

0138Thus, one form of phagocytosis involves interaction between surface ligands and ligand-receptors located at the membranes of the pseudopodia (Shaw and Griffin, 1981). This attachment step, mediated by the specific receptors, is thought to be dependent on bacterial surface adhesins. With respect to less virulent bacteria, such as non-enterotoxigenic E. coli, phagocytosis also may occur in the absence of surface ligands for phagocyte receptors. See Pikaar et al. (1995), for instance. Thus, the present invention encompasses but is not limited to the use of minicells that either possess or lack surface adhesins, in keeping with the nature of their parent bacterial cells, and are engulfed by phagocytes (i.e., "phagocytosis-competent" host cells), of which neutrophils and macrophages are the primary types in mammals.

0139Another engulfing process is endocytosis, by which intracellular pathogens exemplified by species of Salmonella, Escherichia, Shigella, Helicobacter, Pseudomonas and Lactobacilli gain entry to mammalian epithelial cells and replicate there. Two basic mechanisms in this regard are Clathrin-dependent receptor-mediated endocytosis, also known as "coated pit endocytosis" (Riezman, 1993), and Clathrin-independent endocytosis (Sandvig & Deurs, 1994). Either or both may be involved when an engulfing-competent cell that acts by endocytosis (i.e., an "endocytosis-competent" host cell) engulfs minicells in accordance with the invention. Representative endocytosis-competent cells are breast epithelial cells, enterocytes in the gastrointestinal tract, stomach epithelial cells, lung epithelial cells, and urinary tract and bladder epithelial cells.

0140When effecting delivery to an engulfing-competent mammalian cell without the use of a targeting ligand, the nature of the application contemplated will influence the choice of bacterial source for the minicells employed. For example, Salmonella, Escherichia and Shigella species carry adhesins that are recognized by endocytosis-mediating receptors on enterocytes in the gastrointestinal tract, and may be suitable to deliver a drug that is effective for colon cancer cells. Similarly, minicells derived from Helicobacter pylori, carrying adhesins specific for stomach epithelial cells, could be suited for delivery aimed at stomach cancer cells. Inhalation or insufflation may be ideal for administering intact minicells derived from a Pseudomonas species that carry adhesins recognized by receptors on lung epithelial cells. Minicells derived from Lactobacilli bacteria, which carry adhesins specific for urinary tract and bladder epithelial cells, could be well-suited for intraurethral delivery of a drug to a urinary tract or a bladder cancer.

VI.

Formulations

0141The invention includes within its scope compositions, or formulations, comprising (a) an intact minicell and (b) a pharmaceutically acceptable carrier therefor, where the minicell contains a functional nucleic acid molecule or a plasmid comprising a segment that encodes a functional nucleic acid molecule. The functional nucleic acid may be any of those siRNAs, shRNAs, ribozymes or antisense molecules described herein. The functional nucleic acid also may be encoded by another nucleic acid, such as a plasmid, as described herein. The nucleic acid encoding the functional nucleic acid may have any of the regulatory elements or reporter elements, as described herein.

0142The formulation optionally comprise a drug, as described herein. Preferably, the minicell of the formulation contains the drug. Alternatively, the minicell may contain a nucleic acid molecule, such as a plasmid, that encodes the drug.

0143The minicell-containing formulations preferably contain fewer than about 1 contaminating parent bacterial cell per 107 minicells, more preferably contain fewer than about 1 contaminating parent bacterial cell per 108 minicells, even more preferably contain fewer than about 1 contaminating parent bacterial cell per 109 minicells, still more preferably contain fewer than about 1 contaminating parent bacterial cell per 1010 minicells and most preferably contain fewer than about 1 contaminating parent bacterial cell per 1011 minicells.

0144The formulations also optionally contain a bispecific ligand for targeting the minicell to a target cell. The minicell and ligand may be any of those described herein. Thus, the minicell contains a nucleic acid encoding a functional nucleic acid and the bispecific ligand preferably is capable of binding to a surface component of the minicell and to a surface component of a target mammalian cell.

0145A formulation consisting essentially of minicells and, optionally drugs and bispecific ligands, of the present invention (that is, a formulation that includes such minicells, drugs and ligands with other constituents that do not interfere unduly with the nucleic acid or drug-delivering quality of the composition) can be formulated in conventional manner, using one or more pharmaceutically acceptable carriers or excipients.

0146Formulations may be presented in unit dosage form, e.g., in ampules or vials, or in multi-dose containers, with or without an added preservative. The formulation can be a solution, a suspension, or an emulsion in oily or aqueous vehicles, and may contain formulatory agents, such as suspending, stabilizing and/or dispersing agents. A suitable solution is isotonic with the blood of the recipient and is illustrated by saline, Ringer's solution, and dextrose solution. Alternatively, formulations may be in lyophilized powder form, for reconstitution with a suitable vehicle, e.g., sterile, pyrogen-free water or physiological saline. The formulations also may be in the form of a depot preparation. Such long-acting formulations may be administered by implantation (for example, subcutaneously or intramuscularly) or by intramuscular injection.

Administration Routes

0147Formulations of the invention can be administered via various routes and to various sites in a mammalian body, to achieve the therapeutic effect(s) desired, either locally or systemically. Delivery may be accomplished, for example, by oral administration, by application of the formulation to a body cavity, by inhalation or insufflation, or by parenteral, intramuscular, intravenous, intraportal, intrahepatic, peritoneal, subcutaneous, intratumoral, or intradermal administration. The mode and site of administration is dependent on the location of the target cells. For example, cystic-fibrotic cells may be efficiently targeted by inhaled delivery of the targeted minicells. Similarly, tumor metastasis may be more efficiently treated via intravenous delivery of targeted minicells. Primary ovarian cancer may be treated via intraperitoneal delivery of targeted minicells.

Purity

0148Minicells of the invention are substantially free from contaminating parent bacterial cells. Thus, minicell-containing formulations of the invention preferably contain fewer than about 1 contaminating parent bacterial cell per 107 minicells, more preferably contain fewer than about 1 contaminating parent bacterial cell per 108 minicells, even more preferably contain fewer than about 1 contaminating parent bacterial cell per 109 minicells, still more preferably contain fewer than about 1 contaminating parent bacterial cell per 1010 minicells and most preferably contain fewer than about 1 contaminating parent bacterial cell per 1011 minicells.

0149Methods of purifying minicells are known in the art and described in PCT/IB02/04632. One such method combines cross-flow filtration (feed flow is parallel to a membrane surface; Forbes, 1987) and dead-end filtration (feed flow is perpendicular to the membrane surface). Optionally, the filtration combination can be preceded by a differential centrifugation, at low centrifugal force, to remove some portion of the bacterial cells and thereby enrich the supernatant for minicells.

0150Another purification method employs density gradient centrifugation in a biologically compatible medium. After centrifugation, a minicell band is collected from the gradient, and, optionally, the minicells are subjected to further rounds of density gradient centrifugation to maximize purity. The method may further include a preliminary step of performing differential centrifugation on the minicell-containing sample. When performed at low centrifugal force, differential centrifugation will remove some portion of parent bacterial cells, thereby enriching the supernatant for minicells.

0151Particularly effective purification methods exploit bacterial filamentation to increase minicell purity. Thus a minicell purification method can include the steps of (a) subjecting a sample containing minicells to a condition that induces parent bacterial cells to adopt a filamentous form, followed by (b) filtering the sample to obtain a purified minicell preparation.

0152Known minicell purification methods also can be combined. One highly effective combination of methods is as follows:

0153Step A: Differential centrifugation of a minicell producing bacterial cell culture. This step, which may be performed at 2000g for about 20 minutes, removes most parent bacterial cells, while leaving minicells in the supernatant.

0154Step B: Density gradient centrifugation using an isotonic and non-toxic density gradient medium. This step separates minicells from many contaminants, including parent bacterial cells, with minimal loss of minicells. Preferably, this step is repeated within a purification method.

0155Step C: Cross-flow filtration through a 0.45 µm filter to further reduce parent bacterial cell contamination.

0156Step D: Stress-induced filamentation of residual parent bacterial cells. This may be accomplished by subjecting the minicell suspension to any of several stress-inducing environmental conditions.

0157Step E: Antibiotic treatment to kill parent bacterial cells.

0158Step F: Cross-flow filtration to remove small contaminants, such as membrane blebs, membrane fragments, bacterial debris, nucleic acids, media components and so forth, and to concentrate the minicells. A 0.2 µm filter may be employed to separate minicells from small contaminants, and a 0.1 µm filter may be employed to concentrate minicells.

0159Step G: Dead-end filtration to eliminate filamentous dead bacterial cells. A 0.45 um filter may be employed for this step.

0160Step H: Removal of endotoxin from the minicell preparation. Anti-Lipid A coated magnetic beads may be employed for this step.

Administration Schedules

0161In general, the formulations disclosed herein may be used at appropriate dosages defined by routine testing, to obtain optimal physiological effect, while minimizing any potential toxicity. The dosage regimen may be selected in accordance with a variety of factors including age, weight, sex, medical condition of the patient; the severity of the condition to be treated, the route of administration, and the renal and hepatic function of the patient.

0162Optimal precision in achieving concentrations of minicell and drug within the range that yields maximum efficacy with minimal side effects may require a regimen based on the kinetics of the minicell and drug availability to target sites and target cells. Distribution, equilibrium, and elimination of a minicell or drug may be considered when determining the optimal concentration for a treatment regimen. The dosages of the minicells and drugs may be adjusted when used in combination, to achieve desired effects.

0163Moreover, the dosage administration of the formulations may be optimized using a pharmacokinetic/pharmacodynamic modeling system. For example, one or more dosage regimens may be chosen and a pharmacokinetic/pharmacodynamic model may be used to determine the pharmacokinetic/pharmacodynamic profile of one or more dosage regimens. Next, one of the dosage regimens for administration may be selected which achieves the desired pharmacokinetic/pharmacodynamic response based on the particular pharmacokinetic/pharmacodynamic profile. See, e.g.,<patcit id="pcit0025" dnum="WO0067776A"><text>WO 00/67776</text></patcit>.

0164Specifically, the formulations may be administered at least once a week over the course of several weeks. In one embodiment, the formulations are administered at least once a week over several weeks to several months.

0165More specifically, the formulations may be administered at least once a day for about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or 31 days. Alternatively, the formulations may be administered about once every day, about once every 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or 31 days or more.

0166The formulations may alternatively be administered about once every week, about once every 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 weeks or more. Alternatively, the formulations may be administered at least once a week for about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 weeks or more.

0167Alternatively, the formulations may be administered about once every month, about once every 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 months or more.

0168The formulations may be administered in a single daily dose, or the total daily dosage may be administered in divided doses of two, three, or four times daily.

0169In method in which minicells are administered before a drug, administration of the drug may occur anytime from several minutes to several hours after administration of the minicells. The drug may alternatively be administered anytime from several hours to several days, possibly several weeks up to several months after the minicells.

0170More specifically, the minicells may be administered at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24 hours before the drug. Moreover, the minicells may be administered at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or 31 days before the administration of the drug. In yet another embodiment, the minicells may be administered at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 weeks or more before the drug. In a further embodiment, the minicells may be administered at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 months before the drug.

0171In another embodiment, the minicell is administered after the drug. The administration of the minicell may occur anytime from several minutes to several hours after the administration of the drug. The minicell may alternatively be administered anytime from several hours to several days, possibly several weeks up to several months after the drug.

0172The following examples are illustrative only, rather than limiting, and provide a more complete understanding of the invention. The examples demonstrate that drug resistant tumor cells can be effectively treated in-vivo by (1) administration of targeted recombinant minicells carrying RNAi sequences designed to reduce or eliminate expression of drug resistance encoding gene(s), and (2) administration of targeted, drug-packaged minicells carrying the drug to which the cancer cells are made sensitive.

