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

DESCRIPTION

INTACT BACTERIA-DERIVED MINNICELLS UNDERSTANDING PLASMID-FREE FUNCTIONAL NUCLEIC ACIDS FOR IN VIVO ADMINISTRATION IN MAMMALIAN CELLS

BACKGROUND OF THE INVENTION

Recently, a large number of nucleic acid-based strategies have been developed to modulate a variety of cellular functions (Opalinska and Gewirtz, 2002). Oligonucleotide classes, such as aptamers, oligonucleotides that act as baits and bind to transcription factors, ribozymes, oligonucleotides that form triples, immunostimulatory CpG motifs, antisense oligonucleotides (including peptide nucleic acids), small interfering RNAs, and microRNAs, have A lot of attention has been drawn as research tools because of their highly specific mode of action. These oligomeric nucleic acids also have considerable potential as therapeutic agents. However, these therapeutic agents face several obstacles, including free nucleic acid instability, and the safe, efficient and targeted cellular administration of these macromolecules (Dykxhoorn and Lieberman, 2005).

One focus of many nucleic acid-based therapeutic strategies is the phenomenon of interference RNAs (RNAi), where long double stranded RNA (dsRNA) leads to specific degradation of homologous (complementary or partially complementary) gene transcript sequences in a cell. In particular, long dsRNA molecules are processed into smaller RNAs by an endogenous ribonuclease called Dicer (Grishok et al. 2000; Zamore et al. 2000). Smaller RNAs are known as small interfering RNAs (siRNAs) when they are derived from exogenous sources and as microRNAs (miRNAs) when they are produced from genes encoding RNA in the cell's own genome. These two classes of small regulatory RNAs (typically 21 to 23 nucleotides) also differ in that miRNAs show only partial complementarity with messenger RNA (mRNA) targets.

These small regulatory RNAs bind to the so-called RNAinduced silencing complex (RISC), which contains both a helicase activity and an endonuclease activity. Helicase activity unwinds the two strands of RNA molecules, thus 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 site where the antisense chain is attached.

Thus, in RNAi, a single stranded RNA (ssRNA) molecule binds to the target RNA molecule, following Watson-Crick base-pairing rules, and recruits a ribonuclease that degrades the target RNA. In contrast, antisense suppression of gene expression results in ssRNA binding to mRNA, blocking translation without catalyzing mRNA degradation.

As a class, regulatory RNAs have a half-life in human plasma of less than one hour (Layzer et al., 2004), and are rapidly excreted by the kidneys. As a result, several groups have attempted to prepare regulatory RNAs, including siRNAs, which are nuclease resistant. Examples of such efforts include chemical modification of nucleotides (e.g., 2'-F, 2'-0Me, blocked nucleic acids; LNA), or phosphodiester backbone, for example phosphorothioate linkages (Chiu and Rana 2003; Choung et al. , 2006; Czauderna et al., 2003; Elmén et al., 2005; Layzer et al., 2004; Morrissey et al., 2005). Also, to minimize the time that siRNAs or other regulatory RNAs spend in circulation, the researchers conjugated the RNA molecules to proteins and antibodies to target desired mammalian cells. As additional efforts to address concerns about poor stability and rapid renal excretion, the researchers developed vehicles for the administration of regulatory RNAs. Poliplexes (formed by self-assembling polycation nucleic acids), lipopoliplexes (formed by initial condensation of polycation nucleic acid, followed by the addition of cationic lipids), liposomes, and synthetic nanoparticles have also been explored.

These strategies also face a number of problems, such as (a) the rapid elimination of serum transporter proteins by renal excretion, (b) limited number of regulatory RNA molecules that can be conjugated to each carrier protein, (c) difficulty in dissociation intact cellular regulatory RNAs of the carrier protein, (d) rapid deletion due to serum proteins that bind to polypexes, which may act as opsonins (Dash et al., 1999), and (e) liposome instability in vivo, causing serum nucleic acid release, and potential nonspecific transformation.

Viral vectors have also been developed to produce endogenous regulatory RNAs. See, for example,

Devroe and Silver, 2004. These viral vectors, however, present serious safety concerns. Illustrative examples include wild-type virus recombination, insertion potential and oncogenic potential, virus-induced immunosuppression, limited ability of viral vectors to carry large segments of DNA, attenuated virus virulence reversal, manufacturing and distribution difficulties, 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).

Plasmid-based systems have also been developed for recombinant in situ expression of a regulatory RNA, such as a siRNA or a larger precursor (~ 70 nt), a short hairpin RNA (shRNA). A shRNA contains sense and antisense sequences of a target gene, which are linked by a hairpin loop. See, for example, Paddison et al., 2002. ShRNAs may be expressed from a pol-III type promoter or, in the context of a miRNA, by pol II promoters.

As described in international application WO 03/033519, plasmids encoding a shRNA, siRNA or other regulatory RNA can be transformed into a parental bacterial strain, which produces intact minicells due to a mutation that causes asymmetric cell division. This transformation produces recombinant bacteria, in which the plasmid replicates intracellularly, introducing a large number of plasmids into the bacterial cytoplasm. During asymmetric division, some of the plasmids secrete into the minicell cytoplasm. The mini-cells may then administer the plasmid DNA to a mammalian cell, where the plasmid DNA migrates to the cell nucleus. In the nucleus, plasmid DNA expresses shRNA or other regulatory RNA, as appropriate, and the resulting nucleic acid then migrates to the cytoplasm, where it can act as RNAi or gene suppression, depending on the nature of the regulatory RNA involved.

However, since such approaches require host cell machinery, administration of therapeutically effective amounts of nucleic acid via expression-based systems involves complex and prolonged processes, which limit their effectiveness. Thus, a more effective methodology for the delivery of functional nucleic acids, such as regulatory RNAs, to target cells is required.

WO / 2006/021894 describes the administration of RNA molecules to mammalian cells by bacterial derived minicells. More specifically, the examples disclose plasmids encoding an RNA sequence, which plasmids have been transformed into bacterial cells. The resulting recombinant bacterial cells generate plasmid-receiving minicells, and plasmid-containing minicells were in turn assimilated by mammalian cells into which the plasmids were released, resulting in transcription of the RNA sequence into mammalian cells.

SUMMARY OF THE INVENTION

Thus, according to one aspect of the invention, a composition comprising (a) a diversity of intact bacterially derived minicells, wherein each minicell of said diversity comprises regulatory RNA which is packaged in said minicell, and (b) a pharmaceutically carrier acceptable thereof, wherein there is an absence of a construct for in situ expression of said regulatory RNA in said minicells, wherein said regulatory RNA is selected from the group consisting of siRNA, miRNA and shRNA, and wherein said diversity contains a therapeutically effective amount of regulatory RNA.

Functional nucleic acid packaged in the minicell may target RNA transcripts encoding a protein that contributes to drug resistance, apoptosis resistance, or neoplasticity, inter alia. A composition of the invention also further comprises a bispecific ligand, which consists, for example, of a first arm specific for a mini cell surface structure, and a second arm specific for a surface receptor of a non-phagocytic mammalian cell.

The invention allows a method for administering a functional nucleic acid to a target mammalian cell. The methodology of the invention comprises (a) providing a diversity of intact minicells in a pharmaceutically acceptable carrier, each diversity minicell comprising a functional plasmid-free nucleic acid, and (b) contacting the diversity minicells with cells of a mammalian cells such that mammalian cells engulf diversity minicells, and functional nucleic acid is released into the cytoplasm of target cells. As mentioned, regulatory RNAs such as siRNA, miRNA and shRNA may be directed to RNA transcripts encoding a protein that contributes to drug resistance, apoptosis resistance or neoplasticity. In other embodiments, the methodology further comprises administering a drug other than functional nucleic acid to a target mammalian cell. 0 The drug may be administered after or simultaneously with or even prior to administration of the minicell composition.

According to another aspect, the present invention contemplates a method for formulating minicells of the composition of the invention. The method comprises the simultaneous incubation, in a buffer, of (A) a diversity of intact bacterially derived minicells with (B) plasmid-free regulatory RNA, thereby obtaining minicells containing said regulatory RNA, wherein said Regulatory RNA is selected from the group consisting of siRNA, miRNA and shRNA. In some embodiments, simultaneous incubation may involve gentle shaking, while in other embodiments, simultaneous incubation is static. In some aspects, simultaneous incubation lasts about half an hour, while in others, incubation lasts about one hour. In one embodiment, the buffer comprises buffered saline, for example a phosphate buffer solution IX. In another embodiment, the simultaneous incubation is conducted at a temperature of from about 4 ° C to about 37 ° C, from about 20 ° C to about 30 ° C, about 25 ° C or about 37 ° C. . Simultaneous incubation may comprise 10⁷, 10⁸, 10⁹, ΙΟ¹⁰, 10¹¹, 10¹² or 10¹³ mini cells.

Other objects, features and advantages will be apparent from the following detailed description. The detailed description and specific examples are given for illustrative purposes only, as various changes and modifications within the scope of the invention will be apparent to one skilled in the art from this detailed description. In addition, the examples demonstrate the principle of the invention and cannot be expected to specifically illustrate the application of this invention to all examples where it would obviously be useful to the skilled artisan.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 depicts intact minicells packed with Cy3 fluorophore labeled siRNA. Figure 1A is a light microscope, and Figure 1B shows the same slide but observed under fluorescent light with a 515-560 excitation filter revealing strongly fluorescent siRNA molecules that coincide with the minicells.

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directed to EGFR in human breast cancer cells in vitro.

Figure 3 is a graphical representation of the significant antitumor effect obtained by treatment of human breast cancer xenografts (MDA-MB-468) in nude mice, with EGFR-targeted KSP siRNA minicells. Control group 1 (- ♦ -) received sterile saline, and experimental group 2 (- © -) received^FGFRmini cells_siR_NA-Ksp (10⁹) , Four times a week.

nude, with siRNA KSP targeted, with mini cells

Figure 4 is a graphical representation of the significant antitumor effect achieved by the treatment of human colon cancer xenografts (HCT116) in EGFR-packed, carboplatin-packed, mini-cell-packed mice. Group 1 (- ♦ -) received sterile saline, and group 2 (--Δ--), 3 (-) and 4 (-Θ-) rats were treated for the first 10 doses (see Figure 4) with 10^{9th FGFR}mini cellsSiRNApiki, ^EGFRmini cellsSiRNA-Ksp-i / θ ^FGFRmini cells_si_RNA-Ksp-2z respectively.

Figure 5 provides a FACS analysis, at various time points after transfection, of colon cancer cells (HCT116) treated with experimental minicells, ^EGFRmini-cellsSiRNA-pikl, or ^FGFRmini cellsSiR_AT-_Ksp · Figures 5A to 5D provide FACS analysis of samples taken 4 hours after transfection, while Figures 5E to 5H show sample analysis 8 hours after transfection. At

Figures 5A

5E show cell results only, while Figures 5B ^EGFRempty minicells, + and 5F cell results refer to Figures 5C and ^FGFRmini cells_siR_AT-Ksp to + cells

5G show and Figures

5D and 5H concern + cells ^FGFRmini cells_si_RNA-P_lkl,

Figure 6 provides a FACS analysis of various time points after transfection of colon cancer cells (HCT116) treated with experimental minicells, ^EGFRMini-cellsSipNA-pikl, or ^EGFRmini cellsSiR_AT-_Ksp · Figures 6A to 6D provide FACS analysis of samples taken 16 hours after transfection, while Figures 6E to 6H show sample analysis 24 hours after transfection. Figures 6A and 6E show cell results only, while Figures 6B and 6F refer to + cells.^EGFRempty mini cells. Figures 6C and 6G refer to + cells^FGFRMini-cellsSipNA-Ksp, θ Figures 6D and 6H refer to cells + ^FGFRmini cellsSi_RNA-pi_kl.

Figure 7 provides a FACS analysis of various time points after transfection of colon cancer cells (HCT116) treated with experimental minicells, ^FGFRmini-cellsSiRNA-piki, or ^FGFRmini cellsSi_RNA-_Ksp · Figures 7A to 7D provide FACS analysis of samples collected 32 hours after transfection, while Figures 7E to 7H show sample analysis 48 hours after transfection. Figures 7A and 7E show cell results only, while Figures 7B and 7F refer to + cells.^EGFRempty mini cells. Figures 7C and 7G refer to + cells^EGFRSiRNA-KSPz minicells and Figures 7D and 7H refer to + cells ^FGFRmini cellsSi_RNA-pi_kl.

DETAILED DESCRIPTION OF THE INVENTION Aggressive packaged use of chemicals In this sense, it has been

In accordance with the invention, therapeutically effective amounts of regulatory RNA may be in minicells, without or electroporation.

A simple methodology for the direct packaging of these therapeutically effective concentrations of regulatory RNA into intact minicells has been developed, which methodology does not involve plasmid-based expression constructs, nor the expression machinery of a host bacterial cell. Thus, a polynucleotide segment encoding regulatory RNA is not cloned into a plasmid DNA or viral vector. Instead, plasmid-free functional nucleic acids are packaged directly into the mini cells, passing through the intact membrane of the mini cells. In addition, a minicell-based composition of the invention can safely and effectively deliver therapeutically effective amounts of regulatory RNAs such as siRNAs, miRNAs and shRNAs to target mammalian cells.

Definitions

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

For convenience, the meaning of certain terms and phrases

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Antisense oligonucleotide refers to a nucleic acid molecule, complementary to a portion of a transcript of a particular gene, which may hybridize to the transcript and thereby blog its translation. An antisense oligonucleotide may comprise RNA or DNA.

Biomolecular sequence or sequence refers to all or part of a polynucleotide or polynucleotide sequence.

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

Complementary refers to the topological compatibility or matching of the interaction surfaces of two molecules, such as an siRNA molecule and its target mRNA. The molecules may be described as complementary, and furthermore, the characteristics of the contact surfaces are complementary to each other.

