Bacterially derived, intact minicells encompassing regulatory RNA

[0001] Numerous strategies have been recently developed, based on a nucleic acid to modulate various cellular functions (Opalinska and Gewirtz, 2002). Oligonucleotide classes such as aptamers, transcription factor binding oligonucleotide decoys, ribozymes, triplex forming oligonucleotides, CpG immunostimulatory motifs, antisense oligonucleotides (including peptide nucleic acids), small interfering RNAs and microRNAs have attracted much attention as research tools due to their highly specific mode actions. These oligomeric nucleic acids also have significant potential as therapeutics. However, such therapeutics encounter several obstacles, including the instability of free nucleic acids and the safe, efficient 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 the cell leads to sequence-specific degradation of homologous (complementary or partially complementary) gene transcripts. More specifically, long dsRNA molecules are processed into smaller RNAs by endogenous ribonuclease called "Dicer" (Grishok et al., 2000, Zamore et al., 2000). Smaller RNAs are known as "short interfering RNAs" (siRNAs) if they are derived from exogenous sources and "microRNAs" (miRNAs) if they are generated from genes encoding RNA in their own cell genome. These two small classes (typically 21 to 23 nucleotides) of regulatory RNAs also differ in that the miRNAs only show partial complementarity to the purposes of template RNA (mRNA).

[0003] Short regulatory RNAs bind to the so-called "induced RNA silencing complex" (RISC) that has helicase activity 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 hydrolyzes 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 the target RNA molecule according to Watson-Crick base-pairing principles and recruits a ribonuclease that breaks down the target RNA. In contrast, the antisense suppression of gene expression involves the binding of ssRNA to mRNA, blocking translation without catalyzing the degradation of mRNA.

[0005] As a class, regulatory RNAs have a half-life of less than one hour in human plasma (Layzer et al., 2004) and are rapidly secreted by the kidneys. Therefore, several groups have attempted to obtain regulatory RNAs, including siRNAs that are resistant to nuclease. Examples of such aspirations include nucleotide chemical modifications (e.g., 2'-F, 2'-OMe, Locked Nucleic Acids) or phosphodiester backbones, e.g. phosphorothioate linkages (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, in order to minimize the time that siRNAs or other regulatory RNAs remain in the circulation, practitioners have conjugated RNA molecules to proteins and antibodies to target the desired mammalian cells. As a result of further efforts, aimed at solving problems with low stability and rapid excretion by the kidneys, practitioners have developed carriers for the delivery of regulatory RNAs. Polyplexes (formed by autoclaving nucleic acids with polycations), lipopoliplexes (formed by pre-condensation of the nucleic acid with polycations and then by addition of cationic lipids), liposomes and synthetic nanoparticles are also tested.

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

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

[0008] Plasmid-based systems for the recombinant expression of regulatory RNA in situ, such as siRNA or a larger (~ 70 nt) precursor, RNA in the form of a short hairpin (shRNA) have also been developed. shRNA includes sense and antisense sequences from the target gene that are connected through the 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 as a result of a mutation that causes asymmetric cell division. This transformation yields recombinant bacteria in which the plasmid replicates intracellularly, introducing a large number of plasmids into the bacterial cytoplasm. During an asymmetric division, some of the plasmids are segregated into the cytoplasm of the minicell, resulting in recombinant minicells. Minicells can then supply 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,

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

WO / 2006/021894 discloses the use of bacterial minicell vectors to deliver functional nucleic acids to mammalian cells.

WO 2004/022771 discloses compositions and methods for inhibiting gene function using a short interfering nucleic acid or hybrid of nucleic acid analogs in an organism or cell.

Geller, BL et al., Journal of Antimicrobial Chemotherapy, Vol. 55, 4 May 2005, pages 983998, discloses morpholino phosphorodiamide oligomers (PMOs) that inhibit the expression of the Escherichia coli luciferase gene in a sequence dependent manner.

Harth G. et al., Proceedings of the National Academy of Sciences of the United States of America, 4 January 2000, vol 97, no. 1, 4 January 2000, pages 418-423, discloses the effect on M. tuberculosis of phosphorothioate modified antisense oligodeoxyribonucleotides ( PSODNs) against the glutamine synthetase mRNA, and to produce a poly-L-glutamate / glutamine cell wall structure.

Yanagihara Katsunori et al., Journal of Antimcriobial Chemotherapy January 2006, Vol. 57, No. 1, January 2006, pp. 122-125, discloses the efficacy of short interfering RNAs (siRNAs) on coagulase expression.

Summary of the invention [0011] Thus, one aspect of the invention relates to a composition comprising:

(a) a collection of intact minicells of bacterial origin, wherein each minicell of said set comprises a regulatory RNA that is packaged in said minicell, and (b) a pharmaceutically acceptable carrier therefor, wherein (i) the regulatory RNA is selected from the group consisting of from an antisense ssRNA, a ribozyme and an RNA trap, (ii) minicells of a construct for the expression of regulatory RNA in situ are absent, and (iii) the set contains a therapeutically effective amount of regulatory RNA.

[0012] The minicell packed functional nucleic acid can target RNA transcripts encoding a protein that contribute inter alia to drug resistance, resistance to apoptosis or neoplasia. The composition of the invention may also further comprise a bispecific ligand, for example consisting of a first arm specific for the surface structure of the minicell and a second arm specific for the surface receptor of a non-phagocytic mammalian cell.

[0013] In another aspect, the invention relates to a method of providing a regulatory RNA, comprising the steps of:

(a) providing a collection of intact minicells of bacterial origin in a pharmaceutically acceptable carrier, each minicell of the set comprising regulatory RNA; and (b) delivering minicells from the collection for contact with mammalian cells in vitro such that mammalian cells absorb minicells from the set, with the regulatory RNA st released into the cytoplasm of the target cells, wherein (i) the regulatory RNA is selected from the group consisting of antisense ssRNA, ribozyme and RNA traps, (ii) minicells of the construct for the expression of regulatory RNA in situ are absent, and (iii) the set contains a therapeutically effective amount of the regulatory RNA.

As mentioned, regulatory RNA, such as siRNA, miRNA and shRNA, can target RNA transcripts encoding a protein that contributes to drug resistance, resistance to apoptosis or neoplasia. In other embodiments, the methodology further comprises providing a drug, different from the functional nucleic acid, to the target mammalian cell. The drug may be administered after or simultaneously or even before administration of the minicell composition.

According to another aspect, the present invention provides a composition for use in the treatment of cancer, wherein the composition comprises a collection of intact bacterial minicells in a pharmaceutically acceptable carrier, each minicell of the set comprising a regulatory RNA, wherein:

(a) the regulatory RNA is selected from the group consisting of an antisense ssRNA, a ribozyme and an RNA trap;

(b) minicells of the construct for the expression of regulatory RNA in situ are absent, and (c) the collection of minicells contains a therapeutically effective amount of regulatory RNA.

The invention also enables a method of formulating a minicell with functional plasmid-free nucleic acid. The method comprises co-incubating a set of minicells with a functional nucleic acid, such as a regulatory RNA, such as siRNA, miRNA or shRNA, in a buffer. In some embodiments, the co-incubation may include gentle shaking, while in others the co-incubation is static. In some aspects, co-incubation takes 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 phosphate buffer solution IX. 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.

[0015] 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 illustration only, as 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 set forth the principle of the invention and can not be expected to 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 [0016] Figure 1 shows intact minicells packed with siRNA labeled with Cy3 fluorophore. Figure 1A is from a light microscope, while Figure 1B shows the same slide, but displayed under fluorescent light with excitation filter 515-560, revealing highly fluorescent siRNA molecules co-occurring with minicells.

Figure 2 is an image made with confocal fluorescence microscopy and depicts the adhesion and internalization of EGFR-targeted minicells packed with siRNA-Plk1 into human breast cancer cells in-vitro.

Figure 3 graphically illustrates the significant antitumor effect achieved by treating human breast tumor xenografts (MDA-MB-468) of nude mice with EGFR-targeted minicells packed with KSP-siRNA. Control group 1 (- * -) received a sterile saline solution, while experimental group 2 (- -) received^EGFRKSP-minikomórkisiRNA / 10⁹) four times a week.

Figure 4 graphically illustrates the significant antitumor effect achieved by the treatment of human colon carcinoma heterografts (HCT116) of nude mice with EGFR-targeted minicells packed with KSP-siRNA in combination with EGFR-targeted minicells, loaded with carboplatin. Group 1 (- ♦ -) mice received a sterile saline solution, and mice of group 2 (--Δ--), 3 (-) and 4 () were treated for the first 10 doses (see Figure 4) respectively.⁹ EGFRminikomóreksiRNA-PLK1, ^EGFRminikomóreksiRNA-KSP-1 and ^EGFRminikomóreksiRNA-KSP-2.

Figure 5 provides FACS analyzes at various times after transfection of colon carcinoma cells (HCT116) treated with experimental minicells, ^EGFRminikomórkamisiRNA-KSP or ^EGFRminikomórkamisiRNA-Plk1. Figures 5A-5D provide FACS analyzes of samples taken 4 hours after transfection, while Figures 5E-5H show analysis from samples 8 hours after transfection. Figures 5A and 5E show only results from cells, while Figures 5B and 5F relate to empty cells^EGFRminicells. Figures 5C and 5G show results from + cells^EGFR, minikomóreksiRNAKSP and Figures 5D and 5H refer to + cells ^EGFRminikomóreksiRNA-Plk1.

Figure 6 provides FACS analyzes from different times after transfection of colon carcinoma cells (HCT116) treated with experimental minicells, ^EGFRminikomórkamisiRNA-KSP or ^EGFRminikomórkamisiRNA-Plk1. Figures 6A-6D provide FACS analyzes of samples taken 16 hours after transfection, while Figures 6E-6H show analysis from samples 24 hours after transfection. Figures 6A and 6E show only results from cells, while Figures 6B and 6F relate to blank cells^EGFRminicells. Figures 6C and 6G show results from + cells^EGFRminichomóreksiRNA KSP, and Figures 6D and 6H relate to + cells ^EGFRminikomóreksiRNA-Plk1.

Figure 7 provides FACS analyzes at various times after transfection of colon carcinoma cells (HCT116) treated with experimental minicells, ^EGFRminikomórkamisiRNA-KSP or ^EGFRminikomórkamisiRNA-Plk1. Figures 7A-7D provide FACS analyzes of samples taken 32 hours after transfection, while Figures 7E-7H show analysis from samples 48 hours after transfection. Figures 7A and 7E show only results from cells, while Figures 7B and 7F relate to blanks^EGFRminicells. Figures 7C and 7G show results from + cells^EGFRminikomóreksiRNA-KSP, and figures 7D and 7H refer to cells + siRNA-Plk1 ^EGFRminicells siRNA-Plk1.