Example 1. Anti-MDR1 and anti-bcl-2 shRNA expression plasmids and purification of recombinant minicells.

0173Recombinant minicells carrying plasmids encoding shRNA sequences (Mdr1 or bcl-2) were generated as follows. The Mdr1 shRNA sequence used in this study was described by Wu et al., 2003 (5'-TCGA AAGAAACCAACTGTCAGTGTA gagtactg TACACTGACAGTTGGTTTCTT TTTTT-3') (SEQ ID NO: 1) and the bcl-2 shRNA sequence used was described by Wacheck et al., 2003 (5'-TCGATGTGGATGACTGAGTACCTGA gagtactg TCAGGTACTCAGTCATCCACATTTTT-3') (SEQ ID NO: 2). The respective shRNA sequences were synthesized and individually subcloned into plasmid IMG-800 (Imgenex Corp., San Diego, CA, USA) such that the sequences could be expressed from the plasmid U6 promoter. The plasmid carries the pUC origin of replication which enables high plasmid copy numbers in bacterial cells. The recombinant plasmids were sequenced to ensure that the shRNA sequences were correct and in-frame for expression from the U6 promoter. The recombinant plasmids were transformed into the S. typhimurium minCDE- mutant strain and minicells carrying the plasmids were purified as described in <patcit id="pcit0026" dnum="US10602201B"><text>U.S. 10/602,201</text></patcit>. The recombinant minicells were designated minicellsshRNA-MDR1 and minicellsshRNA-bcl2 respectively.

Example 2. Demonstration of receptor-targeted recombinant minicell-mediated shRNA plasmid delivery to drug resistant cancer cells and reversal of drug resistance in-vitro.

0174While siRNAs directed against a range of different drug-resistance encoding transcripts have been shown to reverse drug resistance in cancer cells in-vitro, the critical hurdle is targeted delivery of the siRNAs into cancer cells, particularly in-vivo. Recombinant minicells carrying anti-MDR1 shRNA plasmid (minicellsshRNA-MDR1) and control shRNA against a nonsense RNA sequence (minicellsshRNA-nonsense) were purified and bispecific antibody carrying anti-S. typhimurium O-antigen and anti-human EGFR specificities was prepared and attached to the recombinant minicells, as described in patent application <patcit id="pcit0027" dnum="WO2005056749A"><text>Wo 2005/056749</text></patcit>. The targeted recombinant minicells were designated EGFR minicellsshRNA-MDR-1 and EGFRminicellsshRNA-nonsense. Minicells packaged with chemotherapeutic drugs 5-FU and irinotecan were also prepared and targeted as above and were designated EGFRminicells5-FU and EGFRminicellsIrino respectively.

0175Human colon cancer cell line Caco-2 (ATCC), which is highly resistant to irinotecan and 5-FU, was selected for this in-vitro study to determine firstly, if EGFR-targeted recombinant minicells could successfully deliver the shRNA plasmids to the cancer cells and secondly, if the expression of anti-MDR-1 siRNA could reverse the drug resistance and make the Caco-2 cells sensitive to EGFR-targeted and drug-packaged minicells. Caco-2 cells were seeded at 3 x 106 cells/ flask in Minimum Essential Medium with 10% cosmic calf serum and incubated for 3 hours at 37°C, 5% CO2.

0176The cells were treated with (a)EGFRminicellsshRNA-MDR-1, and (b)EGFRminicellsshRNA-nonsense. A control flask was included that did not receive any treatment. Minicells were added at a concentration of 1010 per flask and all flasks were incubated for 72 hrs. The cells from each treatment were trypsinised and seeded at 1×104 cells/ml/well in 24- well plates and were incubated for 3 hrs at 37°C, 5% CO2. The control untreated cells were then incubated with (6 wells / treatment) (a) free irinotecan (25 µM), (b) free 5-FU (25 µM), (c)EGFRminicellsIrino, and (d)EGFRminicells5-FU.

0177The EGFRminicellsshRNA-MDR-1 treated Caco-2 cells were incubated with (6 wells / treatment) (a)CMVminicells5-FU (non-specifically targeted since the bispecific antibody is targeted to a surface protein on cytomegalovirus), (b) free irinotecan, (c) free 5-FU, (d)EGFRminicellsIrino, and (e)EGFRminicells5-FU. The EGFRminicellsshRNA-nonsense treated Caco-2 cells were then treated with EGFRminicells5-FU,

0178All cells were incubated for a further 72 hrs followed by the colorimetric MTT cell proliferation assay (Cory et al., 1991) using the CellTiter 96 AQueous One Solution Cell Proliferation Assay (Promega Corp., Madison, WI, USA), according to the manufacturer's instructions. The colorimetric measurements were read at 490 nm.

0179The results showed (<figref idref="f0001">Fig. 1</figref>) that the Caco-2 cells were highly resistant to first-line chemotherapy drugs for colon cancer, i.e., irinotecan and 5-FU. Additionally, the cells remained resistant following treatments with EGFRminicellsIrino, EGFRminicells5-FU and EGFRminicellsshRNA-MDR-1. Cells that received the dual treatment, i.e., EGFRminicellsshRNA-MDR-1 followed by EGFRminicellsIrino or EGFRminicells5-FU showed that this treatment was highly successful in reversing the drug resistance and after a single combination treatment > 50% cell death was observed. In contrast, a dual treatment of EGFRminicellsshRNA-nonsense followed by EGFRminicells5-FU had no effect on drug resistance, suggesting that the anti-MDR-1 shRNA expression in the Caco-2 cells was specifically responsible for the reversal of drug resistance. The combination treatment of EGFRminicellsshRNA-MDR-1 followed by free irinotecan or 5-FU was also effective in reversal of drug resistance but to a lesser extent giving ∼ 30% reduction in cell survival. This data also suggests that chemotherapeutic drug delivery via receptor-targeted and drug-packaged minicells may deliver a more potent concentration of drug intracellularly compared to free drug provided in the extracellular environment.

0180This result demonstrated that (a) shRNAs can be effectively delivered to non-phagocytic mammalian cells via receptor-targeted recombinant minicells, (b) functional nucleic acid (shRNA) encoding plasmids escape from intracellular organelles where the minicells are broken down, (c) the plasmid is transported to the mammalian cell nucleus where the shRNA is expressed, (d) the shRNA is effective in degrading the mRNA encoding multi-drug resistance protein, MDR-1, (e) the same mammalian cells are receptive to the next wave of receptor-targeted minicells which now carry a drug instead of a plasmid, (f) the dual treatment protocol, i.e., receptor-targeted minicell-mediated shRNA delivery followed by receptor-targeted minicell-mediated chemotherapeutic drug delivery is highly effective in reversing drug resistance in non-phagocytic mammalian cells.

Example 3. In-vivo demonstration of tumor regression achieved using the method of invention i.e. dual treatment of receptor-targeted minicell-mediated shRNA delivery followed by receptor-targeted minicell-mediated drug delivery.

0181This example demonstrates that receptor-targeted minicells can be used to reverse drug resistance in cancer cells in-vivo.

0182S. typhimurium minCDE-derived minicells were purified and packaged with chemotherapeutic drug irinotecan. 7 x 10 minicells in BSG solution were centrifuged, the supernatant was discarded and the minicells were resuspended in 940 µl BSG and 60 µl of irinotecan solution (1 mg/ml; dissolved in sterile distilled water). The suspension was incubated overnight at 37° C with rotation to allow the irinotecan to diffuse into the minicell cytoplasm. Excess irinotecan non-specifically attached to the outer surface of the minicells was then washed away by stirred cell ultrafiltration as follows. Amicon stirred ultrafiltration cell Model 8010 (Millipore, Billerica, MA, USA) was assembled according to the manufacturer's instructions with an ultrafiltration membrane disc (polyethersulfone; molecular weight cut-off of 300 kDa; Millipore). The cell was washed three times with sterile distilled water followed by a further three washes with BSG. The cell was then filled with 9 ml of fresh BSG and the 1 ml solution of irinotecan-packaged minicells was added. The cell was kept under a pressure of 10 psi, stirred until the volume was reduced to 5 ml and topped-off with 5 ml BSG. Ultrafiltration was continued until the volume again dropped to 5 ml. This topping-off / ultrafiltration procedure was performed 6 times to enable a thorough washing of the exterior surfaces of the irinotecan-packaged minicells. During the last ultrafiltration, the volume was reduced to 1 ml and the sample was transferred to a sterile Eppendorf centrifuge tube, followed by centrifugation at 13,200 rpm for 10 minutes to pellet the irinotecan-packaged minicells.

0183A bispecific antibody was constructed as described above and in <patcit id="pcit0028" dnum="US20040265994A"><text>U.S. published Patent Application No. 2004-0265994</text></patcit>. Briefly, anti-S. typhimurium lipopolysaccharide (Biodesign, Saco, Maine, USA) and anti-human Epidermal Growth Factor Receptor (EGFR) mouse monoclonal antibodies (Oncogene Research Products, Cambridge, MA, USA) were linked to purified recombinant protein A/G via the Fc fragments of each monoclonal antibody. An anti-EGFR monoclonal antibody was selected because the xenografted cells were human colon cancer cells (Caco-2) that are known to overexpress the EGFR on the cell surface (Nyati et al., 2004).

0184Purified recombinant protein A/G (Pierce Biotechnology, Rockford, IL, USA) was diluted to a final concentration of 100 µg/ml in Immunopure binding buffer (Pierce Biotechnology) and 0.5 ml of the solution was incubated overnight at 4°C with a premixed solution containing 20 µg/ml each of anti-S. typhimurium LPS and anti-human EGFR monoclonal antibodies. The excess antibodies unbound to protein A/G were then removed as follows. Dynabeads® Protein G solution (Dynabeads® [2.8 µm] coated with recombinant Protein G covalently coupled to the surface of the magnetic particles; Dynal Biotech, Oslo, Norway) was mixed gently and 100 µl of the solution was transferred into an Eppendorf centrifuge tube. The tube was placed in a Dynal MPC-S (Magnetic Particle Concentrator, type S) to immobilize the beads and the supernatant was discarded. The beads were resuspended in 0.5 ml of washing solution containing 0.1M Na-phosphate buffer (pH 5.0). The bead immobilization and washing steps were repeated three times. The solution containing protein A/G-bispecific antibody mixture was added to the beads and incubated with gentle mixing at room temperature for 40 min. The tube was placed on the MPC-S stand to immobilize the beads and the protein A/G-bispecific antibody was removed with a pipette. This step eliminated the unbound excess monoclonal antibodies and provided a solution that carried the bispecific antibody linked to protein A/G via their Fc fragments. Recombinant minicells were incubated with the protein A/G-bispecific antibody for 1 hr at room temperature, to coat the minicells with the antibody via its anti-LPS Fab region.

0185The mice used in this example were purchased from Animal Resources Centre, Perth, WA, Australia, and all animal experiments were performed in compliance with the guide of care and use of laboratory animals and with Animal Ethics Committee approval. The experiments were performed in the NSW Agriculture accredited small animal facility at EnGeneIC Pty Ltd (Sydney, NSW, Australia). Human colon cancer cells (Caco-2, ATCC) were grown in tissue culture in RPMI 1640 medium supplemented with 5% Bovine Calf Serum (GIBCO-BRL Life Technologies, Invitrogen Corporation, Carlsbad, CA, USA) and glutamine (Invitrogen) in a humidified atmosphere of 95% air and 5% CO2 at 37° C. 1 × 106 cells in 50 µl serum-free media together with 50 µl growth factor reduced matrigel (BD Biosciences, Franklin Lakes, NJ, USA) were injected subcutaneously between the shoulder blades of each mouse using a 23-gauge needle. The tumors were measured twice a week using an electronic digital caliper (Mitutoyo, Japan, precision to 0.001) and mean tumor volume was calculated using the formula, length (mm) x width2 (mm) X 0.5 = volume (mm3). The various treatments commenced once the tumors reached volumes between 50 mm3 and 80 mm3 , and mice were randomized to eight different groups of 11 per group.