Matches or represents, when used in the context of, for example, a polynucleotide or sequence corresponding to or represents a gene, means that a polynucleotide sequence is present in the gene or gene product of the nucleic acid, for example mRNA . 0 The polynucleotide may be present entirely within one exon of a gene genomic sequence, or different portions of the polynucleotide sequence may be present in different exons, for example, such that the contiguous polynucleotide sequence is present in an mRNA before or after splicing, ie a gene expression product.

A baiting RNA is a molecule that can adopt a structure identical to an important functional region of the target RNA. The latter RNA may be native to the mammalian host or a pathogen that has infected a mammalian cell, for example, HIV. The baiting RNA sequester the protein that normally interacts with the target RNA, resulting in a disruption of normal processing of the mammalian host or pathogen.

Drug refers to any physiologically or pharmaceutically active substance which produces a systemic or local effect in animals, particularly mammals and humans.

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

Functional nucleic acid refers to a nucleic acid molecule which, upon introduction into a host cell, specifically interferes with the expression of a protein. In general, functional nucleic acid molecules have the ability to reduce the expression of a protein by interacting directly with a transcript encoding the protein. Regulatory RNAs such as siRNA, shRNA, small RNAs (typically less than 400 bases in length), microRNAs (miRNAs), ribozymes and baiting RNAs, and antisense nucleic acids are examples of functional nucleic acids.

Gene refers to a polynucleotide sequence comprising control and coding sequences necessary for polypeptide production may be precursor. The sequence of a polypeptide is either encoded by a full length coding or by any part of the coding sequence. A gene may constitute an uninterrupted coding sequence, or may include one or more introns, linked through appropriate splicing junctions. In addition, a gene may contain one or more modifications to the coding region or untranslated region which may affect the biological activity or chemical structure of the expression product, the expression rate, or the form of expression control. . These modifications include, but are not limited to, mutations, insertions, detections, and substitutions of one or more nucleotides. In this regard, these modified genes may be referred to as native gene variants.

Host cell refers to a cell that can be, or has been, used as a recipient for a recombinant plasmid or other polynucleotide transfer, and includes the progeny of the original cell that was transfected. The progeny of a single cell may not necessarily be completely identical in morphology or in genomic or total DNA, complementing the original parent, due to mutations, which may be natural, accidental or deliberate.

Hybridization refers to any process whereby a polynucleotide sequence binds to a complementary sequence by base pairing.

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

Marker refers to agents that are capable of providing a detectable signal either directly or by interacting with one or more additional members of a signal producing system. Labels that are directly detectable and which may be used 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 glass beads or colored plastic (eg polystyrene, polypropylene, latex) may also be used. See, for example, US Pat.

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.

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

Mini-cell refers to non-nucleated forms of bacterial cells produced by a disturbance in coordination during binary fission of cell division, with DNA segregation. Mini-cells are distinct from other small vesicles that are spontaneously generated and released in certain situations, and are not due to specific genetic rearrangements or expression of episomal genes. In the context of the present invention, the minicells are intact, since other naked forms, protoplasts, such as leaving spheroplasts, poroplasts, extravasated packaged functional nucleic acid, and would not be therapeutically effective. The intact membrane of the minicell allows the charge to remain within the minicell, and is released intracellularly within the target host mammalian cell.

In this description, chemically modified and modified, refers to oligonucleotides or polynucleotides with one or more chemical changes to the natural molecular structures of all or some of the internucleoside bases, sugar groups and phosphate linkages, as well as molecules with added substitutions, or a combination of changes at these sites. Internucleoside phosphate bonds may be phosphodiester, phosphotriester, phosphoramide, siloxane, carbonate, carboxymethylester, acetamide, carbamate, thioether, phosphoramidate bridge, methylene phosphonate bridge, phosphorothioate, methylphosphonate, phosphorodithioate or sulfonate bond, '-3', 5'-3 'or 5'-5', and combinations of bonds similar thereto. The phosphodiester bond may be substituted by a substitution bond, such as phosphorothioate, methylamino, methylphosphonate, phosphoramidate, and guanidine, and the ribose subunit of polynucleotides may also be substituted (e.g., hexose phosphodiester; peptide nucleic acids). Modifications may be internal (single or repeated) or to or at the ends of the oligonucleotide molecule, and may include additions to the molecule of internucleoside phosphate bonds, such as deoxyribose and phosphate modifications, which cleave or cross-link to opposite chains. , or the associated enzymes, or other proteins. The terms modified oligonucleotides and modified polynucleotides also include oligonucleotides or polynucleotides comprising modifications to sugar groups (for example, 3'-substituted ribonucleotide or deoxyribonucleotide groups), any of which are linked via 5 'to 3' bonds.

The phrase nucleic acid molecules and the term polynucleotides mean polymeric forms of nucleotides of any length, whether ribonucleotides or deoxyribonucleotides. These include DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or a single, double or multiple stranded polymer comprising purine and pyrimidine bases, or other chemically or biochemically unnaturally modified natural nucleotide bases, or derivatives. 0 A polynucleotide backbone may comprise sugars and phosphate groups (as may typically be found in RNA and DNA) or modified or substituted sugars or phosphate groups. Alternatively, the polynucleotide backbone may comprise a polymer of synthetic subunits such as phosphoramidites and thus may be a phosphoramidate oligodeoxynucleoside or a mixed phosphoramidate phosphodiester oligomer. A polynucleotide may comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs, uracil, other sugars, and linking groups, such as fluororibose and thioate, and nucleotide branches. A polynucleotide may be further modified, for example by conjugation to a labeling component. Other types of modifications include capsules, replacement of one or more of the naturally occurring nucleotides with an analog, and introduction of means for binding the polynucleotide to proteins, metal ions, labeling components, other polynucleotides, or a solid support.

Pharmaceutically acceptable concerns physiological compatibility. A pharmaceutically acceptable carrier or excipient does not negate the biological activity of the composition to be administered, is chemically inert and is non-toxic to the organism to which it is to be administered.

Free plasmid qualification means the absence of a construct, such as a plasmid or viral vector, for in situ expression of a functional nucleic acid.

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

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

Regulatory RNA means an inclusive category of RNAs that affect interference RNA expression, suppression of gene expression, or another mechanism. Thus, in addition to antisense shRNA, siRNA, miRNA and ssRNA, the category of regulatory RNAs includes ribozymes and baiting RNAs, inter alia.

Ribozyme refers to an RNA molecule, with an enzymatic activity that can repeatedly cleave other RNA molecules, in a way that is specific to the nucleotide base sequence.

Interference RNA (RNAi) means an RNA-driven mechanism as described above, which involves the degradation of a complementary or partially complementary target RNA for the specific regulation of a sequence or gene in gene expression (protein synthesis).

Sequence identity means a degree of similarity or complementarity. There may be a partial identity or a complete identity. A partially complementary sequence is a sequence that inhibits, at least partially, an identical sequence from hybridizing to a target polynucleotide; is referred to using the substantially identical functional term. Inhibition of sequence hybridization completely complementary to the target sequence can be examined using a hybridization assay (Southern blot or northern blot, solution hybridization, and the like) under stringent conditions. A substantially identical sequence or probe will compete for binding, and inhibit the same (i.e. hybridization) of a sequence or probe completely identical to the target sequence under stringent conditions. This is not to say that low stringency conditions are such that nonspecific binding is permitted; low stringency conditions mean that the binding of two sequences to each other must be a specific (ie selective) interaction. The absence of nonspecific binding can be tested by using a second target sequence, which does not even have a partial level of complementarity (e.g., less than 30% identity); In the absence of non-specific binding, the probe will not hybridize to the second non-complementary target sequence.

Another way of looking at sequence identity in the context of two nucleic acid or polypeptide sequences includes referencing residues in the two sequences that are the same when aligned for maximum match in a specified region. As used herein, percent sequence identity means the value determined by comparing two optimally aligned sequences in a comparison window, wherein the portion of the polynucleotide sequence in the comparison window may comprise additions or deletions (i.e. intervals) , compared to the reference sequence (which does not comprise additions or deletions), for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which identical nucleic acid bases occur in both sequences to generate the number of matching positions by dividing the number of matching positions by the total number of positions in the comparison window, and multiplying the result by 100 to obtain the percent sequence identity.

Small interfering RNAs (siRNA) refers to double-stranded RNA molecules, usually 10 to 30 nucleotides in length, that are capable of mediating RNA interference (RNAi). In general, siRNA molecules have the ability to reduce the expression of a protein through direct interaction with a transcript encoding the protein.

A therapeutically effective amount of a functional nucleic acid is a dose of the molecule in question, for example free siRNA, miRNA or shRNA, which results in a pharmacological response when administered to a subject according to the present invention. However, in the context of the present invention, a therapeutically effective amount may be measured with reference to the prevention or amelioration of a condition or symptom associated with an adverse disease or disorder in an animal model or human subject when functional nucleic acid packed mini cells are administered as described in more detail below. An amount that is found to be 100% effective similar to that although a therapeutically effective amount under a given circumstance, for a particular subject, may not be from the subjects treated in the disease or condition under consideration, the dose is considered therapeutically effective amount by the expert in the art. The appropriate dosage in this regard also varies depending upon, for example, the type, stage, and severity of the disease or condition to be affected. In any case, the present illustration of in vitro test assay (Example 2) and in vivo test assay (Examples 4, 5 and

6) According to the methodology for the present invention, thus quantification of the amount of a functional nucleic acid molecule delivered by the minicells (Example 3), when considered in the light of the entire description, allows one skilled in the art, experienced in Preclinical and clinical tests of drug candidates determine, by routine experimentation, a therapeutically effective amount of functional nucleic acid for a particular indication.

The terms treatment, treating, treating and the like refer to the achievement of a desired pharmacological and / or physiological effect. The effect may be prophylactic in terms of complete or partial prevention of a disease or symptom thereof, and / or may be therapeutic in terms of stabilization or partial or complete cure for a disease and / or adverse effect attributed to the disease. Treatment includes any treatment of a disease in a mammal, in particular a human, and includes: (a) preventing the disease or symptom from occurring in a subject that may have a predisposition to the disease or symptom but has not yet been diagnosed; (b) inhibit the symptom of the disease, ie, stop its development; or (c) alleviate the symptom of the disease, that is, cause regression of the disease or symptom.

Mini Cells

The minicells of the invention are nucleus-free forms of E. coli or other bacterial cells, produced by a disturbance in the coordination, during binary fission, of cell division with DNA segregation. Chromosomal replication in prokaryotes is associated with normal binary fission, which involves the formation of a septum in the middle of the cell. In E. coli, for example, mutation of min genes, such as minCO, can remove inhibition of septum formation at cell poles during cell division, resulting in the production of a normal daughter cell and a nucleic minicell. See de Boer et al. , 1992; Raskin &amp; de Boer, 1999; Hu &amp; Lutkenhaus, 1999; Harry, 2001. Mini-cells are distinct from other small vesicles that are spontaneously generated and released in certain situations and, in contrast to mini-cells, are not due to specific genetic rearrangements or expression of episomal genes. In a preferred embodiment, the mini cells have intact cell walls (intact mini cells).

In addition to mutations in the min operon, nucleic minicells are also generated following a variety of other genetic rearrangements or mutations, which affect septal formation, for example, in divIVBl in B. subtilis. See Reeve and Cornett, 1975. Minicells may also be formed following a disturbance of the levels of gene expression of proteins involved in cell division / chromosome segregation. For example, overexpression of min leads to polar division and minicell production. Similarly, chromosome-free minicells may result from defects in chromosome segregation, for example the smc mutation in Bacillus subtilis (Britton et al., 1998), the spoOJ deletion in B. subtilis (Ireton et al., 1994). , the mukB mutation in E. coli (Hiraga et al., 1989) and the parC mutation in E. coli (Stewart and D'Ari, 1992). Gene products may be provided in trans. For example, when CafA is overexpressed from a high copy number plasmid, it may increase the rate of cell division and / or inhibit chromosome breakdown after replication (Okada et al., 1994), resulting in cell formation. chained and without nuclei (Wachi et al., 1989). Mini cells may be prepared from any bacterial cell of Gram negative or Gram-negative origin.

In one aspect, the mini-cells may contain one or more plasmid-free functional nucleic acids for which an application vehicle is desired. The functional nucleic acid of the invention has the ability to reduce expression of a protein by direct interaction with a transcript encoding the protein.

Packaging of Functional Nucleic Acid in Intact Mini Cells Functional nucleic acid can be packaged directly into intact mini cells. The process avoids the previously required steps of, for example, cloning nucleic acids encoding functional nucleic acid expression plasmids, transformation of parental bacteria that produce minicells with the plasmids, and production of recombinant minicells. Instead, plasmid-free functional nucleic acid can be directly packed into intact minicells by simultaneously incubating a diversity of intact minicells with functional nucleic acid in a buffer. In some embodiments, simultaneous incubation may involve gentle shaking, but in others, simultaneous incubation is static. A simultaneous incubation period of about one hour has been shown to be sufficient, but shorter time periods, such as about half an hour, may also be effective. In one embodiment, the buffer comprises buffered saline, for example a phosphate buffer solution IX. The buffered saline may be in gelatin form. In another embodiment, simultaneous incubation is conducted 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 aspects, simultaneous incubation may comprise about 10⁷, ΙΟ⁸, 10⁹, IO¹⁰, 10¹¹, 10¹² or 10¹³ mini cells. Specific parameters of temperature, time, buffer, minicell concentration, etc. can be optimized for a particular combination of conditions.

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The success of this strategy is now beginning because, over the course of four decades, researchers developed chemical and electrochemical processes (Miller, 1994) to transform acidic bacterial cells. The researchers used aggressive measures because conventional knowledge has always assumed that nucleic acids such as siRNA, miRNA or shRNA without plasmid are too large to passively enter the minicell cytoplasm. For example, porins, which are beta-barrel proteins that typically function as diffusion pores, allow passive transport along the bacterial outer membrane of molecules with molecular weights of 600 daltons or less (Nikaido, 1994).

