Bacterially-derived, intact minicells that encompass plasmid-free functional nucleic acid for in vivo delivery to mammalian cells

[0001] Numerous nucleic acid-based strategies have recently been developed to modulate various cellular functions (Opalinska and Gewirtz, 2002). Oligonucleotide classes, such as aptamers, decoy oligonucleotide binding transcription factor, ribozymes, triplex forming oligonucleotides, CpG immunostimulatory motifs, antisense oligonucleotides (including peptide nucleic acids), small interfering RNA and microRNA have attracted a lot of attention as a research tool, operating mode. These oligomeric nucleic acids also have significant potential as therapeutics. However, such therapies face several obstacles, including free nucleic acid instability and safe, effective and targeted delivery of these macromolecules to the cell (Dykxhoorn and Lieberman, 2005).

[0002] The goal of many nucleic acid-based therapeutic strategies is RNA interference (RNAi), whereby long double-stranded RNA (dsRNA) in a cell leads to sequence-specific degradation of homologous (complementary or partially complementary) gene transcripts. More specifically, long dsRNA molecules are processed to smaller RNAs by an endogenous ribonuclease called "Dicer" (Grishok et al., 2000; Zamore et al., 2000). Smaller RNAs are known as "short interfering RNAs" (siRNAs) if they come from exogenous sources and "microRNAs" (miRNAs) if they are made from genes encoding RNAs in the cell's own genome. These two classes of small (typically 21 to 23 nucleotides) regulatory RNAs are also different in that miRNAs show only partial complementarity for messenger RNA (mRNA) purposes.

[0003] Short regulatory RNA binds to so-called "RNA-induced silencing complex" (RISC), which has helicase and endonuclease activity. Helicase activity develops two strands of the RNA molecule, allowing the antisense strand to bind to the target RNA molecule (Zamore et al., 2000; Zamore, 2002; Vickers et al., 2003). Endonuclease activity hydrolyses the target RNA at the same site where the antisense strand is bound.

[0004] Thus, in RNAi, a single-stranded RNA molecule (ssRNA) binds to a target RNA molecule according to Watson-Crick base pairing principles and recruits a ribonuclease that breaks down the target RNA. In contrast, antisense suppression of gene expression involves binding ssRNA to mRNA, blocking translation without catalyzing mRNA degradation.

[0005] As a class, regulatory RNAs have a half-life of less than an hour in human plasma (Layzer et al., 2004) and are rapidly secreted by the kidneys. Therefore, several groups have attempted to obtain regulatory RNA, including siRNAs, which are nuclease resistant. Examples of such aspirations include nucleotide chemical modifications (e.g., 2'-F, 2'-OMe, Locked Nucleic Acids; LNA), or phosphodiester backbones, e.g. phosphorothioester bonds (Chiu and Rana 2003; Choung et al., 2006; Czauderna et al., 2003; Elmen et al., 2005; Layzer et al., 2004; Morrissey et al., 2005). Also, to minimize the time that siRNA or other regulatory RNAs remain in circulation, practitioners have conjugated RNA molecules to proteins and antibodies to target the desired mammalian cells. As a result of further efforts to address problems with low stability and rapid renal secretion, practitioners have developed carriers for the delivery of regulatory RNAs. Polyplexes (formed by self-assembly of nucleic acids with polycations), lipopoliplexes (formed by initial condensation of nucleic acid with polycations, followed by the addition of cationic lipids), liposomes and synthetic nanoparticles are also studied.

[0006] These approaches also face numerous obstacles, such as (a) rapid clearance of serum proteins from serum by renal secretion, (b) a limited number of RNA regulatory molecules that can be conjugated to any carrier protein, (c) difficulty in intracellular dissociation intact regulatory RNA from the carrier protein, (d) rapid clearance due to serum protein binding polyplexes that may act as opsonins (Dash et al., 1999), and (e) in vivo liposome instability, resulting in the release of nucleic acids into the serum and potential non-specific transformation.

[0007] Viral vectors have also been developed for endogenous production of regulatory RNAs. See, e.g., Devroe and Silver, 2004. However, these viral vectors raise serious security concerns. Exemplary problems include recombination with wild-type viruses, insertion and oncogenic potential, virus-induced immunosuppression, limited ability of viral vectors to transfer large DNA segments, recovery of attenuated viruses in attenuated viruses, difficulty in production and distribution, low stability and adverse reactions (Hacein- Bey-Abina et al., 2003; Kootstra and Verma, 2003; Rapper et al., 2003; Verma and Weitzman, 2005; Check, 2005).

[0008] Plasmid-based systems have also been developed for recombinant expression of regulatory RNA in situ, such as siRNA or larger (~ 70 nt) precursor, RNA in the form of a short hairpin (shRNA). shRNA includes sense and antisense sequences from the target gene that are connected through a hairpin loop. See, e.g., Paddison et al., 2002. shRNA can be expressed from a pol-III promoter or in the context of miRNA, from pol II promoters.

[0009] As described in international application WO 03/033519, plasmids that encode shRNA, siRNA or other regulatory RNA can be transformed into a parent bacterial strain that produces intact minicells, due to a mutation that causes asymmetric cell division. Such transformation results in recombinant bacteria in which the plasmid replicates intracellularly, introducing a large number of plasmids into the bacterial cytoplasm. During asymmetric division, some of the plasmids are segregated into the minicell cytoplasm, resulting in recombinant minicells. Then, minicells can deliver plasmid DNA to a mammalian cell, where plasmid DNA migrates to the cell nucleus. In the nucleus, plasmid DNA expresses shRNA or other regulatory RNA, as the case may be, and then the resulting nucleic acid migrates to the cytoplasm, where it can induce RNAi or gene suppression, depending on the nature of the regulatory RNA involved.

[0010] However, since such approaches require a host mechanism, delivery of therapeutically effective amounts of nucleic acid through expression-based systems involves complex and long processes that limit their effectiveness. Accordingly, a more effective methodology is needed to deliver functional nucleic acids, such as regulatory RNA, to target cells.

[0011] WO / 2006/021894 describes the delivery of RNA molecules to mammalian cells by minicells of bacterial origin. More specifically, examples disclose plasmids encoding the RNA sequence that has been transformed into bacterial cells. The resulting minicells made from recombinant bacterial cells that received plasmids and the minicells containing plasmid were in turn absorbed by mammalian cells into which plasmids were released, resulting in transcription of RNA sequences in mammalian cells.

SUMMARY OF THE INVENTION [0012] According to one aspect of the invention, the composition comprises (a) a set of intact minicells of bacterial origin, wherein each minicell of said set contains regulatory RNA that is packaged in said microcell, and (b) a pharmaceutically acceptable carrier, wherein in said minicells there is no construct for expression in situ of said regulatory RNA, wherein said regulatory RNA is selected from the group consisting of siRNA, miRNA and shRNA, and wherein said set comprises a therapeutically effective amount of said regulatory RNA.

[0013] The functional nucleic acid packaged into a minicell may target RNA transcripts encoding a protein that contributes, inter alia, to drug resistance, resistance to apoptosis or cancer. The composition of the invention may also further comprise a bispecific ligand, for example, consisting of a first arm specific for the minicell surface structure and a second arm specific for the surface receptor of a non-phagocytic mammalian cell.

[0014] The invention allows a method of delivering a functional nucleic acid to a target mammalian cell. The innovative methodology includes (a) providing a set of intact minicells in a pharmaceutically acceptable carrier, each of the minicell sets includes a plasmid-free functional nucleic acid, and (b) bringing the minicells from the set into contact with the mammalian cells so that the mammalian cells absorb the minicells belonging to the set, whereby the functional nucleic acid is released into the cytoplasm of target cells. As mentioned, regulatory RNA, such as siRNA, miRNA and shRNA, may target RNA transcripts encoding a protein that contributes to drug resistance, apoptosis resistance or cancer. In other embodiments, the methodology further comprises delivering the drug, other than functional nucleic acid, to the target mammalian cell. The drug may be administered after or simultaneously or even before administration of the minicell composition.

[0015] In another aspect, the invention contemplates a method of formulating minicells of the composition of the invention. The method includes co-incubating in buffer (A) a set of intact minicells of bacterial origin with (B) free of the RNA regulatory plasmid, thereby obtaining minicells that contain said regulatory RNA, said regulatory RNA being selected from the group consisting of siRNA, miRNA and shRNA. In some embodiments, the co-incubation may include gentle shaking, while in others the co-incubation is static. In some aspects, the co-incubation lasts about half an hour, while in others it takes about an hour. In one embodiment, the buffer comprises a buffered saline solution, e.g., a 1X phosphate buffer solution. In another embodiment, the co-incubation is carried out at a temperature of about 4 ° C to about 37 ° C, about 20 ° C to about 30 ° C, about 25 ° C or about 37 ° C. Co-incubation can include about 10⁷, 10⁸, 10⁹, 10¹⁰, 10¹¹, 10¹² or 10¹³ minicells.

[0016] Other objects, features and advantages of the invention will be apparent from the following detailed description. The detailed description and detailed examples are given for the purpose of illustration only, since various changes and modifications within the scope of the invention will be apparent to those skilled in the art from this detailed description. In addition, the examples illustrate the principle of the invention and it cannot be expected that they will specifically illustrate the use of the invention in all examples where it will of course be useful to those skilled in the art.

BRIEF DESCRIPTION OF THE DRAWINGS [0017]

Figure 1 shows intact minicells packed with Cy3 fluorophore labeled siRNA. Figure 1A is from a light microscope, while Figure 1B shows the same slide, but displayed under fluorescent light with an excitation filter of 515-560, revealing highly fluorescent siRNA molecules coexisting with minicells.

Figure 2 is a picture taken by confocal fluorescence microscopy and shows the adhesion and internalization of minicells targeted at

EGFR, packed siRNA-Plk1 into human breast cancer cells in-vitro.

Figure 3 graphically depicts the significant anti-tumor activity achieved by xenograft treatment of human breast cancer (MDA-MB-468) nude mice with EGFR-targeted minicells packed with KSP-siRNA. Control group! (- ♦ -) received sterile saline, while Experimental group 2 (--o--) received^EGFRminicell RNA-KSP (10⁹) four times a week.

Figure 4 graphically depicts the significant anti-tumor effect achieved by human colon cancer (HCT116) xenograft treatment of nude mice with EGFR-targeted minicells packed with KSP-siRNA in combination with EGFR-targeted minicells packed with carboplatin. Group 1 (- ♦ -) mice received sterile saline, and group 2 (--Δ--), 3 (-) and 4 (-e- ') mice were treated for the first 10 doses (see Figure 4 ) respectively 10^{9 EGFR} minicellesxiRNA-Plk1, ^EGFR siRNA-KSP-1 minicells and ^EGFR minicellesxiRNA-KSP-2.

Figure 5 provides FACS analysis at different times after transfection of colon cancer cells (HCT116) treated with experimental minicells, ^EGFR siRNA-KSP minicells or ^EGFR minicellesxiRNA-Plk1. Figures 5A-5D provide FACS analysis of samples taken 4 hours after transfection, while Figures 5E-5H show analysis of samples 8 hours after transfection. Figures 5A and 5E only show results from cells, while figures 5B and 5F refer to + empty cells^EGFR minicells. Figures 5C and 5G show results from + cells^EGFRsiRNA-KSP minicells and figures 5D and 5H refer to + cells ^EGFR minicellesiRNAPlk1.