0186The various groups received the following treatments: Group 1 (control) received no treatment. Group 2 (control), free irinotecan (1.2 x 104 ng/gm of mouse body weight ∼ 2.4 x 105 ng per mouse) intravenously. This control was included to confirm the in-vitro results that the tumor cells were resistant to the drug. Group 3 (control), EGFR-targeted, irinotecan-packaged minicells (designated EGFRminicellsIrino). Group 4 (control),EGFRminicellsshRNA-MDR-1. Group 5 (control),EGFRminicellsshRNA-bcl-2. Group 6 (control),EGFRminicellsshRNA-MDR-1 followed by free irinotecan. Group 7 (experimental),EGFRminicellsshRNA-MDR-1 followed by EGFRminicellsIrino. Group 8 (expt.),EGFRminicellsshRNA-bcl-2 followed by EGFRminicellsIrino. Irinotecan quantitation studies by HPLC showed that 5 x 108 minicells packaged ∼ 80 ng of the drug. All minicell treatments received 5 x 108 minicells and shRNA treatments were administered on days 9 and 23. All drug treatments were administered on days 15, 18, 29 and 32. This allowed a six day interval between shRNA and drug treatments to ensure that sufficient time was allowed for intracellular and nuclear delivery of shRNA, gene expression and suppression of expression of the drug resistance mediating protein, i.e., either MDR-1 or bcl-2.

0187The results revealed (<figref idref="f0002">Fig. 2</figref>) a striking contrast between the mean tumor volumes in control groups (G 1 to 6) and experimental groups (G 7 and 8). The tumor volumes in the experimental groups were rapidly stabilized and showed significant stabilization in most of the 11 animals in each group. In contrast, the mean tumor volumes in all the different control groups continued to rise and by day 36 post-xenograft establishment the experiment was terminated because the control animals were too sick. The experimental animals, on the other hand, were healthy and did not show any toxic side effects of the treatment. Statistical analysis of the data using one-way ANOVA showed that experimental groups (7 and 8) were highly significant compared to the control groups 1 to 6 (p = 0.0004). This result is a first demonstration of targeted in-vivo delivery of shRNA to address the serious problem of drug resistance in cancer. The result also demonstrated that the invention has general application, because two mechanistically different methods of drug resistance, i.e., over-expressed membrane-associated protein pump (MDR-1) and cytoplasmic anti-apoptosis protein (bcl-2), can be down-regulated in drug-resistant cancer cells in-vivo. Treating the same cells with another wave of receptor-targeted, chemotherapeutic drug-packaged minicells could effectively treat such tumors.

Example 4. Second in-vivo demonstration of tumor regression efficacy achieved using the method of invention i.e. dual treatment of minicell-mediated shRNA delivery followed by minicell-mediated drug delivery.

0188Colorectal cancer cells are also known to be highly resistant to another first-line chemotherapeutic drug, 5-fluorouracil (5-FU), and this example shows that the methods of the invention enable not only the reversal of drug resistance in-vivo but also permit tumor stabilization / regression.

0189As described above, minicells were obtained from an S. typhimurium minCDE- mutant strain and were purified using the gradient centrifugation / filamentation / filtration / endotoxin removal procedure. Similarly recombinant minicells carrying shRNA plasmids, shRNA-MDR-1, shRNA-bcl-2 and shRNA-nonsense were obtained and purified from the respective S. typhimurium minCDE-recombinant strains. The purified empty minicells were packaged with chemotherapeutic drug 5-FU as described for irinotecan in Example 3. HPLC analysis was used to determine the concentration of 5-FU packaged in the mincells.

0190A bispecific antibody comprising anti-human EGFR and anti-S. typhimurium O-antigen dual specificities was constructed as described in Example 3. Recombinant minicells (1010) were incubated with the bispecific antibody for 1 hour at room temperature, to coat the minicells with the antibody via its anti-O-antigen Fab region.

0191Caco-2 cancer cell xenografts were established in Balb/c nude mice and once the tumors reached a volume between 50 mm3 and 80 mm3, mice were randomized into 10 groups (n = 11 mice per group). The 10 intravenous treatments included: (a) G1 - tumor only control. G2 (control), free 5-FU (5 × 104 ng/gm of mouse body weight ∼ 1 × 106 ng per mouse). This control was included to confirm the in-vitro results that the tumor cells were resistant to the drug. G3 (control), EGFR-targeted, 5-FU-packaged minicells (designated EGFRminicells5-FU). G4 (control), EGFRminicellsshRNA-MDR-1. G5 (control),EGFRminicellsshRNA-bcl-2. G6 (control),EGFRminicellsshRNA-MDR-1 followed by CMVminicells5-FU. The CMV antibody is targeted to a surface protein on cytomegalovirus and this serves as a non-specifically targeted control. G7 (control),EGFRminicellsshRNA-nonsense followed by EGFRminicells5-FU. G8 (control),EGFRminicellsshRNA-MDR-1 followed by free 5-FU,. G9 (expt),EGFRminicellsshRNA-MDR-1 followed by EGFRminicells5-FU. G10 (expt),EGFRminicellsshRNA-bcl-2 followed by EGFRminicells5-FU. The shRNA treatments were administered on days 9 and 23 and drug treatments were administered on days 15, 18, 29 and 32. This allowed a six day interval between shRNA and drug treatments to ensure that sufficient time was allowed for intracellular and nuclear delivery of shRNA, gene expression and suppression of expression of the drug resistance mediating protein, i.e., either MDR-1 or bcl-2.

0192The results revealed (<figref idref="f0003">Fig. 3</figref>) a striking contrast between the mean tumor volumes in control groups (G 1 to 8) and experimental groups (G 9 and 10). The tumor volumes in the experimental groups showed significant stabilization in most of the 11 animals in each group. In contrast, the mean tumor volumes in all the different control groups continued to rise and by day 36 post-xenograft establishment the experiment was terminated because the control animals were too sick. The experimental animals, on the other hand, were healthy and did not show any toxic side effects of the treatment. Statistical analysis of the data using one-way ANOVA showed that experimental groups (9 and 10) were highly significant compared to the control groups 1 to 8 (p = 0.0008).

Example 5. In-vivo demonstration of tumor regression achieved in doxorubicin resistant human breast cancer cells using the method of invention.

0193The inventors have shown that human breast adenocarcinoma cell line, MDA-MB-468 is highly sensitive to doxorubicin and that mouse xenografts treated intravenously with EGFRminicellsDox stabilize / regress.

0194In this example, MDA-MB-468 cells were cultivated in tissue culture and treated with increasing concentrations of Dox to develop a Dox-resistant clone. It is well established that such drug treatment in-vitro and in-vivo up-regulates the expression of multi-drug resistance proteins such as MDR-1 and bcl-2. Several Dox-resistant clones were obtained and one was used to establish a xenograft in Balb/c nude mice. The intravenous treatment groups (n = 11 mice per group) included G2 - EGFRminicellsDox and G3 -EGFRminicellsshRNA-MDR-1 followed by EGFRminicellsDox. G1 mice were tumor only control. The shRNA treatment was administered on day 21 and the drug treatments were given on days 27, 34 and 41.

0195The results showed (<figref idref="f0004">Fig. 4</figref>) that the EGFRminicellsshRNA-MDR-1 followed by EGFRminicellsDox treatment in G3 mice was highly effective in reversing Dox resistance in the cancer cells and that the tumors were stabilized. The control treatment with EGFRminicellsDox (G2) showed that the tumor cells were highly resistant to Dox and the tumors grew rapidly.

Example 6. In-vivo demonstration of effect of dosing schedules on reversal of drug resistance and therapeutic effect.

0196This example demonstrates the effect of dosing schedules on the reversal of drug resistance and therapeutic effect. Allowing sufficient time for efficient delivery of shRNA to the tumor cells before the receptor-targeted, drug-packaged minicells are administered improves the results. A time-course experiment was performed, wherein EGFRminicellsshRNA-MDR-1 were administered intravenously in nude mice carrying Caco-2 cell xenograft. In separate groups (n = 11 mice per group), mice were given EGFRminicellsIrino either at 96 hr (G3), 120 hr (G4) or 144 hr (G5) after the EGFRminicellsshRNA-MDR1 treatment. G1 and G2 were tumor only and free irinotecan (∼ 2.4 x 105 ng/dose) controls. The minicells were administered at 5 x 108 per dose and each dose carried ∼ 80 ng of irinotecan packaged in minicells, which is a 3,000-fold lower dose than that administered as free drug. The results showed (<figref idref="f0005">Fig. 5</figref>) a clear correlation between the time allowed for shRNA expression and subsequent administration of EGFRminicellsIrino with 144 hr (G5) being most effective in reversing drug resistance and achieving a significant therapeutic effect.

Example 7. Second in-vivo demonstration of effect of dosing schedules on reversal of drug resistance and therapeutic effect.

0197This example demonstrates that the dosing schedule effect observed in example 6 is broadly applicable.

0198The experiment described in example 6 was repeated with the same controls and experimental groups except that G2 received free 5-FU (1 x 106 ng /dose) and in G3, G4 and G5, the second treatment was carried out with EGFRminicells5-FU. Minicells were administered at 1 x 109 per dose and each dose carried ∼ 80 ng 5-FU, i.e., ∼ 12,500-fold lower than free drug administration in G2 mice.

0199The results showed (<figref idref="f0006">Fig. 6</figref>) that administration of EGFRminicells5-FU at 144 hrs after the administration of EGFRminicellsshRNA-MDR-1 (G5) resulted in maximal efficacy of reversal of drug resistance and therapeutic efficacy. The potency of the invention is evident from the concentration of drug required to effectively treat these highly resistant tumors since the minicells carried 3,000-fold and 12,500-fold less drug compared to free irinotecan and 5-FU treatments respectively. The free drugs had no effect on tumor growth as seen in <figref idref="f0005">Figs 5</figref> and <figref idref="f0006">6</figref>.