However, double stranded plasmid DNA encoding shRNA exceeds one million daltons, and the double stranded siRNA or mRNA exceeds 15,000 daltons.

In addition, once packaged, functional nucleic acids remain within the minicell, and are protected from degradation. In this regard, prolonged incubation studies with packed siRNA minicells in sterile saline showed no leakage of siRNAs. In addition, the simultaneous incubation of nuclease-packed siRNA minicells confirmed that the siRNAs had penetrated the outer membrane of intact minicells, and were protected from degradation. Similarly, despite the fact that they may carry residual nucleases of the parental bacterial cytoplasm minicells, the packed siRNAs are stable in the packed siRNAs also degradation present in the minicell cytoplasm. They prevent phagolysosome manipulation such as acids, free oxygen radicals and acid hydrolases (Conner and Schmid, 2003) for the effect of silencing target mRNA in the host mammalian cell.

In other embodiments, multiple functional nucleic acids directed against different target mRNAs may be packaged in the same minicell. This strategy can be used for exhibit resistance by chemo therapy.

combat drug resistance apoptosis. For example, cancer patients with drug being routinely mediated, resistance. This resistance may overexpress genes such as multiple drug resistance pumps (MDR), and antiapoptotic genes, among others. To combat this resistance, minicells may be packaged with significantly therapeutic concentrations of functional nucleic acids for MDR-associated genes, and administered to a patient prior to chemotherapy. In addition, packaging on the same minicell of multiple functional nucleic acids for different target mRNAs can improve therapeutic success, since most molecular targets are mutated and have multiple alleles.

Thus, packaging plasmid-free functional nucleic acids directly into intact minicells, as described herein, offers numerous advantages. For example, since the approach of the invention does not necessarily require genetic modification of parental bacteria to accommodate expression of functional nucleic acids, a parental bacterium can be used to produce minicells comprising many types of nucleic acids directed to a variety of nucleic acids. indications. Similarly, a mini cell can be loaded with a variety of different RNAs, thereby avoiding or overcoming resistance mechanisms.

Functional Nucleic Acids

As noted above, functional nucleic acids means a category including nucleic acid molecules, which affect expression via interfering RNA, suppression of gene expression, or other mechanism. These molecules are exemplified by single, double or multiple stranded DNA or RNA. Examples of functional nucleic acids include, but are not limited to, regulatory RNA, such as antisense shRNA, siRNA, miRNA, and ssRNA, therefore, ribozymes, and bait RNAs and antisense nucleic acids.

In a preferred embodiment of the invention, intact minicells carry siRNA molecules. Small interference RNA molecules are useful for carrying out RNAi, a mechanism for post-transcriptional genetic silencing. As noted, siRNA generally refers to double stranded RNA molecules, about 10 to 30 nucleotides in length, which are designated for their ability to specifically interfere with protein expression. Preferably, siRNA molecules have a length of 12-28 nucleotides, more preferably a length of 15-25 nucleotides, even more preferably a length of 19-23 nucleotides, and even more preferably a length of 21-23 nucleotides. Thus, siRNA molecules having a length of 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28 or 29 nucleotides are preferred.

The length of a strand determines the length of an siRNA molecule. For example, an siRNA which is described as having a length of 21 ribonucleotides (one 21mer) may comprise two opposite strands of RNA, which hybridize together to 19 contiguous base pairs. The other two ribonucleotides in each chain would in this case form a protrusion. When an siRNA contains two strings of different lengths, the length of the longest string determines the length of the siRNA. For example, a dsRNA containing a 21 nucleotide length strand and a 20 nucleotide length second strand is a 21 mer.

SiRNAs comprising a protrusion are desirable. The protrusion may be at the 5 'end or the 3' end of a chain. Preferably, it is at the 3 'end of the RNA strand. The length of a protrusion may vary, but is preferably from about 1 to about 5 bases, and more preferably, is 2 nucleotides. The siRNA of the present invention comprises a 3 'overhang of preferably about 3 ribonucleotide overhang. Even more ribonucleotides preferably comprising 2 to 4 bases, preferably having a length, 3 'overhang are a

More than 2 the 2 uridine (U).

ShRNAs comprise a single stranded RNA that forms a hairpin-loop structure, wherein the hairpin consists of the complementary sense and antisense strands, which comprise a double stranded siRNA, and the loop is a linker that may have different sizes. The shRNA hairpin structure is generally about 10 to about 30 nucleotides in length. Preferably, the hairpin of shRNA molecules has a length of 12-28 nucleotides, more preferably a length of 15-25 nucleotides, even more preferably a length of 19-23 nucleotides, and even more preferably a length of 21-28 nucleotides. 23 nucleotides. Thus, preferred shRNA molecules comprise hairpins of 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28 or 29 nucleotides in length.

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

Thus, in one aspect of the invention, intact minicells may carry one or more siRNA sequences intended for silencing genes involved in drug resistance or apoptosis resistance. Using minicells encoding multiple siRNAs, cells expressing multiple drug resistance mechanisms can be treated.

Tools that assist in the design of siRNA and regulatory RNAs in general are easily accessible to the public. For example, it is available on the internet at www.dharmacon.com, an siRNA design tool.

Functional Nucleic Acid Targets

Functional nucleic acids according to the invention target the gene or transcript of a protein that promotes drug resistance, inhibits apoptosis, promotes a neoplastic phenotype, inhibits proliferation of pathogens, or inhibits replication or proliferation of virus. Successful application of functional nucleic acid strategies in these contexts is already described in the state of the art, but without the benefits of minicell vectors. See, for example, Sioud (2004), Caplen (2003), Wu et al. (2003), Yague et al. (2004).

Proteins that contribute to drug resistance, or promotion of a neoplastic phenotype, are the preferred targets of functional nucleic acids. Proteins may contribute to acquired drug resistance or intrinsic drug resistance. When diseased cells, such as tumor cells, initially respond to drugs but become resistant in subsequent cycles of treatment, the resistant phenotype is acquired. Useful targets include, but are not limited to, ATP-binding cassette carriers such as Pglycoprotein (P-gp, P-170, PGY1, MDR1, ABCB1, MDR-associated protein, multidrug resistance protein 1, MDR-2 and MDR-3, MRP2 (multidrug resistance-associated protein), BCR-ABL (Abelson protoncogene breakdown region), STI-571 resistance-associated protein, lung resistance-associated protein, cyclooxygenase- 2, kappa nuclear factor, XRCC1 (group 1 x-ray cross-complement), ERCC1 (excision cross-complement gene), GSTP1 (glutathione Stransferase), mutant beta-tubulin, Abcbla (ABCB4), Abccl, Abcc2, Abcc3 (MLP-2), Abcc5, Abcc6, Abcd2, Abcg2, Bax, Bcl2, Bcl21 (bcl-x), Mvp, Rbl, Topl, Top2a, Top2b, Trp53 (p53). Other genes involved in drug resistance also include (a) genes involved in drug metabolism, e.g. , Cyp2el, Dhfr, Ephxl, Ephx2, Gstml (MGST1), Gstpl, Nat2, Nqol, Sodl, Ste, Tpmt, Tyms, Ugcg, (b) genes involved in DNA repair, e.g., Apc, Atm, Brcal, Brca2 , Ercc3 (XPB), Mgmt, M1hl, Xpa, Xpc, (c) genes involved in the cell cycle, for example, Ccndl (cyclin D1), Ccnel (cyclin E1), Cdkl, Cdk2, Cdk4, Cdknla (p21Wafl), Cdknlb (p27Kipl), Cdkn2a (p16Ink4a), Cdkn2d (p19), KSP., (d) genes involved in the growth factor receptors, e.g., Egfr, Erbb2 (Neu, HER2), Erbb3, Erbb4, Fgf2 (bFGF), Met, (e) genes involved in hormone receptors, for example, Ar, Esrl, Esr2, Igf2r, Ppara, Ppard, Pparg, Ppargcl, Rare,

Rarb, Rxra, Rxrb, Rxrg, Srd5a2, and (f) genes involved in transcription factors, for example, Ahr, Aplsl, Apls2, Elkl, Fos (c-fos), Gabpa, Hifla, Mafb, Myc (c-myc ), Nfkbl, Nfkb2, Nfkbib, Nfkbie, Relb (1-rel), TnfrsfllA.

Useful targets also include proteins that contribute to apoptosis resistance. These include Bcl-2 (B-cell leukemia / lymphoma), Bcl-X_L, Al / Bf11, focal adhesion kinase, and mutant p53 protein.

Useful targets further include oncogenic proteins and mutated tumor suppressors. Examples include beta-catenin, PKC-alpha (protein kinase C), C-RAF, K-Ras (V12), h-Ras, DP97 Dead box RNA helicase, DNMT1 (DNA methyltransferase 1), FLIP (Flice inhibitory protein), C-Sfc, 53BPI, Polycomb group protein EZH2 (zest homolog enhancer), ErbBl, HPV-16 E5 and E7 (early 5 and early 7 human papillomavirus), Fortilin &amp; MCI1P (myeloid cell leukemia protein 1) , DIP13a (DCC-interacting protein 13a), MBD2 (methylated CpG binding domain), p21, KLF4 (Kruppel-like factor 4), tpt / TCTP (translation controlled tumor protein), SPK1 &amp; SPK2 (sphingosine kinase), P300, PLK1 (Polo-like kinase-1), Trp53, Ras, ErbBl, VEGF endothelial vascular growth), and BAG-1 (BCL2-associated athanogene 1).

A large number of molecular targets for cancer treatment have been identified, and RNAi discovery platforms are rapidly identifying a variety of different new targets. Examples of such molecular targets useful in this invention include tyrosine kinases (variant), Akt (protein kinase Β, PKB), Aktl, AlfaLbeta2 integrin, aminopeptidase, androgen receptor, Aurora A, Aurora B, fibroblast growth factor receptor basic (bFGF) (bFGFr), BRaf, carcino-embryonic antigen (CEA), CD142, CD37, CD44, CD5, CD74, CD77, Chkl, CHK2, CHras, ​​CSFlr, CXCR4, cyclin D1 (CCND1), cyclin dependent kinase 1 (CDK1), cyclin 2-dependent kinase (CDK2), cyclin-dependent kinase inhibitor 1B (CDKN1B, p27, KIP1), CYP26, fibroblast growth factor 3 receptor (FGFr3), fibroblast growth factor 4 receptor (FGFr4), G250, hedgehog signaling pathway (Hh) , hepatocyte growth factor / scatter factor (HGF / SF or SF / HGF), HEr4 (ErbB4), HIF, histone deacetylase 9 (HDAC9), Homeobox gene (H0XB7), hyaluronan (HA), insulin-like growth factor (IGF), insulin-like growth factor 1 receptor (IGFlr, IGFlr, IGFIr, IGFlr), insulin-like growth factor 2 binding protein (IGFBP2), insulin-like growth factor 5 binding protein (IGFBP5), kinase integrin-like (ILK), interleukin receptor (IL6), interleukin 1 (IL1) receptor type II, interleukin 10 (IL10), interleukin 4 (IL4) receptor, interleukin 6 (IL6), interleukin 15 (IL15) , interleukin 3 receptor alpha chain (IL3r alpha), JAK, JAK3, JNK1, JNK2, Kinesin Spindle Protein (KSP), laminin 5, Lewis (b), lymphotoxin beta receptor (LTBr), lysophosphatidic acid receptors (LPA) (LPAr), lysophosphatidic acid acetyltransferase, macrophage migration inhibitory factor (MIF), MAGE3, microtubules, MUC2, Notch 1 (TAN1), P38 (p38 MAPK) -mediated apogenesis mediator protein kinase p53 (PUMA), PDGF tyrosine kinase (TK) signaling pathway, phosphatase and tensin homolog (PTEN), phosphatidylinositol 3'kinase (PI3K) signaling, plasminogen activator, urine kinase receptor (PLAU), Pololike kinase 1 (Plkl), Poly (ADP ribose), polymerase (PARP), cell proliferation nuclear antigen (PCNA), prostate stem cell antigen (PSCA), prostate specific antigen (PSA) 773, protein tyrosine phosphatase (PTP), Rad51 protein, RAF1, alpha retinoic acid (RAr) receptor, retinoic acid gamma receptor (RAr), retinoid beta X receptor (RXr), serine (or cysteine) proteinase inhibitor, telomerase reverse transcriptase (TERT, hTERT), telomeres, Thomsen Friedenreich (TF) antigen, thrombospondin 1 (TSP1) ), transferrin, tumor necrosis factor alpha (TNFα, TNFA), tumor necrosis factor alpha receptor (TNFr, TNFr), tumor-associated carbonic anhydrase (CA) IX (CA9), interferon type I, ubiguitin ligase, molecule vascular cell adhesion 1 (VCAM1, CD106), vascular endothelial growth factor (VEGF, VEGFA), vascular endothelial growth factor D (VEGFD), vitronectin (VTN), Wilms' tumor 1 (WT1) etc.

Regarding HIV infection, targets include HIV-Tat, HIV-Rev, HlV-Vif, HIV-Nef, HIV-Gag, HIV-Env, LTR, CD4, CXCR4 (guimiocin receptor) and CCR5 (guimiocin receptor). ).

Due to the heterogeneity of tumor cells, a number of different drug resistance or apoptosis resistance pathways may be active in the target cells. Therefore, functional nucleic acids used in the methods of the invention may need to be altered over time. For example, if biopsy samples reveal new mutations that result in drug resistance, specific functional nucleic acids can be designed that are packaged into intact minicells that are administered to the mammalian host to attack drug resistance.

Administration of Functional Nucleic Acids via Intact Mini Cells

The invention allows a method of administering functional nucleic acids comprising (a) providing a diversity of intact minicells in a pharmaceutically acceptable carrier, each minicell of the diversity comprising plasmid free functional nucleic acids, and (b) placing the minicells. diversity in contact with mammalian cells, such that mammalian cells engulf diversity mini-cells, the nucleic acids being released into the cytoplasm of the target cells. Mini cells are contacted with the mammalian cell by bispecific ligands as described in PCT published application WO 05/056749. Contact between the mini cells and the target mammalian cell may occur in vitro or in vitro.