Detailed Description of the Invention [0017] According to the present invention, therapeutically effective amounts of regulatory RNA can be packaged into minicells without the use of harsh chemicals or electroporation. In this respect, a simple methodology has been developed for directly packaging such therapeutically effective regulatory RNA concentrations into intact minicells that does not include plasmid-based expression constructs or a host cell bacterial expression mechanism. Accordingly, the polynucleotide segment that encodes the regulatory RNA is not cloned into the plasmid DNA or the viral vector. Instead, functional plasmid-free nucleic acids are packaged directly into minicells through the minicell membrane through the intact membrane.

Definitions [0018] Unless defined otherwise, all technical and scientific terms used in the description have the same meaning as commonly understood by those skilled in the art.

[0019] For convenience, the meanings of specific terms and phrases used in the description, examples and appended claims are set out below. Other terms and phrases are defined throughout the description. [0020] The singular forms "a," "an" and "the" include plural aspects unless the context clearly dictates otherwise.

[0021] An "antisense oligonucleotide" is a nucleic acid molecule complementary to a portion of a particular gene transcript that can hybridize to a transcript and block its translation. The antisense oligonucleotide may contain RNA or DNA.

[0022] A "biomolecular sequence" or "sequence" means all or part of a polynucleotide or polypeptide sequence.

[0023] "Cancer", "cancer", "tumor" and "malignant tumor" as used interchangeably herein, mean 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 particularly applicable to malignant, pre-metastatic, metastatic and non-metastatic cells.

[0024] "Complementary" means the topological compatibility or matching of the interacting surface of two molecules, such as the siRNA molecule and its target mRNA. The molecules can be described as complementary, and the characteristics of the contact surfaces are complementary to each other.

[0025] "Respond" or "represent" when used in the context of a polynucleotide or a sequence that "responds" or "represents" a gene, for example, means that the polynucleotide sequence is present in the gene or gene product of the nucleic acid, e.g. mRNA . The polynucleotide may be present entirely within the exon of the genomic gene sequence or different portions of the polynucleotide sequence may be present in different exons, e.g. so that the adjacent polynucleotide sequence is present in the mRNA, either before or after splicing, which is the gene expression product.

[0026] An "RNA trap" is a molecule that can assume an identical structure to an important functional RNA region to be targeted. This latter RNA may be native to a mammalian host or pathogen that has infected a mammalian cell, e.g. HIV. The RNA trap restricts access to a protein that normally interacts with the target RNA, resulting in a disruption of normal processing in a mammalian or pathogenic host.

[0027] "Drug" means any physiologically or pharmacologically active substance that causes the desired local or systemic effects in animals, particularly mammals and humans.

[0028] "Expression" generally means a process by which a polynucleotide sequence is successfully transcribed and translated such that detectable levels of the amino acid sequence or protein are expressed. In the present description, in some contexts, expression refers to the generation of mRNA. In other contexts, expression refers to protein production.

[0029] A "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 codes for the protein. Regulatory RNAs such as siRNA, shRNA, short RNA (typically less than 400 bases long), micro-RNA (miRNA), ribozymes and RNA trap, and antisense nucleic acids are exemplary functional nucleic acids.

[0030] A "gene" refers to a polynucleotide sequence that contains the control and coding sequences necessary to produce a polypeptide or precursor. The polypeptide may be encoded by a full-length coding sequence or any part of the coding sequence. The gene may be an uninterrupted coding sequence or may comprise one or more introns connected through appropriate splice nodes. In addition, the gene may contain one or more modifications in coding or non-translated regions that can affect the biological activity or chemical structure of the expression product, the rate of expression or the way the expression is controlled. Such modifications include, but are not limited to, mutations, insertions, deletions, and substitutions of one or more nucleotides. Therefore,

[0031] A "host cell" means a cell that can be or has been used as a recipient of a recombinant plasmid or other polynucleotide transfer and contains progeny of a basal cell that has been transfected. The progeny of a single cell does not necessarily have to be completely identical in terms of morphology or genomic or total DNA to the parent cell, due to a natural, accidental or intentional mutation.

[0032] "Hybridization" means any process by which a polynucleotide sequence binds to a complementary sequence by combining bases into pair.

[0033] "Subject", "subject", "host" and "patient", used interchangeably in this specification mean any mammalian subject for whom diagnosis, treatment or therapy is desired. In one preferred embodiment the subject, subject, host or patient is a human. Other subjects may include, but are not limited to, cattle, horses, dogs, cats, guinea pigs, rabbits, rats, primates and mice.

[0034] "Tag" means agents that are capable of delivering a detectable signal, either directly or by interacting with one or more additional members of the signal production system. Tags 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 the like as markers. Colorimetric markers such as colloidal gold or spheres 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.

[0035] "Oligonucleotide" means a polynucleotide comprising, for example, about 10 nucleotides (nt) to about 1000 nt. Oligonucleotides for use in the invention preferably have a length of about 10 nt to about 150 nt. The oligonucleotide may be a naturally occurring oligonucleotide or a synthetic oligonucleotide. Oligonucleotides can be modified.

[0036] A "minicell" means non-nuclear forms of bacterial cells arising from a disorder in coordination during fission of binary division of a cell with DNA segregation. Minicells are different from other small vesicles, which are produced and released spontaneously in specific situations and are not subject to specific genetic reorganisations 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 leakage of packed functional nucleic acid and would not be therapeutically effective. The intact cell membrane allows the charge to be retained within the minicell and to be released intracellularly within the target mammalian host cell.

[0037] As used herein, "modified" and "chemically modified" means oligonucleotides or polynucleotides with one or more chemical changes relative to the natural molecular structure of all or any of the bases, sugar residues and internucleoside phosphate linkages as well as molecules having substitutions added or combinations of modifications in these places. Internucleoside phosphate bonds can be phosphodiester, phosphotriester, phosphoramidate, siloxane, carbonate, carboxymethylester, acetamide, carbamate, thioether, bridged phosphoramidate, bridged methylene phosphonate, phosphorothioate, methylphosphonate, phosphorodithioate, bridged phosphorothioate or sulfon internucleotide linkages or 3'-3 'bonds, 5'-3 'or 5'-5' and combinations of such similar bonds. The phosphodiester bond may be replaced by a replacement bond such as phosphorothioate, methylamine, methylphosphonate, phosphoramidate and guanidino, and the ribose subunit of polynucleotides may also be substituted (e.g., phosphodiester hexoses, peptide nucleic acids). Modifications may be internal (single or repeated) or end (s) of oligonucleotide molecules and may include additions to molecules of internucleoside phosphate linkages, such as deoxyribose and phosphate modifications that cleave or cross-link 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 '

[0038] The expression "nucleic acid molecule" and the term "polynucleotide" means polymeric forms of nucleotides of any length, either ribonucleotides or deoxynucleotides. These include one-, two- 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 derived nucleotide bases. The polynucleotide backbone may contain sugars and phosphate groups (as typically found in RNA or DNA) or modified or substituted sugar or phosphate groups. Alternatively, the polynucleotide backbone may contain polymer sythetic subunits such as phosphoramidates and thus can be oligodeoxynucleoside phosphoramidate or a mixed oligomer of phosphoramidoposfestherster. The polynucleotide may comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs, uracyl, other sugars and linking groups such as fluororibose and thioesters and nucleotide branches. The polynucleotide may be further modified, such as by conjugation with the labeling component. Other types of modifications include caps, substitution of one or more of the naturally occurring nucleotides with an analog and introduction of means for attaching the polynucleotide to proteins, metal ions, labeling components, other polynucleotides, or a solid support.

[0039] "Pharmaceutically acceptable" means physiological compatibility. The pharmaceutically acceptable carrier or excipient does not abrogate the biological activity of the composition administered, is chemically inert and is not toxic to the organism to which it is administered.

[0040] A "plasmid-free" qualifier means the absence of a construct, such as a plasmid or viral vector, for the in situ expression of a functional nucleic acid.

[0041] The "polypeptide" and "protein" used herein interchangeably mean a polymeric form of amino acids of any length, which may include translated, untranslated, chemically modified, biochemically modified and derived amino acids. The polypeptide or protein may be naturally occurring, recombinant or synthetic, or any combination thereof. In addition, the polypeptide or protein may comprise a fragment of a naturally occurring protein or peptide. The polypeptide or protein may be a single molecule or it may be a complex multiparticulate. In addition, such polypeptides or proteins may have modified peptide backbones. The terms include fusion proteins, including fusion proteins with a heterologous amino acid sequence, fusions with a heterologous and homologous leader sequence,

[0042] "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. [0043] "Regulatory RNA" means a category including RNA that affects expression by 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, but is not limited to, ribozymes and RNA traps.

[0044] "Ribozyme" means an RNA molecule having enzymatic activity that repeatedly cleaves other RNA molecules in a manner specific to the nucleotide base sequence:

[0045] "RNA interference" (RNAi) means an RNA-guided mechanism as described above, including the degradation of a complementary or partially complementary target RNA, for the regulation of gene expression (protein synthesis), sequence specific or gene specific.

[0046] "Sequence identity" means the degree of similarity or complementarity. There may be partial identity or complete identity. Partial sequence complementarity means one that prevents at least partial hybridization of the identical sequence to the target polynucleotide; this means the use of the functional term "substantially identical". Inhibition of the hybridization of a completely complementary sequence to a target sequence can be tested by using a hybridization assay (type hybridization analysis)

Southern or Northern, solution hybridization and the like) under low stringency conditions. In general, an 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 stringency conditions are those in which non-specific binding is allowed; non-stringent conditions require that the binding of two sequences to each other is a specific (i.e., selective) interaction. The absence of non-specific binding can be tested by using a second target sequence in which even a partial degree of complementarity (e.g., less than about 30% identity) is lacking; in the absence of non-specific binding, the probe will not hybridize to the second non-complementary target sequence.

[0047] Another way of examining sequence identity, in the context of two nucleic acid or polypeptide sequences, involves the reference of residues in two sequences that are the same when aligned for maximum compliance in a particular region. As used herein, "percentage of sequence identity" means a value determined by comparing two optimally matched sequences within a comparison window, wherein a portion 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 deletion) to optimally match the two sequences. The percentage is calculated by determining the number of positions in which the identical nucleic acid bases occur in both sequences in order to obtain the number of matching positions,

[0048] "Short interfering RNAs" (siRNA) means double-stranded RNA molecules, typically in the length of about 10 to about 30 nucleotides that are capable of mediating RNA interference (RNAi). Typically, siRNA molecules have the ability to reduce protein expression by directly interacting with the transcript that codes for that protein.