REFERENCES

0200All publications and patents mentioned in this specification are incorporated herein by reference. Reference to a publication or patent, however, does not constitute an admission as to prior art. <ul id="ul0001" list-style="none" compact="compact"><li><nplcit id="ncit0001" npl-type="s"><text>Ambudkar, et al., Annu. Rev. Pharmacol. Toxicol. 39: 361 (1999</text></nplcit>).</li><li><nplcit id="ncit0002" npl-type="s"><text>Antoku, et al., Biochem. Biophys. Res. Commun., 286: 1003 (2001</text></nplcit>).</li><li><nplcit id="ncit0003" npl-type="s"><text>Ayesh et al., Anticancer Drugs, 7(6): 678-86 (1996</text></nplcit>)</li><li><nplcit id="ncit0004" npl-type="s"><text>Ayesh et al., Biochim. Biophys. Acta, 1316(1): 8-18 (1996</text></nplcit>)</li><li><nplcit id="ncit0005" npl-type="s"><text>Bakhshi, et al., Cell, 41: 899 (1985</text></nplcit>).</li><li><nplcit id="ncit0006" npl-type="s"><text>Bargou, et al., Int. J. Cancer, 60: 854 (1995</text></nplcit>).</li><li><nplcit id="ncit0007" npl-type="s"><text>Batra et al., Gene Ther. 1(4): 255-60 (1994</text></nplcit>).</li><li><nplcit id="ncit0008" npl-type="s"><text>Becker et al., Cancer Biol Ther., 1(5):548-53 (2002</text></nplcit>).</li><li><nplcit id="ncit0009" npl-type="s"><text>Bredel, Brain Res. Rev., 35: 161 (2001</text></nplcit>).</li><li><nplcit id="ncit0010" npl-type="s"><text>Britton et al., Genes Dev., 12: 1254 (1998</text></nplcit>).</li><li><nplcit id="ncit0011" npl-type="s"><text>Broxterman et al., Cancer Lett., 35(1): 87-95 (1987</text></nplcit>).</li><li><nplcit id="ncit0012" npl-type="s"><text>Brummelkamp, et al., Science 296:550 (2002</text></nplcit>).</li><li><nplcit id="ncit0013" npl-type="s"><text>Caplen, Expert Opin. Biol. Ther., 3(4): 575-86 (2003</text></nplcit>).</li><li><nplcit id="ncit0014" npl-type="s"><text>Caplen and Mousses, Ann. N.Y. Acad. Sci., 1002: 56-62 (2003</text></nplcit>).</li><li><nplcit id="ncit0015" npl-type="s"><text>Carter, P. Nat Rev Cancer, 1(2): 118-29 (2001</text></nplcit>).</li><li><nplcit id="ncit0016" npl-type="s"><text>Chen, et al., Int. J. Cancer, 93: 107 (2001</text></nplcit>).</li><li><nplcit id="ncit0017" npl-type="s"><text>Ciliberto et al., Cell. 41: 531 (1985</text></nplcit>).</li><li><nplcit id="ncit0018" npl-type="s"><text>Cole et al., Science 258:1650-1654 (1992</text></nplcit>).</li><li><nplcit id="ncit0019" npl-type="s"><text>Collins & Olive, 32 Biochem. 2795-99 (1993</text></nplcit>).</li><li><nplcit id="ncit0020" npl-type="s"><text>Cory et al., Cancer Commun. 3: 207-212 (1991</text></nplcit>).</li><li><nplcit id="ncit0021" npl-type="s"><text>Cory, S. Annu. Rev. Immunol., 13: 513-543 (1995</text></nplcit>).</li><li><nplcit id="ncit0022" npl-type="s"><text>de Boer et al., J. Bacteriol. 174: 63 (1992</text></nplcit>).</li><li><nplcit id="ncit0023" npl-type="s"><text>de Haard, H. J. et al. J. Biol. Chem. 274, 18218-18230 (1999</text></nplcit>).</li><li><nplcit id="ncit0024" npl-type="s"><text>Doige et al., Ann. Rev. Microbiol., 47: 291-319 (1993</text></nplcit>).</li><li><nplcit id="ncit0025" npl-type="s"><text>Dubel et al., J. Immunol. Methods, 178: 201-209 (1995</text></nplcit>).</li><li><nplcit id="ncit0026" npl-type="s"><text>Duan et al., Mol. Cancer Therapeutics, 3(7): 833-38 (2004</text></nplcit>).</li><li><nplcit id="ncit0027" npl-type="s"><text>Duxbury et al., J. Am. Coll. Surg., 198: 953-59 (2004</text></nplcit>).</li><li><nplcit id="ncit0028" npl-type="s"><text>Elbashir et al., Genes Dev., 15(2):188-200 (2001</text></nplcit>).</li><li><nplcit id="ncit0029" npl-type="s"><text>Elbashir et al., Nature, 411(6836):494-8 (2001</text></nplcit>).</li><li><nplcit id="ncit0030" npl-type="s"><text>Endicott et al., Ann. Rev. Biochem., 58 : 137-71 (1989</text></nplcit>).</li><li><nplcit id="ncit0031" npl-type="s"><text>Fardel et al., Gen Pharmacol., 27(8):1283-1291 (1996</text></nplcit>).</li><li><nplcit id="ncit0032" npl-type="s"><text>Forbes, Australian J. Biotechnology 1: 30 (1987</text></nplcit>).</li><li><nplcit id="ncit0033" npl-type="s"><text>Frain et al., Mol. Cell Biol.., 10: 991 (1990</text></nplcit>).</li><li><nplcit id="ncit0034" npl-type="s"><text>Frankel et al., Leukemia, 14: 576-585 (2000</text></nplcit>).</li><li><nplcit id="ncit0035" npl-type="s"><text>Gariboldi et al., Int. J. Oncology, 22: 1057-64 (2003</text></nplcit>).</li><li><nplcit id="ncit0036" npl-type="s"><text>Glennie et al., J. Immunol., 139(7):2367-75 (1987</text></nplcit>).</li><li><nplcit id="ncit0037" npl-type="s"><text>Gosselin et al., Biotechnol. Annu. Rev., 8:103-31 (2002</text></nplcit>).</li><li><nplcit id="ncit0038" npl-type="s"><text>Gottesman, et al., Nat. Rev. Cancer 2: 48 (2002</text></nplcit>).</li><li><nplcit id="ncit0039" npl-type="s"><text>Griffiths et al. , EMBO J. 13: 3245-3260 (1994</text></nplcit>).</li><li><nplcit id="ncit0040" npl-type="s"><text>Guerrier-Takada et al., Cell, 35: 849 (1983</text></nplcit>).</li><li><nplcit id="ncit0041" npl-type="s"><text>Hampel and Tritz, Biochem., 28: 4929 (1989</text></nplcit>).</li><li><nplcit id="ncit0042" npl-type="s"><text>Hampel et al., Nucleic Acids Research: 299 (1990</text></nplcit>)</li><li><nplcit id="ncit0043" npl-type="s"><text>Hanada, et al., J. Biol. Chem., 270: 11962 (1995</text></nplcit>).</li><li><nplcit id="ncit0044" npl-type="s"><text>Hanahan, Nature, 315: 115 (1985</text></nplcit>).</li><li><nplcit id="ncit0045" npl-type="s"><text>Hannon, Nature (Lond.), 418: 244 (2002</text></nplcit>).</li><li><nplcit id="ncit0046" npl-type="s"><text>Harry, Mol. Microbiol., 40: 795 (2001</text></nplcit>).</li><li><nplcit id="ncit0047" npl-type="s"><text>Hart, Semin. Oncol., 23: 154 (1996</text></nplcit>).</li><li><nplcit id="ncit0048" npl-type="s"><text>Heim et al., Proc. Nat'1. Acad. Sci. USA, 91: 12501 (1994</text></nplcit>).</li><li><nplcit id="ncit0049" npl-type="s"><text>Hiraga et al., J. Bacteriol., 171: 1496 (1989</text></nplcit>).</li><li><nplcit id="ncit0050" npl-type="s"><text>Hoshida et al., Pancreas, 25(2):111-21 (2002</text></nplcit>).</li><li><nplcit id="ncit0051" npl-type="s"><text>Hu et al., Cancer Res., 56: 3055-3061 (1996</text></nplcit>).</li><li><nplcit id="ncit0052" npl-type="s"><text>Hu et al., Mol. Microbiol., 34:82-90 (1999</text></nplcit>).</li><li><nplcit id="ncit0053" npl-type="s"><text>Hunter et al., Mol. Cell. Biol., 16: 877 (1996</text></nplcit>).</li><li><nplcit id="ncit0054" npl-type="s"><text>Hudson & Souriau, Expert Opin. Biol. Ther. 1: 845-855 (2001</text></nplcit>).</li><li><nplcit id="ncit0055" npl-type="s"><text>Hudson & Souriau, Nat. Med., 9 (1):129-34 (2003</text></nplcit>).</li><li><nplcit id="ncit0056" npl-type="s"><text>Hung et al., Adv. Exp. Med. Biol., 465:171-80 (2000</text></nplcit>).</li><li><nplcit id="ncit0057" npl-type="s"><text>Hutvagner & Zamore, Curr. Opin. Genet. Dev., 12: 225 (2002</text></nplcit>).</li><li><nplcit id="ncit0058" npl-type="s"><text>Ireton et al., J. Bacteriol. 176: 5320 (1994</text></nplcit>).</li><li><nplcit id="ncit0059" npl-type="s"><text>Jones et al., Nature 321: 522-525 (1986</text></nplcit>).</li><li><nplcit id="ncit0060" npl-type="s"><text>Juliano & Ling, Biochimica et Biophysica Acta, 455:152 (1976</text></nplcit>).</li><li><nplcit id="ncit0061" npl-type="s"><text>Kaetzel et al., Biochem. Soc. Trans. 25:475-480 (1997</text></nplcit>).</li><li><nplcit id="ncit0062" npl-type="s"><text>Karpovsky et al., J. Exp. Med., 160(6):1686-701 (1984</text></nplcit>).</li><li><nplcit id="ncit0063" npl-type="s"><text>Katabi et al., Human Gene Therapy 10: 155 (1999</text></nplcit>).</li><li><nplcit id="ncit0064" npl-type="s"><text>Kelsey et al., Genes and Devel. 1: 161 (1987</text></nplcit>).</li><li><nplcit id="ncit0065" npl-type="s"><text>Kleeff et al., Cancer Gene Ther., 9(6):522-32 (2002</text></nplcit>).</li><li><nplcit id="ncit0066" npl-type="s"><text>Knappik et al., J. Mol. Biol. 296: 57-86 (2000</text></nplcit>).</li><li><nplcit id="ncit0067" npl-type="s"><text>Kostelny et al., J. Immunol. 148(5):1547-53 (1992</text></nplcit>).</li><li><nplcit id="ncit0068" npl-type="s"><text>Kroemer, Nat. Med., 3: 614 (1997</text></nplcit>).</li><li><nplcit id="ncit0069" npl-type="s"><text>Kurane et al., Jpn. J. Cancer Res. 89: 1212 (1998</text></nplcit>).</li><li><nplcit id="ncit0070" npl-type="s"><text>Leder et al., Cell 45: 485 (1986</text></nplcit>).</li><li><nplcit id="ncit0071" npl-type="s"><text>Lehnert, Eur. J. Cancer, A(6): 912-20 (1996</text></nplcit>).</li><li><nplcit id="ncit0072" npl-type="s"><text>Levin et al., J. Bacteriol. 174: 6717 (1992</text></nplcit>).</li><li><nplcit id="ncit0073" npl-type="s"><text>List, Oncology, 7(10): 23-8, 32, 35-38 (1993</text></nplcit>).</li><li><nplcit id="ncit0074" npl-type="s"><text>List et al., J. Clin. Oncol., 11(9): 1652-60 (1993</text></nplcit>).</li><li><nplcit id="ncit0075" npl-type="s"><text>Litman, et al., J. Cell Sci., 113: 2011 (2000</text></nplcit>).</li><li><nplcit id="ncit0076" npl-type="s"><text>Litman et al., Cell Mol. Life Sci., 58(7): 931-59 (2001</text></nplcit>).</li><li><nplcit id="ncit0077" npl-type="s"><text>MacDonald et al., Hepatology 7: 425 (1987</text></nplcit>).</li><li><nplcit id="ncit0078" npl-type="s"><text>Mason et al., Science 234: 1372 (1986</text></nplcit>).