BACKGROUND OF THE INVENTION

The present invention relates to targeted delivery, by means of intact killed bacterial cells, of bioactive molecules, including therapeutic nucleic acids, functional nucleic acids, drugs, peptides, proteins, carbohydrates and lipids, to mammalian host cells.

A number of hurdles continue to challenge targeted delivery of bioactive molecules to mammalian cells (e.g., cancer cells), particularly in-vivo. Those hurdles include (a) composition, functional characteristics and stability of delivery vehicles, (b) packaging therapeutically significant concentrations of bioactive molecules, (c) targeting desired diseased cells in-vivo, (d) overcoming a series of intracellular barriers and successfully deliver therapeutic concentrations of bioactive molecules to intracellular targets, (e) avoiding a range of host immune elements such as antibodies, complement, and macrophages that may destroy a vector before it reaches a target, (f) crossing the endothelial barrier of blood vessel walls, particularly at the site of a tumor mass, (g) migrating through several layers of cells to reach a target (e.g., it is known that a solid tumor is an organized structure containing both tumor cells and normal cells; hence a vector must cross several layers of normal cells to access malignant cells), (h) migrating through an extracellular matrix (ECM) comprised of glycoproteins, sulfated glycosaminoglycans, hyaluronan, proteoglycans and collagen that fills the space between cells and therefore hampers transport of a vector, and (i) addressing high interstitial hypertension (elevated hydrostatic pressure outside blood vessels) in the tumor microenvironment, which may limit the access of bioactive molecules.

A number of different vectors have been proposed for both nucleic acid and drug delivery, including viral, non-viral non-living, and non-viral living vectors. The non-viral non-living vectors have been adapted for both nucleic acid and drug delivery. The other two types of vectors have been adapted for nucleic acid delivery. Non-viral living vectors are mainly being developed for direct tumor-cell killing capabilities. While all these vectors have advantages, they also have drawbacks.

Viral vectors, such as retrovirus, adenovirus, adeno-associated virus, pox virus, herpes simplex virus, and lentivirus, have been developed for gene delivery. However, viral vectors are unable to deliver genes systemically and specifically to primary and/or metastasized tumor cells without infecting normal tissues (Akporiaye and Hersh, 1999; Biederer et al., 2002; Green & Seymour, 2002). Additionally, the extremely limited diffusibility of virions within extracellular spaces significantly hinders the dissemination of viral vectors. Moreover, viruses are antigenic, and therefore give rise to host immune responses. Such immune responses include both specific adaptive responses and non-specific innate responses (Chen et al., 2003; Ferrari et al., 2003; Wakimoto et al., 2003). The latter plays an important role in eliminating adenoviral vectors (Liu and Muruve, 2003) and HSV (Wakimoto et al., 2003).

Non-viral non-living vectors are exemplified by cationic polymers (polyplexes), cationic lipids (liposomes, lipoplexes) and synthetic nanoparticles (nanoplexes). They are more versatile than viral vectors, and offer several distinct advantages because their molecular composition can be controlled, manufacturing and analysis of such vectors is fairly simple, they can accommodate a range of transgene sizes (Kreiss et al., 1999; de Jong et al., 2001) and they are less immunogenic (Whitmore et al., 1999, 2001; Dow et al., 1999; Ruiz et al., 2001). The efficiency of gene delivery with non-viral non-living vectors is significantly less, however, than with viral vectors. At least 106plasmid copies are needed to transfect a single cell, with approximately 102-104copies actually making it to the nucleus for transgene expression (Feigner and Ringold, 1989; James and Giorgio, 2000; Tachibana et al., 2002). This inefficiency is attributable to the inability of non-viral non-living vectors to overcome the numerous challenges encountered between a site of administration and localization in a target cell nucleus, including, (a) the physical and chemical stability of DNA and its delivery vehicle in the extracellular space, (b) cellular uptake by endocytosis, (c) escape from the endosomal compartments prior to trafficking to lysosomes and cytosolic transport, and (d) nuclear localization of the plasmid for transcription. In addition to these physical and chemical obstacles, biological barriers, such as immunogenic responses to the vector itself and immune stimulation by certain DNA sequences containing a central unmethylated CpG motif exist (Yew et al., 1999; Scheule, 2000; Ruiz et al., 2001).