[0049] A "therapeutically effective" amount of a functional nucleic acid is a dose of a test molecule, e.g., siRNA, miRNA or free shRNA, which elicits a pharmacological response when administered to a subject of the present invention. Thus, in the context of the present invention, a therapeutically effective amount can be quantified by reference to the prevention or amelioration of an undesirable condition or symptom associated with a disease or disorder, or animal model or human subject if minicells with packed functional nucleic acid are administered, as described more detailed below. The amount that turns out to be the "therapeutically effective amount" in a given case for a particular subject does not have to be effective for 100% of subjects treated similarly because of the disease or condition being considered, even if such a dose is considered to be a "therapeutically effective amount" by those skilled in the art. The appropriate doses will also vary in this respect as a function, e.g. of the type, stage and severity of the disease or disease to be affected. In each case, the present in vitro study (Example 2) and in vivo studies (Examples 4, 5 and 6) in accordance with the present invention, as well as a methodology for quantifying a minicell derivative amount of a functional nucleic acid molecule (Example 3), when contemplated in the light of the entire description, empower the skilled person in the field of preclinical and clinical trials of drug candidates to determine, by routine experimentation, a therapeutically effective amount of a functional nucleic acid for a particular indication. [0050] The terms "therapy", "Treating", "treating" and the like means obtaining the desired pharmacological and / or physiological effect. The effect may be prophylactic for the total or partial prevention of a disease or symptom thereof and / or may be therapeutic for partially or completely stabilizing or treating a disease and / or an adverse effect attributed 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 that 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 symptom of disease, i.e. causing regression of the disease or symptom. "Treat" and the like mean obtaining the desired pharmacological and / or physiological effect. The effect may be prophylactic for the total or partial prevention of a disease or symptom thereof and / or may be therapeutic for partially or completely stabilizing or treating a disease and / or an adverse effect attributed 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 that 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 symptom of disease, i.e. causing regression of the disease or symptom. "Treat" and the like mean obtaining the desired pharmacological and / or physiological effect. The effect may be prophylactic for the total or partial prevention of a disease or symptom thereof and / or may be therapeutic for partially or completely stabilizing or treating a disease and / or an adverse effect attributed 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 that 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 symptom of disease, i.e. causing regression of the disease or symptom. The effect may be prophylactic for the total or partial prevention of a disease or symptom thereof and / or may be therapeutic for partially or completely stabilizing or treating a disease and / or an adverse effect attributed 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 that 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 symptom of disease, i.e. causing regression of the disease or symptom. The effect may be prophylactic for the total or partial prevention of a disease or symptom thereof and / or may be therapeutic for partially or completely stabilizing or treating a disease and / or an adverse effect attributed 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 that 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 symptom of disease, i.e. causing regression of the disease or symptom.

Minicells [0051] The minicells of the invention are non-nucleated forms of E. coli or other bacteria, caused by a disorder in coordination, during binary cleavage, of cell division with DNA segregation. Prokaryotic chromosomal replication is associated with normal binary fission, which involves creating a septum in the middle of the cell. For example, in E. coli, a mutation of min genes, such as minCDs, can remove inhibition of cell division formation at cell polices during cell division, resulting in the production of a normal progeny cell and a non-nuclear minicell. See Boer et al., 1992; Raskin and de Boer, 1999; Hu and Lutkenhaus, 1999; Harry, 2001. Minicells are different from other small vesicles that are produced and released spontaneously in specific situations, and which, unlike minicells, they are not subject to specific genetic reorganisations or expression of episomal genes. In a preferred embodiment, the minicells have intact cell walls ("intact minicells").

[0052] In addition to the operon mutation min, the non-nuclear minicells are also produced after many other genetic rearrangements or mutations that disrupt the formation of the septum, for example in divIVB1 in B. subtilis. See Reeve and Cornett, 1975. Minicells can also be created after interference at the gene expression levels of proteins involved in cell division / chromosomal segregation. For example, overexpression of minE leads to polar division and production of minicells. Similarly, chromosome-depleted minicells can result in chromosomal segregation defects, such as the smc mutation in Bacillus subtilis (Britton et al., 1998), the deletion of 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 trance. If it is overexpressed from a high-copy plasmid, for example, CafA can increase cell division rate and / or inhibit chromosome division after replication (Okada et al., 1994), resulting in the formation of cell-linked and non-nuclear minicells (Wachi et al., 1989). Minicells can be obtained from any bacterial cells of Gram-positive or Gram-negative origin.

[0053] In one aspect, the minicells may contain one or more plasmid-free functional nucleic acids whose delivery is desirable. The functional nucleic acid of the invention has the ability to reduce protein expression by directly interacting with the transcript that codes for the protein.

Packaging of functional nucleic acid into intact minicells [0054] A functional nucleic acid can be packaged directly into intact minicells. This method celebrates the previously required steps, for example, cloning of nucleic acids encoding functional nucleic acid into expression plasmids, transformation of minicell producing bacteria into the plasmids and production of recombinant minicells. Instead, a plasmid-functional functional nucleic acid can be directly packaged into intact minicells by co-incubating a set of 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 around one hour has proven to be sufficient, but shorter periods, such as about half an hour, can also be effective. In one embodiment, the buffer comprises a buffered saline solution, e.g. a solution of 1X buffered phosphate. The buffered saline solution 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; from about 20 ° C to about 30 ° C, about 25 ° C or about 37 ° C. In other respects, co-incubation can include about 10 In another embodiment, the co-incubation is carried out at a temperature of about 4 ° C to about 37 ° C; from about 20 ° C to about 30 ° C, about 25 ° C or about 37 ° C. In other respects, co-incubation can include about 10 In another embodiment, the co-incubation is carried out at a temperature of about 4 ° C to about 37 ° C; from about 20 ° C to about 30 ° C, about 25 ° C or about 37 ° C. In other respects, co-incubation can include about 10⁷, 10⁸, 10⁹, 10¹⁰, 10¹¹, 10¹² or 10¹³ minicells. Specific parameters of temperature, time, buffer, concentration of minicells, etc. can be optimized for a specific combination of conditions.

[0055] The success of this approach is surprising because practitioners have developed various chemical and electrochemical processes (discussed in the review by Miller, 1994) for over forty years to transform nucleic acids into bacterial cells. Practitioners have used such strong measures because the conventional view was held that nucleic acids such as siRNA, miRNA or plasmid-free shRNA are too large to pass passively to the cytoplasm of the minicell. For example, porins, which are β-barrel proteins that typically act as diffusion pores, allow passive transport through the outer bacterial membrane of molecules with molecular weights of 600 daltons or less (Nikaido, 1994). Meanwhile, double-stranded DNA plasmid encoding shRNA exceeds one million daltons,

[0056] Furthermore, once packaged, the functional nucleic acid remains inside the minicell and is protected against degradation. In this respect, studies of prolonged incubation with minicells packed with siRNA incubated in sterile saline did not show any siRNA leakage. In addition, co-incubation of minicells of packed siRNA with nucleases confirmed that siRNAs penetrated the outer membrane of intact minicells and were protected from degradation. Similarly, despite the fact that minicells can be expected to transfer residual nucleases from the parent bacterial cytoplasm, packaged siRNAs are stable in the cytoplasm of minicells. Packed siRNA also escapes the degradation mechanisms present in phagolysosomes, such as acids, free oxygen radicals and acid hydrolases (Conner and Schmid, 2003), in order to reduce the expression of target mRNA within a mammalian cell. [0057] In other embodiments, a plurality of functional nucleic acids targeting 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, inter alia, multidrug resistance (MDR) pumps and antiapoptotic genes. To combat this resistance, minicells can be packed with therapeutically significant concentrations of functional nucleic acid for MDR-associated genes and administered to the patient prior to chemotherapy.

[0058] Thus, packaging of functional plasmid-free nucleic acid into intact minicells as described herein provides numerous advantages. For example, since the approach of the invention does not require a genetic modification of the parent bacteria to match the expression of a functional nucleic acid, one parent bacterium can be used to produce minicells containing multiple types of nucleic acids targeting multiple indications. Similarly, the minicell may be packed with many different RNAs, thereby avoiding or overcoming resistance mechanisms.

Functional nucleic acids [0059] As mentioned above, a functional nucleic acid is a category that includes nucleic acid molecules that affect expression by RNA interference, gene expression suppression or other mechanism. Examples of such molecules are one-, two- 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 RNA traps and antisense nucleic acids.

[0060] In a preferred embodiment of the invention, the intact minicells carry siRNA molecules. Short-circulating RNA molecules are useful for RNAi, a post-transcriptional gene silencing mechanism. As mentioned, "siRNA" typically means double-stranded RNA molecules with a length of about 10 to about 30 nucleotides that have been named because of their particular ability to interfere with protein expression. Preferably, the 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 21-23 nucleotides in length. Therefore, preferred siRNA molecules have lengths 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28 or 29 nucleotides.

[0061] The length of one strand determines the length of the siRNA molecule. For example, siRNA, which is described as having a length of 21 ribonucleotides (21-mers), may contain two opposite RNA strands that have joined together at the length of 19 adjacent base pairs. The other two ribonucleotides on each strand will be an "unpaired end". When the siRNA contains two threads of different lengths, the longer of the strands determines the length of the siRNA. For example, dsRNA containing one strand that is 21 nucleotides in length and the other 20 nucleotides long strand is 21 milliseconds.

[0062] siRNAs that contain an unpaired end are desirable. The unpaired end may be present at the 5 'or 3' end of the strand. Preferably, it is at the 3 'end of the RNA strand. The length of the unpaired end may vary, but is preferably from about 1 to about 5 bases and more preferably is about 2 nucleotides in length. Preferably, the siRNAs of the present invention will contain an unpaired 3 'end having from about 2 to 4 bases. More preferably, the unpaired 3 'end has a length of 2 ribonucleotides. Even more preferably, the 2 ribonucleotides containing the unpaired 3 'end are uridine (U).

[0063] shRNA contains a single strand of RNA that forms a stem-loop structure where the trunk consists of complementary sense and antisense strands that contain double-stranded siRNA and the loop is a variable link. The shRNA stem structure is usually about 10 to about 30 nucleotides in length. Preferably, the shRNA molecule has a length of 12-28 nucleotides, more preferably 15-25 nucleotides in length, even more preferably 19-23 nucleotides in length and most preferably 21-23 nucleotides in length. Therefore, preferred shRNA molecules contain stems that have lengths 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28 or 29 nucleotides.

[0064] 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 of 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,

[0065] Accordingly, in one aspect of the invention, intact minicells may carry one or more sequences of siRNA directed to silencing drug resistance or apoptosis resistance genes. Using minicells that encode multiple siRNAs, it is possible to treat cells that express multiple drug resistance mechanisms.

[0066] Tools supporting the design of siRNA and regulatory RNAs 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>.

Aims of functional nucleic acids [0067] The functional nucleic acids of the invention are preferably targeted to a gene or transcript of a protein 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 benefits of minicell vectors. See, e.g., Sioud (2004), Caplen (2003), Wu et al. (2003), Yague et al. (2004).