Figure 6 provides FACS analysis at various times after transfection of colon cancer cells (HCT116) treated with experimental minicells, ^EGFR siRNA-KSP minicells or ^EGFR minicell cell RNA-Plk1. Figures 6A-6D provide FACS analysis of samples taken 16 hours after transfection, while Figures 6E6H show analysis of samples 24 hours after transfection. Figures 6A and 6E only show results from cells, while figures 6B and 6F refer to + empty cells^EGFR minicells. Figures 6C and 6G show results from + cells^EGFRKSP. minicell axiRNA, and figures 6D and 6H refer to + cells ^EGFR minicellesxiRNA-Plk1.

Figure 7 provides FACS analysis at various times after transfection of colon cancer cells (HCT116) treated with experimental minicells, ^EGFR siRNA-KSP minicells or ^EGFR minicell cell RNA-Plk1. Figures 7A-7D provide FACS analysis of samples taken 32 hours after transfection, while Figures 7E7H show analysis from samples 48 hours after transfection. Figures 7A and 7E only show results from cells, while figures 7B and 7F refer to empty^EGFR minicells. Figures 7C and 7G show results from + cells^EGFR minicell-KSR-KSP, and figures 7D and 7H refer to + siRNA-Plk1 cells ^EGFR siRNA-Plk1 minicells.

DETAILED DESCRIPTION OF THE INVENTION [0018] According to the invention, therapeutically effective amounts of regulatory RNA can be packaged into minicells without the use of corrosive chemicals or electroporation. In this regard, a simple methodology has been developed for directly packaging such therapeutically effective regulatory RNA concentrations into intact minicells that does not include expression constructs based on plasmid or the expression mechanism of the host bacterial cell. Accordingly, the polynucleotide segment that encodes regulatory RNA is not cloned into plasmid DNA or viral vector. Instead, plasmid-free functional nucleic acids are packaged directly into the minicells by passing through the intact minicell membrane. In addition, the minicell composition of the invention can deliver therapeutically effective amounts of regulatory RNA, such as siRNA, miRNA, and shRNA, to target mammalian cells safely and efficiently.

Definitions [0019] Unless defined otherwise, all technical and scientific terms used in the description have the same meaning as commonly understood by those of skill in the relevant field.

[0020] For convenience, the meanings of the specific terms and phrases used in the description, examples and appended claims are given below. Other terms and phrases are defined throughout the description.

[0021] Singular forms include plural aspects, unless the context clearly indicates otherwise.

[0022] "Antisense oligonucleotide" means a nucleic acid molecule complementary to a portion of a particular transcript of a gene that can hybridize to the transcript and block its translation. The antisense oligonucleotide may contain RNA or DNA.

[0023] "Biomolecular sequence" or "sequence" means all or part of a polynucleotide or polypeptide sequence.

[0024] "Cancer", "cancer", "tumor", "malignant tumor" and "cancer", as used interchangeably herein, means cells or tissues that have an abnormal growth phenotype characterized by a significant loss of control over cell proliferation. The methods and compositions of the invention are used in particular for malignant, pre-metastatic, metastatic and non-metastatic cells.

[0025] "Complementary" means the topological compatibility or matching of the interacting surfaces of two molecules, such as the siRNA molecule and its target mRNA. The molecules can be described as complementary and furthermore the contact surface characteristics are complementary to each other.

[0026] "Reply" or "present" when used, for example, in the context of a polynucleotide or sequence that "corresponds" or "represents" a gene means that the polynucleotide sequence is present in the gene or in the nucleic acid gene product, e.g. mRNA . The polynucleotide may be present entirely within the exon of the genomic sequence of the gene or different parts of the polynucleotide sequence may be present in different exons, e.g. such that an adjacent polynucleotide sequence is present in mRNA, either before or after splicing, which is a gene expression product.

[0027] A "decoy RNA" is a molecule that can adopt a structure identical to the important RNA functional region to be targeted. The latter RNA may be native to a mammalian host or pathogen that has infected the mammalian cell, e.g., HIV. Decoy RNA restricts access to a protein that typically interacts with a target RNA, resulting in a disruption of normal processing in a mammalian or pathogenic host.

[0028] "Drug" means any physiologically or pharmacologically active substance that causes a desired local or systemic effect in animals, especially mammals and humans.

[0029] "Expression" generally means the process by which a polynucleotide sequence is successfully transcribed and translated such that detectable levels of the amino acid sequence or protein are expressed. As used herein in some contexts, expression refers to the production of mRNA. In other contexts, expression refers to protein production.

[0030] "Functional nucleic acid" refers to a nucleic acid molecule that, when introduced into a host cell, specifically interferes with protein expression. Typically, functional nucleic acid molecules have the ability to reduce protein expression by directly interacting with the transcript that encodes that protein. Regulatory RNAs, such as siRNA, shRNA, short RNA (typically less than 400 bases long), micro-RNA (miRNA), ribozymes and decoy RNA, and antisense nucleic acids are exemplary functional nucleic acids.

[0031] "Gene" refers to a polynucleotide sequence that contains control and coding sequences that are necessary for the production of a polypeptide or precursor. The polypeptide may be encoded by the full-length coding sequence or any part of the coding sequence. The gene may be an uninterrupted coding sequence, or it may comprise one or more introns connected by appropriate splice nodes. In addition, the gene may contain one or more modifications in the coding or non-translated regions that may affect the biological activity or chemical structure of the expression product, expression rate, or expression control method. 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.

[0032] "Host cell" means a cell that may or has been used as a recipient of a recombinant plasmid or other polynucleotide transfer and contains progeny of the primary cell that has been transfected. Single cell progens may not necessarily be completely identical in morphology or in genomic or total DNA to the parent cell due to natural, accidental or deliberate mutation.

[0033] "Hybridization" means any process by which a polynucleotide sequence binds to a complementary sequence by pairing the bases.

[0034] "Subject", "subject", "host" and "patient", as used interchangeably herein, means any mammalian subject for which diagnosis, treatment or therapy is desired. In a preferred embodiment, the subject, subject, host or patient is a human. Other entities may include, but are not limited to, cattle, horses, dogs, cats, guinea pigs, rabbits, rats, primates and mice.

[0035] "Label" means substances that are capable of providing a detectable signal, either directly or by interacting with one or more additional members of the signal generating system. Labels that are directly detected 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 as³⁵S ³²P ³H and similar as tags. Colorimetric markers such as colloidal gold or balls of colored glass or plastic (e.g. polystyrene, polypropylene, latex) may also be used. For example, see U.S. Patents No. 4,366,241, No. 4,277,437, No. 4,275,149, No. 3,996,345, No. 3,939,350, No. 3,850,752 and No. 3,817,837.

[0036] "Oligonucleotide" means a polynucleotide containing, 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 in length. The oligonucleotide may be a naturally occurring oligonucleotide or synthetic oligonucleotide. Oligonucleotides can be modified.

[0037] "Minicell" means the non-nuclear forms of bacterial cells resulting from a disorder of coordination during the cleavage of binary cell division with DNA segregation. Minicells are different from other small vesicles that are produced and released spontaneously in specific situations and are not subject to specific genetic reorganization or expression of episomal genes. In the context of this invention, minicells are intact because other "exposed" forms such as spheroplasts, poroplasts, protoplasts would allow the packed functional nucleic acid to leak and would not be therapeutically effective. The intact cell membrane allows the minicell to retain charge and release it intracellularly within the target mammalian host cell.

[0038] In this specification, "modified" and "chemically modified" means oligonucleotides or polynucleotides with one or more chemical changes relative to the natural molecular structure of all or any bases, sugar residues and internucleoside phosphate bonds, as well as molecules having an added substitution or combinations of modifications in these places. The internucleoside phosphate bonds can be phosphodiester, phosphotriester, phosphoramidate, siloxane, carbonate, carboxymethyl ester, acetamidate, carbamate, thioether, bridged phosphoramidates, bridged methylene phosphonates, phosphorothioate, phosphorothioate, phosphorothioate, phosphonothioate, phosphonothioate, phosphonothioate 5'-3 'or 5'-5' and combinations of such similar bonds. The phosphodiester linkage may be replaced with a replacement linkage such as phosphorothioester, methylamino, methylphosphonate, phosphoramidate and guanidine, and the ribose subunit of polynucleotides may also be substituted (e.g., hexose phosphodiester; peptide nucleic acids). Modifications may be internal (single or repeated) or at the end (ends) of the oligonucleotide molecule and may include addition to the molecule of internucleoside phosphate linkages, such as deoxyribose and phosphate modifications that cleave or crosslink to opposite chains or related enzymes or other proteins. The terms "modified oligonucleotides" and "modified polynucleotides" also include oligonucleotides or polynucleotides including modifications to sugar residues (e.g., 3'-substituted ribonucleotides or deoxyribonucleotide monomers), any of which are joined together through 5 'to 3' bonds.

[0039] The expression "nucleic acid molecules" and the term "polynucleotides" means polymeric forms of nucleotides of any length, either ribonucleotides or deoxynucleotides. These include single, double or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids or a polymer containing purine and pyrimidine bases or other natural, chemically or biochemically modified, unnatural or derivatized nucleotide bases. The polynucleotide backbone may contain sugars and phosphate groups (which can typically be found in RNA and DNA) or modified or substituted sugar or phosphate groups. Alternatively, the polynucleotide backbone may comprise a polymer of synthetic subunits such as phosphoramidites and thus may be an oligodeoxynucleoside phosphoramidate or a mixed phosphoramidite phosphodiester phosphate oligomer. The polynucleotide may include modified nucleotides such as methylated nucleotides and nucleotide analogues, uracil, other sugars and linking groups such as fluororibose and thioesters and nucleotide branching. The polynucleotide may be further modified, such as by conjugation with a labeling component. Other types of modification include caps, substitution of one or more naturally occurring nucleotides with an analog, and the introduction of agents for attachment of the polynucleotide to proteins, metal ions, labeling components, other polynucleotides or a solid support.

[0040] "Pharmaceutically acceptable" means physiological compatibility. The pharmaceutically acceptable carrier or excipient does not tolerate the biological activity of the administered composition, is chemically inert and is not toxic to the organism to which it is administered.

[0041] A "plasmid-free" qualifier means the absence of a construct, such as a plasmid or viral vector, for in situ expression of a functional nucleic acid.

[0042] "Polypeptide" and "protein" as used herein interchangeably means the polymeric form of amino acids of any length, which may include translated, non-translated, chemically modified, biochemically modified and derivatized amino acids. The polypeptide or protein may be naturally occurring, recombinant or synthetic, or any combination thereof. In addition, the polypeptide or protein may contain a fragment of a naturally occurring protein or peptide. The polypeptide or protein may be a single molecule or may be a complex multiparticulate complex. Additionally, 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 a heterologous and homologous leader sequence, with or without N-terminal methionine residues, immunologically labeled proteins and the like.

[0043] "Purified" means 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 ingredients with which it is naturally associated.

[0044] "regulatory RNA" means a category including RNA that affect expression through RNA interference, suppression of gene expression, or other mechanism. Accordingly, in addition to shRNA, siRNA, miRNA and antisense ssRNA, the category of regulatory RNA includes ribozymes and decoy RNA, among others.

[0045] "Ribozyme" means an RNA molecule having enzymatic activity that repeatedly cleaves other RNA molecules in a manner specific to the nucleotide base sequence:

[0046] "RNA interference" (RNAi) means an RNA-driven mechanism, as described above, involving the degradation of a complementary or partially complementary target RNA to regulate gene expression (protein synthesis). Sequence specific or gene specific.