</li><li><nplcit id="ncit0079" npl-type="b"><text>McCubrey, et al., Cell Cycle Checkpoints and Cancer, pp. 17-53. Georgetown, TX: Landes Bioscience, (2001 a</text></nplcit>).</li><li><nplcit id="ncit0080" npl-type="s"><text>McCubrey, et al., Leukemia, 15: 1203 (2001</text></nplcit>).</li><li><nplcit id="ncit0081" npl-type="s"><text>Miller et al., J. Clin. Oncol., 9(1): 17-24 (1991</text></nplcit>).</li><li><nplcit id="ncit0082" npl-type="s"><text>Morton & Potter, J. Pharmacology & Exper. Therapeutics 286: 1066 (1998</text></nplcit>).</li><li><nplcit id="ncit0083" npl-type="s"><text>Nieth et al., FEBS Letters, 545: 144-50 (2003</text></nplcit>).</li><li><nplcit id="ncit0084" npl-type="s"><text>Nyati et al., Clin Cancer Res. 10; 691-700 (2004</text></nplcit>).</li><li><nplcit id="ncit0085" npl-type="s"><text>Okada et al., Sci. Prog. 77: 253 (1993-94</text></nplcit>).</li><li><nplcit id="ncit0086" npl-type="s"><text>Okada et al., J. Bacteriol. 176: 917 (1994</text></nplcit>).</li><li><nplcit id="ncit0087" npl-type="s"><text>Osbourn et al., Drug Delivery Tech., 8: 845-851 (2003</text></nplcit>).</li><li><nplcit id="ncit0088" npl-type="s"><text>Pack et al., Biochemistry, 31(6):1579-84 (1992</text></nplcit>).</li><li><nplcit id="ncit0089" npl-type="s"><text>Perrotta and Been, Biochem., 31: 16 (1992</text></nplcit>)</li><li><nplcit id="ncit0090" npl-type="s"><text>Pikaar et al., J. Infect. Dis. 172: 481 (1995</text></nplcit>).</li><li><nplcit id="ncit0091" npl-type="s"><text>Pinkert et al., Genes and Devel. 1: 268 (1987</text></nplcit>).</li><li><nplcit id="ncit0092" npl-type="s"><text>Prasher et al., Trends in Genetics 11: 320 (1995</text></nplcit>).</li><li><nplcit id="ncit0093" npl-type="s"><text>Raskin & de Boer, J. Bacteriol. 181: 6419 (1999</text></nplcit>).</li><li><nplcit id="ncit0094" npl-type="s"><text>Readhead et al., Cell 48: 703 (1987</text></nplcit>).</li><li><nplcit id="ncit0095" npl-type="s"><text>Reeve & Cornett, J. Virol. 15: 1308 (1975</text></nplcit>).</li><li><nplcit id="ncit0096" npl-type="s"><text>Ridgway et al., Protein Eng., 9(7): 617-21 (1996</text></nplcit>).</li><li><nplcit id="ncit0097" npl-type="s"><text>Riezman, Trends in Cell Biology, 3: 330 (1993</text></nplcit>).</li><li><nplcit id="ncit0098" npl-type="s"><text>Rossi et al., Aids Research and Human Retroviruses, 8: 183 (1992</text></nplcit>)</li><li><nplcit id="ncit0099" npl-type="s"><text>Salomon et al., Crit. Rev. Oncol. Hematol., 19: 183-232 (1995</text></nplcit>).</li><li><nplcit id="ncit0100" npl-type="s"><text>Salveson et al., Cell, 91: 443 (1997</text></nplcit>).</li><li><nplcit id="ncit0101" npl-type="s"><text>Sandvig & Deurs, Trends in Cell Biology, 4: 275 (1994</text></nplcit>).</li><li><nplcit id="ncit0102" npl-type="s"><text>Sato, et al., Proc. Natl. Acad. Sci. USA, 91: 9238 (1994</text></nplcit>).</li><li><nplcit id="ncit0103" npl-type="s"><text>Sattler, et al., Science (Wash. DC), 275: 983 (1997</text></nplcit>).</li><li><nplcit id="ncit0104" npl-type="s"><text>Saville & Collins, Cell, 61: 685-96 (1990</text></nplcit>).</li><li><nplcit id="ncit0105" npl-type="s"><text>Saville & Collins, PNAS (USA), 88: 8826-30 (1991</text></nplcit>).</li><li><nplcit id="ncit0106" npl-type="s"><text>Scheffer, et al., Curr. Opin. Oncol., 12: 550 (2000</text></nplcit>).</li><li><nplcit id="ncit0107" npl-type="s"><text>Sellers & Fisher, J. Clin. Invest., 104: 1655 (1999</text></nplcit>).</li><li><nplcit id="ncit0108" npl-type="s"><text>Sharp, Genes Dev., 15: 485 (2001</text></nplcit>).</li><li><nplcit id="ncit0109" npl-type="s"><text>Shaw & Griffen, Nature 289: 409 (1981</text></nplcit>).</li><li><nplcit id="ncit0110" npl-type="s"><text>Sheets et al., Proc. Natl Acad. Sci. USA, 95: 6157-6162 (1998</text></nplcit>).</li><li><nplcit id="ncit0111" npl-type="s"><text>Siould, "Therapeutic siRNAs," Trends in Pharmacological Sciences, 25(1): 22-28 (2004</text></nplcit>).</li><li><nplcit id="ncit0112" npl-type="s"><text>Speert et al., J. Clin. Invest., 82: 872 (1988</text></nplcit>).</li><li><nplcit id="ncit0113" npl-type="s"><text>Stewart & D'Ari, J. Bacteriol., 174: 4513 (1992</text></nplcit>).</li><li><nplcit id="ncit0114" npl-type="s"><text>Sun, et al., Biochem. Biophys. Res. Commun., 280: 788 (2001</text></nplcit>).</li><li><nplcit id="ncit0115" npl-type="s"><text>Swift et al., Cell, 38: 639 (1984</text></nplcit>).</li><li><nplcit id="ncit0116" npl-type="s"><text>Thurnher et al., Glycobiology, 4(4):429-35 (1994</text></nplcit>).</li><li><nplcit id="ncit0117" npl-type="s"><text>Todorovska et al., J. Immunol. Methods, 248: 47-66 (2001</text></nplcit>).</li><li><nplcit id="ncit0118" npl-type="s"><text>Tomlinson & Holliger, Methods Enzymol., 326: 461-479 (2000</text></nplcit>).</li><li><patcit id="pcit0029" dnum="US3817837A"><text>U.S. patent No. 3,817,837</text></patcit></li><li><patcit id="pcit0030" dnum="US3850752A"><text>U.S. patent No. 3,850,752</text></patcit></li><li><patcit id="pcit0031" dnum="US3939350A"><text>U.S. patent No. 3,939,350</text></patcit></li><li><patcit id="pcit0032" dnum="US3996345A"><text>U.S. patent No. 3,996,345</text></patcit></li><li><patcit id="pcit0033" dnum="US4275149A"><text>U.S. patent No. 4,275,149</text></patcit></li><li><patcit id="pcit0034" dnum="US4277437A"><text>U.S. patent No. 4,277,437</text></patcit></li><li><patcit id="pcit0035" dnum="US4366241A"><text>U.S. patent No. 4,366,241</text></patcit></li><li><patcit id="pcit0036" dnum="US4496778A"><text>U.S. patent No. 4,496,778</text></patcit></li><li><patcit id="pcit0037" dnum="US4987071A"><text>U.S. patent No. 4,987,071</text></patcit></li><li><patcit id="pcit0038" dnum="US4975278A"><text>U.S. Patent No. 4,975,278</text></patcit></li><li><patcit id="pcit0039" dnum="US5037743A"><text>U.S. patent No. 5,037,743</text></patcit></li><li><patcit id="pcit0040" dnum="US5143830A"><text>U.S. patent No. 5,143,830</text></patcit></li><li><patcit id="pcit0041" dnum="US5260203A"><text>U.S. patent No. 5,260,203</text></patcit></li><li><patcit id="pcit0042" dnum="US5455030A"><text>U.S. patent No. 5,455,030</text></patcit></li><li><patcit id="pcit0043" dnum="US5530101A"><text>U.S. patent No. 5,530,101</text></patcit></li><li><patcit id="pcit0044" dnum="US5585089A"><text>U.S. patent No. 5,585,089</text></patcit></li><li><patcit id="pcit0045" dnum="US6025197A"><text>U.S. patent No. 6,025,197</text></patcit></li><li><patcit id="pcit0046" dnum="US6639055B"><text>U.S. patent No. 6,639,055</text></patcit></li><li><patcit id="pcit0047" dnum="US20040265994A"><text>U.S. published patent application No. 2004-0265994</text></patcit></li><li><nplcit id="ncit0119" npl-type="s"><text>Van Bambeke, et al., Biochem. Pharmacol. , 60: 457 (2000</text></nplcit>).</li><li><nplcit id="ncit0120" npl-type="s"><text>Vaughan, et al., Nature Biotechnol., 14: 309-314 (1996</text></nplcit>).</li><li><nplcit id="ncit0121" npl-type="s"><text>Vaughan et al., Nature Biotechnol., 16: 535-539 (1998</text></nplcit>).</li><li><nplcit id="ncit0122" npl-type="s"><text>Wachi et al., J. Bacteriol., 171: 6511 (1989</text></nplcit>).</li><li><nplcit id="ncit0123" npl-type="s"><text>Wang, et al., Nat. Med., 5: 412 (1999</text></nplcit>).</li><li><nplcit id="ncit0124" npl-type="s"><text>Wang et al., Proc. Natl. Acad. Sci. USA, 93: 7063-7068 (1996</text></nplcit>).</li><li><nplcit id="ncit0125" npl-type="s"><text>White & McCubrey, Leukemia, 15: 1011-1021 (2001</text></nplcit>).</li><li><patcit id="pcit0048" dnum="WO8101145A"><text>WO 81/01145</text></patcit></li><li><patcit id="pcit0049" dnum="WO8807378A"><text>WO 88/07378</text></patcit></li><li><patcit id="pcit0050" dnum="WO9521191A"><text>WO 95/21191</text></patcit></li><li><patcit id="pcit0051" dnum="WO0067776A"><text>WO 00/67776</text></patcit></li><li><patcit id="pcit0052" dnum="WO05056749A"><text>WO 05/056749</text></patcit></li><li><nplcit id="ncit0126" npl-type="s"><text>Wu, et al., Cancer Res., 63: 1515-19 (2003</text></nplcit>).</li><li><nplcit id="ncit0127" npl-type="s"><text>Wacheck, et al., Oligonucleotides, 13: 393 (2003</text></nplcit>).</li><li><nplcit id="ncit0128" npl-type="s"><text>Wright & Jong, Experimental Medi., 163: 1245 (1986</text></nplcit>).</li><li><nplcit id="ncit0129" npl-type="s"><text>Yague et al., Gene Therapy, 11: 1170-74 (2004</text></nplcit>).</li><li><nplcit id="ncit0130" npl-type="s"><text>Ziady et al., Am. J. Physiol., 273(2 Pt 1):G545-52 (1997</text></nplcit>).</li></ul> Items: <ul id="ul0002" list-style="none"><li>Item 1. A composition comprising (a) an intact minicell that contains a functional nucleic acid molecule or a plasmid comprising a segment that encodes a functional nucleic acid molecule, and (b) a pharmaceutically acceptable carrier therefore, wherein said functional nucleic acid molecule targets the transcript of a protein that contributes to drug resistance, apoptosis resistance, or neoplasticity.</li><li>Item 2. The composition of item 1, wherein said plasmid comprises a regulatory element operably linked to the segment that encodes a functional nucleic acid.</li><li>Item 3. The composition of item 2, wherein said regulatory element is a promoter dependent on RNA polymerase.</li><li>Item 4. The composition of item 3, wherein said promoter is the RNA polymerase III promoter H1 or U6 or 7SK or the RNA polymerase II promoter CMV immediate early promoter.