[0068] Proteins that contribute to drug resistance or stimulation of the tumor phenotype are preferred targets of a functional nucleic acid. Proteins may contribute to acquired drug resistance or to internal drug resistance. Acquisition of a resistant phenotype occurs when diseased cells, such as cancer cells, initially respond to drugs but become resistant after subsequent treatment cycles. Useful targets involved in acquired or internal drug resistance include, but are not limited to, ATP-binding tape transporters, such as Pglycoprotein (P-gp, P-170, PGY1, MDR1, ABCB1, MDR-related protein, multidrug resistance protein 1, MDR-2 and MDR -3, MRP2 (a protein associated with multidrug resistance), BCRABL (region of fracture clusters - Abelson's protooncogene), STI-571 resistance-related protein, a protein associated with lung resistance, cyclooxygenase 2, kappa nuclear factor, XRCC1 (cross-complementation of X-related damage, group 1), ERCC1 (cross-complementation exclusion gene), GSTP1 (glutathione S-transferase), β-tubulin mutant, Abcbla (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 the drug metabolism, e.g. Arnt, Blmh, C130052I12Rik (CRR9p), Comt, Crabp1, Cyp1a1, Cyp1a2, Cyp2b19, Cyp2b20, Cyp2c29, Cyp2c, Cyp2c70, Cyp2d22, Cyp2e1, Dhfr , Ephx1, Ephx2, Gstm1 (MGST1), Gstp1, Nat2, Nqo1, Sod1, Ste, Tpmt, Tyms, Ugcg, (b) genes involved in DNA repair, for example Apc, Atm, Brca1, Brca2, Ercc3 (XPB), Mgmt, Mlh1, Xpa, Xpc, (c) genes involved in the cell cycle,

[0069] Suitable targets also include proteins that contribute to resistance to apoptosis. These include Bcl-2 (B cell leukemia / lymphoma), Bcl-XL, A1 / Bfl 1, focal adhesion kinase and mutant p53 protein.

[0070] Useful targets further include oncogenic and mutant tumor suppressor proteins. Examples include β-catenin, PKC-α (C-protein kinase), C-RAF, K-Ras (V12), h-Ras, Dead box RNA helicase DP97, DNMT1 (DNA methyl transferase 1), FLIP (inhibitory protein similar to Flice), C-Sfc, 53BPI, Polycomb EZH2 group protein (zeste homologue amplifier), ErbB 1, HPV-16 E5 and E7 (human early 5 and early 7 papilloma virus), fortilinin and MCI1P (myeloid leukemia protein 1), DIP13a (DDC-reactive protein 13a), MBD2 (methyl CpG binding domain), p21, KLF4 (Kruppel-like factor 4), tpt / TCTP (tumor-driven tumor protein), SPK1 and SPK2 (sphingosine kinase), P300, PLK1 (Polo-like kinase 1), Trp53, Ras, ErbB1, VEGF (vascular endothelial growth factor), and BAG-1 (atlanogen 1 associated with BCL2). [0071] Numerous molecular targets have been identified for the treatment of cancer, and RNAi exploration platforms quickly identify many different new targets. Examples of such molecular targets useful in the invention include tyrosine kinase (variant), Akt (B protein kinase, PKB), Akt1, AlphaLbeta2 integrin, aminopeptidase, androgen receptor, Aurora A, AuroraB, primary fibroblast growth factor receptor (bFGF) (bFGFr), BRaf, carcinoembryonic 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 inhibitor 1B (CDKN1B, p27, KIP1), CYP26, fibroblast growth factor receptor 3 (FGFr3), fibroblast growth factor receptor 4 (FGFr4), G250, Hedgehog signaling pathway (Hh),

[0072] With regard to HIV infection, the targets include HIV-Tat, HIV-Rev, HIV-Vif, HIV-Nef, HIVGag, HIV-Env, LTR, CD4, CXCR4 (chemokine receptor) and CCR5 (chemokine receptor).

[0073] Due to the heterogeneity of tumor cells, multiple drug resistance or apoptosis resistance pathways can be activated in target cells. Thus, the functional nucleic acids used in the methods of the invention may need to change over time. For example, if the biopsy samples reveal new mutations that lead to acquired drug resistance, then a specific functional nucleic acid can be designed and packaged into intact minicells that are administered to the mammalian host to respond to the acquired drug resistance.

Delivery of functional nucleic acid by intact minicells [0074] The invention enables a method of providing a functional nucleic acid that comprises (a) providing a collection of intact minicells in a pharmaceutically acceptable carrier, each minicell of the set comprising a functional plasmid-free nucleic acid, and (b) contact of a collection of minicells with mammalian cells such that the mammalian cells absorb the minicells belonging to the harvest, wherein the functional nucleic acid is released into the cytoplasm of the target cells. The minicells are contacted with a target mammalian cell by bispecific ligands as described in published PCT application WO 05/056749. The contact between the minicell and the target mammalian cells may occur in vitro or in vivo.

The method of overcoming drug resistance and treatment of a disease [0075] The invention further provides a method of overcoming drug resistance and treating a disease such as cancer or AIDS in a subject. The method comprises (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 a functional nucleic acid with a target mammalian cell such that the cell the mammal absorbs the minicell as described in the '749 PCT application cited above, and (c) delivery of the drug to the mammalian target cell as described in published PCT application WO 05/079854. Preferably, step (c) is performed after steps (a) and (b), for allowing a functional nucleic acid to reduce drug resistance before drug administration. The delivery of the drug and the introduction of a functional nucleic acid can occur in succession, in any order or simultaneously.

[0076] Drugs can be delivered by any conventional means. For example, the medicaments may be delivered orally, parenterally (including subcutaneously, intravenously, intramuscularly, intraperitoneally, and by infusion), topically, percutaneously, or by inhalation. The appropriate mode of delivery and dosage of each drug is easily determined by those skilled in the medical field.

Drug Delivery by Minicells [0077] Although drug delivery may be via conventional means, minicell delivery is preferred as described in published PCT application WO 05/079854. In this regard, the applicants have found that the same mammalian cells can be effectively re-transfected by target intact minicells that are packaged with different charges. For example, minicells packed with a functional nucleic acid can transfect a mammalian cell, whereupon the drug-packed minicells can deliver the drug to the same mammalian cell, to achieve a complementary or synergistic antitumor effect.

[0078] The medicament may be packed into a separate minicell relative to that of the functional nucleic acid. Alternatively, the drug may be packed into the same minicell as the functional nucleic acid. Some drugs can interact with nucleic acids and prevent the drug and nucleic acid from co-packing into the same minicell. For example, it is known that doxorubicin interacts with DNA.

[0079] Preferably, the minicells of the invention contain a sufficient amount of a drug to exert the physiological or pharmacological effect of the drug on the target cell. Also preferably, the medicaments contained within the minicells are heterologous or foreign to minicells, meaning that the parental bacterial cells of minicells normally do not produce the drug.

[0080] Both hydrophilic and hydrophobic drugs can be packaged into minicells by creating a concentration gradient of drug between the extracellular environment containing the minicells and the cytoplasm of the minicell. When the extracellular environment contains a higher drug concentration than the minicell cytoplasm, the drug naturally moves down this concentration gradient to the minicell cytoplasm. However, when the concentration gradient is reversed, the drug does not go outside of the minicells. The procedure and mechanisms for loading the drug into minicells are as described in published PCT application WO 05/079854.

[0081] To pack minicells with drugs that are not usually water-soluble, the drugs can be pre-dissolved in a suitable solvent. For example, paclitaxel may be dissolved in a 1: 1 mixture of ethanol and Cremophor EL (polyethoxylated castor oil) followed by dilution in PBS to achieve a solution of paclitaxel that is partially dissolved in aqueous media and brings minimal amounts of organic solvent to ensure that the drug remains in solution. Minicells can be incubated in this final environment to package the drug. Thus, applicants have reported that even hydrophobic drugs can diffuse into the cytoplasm of minicells, in order to achieve high and therapeutically significant packaging of the cytoplasm drug. This is unexpected,

[0082] Another method of charging minicells with a drug involves culturing recombinant parent bacterial 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 a cell biosynthetic pathway of a desired drug can be cloned and transferred to a parent bacterial strain that is capable of producing minicells. Gene transcription and translation of the gene cluster results in biosynthesis of the drug within the cytoplasm of parent bacterial cells, filling the bacterial cytoplasm with the drug. When the parent bacterial cell divides and forms progeny minicells, the minicells also contain the drug in their cytoplasm.

[0083] Similarly, another method of packing 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 [0084] Drugs useful in the invention can mean any physiologically or pharmacologically active substance that results in a desired local or systemic effect in animals, particularly mammals and humans. Drugs may be inorganic or organic compounds, but not limited to, including peptides, proteins, nucleic acids and fine particles, 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, sulphate, laurate, palmitate, phosphate (V), nitrate (III), nitrate (V), borate, acetate, maleate. tartrate, oleate, salicylate and the like. In the case of acidic drugs, metal salts, amines or organic cations may 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 is a water-soluble derivative thereof, or as its basic derivative, which in any case, or by its delivery, is converted by enzymes, hydrolysed at the pH of the body, or by means of other metabolic processes to the starting therapeutically active form.

[0085] 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 which are affected by classical multidrug resistance are of particular use 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).

[0087] In general, cancer chemotherapy agents are preferred drugs. Useful cancer chemotherapy drugs include nitrogen mustards, nitrosoules, ethyleneimines, alkanesulfonates, tetrazines, platinum compounds, pyrimidine analogs, purine analogs, antimetabolites, folic acid analogs, anthracyclines, taxanes, vinca alkaloids, topoisomerase inhibitors and hormonal agents. Examples of drugs for chemotherapy include actinomycin-D, alkerate, Ara-C, anastrozole, asparaginase, BiCNU, bicalutamide, bleomycin, busulfan, capecitabine, carboplatin, carmustine, CCNU, chlorambucil, cisplatin, cladribine, CPT-11, cyclophosphamide, cytarabine, arabinoside cytosine, cytoxan, dacarbazine, dactinomic, daunorubicin, dexrazoxane, docetaxel, doxorubicin, DTIC, epirubicin, ethyleneimine, etoposide, floxuridine, fludarabine, fluorouracil, flutamide,