[0047] "Sequence identity" means the degree of similarity or complementarity. There may be partial or complete identity. Partial sequence complementarity is one that prevents at least partially hybridization of an identical sequence to a target polynucleotide; this means that the functional term 'essentially identical' is used. Inhibition of hybridization of a completely complementary sequence to a target sequence can be tested by using a hybridization assay (Southern or Northern hybridization analysis, solution hybridization and the like) under low stringency conditions. A substantially identical sequence or probe will compete and inhibit the binding (i.e., hybridization) of a completely identical sequence or probe to the target sequence under low stringency conditions. This does not mean that low stringent conditions mean those in which non-specific binding is allowed; low stringent conditions require that the binding of the two sequences to each other be a specific (i.e., selective) interaction. The lack of non-specific binding can be tested by using a second target sequence that 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 a second non-complementary target sequence.

[0048] Another way of considering sequence identity, in the context of two nucleic acid or polypeptide sequences, involves referencing residues in two sequences that are the same when matched to the maximum match in a particular region. As used herein, "percentage sequence identity" means the value determined by comparing two optimally matched sequences within a comparison window, wherein part of the polynucleotide sequence in the comparison window may include additions or deletions (i.e. gaps) compared to the reference sequence (which does not include addition). or deletions) to optimally match two sequences. The percentage is calculated by determining the number of positions that have identical nucleic acid bases in both sequences to obtain the number of matches, dividing the number of matches by the total number of positions in the comparison window, and multiplying this result by 100 to get the percentage of sequence identity.

[0049] "Short interfering RNA" (siRNA) means double-stranded RNA molecules, typically from about 10 to about 30 nucleotides in length, that are capable of mediating RNA interference (RNAi). Typically, siRNA molecules have the ability to reduce the expression of a protein by directly interacting with the transcript that code 9 them.

[0050] A "therapeutically effective" amount of a functional nucleic acid is the dose of the molecule tested, eg, siRNA, miRNA or free shRNA, which elicits a pharmacological response when administered to a subject of the invention. Thus, in the context of the invention, a therapeutically effective amount can be quantified by reference to preventing or improving an undesirable condition or symptom associated with the disease or disorder, either in an animal model or in a human subject, if minicells with packed functional nucleic acid are administered as described in more detail below. An amount that turns out to be a "therapeutically effective amount" in a given case for a particular subject may not be effective for 100% of subjects treated similarly for the disease or condition under consideration, even if such a dose is considered by the skilled person to be a "therapeutically effective amount." Appropriate dosages will also vary in this regard as a function, for example, of the type, stage and severity of the disease or condition to be influenced. In each case, the presentation of an in vitro (Example 2) and in vivo (Examples 4, 5 and 6) tests according to the invention as well as a methodology for quantifying a minicell amount of functional nucleic acid (Example 3) when considered in light of the entire description, authorized by an expert in the field of pre-clinical and clinical examination of drug candidates, for determining by routine experimentation a therapeutically effective amount of a functional nucleic acid for a particular indication.

[0051] The terms "therapy", "treatment", "treat" and the like mean obtaining the desired pharmacological and / or physiological effect. The effect may be prophylactic in terms of completely or partially preventing the disease or symptom thereof and / or may be therapeutic in terms of partial or complete stabilization or treatment of the disease and / or adverse effect attributable to the disease. "Treatment" includes any treatment of a disease in a mammal, particularly a human, and includes: (a) preventing the occurrence of a disease or symptom in a subject who may be predisposed to the disease or condition but has not yet been diagnosed as having it, (b) inhibiting the symptom diseases, i.e. stopping its development; or (c) relieving the disease symptom, i.e., causing regression of the disease or symptom.

Minicells [0052] Minicells of the invention are non-nuclear forms of E. coli cells or other bacteria that result from a disorder of coordination, during binary cleavage, of cell division with DNA segregation. Prokaryotic chromosomal replication is associated with normal binary cleavage that involves the formation of a septum in the middle of the cell. For example in E. coli, mutation of min genes, such as minCD, may remove inhibition of septum formation at cell poles during cell division, resulting in the production of a normal daughter cell and a non-nuclear minicell. See Boer et al., 1992; Raskin & de Boer, 1999; Hu & Lutkenhaus, 1999; Harry, 2001. Minicells are different from other small vesicles that are produced and released spontaneously in specific situations and which, unlike minicells, are not subject to specific genetic reorganization or expression of episomal genes. In a preferred embodiment, the minicells have intact cell walls ("intact minicells").

[0053] In addition to the operon mutation, non-nuclear minicells are also produced after many other genetic rearrangements or mutations that interfere with septum formation, for example in divIVB1 in B. subtilis. See. Reeve and Cornett, 1975. Minicells can also be formed after interference at 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, minicells lacking a chromosome may result in chromosome segregation defects, for example, 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 the parC mutation in E. coli (Stewart and D'Ari, 1992). Gene products can be delivered in trans. If overexpressed from a high-copy plasmid, for example, CafA may increase the rate of cell division and / or inhibit chromosome division after replication (Okada et al., 1994), resulting in the formation of chain-linked and non-nuclear minicells (Wachi et al., 1989) . Minicells can be obtained from any bacterial cells of Gram-positive or Gram-negative origin.

[0054] In one aspect, the minicells may contain one or more plasmid-free nucleic acids whose delivery is desired. The functional nucleic acid of the invention has the ability to reduce the expression of a protein by directly interacting with the transcript that encodes the protein.

Packaging of functional nucleic acid into intact minicells [0055] Functional nucleic acid can be packaged directly into intact minicells. This method bypasses the previously required steps, for example, cloning of nucleic acids encoding functional nucleic acid into expression plasmids, transformation of parent minicell-producing bacteria with plasmids and production of recombinant minicells. Instead, the functional nucleic acid without plasmid can be directly packaged into intact minicells by co-incubating intact minicells with functional nucleic acid in buffer. In some embodiments, the co-incubation may include gentle shaking, while in others the co-incubation is static. A co-incubation period of about one hour has proven to be sufficient, but shorter periods such as about half an hour may also be effective. In one embodiment, the buffer comprises a buffered saline solution, e.g., a 1X phosphate buffered solution. Buffered saline may be in the form of gelatin. In another embodiment, the co-incubation is carried out at a temperature of about 4 ° C to about 37 ° C; about 20 ° C to about 30 ° C, about 25 ° C or about 37 ° C. In other aspects, co-incubation may include about 10⁷, 10⁸, 10⁹, 10¹⁰, 10¹¹, 10¹² or 10¹³ minicells. Specific parameters of temperature, time, buffer, minicell concentration etc. can be optimized for a specific combination of conditions.

[0056] The success of this approach is surprising because for more than forty years practitioners have developed various chemical and electrochemical processes (discussed in a review by Miller, 1994) to transform nucleic acids into bacterial cells. Practitioners have used such strong measures because the conventional view has been maintained that plasmid-free nucleic acids such as siRNA, miRNA or shRNA are too large to passively pass into the microcell cytoplasm. For example, porins, which are β-barrel proteins that typically act as diffusion pores, allow passive transport of molecules with molecular weights of 600 Daltons or less across the outer bacterial membrane (Nikaido, 1994). Meanwhile, the double-stranded DNA plasmid encoding shRNA exceeds one million daltons, and the double-stranded siRNA or miRNA exceeds 15,000 daltons.

[0057] Furthermore, after packaging, the functional nucleic acid remains inside the minicell and is protected from degradation. In this regard, studies on prolonged incubation with siRNA-packed minicells incubated in sterile saline did not show siRNA leakage. In addition, co-incubation of minicells packed with siRNA with nucleases confirmed that siRNAs had penetrated through the outer membrane of intact minicells and were protected from degradation. Similarly, despite the fact that minicells bearing residual nucleases from parent bacterial cytoplasm can be expected to be packed with siRNAs are stable in the minicell cytoplasm. Packaged siRNA also escapes the degradation mechanisms present in phagolysosomes, such as acids, free oxygen radicals and acid hydrolases (Conner and Schmid, 2003), for the purpose of reducing the expression of target mRNA within a mammalian cell.

[0058] In other embodiments, multiple functional nucleic acids directed to different target mRNAs can be packaged into the same minicell. This approach can be used to combat drug resistance and resistance to apoptosis. For example, cancer patients routinely show resistance to chemotherapeutic drugs. Such resistance may be caused by overexpression of genes such as multi-drug resistance pumps (MDRs) and anti-apoptotic genes, among others. To combat this resistance, minicells may be packed with significant concentrations of therapeutically functional nucleic acid for MDR-associated genes and administered to a patient prior to chemotherapy. In addition, the packaging of different functional nucleic acids into different minicells targeting different mRNA targets can enhance therapeutic success because molecular targets are mutated and have different alleles.

[0059] Thus, the packaging of a plasmid-free functional nucleic acid into intact minicells as described herein has numerous advantages. For example, because the inventive approach does not require the genetic modification of parent bacteria to match functional nucleic acid expression, one parent bacterium can be used to produce minicells containing multiple types of nucleic acids directed for multiple indications. Similarly, a minicell can be packed with many different RNAs, thereby avoiding or overcoming resistance mechanisms.

Functional nucleic acids [0060] As mentioned above, functional nucleic acid means a category comprising nucleic acid molecules that affect expression by RNA interference, suppression of gene expression, or other mechanism. Examples of such molecules are single, double or multi-stranded DNA or RNA. Examples of functional nucleic acids include, but are not limited to, regulatory RNAs such as shRNA, siRNA, miRNA and antisense ssRNA, and thus ribozymes and decoy RNA, and antisense nucleic acids.

[0061] In a preferred embodiment of the invention, the intact minicells carry siRNA molecules. Short interfering RNA molecules are useful for conducting RNAi, a post-transcriptional gene silencing mechanism. As mentioned, "siRNA" typically means double-stranded RNA molecules from about 10 to about 30 nucleotides in length, which have been named because of their particular ability to interfere with protein expression. Preferably siRNA molecules are 12-28 nucleotides in length, more preferably 15-25 nucleotides in length, even more preferably 19-23 nucleotides in length and most preferably 23-21 nucleotides in length. 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.

[0062] One strand length determines the length of the siRNA molecule. For example, siRNA, which is described as having 21 ribonucleotides (21-mer), may contain two opposing RNA strands that have joined each other over 19 adjacent base pairs. The other two ribonucleotides on each strand will form the "unpaired end". When siRNA contains two strands of different lengths, the longer strand determines the length of the siRNA. For example, a dsRNA containing one strand that is 21 nucleotides long and another strand that is 20 nucleotides long is a 21-mer.

[0063] SiRNAs that contain an unpaired end are desirable. The unpaired end may be at the 5 'or 3' end of the thread. Preferably, it is at the 3 'end of the RNA strand. The unpaired end length can vary, but is preferably from about 1 to about 5 bases and more preferably is about 2 nucleotides in length. Preferably, the siRNA of the invention will have an unpaired 3 'end having about 2 to 4 bases. More preferably, the unpaired 3 'end is 2 ribonucleotides in length. Even more preferably, 2 ribonucleotides having an unpaired 3 'end are uridine (U).

[0064] shRNAs contain a single RNA strand that forms a trunk-loop structure, where the trunk consists of complementary sense and antisense strands that contain double-stranded siRNA, and the loop is a variable-size connector. The structure of the shRNA trunk is usually from about 10 to about 30 nucleotides in length. Preferably, the trunk of the shRNA molecules is 12-28 nucleotides in length, more preferably 15-25 nucleotides in length, even more preferably 19-23 nucleotides in length and most preferably 23-21 nucleotides in length. Therefore, preferred shRNA molecules contain 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.