Method to Overcome Resistance to Disease Treatment

Drugs put them functional in

The invention further allows a method for overcoming drug resistance and treating a disease, such as cancer or AIDS, in an individual. The method comprises (a) packaging one or more targeting nucleic acids or target protein transcripts, which promote drug resistance, in intact purified minicells, (b) nucleic acid-containing minicells in contact with a target mammalian cell such that the mammalian cell engulfs the minicell as described in the aforementioned PCT application 749, and (c) administering a drug to the target mammalian cell, as described in PCT published application WO 05/079854. Preferably, step (c) is performed after steps (a) and (b) to allow the functional nucleic acid to decrease drug resistance prior to administration thereof. Drug administration and introduction of functional nucleic acids may occur consecutively, in any order, or simultaneously.

The drugs may be administered by any conventional route. For example, drugs may be administered orally, parenterally (including subcutaneously, intravenously, intramuscularly, intraperitoneally, and by infusion), topically, transdermally or by inhalation. The appropriate mode of administration and dosage for each drug is readily determined by those skilled in the art.

Drug Administration through Minicell Intermediate

Although drug administration may be by conventional routes, administration via minicells is preferred as described in PCT published application WO 05/079854. In this regard, the inventors have found that the same mammalian cells can be successfully transfected again by intact minicells which are packaged with different charges. For example, functional nucleic acid-packed mini-cells may transfect a mammalian cell, after which drug-packed mini-cells may deliver the drug to the same mammalian cell for a complementary or synergistic antitumor effect.

The drug may be packaged in a different minicell than that containing the functional nucleic acid. Alternatively, the drug may be packaged in the same mini-cell as the functional nucleic acid. Certain drugs may interact with nucleic acids and prevent simultaneous packaging of the drug and nucleic acid in the same minicell. For example, it is known that doxorubicin interacts with DNA.

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

Hydrophilic and hydrophobic drugs may be packaged in minicells, creating a gradient of drug concentration between an extracellular medium containing minicells and the minicell cytoplasm. When the extracellular medium contains a higher drug concentration than the minicell cytoplasm, the drug will move in this concentration gradient towards the lowest concentration to the minicell cytoplasm. When the concentration gradient is reversed, however, the drug does not move out of the mini cells. The procedure and mechanisms for drug loading into mini cells corresponds to that described in PCT published application WO 05/079854.

To load mini cells with drugs that are not normally soluble in water, drugs may initially be dissolved in an appropriate solvent. For example, paclitaxel may be dissolved in a 1: 1 mixture of ethanol and cremofor EL (polyethoxylated castor oil), followed by a dilution in PBS to achieve a solution of paclitaxel which is partially diluted in aqueous medium and which carries amounts minimum organic solvent to ensure that the drug remains in solution. Mini-cells may be incubated in this final medium for drug loading. Thus, the inventors have found that even hydrophobic drugs can diffuse into the minicell cytoplasm to achieve high and therapeutically significant cytoplasm drug loading. This is unexpected because the minicell membrane is composed of a bilayer of hydrophobic phospholipids, which would be expected to prevent the spread of hydrophobic molecules to the cytoplasm.

Another method for loading minicells with a drug involves culturing a parental bacterial cell, under conditions such that the parental bacterial cell transcribes and translates a nucleic acid encoding the drug, and the drug is released into the parental bacterial cell cytoplasm. For example, a set of genes encoding the cellular biosynthetic pathway for a desired drug may be cloned and transferred to a parent bacterial strain which is in turn capable of producing the mini-cells. Genetic transcription and translation of the gene pool results in drug biosynthesis within the parental bacterial cell cytoplasm, filling the bacterial cytoplasm with the drug. When the parental bacterial cell divides and forms descending minicells, the minicells also contain the drug in their cytoplasm. Prepackaged minicells may be purified by any minicell purification process, including the methodology described above.

Equivalently, another method for loading minicells with a drug involves culturing a recombinant minicell containing a drug-encoding expression plasmid under conditions such that the gene encoding the drug is transcribed and translated into the minicell. .

Pharmaceuticals

Drugs useful for the invention may be any physiological or pharmacologically active substance which produces a local or systemic effect on animals, particularly mammals and humans. The drugs may be inorganic or organic compounds, without limitation, including peptides, proteins, nucleic acids, and small molecules, any of which may be characterized or not. They may exist in various forms, such as unmodified molecules, molecular complexes, pharmacologically acceptable salts, such as hydrochloride, hydrobromide, sulfate, laurate, palmitate, phosphate, nitrite, nitrate, borate, acetate, maleate, tartrate, oleate, salicylate, and others. For acidic drugs, salts of metals, amines or organic cations, for example, quaternary ammonium may be used. Derivatives of drugs such as bases, esters and amides may also be used. A drug that is water-insoluble may be used in a form that is a water-soluble derivative thereof, or a derivative base thereof, which in any case, or by its administration, is converted by enzymes, hydrolysed to body pH, or by other metabolic processes to the original therapeutically active form.

Useful drugs include chemotherapy agents, immunosuppressive agents, cytokines, cytotoxic agents, nucleolytic compounds, radioactive isotopes, receptors, and prodrug activating enzymes which may occur naturally or be produced by recombinant methods.

Drugs that are affected by multidrug resistance have a special utility in the invention; vinca alkaloids (eg vinblastine and vincristine), anthracyclines (eg doxorubicin and daunorubicin), RNA transcription inhibitors (eg actinomycin D) and microtubule stabilizing drugs (eg paclitaxel).

In general, chemotherapy agents for cancer are the preferred drugs. Cancer chemotherapy drugs which are useful include nitrogen mustards, nitrosoureas, ethyleneimine, alkane sulfonates, tetrazine, platinum compounds, pyrimidine analogs, purine analogs, antimetabolites, folate analogs, anthracyclines, taxanes, vinca alkaloids, topoisomerase and hormonal agents. Examples of chemotherapy drugs are actinomycin D, alkeran, ara-C, anastrozole, asparaginase, BiCNU, bicalutamide, bleomycin, busulfan, capecitabine, carboplatin, carboplatin, carmustine, CCNU, chlorambucil, cisplatin, cladribine, CPT-11, cyclopham, CPT-11, , cytosine arabinoside, cytoxan, dacarbazine, dactinomycin, daunorubicin, dexrazoxane, docetaxel, doxorubicin, DTIC, epirubicin, ethyleneimine, etoposide, floxuridine, fludarabine, fotemustine, gemcitabine, hydroxyurea, idarubicin, lomustine, mechlorethamine, methotrexate, oxaliplatin, plicamycin, flutamide, hexamethylamine, irinotecan, mercaptopurine, my toxantrone, pentostatin, steroids, streptozocin, mitomycum, paclitaxel, procarbazine, herpetranephylamide, hydrothexamide, ,

STI-571, streptozocin, tamoxifen, temozolomide, teniposide, tetrazine, thioguanine, thiotepa, tomudex, topotecan, treosulfan, trimetrexate, vinblastine, vindesine, vinorelbine, VP 16, and xeloda.

Useful cancer chemotherapy drugs also include alkylating agents, such as thiotepa and cyclophosphamide; alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamellamines including altretamine, triethylenomelamine, triethylenophosphoramide, triethylenephosphoramide and trimethylolomelamine; mustards

<td>of nitrogen</td><td>such as chlorambucil, chlornafazine,</td>

<td>colophosphamide,</td><td>estramustine, ifosfamide, mechlorethamine,</td>

<td>hydrochloride</td><td>mechlorethamine oxide, melphalan,</td>

<td>novembiehin,</td><td>phenesterine, prednimustine, trophosphamide,</td>

<td>mustard</td><td>uracil; nitroureas like carmustine,</td>

<td>chlorozotocin,</td><td>fotemustine, lomustine, nimustine, and</td>

<td>ranimustine;</td><td>antibiotics such as aclacinomisins,</td>

<td>actinomycin,</td><td>autramycin, azaserine, bleomycin,</td>

<td>cactinomycin,</td><td>caligeamycin, carbine, Carminomycin,</td>

<td>carzinophylline,</td><td>chrominicin, dactinomycin, daunorubicin,</td>

<td>detorubicin,</td><td>6-diazo-5-oxo-L-norleucine, doxorubicin,</td>

<td>epirubicin,</td><td>esorubicin, idambicin, marcelomycin,</td>

mitomycins, mycophenolic acid, nogalamycin, olivomycin,

<td>peplomycin,</td><td>potfiromycin, puromycin, guelamycin,</td>

<td>rhodubicin,</td><td>streptonigrine, streptozocin,</td>

<td>tubercidine,</td><td>ubenimex, zinostatin and zorubicin;</td>

antimetabolites such as methotrexate and 5-fluorouracil (5FU); folic acid analogs such as denopterin, methotrexate, pteropterin, and trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine, and thioguanine; pyrimidine analogs such as ancitabine, azacytidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocytabine, floxuridine, and 5-FU; androgens such as calusterone, dromostanolone propionate, epithiostanol, Rnepitiostane, and Testolactone; anti-adrenals such as aminoglutethimide, mitotane, and trilostane; folic acid replenisher, such as frolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcin; diaziguone; elfornitinea; elliptin acetate; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidamine; mitoguazone; mitoxantrone; mopidamol; nitracrine; pentostatin; fenamet; pirarrubicin; podophilic acid; 2-ethylhydrazide; procarbazine; PSK®; razoxane; sizofran; spirogermanium; tenuazonic acid; triaziquone; 2,2 ', 2-trichlorotriethylamine; urethane vindesine; dacarbazine; manomustine; mitobronitol, mitolactol; pipobroman; gacytosine; arabinoside (Ara-C); cyclophosphamide; thiotepa; taxoids, for example paclitaxel (TAXOL®, Bristol Myers Squibb Oncology, Princeton, NJ) and doxetaxel (TAXOTERE®, Rhone-Poulenc Rorer, Antony, France); chlorambucil; gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitomycin C; mitoxantrone; vincristine; vinorelbine; navelbine; new chair; teniposide; daunomycin; aminopterin; xeloda; ibandronate; CPT-11; topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO); retinoic acid; speramycin; capecitabine; and pharmaceutically acceptable salts, acids or derivatives thereof. Also included are antihormonal agents that act to regulate or inhibit the action of hormones in tumors, such as antiestrogens including, for example, tamoxifen, raloxifene, aromatase inhibiting 4 (5) -imidazoles, 4-hydroxy tamoxifen, trioxifene, keoxifene, onapristone, and toremifene (Fareston); and antiandrogens such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; and pharmaceutically acceptable salts, acids or derivatives thereof.

Useful drugs also include cytokines. Examples of such cytokines are lymphokines, monokines, and traditional polypeptide hormones. Included among the cytokines are growth factors such as methionyl and parathyroid growth hormone; prorelaxine;

human, human growth hormone Bovine growth hormone; thyroxine hormone; insulin; proinsulin; relaxin; glycoprotein hormones, such as follicle stimulating hormone (FSH); thyrostimulant hormone (TSH) and luteinizing hormone (LH); liver growth factor; fibrobast growth factor; prolactin; placental lactogen hormone; tumor necrosis factor alpha and beta; mulerian inhibitory substance; rat gonadotropin-associated peptide; inhibin; activin; vascular endothelial growth factor; integrin; thrombopoietin (TPO); nerve growth factors such as NGF-beta; platelet growth factor; transforming growth factors (TGFs) such as TGF-alpha and TGF-beta; similar growth factor

II; erythropoietin (EPO); insulin I and osteoinductive factors; beta and gamma;

interferons such as interferon alfa, colony stimulating factors (CSFs) such as macrophage CSF (M-CSF), granulocyte macrophage CSF (GMCSF); and granulocyte CSF (G-CSF); interleukins (ILs) such as IL-1, IL-1a, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7,

IL-8, IL-9, IL-11, IL-12, IL-15; a tumor necrosis factor, such as TNF-alpha or TNF-beta; and other polypeptide factors, including LIF and the ligand kit (KL). As used herein, the term cytokine includes proteins from natural sources, or from recombinant cell cultures, biologically active equivalents of native sequence cytokines.

The drugs may be prodrugs, subsequently activated, for example, by a prodrug activating enzyme, which converts a prodrug, such as a peptidyl chemotherapeutic agent, into an active anticancer drug. For example, see WO 88/07378, WO 81/01145 and US Patent No. 4,975,278. In general, the enzyme component includes any enzyme capable of acting on a prodrug such that it converts it to its most active cytotoxic form.

Drive Mini-Cells for Specific Mammalian Cells

In one aspect of the invention, a minicell is directed to a target mammalian cell via a bispecific ligand as described in PCT published applications WO 05/056749 and WO 05/079854. The bispecific ligand, which has specificity for both the mini cell component and the mammalian cell component, causes the mini cell to bind to the mammalian cell such that the mini cell is engulfed by the mammalian cell and the functional nucleic acid It is released into the mammalian cell cytoplasm. This directed administration method may be performed in vivo or in vitro, or in vivo and in vitro.

Contact between the bispecific ligand, the mini-cell and the mammalian cell can occur in a variety of different ways. For in vivo administration, it is preferable to administer a minicell that already has the bispecific ligand attached to it. Thus, the mini-cell, bispecific ligand and target cell are contacted when the bispecific-mini-cell ligand complex arrives at the target cell in vivo. Alternatively, the bispecific ligand and minicell may be administered separately in vitro.

Contact between bispecific ligands, mini-cells and mammalian cells may also occur during one or more in vitro incubations. In one embodiment, the three elements are incubated together at one time. Alternatively, step by step incubations may be performed.

In an example of a step-by-step approach, the mini-cells and bispecific ligands are first incubated together to form a bispecific target cell-linked complex, which is then incubated with the target cells. In another example, bispecific ligands are first incubated with target cells, followed by an incubation with minicells. A combination of one or more in vitro incubations and in vivo administrations may also contact bispecific ligands, minicells and target mammalian cells.