As an alternate to non-living nucleic acid/drug delivery vehicles, live bacterial vectors have also been developed for tumor targeted therapy (Pawalek et al., 2003; Soghomonyan et al., 2005). These vectors do not carry a payload of nucleic acids or drugs, but preferentially accumulate in tumor cells, replicate intracellularly and kill the infected cells (Pawelek et al., 1997). This phenomenon is thought to be facilitated by a complex bacterial system for introducing bacterial proteins directly into mammalian cells, which can result in the induction of apoptosis (Chen et al., 1996; Monack et al., 1996; Zhou et al., 2000). Currently,Bifidobacterium(Yazawa et al., 2000; 2001; Li et al., 2003),Clostridium(Minton et al., 1995; Fox et al., 1996; Lemmon et al., 1997; Theys et al., 2001; Dang et al., 2001; Nuyts et al., 2002a; 2002b; Liu et al., 2002)Salmonella(Pawelek et al., 1997; Low et al., 1999; Platt et al., 2000; Luo et al., 2001; Rosenberg et al., 2002) andVibrio(Yu et al., 2004) are under investigation as tumor-selective live bacterial vectors.

Live attenuated bacteria have also been explored as vehicles for delivering nucleic acids (Paglia et al., 2000; Weiss and Chakraborty 2001; Yuhua et al., 2001), which may encode angiogenic inhibitors (Lee et al., 2005a; 2005b; Li et al., 2003), prodrug-converting enzymes (King et al., 2002) or cytokines (Yamada et al., 2000). Significant drawbacks of this approach include (a) live recombinant bacteria gradually lose plasmid DNA in vivo, mainly due to the absence of selection pressure and associated plasmid segregation, (b) bacteria carrying plasmid DNA tend to have a lower growth rate and appear to accumulate at lower levels and reside for a shorter period of time within tumors than bacteria without plasmids, (c) live Gram-negative bacterial vectors can cause severe endotoxin response in mammalian hosts, possibly due to in-vivo shedding of endotoxin (lipopolysaccharide; LPS), and evoke a Toll-like receptor response due to cellular invasion, (d) most of the tumor-targeting live bacteria accumulate and grow in the necrotic and relatively hypoxic foci within tumors, but not in well-oxygenated tumors at the rim of the growing nodules where tumor cells are normally most aggressive, (e) the risk associated with possible reversion to a virulent phenotype of these bacteria is a major concern (Dunham, 2002), and (f) the risk of infecting normal cells may lead to bacteremia and associated septic shock. The latter may particularly be a problem in immuno-compromised patients, such as late stage cancer patients.

Because problems continue to hamper the success of cancer therapeutics in particular, an urgent need exists for targeted delivery strategies that will either selectively deliver bioactive agents to tumor cells and target organs, or protect normal tissues from administered antineoplastic agents. Such strategies should improve the efficacy of treatment by increasing the therapeutic indexes of anticancer agents, while minimizing the risks of therapy-related toxicity.

The present invention provides a versatile delivery vehicle for improved drug, therapeutic nucleic acid and functional nucleic acid delivery strategies, especially but not exclusively in the context of cancer chemotherapy.

SUMMARY OF THE INVENTION

To address these and other needs, the present invention provides, in one aspect, a composition that comprises a plurality of intact killed bacterial cells and a pharmaceutically acceptable carrier. The killed bacterial cells contain a therapeutic nucleic acid, a drug or a functional nucleic acid. With respect to the latter, in one embodiment the functional nucleic acid is plasmid-free. In this regard, functional nucleic acids are packaged directly into killed bacterial cells by passing through the bacterial cell's intact membrane, without using plasmid-based expression constructs or the expression machinery of a host cell. Such plasmid-free functional nucleic acids are exemplified by single-, double-, or multi-stranded DNA or RNA. In one embodiment, killed bacterial cells contain plasmid-free functional nucleic acid that is regulatory RNA. In a preferred embodiment, the composition is essentially free of endotoxin.

The invention also provides bispecific ligands useful for targeting killed bacterial cells to mammalian host cells. The bispecific ligand may be polypeptide, carbohydrate or glycopeptide, and may comprise an antibody or antibody fragment. In preferred embodiments, the bispecific ligand has a first arm that carries specificity for a bacterial surface structure and a second arm that carries specificity for a mammalian cell surface structure. Further, the first arm and the second arm of the bispecific ligand may be monospecific or multivalent. A desirable bacterial surface structure for ligand binding is an O-polysaccharide component of a lipopolysaccharide (LPS). Desirable mammalian cell surface structures for ligand binding are receptors, preferably those capable of activating receptor-mediated endocytosis.