[0065] siRNAs of the invention are intended to interact with the target ribonucleotide sequence, which means that they complement the target sequence sufficiently to hybridize to the target sequence. In one embodiment, the invention provides siRNA molecules comprising a ribonucleotide sequence at least 70%, 75%, 80%, 85% or 90% identical to the target ribonucleotide sequence or complementary to the 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 complementary to the target ribonucleotide sequence. Most preferably, the siRNA will be 100% identical to the target nucleotide sequence or complementary to the ribonucleotide sequence. However, siRNA molecules with insertions, deletions or single point mutations relative to the target may also be effective.

[0066] Accordingly, in one aspect of the invention, intact minicells may carry one or more siRNA sequences directed to silencing drug resistance or apoptosis resistance genes. By using minicells that encode many siRNAs, it is possible to treat cells that express many drug resistance mechanisms. [0067] Tools supporting siRNA and regulatory RNA design in general are readily available to the public. For example, the siRNA computer design tool is available on the Internet at <a href="http://www.dharmacon.com">www.dharmacon.com</a>.

Objectives of functional nucleic acids [0068] The functional nucleic acids of the invention are preferably directed to a gene or protein transcript that stimulates drug resistance, inhibits apoptosis, stimulates the tumor phenotype, inhibits pathogen proliferation or inhibits viral replication or proliferation. The successful application of functional nucleic acid strategies in such situations has already been achieved in this field, but without the benefit of minicell vectors. See. for example. Sioud (2004), Caplen (2003), Wu et al. (2003), Yague et al. (2004).

[0069] Proteins that contribute to drug resistance or stimulation of the tumor phenotype are preferred targets for functional nucleic acid. Proteins can contribute to acquired drug resistance or to internal drug resistance. Acquiring a resistant phenotype occurs when diseased cells, such as cancer cells, initially respond to drugs but become refractory after successive cycles of treatment. Useful targets involved in acquired or intrinsic drug resistance include, but are not limited to, ATP binding cassette transporters such as P-glycoprotein (P-gp, P-170, PGY1, MDR1, ABCB1, MDR-related protein, multi-drug resistance protein 1, MDR-2 and MDR-3, MRP2 (multi-drug resistance protein), BCRABL (fracture cluster region - Abelson protooncogene), STI-571 resistance associated protein, lung resistance associated protein, cyclooxygenase 2, kappa nuclear factor, XRCC1 (cross-complementation of X-ray damage; group 1), ERCC1 (cross-complement and excision gene), GSTP1 (glutathione S-transferase), β-tubulin mutant, Abcb1a (ABCB4), Abcc1, Abcc2, Abcc3 (MLP-2) , Abcc5, Abcc6, Abcd2, Abcg2, Bax, Bcl2, Bcl21 (bcl-x), Mvp, Rb1, Top1, Top2a, Top2b, Trp53 (p53). Other genes involved in drug resistance are also (a) genes involved in drug metabolism, e.g. Arnt, Blmh, C130052I12Rik (CRR9p), Comt, Crabp1, Cyp1a1, Cyp1a2, Cyp2b19, Cyp2b20, Cyp2c29, Cyp2c40, Cyp2 , Ephx1,

Ephx2, Gstm1 (MGST1), Gstp1, Nat2, Nqo1, Sod1, Ste, Tpmt, Tyms, Ugcg, (b) genes involved in DNA repair, e.g. Apc, Atm, Brca1, Brca2, Ercc3 (XPB), Mgmt, Mlh1 , Xpa, Xpc, (c) genes involved in the cell cycle, e.g. Ccnd1 (cyclin D1), Ccne1 (cyclin E1), Cdk1, Cdk2, Cdk4, Cdkn1a (p2IWaf1), Cdkn1b (p27Kip1), Cdkn2a (p16Ink4a) (p19), KSP., (d) genes involved in growth factor receptors, e.g. Egfr, Erbb2 (Neu, HER2), Erbb3, Erbb4, Fgf2 (bFGF), Met, (e) genes involved in hormone receptors, e.g. Ar, Esr1, Esr2, Igf2r, Ppara, Ppard, Pparg, Ppargc1, Rara, Rarb, Rxra, Rxrb, Rxrg, Srd5a2, and (f) genes involved in transcription factors, on example Ahr, Ap1s1, Ap1s2, Elk1, Fos (c-fos), Gabpa, Hif1a, Mafb, Myc (c-myc), Nfkb1, Nfkb2, Nfkbib, Nfkbie, Relb (1-rel), Tnfrsf11A.

[0070] Useful targets also include proteins that contribute to apoptosis resistance. These include Bcl-2 (B-cell leukemia / lymphoma), Bcl-XL, A1 / Bfl 1, adhesion-focal kinase and the mutated p53 protein.

[0071] Useful targets further include oncogenic and mutated tumor suppressor proteins. Examples include β-catenin, PKC-α (protein kinase C), C-RAF, K-Ras (V12), hRas, Dead box DP97 helicase RNA, DNMT1 (DNA 1 methyltransferase), FLIP (Flice-like inhibitory protein) , C-Sfc, 53BPI, Polycomb group protein EZH2 (homologue enhancer zeste), ErbB 1, HPV-16 E5 and E7 (human papillomavirus early 5 and early 7), fortilinin and MCI1P (myeloid leukemia protein 1), DIP13a ( DDC-reactive protein 13a), MBD2 (methyl CpG binding domain), p21, KLF4 (Kruppel 4-like factor), tpt / TCTP (tumor-controlled translation protein), SPK1 and SPK2 (sphingosine kinase), P300, PLK1 (Polo-like kinase 1), Trp53, Ras, ErbB1, VEGF (growth factor endothelium), and BAG-1 (atanogen 1 bound to BCL2).

[0072] Numerous molecular targets have been identified for the treatment of cancer, and RNAi discovery platforms quickly identify many different new targets. Examples of such molecular targets useful in the invention include tyrosine kinase (variant), Akt (protein kinase B, PKB), Akt1, integrin AlphaLbeta2, aminopeptidase, androgen receptor, Aurora A, AuroraB, primary fibroblast growth factor receptor (bFGFr) (bFGFr), Braf, carcinembryonic antigen (CEA), CD142, CD37, CD44, CD5, CD74, CD77, Chk1, CHK2, CHras, ​​CSF1r, CXCR4, Cyclin D1 (CCND1), cyclin dependent kinase 1 (CDK1), cyclin dependent kinase 2 (CDK2) cyclin dependent kinase 1B inhibitor (CDKN1B, p27, KIP1), CYP26, fibroblast growth factor 3 receptor (FGFr3), fibroblast growth factor 4 receptor (FGFr4), G250, Hedgehog signaling pathway (Hh), growth factor / hepatocyte scattering factor ( HGF / SF or SF / HGF), HEr4 (ErbB4), HIF, histone deacetylase 9 (HDAC9), Homeobox gene (HOXB7), hyaluronan (HA), insulin-like growth factor (IGF), insulin-like growth factor receptor 1 (IGF1r, IGF1r, IGFIr, IGFIr), insulin-like growth factor 2 binding protein (IGFBP2), insulin-like growth factor binding protein 5 (IGFBP5), integrin-like kinase (ILK) interleukin receptor (IL6), type II interleukin 1 receptor (IL1), interleukin 10 (IL10) ), interleukin 4 (IL4) receptor, interleukin 6 (IL6), interleukin 15 (IL15), interleukin 3 receptor alpha chain (IL3r alpha), JAK, JAK3, JNK1, JNK2, spindle kinesin protein (KSP), laminin 5, Lewis (b) beta receptor (LTBr) lymphotoxin (LT), receptors (LPAr) lysophosphatidic acid (LPA), lysophosphatidic acid acyltransferase, inhibitory factor for macrophage migration (MIF), MAGE3, microtubules, MUC2, Notch 1 (TAN1), mitogen-activated protein kinase p38 MAPK), up-regulated by P53 mediator of apoptosis (PUMA), PDGF tyrosine kinase (TK) signaling pathway, phosphatase and tensine homologue (PTEN), phosphatidylinositol 3 'kinase (PI3K), plasminogen activator urokinase (PLAUr) receptor (PLAU), Polo 1-like kinase (Plk1), Poly (ADP ribose), polymerase (PARP), nuclear cellular proliferation antigen (PCNA), prostate stem cell antigen (PSCA), gland-specific antigen stepper (PSA) 773, protein tyrosine phosphatase (PTP), Rad51 protein, RAF1, retinoic acid receptor (RAr) alpha, retinoic acid receptor (RAr) gamma, retinoid X receptor (RXr) beta, serine (or cysteine) proteinase inhibitor . telomerase reverse transcriptase (TERT, hTERT), telomeres, Thomsen Friedenreich antigen (TF), thrombospondin 1 (TSP1), transferrin, tumor necrosis factor alpha (TNFa, TNFA), tumor necrosis factor receptor (TNFr, TNFr), carbonic anhydrase associated tumor (CA) IX (CA9), type I interferon, ubiquitin ligase, vascular cell 1 (VCAM1, CD106) adhesion molecule, vascular endothelial growth factor (VEGF, VEGFA), vascular endothelial growth factor D (VEGFD), vitronectin (VTN), Wilms 1 tumor (WT1) etc.

[0073] With respect to HIV infection, targets include HIV-Tat, HIV-Rev, HIV-Vif, HIV-Nef, HIV-Gag, HIV-Env, LTR, CD4, CXCR4 (chemokine receptor) and CCR5 (receptor The chemokine).

[0074] Due to the heterogeneity of the tumor cells, many different drug resistance or apoptosis resistance pathways can be triggered in target cells. Thus, functional nucleic acids used in the methods of the invention may need to change over time. For example, if biopsy samples reveal new mutations that lead to acquired drug resistance, then specific functional nucleic acid can be designed and packed into intact minicells that are administered to a mammalian host to respond to acquired drug resistance.

Functional nucleic acid delivery by intact minicells [0075] The invention enables a functional nucleic acid delivery method that includes (a) providing a set of intact minicells in a pharmaceutically acceptable carrier, each minicell includes plasmid-free functional nucleic acid, and (b) bringing the set into contact minicells with mammalian cells, so that mammalian cells absorb minicells belonging to the harvest, wherein the functional nucleic acid is released into the cytoplasm of target cells. Minicells are contacted with the target mammalian cell by bispecific ligands as described in published PCT Application WO 05/056749. Contact between the minicell and target mammalian cells may occur in vitro or in vivo.

Method of overcoming drug resistance and treating disease [0076] The invention further allows a method of overcoming drug resistance and treating a disease such as cancer or AIDS, in a subject. The method includes (a) packaging one or more functional nucleic acids that target genes or transcripts of proteins that promote drug resistance to intact purified minicells, (b) contacting minicells containing functional nucleic acid with the target mammalian cell such that the mammalian cell absorbs the minicell as described in the '749 PCT application cited above, and (c) delivering the drug to the target mammalian cell, as described in published PCT application WO 05/079854. Preferably, step (c) is performed after steps (a) and (b) to allow the functional nucleic acid to reduce resistance to drug administration. Drug delivery and incorporation of a functional nucleic acid may be consecutive, in any order or simultaneously.

[0077] 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 for each drug is easily determined by those skilled in the medical field.

Drug delivery by minicells [0078] Although drug delivery can be by conventional means, delivery by minicells is preferred as described in published PCT Application WO 05/079854. In this regard, applicants have found that the same mammalian cells can be successfully re-transfected by targeted intact minicells that are packed with different charges. For example, functional nucleic acid-packed minicells can transfect a mammalian cell, after which the drug-packed minicells can deliver the drug to the same mammalian cell to achieve complementary or synergistic anti-cancer effects.