[0088] Useful cancer chemotherapy drugs also include alkylating agents such as thiotepa and cyclophosphamide; alkylsulfonates such as busulfan, improsulfan and piposulfan; aziridines, such as benzodopa, carboquone, meturedopa and uredopa; ethyleneimines and methylamines, including altretamine, triethylenemethylamine, triethylenephosphoramide, triethylenethiophosphoramide and trimethylol melamine; nitrogen mustard derivatives such as chlorambucil, chloraphosphine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, Novembiehin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitroureas such as kannustine, chlorozotocin, fotemustine, lomustine, nimustine and ranimustine; antibiotics, such as aclacinomizines, actinomycin, autramycin, azaserine, bleomycin, cactinomycin, calicheamicin, carabicin, carminomycin, carzinophylline, chromoinycins, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin, epirubicin, eorubicin, idambicin, marcellomycin, mitomycin, mycophenolic acid, nogalamycin, olanzomycin, peplomycin, potfiromycin, puromycin , quelamycin, rhodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin and zinc bicin; anti-metabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogs such as denopterin, methotrexate, pteropterin and trimetrexate, purine analogs such as fludarabine, 6-mercaptopurine, thiamiprin, and thioguanine; pyrimidine analogs such as ancitabine, azacytidine, 6-azauridine, karmofur, cytarabine, dideoxyuridine, doxifluridine, enocytabine, floxuridine and 5-FU; androgens, such as calusterone, dromostanolone propionate, epithostanol, rivupostate, testolactone; antagonists of adrenocortical hormones, such as aminoglutethimide, mitotane, and trilostane; folic acid supplements such as frolinic acid; aceglatone; aldofosfamide glycoside, aminolevulinic acid;

amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elfornithine; elliptic acetate; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidamine; mitoguazone; mitoxantrone; mopidamol; nitracrine; pentostatin; phenamet; pirarubicin; podophyllinic acid; 2etylohydrazyd; procarbazine; PSK®; razoxane; sizofran; spirogermanium; tenuazonic acid; triaziquone; 2,2 ', 2 "-trichlorotriethylamine; urethane; vindesine, dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside (' Ara-C '); cyclophosphamide; thiotepa; taxoids, e.g. paclitaxel (TAXOL®, Bristol); Myers Squibb Oncology, Princeton, NJ) and doksetaxel (TAXOTERE®, Rhone-Poulenc Rorer, Antony, France); chlorambucil; gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitomycin C; mitoxantrone; vincristine; vinorelbine; navelbine; edatrexate; teniposide; daunomycin; aminopterin; Xeloda; ibandronate; CPT-11; an RFS 2000 topoisomerase inhibitor; difluoromethylornithine (DMFO); retinoic acid; esperamicins; capecitabine; and pharmaceutically acceptable salts, acids or derivatives of any of the foregoing. Also included are antihormonal agents that act to regulate or inhibit the activity of the hormone on tumors, such as anti-estrogens, including for example tamoxifen, raloxifene, aromatase inhibitory 4 (5) -imidazole, 4-hydroxy tamoxifen, trioxifene, keoxifene, onapristone and toremifen (Fareston); and anti-androgens, such as flutamide, nilutamide, bicalutamide, leuprolide and goserelin; and pharmaceutically acceptable salts, acids or derivatives of any of the foregoing. ifosfamide; mitomycin C; mitoxantrone; vincristine; vinorelbine; navelbine; edatrexate; teniposide; daunomycin; aminopterin; Xeloda; ibandronate; CPT-11; an RFS 2000 topoisomerase inhibitor; difluoromethylornithine (DMFO); retinoic acid; esperamicins; capecitabine; and pharmaceutically acceptable salts, acids or derivatives of any of the foregoing. Also included are antihormonal agents that act to regulate or inhibit the activity of the hormone on tumors, such as anti-estrogens, including for example tamoxifen, raloxifene, aromatase inhibitory 4 (5) -imidazole, 4-hydroxy tamoxifen, trioxifene, keoxifene, onapristone and toremifen (Fareston); and anti-androgens, such as flutamide, nilutamide, bicalutamide, leuprolide and goserelin; and pharmaceutically acceptable salts, acids or derivatives of any of the foregoing. ifosfamide; mitomycin C; mitoxantrone; vincristine; vinorelbine; navelbine; edatrexate; teniposide; daunomycin; aminopterin; Xeloda; ibandronate; CPT-11; an RFS 2000 topoisomerase inhibitor; difluoromethylornithine (DMFO); retinoic acid; esperamicins; capecitabine; and pharmaceutically acceptable salts, acids or derivatives of any of the foregoing. Also included are antihormonal agents that act to regulate or inhibit the activity of the hormone on tumors, such as anti-estrogens, including for example tamoxifen, raloxifene, aromatase inhibitory 4 (5) -imidazole, 4-hydroxy tamoxifen, trioxifene, keoxifene, onapristone and toremifen (Fareston); and anti-androgens, such as flutamide, nilutamide, bicalutamide, leuprolide and goserelin; and pharmaceutically acceptable salts, acids or derivatives of any of the foregoing. vincristine; vinorelbine; navelbine; edatrexate; teniposide; daunomycin; aminopterin; Xeloda; ibandronate; CPT-11; an RFS 2000 topoisomerase inhibitor; difluoromethylornithine (DMFO); retinoic acid; esperamicins; capecitabine; and pharmaceutically acceptable salts, acids or derivatives of any of the foregoing. Also included are antihormonal agents that act to regulate or inhibit the activity of the hormone on tumors, such as anti-estrogens, including for example tamoxifen, raloxifene, aromatase inhibitory 4 (5) -imidazole, 4-hydroxy tamoxifen, trioxifene, keoxifene, onapristone and toremifen (Fareston); and anti-androgens, such as flutamide, nilutamide, bicalutamide, leuprolide and goserelin; and pharmaceutically acceptable salts, acids or derivatives of any of the foregoing. vincristine; vinorelbine; navelbine; edatrexate; teniposide; daunomycin; aminopterin; Xeloda; ibandronate; CPT-11; an RFS 2000 topoisomerase inhibitor; difluoromethylornithine (DMFO); retinoic acid; esperamicins; capecitabine; and pharmaceutically acceptable salts, acids or derivatives of any of the foregoing. Also included are antihormonal agents that act to regulate or inhibit the activity of the hormone on tumors, such as anti-estrogens, including for example tamoxifen, raloxifene, aromatase inhibitory 4 (5) -imidazole, 4-hydroxy tamoxifen, trioxifene, keoxifene, onapristone and toremifen (Fareston); and anti-androgens, such as flutamide, nilutamide, bicalutamide, leuprolide and goserelin; and pharmaceutically acceptable salts, acids or derivatives of any of the foregoing. aminopterin; Xeloda; ibandronate; CPT-11; an RFS 2000 topoisomerase inhibitor; difluoromethylornithine (DMFO); retinoic acid; esperamicins; capecitabine; and pharmaceutically acceptable salts, acids or derivatives of any of the foregoing. Also included are antihormonal agents that act to regulate or inhibit the activity of the hormone on tumors, such as anti-estrogens, including for example tamoxifen, raloxifene, aromatase inhibitory 4 (5) -imidazole, 4-hydroxy tamoxifen, trioxifene, keoxifene, onapristone and toremifen (Fareston); and anti-androgens, such as flutamide, nilutamide, bicalutamide, leuprolide and goserelin; and pharmaceutically acceptable salts, acids or derivatives of any of the foregoing. aminopterin; Xeloda; ibandronate; CPT-11; an RFS 2000 topoisomerase inhibitor; difluoromethylornithine (DMFO); retinoic acid; esperamicins; capecitabine; and pharmaceutically acceptable salts, acids or derivatives of any of the foregoing. Also included are antihormonal agents that act to regulate or inhibit the activity of the hormone on tumors, such as anti-estrogens, including for example tamoxifen, raloxifene, aromatase inhibitory 4 (5) -imidazole, 4-hydroxy tamoxifen, trioxifene, keoxifene, onapristone and toremifen (Fareston); and anti-androgens, such as flutamide, nilutamide, bicalutamide, leuprolide and goserelin; and pharmaceutically acceptable salts, acids or derivatives of any of the foregoing. and pharmaceutically acceptable salts, acids or derivatives of any of the foregoing. Also included are antihormonal agents that act to regulate or inhibit the activity of the hormone on tumors, such as anti-estrogens, including for example tamoxifen, raloxifene, aromatase inhibitory 4 (5) -imidazole, 4-hydroxy tamoxifen, trioxifene, keoxifene, onapristone and toremifen (Fareston); and anti-androgens, such as flutamide, nilutamide, bicalutamide, leuprolide and goserelin; and pharmaceutically acceptable salts, acids or derivatives of any of the foregoing. and pharmaceutically acceptable salts, acids or derivatives of any of the foregoing. Also included are antihormonal agents that act to regulate or inhibit the activity of the hormone on tumors, such as anti-estrogens, including for example tamoxifen, raloxifene, aromatase inhibitory 4 (5) -imidazole, 4-hydroxy tamoxifen, trioxifene, keoxifene, onapristone and toremifen (Fareston); and anti-androgens, such as flutamide, nilutamide, bicalutamide, leuprolide and goserelin; and pharmaceutically acceptable salts, acids or derivatives of any of the foregoing. Aromatase 4 (5) -imidazoles, 4-hydroxy tamoxifen, trioxifene, keoxifene, onapristone and toremifene (Fareston); and anti-androgens, such as flutamide, nilutamide, bicalutamide, leuprolide and goserelin; and pharmaceutically acceptable salts, acids or derivatives of any of the foregoing. Aromatase 4 (5) -imidazoles, 4-hydroxy tamoxifen, trioxifene, keoxifene, onapristone and toremifene (Fareston); and anti-androgens, such as flutamide, nilutamide, bicalutamide, leuprolide and goserelin; and pharmaceutically acceptable salts, acids or derivatives of any of the foregoing.

[0089] 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; substance that inhibits the functions of Mϋller wires; mouse gonadotropin-associated peptide; inhibin; activin; vascular endothelial growth factor; integrin; thrombopoetin (TPO); nerve growth factors such as NGF-β; platelet growth factor; transforming growth factors (TGFs) such as TGF-α and TGF-β; insulin-like growth factors -I and -II; erythropoietin (EPO); osteoinductive factors; interferons, such as interferon-α, -β and -γ; growth promoting factors (CSFs) such as macrophage-CSF (MCSF); granulocyte-macrophage-CSF (GM-CSF); and granulocyte-CSF (G-CSF); interleukins (IL) such as IL1, IL-1α, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-11, IL-1 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. erythropoietin (EPO); osteoinductive factors; interferons, such as interferon-α, -β and -γ; growth promoting factors (CSFs) such as macrophage-CSF (MCSF); granulocyte-macrophage-CSF (GM-CSF); and granulocyte-CSF (G-CSF); interleukins (IL) such as IL1, IL-1α, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-11, IL-1 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. erythropoietin (EPO); osteoinductive factors; interferons, such as interferon-α, -β and -γ; growth promoting factors (CSFs) such as macrophage-CSF (MCSF); granulocyte-macrophage-CSF (GM-CSF); and granulocyte-CSF (G-CSF); interleukins (IL) such as IL1, IL-1α, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-11, IL-1 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. interleukins (IL) such as IL1, IL-1α, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-11, IL-1 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. interleukins (IL) such as IL1, IL-1α, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-11, IL-1 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.

[0090] Drugs can be prodrugs, subsequently activated, e.g. by a prodrug activating enzyme, which converts a prodrug, such as a peptidyl chemotherapeutic agent, into an active antitumor drug. For example, see WO 88/07378, WO 81/01145, and U.S. Patent No. 4,975,278. In general, the enzyme component includes any enzyme capable of affecting the prodrug in such a way that it is converted to its more active, cytotoxic form.

Targeting minicells to specific mammalian cells.

[0091] In one aspect of the invention, the minicell is targeted to a mammalian target 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 the minicell and mammalian cell components results in the binding of the minicell to a mammalian cell such that the minicell is absorbed by the mammalian cell, whereby the functional nucleic acid is released into the mammalian cell cytoplasm. This targeted delivery method can be carried out in vivo or in vitro, or both in vivo and in vitro.