The inventors have found that the targeted administration approach is broadly applicable to a variety of resistant, mini-cell mammalian cells.

typically are endocytosis of antibodies including cells that specific adhesion and For example, bispecific ligands with anti-O-polysaccharide specificity in one arm and anti-HER2 receptor or anti-EGFR receptor specificity in the other arm efficiently bind minicells to the respective receptors in a variety of non-phagocytic target cells. These cells include lung, ovarian, brain, breast, prostate and skin cancer cells. In addition, efficient binding precedes rapid endocytosis of minicells by each non-phagocytic cell.

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

Ligands useful in the directed administration methods of this invention include any agent that binds to a surface component in a target cell and to a surface component in a mini cell. Preferably, the surface component in a target cell is a receptor, especially a receptor capable of mediating endocytosis. Ligands may comprise a polypeptide and / or carbohydrate component. Antibodies are preferred ligands. For example, a bispecific antibody that contains two specificities, one for a surface component in intact bacterial-derived minicells, and another for a surface component in target mammalian cells, can be efficiently used to target the minicells to cells. of mammalian target in vitro and in vivo. Useful ligands also include receptors, enzymes, binding peptides, fusion / chimeric proteins and small molecules.

Selection of a particular ligand is based on two 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 target cell surface. Thus, the ligands preferably have a first arm that has a specificity for a surface structure of an intact bacterially derived minicell, and a second arm that has a specificity for a structure on the surface of a mammalian cell. Each arm can be multivalent. Preferably, each arm is monospecific, even if multivalent.

For the binding of bacterially derived minicells, it is desirable that a ligand arm be specific for the O-polysaccharide component of a lipopolysaccharide found in the parental bacterial cell. Other surface structures of minicells that can be exploited for ligand binding include exposed cell surface polypeptides and carbohydrates present on the outer membranes such as outer membrane proteins, pili exposed peptide segments, fimbriae and flagella, exposed to the cell surface.

For binding to target cells, a ligand arm is specific for a surface component of a mammalian cell. These components include cell surface proteins, peptides and carbohydrates, whether characterized or not. Surface receptors, especially those capable of activating receptor-mediated endocytosis, are preferred cell surface components to target. These receptors, if overexpressed on the surface of a target cell, confer additional selectivity to identify the cells to be treated, thereby reducing the possibility of administration to non-target cells.

By way of example, it is possible that the target cells are tumor cells, metastasis cells, vascular cells such as endothelial cells and smooth muscle cells, lung cells, kidney cells, blood cells, bone marrow cells, brain cells, liver cells, and so forth, or precursors of any selected cell, by selecting a ligand that specifically binds a motif of a cell surface receptor on the desired cells. Examples of cell surface receptors include carcinoembryonic antigen (CEA), which is overexpressed in most colon, rectum, breast, lung, pancreas and gastrointestinal carcinomas (Marshall, 2003); heregulin receptors (HER-2, neu or c47 erbB-2), which is often overexpressed in breast, ovarian, lung, prostate, and cervical cancers (Hung et al., 2000), epidermal growth (EGFR), which is highly expressed in a variety of solid tumors, including breast, head and neck, non-small cell lung and prostate tumors (Salomon et al., 1995); asialoglycoprotein receptor (Stockert, 1995); transferrin receptor (Singh, 1999); receptor for the serpine enzyme complex, which is expressed in hepatocytes (Ziady et al., 1997); fibroblast growth factor receptor (FGFR), which is overexpressed in pancreatic ductal adenocarcinoma cells (Kleeff et al. , 2002); vascular endothelial growth factor receptor (VEGFR) for anti-angiogenesis gene therapy (Becker et al., 2002; Hoshida et al., 2002); folate receptor, which is selectively overexpressed in 90% of non-mucinous ovarian carcinomas (Gosselin and Lee, 2002); cell surface glycocalix (Batra et al. , 1994); carbohydrate receptors (Thurnher et al., 1994); and polymeric immunoglobulin receptor, which is useful for the administration of genes to respiratory epithelial cells, and interesting for the treatment of lung diseases such as cystic fibrosis (Kaetzel et al., 1997).

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

Polypeptide antibodies, wild-type light chains, have four identical and two identical heavy chains. Both types of polypeptide chains have constant regions, which do not vary or vary very little between antibodies of the same class, and variable regions. The variable regions are unique to a particular antibody, and comprise an antigen binding domain, which recognizes a specific epitope. The regions of the antigen binding domain that are most directly involved in a binding of are the complementarity determining antibody regions (CDRs). The term antibody also comprises antibody derivatives such as antibody fragments which retain the ability to specifically bind to antigens. Such antibody fragments include Fab fragments (a fragment containing the antigen binding domain, and comprising a light chain and part of a heavy chain, linked by a disulfide bond), Fab '(an antibody fragment containing a single antigen-binding domain comprising a Fab portion and an additional portion of the heavy chain through the hinge region), F (ab ') 2 (two Fab' molecules joined by interchain disulfide bridges, in the heavy chain articulated regions), a bispecific Fab (a Fab molecule with two antigen binding domains, each of which can be directed to a different epitope), and an scFv (the antigen binding determinant variable region, of a single light and heavy chain of an antibody, linked by an amino acid chain).

When antibodies, including antibody fragments, constitute part or all of the ligands, they are preferably of human origin, or are modified to be usable in humans. The so-called humanized antibodies are well known in the prior art. See, for example, Osbourn et al., 2003. They have been modified by genetic manipulation and / or in vitro treatment to reduce their affinity for antigens in humans. Methods for antibody humanization are described, for example, in US Patent Nos. 6,639,055, No. 5,585,089 and No. 5,530,101. In the simplest case, humanized antibodies are formed by grafting antigen-binding loops, known as complementarity determining regions (CDRs), of a rat mAb into a human IgG. See Jones et al. 1968; Riechmann et al., 1988; Verhoeyen et al. , 1988. Generation of high affinity humanized antibodies generally, however, requires the transfer of one or more additional residues from the so-called framework regions (FRs) of rat parental mAb. Many variants of humanization technology have already been developed. See Vaughan et al., 1998.

Instead of humanized antibodies, human antibodies may also be used in this invention. These have a high affinity for their respective antigens, and are routinely obtained from very large single variable chain fragments (scFvs) or from Fab phage display libraries. See Griffiths et al., 1994; Vaughan et al., 1996; Sheets et al., 1998; de Haard et al., 1999; and Knappik et al. , 2000.

Useful ligands also include bispecific single chain antibodies, which are typically recombinant polypeptides consisting of a variable light chain moiety covalently linked through a linker molecule to a corresponding variable heavy chain moiety. See US patents No. 5,455,030, No. 5,260,203 and No. 4,496,778. Bispecific antibodies may also be prepared by other methods. For example, chemical heteroconjugates may be created by chemically linking antibodies or intact antibody fragments of different species. See Karpovsky et al., 1984. However, these heteroconjugates are difficult to reproducibly make, and are at least twice as large as normal monoclonal antibodies. Bispecific antibodies can also be created by disulfide exchange, which involves enzymatic cleavage and re-association of antibody fragments. See Glennie et al., 1987.

Since Fab and scFv fragments are monovalent, they often have low affinity for target structures. Preferred ligands are therefore prepared from these components, and engineered to form dimeric, trimeric or tetrameric conjugates to increase functional affinity. See Tomlinson and Holinger, 2000; Carter, 2001; Hudson and Souriau, 2001; and Todorovska et al., 2001. These conjugate structures may be created by chemical and / or genetic crosslinking.

The bispecific ligands of the invention are preferably monospecific at each end, that is, they are specific for only one component of the mini cells at one end, and specific for only one component of the target cells at the other end. Ligands may be multivalent at one or both ends, for example, in the form of so-called diantibodies, triamibodies and tetraantibodies. See Hudson and Souriau, 2003. A di-antibody is a bivalent dimer formed by a non-covalent association of two scFvs, which gives rise to two Fv binding sites. Similarly, a tri-antibody results from the formation of a trivalent trimer of three scFvs, resulting in three binding sites, and a tetraantibody results from the formation of a tetravalent tetravalent of four scFvs generating four binding sites.

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

Antibody variable regions may also be fused to a wide variety of protein domains. Fusion to human immunoglobulin domains, such as IgG1 CH3, adds mass and promotes dimerization. See Hu et al., 1996. Fusion to Fc-articulated regions of human Ig may add effector functions. In addition, fusion of heterologous protein domains of multimeric proteins promotes multimerization. For example, fusion of a small scFv to antipathetic small propellers has been used to produce minibodies. See Pack and Pluckthun, 1992. Heterodimer-forming protein domains, such as fos / jun, can be used to produce bispecific molecules (Kostelny et al., 1992) and alternatively, homodimerization domains can be engineered to form heterodimers. , through manipulation strategies such as knobs into holes (Ridgway et al., 1996). Finally, fusion protein partners can be selected which provide multimerization and an additional function, for example streptavidin. See Dubel et al., 1995.

Administration to Phagocytosis or Endocytosis Competent Cells

The invention further provides administration by placing bacterially derived minicells in contact with mammalian cells which are competent for phagocytosis or endocytosis. These mammalian cells, which are capable of engulfing parental bacterial cells as intracellular bacterial pathogens, also engulf the mini-cells, which release their charge on the cytoplasm of mammalian cells. This administration approach can be accomplished without the use of target ligands.

A variety of mechanisms may be involved in engulfing minicells by a particular cell type, and the present invention is not dependent on any particular mechanism. For example, phagocytosis is a well-documented process in which macrophages and other phagocytic cells, such as neutrophils, ingest particles by extending pseudopods across the particle surface until the particle is completely enveloped. Although described as non-specific phagocytosis, the involvement of specific receptors in the process has already been demonstrated. See Wright et al. , (1968); Speert et al. (1988).

Thus, a form of phagocytosis involves the interaction between surface ligands and ligand receptors located on the pseudopod membranes. This binding step, mediated by specific receptors, is thought to be dependent on adhesins present on the surface of bacteria. With respect to less virulent bacteria such as non-enterotoxigenic E. coli, phagocytosis can also occur in the absence of surface ligands for phagocyte receptors. See Pikaar et al. (1995), for example. Thus, the present invention comprises, but is not limited to the use of minicells having or without surface adhesins, according to the nature of their parent bacterial cells, and are engulfed by phagocytes (i.e. phagocytosis-competent host cells) , being neutrophils and macrophages the main types in mammals.

Another process of engulfment is endocytosis, through which intracellular pathogens exemplified by Salmonella, Escherichia, Shigella, Helicobacter, Pseudomonas and Lactobacilli species can enter mammalian epithelial cells where they replicate. Two basic mechanisms in this regard are clathrin-dependent receptor-mediated endocytosis, also known as coated pit endocytosis (Riezman, 1993), and clathrin-independent endocytosis (Sandvig &amp; Deurs, 1994). Each or both of these may be involved when an engulfing competent cell acting through endocytosis (i.e. an endocytosis competent host cell) engulfs minicells according to the invention. Representative endocytosis competent cells are breast epithelial cells, gastrointestinal tract enterocytes, gastric epithelial cells, lung epithelial cells, and urinary bladder epithelial cells.

When administered to a swallowing competent mammalian cell without the use of a target ligand, the nature of the contemplated application will influence the choice of bacterial source for the minicells employed. For example, Salmonella, Escherichia and Shigella species have adhesins that are recognized by receptors that mediate endocytosis in enterocytes in the gastrointestinal tract, and may be suitable for administering a drug that is effective for colon cancer cells. Similarly, Helicobacter pylori-derived minicells containing gastric epithelial cell specific adhesins may be suitable for administration to stomach cancer cells. Inhalation or insufflation may be ideal for the administration of intact minicells derived from Pseudomonas species that have receptor-recognized adhesins on lung epithelial cells. Lactobacilli bacteria-derived minicells, which have urinary tract-specific adhesins and bladder epithelial cells, may be suitable for intrauretal administration of a drug in urinary tract or bladder cancer.

Formulations

As indicated, in one aspect, there is provided a composition comprising (a) a diversity of intact bacterially derived minicells, wherein each minicell of said diversity comprises regulatory RNA, which is packaged in said minicell, and (b) a carrier. pharmaceutically acceptable thereof, wherein there is the absence of a construct for in situ expression of said regulatory RNA in said minicells, wherein said regulatory RNA is selected from the group consisting of siRNA, miRNA and shRNA, and wherein said diversity contains a therapeutically effective amount of said regulatory RNA.

The formulation optionally comprises a drug. In one example, the formulation mini cell contains the drug, while in another example the mini cell may contain a nucleic acid molecule, such as a plasmid, that encodes the drug.

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

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

Routes of Administration

The formulations described herein may be administered via various routes and at various locations within a mammalian body to achieve the desired therapeutic effect or effects locally or systemically. Administration may be achieved, for example, by oral administration, by applying the formulation to a body cavity, by inhalation or insufflation, or by parenteral, intramuscular, intravenous, intrahepatic, peritoneal, subcutaneous, or intradermal administration. The mode and place of administration is dependent on the location of the target cells. For example, cystic-fibrotic cells may be affected by inhalation administration of the affected minicells. Similarly, tumor metastasis cells can be treated more efficiently by intravenous administration of affected minicells. Primary ovarian cancers can be treated by intraperitoneal administration of affected minicells.

intraportal, intratumoral,

Purity

In one aspect, the mini-cells are substantially contaminant-free parental bacterial cells. Thus, the

<td>formulations that</td><td colspan="2">contain</td><td colspan="2">mini cells,</td><td></td><td>contain</td>

<td>preferably less</td><td>of</td><td>what</td><td>fence</td><td>1 cell</td><td colspan="2">bacterial</td>

<td>contaminating parent</td><td></td><td>per</td><td> 10⁷</td><td colspan="2">mini cells,</td><td>more</td>

<td colspan="2">preferably less</td><td>of</td><td>what</td><td>about</td><td> 1</td><td>cell</td>

contaminating parental bacterial by 10⁸ even more preferably less than about 1 contaminating parental bacterial cell per 10⁹ more preferably less than about 1 parent bacterial cell contaminated by IO¹⁰It is absolutely preferred that they contain less than about 1 contaminating parental bacterial cell per 10¹¹ mini cells.