</li><li>Item 5. The composition of item 1, wherein said functional nucleic acid is an siRNA, shRNA or miRNA molecule that targets the transcript of a protein that contributes to drug resistance.</li><li>Item 6. The composition of item 1, wherein said functional nucleic acid is an antisense molecule that targets the transcript of a protein that contributes to drug resistance.</li><li>Item 7. The composition of item 1, wherein said functional nucleic acid molecule is a ribozyme that targets the transcript of a protein that contributes to drug resistance.</li><li>Item 8. The composition of item 1, wherein said functional nucleic acid molecule targets the transcript of P-glycoprotein, MDR-2 or MDR-3.</li><li>Item 9. The composition of item 1, wherein said functional nucleic acid molecule targets the transcript of MRP2, BCR-ABL, STI-571 resistance-associated protein, lung resistance-related protein, cyclooxygenase-2, nuclear factor kappa, XRCC1, ERCC1, GSTP1, mutant β-tubulin, or a growth factor.</li><li>Item 10. The composition of item 1, wherein said functional nucleic acid molecule targets the transcript of a protein that contributes to apoptosis resistance.</li><li>Item 11. The composition of item 10, wherein said functional nucleic acid molecule targets a transcript of Bcl-2, Bcl-XL, A1/Bfl 1, focal adhesion kinase or p53 protein..</li><li>Item 12. The composition of item 1, wherein said functional nucleic acid molecule targets a transcript of a protein that contributes to neoplasticity.</li><li>Item 13. The composition of item 12, wherein said functional nucleic acid molecule targets a transcript of β-Catenin, PKC-α, C-RAF, K-Ras, DP97 Dead box RNA helicase, DNMT1, FLIP, C-Sfc, 53BPI, Polycomb group protein EZH2, ErbB1, HPV-16 E5 and E7, Fortilin & MCI1P, DIP13α, MBD2, p21, KLF4, tpt/TCTP, SPK1 & SPK2, P300, PLK1, Trp53, Ras, ErbB1, VEGF, or BAG-1.</li><li>Item 14. The composition of item 1, wherein said plasmid encodes multiple functional nucleic acid molecules.</li><li>Item 15. The composition of item 14, wherein said plasmid further comprises a promoter for each encoded functional nucleic acid molecule.</li><li>Item 16. The composition of item 1, further comprising a drug.</li><li>Item 17. The composition of item 16, wherein said functional nucleic acid molecule targets the transcript of a protein that contributes to resistance to said drug.</li><li>Item 18. The composition of item 16, wherein said drug is packaged in an intact minicell.</li><li>Item 19. The composition of item 18, wherein said functional nucleic acid molecule or plasmid, and said drug are packaged within the same minicell.</li><li>Item 20. The composition of item 1, further comprising a bispecific ligand.</li><li>Item 21. The composition of item 20, wherein said bispecific ligand comprises a first arm that carries specificity for a minicell surface structure and a second arm that carries specificity for a non-phagocytic mammalian cell surface receptor.</li><li>Item 22. The composition of item 21, wherein said minicell surface structure is an O- polysaccharide component of a lipopolysaccharide on said minicell surface.</li><li>Item 23. The composition of item 21, wherein said mammalian cell surface receptor is capable of activating receptor-mediated endocytosis of said minicell.</li><li>Item 24. The composition of item 20, wherein said bispecific ligand comprises an antibody or antibody fragment.</li><li>Item 25. The composition of item 1, wherein said composition contains fewer than about 1 contaminating parent bacterial cell per 107 minicells.</li><li>Item 26. The composition of item 1, wherein said composition contains fewer than about 1 contaminating parent bacterial cell per 108 minicells</li><li>Item 27. The composition of item 1, wherein said composition contains fewer than about 1 contaminating parent bacterial cell per 109 minicells</li><li>Item 28. The composition of item 1, wherein said composition contains fewer than about 1 contaminating parent bacterial cell per 1010 minicells</li><li>Item29. The composition of item 1, wherein said composition contains fewer than about 1 contaminating parent bacterial cell per 1011 minicells.</li><li>Item 30. A method of delivering a functional nucleic acid, comprising (a) providing an intact minicell that contains (i) a functional nucleic acid molecule or (ii) a plasmid comprised of a segment that encodes a functional nucleic acid molecule and then (b) bringing said minicell into contact with a target mammalian cell, such that said mammalian cell engulfs said minicell, whereby said functional nucleic acid is released into the cytoplasm of the target cell.</li><li>Item 31. The method of item 30, wherein said functional nucleic acid is expressed by the target cell.</li><li>Item 32. The method of item 30, wherein said minicell contains a plasmid comprised of a regulatory element that is operably linked to said segment.</li><li>Item 33. The method of item 31, wherein said regulatory element is a promoter dependent on RNA polymerase.</li><li>Item 34. The method of item 33, wherein said promoter is the RNA III polymerase promoter H1 or U6.</li><li>Item 35. The method of item 30, wherein contact between said minicell and said mammalian cell occurs in vitro.</li><li>Item 36. The method of item 30, wherein contact between said minicell and said mammalian cell occurs in vivo.</li><li>Item 37. The method of item 30, wherein said functional nucleic acid molecule is an siRNA molecule that targets the transcript of a protein that contributes to drug-resistance.</li><li>Item 38. The method of item 30, wherein said functional nucleic acid molecule is an antisense molecule that targets the transcript of a protein that contributes to drug-resistance.</li><li>Item 39. The method of item 30, wherein said functional nucleic acid molecule is a ribozyme that targets the transcript of a protein that contributes to drug-resistance.</li><li>Item 40. The method of item 30, wherein said functional nucleic acid molecule targets the transcript of P-glycoprotein, MDR-2 or MDR-3.</li><li>Item 41. The method of item 30, wherein said functional nucleic acid molecule targets the transcript of MRP2, BCR-ABL, STI-571 resistance-associated protein, lung resistance-related protein, cyclooxygenase-2, nuclear factor kappa, XRCC1, ERCC1, GSTP1, mutant β-tubulin, or a growth factor.</li><li>Item 42. The method of item 30, wherein said functional nucleic acid molecule targets the transcript of a protein that contributes to apoptosis resistance.</li><li>Item 43. The method of item 42, wherein said functional nucleic acid molecule targets a transcript of Bcl-2, Bcl-XL, A1/Bfl 1, focal adhesion kinase or p53 protein.</li><li>Item 44. The method of item 30, wherein said functional nucleic acid molecule targets a transcript of a protein that contributes to neoplasticity.</li><li>Item 45. The method of item 44, wherein said functional nucleic acid molecule targets a transcript of β-Catenin, PKC-α, C-RAF, K-Ras, DP97 Dead box RNA helicase, DNMT1, FLIP, C-Sfc, 53BPI, Polycomb group protein EZH2, ErbB1, HPV-16 E5 and E7, Fortilin & MCI1P, DIP13α, MBD2, p21, KLF4, tpt/TCTP, SPK1 & SPK2, P300, PLK1, Trp53, Ras, ErbB1, VEGF, or BAG-1.</li><li>Item 46. The method of item 30, wherein said plasmid encodes multiple functional nucleic acid molecules.</li><li>Item 47. The method of item 46, wherein said plasmid further comprises a promoter for each encoded functional nucleic acid molecule.</li><li>Item 48. The method of item 30, further comprising the step of (c) delivering a drug to said target mammalian cell.</li><li>Item 49. The method of item 48, wherein said functional nucleic acid molecule targets the transcript of a protein that contributes to resistance to said drug.</li><li>Item 50. The method of item 48, wherein said drug is packaged in an intact minicell.</li><li>Item 51. The method of item 50, wherein said functional nucleic acid molecule or plasmid, and said drug are packaged within the same minicell.</li><li>Item 52. The method of item 48, wherein step (c) occurs subsequent to steps (a) and (b).</li><li>Item 53. The method of item 48, wherein said drug is delivered concurrently with step (b).</li><li>Item 54. The method of item 30, wherein step (b) entails bringing a bispecific ligand into contact with said intact minicell and said target mammalian cell, such that (i) said bispecific ligand causes said minicell to bind to said mammalian cell.</li><li>Item 55. The method of item 54, wherein said target mammalian cell is a non-phagocytic cell.</li><li>Item 56. The method of item 54, wherein said bispecific ligand comprises a first arm that carries specificity for a minicell surface structure and a second arm that carries specificity for a non-phagocytic mammalian cell surface receptor.</li><li>Item 57. The method of item 56, wherein said minicell surface structure is an O-polysaccharide component of a lipopolysaccharide on said minicell surface.</li><li>Item 58. The method of item 56, wherein said mammalian cell surface receptor is capable of activating receptor-mediated endocytosis of said minicell.</li><li>Item 59. The method of item 54, wherein said bispecific ligand comprises an antibody or antibody fragment.</li><li>Item 60. The method of item 30, wherein said mammalian cell is phagocytosis- or endocytosis-competent.</li></ul>