[0092] The contact between a bispecific ligand, a minicell and a mammalian cell can occur in a number of different ways. For in vivo delivery, it is preferred to administer a minicell that already has a bispecific ligand attached to it. Hence, all of a minicell, a bispecific ligand and a target cell come into contact when the bispecific directed minicell target reaches the target cell in vivo. Alternatively, the bispecific ligand and the minicell can be administered separately in vivo.

[0093] Contact between bispecific ligands, minicells and mammalian cells may also take place 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 staged approach, minicells and bispecific ligands are first incubated together to form a bispecific ligand-targeted minicell that is then incubated with target cells. In a further 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 the contact of bispecific ligands, minicells and mammalian target cells.

[0094] The inventors have found that a targeted delivery approach is generally applicable to a variety of mammalian cells, including cells that are normally resistant to specific adhesion and endocytosis of minicells. For example, antibody bispecific ligands with anti-O-polysaccharide specificity on one arm and the anti-HER2 receptor or anti-EGF receptor specificity on the other arm efficiently bind minicells to the 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 the rapid endocytosis of the minicells by each of the non-phagocytic cells.

[0095] Target cells of the invention include any cells to which a functional nucleic acid is to be introduced. Desirable target cells are characterized by the expression of the cell surface receptor, which, after binding of the ligand, facilitates endocytosis. Preferred target cells are non-phagocytic, meaning that the cells are not specialized phagocytes, such as macrophages, dendritic cells and natural killer (NK) cells. Preferred target cells are also mammalian.

[0096] Ligands useful in the targeted delivery methods of the invention include any agents that bind to the surface component on the target cell and the surface component on the 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 double specificities for a surface component on intact bacterial minicells and a 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,

[0097] The selection of a specific ligand is made on the basis of 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, the ligands preferably have a first arm that carries specificity for the surface structure of the intact minicell of bacterial origin and a second arm that carries specificity for the surface structure of the mammalian cell. Each of the first and second arm can be multi-valued. Preferably, each arm is monospecific, even if multivalent.

[0098] For binding to bacterial minicells, it is desirable for one arm of the ligand 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 ligand binding include cell-surface exposed polypeptides and external carbohydrates, such as outer membrane proteins, exposed cell surface fragments of pilli peptide, fimbriae and flagellum.

[0099] For binding to target cells, one arm of the ligand is specific for a surface component of a mammalian cell. Such components include cell surface proteins, peptides and carbohydrates, whether characterized or non-characted. Cell surface receptors, particularly those capable of activating receptor-mediated endocytosis, are desirable surface components of the targeting cell. Such receptors, if overexpressed on the surface of the target cell, give additional selectivity for targeting cells for treatment, thereby reducing the possibility of delivery to non-target cells.

[0100] By way of example, tumor cells, metastatic cells, vascular cells such as endothelial cells and smooth muscle cells, lung cells, renal cells, blood cells, bone marrow cells, brain cells, liver cells, etc. may be targeted. , or precursors of any selected cells, by selection of a ligand that specifically binds to the cell surface receptor motif on desired cells. Examples of cell surface receptors include carcinoembryonic antigen (CEA), which is overexpressed in most tumors of the colon, anus, 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 cancers (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 cancer and prostate cancer (Salomon et al., 1995); asjoglycoprotein receptor (Stockert, 1995); transferrin receptor (Singh, 1999); serpin enzymatic complex receptor that is expressed on hepatocytes (Ziady et al., 1997); fibroblast growth factor receptor (FGFR), which is overexpressed on pancreatic ductal adenocarcinoma cells (Kleeff et al., 2002); vascular endothelial growth factor receptor (VEGFR) for gene therapy against angiogenesis (Becker et al., 2002; Hoshida et al., 2002); a folate receptor that is selectively overexpressed in 90% of ovarian-noncubic 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 in delivering genes to respiratory epithelial cells and is attractive for the treatment of lung diseases such as cystic fibrosis (Kaetzel et al., 1997).

[0101] Preferred ligands include antibody and / or antibody derivatives. As used herein, the term "antibody" includes an immunoglobulin molecule obtained by generating an immunogenic response in vitro or in vivo. 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.

[0102] Wild type antibodies have four polypeptide chains, two identical heavy chains and two identical light chains. Both types of polypeptide chains have constant regions that are not differentiated or differ minimally between antibodies of the same class, and variable regions. The variable regions are unique to a particular antibody and contain an antigen binding domain that recognizes a specific epitope. Antigen-binding domain regions that are most directly involved in antibody binding are &amp; quot; complementarity determining regions &amp; quot; (CDRs).

[0103] 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 comprise a light chain and a heavy chain portion linked by a disulfide bond bridge) Fab '(an antibody fragment comprising a single antigen-binding domain comprising a Fab and an additional heavy chain portion through the hinge region , F (ab ') 2 (two Fab' molecules linked by disulfide bonds between the chains in heavy chain hinge regions), bispecific Fabs (Fab molecule having two antigen binding domains, each of which can be targeted to another epitope), and scFv ( variable, binding antigen determining the single light and heavy chain region of the antibody, linked by an amino acid chain). [0104] When 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 means of genetic manipulation and / or in vitro treatment to reduce their antigenicity in humans. Methods for humanizing antibodies are described, e.g. in US patents 6 639 055, No. 5 585 089 and 5 530101. In the simplest case, humanized antibodies are formed by grafting 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 humanized antibodies with high affinity, however, generally requires the transfer of one or more additional residues from the so-called framework regions (FR) of the murine parent mAb. Several variants of humanizing technology have also been developed. See Vaughan et al., 1998.

[0105] 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 (scFv) or Fab libraries for phage display. See Griffiths et al., 1994; Vaughan et al., 1996; Sheets et al., 1 998; de Haard et al., 1999; and Knappik et al., 2000.

[0106] Useful ligands also include single-chain bispecific antibodies that are typically recombinant polypeptides consisting of a light chain variable part covalently attached through the linker molecule to the corresponding part of the variable heavy chain. See U.S. Patent Nos. 5 455 030, No. 5 260 203 and 4 496 778. Bispecific antibodies may also be obtained by other methods. For example, chemical heteroconjugates can be generated by chemically combining 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.

[0107] Since Fab and scFv fragments are monovalent, they often have low affinity for target structures. Thus, preferred ligands obtained from these components are derived by engineering into dimeric, trimer or tetramer 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 crosslinking.

[0108] 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, e.g. in the form of so-called he diciał, triciał and tetraciał. See Hudson and Souriau, 2003. Diastiało denotes a divalent dimer formed by the non-covalent association of two scFvs that gives two Fv binding sites. Similarly, the tricuspid is the result of the formation of a trivalent trimer of three scFvs, resulting in three binding sites, and the tetrabilate is the result of the formation of a four-valent tetramer of four scFvs, giving four binding sites.

[0109] 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 rapidly growing. See Hudson and Souriau, 2003. An example of such an antibody that can be used to form one arm of a bispecific ligand has specificity for HER2: Herceptin ™; Trastuzumab.

[0110] Antibody variable regions can also be coupled 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 with human Ig Fc hinge regions can add effector functions. Also, the fusion of heterologous protein domains from multimeric proteins stimulates multimerization. For example, a short scFv fusion was used for short amphipathic helixes to produce mini-antibodies. 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, the homodimerization domains can be modified by engineering, to form heterodimers using engineering strategies such as knobs in holes (Ridgway et al., 1996). Finally, a selection of fusion protein partners may 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 [0111] The invention further provides delivery by contacting minicells of bacterial origin with mammalian cells that are competent for phagocytosis or endocytosis. Such mammalian cells, which are capable of absorbing parent bacterial cells by intracellular bacterial pathogenesis, likewise absorb minicells that release their content in the cytoplasm of mammalian cells. This delivery approach can be carried out without the use of targeted ligands.

[0112] A variety 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 respect. For example, phagocytosis is a well-documented process in which macrophages and other phagocyte cells, such as neutrophils, absorb particles by lengthening the nibinodes above the surface of the particle until the particle is completely absorbed. Although this is described as "unspecific" phagocytosis, the participation of specific receptors in the process has been demonstrated. See Wright et al. (1986); Speert et al. (1988).

[0113] Hence, one form of phagocytosis involves interactions between surface ligands and ligand receptors located in the nibinate membranes. This attachment step, mediated by specific receptors, is considered to be dependent on bacterial surface adhesins. With respect to less virulent bacteria, such as non-epoxidogenic E. coli, phagocytosis may also occur in the absence of surface ligands for phagocytic receptors. See, e.g., Pikaar et al. (1995). Hence, the present invention includes, but is not limited to, use of minicells that have- or not - surface adhesins, in accordance with the nature of their parent bacterial cells, and are absorbed by phagocytes (i.e., "phagocytosis-competent" host cells) from which neutrophils and macrophages are the main types in mammals.

[0114] Another absorption process is endocytosis, by means of which intracellular pathogens, on the example of Salmonella species, Escherichia, Shigella, Helicobacter, Pseudomonas and Lactobacilli, obtain access to mammalian epithelial cells where they are replicated. Two basic mechanisms in this regard are receptor-mediated endocytosis, which is mediated by the receptor, also known as the "endocytosis of shell [clathrinic] pits" (Riezman, 1993) and clathrin-independent endocytosis (Sandvig and Deurs, 1994). One or both may be involved when a cell-competent cell for absorption works by endocytosis (i.e., a "competent for endocytosis" host cell)minicell according to the invention. Representative cell-competent for endocytosis are mammary epithelial cells, enterocytes in the gastrointestinal tract, gastric epithelial cells, lung epithelial cells and epithelial cells of the urinary tract and bladder.

[0115] When delivering to a mammalian cell susceptible for absorption without a targeting ligand, the nature of the application contemplated will influence the choice of the bacterial source of the minicells used. For example, Salmonella, Escherichia and Shigella species carry adhesins that are recognized by endocytosis-mediated receptors on enterocytes in the digestive tract, and may be suitable for delivery of a drug that is effective against colon cancer cells. Likewise, minicells derived from Helicobacter pylori, carrying adhesins specific for gastric epithelial cells, may be adapted to deliver gastric-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 bacteria that carry adhesins specific for urinary and bladder epithelial cells may be well suited for delivering drug to the urethra of urinary tract cancer or bladder cancer.

Formulations [0116] As indicated, in one aspect, there is provided a composition comprising:

(a) a collection of intact minicells of bacterial origin, wherein each minicell of the set comprises a regulatory RNA that is packaged in a minicell, and (b) a pharmaceutically acceptable carrier therefor, wherein (i) the regulatory RNA is selected from the group consisting of antisense ssRNA, ribozyme and RNA traps, (ii) minicells of the construct for the expression of regulatory RNA in situ are absent, and (iii) the set contains a therapeutically effective amount of regulatory RNA.

[0117] The formulation optionally comprises a drug. In one example, the minicell of the preparation contains the drug, while in another minicell it may contain a nucleic acid molecule, such as a plasmid, which encodes the drug.

[0118] The formulations also optionally comprise a bispecific ligand for targeting the minicell to the target cell. The minicell and ligand can be any of those described herein. Hence, the minicell comprises a nucleic acid encoding a functional nucleic acid and the bispecific ligand is preferably capable of binding to the surface component of the minicell and to the surface component of the target mammalian cell.