Methods that can be used for purification of minicells are known and are described in international publication number WO 03/033519. One of these methods combines cross flow filtration (feed flow is parallel to a membrane surface; Forbes, 1987) and conventional filtration (feed flow is perpendicular to the membrane surface). Optionally, the combination of filtrations may be preceded by differential centrifugation at low centrifugation force to remove some portion of the bacterial cells, thereby enriching the supernatant for the mini cells.

Another purification method employs density gradient centrifugation in a biologically compatible medium. After centrifugation, a strip of minicells is collected from the gradient and optionally, the minicells are subjected to a series of additional density gradient centrifugations to maximize purity. The method may further include a preliminary step of performing a differential centrifugation on a sample containing minicells. When performed at a low centrifugal force, differential centrifugation will remove some part of the parental bacterial cells, thereby enriching the supernatant for the mini cells.

Particularly effective purification methods exploit bacterial filamentation to increase the purity of minicells. Thus, a method for purifying minicells may include the steps of (a) subjecting a sample containing minicells to a condition that induces parental bacterial cells to take a filamentous form, followed by (b) filtering the sample to obtain a preparation of purified mini cells.

Known minicell purification methods may also be combined. A highly effective combination of methods is as follows:

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

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

Step C: Cross-flow filtration through a 0.45 pm filter to further reduce parental bacterial cell contamination.

Step D: Stress-induced filamentation of residual parental bacterial cells. This can be achieved by subjecting the mini-cell suspension to any stress-inducing environmental condition.

Step E: Antibiotic treatment to kill parental bacterial cells.

Step F: Cross - flow filtration to remove small contaminants such as membrane blisters, membrane fragments, bacterial debris, nucleic acids, mini - cell components separate the mini - cell filters from the medium. A 0.1 pm minicell filter can etc., and to concentrate 0.2 pm can be used for small contaminants, and one to concentrate the

Step G: Conventional filtration to eliminate dead filamentous bacterial cells. A 0.45 pm filter may be used for this step.

Step H: Removal of Endotoxins from Mini Cell Preparation. Anti-lipid A coated magnetic beads may be used for this step.

Administration Schemes

In general, the formulations disclosed herein may be used at appropriate dosages, defined by routine testing, to obtain an optimal physiological effect, while any possible toxicities are minimized. The dosage regimen may be selected according to a variety of factors including age, weight, gender, medical condition of the patient; the severity of the condition being treated, the route of administration, and the renal and hepatic function of the patient.

Optimal accuracy to achieve minicell and drug concentrations within the range that yields maximum efficacy with minimal side effects may require a regimen based on functional nucleic acid kinetics and drug availability at target sites and target cells. The distribution, balance and elimination of a minicell or drug may be considered when determining the optimal concentration required for a treatment regimen. Dosages of minicells and drugs may be adjusted when used in combination to achieve the desired effects.

Additionally, dosage administration of the formulations may be optimized using a pharmacokinetic / pharmacodynamic modeling system. For example, one or more dosage regimens may be chosen, and a pharmacokinetic / pharmacodynamic model may be used to determine the pharmacokinetic / pharmacodynamic profile of one or more dosage regimes. Next, one of the dosage regimens for administration can be selected which results in a desired pharmacokinetic / pharmacodynamic response based on a particular pharmacokinetic / pharmacodynamic profile. See, for example, WO 00/67776. In this regard, a dosage regimen for any indication may be determined using the approach and model described herein in Example 6, modified for the particular cell of interest.

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 over several weeks to several months.

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 daily, 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.

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

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.

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

In a method in which minicells are administered prior to a drug, drug administration can occur at any time, from a few minutes to a few hours after minicell administration. Alternatively, the drug may be administered at any time from several hours to several days, possibly several weeks to several months after the minicells.

More specifically, functional nucleic acid packed mini cells may be administered at least 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. Additionally, the minicells may be administered at least 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 mini cells may be administered at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19. or 20 weeks or more before the drug.

In another embodiment, the minicells may be administered at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 months prior to the drug.

In another embodiment, the minicells are administered after the drug. Mini-cell administration can be done at any time, from several minutes to several hours after drug administration. The minicell may alternatively be administered at any time from several hours to several days, possibly several weeks to several months after the drug.

The following Examples are merely illustrative, and not limiting, and allow a more complete understanding of the invention.

Examples

1. Direct packaging of regulatory RNA in intact minicells in vitro

Bacteria-derived intact minicells were prepared and purified as described in US Published Patent Application No. US 2004/0265994. Cy3-labeled glyceraldehyde-3-phosphate dehydrogenase siRNA (GAPDH) was obtained (max._max) 547 nm, max. (THE_max) 563 nm), product from Ambion (Austin, Texas, USA), and was reconstituted in nuclease free water to a final concentration of 50 μΜ.

About 10⁷ Mini-cells were suspended in phosphate buffer solution IX (PBS) (Gibco), and incubated along with 1 μΜ of Cy3 labeled GAPDH siRNA. Incubation was performed for 2 hours at 37 ° C with gentle mixing. Control minicells were incubated with PBS IX only. After loading, the minicells were washed twice with PBS IX by centrifugation for 10 minutes at 16,200 x g. Experimental and control minicells were observed on a DMLB fluorescence microscope, product from Leica (Germany), with an attached D70 chamber, product from Olympus Microscopes (Germany). Images were acquired using a 100x oil immersion lens.

The above simultaneous incubation experiments were also performed under different experimental conditions, such as incubation at room temperature, 37 ° C, and 4 ° C. Additionally, incubation times varied to include 1 hour, respectively.

hours, hours hours,

As shown in Figure 1B, siRNA molecules rapidly diffuse into intact minicells. Incubation of 2 hours at 37 ° C was sufficient to achieve very significant packaging of the mini cells.

In order to determine whether siRNA molecules were within or adhering to the surface of the mini-cells, the Cy3-labeled siRNA mini-cells were incubated with exonuclease overnight, followed by further fluorescence microscopy analysis. The results were identical to those shown in Fig. 1B, indicating that siRNAs were internalized by the mini cells and are not adhering to their surface.

2. In vitro transfection of human breast cancer cells with bispecific antibody-recognized minicells packed with regulatory RNA

To demonstrate that RNA-carrying minicells are stable in serum in vitro and can be internalized by specifically recognized mammalian cells, the following experiment was performed.

A polynase kinase 1-directed siRNA (Plkl) was synthesized with a 5'GGTGGATGTGTGGTCCATTTT-3 'target sequence, and labeled with a fluorescence marker, AlexaFluor 488. , bipolar spindle formation, chromosome segregation and cytokinesis and, as a crucial function, fidelity monitoring of checkpoint control (Glovar et al., 1998; Barr et al., 2004; van de Weerdt and

Medema 2006). In humans, Plkl is the best characterized member of this family. Plkl is associated with tumorigenesis and belongs to the serine / threonine kinases family, which represents an interesting target for new chemotherapy agents. Thus, Plkl is regarded as a promising target for the development of anticancer drugs (Strebhardt and Ullrich, 2006).

The mini cells were purified, and 10⁹ mini-cells were packaged with anti-siRNA^AF488 Plkl as described in Example 1. A bispecific antibody (BsAb) was prepared with specificities for S. typhimurium antigen O and human anti-EGFR; it was bound to the anti-AF488-piki-siRNAz minicells as described in published PCT Application WO 05/056749. The resulting minicells were called^EGFRmini cellsAF488-pi_kl-_si_RNA. These mini cells (10⁹) were incubated with tissue culture human breast cancer cells at a density of 10,000 minicells: 1 tumor cell. Incubation was performed for 1 hour, 2 hours, 4 hours and 24 hours. At each time point, cells were harvested and stained with DAPI (nuclear staining, blue fluorescence). Cells were observed using the 1X81 confocal microscope (Olympus) and CellR software.

After 1 hour, the fluorescent siRNA minicells had adhered to the MDA-MB-468 cells (see Figure 2). This binding was believed to be due to binding to the minicell-bound BsAb which is directed to the EGFR receptor in MDA-MB-468 cells as incubation controls with the minicells._AF488__Plkl__siRNA without BsAb showed that these were removed and showed no green fluorescence associated with MDA-MB-468 cells. 2 hours after incubation, the^EGFRmini cells_AF488-piki-_siRNAs were internalized within MDA-MB-468 cells and exhibited intense green fluorescence. After 24 hours, most of the green fluorescence had disappeared, indicating that internalized minicells had presumably been destroyed within phagolysosomes.

3 SiRNA extraction and quantification of intact minicells

Since siRNAs do not occur in nature in bacterial cells or bacterial-derived minicells, it is not surprising that there is no established methodology for the extraction of siRNAs from these particles. Thus, the present inventors have developed a method for the quantitative extraction of siRNAs that are packaged in intact minicells in accordance with the invention.

Spindle kinesin protein (KSP), also known as kinesin-5 and Eg5, is a microtubule motor protein. This protein is essential for the formation of bipolar spindles and proper segregation of sister chromatids during mitosis (Enos and Morris, 1990; Blangy et al., 1995; Sawin and Mitchison, 1995; Dagenbach and Endow, 2004). KSP inhibition causes monopolar mitotic spindle formation, activates the spindle formation checkpoint, and retains cells in mitosis, which leads to subsequent cell death (Blangy et al., 1995, Caner et al., 1999; Kapoor et al., 2000; Tao et al., 2005).

An anti-KSP siRNA was selected for packaging in minicells to provide information on optimizing the extraction of minicell siRNAs according to the invention. More specifically, KSP-1-siRNA (sense strand; 5'-AAC TGG ATC GTA AGA AGG CAG-3 ') oligonucleotide sequences were synthesized and packaged in minicells according to the procedures set forth in Example 1, supra.

The mini cells_sgo_AT-Ksp (10¹⁰) and a comparable number of empty control minicells were processed using a variety of commercially available nucleic acid extraction kits. The results showed that the mirVana (Ambion) miRNA isolation kit provided quantitative extraction of siRNA-KSP from intact minicells. The procedure was performed according to the manufacturer's instructions.

Purified siRNAs were first stained with an ultrasensitive dye, Inc. (Eugene, nucleic acid fluorescent, RiboGreen ™, Molecular Probes Oregon product, USA), followed by quantification using NanoDrop ND-3300 Fluorospectrometer, in

NanoDrop Technologies Inc. also in accordance with (Wilmington, Delaware, USA) manufacturer's instructions. O

RNA-bound RiboGreen ™ has a maximum excitation of -500 nm and a maximum emission of -525 nm.

The results show that the mini cells were able to carry siRNAs. IO¹⁰ empty mini cells contained -1.4 pg of RNA, possibly a basal level of endogenously formed bacterial RNA. Same number of mini cells_sgo_AT-KPs contained -2.7 pg RNA, comprising endogenous bacterial RNA plus packaged exogenous siRNA-KPS. Thus, these data demonstrate that IO¹⁰ mini-cells can package at least 1.3 pg of exogenously packaged siRNA.

4 In vivo demonstration of antitumor effects obtained by regulatory RNA-packed minicells

The following studies were conducted to show that regulatory RNA packaged minicells can deliver intact regulatory RNA at therapeutically effective concentrations to tumor cells in vivo.

An siRNA against KSP, as described in Example 3, was selected to package into the mini cells according to the present invention. The mini cells were purified, and 10⁹mini-cells were packaged with anti-KSP siRNA as described in Example 1. A BsAb was prepared which was attached to the SiRNA-Kspz mini cells as described in Example 2 to produce ^EGFRmini cell_AT-_Ksp ·

The mice used in this Example were purchased from the Animal Resources Center (Perth, WA, Australia), and all animal experiments were performed in accordance with the Animal Care and Use Guide approved by the Animal Ethics Committee. The experiments were performed at the NSG Agriculture accredited small animal facility at EnGenelC Pty Ltd (Sydney, New South Wales, Australia).

Human breast cancer cells (MDA-MB-468, ATCC) were cultured in tissue culture in RPMI 1640 medium supplemented with 5% GIBCO-BRL bovine serum, product of Invitrogen Corporation (Carlsbad, California, USA). , and glutamine (Invitrogen) in a humidified atmosphere of 95% air and 5% CO₂at 37 ° C. lx10⁶ Cells in 50 µl of serum free medium were mixed with 50 µl of low growth factor content matrigel, product of BD Biosciences (Franklin Laques, New Jersey, USA). Through a 23 gauge needle, cells were injected subcutaneously between the shoulder blades of each rat. Tumors were measured twice weekly using an electronic digital caliper (accuracy 0.001), Mitutoyo product (Japan), and the average tumor volume was calculated using the following formula: length (mm) x width² (mm) x 0.5 = volume (mm³) .

The various treatments began when the tumors reached volumes between 170 mm³ and 200 mm³, and the rats were randomly divided into two groups of 8 rats. Control group 1 received sterile saline and experimental group 2 received^EGFRmini cells_si_RNA-_Ksp (10⁹) , Four times a week.

As shown in Figure 3, the ³siRNA-KSP minicells resulted in a very significant antitumor effect compared to saline controls.

The results demonstrate that (a) siRNAs are stable within

Fully functional cells were administered to tumor cells in vivo, and (c) the mini-cell administered therapeutically significant siRNA concentrations to those of the intact rn (b) siRNA mini-cells in vivo.

5 Demonstration of tailor-made cancer therapy by treatment with regulatory RNA-packed mini-cells, followed by drug-packed mini-cells

Most anticancer therapies are associated with drug resistance. The same is true for regulatory RNA treatment, since gene mutations in tumor cells can render regulatory RNAs ineffective if the target gene mutates within the sequence that is targeted by regulatory RNAs.