[0079] The drug may be packaged in a separate minicell relative to that of the functional nucleic acid. Alternatively, the drug may be packaged in the same minicell as the functional nucleic acid. Some drugs may interact with nucleic acids and prevent the medicine and nucleic acid from being packed into the same minicell together. For example, doxorubicin is known to interact with DNA.

[0080] Preferably, the minicells of the invention contain a sufficient amount of drug to exert a physiological or pharmacological effect of the drug on the target cell. Also preferably, the drugs contained within the minicells are heterologous or foreign to the minicells, which means that the bacterial stem cells of the minicells do not normally produce the drug.

[0081] Both hydrophilic and hydrophobic drugs can be packaged into minicells by creating a drug concentration gradient between the extracellular environment containing the minicells and the minicell cytoplasm. When the extracellular environment contains a higher concentration of drug than the minicell cytoplasm, the drug naturally shifts down this concentration gradient to the minicell cytoplasm. However, when the concentration gradient is reversed, the drug does not come out of the minicells. The procedure and mechanisms for loading the drug into minicells are as described in published PCT Application WO 05/079854.

[0082] In order to package minicells with drugs that are not usually water-soluble, the drugs can be pre-dissolved in a suitable solvent. For example, paclitaxel can be dissolved in a 1: 1 mixture of ethanol and cremophore EL (polyethoxylated castor oil) followed by dilution in PBS to achieve a solution of paclitaxel that is partially dissolved in aqueous media and carries minimal amounts of organic solvent to ensure that the medicine remains in solution. Minicells can be incubated in this final environment for drug packaging. Thus, applicants have found that even hydrophobic drugs can diffuse into the cytoplasm of minicells in order to achieve high and therapeutically significant drug packaging of the cytoplasm. This is surprising because the minicell membrane is composed of a hydrophobic phospholipid bilayer, which was expected to prevent the diffusion of hydrophobic molecules into the cytoplasm.

[0083] Another method of loading minicells with a drug involves culturing the recombinant bacterial stem cells under conditions such that the parent bacterial cell transcribes and translates the nucleic acid encoding the drug and the drug is released into the cytoplasm of the parent bacterial cell. For example, a gene cluster encoding the cellular biosynthesis pathway of a desired drug may be cloned and transferred to a parent bacterial strain that is capable of producing minicells. Gene transcription and gene cluster translation results in drug biosynthesis within the cytoplasm of stem bacterial cells, filling the bacterial cytoplasm with the drug. When the parent bacterial cell divides and creates daughter minicells, the minicells also contain the drug in their cytoplasm. Pre-packed minicells can be purified by any suitable minicell purification method, including the methodology described above.

[0084] Similarly, another method of packaging minicells with a drug involves culturing a recombinant minicell that contains the expression plasmid encoding the drug under conditions such that the gene encoding the drug is transcribed and translated within the minicell.

Drugs [0085] Drugs useful in the invention can be a physiologically or pharmacologically active substance that produces the desired local or systemic effect in animals, especially mammals and humans. Drugs may be inorganic or organic compounds, including but not limited to, peptides, proteins, nucleic acids and small molecules, all of which may be characterized or uncharacterized. They may exist 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, metal salts, amines or organic cations can be used, for example quaternary ammonium compounds. Drug derivatives such as bases, esters and amides can also be used. A drug that is insoluble in water can be used in a form that means its water-soluble derivative, or as its basic derivative, which in any case, or through delivery, is converted by enzymes, hydrolyzed at the body's pH, or by other means metabolic processes to their original therapeutically active form.

[0086] Useful drugs include chemotherapeutic agents, immunosuppressants, cytokines, cytotoxic agents, nucleolytic compounds, radioactive isotopes, receptors, and pro-drug activating enzymes that can be naturally occurring or produced by recombinant means.

Drugs that are affected by classic multi-drug resistance have particular utility in the invention, such as vinca alkaloids (e.g. vinblastine and vincristine), anthracyclines (e.g. doxorubicin and daunorubicin), RNA transcription inhibitors (e.g. actinomycin-D) and drugs stabilizing microtubules (e.g. paclitaxel).

[0088] In general, cancer chemotherapy agents are preferred drugs. Useful drugs for the treatment of cancer chemistry include nitrogen mustards, nitrosoureas, ethyleneimines, alkanesulfonates, tetrazine, platinum compounds, pyrimidine analogs, purine analogues, antimetabolites, folic acid analogs, anthracyclines, taxanes, vinca alkaloids, hormone inhibitors and topoisomerase agents. Examples of chemotherapy drugs are actinomycin-D, alkeran, Ara-C, anastrozole, asparaginase, BiCNU, bicalutamide, bleomycin, busulfan, capecitabine, carboplatin, carmustine, CCNU, chlorambucil, cisplatin, cladribine, CPT-11 cytosines, cytoxane, dacarbazine, dactinomycin, daunorubicin, dexrazoxane, docetaxel, doxorubicin, DTIC, epirubicin, ethyleneimine, etoposide, floxuridine, fludarabine, fluorouracil, flutamide, photemustine, gemcitabine herceptin, hexamethylamine, hydroxycarbamide, idarubicin, ifosfamide, irinotecan, lomustine, mechlorethamine, melphalan, mercaptopurine, methotrexate, mitomycin, mitotane, mitoxantrone, oxaliplatin, paclitaxel, pamidronate, pentristatin, stentristatin streptozocin, tamoxifen, temozolomide, teniposide, tetrazine, thioguanine, thiotepa, tomudex, topotecan, treosulfan, trimetrexate, vinblastine, vincristine, vindesine, vinorelbine, VP-16 and Xeloda.

[0089] Useful drugs for cancer chemotherapy also include alkylating agents such as thiotepa and cyclophosphamide; alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carbocarbone, meturedopa and uredopa; ethyleneimines and methylamelamines, including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide and trimethylol melamine; nitrogen mustard derivatives such as chlorambucil, chlornafazine, cholophosphamide, estramustine, ifosfamide, mechloretamine, mechloretamine oxide hydrochloride, melphalan, Novembiehin, fenesterine, prednimustine, trophosphamide, uracil mustard; nitroureas such as carmustine, chlorozotocin, photemustine, lomustine, nimustine and ranimustine; antibiotics such as aclacinomizins, actinomycin, autramycin, azaserin, bleomycin, cactinomycin, calicheamycin, carabicin, carminomycin, carzinophylline, chromomycin, dactinomycin, daunorubi17 tin, detorubicin, 6-ducinose , idarubicin, marcellomycin, mitomycin, mycophenolic acid, nogalamycin, olivomycin, peplomycin, potfiromycin, puromycin, quelamycin, rhodorubicin, streptonigrine, streptozocin, tubercidin, ubenimex, zynostatin and zorubicin; anti-metabolites such as methotxate and 5-fluorouracil (5-FU); folic acid analogues such as denopterin, methotrexate, pteropterin and trimetrexate, purine analogues such as fludarabine, 6-mercaptopurine, thiprin, thioguanine; pyrimidine analogues such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocytabine, floxuridine and 5-FU; androgens such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone; adrenocortical hormone antagonists such as aminoglutethimide, mitotane, trilostane; folic acid supplements such as folinic acid; aceglatone; aldophosphamide glycoside, aminolevulinic acid; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elfornithine; elliptin acetate; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidamine; mitoguazone; mitoxantrone; mopidamol; nitracrine; pentostatin; phenamet; pirarubicin; podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK®; razoxane; sizofiran; spirogermanium; tenuazonic acid; triaziquone; 2 ', 2 "-trichlorotriethylamine; urethane; vindesine, dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside (" Ara-C "); 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; platinine analogues such as cisplatin and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitomycin C; mitoxantrone; vincristine; vinorelbine; navelbine; edatrexate; teniposide; daunomycin; aminopterin; Xeloda; ibandronate; CPT-11; RFS 2000 topoisomerase inhibitor; 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 the hormone's effects on tumors, such as anti-estrogens, including, for example, tamoxifen, raloxifene, 4 (5) -imidazole aromatase inhibitors, 4-hydroxy tamoxifen, trioxyfen, keoxyfen, 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.

[0090] Useful drugs also include cytokines. Examples of such cytokines are lymphokines, monokines and traditional polypeptide hormones. 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; a substance that inhibits the functions of Muller's ducts; mouse gonadotropin-related peptide; inhibin; activin; vascular endothelial growth factor; integrin; thrombopoietin (TPO); nerve growth factors such as NGF-β; platelet-derived growth factor; transforming growth factors (TGFs) such as TGF-α and TGF-β; insulin-like growth factors -I and -II; erythropoietin (EPO); osteoinductive factors; interferons such as interferon -α, -β and -γ; growth-promoting factors (CSF) such as macrophage-CSF (M-CSF); granulocyte-macrophage-CSF (GM-CSF); and granulocyte-CSF (G-CSF); interleukins (IL) 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; tumor necrosis factor such as TNF-α or TNF-β; and other polypeptide factors, including LIF and kit ligand (KL). As used herein, the term cytokine includes proteins from natural sources or from recombinant cell culture and biologically active equivalents of native cytokine sequences.

[0091] Drugs can be prodrugs that are subsequently activated, e.g., by a prodrug activating enzyme that converts a prodrug, such as a peptidyl chemotherapeutic agent, to the form of an active anti-cancer drug. For example, see WO 88/07378, WO 81/01145, and US Patent No. 4,975,278. In general, the enzyme component includes any enzymes capable of interacting with a prodrug in such a way that it undergoes conversion to a more active, cytotoxic form.

Targeting minicells to specific mammalian cells [0092] In one aspect of the invention, the minicell is targeted to the target mammalian cell via a bispecific ligand as described in published PCT patent applications WO 05/056749 and WO 05/079854. A bispecific ligand, with specificity for both minicell and mammalian cell components, causes the minicell to bind to the mammalian cell, such that the minicell is absorbed by the mammalian cell, whereby functional nucleic acid is released into the mammalian cell cytoplasm. This method of targeted delivery can be carried out in vivo or in vitro, or both in vivo and in vitro.

[0093] Contact between the bispecific ligand, minicell and mammalian cell can occur in a number of different ways. For in vivo delivery, it is preferable to administer a minicell that already has a bispecific ligand attached thereto. Hence the contact of all: minicell, bispecific ligand and target cell when the bispecific ligand-targeted minicell reach the target cell in vivo. Alternatively, the bispecific ligand and minicell may be administered separately in vivo. [0094] Contact between bispecific ligands, minicells and mammalian cells may occur during one or more in vitro incubations. In one embodiment, three elements are incubated with each other all at once. Alternatively, incubations can be carried out in stages. In one example of a stepwise approach, minicells and bispecific ligands are first incubated together to form a bispecific ligand-targeted minicells that are then incubated with target cells. In another example, bispecific ligands are first incubated with target cells, followed by incubation with minicells. The combination of one or more in vitro incubations and in vivo administration may also result in contact of bispecific ligands, minicells and mammalian target cells.

[0095] The inventors have found that the targeted delivery approach finds general application to mammalian cells, including cells that are normally resistant to specific adhesion and endocytosis of minicells. For example, bispecific antibody ligands with anti-O-polysaccharide specificity on one arm and anti-HER2 receptor specificity or anti-EGF receptor on the other arm effectively bind minicells to relevant receptors on a number of non-phagocytic target cells. These cells include lung, ovarian, brain, breast, prostate and skin cancer cells. In addition, effective binding precedes rapid endocytosis of minicells by each of the non-phagocytic cells.