[0119] The formulations may be presented in a unit dosage form, e.g. in ampoules or vials, in multi-dose containers, with or without a preservative. The formulation may be in the form of a solution, suspension or emulsion in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and / or dispersing agents. The appropriate solution is isotonic with the blood of the recipient and is illustrated by saline solution, Ringer's solution and dextrose solution. Alternatively, the formulations may be in the form of a lyophilized powder, for reconstitution with a suitable carrier, e.g. sterile pyrogen-free water or physiological saline solution. The formulations may also be in the form of a preparation for affixing to the tissues.

Routes of Administration [0120] The formulations described herein can be administered by various routes and to various sites in the mammalian body to achieve the desired therapeutic (s) to be performed, either locally or systemically. Delivery can be accomplished, for example, by oral administration, by administration of the formulation to the body cavity, by inhalation or insufflation, or by parenteral, intramuscular, intravenous, portal vein, intrahepatic, peritoneal, subcutaneous, intracutaneous or intradermal administration. The mode and place of administration depend on the location of the target cells. For example, fibrotic cyst cells may be an effective target for delivery of targeted minicells by inhalation. Similarly, tumor metastasis can be more effectively treated by intravenously delivering targeted minicells. Primary ovarian cancer can be treated by intraperitoneal administration of targeted minicells. Purity [0121] In one aspect, the minicells are substantially free of contaminating parent bacterial cells. Thus, minicell preparations preferably contain less than about 1 contaminating parent bacterial cell per 10⁷ minicells, more preferably contain less than about 1 contaminating parent bacterial cell per 10⁸ minicells, even more preferably contain less than about 1 contaminating parent bacterial cell per 10⁹ minicells, even more preferably contain less than about 1 contaminating parent bacterial cell per 10¹⁰ minicells and most preferably contain less than about 1 contaminating parent bacterial cell per 10¹¹minicells.

[0122] Methods for purifying minicells are known in the art and described in International Publication No. WO03 / 033519. One such method combines cross-flow filtration (feed flow is parallel to the membrane surface, Forbes, 1987) and dead-end filtration (feed flow is perpendicular to the membrane surface). Optionally, the filtration combination may be preceded by differential centrifugation at low centrifugal force to remove some of the bacterial cells and, as a consequence, enrich the supernatant in a minicell.

[0123] A further purification method uses density gradient centrifugation in a biologically compatible medium. After centrifugation, the minicell band collects from the gradient, and, optionally, minicells are subjected to subsequent rounds of density gradient centrifugation to maximize frequency. The method may further include a preliminary step of performing differential centrifugation on a sample containing minicells. When operated at low centrifugal force, differential centrifugation will remove some of the parent bacterial cells, thereby enriching the supernatant into minicells.

[0124] 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 the minicell sample to a condition that induces in parent bacterial cells to assume a filamentous form, followed by (b) filtering the sample, to obtain a purified preparation of minicells.

[0125] Known methods for purifying minicells can also be combined. One highly effective combination of methods is as follows:

Stage A: Differential centrifugation of the minicell producing bacterial cell culture. This step, which can be carried out at 2,000 g for about 20 minutes, removes most of the parent bacterial cells while leaving minicells in the supernatant.

Step B: Density gradient centrifugation using an isotonic and non-toxic medium for the density gradient. This stage separates the minicells from many impurities, including parent bacterial cells, with minimal loss of minicells. Preferably, this step is repeated as part of the purification process.

Step C: Filtration in cross flow through a 0.45 μm filter to further reduce contamination with parent bacterial cells.

Stage D: Stress-induced fibril formation in the remaining parent bacterial cells. This can be done by suspending the minicells by any of several stressful environmental conditions.

Step E: Antibiotic treatment to kill parent bacterial cells.

Step F: Crossflow filtration to remove fine impurities such as membrane vesicles, membrane fragments, bacterial residues, nucleic acids, nutrient components, etc., and to concentrate the minicells. A 0.2 μm filter can be used to separate minicells from fine impurities, and a 0.1 μm filter can be used to concentrate the minicells.

Step G: Dead-end filtration to remove fibrillar dead bacterial cells. In this step, a 0.45 μπ filter can be used.

Step H: Removal of endotoxins from the minicell preparation. In this step, magnetic beads coated with anti-lipid A may be used.

Schedules of Administration [0126] In general, the formulations disclosed herein can be used at appropriate dosages, as defined in routine tests, to obtain the 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 health status of the patient; severity of the disease for treatment, administration route, and renal and liver function of the patient.

[0127] Optimal precision in achieving concentrations of minicells and drug in the range that yields maximum efficacy with minimal side effects may require a scheme based on functional nucleic acid kinetics and drug availability to target sites and target cells. Distribution, balance and removal of minicells or a drug may be considered in determining the optimal concentration for the treatment regimen. Dosage of minicells and drugs can be adjusted when used in combination to achieve the desired effect. [0128] Furthermore, the administration of the doses of the preparations can be optimized using a pharmacokinetic / pharmacodynamic modeling system. For example, one or more dosing schedules can be selected, and a pharmacokinetic / pharmacodynamic model can be used to determine the pharmacokinetic / pharmacodynamic profile of one or more dosing schedules. One dose regimen may 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, a dosage regimen for any indication can be determined using the approach and model described in Example 6, with modifications for a particular target cell. for which a desired pharmacokinetic / pharmacodynamic response is achieved based on a particular pharmacokinetic / pharmacodynamic profile. See, e.g., WO 00/67776. In this regard, a dosage regimen for any indication can be determined using the approach and model described in Example 6, with modifications for a particular target cell. for which a desired pharmacokinetic / pharmacodynamic response is achieved based on a particular pharmacokinetic / pharmacodynamic profile. See, e.g., WO 00/67776. In this regard, a dosage regimen for any indication can be determined using the approach and model described in Example 6, with modifications for a particular target cell.

[0129] In particular, the formulations may be administered at least once a week over several weeks. In one embodiment, the formulations are administered at least once a week for from several weeks to several months.

[0130] More specifically, the formulations may be administered at least once a day for about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or 31 days. Alternatively, the formulations may be administered about once a day, about once every 2, 3, 4, 5,

6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or 31 days or more.

[0131] 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.

[0132] 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.

[0133] The formulations may be administered in a single daily dose, or the total daily dosage may be administered in divided doses of two, three or four times a day.

[0134] In the method where the minicells are administered before the drug, administration of the drug can take place at any time, from a few minutes to a few hours after minicell administration. The drug can alternatively be administered at any time, from several hours to several days, potentially several weeks up to several months after minicells.

[0135] More specifically, minicells with packed functional nucleic acid may be administered at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 , 18, 19, 20, 21, 22, 23 or 24 hours before the drug. In addition, minicells 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, 24, 25, 26, 27, 28, 29, 30 or 31 days prior to drug administration. In yet another embodiment, the minicells 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 or 20 weeks or more before the drug. In a further embodiment, the minicells can be administered at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 months before the drug.

[0136] In another embodiment, the minicell is administered after the drug. Administration of minicells may occur at any time, from a few minutes to several hours after drug administration. The minicell can alternatively be administered at any time, from several hours to several days, potentially several weeks up to several months after the drug.

[0137] The following examples are illustrative only and non-limiting, and provide a more complete understanding of the invention.

Examples

1. Direct packaging of regulatory RNA to intact minicells in vitro [0138] Intact minicells of bacterial origin were prepared and purified as described in published US Patent Application No. 2004/0265994. Cy3 labeled siRNAs of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (maximum excitation ^ max) 547 nm, emission maximum (? Max) 563 nm), Ambion product (Austin, Texas USA) were obtained and reconstituted in nuclease-free water to the final 50 μM concentration.

[0139] Approximately 10⁷ minicells were resuspended in 1x phosphate buffer solution (PBS) (Gibco) and co-incubated with 1 μ of Cy3 labeled GAPDH siRNA. Incubation was carried out for 2 hours at 37 ° C with gentle agitation. Control minicells were apparently loaded by incubation with 1 x PBS only. After loading the minicells were pelleted and washed twice with 1 x PBS by centrifugation for 10 minutes at 16200 x g. Experimental and control minicells were observed under a DMLB fluorescence microscope, a Leica product (Germany) with attached D70 apparatus, a product of Olympus Microscopes (Germany). The images were obtained using a 100 x lens with oil immersion.

[0140] The above co-incubation experiments were also carried out under other experimental conditions, such as incubation at room temperature, 37 ° C, and 4 ° C. Additionally, co-increment times varied, including 1 hour, 2 hours, 4 hours and 12 hours, respectively.

[0141] As shown in Figure 1B, the intact siRNA molecules rapidly diffused into the minicells. A 2-hour incubation at 37 ° C was sufficient to achieve a highly significant packaging of minicells.

[0142] To determine if siRNA molecules were inside minicells or adhered to the minicell surface, minicells with Cy3-fluorescently labeled siRNA were incubated with exonucleases overnight and then fluorescence microscopy was repeated. The results were identical to those shown in Figure 1B, indicating that siRNAs were internalized by minicells and did not adhere to the surface of minicells.

2. In vitro transfection of human breast cancer cells with minicells packed with regulators

RNA, targeting the bispecific antibody [0143] 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 experiment was performed.

[0144] SiRNA directed to polynucleotide 1 (Plk1) with target sequence 5'GGTGGATGTGTGGTCCATTTT-3 'was synthesized and labeled with the fluorescent label AlexaFluor 488. Polyokinase has various functions during mitosis, centrosome maturation, bipolar spindle formation, chromosomal segregation and cytokinesis, and, which is crucial, monitoring the reproduction fidelity for the checkpoint (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. Pll1 is associated with cancer and belongs to the family of serine / threonine kinases, which are attractive targets for new chemotherapeutics. Accordingly, Pll1 is considered a promising target in the development of anti-cancer drugs (Strebhardt and Ullrich, 2006).

[0145] The minicells were purified, and 10⁹ minicells were packed anti^AF488Pl1 siRNA, as described in Example 1. A bispecific antibody (BsAb), carrying specificity for S. typhimurium anti-human antigen EGFR, was prepared, and minicells of AF488-Plk1-siRNA were added as described in published PCT application WO 05/056749. The resulting minicells were named^EGFRminikomórkamiAF488-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, the cells were harvested and stained with DAPI (nuclear staining, blue fluorescence). Cells were observed using confocal microscopy IX81 (Olympus) and CellR software.

[0146] After 1 hour the fluorescent, siRNA bearing minicells adhered to MDA-MB468 cells (see Figure 2). This attachment was believed to be due to binding to the minicells BsAb, which were targeting the EGF receptor on MDAMB-468 cells, as the control of incubation with non-target minicells of AF478-Plk1-siRNA was followed by washing and no green fluorescence was observed. associated with MDA-MB468 cells. After 2 hours from the end of incubation,^EGFRminicells AF488-Plk1-siRNA 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 decomposed, presumably in phagolysosomes.