Contents5

Every citation, both ways

Citations
Document	Relation	Office	Cited during
US10888529B2	Cited by	United States of America	Applicant
WO2017174847A1	Cited by	World Intellectual Property Organization (WIPO)	Applicant
WO0067776A1	Cites	World Intellectual Property Organization (WIPO)	Applicant
WO0217852A2	Cites	World Intellectual Property Organization (WIPO)	Search report
WO03033519A2	Cites	World Intellectual Property Organization (WIPO)	Search report
US2003203481A1	Cites	United States of America	Search report
US2004265994A1	Cites	United States of America	Applicant
US2004265994A1	Cites	United States of America	Applicant
WO2005056749A2	Cites	World Intellectual Property Organization (WIPO)	Applicant
WO2005056749A2	Cites	World Intellectual Property Organization (WIPO)	Applicant
WO2005056749A2	Cites	World Intellectual Property Organization (WIPO)	Search report
WO2005056749A2	Cites	World Intellectual Property Organization (WIPO)	Applicant
WO2005079854A1	Cites	World Intellectual Property Organization (WIPO)	Search report
US3817837A	Cites	United States of America	Applicant
US3850752A	Cites	United States of America	Applicant
US3939350A	Cites	United States of America	Applicant
US3996345A	Cites	United States of America	Applicant
US4275149A	Cites	United States of America	Applicant
US4277437A	Cites	United States of America	Applicant
US4366241A	Cites	United States of America	Applicant
US4496778A	Cites	United States of America	Applicant
US4975278A	Cites	United States of America	Applicant
US4987071A	Cites	United States of America	Applicant
US5037743A	Cites	United States of America	Applicant
US5143830A	Cites	United States of America	Applicant
US5260203A	Cites	United States of America	Applicant
US5455030A	Cites	United States of America	Applicant
US5530101A	Cites	United States of America	Applicant
US5530101A	Cites	United States of America	Applicant
US5530101A	Cites	United States of America	Applicant
US5585089A	Cites	United States of America	Applicant
US5585089A	Cites	United States of America	Applicant
US5585089A	Cites	United States of America	Applicant
US60220103A	Cites	United States of America	Applicant
US60220103A	Cites	United States of America	Applicant
US6025197A	Cites	United States of America	Applicant
US6639055B1	Cites	United States of America	Applicant
WO8101145A1	Cites	World Intellectual Property Organization (WIPO)	Applicant
WO8807378A1	Cites	World Intellectual Property Organization (WIPO)	Applicant
WO9521191A1	Cites	World Intellectual Property Organization (WIPO)	Applicant
WO9521191A1	Cites	World Intellectual Property Organization (WIPO)	Applicant
WO9521191A1	Cites	World Intellectual Property Organization (WIPO)	Applicant
AMBUDKAR ET AL., ANNU. REV. PHARMACOL. TOXICOL., vol. 39, 1999, pages 361	Non-patent	–	Applicant
ANTOKU ET AL., BIOCHEM. BIOPHYS. RES. COMMUN., vol. 286, 2001, pages 1003	Non-patent	–	Applicant
AYESH ET AL., ANTICANCER DRUGS, vol. 7, no. 6, 1996, pages 678 - 86	Non-patent	–	Applicant
AYESH ET AL., BIOCHIM. BIOPHYS. ACTA, vol. 1316, no. 1, 1996, pages 8 - 18	Non-patent	–	Applicant
BAKHSHI ET AL., CELL, vol. 41, 1985, pages 899	Non-patent	–	Applicant
BARGOU ET AL., INT. J. CANCER, vol. 60, 1995, pages 854	Non-patent	–	Applicant
BATRA ET AL., GENE THER, vol. 1, no. 4, 1994, pages 255 - 60	Non-patent	–	Applicant
BECKER ET AL., CANCER BIOL THER., vol. 1, no. 5, 2002, pages 548 - 53	Non-patent	–	Applicant
BREDEL, BRAIN RES. REV., vol. 35, 2001, pages 161	Non-patent	–	Applicant
BRITTON ET AL., GENES DEV., vol. 12, 1998, pages 1254	Non-patent	–	Applicant
BROXTERMAN ET AL., CANCER LETT., vol. 35, no. 1, 1987, pages 87 - 95	Non-patent	–	Applicant
BRUMMELKAMP ET AL., SCIENCE, vol. 296, 2002, pages 550	Non-patent	–	Applicant
CAPLEN, EXPERT OPIN. BIOL. THER., vol. 3, no. 4, 2003, pages 575 - 86	Non-patent	–	Applicant
CAPLEN, MOUSSES, ANN. N.Y. ACAD. SCI., vol. 1002, 2003, pages 56 - 62	Non-patent	–	Applicant
CARTER, P., NAT REV CANCER, vol. 1, no. 2, 2001, pages 118 - 29	Non-patent	–	Applicant
CHEN ET AL., INT. J. CANCER, vol. 93, 2001, pages 107	Non-patent	–	Applicant
CILIBERTO ET AL., CELL, vol. 41, 1985, pages 531	Non-patent	–	Applicant
COLE ET AL., SCIENCE, vol. 258, 1992, pages 1650 - 1654	Non-patent	–	Applicant
COLLINS, OLIVE, BIOCHEM., vol. 32, 1993, pages 2795 - 99	Non-patent	–	Applicant
CORY ET AL., CANCER COMMUN, vol. 3, 1991, pages 207 - 212	Non-patent	–	Applicant
CORY, S., ANNU. REV. IMMUNOL., vol. 13, 1995, pages 513 - 543	Non-patent	–	Applicant
DE BOER ET AL., J. BACTERIOL., vol. 174, 1992, pages 63	Non-patent	–	Applicant
DE HAARD, H. J. ET AL., J. BIOL. CHEM., vol. 274, 1999, pages 18218 - 18230	Non-patent	–	Applicant
DOIGE ET AL., ANN. REV. MICROBIOL., vol. 47, 1993, pages 291 - 319	Non-patent	–	Applicant
DUBEL ET AL., J. IMMUNOL. METHODS, vol. 178, 1995, pages 201 - 209	Non-patent	–	Applicant
DUAN ET AL., MOL. CANCER THERAPEUTICS, vol. 3, no. 7, 2004, pages 833 - 38	Non-patent	–	Applicant
DUXBURY ET AL., J. AM. COLL. SURG., vol. 198, 2004, pages 953 - 59	Non-patent	–	Applicant
ELBASHIR ET AL., GENES DEV., vol. 15, no. 2, 2001, pages 188 - 200	Non-patent	–	Applicant
ELBASHIR ET AL., NATURE, vol. 411, no. 6836, 2001, pages 494 - 8	Non-patent	–	Applicant
ENDICOTT ET AL., ANN. REV. BIOCHEM., vol. 58, 1989, pages 137 - 71	Non-patent	–	Applicant
FARDEL ET AL., GEN PHARMACOL., vol. 27, no. 8, 1996, pages 1283 - 1291	Non-patent	–	Applicant
FORBES, AUSTRALIAN J. BIOTECHNOLOGY, vol. 1, 1987, pages 30	Non-patent	–	Applicant
FRAIN ET AL., MOL. CELL BIOL.., vol. 10, 1990, pages 991	Non-patent	–	Applicant
FRANKEL ET AL., LEUKEMIA, vol. 14, 2000, pages 576 - 585	Non-patent	–	Applicant
GARIBOLDI ET AL., INT. J. ONCOLOGY, vol. 22, 2003, pages 1057 - 64	Non-patent	–	Applicant
GLENNIE ET AL., J. IMMUNOL., vol. 139, no. 7, 1987, pages 2367 - 75	Non-patent	–	Applicant
GOSSELIN ET AL., BIOTECHNOL. ANNU. REV., vol. 8, 2002, pages 103 - 31	Non-patent	–	Applicant
GOTTESMAN ET AL., NAT. REV. CANCER, vol. 2, 2002, pages 48	Non-patent	–	Applicant
GRIFFITHS ET AL., EMBO J., vol. 13, 1994, pages 3245 - 3260	Non-patent	–	Applicant
GUERRIER-TAKADA ET AL., CELL, vol. 35, 1983, pages 849	Non-patent	–	Applicant
HAMPEL, TRITZ, BIOCHEM., vol. 28, 1989, pages 4929	Non-patent	–	Applicant
HAMPEL ET AL., NUCLEIC ACIDS RESEARCH, 1990, pages 299	Non-patent	–	Applicant
HANADA ET AL., J. BIOL. CHEM., vol. 270, 1995, pages 11962	Non-patent	–	Applicant
HANAHAN, NATURE, vol. 315, 1985, pages 115	Non-patent	–	Applicant
HANNON, NATURE (LOND., vol. 418, 2002, pages 244	Non-patent	–	Applicant
HARRY, MOL. MICROBIOL., vol. 40, 2001, pages 795	Non-patent	–	Applicant
HART, SEMIN. ONCOL., vol. 23, 1996, pages 154	Non-patent	–	Applicant
HEIM ET AL., PROC. NAT'L. ACAD. SCI. USA, vol. 91, 1994, pages 12501	Non-patent	–	Applicant
HIRAGA ET AL., J. BACTERIOL., vol. 171, 1989, pages 1496	Non-patent	–	Applicant
HOSHIDA ET AL., PANCREAS, vol. 25, no. 2, 2002, pages 111 - 21	Non-patent	–	Applicant
HU ET AL., CANCER RES., vol. 56, 1996, pages 3055 - 3061	Non-patent	–	Applicant
HU ET AL., MOL. MICROBIOL., vol. 34, 1999, pages 82 - 90	Non-patent	–	Applicant
HUNTER ET AL., MOL. CELL. BIOL., vol. 16, 1996, pages 877	Non-patent	–	Applicant
HUDSON, SOURIAU, EXPERT OPIN. BIOL. THER., vol. 1, 2001, pages 845 - 855	Non-patent	–	Applicant
HUDSON, SOURIAU, NAT. MED., vol. 9, no. 1, 2003, pages 129 - 34	Non-patent	–	Applicant
HUNG ET AL., ADV. EXP. MED. BIOL., vol. 465, 2000, pages 171 - 80	Non-patent	–	Applicant
HUTVAGNER, ZAMORE, CURR. OPIN. GENET. DEV., vol. 12, 2002, pages 225	Non-patent	–	Applicant
IRETON ET AL., J. BACTERIOL., vol. 176, 1994, pages 5320	Non-patent	–	Applicant

Showing 100 of 172

106 members in 20 offices

This recordMember, by publication datePriority claim10 more offices are in the list below

Priority claims9

Priority claims
Document	Office	Kind	Date
604433P	United States of America	–	2004-08-26
60443304	United States of America	P	2004-08-26
60443304	United States of America	P	2004-08-26
05806875	European Patent Office (EPO)	A	2005-08-25
05806875	European Patent Office (EPO)	A	2005-08-25
05806875	–	–	–
604433P	–	–	–
EP20050806875	–	–	–
US20040604433P	–	–	–

Members106

Family members, oldest first
Document	Office	Kind
AU2005276145A1	Australia	A1	2006-03-02
CA2577938A1	Canada	A1	2006-03-02
CA2803995A1	Canada	A1	2006-03-02
WO2006021894A2	World Intellectual Property Organization (WIPO)	A2	2006-03-02
WO2006021894A3	World Intellectual Property Organization (WIPO)	A3	2006-05-04
EP1791959A2	European Patent Office (EPO)	A2	2007-06-06
MX2007002294A	Mexico	A	2007-10-19
CN101072876A	China	A	2007-11-14
US2007298056A1	United States of America	A1	2007-12-27
JP2008510794A	Japan	A	2008-04-10
US2008299084A1	United States of America	A1	2008-12-04
AU2008291833A1	Australia	A1	2009-03-05
CA2682704A1	Canada	A1	2009-03-05
CA2844647A1	Canada	A1	2009-03-05
CA2933978A1	Canada	A1	2009-03-05
WO2009027830A2	World Intellectual Property Organization (WIPO)	A2	2009-03-05
EP1791959A4	European Patent Office (EPO)	A4	2009-03-18
WO2009027830A3	World Intellectual Property Organization (WIPO)	A3	2009-05-14
NZ553910A	New Zealand	A	2009-05-31
MX2009010411A	Mexico	A	2009-11-23
EP2145002A2	European Patent Office (EPO)	A2	2010-01-20
CN101715489A	China	A	2010-05-26
AU2005276145B2	Australia	B2	2010-05-27
JP2010523487A	Japan	A	2010-07-15
HK1138326A1	Hong Kong, China	A1	2010-08-20
AU2010212520A1	Australia	A1	2010-09-16
AU2005276145C1	Australia	C1	2011-01-06
US2011111041A1	United States of America	A1	2011-05-12
EP2386640A2This record	European Patent Office (EPO)	A2	2011-11-16
EP2386640A9	European Patent Office (EPO)	A9	2011-12-28
NZ580487A	New Zealand	A	2012-04-27
EP2386640A3	European Patent Office (EPO)	A3	2012-06-27
JP4965447B2	Japan	B2	2012-07-04
HK1163160A1	Hong Kong, China	A1	2012-09-07
AU2008291833B2	Australia	B2	2012-11-08
CN101072876B	China	B	2012-11-28
EP2532746A2	European Patent Office (EPO)	A2	2012-12-12
AU2010212520B2	Australia	B2	2013-01-31
CN102921019A	China	A	2013-02-13
JP5167338B2	Japan	B2	2013-03-21
CA2577938C	Canada	C	2013-04-16
AU2013203202A1	Australia	A1	2013-05-02
EP2532746A3	European Patent Office (EPO)	A3	2013-08-14
US2013261170A1	United States of America	A1	2013-10-03
US8669101B2	United States of America	B2	2014-03-11
US8691963B2	United States of America	B2	2014-04-08
US8735566B2	United States of America	B2	2014-05-27
CA2803995C	Canada	C	2014-06-10
CA2682704C	Canada	C	2014-07-08
US2014221459A1	United States of America	A1	2014-08-07
US2014315976A1	United States of America	A1	2014-10-23
EP2145002B1	European Patent Office (EPO)	B1	2015-01-07
EP2386640B1	European Patent Office (EPO)	B1	2015-01-28
US8956864B2	United States of America	B2	2015-02-17
CN102921019B	China	B	2015-04-01
DK2145002T3	Denmark	T3	2015-04-20
ES2534435T3	Spain	T3	2015-04-22
DK2386640T3	Denmark	T3	2015-04-27
EP2865755A2	European Patent Office (EPO)	A2	2015-04-29
PT2145002E	Portugal	E	2015-04-29
EP2865755A3	European Patent Office (EPO)	A3	2015-05-06
ES2535235T3	Spain	T3	2015-05-07
PT2386640E	Portugal	E	2015-05-18
HRP20150328T1	Croatia	T1	2015-05-22
SI2145002T1	Slovenia	T1	2015-05-29
PL2145002T3	Poland	T3	2015-06-30
SI2386640T1	Slovenia	T1	2015-06-30
US9066982B2	United States of America	B2	2015-06-30
PL2386640T3	Poland	T3	2015-08-31
SG10201507969PA	Singapore	A	2015-10-29
US9242007B2	United States of America	B2	2016-01-26
US2016051687A1	United States of America	A1	2016-02-25
HUE024999T2	Hungary	T2	2016-02-29
HK1209786A1	Hong Kong, China	A1	2016-04-08
US2016113883A1	United States of America	A1	2016-04-28
CN101715489B	China	B	2016-05-25
CA2844647C	Canada	C	2016-07-26
CN105854029A	China	A	2016-08-17
EP2865755B1	European Patent Office (EPO)	B1	2017-02-01
CY1116201T1	Cyprus	T1	2017-02-08
CY1116310T1	Cyprus	T1	2017-02-08
AU2013203202B2	Australia	B2	2017-02-16
DK2865755T3	Denmark	T3	2017-05-15
PT2865755T	Portugal	T	2017-05-26
AU2017203234A1	Australia	A1	2017-06-08
SI2865755T1	Slovenia	T1	2017-06-30
HRP20170662T1	Croatia	T1	2017-07-14
ES2626179T3	Spain	T3	2017-07-24
LT2865755T	Lithuania	T	2017-07-25
US9730897B2	United States of America	B2	2017-08-15
EP3205724A1	European Patent Office (EPO)	A1	2017-08-16
PL2865755T3	Poland	T3	2017-08-31
US2017258734A1	United States of America	A1	2017-09-14
CY1119031T1	Cyprus	T1	2018-01-10
US2018028456A1	United States of America	A1	2018-02-01
SG10201804257RA	Singapore	A	2018-06-28
CA2933978C	Canada	C	2018-08-21
US10098847B2	United States of America	B2	2018-10-16
US2019091160A1	United States of America	A1	2019-03-28
EP3205724B1	European Patent Office (EPO)	B1	2019-05-08