[0096] Target cells of the invention include any cells into which a functional nucleic acid is to be introduced. Desired target cells are characterized by cell surface receptor expression, which, after ligand binding, facilitates endocytosis. Preferred target cells are non-phagocytic, which means that the cells are not specialized phagocytes such as macrophages, dendritic cells and natural killer (NK) cells. Preferred target cells are also mammalian.

[0097] Ligands useful in targeted delivery methods of the 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 the target cell is a receptor, particularly a receptor capable of mediating endocytosis. The ligands may contain a polypeptide and / or a carbohydrate component. Antibodies are preferred ligands. For example, a bispecific antibody that carries dual specificities for the surface component on intact bacterial minicells and for the surface component on mammalian target cells can be effectively used to target minicells to mammalian target cells in vitro and in vivo. Useful ligands also include receptors, enzymes, binding peptides, fusion / chimeric proteins and small molecules.

[0098] Selection of a particular ligand is based on two main 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 target cells. Thus, ligands preferably have a first arm that carries the specificity for the surface structure of an intact minicell of bacterial origin and a second arm that carries the specificity for the surface structure of a mammalian cell. Each of the first and second arms can be multivalent. Preferably, each arm is monospecific, even if multivalent.

[0099] For the purpose of binding to minicells of bacterial origin, it is desirable for one ligand arm to be specific for the O-polysaccharide component of the lipopolysaccharide found on the parent bacterial cell. Other minicell surface structures that can be used to bind ligand include cell surface exposed polypeptides and carbohydrates on outer membranes, such as outer membrane proteins, cell surface exposed pilli, fimbri and flagella peptides.

[0100] For binding to target cells, one arm of the ligand is specific for the surface component of a mammalian cell. Such components include cell surface proteins, peptides and carbohydrates, whether characterized or non-characterized. Cell surface receptors, especially those capable of activating receptor-mediated endocytosis, are desirable cell surface components for targeting. Such receptors, if overexpressed on the surface of the target cell, confer additional selectivity for targeting the cells for treatment, thereby reducing the possibility of delivery to non-target cells.

[0101] By way of example, cancer cells, metastatic cells, vascular cells such as endothelial cells and smooth muscle cells, lung cells, kidney cells, blood cells, bone marrow cells, brain cells, liver cells etc. can be targeted. , or precursors to any selected cells, by selecting a ligand that specifically binds the cell surface receptor motif on the desired cells. Examples of cell surface receptors include carcinoembryonic antigen (CEA), which is overexpressed in most cancers of the colon, rectum, breast, lung, pancreas and gastrointestinal tract (Marshall, 2003); heregulin receptors (HER-2, neu or c-erbB-2), which are often overexpressed in breast, ovarian, colon, lung, prostate and cervical cancer (Hung et al., 2000); epidermal growth factor receptor (EGFR), which is highly expressed in many solid tumors, including breast, head and neck, non-small cell lung and prostate cancer (Salomon et al., 1995); asjaloglikoprotein receptor (Stockert, 1995); transferrin receptor (Singh, 1999); a serpine enzyme complex receptor that is expressed on hepatocytes (Ziady et al., 1997); fibroblast growth factor receptor (FGFR) that is overexpressed on pancreatic ductal adenocarcinoma cells (Kleeff et al., 2002); vascular endothelial growth factor receptor (VEGFR), for gene therapy against angiogenesis (Becker et al., 2002; Hoshida et al., 2002); folate receptor that is selectively overexpressed in 90% of non-mucosal ovarian cancers (Gosselin and Lee, 2002); cell surface glycocalyx (Batra et al., 1994); carbohydrate receptors (Thurnher et al., 1994); and a polymeric immunoglobulin receptor that is useful for delivering genes to respiratory epithelial cells and attractive for the treatment of lung diseases such as cystic fibrosis (Kaetzel et al., 1997).

[0102] Preferred ligands include antibodies and / or antibody derivatives. As used herein, the term "antibody" includes an immunoglobulin molecule obtained by generating in vitro or in vivo immunogenic responses. 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 invention can be obtained by recombinant DNA techniques.

[0103] Wild type antibodies have four polypeptide chains, two identical heavy chains, and two identical light chains. Both types of polypeptide chain have constant regions that are not differentiated or differ slightly between antibodies of the same class, and variable regions. Variable regions are unique to a particular antibody and contain an antigen binding domain that recognizes a specific epitope. The antigen binding domain regions that are most directly involved in antibody binding are "complementarity determining regions" (CDRs).

[0104] The term "antibody" also includes antibody derivatives, such as antibody fragments, that retain the ability to specifically bind to antigens. Such antibody fragments include Fab fragments (a fragment that contains an antigen binding domain and includes a light chain and a portion of a heavy chain connected by a disulfide bond bridge), Fab '(an antibody fragment containing a single antigen binding domain containing Fab and an additional heavy chain, through the region, F (ab ') 2 (two Fab' molecules connected by disulfide bonds between chains in the hinge regions of heavy chains), bispecific Fab (Fab molecule having two antigen binding domains, each of which can be directed to a different epitope), and scFv (variable, antigen binding determining the single light and heavy chain region of the antibody, linked by an amino acid chain).

[0105] When the antibodies, including antibody fragments, form part or all of the ligands, they are preferably of human origin or are modified to be suitable for human use. The 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 humans. Methods for humanizing antibodies are described, e.g. in US Patent Nos. 6,639,055, No. 5,585,089 and No. 5,530,101. In the simplest case, humanized antibodies are created by transplanting antigen binding loops, known as complementarity determining regions (CDRs), from mouse mAb to human IgG. See. Jones et al., 1986; Riechmann et al., 1988; Verhoeyen et al., 1988. Generation of high affinity humanized antibodies, however, generally requires the transfer of one or more additional residues from so-called framework regions (FR) of mouse parent mAb. Several variants of humanization technology have also been developed. See. Vaughan et al., 1998.

[0106] Human antibodies, instead of "humanized antibodies", can also be used in the invention. They have high affinity for their respective antigens and are routinely obtained from very large, single chain variable fragments (scFvs) or 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.

[0107] Useful ligands also include bispecific single chain antibodies that typically denote recombinant polypeptides consisting of the light chain variable portion covalently attached by the linker molecule to the corresponding heavy chain variable portion. See. U.S. Patents No. 5,455,030, No. 5,260,203 and No. 4,496,778. Bispecific antibodies can also be obtained by other methods. For example, chemical heteroconjugates can be generated by chemically linking intact antibodies or antibody fragments with different specificities.

See. Karpovsky et al., 1984. However, such heteroconjugates are difficult to obtain in a reproducible manner and are at least twice as large as normal monoclonal antibodies. Bispecific antibodies can also be generated by disulfide exchange, which is associated with enzymatic cleavage and reassociation of antibody fragments. See. Glennie et al., 1987.

[0108] Because Fab and scFv fragments are monovalent, they often have low affinity for target structures. Thus, preferred ligands obtained 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 can be generated by chemical and / or genetic cross-linking.

[0109] The bispecific ligands of the invention are preferably 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. Ligands can be multivalent at one or both ends, for example in the form of so-called diclassus, tricolor and tetracytal. See. Hudson and Souriau, 2003. Diabody means a divalent dimer formed by non-covalent association of two scFvs that results in two Fv binding sites. Similarly, the trichody is the result of the formation of a three-valent three scFv trimer, resulting in three binding sites, and the tetrabody is the result of the formation of a tetravalent four scFv tetramer, resulting in four binding sites.

[0110] Several humanized, human and mouse monoclonal antibodies and fragments thereof that have specificity for receptors on mammalian cells have been approved for therapeutic use in humans, and this 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 HER2 specificity: Herceptin^™; Trastuzumab.

[0111] Antibody variable regions can also be conjugated to a wide range of protein domains. Fusion with human immunoglobulin domains such as CH3 IgG1 both increases mass and stimulates dimerization. See. Hu et al., 1996. Fusion to human Ig Fc hinge regions may add effector functions. Also, fusion to heterologous protein domains from multimeric proteins stimulates multimerization. For example, short scFv fusion to short amphipathic helices has been used for the production of 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, alternatively, homodimerization domains can be engineered to form heterodimers using engineering strategies such as " knobs-intoholes ”[creating protrusions matching the holes] (Ridgway et al., 1996). Finally, fusion protein partner selection can be carried out that will provide both multimerization as well as an additional function, e.g. streptavidin. See. Dubel et al., 1995.

Delivery to cells competent for phagocytosis or endocytosis [0112] The invention further provides delivery by bringing minicells of bacterial origin into contact with mammalian cells that are competent for phagocytosis or endocytosis. Such mammalian cells that are able to absorb parent bacterial cells by intracellular bacterial pathogens similarly absorb minicells that release their content in the mammalian cell cytoplasm. This delivery approach can be carried out without the use of targeted ligands.

[0113] A number of mechanisms may be involved in the absorption of minicells by a given cell type, and the 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, absorb particles by extending the pseudo-leg above the surface of the particle until the particle is completely absorbed. Although described as "non-specific" phagocytosis, specific receptors have been shown to be involved in the process. See. Wright et al., (1986); Speert et al., (1988).

[0114] Thus, one form of phagocytosis requires interactions between surface ligands and ligand receptors located in pseudocereal membranes. This attachment step, regulated by specific receptors, is considered to be dependent on bacterial surface adhesives. For less virulent bacteria, such as nonenterotoxinogenic E. coli, phagocytosis may also occur in the absence of surface ligands for phagocyte receptors. See. e.g. Pikaar et al. (1995). Hence, the invention includes, but is not limited to, the use of minicells that possess - or are - surface adhesins, in harmony with the nature of their bacterial parent cells, and are absorbed by phagocytes (i.e., "phagocytic competent" host cells) from which neutrophils and macrophages are the main types in mammals.

[0115] Another absorption process is endocytosis, by which intracellular pathogens, by the example of Salmonella, Escherichia, Shigella, Helicobacter, Pseudomonas and Lactobacilli, gain access to mammalian epithelial cells, where they are replicated. Two basic mechanisms in this regard are clrin-dependent endocytosis, regulated by the receptor, also known as "endocytosis of [clrin] cavities" (Riezman, 1993) and clrin-independent endocytosis (Sandvig &amp; Deurs, 1994). One or both may be involved when the absorption-competent cell that acts by endocytosis (i.e., the "endocytosis-competent" host cell) absorbs minicells in accordance with the invention. Representative endocytosis-competent cells are breast epithelial cells, gastrointestinal enterocytes, gastric epithelial cells, lung epithelial cells, and urinary tract and bladder epithelial cells.

[0116] When conducting delivery to an absorbent competent mammalian cell without using a targeting ligand, the nature of the application contemplated will affect the choice of the bacterial source of the minicells used. For example, Salmonella, Escherichia and Shigella species carry adhesins, which are recognized by endocytosis-mediated receptors on enterocytes in the gastrointestinal tract, and may be suitable to provide a drug that is effective against colon cancer cells. Similarly, minicells derived from Helicobacter pylori, carrying adhesins specific for gastric epithelial cells, may be adapted to deliver cell-targeted gastric cancer. Inhalation or insufflation may be ideal for administering intact minicells derived from the Pseudomonas species, which carries adhesins recognized by receptors on lung epithelial cells. Minicells derived from Lactobacilli that carry adhesins specific for the epithelial cells of the urinary tract and the bladder can be well adapted to deliver the drug to the urethra for urinary tract cancer or bladder cancer.