3. Extraction and quantification of siRNAs from intact minicells [0147] As siRNAs do not occur naturally in bacterial cells or minicells of bacterial origin, it is not surprising that there is no established methodology for extracting siRNA from such particles. Accordingly, the present applicants have developed a method for quantitatively extracting siRNAs that are packaged in intact minicells according to the invention.

[0148] Kinesin spindle protein (KSP), also known as "kinesin-5" and "Eg5", is the motor protein of microtubules. It is of key importance for the creation 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 folding control point, and stops cells in the mitotic phase, which leads to cell death (Blangy et al., 1995, Caner et al., 1999; Kapoor et al., 2000; Tao et al. , 2005).

[0149] Selected siRNA against KSP for packaging in minicells was chosen to direct the optimization of siRNA extraction from minicells in accordance with the present invention. More specifically, 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 above.

[0150] Minicells / RNA-KSP (10¹⁰) and a comparable number of empty control minicells were processed using a range of commercially available kits for nucleic acid extraction. The results showed that the mirVana miRNA isolation kit (Ambion) ensured the quantitative extraction of siRNA-KSP from intact minicells. The procedure was carried out according to the manufacturer's instructions.

[0151] The purified siRNAs were first stained with an ultra-sensitive fluorescent dye, RiboGreen ™, Molecular Probes Inc. (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. RiboGreen ™ -linked RNA has a maximum excitation of ~ 500 nm and a maximum emission of ~ 525 nm.

[0152] The results showed that minicells were capable of transferring siRNA. 10¹⁰ empty minicells carried ~ 1.4 μg of RNA, presumably the background level of endogenously formed bacterial RNA. The same number of minikomóreksiRNA-KSP carried ~ 2.7 μg RNA, including endogenous bacterial RNA plus exogenously packed siRNA-KSP. Thus, these data demonstrate that 10¹⁰ minicells may pack at least ~1.3 μg of exogenously packed siRNA.

4. Demonstration in vivo of anti-tumor effect achieved by minicells with packed regulatory RNA [0153] The following studies were performed to show that minicells with packed RNA regulatory can deliver intact regulatory RNA at therapeutically effective concentrations to tumor cells in vivo.

[0154] Selection of siRNA against KSP, as described in Example 3, was performed for packaging in minicells of the present invention. The minicells were purified, and 10⁹ minicells were packed with anti-KSP siRNA as described in Example 1. BsAb was also prepared and attached to minicomicon XRNA-KSP as described in Example 2 to generate ^EGFRminikomórkisiRNA-KSP.

[0155] Mice used in this example were purchased from the Animal Resources Center (Perth, WA, Australia), and all animal experiments were carried out in accordance with the guidelines for the care and use of laboratory animals, following the approval of the Animal Ethics Committee. The experiments were conducted in a small animal facility, accredited by NSW Agriculture at EnGeneIC Pty Ltd (Sydna, New South Wales, Australia).

[0156] Human breast cancer cells (MDA-MB-468, ATCC) were grown in tissue culture in RPMI 1640 medium supplemented with GIBCO-BRL 5% bovine calf serum, Invitrogen Corporation (Carlsbad, Calif. USA) and glutamine (Invitrogen) in humidified atmosphere, with 95% air and 5% CO² at 37 ° C. 1 x 10⁶ The cells in 50 μΐ of serum free medium were mixed with 50 μΐ of reduced growth factor matrizelet, a product of BD Biosciences (Franklin Lakes, New Jersey USA). Using a 23 G needle, the cells were injected subcutaneously between the blades of each mouse. Tumors were measured twice a week using an electric digital caliper (accuracy 0.001), product Mitutoyo (Japan), and the average tumor volume calculated using the formula: length (mm) x width² (mm) X 0.5 = volume (mm³).

[0157] Various therapies were initiated after tumors reached volumes between 170 mm³a 200 mm 3, and mice were randomized into two different groups of eight per group. Control group 1 received a sterile saline solution while the experimental group 2 was receiving^EGFRminikomórkisiRNA-KSP. (10⁹), four times a week.

[0158] As shown in Figure 3, ^EGFRminicellsRNA-KSP provided a highly significant antitumor effect compared to controls with saline. 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 supplied therapeutically relevant concentrations of siRNA to tumor cells in vivo.

5. Demonstration of adapted anti-cancer therapy by treatment with minicells with packed regulatory RNA followed by minicells with packed drug [0159] Most anticancer therapies are associated with drug resistance. The same applies to RNA regulatory treatment, as genetic mutations in cancer cells may render the regulatory RNA ineffective if the target gene is mutated within the sequence that is the target of the regulatory RNA.

[0160] 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 encounters serious difficulties, however, since most anticancer drugs are highly toxic, and combination therapies increase this toxicity, resulting in a dose reduction and frequent interruption of therapy when the patient ceases to deal with toxicity. The following study was carried out to assess the efficacy of minicells with packed regulatory RNA in combating such resistance.

[0161] As described above, minicells were purified and packed (10⁹) antiKSP or anti-Plk1 siRNA. Also, as described before, a bispecific antibody, carrying the anti-human S. typhimuriumi anti-human EGFR specific antigen, was prepared and attached to minikomóreksiRNA-KSP to generate^EGFRminikomóreksiRNA-KSP.

[0162] Human colon carcinoma xenografts (HCT116; ATCC) were established in nude mice as described in Example 3, and were treated as follows: Group 1 mice received sterile saline and mice of groups 2, 3 and 4 were treated, for the first 10 doses (see Figure 4) 10^{9 EGFR}minikomóreksiRNA-Plkl, ^EGFRminikomóreksiRNA-KSP-1 and ^EGFRminikomóreksiRNA-KSP-2. The sequences of Plk1 and KSP-1 were as shown in the examples above. SRNA-KSP-2 (sense strand; 5 'CTGAAGACC TGAAGACAAT 3') is directed to another section of the KSP mRNA. After day 33, mice in groups 2, 3 and 4 were treated with two doses^EGFRminikomórekkarboplatyna.

[0163] The results showed that (Figure 4) after day 26, the tumors became refractory to treatment with siRNA. Accordingly, mice in groups 2, 3 and 4 were treated for four consecutive doses, with all three doses^EGFRminikomóreksiRNA-PLK1 + ^EGFRminikomórkisiRNAKSP-1 + ^EGFRminikomórkisiRNA-KSP-2) combined in equal amounts, i.e. ~ 3 x 10⁸ any type of minicell. In addition, until day 33, tumors were highly resistant to all siRNAs (Figure 4). After administration^EGFRminicellular carcinoma, tumor growth in groups of 3 and 4 mice was significantly slowed down. After administration^EGFRminicellular carcinoma, a significant reduction in tumor volume was found in the 3 mice group.

[0164] These data show that drug-resistant cancer cells can be treated effectively in vivo in accordance with the present invention. In particular, (1) sequentially administering targeted minicells carrying regulatory RNA sequences designed to reduce tumor burden to a significant extent when tumor cells become resistant to siRNA-regulated antitumor activity, using (2) targeted minicells harboring a drug that is it does not affect the same protein that regulatory RNA targets.

6. Demonstration of knockdown of target protein in tumor cells and the resulting arrest of cell growth after targeted delivery of a therapeutically effective amount of regulatory RNA packaged in intact minicells. To demonstrate that innovative methods result in the packaging of therapeutically effective amounts of regulatory RNA in intact minicells , it was necessary to demonstrate that targeting a bispecific antibody, minicells with regulatory RNA, can efficiently and efficiently trigger the arrest of tumor cell growth and induce apoptotic cell death.

[0166] In a humidified atmosphere, 95% air and 5% CO2 at 37 ° C, human epithelial colon carcinoma cells (HCT116) were grown in tissue culture in RPMI 1640 medium, with 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 was specific for S. typhimurium anti-human EGFR antigen. Hence, from minikomóreksiRNAKSP, minikomóreksiRNA-Plk1 and minicells (control) were generated^EGFRminikomórkisiRNA-KSP ^EGFRminikomórkisiRNA-Plk1 and ^EGFRminicells. HCT116 cells were seeded in six-well plates, and the experimental and control groups were transfected at a ratio of 5,000 minicells: 1 cell of HCT116. In addition, control from the cells themselves was included.

[0167] After 2 hours 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. The cells were washed twice in citrate phosphate buffer (pH 7.8) and treated with 100 mg / ml RNAse to ensure that only DNA was stained. The cells were stained with propidium iodide (staining for nucleic acids) and then analyzed using a FACSCalibur ™ flow cytometer, Becton Dickinson (Franklin Lakes, New Jersey USA), Macquarie University (Sydna, Australia), and CELL Quest acquisition-and-analysis software. , also a Becton Dickinson product.

[0168] Cell FACS analysis showed that, at 4 and 8 hours after transfection (Figure 5A), treated cells ^EGFRminikomórkamisiRNA-KSP or ^EGFRminicells + 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 the normal G1, S and G2 phases of the cell cycle. After 16 and 24 hours, the experimental cells showed not only a strong retention in the G2 phase, 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 converted to cellular debris (see in particular the brackets in Figure 7).

[0169] These results demonstrate that targeted intact minicells have a therapeutically effective amount of regulatory RNA packaged, and that the minicells of the invention were highly effective and significantly efficient in the targeted induction of silencing of the protein within the tumor cells, resulting in apoptotic cell death.

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SEQUENCE LIST &lt;110&gt; BRAHMBHATT, HIMANSHU MACDIARMID, JENNIFER HULF, TOBY &lt;120&gt; NON-INFRINGED BACTERIAL CELLS, INCLUDING FREE NEPTIC ACID FOR PLATING INTO NUCLEIC ACID FOR IN VIVO CELLS &lt;130&gt; 060348-0179 &lt;140&gt; 12 / 053,197 &lt;141&gt; 2008-03-21 &lt;150&gt; 60 / 909,074 &lt;151&gt; 2007-03-30 &lt;160&gt; 3 &lt;170&gt; Patentln Ver. 3.3 &lt;210&gt; 1 &lt;211&gt; 21 &lt;212&gt; DNA &lt;213&gt; Artificial sequence &lt;220&gt;

&lt;223&gt; Sequence description: synthetic oligonucleotide &lt;40θ&gt; 1 ggtggatgtg tggtccattt t 21 &lt;210&gt; 2 &lt;211&gt; 21 &lt;212&gt; DNA &lt;213&gt; Artificial sequence &lt;220&gt;

&lt;223&gt; Sequence description: synthetic oligonucleotide &lt;400&gt; 2 aactggatcg taagaaggca g 21 &lt;210&gt; 3 &lt;211&gt; 19 &lt;212&gt; DNA &lt;213&gt; Artificial sequence &lt;220&gt;

&lt;²²'^J&gt;&gt; Description of the sequence:

&lt;400&gt; 3 ctgaagacct gaagacaat synthetic oligonucleotide

EnGeneIC Molecular Delivery Pty Ltd Agent:

PL-PAT-2012-965

EP 2 865 755 B1