There has been no effective strategy to address the problem of drug resistance in cancer patients. Instead, new drugs must be administered to overcome the mutation. However, this approach faces serious difficulties, as most anticancer drugs are highly toxic, and combined therapies increase this toxicity, resulting in dose limitation and frequent abandonment of therapy when the patient can no longer withstand the toxicity. . 0 The following study was conducted to determine the effectiveness of regulatory RNA-packed mini cells in resolving this type of resistance.

As described above, the minicells were purified and packaged (10⁹) with anti-KSP or antiPlk1 siRNA. Also as previously described, bispecific antibody with specificities for S. typhimurium anti-antigen 0 and human anti-EGFR was preferred, which was bound to the SiRNA-Ksp minicells to produce^EGFRmini-cellassiRNA-Ksp ·

Human colon cancer xenografts (HCT116; ATCC) have been established in nude mice as described in

Example 3; these were treated iv as follows: group 1 rats received sterile saline, and groups 2, 3 and 4 rats were treated for the first 10 doses (see Figure 4) with 10^{9th FGFR}mini-cellsSiRNA-pikl, ^FGFRmini cellsSi_RNA-Ksp-i, θ ^FGFRmini cells_si_RN_THE-Ksp-2z respectively. The sequences Plk1 and KSP-1 are described in the previous Examples. SiRNA-KSP-2 (sense strand; 5 'CTGAAGACC TGAAGACAAT 3') is directed to a different segment than KSP mRNA. After day 33, rats in groups 2, 3 and 4 were treated with two doses of^FGFRmini cells_carbOpi_Thetina ·

The results showed (Figure 4) that after day 26, the tumors became resistant to siRNA treatments. Thus, rats in groups 2, 3 and 4 were treated for the four subsequent doses with the three doses of^Rmini cells

SYNRNA-Plkl + ^FGFRsiRNA-KSP-l mini cells ^FGFRmini cells_si_RNA-Ksp-2z combined in equal doses, ie ~ 3 x 10⁸ of each type of mini cell. Additionally, by day 33, the tumors were highly resistant to all siRNAs (Figure following administration of^EGFRmini cells_carbOpiatinaz a group of 3 mice showed a significant regression of tumor volume.

These results show that drug resistant tumor cells can be efficiently treated in vivo in accordance with the present invention. In particular, (1) sequential administrations of target minicells containing regulatory RNA sequences designed to significantly reduce tumor burden are followed when tumor cells become resistant to siRNA-mediated antitumor effect by (2) minicells. containing a drug that does not act on the same target protein as regulatory RNA.

6 Demonstration of target protein knockdown in tumor cells, and consequent arrest of cell growth following targeted administration of a therapeutically effective amount of regulatory RNA packaged into intact minicells.

To demonstrate that the method of the invention allows for the packaging of therapeutically effective amounts of regulatory RNA into intact minicells, it was necessary to demonstrate that regulatory RNA-packed minicells, targets of bispecific antibodies, were capable of stopping cell growth efficiently and effectively, and induce apoptosis cell death.

So were ^EGFRmini cells_siRNA-pi_kl mini cells_sgo_AT-KSPz

In a humid atmosphere of 95% air and 5% CO₂At 37 ° C, human colon cancer cells (HCT116) were cultured in tissue culture in RPMI 1640 medium supplemented with 5% bovine serum and glutamine. As described above, the minicells were purified, packed with siRNAs directed against Plk1 or KSP, and bound to a BsAb that had specificities for S. typhimurium antigen O and human anti-EGFR.

generated ^EGERmini cells_sgo_AT-_Kspz e ^EGFRminicells from the SsiRNA-pikx minicells and minicells (control). HCT116 cells were seeded in 6-well plates, and control and experimental groups were transfected at a ratio of 5,000 minicells: 1 cell.

HCT116. An additional control consisting of cells only was included.

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

FACS cell analysis showed that at 4 and 8 hours after transfection (Figure 5A), cells treated with ^EGFRmini-cellsSiRNA-KSPz or ^FGFRmini cellsSi_RNA-pi_kl they were characterized by a robust cell cycle arrest at the G2 phase. Control cells, whether they are only cells or cells treated with^FGFRminicells showed no adverse effects. These cells showed normal cell cycle phases G1, G2 and G2. At 16 and 24 hours, the experimental cells showed not only a robust G2 arrest but also a high number of apoptotic cells (Figure 6). At 32 and 48 hours, most cells in the experimental groups were apoptotic and had become cell debris (see, in particular, keys in Figure 7).

These results demonstrate that the target intact minicells were packaged with a therapeutically effective amount of regulatory RNA, and that the minicells of the invention were highly efficient in achieving protein knockdown in tumor cells, which resulted in apoptotic cell death.

CITED PUBLICATIONS

Barr, FA, Sillje, HH, and Nigg, EA Pololike kinases and the orchestration of cell division. Nat. Rev. Mol. Cell. Biol. 5: 429-440 (2004).

Batra, RK, Wang-Johanning, F., Wagner, E., Garver, RI Jr., Curiel, DT Receptor-mediated gene delivery employing lectin-binding specificity. Gene Ther. 1: 255-260 (1994).

Becker, CM, Farnebo, FA, Iordanescu, I., Behonick, DJ, Shih, MC, Dunning, P., Christofferson, R., Mulligan, RC, Taylor, GA, Kuo, CJ, Zetter, BR Gene therapy of prostate cancer with the soluble vascular endothelial growth factor receptor Flkl. Biol Cancer. The R. 1: 548-553 (2002).

Bertrand, JR., Pottier, M., Vekris, A., Opolon, P., Maksimenko, A., Malvy, C. Comparison of antisense oligonucleotides and siRNAs in cell culture and in vivo. Biochem. Biophys. Res. Commun. 296: 1000-1004 (2002).

Blangy, A., Lane, HA, d'Herin, P., Harper, M., Kress, M., Nigg, EA Phosphorylation by p34cdc2 regulates spindle association of human Eg5, a kinesin-related motor essential for bipolar spindle formation in alive. Cell 83: 1159-1169 (1995).

Borst, P. and Elferink, RO Mammalian ABC transporters in health and disease. Annu Rev. Biochem. 71: 537-592 (2002).

Bredel, M. Anticancer drug resistance in primary human brain tumors. Brain Res. Rev. 35: 161-204 (2001).

Britton, RA, Lin, DC, Grossman, AD Characterization of a prokaryotic SMC protein involved in chromosome partitioning. Genes Dev. 12: 1254-1259 (1998).

Caplen, NJ RNAi as a gene therapy approach. Expert Opin. Biol. The R. 3: 575-586 (2003).

Crankcase, P. Improving the efficacy of antibody-based cancer therapies. Nat. Rev. Cancer 1: 118-129 (2001).

Check, E. Gene therapy put on hold as third child develops cancer. Nature, 433: 561 (2005).

Chen, ZS et al. Reversal of drug resistance mediated by multidrug resistance protein (MRP) 1 by dual effects of agosterol A on MRP 1 function. Int. J. Cancer 93: 107-113 (2001).

Chiu, YL., Rana, TM modification analysis. Choung, S., Kim, YJ

siRNA function in RNAi: a Chemical RNA 9: 1034-1048 (2003).

, Kim, S., Park, HO, Choi, YC

Dash, PR, Seymour, LW distribution

Chemical modification of siRNAs to improve serum stability without loss of efficacy. Biochem. Biophys. Res. Commun. 342: 919-927 (2006).

Conner, SD and Schmid, SL Regulated portals of entry into the cell. Nature 422: 37-44 (2003).

Czauderna, F., Fechtner, M., Dames, S., Aygun, H.A., Pronk, GJ, Giese, K., Kaufmann, J. Variations and stabilizing modifications of siRNAs in mammalian cells. Nucleic Acids Res. 31 (2003).

Dagenbach, EM, and Endow, SA A new kinesin tree. J. Cell Sci. 117: 3-7 (2004).

Read, ML, Barrett, LB, Wolfert, MA Factors affecting blood clearance and in vivo of polyelectrolyte complexes for gene delivery. Gene. The R. 6: 643-650 (1999).

from Boer, PA, Crossley, RE, Rothfield, LI Roles of MinC and MinD in the site-specific septation block mediated by the MinCDE system of Escherichia coli. J. Bacteriol. 174: 63-70 (1992).

de Haard, HJ et al. A large non-immunized human Fab fragment phage library that allows rapid isolation and kinetic analysis of high affinity antibodies. J. Biol. Chem. 274: 18218-18230 (1999).

Devroe, E. and PA Silver Therapeutic potential of retroviral RNAi vectors. Expert Opn. Biological Therapy 4: 319 (2004).

, Klippel, Structural synthetic 2705-2716

Doige, CA and Ames, G bacteria and humans: multidrug resistance.

(1993).

F. ATP-dependent transport systems in relevance to cystic fibrosis and Annu. Rev. Microbiol. 47: 291-319

Dubel, S., Breitling, F., Kontermann, R., Schmidt, T., Skerra, A., Little, M. Bifunctional and multimeric complexes of streptavidin fused to single chain antibodies (scFv). J. Immunol. Methods 178: 201-209 (1995).

Dykxhoorn, DM and Lieberman, J. The silent revolution: RNA interference as basic biology, research tool, and therapeutic. Annu Rev. Med. 56: 401-423 (2005).

Dykxhoorn, MD, Palliser, D., Lieberman, J. The silent treatment: siRNAs as small molecule drugs. Gene Ther. 13:

541-552 (2006) .

Elmén, J., Thonberg, H., Ljungberg, K., Frieden, M., Westergaard, M., Xu, Y., Wahren, B., Liang, Z., Orum, H., Koch, T. and others. Locked nucleic acid (LNA) mediated improvements in siRNA stability and functionality. Nucleic Acids Res. 33: 439-447 (2005).

Enos, AP, and Morris, NR Mutation of a gene that encodes kinesin-like protein blocks nuclear division in A. nidulans. Cell 60: 1019-1027 (1990).

Glennie, MJ, McBride, HM, Worth, AT, Stevenson, GT Preparation and performance of bispecific F (ab 'gamma) 2 antibody containing thioether-linked Fab' gamma fragments. J. Immunol. 39: 2367-2375 (1987).

Glover, DM, Hagan, IM, and Tavares, AA Polo-like kinases: a team that plays throughout mitosis. Genes Dev. 12: 3777-3787 (1998).

Gosselin, MA and Lee, RJ Folate receptor-targeted liposomes as vectors for therapeutic agents. Biotechnol. Annu Rev. 8: 103-131 (2002).

Gottesman, M., Fojo, T. and Bates, S. Multidrug resistance in cancer: role of ATP-dependent transporters. Nature Rev.

Cancer 2: 48-58 (2002).

Griffiths, AD et al. Isolation of high affinity human antibodies directly from large synthetic repertoires. EMBO J. 13: 3245-3260 (1994).

Grishok, A., Tabara, H., Mello, CC Genetic requirements for inheritance of RNAi in C. elegans. Science 287: 24942497 (2000).

Guerrier-Takada, C., Gardiner, K., Marsh, T., Altman, S. The RNA moiety of ribonuclease

Pace, N., P is the catalytic subunit of the enzyme. Cell 35: 849-857 (1983)

<td>Hacein-beyond-</td><td>Abina, S. ,</td><td>Von Kalle, C.,</td><td colspan="2">Schmidt, Μ. ,</td>

<td>McCormack,</td><td colspan="2">MP Wulffraat N. Leboulch,</td><td>P.,</td><td>Lime.,</td>

<td>Osborne, C</td><td>. S . Pawliuk</td><td>R., Morillon, E.</td><td>et</td><td>al. LMO2-</td>

<td>associated</td><td colspan="2">clonal T cell proliferation in</td><td>two</td><td>patients</td>

<td>after gene</td><td>therapy for</td><td>SCID-X1. Science,</td><td> 302</td><td> : 415-419</td>

<td> (2003) .</td><td></td><td></td><td></td><td></td>

<td>Hampel, A.</td><td>and Tritz, R.</td><td colspan="3">RNA catalytic properties of the</td>

<td>minimum (-)</td><td>sTRSV sequence</td><td>. Biochem. 28: 4929-</td><td> 4933</td><td> (1989) .</td>

<td>Hampel, A.</td><td>, Tritz, R.,</td><td>Hicks Μ. Cross</td><td>P.</td><td>Hairpin</td>

<td>catalytic:</td><td colspan="2">RNA model: evidence for helices</td><td>and</td><td>sequence</td>

<td>requirement</td><td>for substrate</td><td>RNA. Nucleic Acids</td><td>Res.</td><td> 18: 299-</td>

<td> 304 (1990).</td><td></td><td></td><td></td><td></td>

<td>Hanada, Μ.,</td><td>Aime-Sempe, C</td><td>. Sappho T. Reed,</td><td>JC</td><td>Structure</td>

function analysis of Bcl-2 protein. Identification of conserved domains important for homodimerization with Bcl-2 and heterodimerization with Bax. J. Biol. Chem. 270: 1196211969 (1995).

Harry, EJ Bacterial cell division: Regulating Z-ring formation. Mol. Microbiol. 40: 795-803 (2001).

Higgins, CF ABC transporters: physiology, structure and mechanism - an overview. Res. Microbiol. 152: 205-210 (2001).

Hiraga, S., Niki, H., Ogura, T., Ichinose, C., Mori, H., Ezaki, B., Jaffe, A. Chromosome partitioning in Escherichia coli: novel mutants producing anucleate cells. J. Bacteriol. 171: 1496-1505 (1989).

Hoshida, T., S., Shineha Matsuno, S. endothelial adenovirus

Sunamura, M., Duda, DG, Egawa, S., Miyazaki, R., Hamada, H., Ohtani, H., Satomi, S.,

Gene therapy for pancreatic cancer using a vector encoding soluble flt-1 vascular growth factor receptor. Pancreas 25: 111-121 (2002).