Formulations [0117] As indicated, in one aspect there is provided a composition comprising (a) a set of intact minicells of bacterial origin, wherein each minicell of said set contains regulatory RNA that is packaged in said minicell, and (b) a pharmaceutically acceptable carrier therefor, wherein in said minicells there is no construct for expression in situ of said regulatory RNA, wherein said regulatory RNA is selected from the group consisting of siRNA, miRNA and shRNA, and wherein said set comprises a therapeutically effective amount of said regulatory RNA.

[0118] The formulation optionally comprises a drug. In one example, the minicell of the formulation contains the drug, while in another the minicell may contain a nucleic acid molecule, such as a plasmid that encodes the drug.

[0119] The formulations also optionally contain a bispecific ligand for targeting the minicell to the 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 a bispecific ligand preferably is capable of binding to the surface component of the minicell and to the surface component of the target mammalian cell.

[0120] The formulations can be presented in unit dosage form, e.g., in ampoules or vials, in multi-dose containers, with or without a preservative added. The formulation may be in the form of a solution, suspension or emulsion in oily or aqueous carrier liquids, and may contain formulatory agents such as suspending, stabilizing and / or dispersing agents. A suitable solution is isotonic with the recipient's blood and is illustrated by saline solution, Ringer's solution and dextrose solution. Alternatively, the formulations may be in the form of a lyophilized powder, to be reconstituted with a suitable carrier, e.g. sterile pyrogen-free water or physiological saline. The formulations can also be in the form of a tissue preparation. Such long-acting formulations can be administered by implantation (e.g., subcutaneous or intramuscular) or by intramuscular injection.

Routes of administration [0121] The formulations described herein can be administered by various routes and to different locations in the mammalian body to achieve the desired therapeutic effect, locally or systemically. Delivery can be achieved, for example, by oral administration, by administration of the formulation into the body cavity, by inhalation or insufflation, or by parenteral, intramuscular, intravenous, portal vein, intrahepatic, peritoneal, subcutaneous, intraocular or intradermal administration. The mode and place of administration depend on the location of the target cells. For example, cystic cells with fibrosis may be an effective target for inhaled delivery of targeted minicells. Similarly, tumor metastasis can be more effectively treated by intravenous delivery of targeted minicells. Primary ovarian cancer can be treated by intraperitoneal administration of targeted minicells.

Purity [0122] In one aspect, the minicells are substantially free of contaminating bacterial stem cells. Thus, formulations containing a minicell preferably contain less than about 1 contaminating bacterial parent cell per 10⁷ minicells, more preferably contain less than about 1 contaminating parent bacterial cell out of 10⁸ minicells, even more preferably contain less than about 1 contaminating parent bacterial cell out of 10⁹ minicells, even more preferably contain less than about 1 contaminating parent bacterial cell out of 10¹⁰ minicells and most preferably contain less than about 1 contaminating parent bacterial cell in 10¹¹ minicells.

[0123] Methods for purifying minicells are known in the art and described in International Publication No. WO03 / 033519. One such method combines cross-flow filtration (the feed flow is parallel to the membrane surface; Forbes, 1987) and dead-end filtration [with unidirectional flow] (the overflow is perpendicular to the membrane surface). Optionally, the combination of filtration may be preceded by differential centrifugation, at low centrifugal force, to remove some of the bacterial cells and, as a consequence, enrich the supernatant with minicells.

[0124] Another purification method uses density gradient centrifugation in a biologically compatible medium. After centrifugation, the minicell band collects from the gradient, and, optionally, the minicells are subjected to subsequent rounds of density gradient centrifugation to maximize frequency. The method may further comprise the initial step of conducting differential centrifugation on the sample containing minicells. When running at low centrifugal force, differential centrifugation will remove some of the parent bacterial cells, thereby enriching the supernatant with minicells.

[0125] Particularly effective purification methods use bacterial filaments to increase the purity of minicells. Hence, the minicell purification method may comprise the steps of (a) subjecting a minicell-containing sample to a condition that induces the bacterial cell to assume a filamentous form, followed by (b) filtering the sample, to obtain a purified minicell preparation.

[0126] Known methods for purifying minicells may also be combined. One highly effective combination of methods is as follows:

[0127] Step A: Differential centrifugation of minicells producing bacterial cells. This step, which can be carried out at 2000 g for about 20 minutes, removes most parent bacterial cells while leaving the minicells in the supernatant.

[0128] Step B: Density gradient centrifugation using an isotonic and non-toxic medium for making a density gradient. This stage separates minicells from many contaminants, including stem bacterial cells, with minimal loss of minicells. Preferably, this step is repeated as part of the purification method.

[0129] Step C: Cross-flow filtration through a 0.45 gm filter to further reduce contamination with parent bacterial cells.

[0130] Step D: Stress-induced fibril formation in remaining bacterial stem cells. This can be done by subjecting the minicell suspension to any of several stressful environmental conditions.

[0131] Step E: Antibiotic treatment to kill parent bacterial cells.

[0132] Step F: Cross-flow filtration to remove fine impurities such as membrane vesicles, membrane fragments, bacterial residues, nucleic acids, nutrient components etc., and to concentrate minicells. A 0.2 gm filter can be used to separate minicells from fine contaminants, and a 0.1 gm filter can be used to concentrate minicells.

[0133] Step G: Dead-end filtration to remove filamentous dead bacterial cells. A 0.45 m filter can be used at this stage.

[0134] Step H: Removal of endotoxins from the minicell preparation. Anti-lipid A coated magnetic beads can be used at this stage.

Administration regimens [0135] In general, the formulations disclosed herein can be used at appropriate dosages, as defined in routine tests, to obtain an optimal physiological effect, while minimizing any potential toxicity. The dosage regimen can be selected in accordance with a number of factors, including age, weight, sex, and patient's condition; severity of the condition for treatment, route of administration, and renal and hepatic function of the patient.

[0136] Optimal precision in achieving minicell and drug concentrations in a range that gives maximum efficacy with minimal adverse effects may require a regimen based on functional nucleic acid kinetics and drug availability for target sites and target cells. The distribution, balance and removal of minicells or the drug may be considered when determining the optimal concentration for the treatment regimen. The dosage of minicells and drugs can be adjusted, when used in combination, to achieve the desired effect.

[0137] In addition, the dosing of the formulation can be optimized using a pharmacokinetic / pharmacodynamic modeling system. For example, one or more dosage regimens can be selected, and a pharmacokinetic / pharmacodynamic model can be used to determine the pharmacokinetic / pharmacodynamic profile of one or more dosage regimens. One dosage regimen can then be selected for administration for which the desired pharmacokinetic / pharmacodynamic response is achieved based on the particular pharmacokinetic / pharmacodynamic profile. See. e.g. WO 00/67776. In this regard, the dosage regimen for any indication can be determined using the approach and model described in Example 6, with modifications for a particular target cell.

[0138] Specifically, the formulations can be administered at least once a week over several weeks. In one embodiment, the formulations are administered at least once a week for several weeks to several months.

[0139] More specifically, the formulations can 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 can be administered about once a day, about once in 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.

[0140] The formulations may alternatively be administered about once a 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.

[0141] Alternatively, the formulations may be administered about once a month, about once every 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 months or more.

[0142] The formulations can be administered in a single daily dose, or the total daily dosage can be administered in divided doses of two, three or four times a day.

[0143] In a method in which minicells are administered prior to drug delivery of the drug may occur at any time, from several minutes to several hours after administration of the minicells. Alternatively, the drug can be administered at any time, from several hours to several days, potentially several weeks up to several months after the minicells.

[0144] More specifically, minicells packed with functional nucleic acid can 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. In addition, 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 administration. 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 another embodiment, the minicells may be administered at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 months before drug.

[0145] In another embodiment, the minicell is administered after the drug. Minicells may be administered at any time, from a few minutes to several hours after administration. Alternatively, the minicell can be given at any time, from several hours to several days, potentially several weeks up to several months after the drug.

[0146] The following examples are illustrative only, and are not limiting, and provide a more complete understanding of the invention.

Examples

1. Direct Packaging of Regulatory RNA into Intact Minicells In Vitro [0147] Intact bacterial-derived minicells were obtained and purified as described in US Patent Application Publication No. US 2004/0265994. Cy3-labeled glyceraldehyde 3-phosphate dehydrogenase (GAPDH) siRNA labeled (excitation maximum (Xmx) 547 nm, emission maximum (Xmx) 563 nm), product Ambion (Austin, Texas USA) and reconstituted in nuclease-free water to a final concentration of 50 μΜ .

[0148] Approximately 10⁷ the minicells were resuspended in 1x phosphate buffer solution (PBS) (Gibco) and co-incubated with 1 μ Cy Cy3-labeled GAPDH siRNA.

Incubation was carried out for 2 hours at 37 ° C, with gentle mixing. Control minicells were apparently loaded by incubation with only 1 x PBS. After loading, the minicells were pelleted and washed twice with 1 x PBS by centrifugation for 10 minutes at 16,200 x g. Experimental and control minicells were observed under a DMLB fluorescence microscope, product Leica (Germany) with D70 attached, product Olympus Microscopes (Germany). Images were obtained using a 100 x lens with oil immersion.

[0149] The above co-incubation experiments were also carried out under other experimental conditions, such as incubation at room temperature, 37 ° C, and 4 ° C. In addition, the co-incubation times varied, including 1 hour, 2 hours, 4 hours and 12 hours, respectively.

[0150] As shown in Figure 1B, intact siRNA molecules quickly diffused into minicells. A 2 hour incubation at 37 ° C was sufficient to achieve highly significant minicell packaging.

[0151] To determine whether siRNA molecules were inside the minicells or adhered to the surface of the minicell, minicells with Cy3 fluorescently labeled siRNA were incubated with exonucleases overnight, followed by fluorescence microscopy. The results were identical to those shown in Figure 1B, indicating that siRNAs were internalized by the minicells and did not adhere to the surface of the minicells.

2. In vitro transfection of human breast cancer cells with microcells packed with regulatory RNA, with bispecific antibody targeting [0152] To demonstrate that minicells carrying regulatory RNA are stable in serum in vitro and that they can be internalized by specifically-targeted mammalian cells, the following were performed experience.

[0153] Polynuclease 1 (Plk1) targeted siRNA synthesized with the target sequence of 5'-GGTGGATGTGTGGTCCATTTT-3 'were synthesized and labeled with a fluorescent tag, AlexaFluor 488. Polo-kinases have different functions during mitosis, centrosunomic maturation, bilateral formation , chromosome segregation and cytokinesis, and, which is crucial, monitoring checkpoint reproduction fidelity (Glover et al., 1998; Barr et al., 2004; van de Weerdt and Medema 2006). In humans, Plk1 is the best-characterized member of this family. Plk1 is associated with cancer and belongs to the serine / threonine kinase family, which are attractive targets for new chemotherapeutics. Accordingly, Plk1 is considered a promising target in the development of anti-cancer drugs (Strebhardt and Ullrich, 2006).

[0154] Minicells were purified, and 10⁹ minicells packed with anti^AF488Plk1 siRNA as described in Example 1. A bispecific antibody (BsAb) bearing specificity for the S. typhimurium anti-antigen and anti-human EGFR was obtained, and AF488-Plk1-siRNA minicells were attached as described in published PCT application WO 05/056749. The resulting minicells are named^EGFRminikomórkiAF488-PLK1-siRNA. These minicells (10⁹) were incubated with human breast cancer cells in tissue culture at a density of 10,000 minicells: 1 tumor cell. Incubation was carried out for 1 hour, 2 hours, 4 hours and 24 hours. At each time point, cells were harvested and stained with DAPI (nuclear staining, blue fluorescence). Cells were observed using an iX81 confocal microscope (Olympus) and CellR software.