Showing 100 of 106

118 legal events, as 23 offices reported them to INPADOC

Over the term

Point at a mark for the event

Events

Legal events, newest first
	Event	Code	Office
2025-10-09	Lapsed in a contracting state [announced via postgrant information from national office to epo]LapsedPG25	PG25	EP
2025-10-03	Announcement of lapse in spainLapsedFD2A	FD2A	ES
2025-09-17	Patent expired after termination of 20 yearsExpiredPE20	PE20	GB
2025-08-25	Expiry of rightR071	R071	DE
2024-10-31	Annual fee paid to national office [announced via postgrant information from national office to epo]GrantedPGFP	PGFP	EP
2024-10-21	Annual fee paid to national office [announced via postgrant information from national office to epo]GrantedPGFP	PGFP	EP
2024-10-16	Annual fee paid to national office [announced via postgrant information from national office to epo]GrantedPGFP	PGFP	EP
2024-10-14	Annual fee paid to national office [announced via postgrant information from national office to epo]GrantedPGFP	PGFP	EP
2024-10-10	Annual fee paid to national office [announced via postgrant information from national office to epo]GrantedPGFP	PGFP	EP
2023-06-14	Opt-out of the competence of the unified patent court (upc) registeredP01	P01	EP
2022-11-30	Lapsed in a contracting state [announced via postgrant information from national office to epo]LapsedPG25	PG25	EP
2022-06-30	Lapsed in a contracting state [announced via postgrant information from national office to epo]LapsedPG25	PG25	EP
2021-10-29	Lapsed in a contracting state [announced via postgrant information from national office to epo]LapsedPG25	PG25	EP
2021-09-30	Lapse of patentLapsedKO00	KO00	SI
2021-08-31	Lapsed in a contracting state [announced via postgrant information from national office to epo]LapsedPG25	PG25	EP
2021-08-31	Lapsed in a contracting state [announced via postgrant information from national office to epo]LapsedPG25	PG25	EP
2021-08-31	Lapsed in a contracting state [announced via postgrant information from national office to epo]LapsedPG25	PG25	EP
2021-08-31	Lapsed in a contracting state [announced via postgrant information from national office to epo]LapsedPG25	PG25	EP
2021-07-30	Lapsed in a contracting state [announced via postgrant information from national office to epo]LapsedPG25	PG25	EP
2021-06-30	Lapsed in a contracting state [announced via postgrant information from national office to epo]LapsedPG25	PG25	EP
2021-06-25	Lapse european patentLapsedMM4D	MM4D	LT
2021-05-31	Lapsed in a contracting state [announced via postgrant information from national office to epo]LapsedPG25	PG25	EP
2021-05-31	Lapsed in a contracting state [announced via postgrant information from national office to epo]LapsedPG25	PG25	EP
2021-05-31	Lapsed in a contracting state [announced via postgrant information from national office to epo]LapsedPG25	PG25	EP
2021-05-31	Lapsed in a contracting state [announced via postgrant information from national office to epo]LapsedPG25	PG25	EP
2021-05-26	Lapsed because of non-payment of the annual feeLapsedMM	MM	BE
2021-04-30	Lapsed in a contracting state [announced via postgrant information from national office to epo]LapsedPG25	PG25	EP
2021-04-30	Lapsed in a contracting state [announced via postgrant information from national office to epo]LapsedPG25	PG25	EP
2021-04-30	Lapsed in a contracting state [announced via postgrant information from national office to epo]LapsedPG25	PG25	EP
2021-04-30	Lapsed in a contracting state [announced via postgrant information from national office to epo]LapsedPG25	PG25	EP
2021-04-30	Lapsed in a contracting state [announced via postgrant information from national office to epo]LapsedPG25	PG25	EP
2021-04-30	Lapsed in a contracting state [announced via postgrant information from national office to epo]LapsedPG25	PG25	EP
2021-04-30	Lapsed in a contracting state [announced via postgrant information from national office to epo]LapsedPG25	PG25	EP
2021-04-30	Lapsed in a contracting state [announced via postgrant information from national office to epo]LapsedPG25	PG25	EP
2021-04-30	Lapsed in a contracting state [announced via postgrant information from national office to epo]LapsedPG25	PG25	EP
2021-04-30	Lapsed in a contracting state [announced via postgrant information from national office to epo]LapsedPG25	PG25	EP
2021-04-30	Lapsed in a contracting state [announced via postgrant information from national office to epo]LapsedPG25	PG25	EP
2021-04-28	Patent lapsed due to non-payment of maintenance feesLapsedMM4A	MM4A	SK
2021-04-15	Lapse because of not paying annual feesLapsedMM01	MM01	AT
2021-04-07	Lapsed because of non-payment of the annual feeLapsedMM	MM	NL
2021-03-31	Lapsed in a contracting state [announced via postgrant information from national office to epo]LapsedPG25	PG25	EP
2021-03-31	Patent ceasedCeasedPL	PL	CH
2021-03-30	Ep patent has lapsedLapsedEUG	EUG	SE
2021-03-29	Ep patent lapsedLapsedEBP	EBP	DK
2021-03-24	Ep patent expiredExpiredMAE	MAE	FI
2021-03-15	Lapsed by not paying the annual feesLapsedMM4A	MM4A	EE
2020-01-31	Annual fee paid to national office [announced via postgrant information from national office to epo]GrantedPGFP	PGFP	EP
2019-12-31	Annual fee paid to national office [announced via postgrant information from national office to epo]GrantedPGFP	PGFP	EP
2019-11-29	Annual fee paid to national office [announced via postgrant information from national office to epo]GrantedPGFP	PGFP	EP
2019-11-29	Annual fee paid to national office [announced via postgrant information from national office to epo]GrantedPGFP	PGFP	EP
2019-11-29	Annual fee paid to national office [announced via postgrant information from national office to epo]GrantedPGFP	PGFP	EP
2019-11-29	Annual fee paid to national office [announced via postgrant information from national office to epo]GrantedPGFP	PGFP	EP
2019-11-29	Annual fee paid to national office [announced via postgrant information from national office to epo]GrantedPGFP	PGFP	EP
2019-11-29	Annual fee paid to national office [announced via postgrant information from national office to epo]GrantedPGFP	PGFP	EP
2019-10-31	Annual fee paid to national office [announced via postgrant information from national office to epo]GrantedPGFP	PGFP	EP
2019-10-31	Annual fee paid to national office [announced via postgrant information from national office to epo]GrantedPGFP	PGFP	EP
2019-10-31	Annual fee paid to national office [announced via postgrant information from national office to epo]GrantedPGFP	PGFP	EP
2019-10-31	Annual fee paid to national office [announced via postgrant information from national office to epo]GrantedPGFP	PGFP	EP
2019-10-31	Annual fee paid to national office [announced via postgrant information from national office to epo]GrantedPGFP	PGFP	EP
2019-10-31	Annual fee paid to national office [announced via postgrant information from national office to epo]GrantedPGFP	PGFP	EP
2019-10-31	Annual fee paid to national office [announced via postgrant information from national office to epo]GrantedPGFP	PGFP	EP
2019-10-31	Annual fee paid to national office [announced via postgrant information from national office to epo]GrantedPGFP	PGFP	EP
2019-10-31	Annual fee paid to national office [announced via postgrant information from national office to epo]GrantedPGFP	PGFP	EP
2019-10-31	Annual fee paid to national office [announced via postgrant information from national office to epo]GrantedPGFP	PGFP	EP
2019-10-31	Annual fee paid to national office [announced via postgrant information from national office to epo]GrantedPGFP	PGFP	EP
2019-10-31	Annual fee paid to national office [announced via postgrant information from national office to epo]GrantedPGFP	PGFP	EP
2019-10-31	Annual fee paid to national office [announced via postgrant information from national office to epo]GrantedPGFP	PGFP	EP
2019-10-31	Annual fee paid to national office [announced via postgrant information from national office to epo]GrantedPGFP	PGFP	EP
2019-09-30	Annual fee paid to national office [announced via postgrant information from national office to epo]GrantedPGFP	PGFP	EP
2019-09-30	Annual fee paid to national office [announced via postgrant information from national office to epo]GrantedPGFP	PGFP	EP
2018-08-27	Fee paymentPLFP	PLFP	FR
2017-08-22	Fee paymentPLFP	PLFP	FR
2016-11-29	Amendment of ipc main classPREVIOUS MAIN CLASS: A61K0047480000R079	R079	DE
2016-08-22	Fee paymentPLFP	PLFP	FR
2016-03-15	Publication of translation of european patent specificationUEP	UEP	AT
2016-02-29	Filing of the translation of the text of european patentsAG4A	AG4A	HU
2016-01-06	No opposition filedOpposition26N	26N	EP
2015-12-04	No opposition filed within time limitOppositionORIGINAL CODE: 0009261PLBE	PLBE	EP
2015-12-04	Information on the status of an ep patent application or granted ep patentGrantedSTATUS: NO OPPOSITION FILED WITHIN TIME LIMITSTAA	STAA	EP
2015-11-20	Standard patents granted in hong kongGrantedGR	GR	HK
2015-11-09	Ep patent valid in romaniaEPE	EPE	RO
2015-10-29	No opposition filed against granted patent, or epo opposition proceedings concluded without decisionGrantedR097	R097	DE
2015-08-31	Lapsed in a contracting state [announced via postgrant information from national office to epo]LapsedPG25	PG25	EP
2015-08-31	Translation of ep patentT3	T3	PL
2015-08-04	Translation of european patent specification into slovakT3	T3	SK
2015-06-26	Fee paymentPLFP	PLFP	FR
2015-06-24	Ep patent validated in greeceEP	EP	GR
2015-06-15	Translation of the specification of european patent valid in estoniaFG4A	FG4A	EE
2015-05-18	Translation is availableAVAILABILITY OF NATIONAL TRANSLATIONSC4A	SC4A	PT
2015-05-12	Translation of granted ep patentGrantedTRGR	TRGR	SE
2015-05-07	Definitive protectionFG2A	FG2A	ES
2015-04-29	Translation filed for an european patent granted for nl, confirming art. 52 par. 1 or 6 of the patents act 1995GrantedT3	T3	NL
2015-04-27	Ep patent with danish claimsT3	T3	DK
2015-04-15	New agentNV	NV	CH
2015-03-15	Reference to at number (ep patent validated in austria)REF	REF	AT
2015-03-12	Dpma publication of mentioned ep patent grantGrantedR096	R096	DE
2015-02-25	European patents granted designating irelandGrantedFG4D	FG4D	IE
2015-01-30	European patent takes effect as a national patent in ch/liEP	EP	CH
2015-01-28	Divisional application: reference to earlier applicationAC	AC	EP
2015-01-28	Designated contracting statesAK	AK	EP

Showing 100 of 118