Hu, Z. and Lutkenhaus, J. Topological regulation of cell division in Escherichia coli involves rapid pole to pole oscillation of the division inhibitor MinC under the control of MinD and MinE. Mol. Microbiol. 34: 82-90 (1999).

Recombinant antibodies for Expert Opin. Biol. The R. 1:

Development of

Hudson, PJ and Souriau, C. cancer diagnosis and therapy,

845-855 (2001) .

Hung, MC, Hortobagyi, GN, Ueno, NT clinical trial of EIA gene therapy targeting HER-2 / neuoverexpressing breast and ovarian cancer. Adv. Exp. Med. Biol. 465: 171-180 (2000).

Ireton, K., Gunther, NW 4th., Grossman, AD spoOJ is required for normal chromosome segregation as well as the initiation of sporulation in Bacillus subtilis. J. Bacteriol. 176: 5320-5329 (1994).

Karpovsky, B., Titus, JA, Stephany, DA, Segai, MD Production of target-specific effector cells using heterocross-linked aggregates containing anti-target cell and anti-Fc gamma receptor antibodies. J. Exp. Med 160: 16861701 (1984).

Knappik, A. et al. Fully synthetic human combinatorial antibody libraries (HuCAL) based on modular consensus frameworks and randomized CDRs with trinucleotides. J. Mol. Biol. 296: 57-86 (2000).

Jones, PT, Dear, PH, Foote, J., Neuberger, MS, Winter, G. Replacing the complementarity-determining regions in a human antibody with those from a mouse. Nature

321: 522-525 (1986) .

Kaetzel, CS, Blanch, VJ, Hempen, PM, Phillips, KM, Piskurich, JF, Youngman, KR The polymeric immunoglobulin receptor: structure and synthesis. Biochem. Soc. Trans. 25: 475-480 (997).

Kapoor, TM, Caner, TU, Coughlin, ML, Mitchison, TJ 2000. Probing spindle assembly mechanisms with monastrol, a small molecule inhibitor of the mitotic kinesin, Eg5. J. Cell Biol. 150: 975-988 (2000).

Kleeff, J., Fukahi, K., Lopez, ME, Friess, H., Buchler, MW, Sosnowski, BA, Korc, M. Targeting of suicide gene delivery in pancreatic cancer cells via FGF receptors. Cancer Gene Ther. 9: 522-532 (2002).

Kootstra, NA and Verma, IM (2003) Gene therapy with viral vectors. Annu Rev. Pharmacol. Toxicol. 43: 413-439 (2003). Kostelny, SA, Cole, MS, Tso, JY Formation of a bispecific antibody by use of leucine zippers. J. Immunol. 148: 1547-1553 (1992).

Kruh, GD and Belinsky, MG The MRP family of drug efflux pumps. Oncogene 22: 7537-7552 (2003).

Layzer, JM, McCaffrey, AP, Tanner, AK, Huang, Z., Kay, MA, Sullenger, BA In vivo activity of nucleaseresistant siRNAs. RNA 10: 766-771 (2004).

Lee, RC, Feinbaum, RL, Ambros, V. The C. elegans heterochronic gene lin-4 encodes small RNAs with antisense complementarity to lin-14. Cell 75: 843-854 (1993).

Marshall, J. Carcinoembryonic antigen-based vaccines. Semin Oncol. 30: 30-36 (2003).

Caner, TU, Kapoor, TM, Haggarty, SJ, King, RW, Schreiber, SL, Mitchison, TJ Small molecule inhibitor of mitotic spindle bipolarity identified in a phenotypebased screen. Science 286: 971-974 (1999).

McManus, Μ. T. and Sharp, PA Gene silencing in mammals by small interfering RNAs. Nat. Rev. Genet. 3: 737-747 (2002).

Miller, JF Bacterial transformation by electroporation. Methods Enzymol. 235: 375-385 (1994).

Modok, S., Mellor, HR, Callaghan, R. Modulation of multidrug resistance efflux pump activity to overcome chemoresistance in cancer. Curr. Open Pharmacol. 6: 350354 (2006).

Monia, BP, Johnston, JF, Sasmor, H., Cummins, LL Nuclease resistance and antisense activity of modified oligonucleotides targeted to Ha-ras. J. Biol. Chem. 271: 14533-14540 (1996).

Morrissey, DV, Blanchard, K., Shaw, L., Jensen, K., Lockridge, J., Dickinson, B., McSwiggen, J., Vargeese, C., Bowman, K., Shaffer, CS, and others. Activity of stabilized short interfering RNA in a mouse model of hepatitis B virus replication. Hepatology 41: 1349-1356 (2005).

Nicholson, RI, Gee, JM, Harper, ME EGFR and cancer prognosis. Eur. J. Cancer 37: S9-S15 (2001).

Nikaido, H. Prevention of drug access to bacterial targets: permeability barriers and active efflux. Science 264: 382388. (1994).

Okada, Y., Wachi, M., Hirata, A., Suzuki, K., Nagai, K., Matsuhashi, M. Cytoplasmic axial filaments in Escherichia coli cells: possible function in the mechanism of chromosome segregation and cell division. J. Bacteriol. 176: 917-922 (1994).

Opalinska, JB and Gewirtz, AM Nucleic-acid therapeutics: basic principles and recent applications. Nat. Rev. Drug Discov. 1: 503-514 (2002).

Osbourn, J., Jermutus, L., Duncan, A. Current methods for the generation of human antibodies for the treatment of autoimmune diseases. Drug Del. Tech. 8: 845-851 (2003).

Pack, P. and Pluckthun, A. Miniantibodies: use of amphipathic helices to produce functional, flexibly linked dimeric Fv fragments with high avidity in Escherichia coli. Biochemistry 31: 1579-1584 (1992).

Paddison, PJ, A. Caudy, E. Bernstein, GJ Hannon, DC Conklin Short hairpin RNAs (shRNAs) induce specific sequences silencing in mammalian cells. Genes &amp; Development 16: 948-958 (2002).

Perrotta, AT and Been, MD Cleavage of oligoribonucleotides by a ribozyme derived from the hepatitis delta virus RNA sequence. Biochem. 31: 16-21 (1992).

Pikaar, JC, Voorhout, WF, van Golde, LM, Verhoef, J., Van Strijp, JA, Van Iwaarden, JF Opsonic activities of surfactant proteins A and D in phagocytosis of gramnegative bacteria by alveolar macrophages. J Infect. Dis. 172: 481-489 (1995).

Raper, SE, Chirmule, N., Lee, FS, Wivel, NA:, Bagg, A., Gao, GP, Wilson, JM and Batshaw, ML Fatal systemic inflammatory response syndrome in an ornithine transcarbamylase deficient patient following adenoviral gene transfer. Mol. Genet. Metab. 80: 148-158 (2003).

Raskin, DM and Boer, PA MinDE-dependent pole-to-pole oscillation of division inhibitor MinC in Escherichia coli. J. Bacteriol. 181: 6419-6424 (1999).

Reeve, JN and Cornett, JB Bacteriophage SPO1-induced macromolecular synthesis in minicells of Bacillus subtilis. J. Virol. 15: 1308-1316 (1975).

Ridgway, JB, Presta, LG, Crankcase, P. Knobs-into-holes engineering of CH3 antibody domains for heavy chain heterodimerization. Protein Eng. 9: 617-621 (1996).

Riechmann, L., Clark, M., Waldmann, H., Winter, G. Reshaping human antibodies for therapy. Nature 332: 323-327 (1988).

Riezman, H. Three clathrin-dependent budding steps and cell polarity. Trends in Cell Biol. 3: 330-332 (1993).

Rossi, JJ, Elkins, D., Zaia, JA Sullivan, S. Ribozymes as anti-HIV-1 therapeutic agents: principles, applications, and problems. Aids Res. Human Retroviruses 8: 183-189 (1992).

Salomon, DS, Brandt, R., Ciardiello, F., Normanno, N. Epidermal growth factor-related peptides and their receptors in human malignancies. Crit. Rev. Oncol. Hematol 19: 183-232 (1995).

Sandvig, K. and Deurs, B. Endocytosis without clathrin. Trends in Cell Biol. 4: 275-277 (1994).

Sawin, KE and Mitchison, TJ Mutations in the kinesin-like protein Eg5 disrupting localization to the mitotic spindle. Proc. Natl. Acad. Know. USA 92: 4289-4293 (1995).

Saville, BJ and Collins, RA A site-specific self-cleavage reaction performed by the novel RNA in Neurospora mitochondria. Cell 61: 685-696 (1990).

Saville, BJ and Collins, RA RNA-mediated ligation of self-cleavage products of a Neurospora mitochondrial plasmid transcript. PNAS (USA) 88: 8826-8830 (1991).

Sellers, WR and Fisher, DE Apoptosis in cancer drug targeting. J. Clin. Invest. 104: 1655-1661 (1999).

Sheets, MD et al. Efficient construction of a large nonimmune phage antibody library: production of highaffinity human single-chain antibodies to protein antigens. Proc. Natl Acad. Know. USA 95: 6157-6162 (1998).

Singh, M. Transferrin As A targeting ligand for liposomes and anticancer drugs. Curr. Pharm. Off 5: 443-451 (1999).

Sioud, M. Therapeutic siRNAs. Trends Pharmacol. Know. 25: 22-28 (2004).

Speert, DP, Wright, SD, Silverstein, SC, Mah, B. Functional characterization of macrophage receptors for Invitro phagocytosis of unopsonized pseudomonas aeruginosa. J. Clin. Invest. 82: 872-879 (1988).

Stewart, PS and D'Ari, R. Genetic and morphological characterization of an Escherichia coli chromosome segregation mutant. J. Bacteriol. 174: 4513-4516 (1992). Stockert, RJ The receptor asialoglycoprotein: relationships between structure, function, and expression. Physiol. Rev. 75: 591-609 (1995).

Strebhardt, K., and Ullrich, A. Targeting Pololike kinase 1 for cancer therapy. Nat. Rev. Cancer 6: 321-330 (2006).

Sun et al apoptosis Overexpression inducing ligand of TNF-related Bcl-2 blocks (TRAIL) -induced apoptosis in human lung cancer cells. Biochem. Biophys. Res. Commun., 280: 788 (2001).

Tao, W., South, VJ, Zhang, Y., Davide, JP, Farrell, L., Kohl, NE, Sepp-Lorenzino, L., Lobell, RB Induction of apoptosis by an inhibitor of the mitotic kinesin KSP reguires both activation of the spindle assembly checkpoint and mitotic slippage. Cancer Cell 8: 49-59 (2005).

Thurnher, M., Wagner, E., Clausen, H., Mechtler, K., Rusconi, S., Dinter, A., Bimstiel, ML, Berger, EG, Cotton, M. Carbohydrate receptor-mediated gene transfer to human T leukemic cells. Glycobiology 4: 429-435 (1994). Todorovska, A. et al. Design and application of diabodies, triabodies and tetrabodies for cancer targeting. J. Immunol. Methods 248: 47-66 (2001).

Tomlinson, I. and Holliger, P. Methods for generating multivalent and bispecific antibody fragments. Methods Enzymol. 326: 461-479 (2000).

van de Weerdt, BC, and Medema, RH Polo-like kinases: a team in control of the division. Cell Cycle 5: 853-864 (2006).

Vaughan, TJ, antibodies by design (1998).

Osbourn, JK, Nature

Tempest, PR Human

Biotechnol. 16: 535-539

Verhoeyen, M., Milstein, C., Winter, G. Reshaping human antibodies: grafting an antilysozyme activity. Science 239: 1534-1536 (1988).

Verma, IM and Weitzman, MD Gene therapy: twenty-first century medicine. Annu Rev. Biochem. 74: 711-738 (2005).

Vickers, TA, Koo, S., Bennett, CF, Crooke, ST, Dean, Ν. M., Baker, BF Efficient Reduction of Target RNAs by Small Interfering RNA and RNase H-dependent Antisense Agents. A COMPARATIVE ANALYSIS J. Biol. Chem. 278: 7108-7118 (2003).

Wachi, M., Doi, M., Okada, Y., Matsuhashi, M. New mreC and mreD genes, responsible for forming the rod shape of Escherichia coli cells. J. Bacteriol. 171: 6511-6516 (1989).

Wang, CY, Cusack, JC Jr., Liu R., Baldwin, AS Jr. Control of inducible chemoresistance: Enhanced anti-tumor therapy through increased apoptosis by inhibition of NF-kB. Nat. Med. 5: 412-417 (1999).

White, MK, and McCubrey, JA Suppression of apoptosis: role in cell growth and neoplasia. Leukemia 15: 1011-1021 (2001).

Wright, SD, Detmers, PA, Jong, MT, Meyer, BC Interferon-gamma depressants binding of ligand by c3b and c3bi receptors on cultured human monocytes, an effect reversed by fibronectin. J. Exp. Med. 163: 1245-1259 (1986).

Wu, H., Hait, WN, Yang, JM Small interfering RNA suppression of MDR1 (P-glycoprotein) restores sensitivity to multidrug-resistant cancer cells. Cancer Res. 63: 1515-1519 (2003).

Yague, E., Higgins, CF, Raguz, S. Complete reversal of multidrug resistance by stable expression of small interfering RNAs targeting MDR1. Gene Ther. 11: 1170-1174 (2004).

Zamore, PD, Tuschl, T., Sharp, PA, Bartel, DP RNAi: double-stranded RNA directs the ATP-dependent cleavage of mRNA at 21 to 23 nucleotide intervals. Cell 101: 25-33 (2000).

Zamore, PD Ancient pathways programmed by small RNAs. Science 296: 1265-1269 (2002).

Ziady, AG, Perales, JC, Ferkol, T., Gerken, T., Beegen, H., Perlmutter, DH, Davis, PB Gene transfer into hepatoma cell lines via the serpin enzyme complex receptor. Am. J. Physiol. 273: G545-G552 (1997).