[0155] After 1 hour, fluorescent siRNA-bearing minicells adhered to MDA-MB-468 cells (see Figure 2). This attachment was thought to be due to binding to the attached minicells - BsAb, which were directed to the EGF receptor on MDA-MB-468 cells, as in incubation with non-target minicells AF488-Plk1-siRNA, washing was not observed and no green fluorescence associated with MDA-MB-468 cells. After 2 hours from the end of incubation, ^EGFRAF488-Plk1-siRNA minicells were internalized within mDA-MB-468 cells and showed intense green fluorescence. After 24 hours, most of the green fluorescence has disappeared, indicating that internalized minicells have degraded, presumably in phagolysosomes.

3. Extraction and quantification of siRNA from intact minicells [0156] As siRNAs do not naturally occur in bacterial cells or minicells of bacterial origin, it is not surprising that there are no well-established methodologies for siRNA extraction from such particles. Accordingly, applicants have developed a method for the quantitative extraction of siRNAs that are packaged in intact minicells in accordance with the invention.

[0157] Kinesin spindle protein (KSP), also known as "kinesin-5" and "Eg5", is a microtubule motor protein. It is crucial for the formation of bipolar spindles and for the correct segregation of sister chromatids during mitosis (Enos and Morris, 1990; Blangy et al., 1995; Sawin and Mitchison, 1995; Dagenbach and Endow, 2004). Inhibition of KSP causes the formation of unipolar mitotic spindles, activates the spindle assembly checkpoint, and stops cells in the mitosis phase, which leads to cell death (Blangy et al., 1995, Caner et al., 1999; Kapoor et al., 2000; Tao et al., 2005).

[0158] Anti-KSP siRNA was selected for packaging in minicells to direct optimization of siRNA extraction from minicells in accordance with the invention. More specifically, the double-stranded KSP-1-siRNA oligonucleotide sequences (sense strand; 5'-AAC TGG ATC GTA AGA AGG CAG-3 ') were synthesized and packaged into minicells according to the procedures detailed in Example 1, supra.

[0159] Minicell RNA-KSP (10¹⁰) and a comparable number of empty control cells were processed using a number of commercially available nucleic acid extraction kits. The results showed that the mirVana miRNA isolation kit (Ambion) provided the quantitative extraction of siRNA-KSP from intact minicells. The procedure was carried out according to the manufacturer's instructions.

[0160] Purified siRNAs were first stained with an ultra-sensitive fluorescent dye for nucleic acids, RiboGreen^™, Molecular Probes Inc. product (Eugene, Oregon USA), followed by quantitative analysis using a NanoDrop ND-3300 fluorospectrometer, a product of NanoDrop Technologies Inc. (Wilmington, Delaware USA), also according to the manufacturer's instructions. Associated with RNA RiboGreen^™ has a maximum excitation of ~ 500 nm and a maximum emission of ~ 525 nm.

[0161] The results showed that minicells were capable of carrying siRNA. 10¹⁰ empty minicells carried ~ 1.4 μg RNA, presumably the background level of endogenously formed bacterial RNA. The same number of minicell axiRNA-KSP carried ~ 2.7 μg RNA, including endogenous bacterial RNA plus exogenously packaged siRNA-KSP. Thus, these data demonstrate that¹⁰ minicells may pack at least ~ 1.3 μg of exogenously packed siRNA.

4. In vivo demonstration of the antitumor effect achieved by minicells packed with regulatory RNA [0162] The following studies were performed to demonstrate that cells with packed regulatory RNAs can deliver intact regulatory RNA at therapeutically effective concentrations to tumor cells in vivo.

[0163] SiRNA selection against KSP was performed as described in Example 3 for packaging in minicells according to the invention. Minicells were purified, and 10⁹ minicells were packaged with anti-KSP siRNA as described in Example 1. BsAb was also prepared and attached to minicelliRNA-KSP as described in Example 2 to generate ^EGFRminikomórkisiRNA-KSP.

[0164] The mice used in this example were purchased from the Animal Resources Center (Perth, WA, Australia), and all animal experiments were conducted in accordance with the guidelines for the care and use of laboratory animals, after approval by the Animal Ethics Committee. The experiments were carried out in a small animal facility with NSW Agriculture accreditation at EnGeneIC Pty Ltd (Sydney, New South Wales, Australia).

[0165] Human breast cancer cells (MDA-MB-468, ATCC) were cultured in tissue culture in RPMI 1640 medium supplemented with GIBCO-BRL 5% bovine calf serum, product from Invitrogen Corporation (Carlsbad, California USA) and glutamine (Invitrogen) in humidified atmosphere, with 95% air and 5% CO2 at 37 ° C. 1 x 10⁶ cells in 50 μl serum-free medium were mixed with 50 μl growth factor with reduced matrix gel, product BD Biosciences (Franklin Lakes, New Jersey USA). Using a 23G needle, cells were injected subcutaneously between the shoulder blades of each mouse. Tumors were measured twice a week using an electric digital caliper (accuracy of 0.001), product Mitutoyo (Japan), and the mean tumor volume was calculated using the formula: length (mm) x width² (mm) X 0.5 = volume (mm³).

[0166] Various therapies were initiated after tumors reached volumes between 170 mm³ and 200 mm³, and mice were randomized into two different groups of eight per group. Control group 1 received sterile saline, while test group 2 received^EGFRminikomórkisiRNA-KSP. (10⁹), four times a week.

[0167] As shown in Figure 3, ^EGFRminicells RNA-KSP provided a highly significant anti-tumor activity compared to the salt solution control. The results demonstrate that (a) siRNAs were stable within minicells in vivo, (b) intact and fully functional siRNAs were delivered to tumor cells in vivo, and (c) the minicell delivered therapeutically significant concentrations of siRNA into tumor cells in vivo.

5. Demonstration of adaptive cancer therapy by treatment with minicells packed with regulatory RNA followed by minicells with packaged drug [0168] Most cancer therapies are associated with drug resistance. The same applies to regulatory RNA treatment as genetic mutations in tumor cells may render regulatory RNA ineffective if the target gene mutates within the regulatory target RNA sequence.

[0169] There was no effective strategy for drug resistance in cancer patients. Instead, it is necessary to administer new drugs to bypass the mutation. This approach faces serious difficulties, however, since most anti-cancer drugs are highly toxic, and combination therapies increase this toxicity, resulting in reduced dose and frequent discontinuation of therapy when the patient stops dealing with toxicity. The following study was performed to assess the effectiveness of minicells packed with regulatory RNA in the treatment of such resistance.

[0170] As described above, minicells were purified and packaged (10⁹) anti-KSP or anti-Plk1 siRNA. Also, as previously described, a bispecific antibody that produced the specificity of the S. typhimurium anti-antigen and anti-human EGFR was prepared and attached to minicell XRNA-KSP to generate^EGFRminikomóreksiRNA-KSP.

[0171] Human colon cancer xenografts (HCT116; ATCC) were established in nude mice as described in Example 3, and treated iv as follows: Group 1 mice received sterile saline, and mice from groups 2, 3 and 4 were treated, for the first 10 doses (see Figure 4) of 10, respectively^{9 EGFR}minikomóreksiRNA-Plkl, ^EGFRminicellesXiRNA-KSP-1 and ^EGFRminikomóreksiRNA-KSP-2. The Plk1 and KSP-1 sequences were as shown in the examples above. siRNA-KSP-2 (sense strand; 5 'CTGAAGACC TGAAGACAAT 3') is directed at another section of KSP mRNA. After day 33, mice in groups 2, 3 and 4 were treated with two doses^EGFRminikomórekkarboplatyna.

[0172] The results showed that (Figure 4) after day 26, tumors became resistant to siRNA treatment. Accordingly, mice in groups 2, 3 and 4 were treated for four successive doses with all three doses^EGFRminicellesxiRNA-Plk1, + ^EGFRminicells RNAksp-1, + ^EGFR minicell RNA-KSP-2) combined in equal amounts, i.e. ~ 3 x 10⁸ any type of minicells. In addition, by day 33 tumors were highly resistant to all siRNAs (Figure 4). After administration^EGFRminicell carboplatin, tumor growth in groups of 3 and 4 mice slowed significantly. After administration^EGFRminicell carboplatin, a significant reduction in tumor volume was found in a group of 3 mice.

[0173] These data show that drug-resistant tumor cells can be treated effectively in vivo according to the invention. In particular, (1) sequential administration of targeted minicells carrying regulatory RNA sequences designed to significantly reduce tumor burden when the tumor cells become resistant to siRNA-regulated antitumor activity is performed using (2) targeted minicells carrying a drug that it does not act on the same protein targeted by regulatory RNA.

6. Demonstration of knockdown of the target protein in tumor cells and the resulting arrest of cell union after targeted delivery of a therapeutically effective amount of regulatory RNA packaged in intact minicells [0174] To demonstrate that innovative methods pack therapeutically effective amounts of regulatory RNA in intact minicells, it was necessary demonstration that directed by a bispecific antibody, minicells with regulatory RNA can effectively and efficiently trigger tumor cell growth arrest and induce apoptotic cell death.

[0175] In humidified atmosphere, in 95% air and 5% CO2 at 37 ° C, human epithelial colon cancer cells (HCT116) were cultured in tissue culture in RPMI 1640 medium, with the addition of 5% bovine calf serum and glutamine. As described above, minicells were purified, siRNA directed against Plk1 or KSP was packaged, and attached to BsAb, which carried specificity for the S. typhimurium anti-antigen and anti-human EGFR. Hence, minicellsXiRNA-KSP, minicellsXiRNA-Plk1 and minicells (control) were generated ^EGFRminikomórkisiRNA-KSP ^EGFRminicell RNA-Plk1 and ^EGFRminicells. HCT116 cells were inoculated into six-well plates, and experimental and control groups were transfected at a ratio of 5,000 minicells: 1 HCT116 cell. In addition, control from cells alone was included.

[0176] After 2 hours of incubation with minicells, the wells were washed three times with fresh PBS. The wells were emptied after 4 hours, 8 hours, 16 hours, 24 hours, 32 hours and 48 hours after transfection, and the cells were fixed in cold 70% ethanol and incubated at 4 ° C for 30 minutes. Cells were washed twice in lemon phosphate buffer (pH 7.8) and treated with 100 mg / ml RNAse to ensure that only DNA was stained. Cells were stained with propidium iodide (nucleic acid staining) and then analyzed using a FACSCalibur ™ flow cytometer, Becton Dickinson product (Franklin Lakes, New Jersey USA), at Macquarie University (Sydney, Australia), and CELL Quest acquisition-andanalysis software, also Becton Dickinson product.

[0177] FACS analysis of the cells showed that, at 4 and 8 hours after transfection (Figure 5A), treated cells ^EGFRminicell cell RIS-KSP or ^EGFRminicell cells RNA-Plk1 were characterized by strong cell cycle arrest in the G2 phase. Control cells, cells alone or treated^EGFRminicells, did not show any side effects. These cells presented normal G1, S and G2 phases of the cell cycle. After 16 and 24 hours, the experimental cells displayed not only strong G2 phase arrest, but also a large number of apoptotic cells (Figure 6). After 32 and 48 hours, most of the cells in the experimental groups were apoptotic and were converted to cellular residues (see in particular the brackets in Figure 7).

[0178] These results demonstrate that the targeted intact minicells had a therapeutically effective amount of regulatory RNA packed, and that the minicells of the invention were highly effective and significantly efficient in targeted knockdown of proteins within cancer cells, resulting in apoptotic cell death .

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