According to another aspect, the invention provides a delivery method that comprises bringing a plurality of killed bacterial cells into contact with mammalian cells that are phagocytosis- or endocytosis-competent, such that the killed bacterial cells are engulfed by the mammalian cells and release their payload intracellularly. The payload may comprise a therapeutic nucleic acid, a functional nucleic acid or a drug.

In one embodiment, a method of delivering a functional nucleic acid, comprises (a) providing a plurality of killed bacterial cells in a pharmaceutical carrier, each killed bacterial cell of the plurality encompassing (i) a functional nucleic acid or (ii) a plasmid comprised of a segment that encodes a functional nucleic acid and then (b) bringing said killed bacterial cells of the plurality into contact with target mammalian cells, such that said mammalian cells engulf said killed bacterial cell, whereby said functional nucleic acid is released into the cytoplasm of the target cell. In one aspect, the killed bacterial cells are plasmid-free, while in another the functional nucleic acid is regulatory RNA.

According to another aspect, the invention provides a targeted delivery method that comprises bringing bispecific ligands into contact with (i) intact killed bacterial cells that contain a desired payload and (ii) mammalian cells, preferably non-phagocytic mammalian cells. The bispecific ligands have specificity for both a surface component on the intact killed bacterial cells and a surface component on the mammalian cells, such as a receptor. As a result, the ligands cause the killed bacterial cell to bind to the mammalian cells, the killed bacterial cells are engulfed by the mammalian cells, and the payload contained in the killed bacterial cells is released into the cytoplasm of the mammalian cell. The payload may comprise a therapeutic nucleic acid, a functional nucleic acid or a drug.

In yet another aspect, the invention provides a method of overcoming drug resistance or apoptosis resistance and treating a malignancy in a subject by delivering a functional nucleic acid to a target cell. The method comprises bringing a killed bacterial cell that contains (i) a functional nucleic acid molecule or (ii) a plasmid comprised of a segment that encodes a functional nucleic acid molecule into contact with a target mammalian cell. The mammalian cell engulfs the killed bacterial cell, the functional nucleic acid is released into the cytoplasm, transported to the nucleus and expressed by the target cell.

In relation to this invention, the contact between killed bacterial cells and mammalian cells may be in vitro or in vivo.

The invention further provides methods for loading killed bacterial cells with a drug. One such method involves creating a concentration gradient of the drug between an extracellular medium containing the killed bacterial cells and the killed bacterial cell cytoplasm. The drug naturally moves down this concentration gradient, into the killed bacterial cell cytoplasm. Leakage of the drug from the bacterial cytoplasm is prevented due to the bacterial cells being metabolically inactive.

Another method of loading killed bacterial cells with a drug involves culturing a bacterial cell under conditions, such that the bacterial cell transcribes and translates a therapeutic nucleic acid encoding the drug, such that the drug is released into the cytoplasm of the bacterial cell, and then killing the bacterial cell to form one or more killed bacterial cells containing the drug in their cytoplasm.

In accordance with another aspect, the present invention contemplates a method for formulating a killed bacterial cell with a plasmid-free functional nucleic acid. The method comprises co-incubating a plurality of killed bacterial cells with a functional nucleic acid, such as regulatory RNA like siRNA, miRNA or shRNA, in a buffer. In some embodiments, the co-incubation may involve gentle shaking, while in others the co-incubation is static. In some aspects, the co-incubation lasts about half an hour, while in others it lasts about an hour. In one embodiment, the buffer comprises buffered saline, for example, a 1X phosphate buffer solution. In another embodiment, the co-incubation is conducted at a temperature of about 4° C. to about 37° C., about 20° C. to about 30° C., about 25° C., or about 37° C. The co-incubation can comprise about 107, 108, 109, 1010, 1011, 1012or 1013killed bacterial cells.

The present invention contemplates a use of intact killed bacterial cells and bispecific ligands in the preparation of a medicament, for use in a method of treating a disease or modifying a trait by administration of the medicament to a cell, tissue, or organ. In the medicament, the killed bacterial cells contain a therapeutic nucleic acid molecule, a drug or a functional nucleic acid molecule, and, optionally, bispecific ligands that are capable of binding to the killed bacterial cells and to target non-phagocytic mammalian cells. Such medicaments are useful to treat various conditions and diseases by increasing expression or function of a desired protein, or by inhibiting expression or function of a target protein. Illustrative of such conditions and diseases are a cancer and an acquired disease, such as AIDS, pneumonia emphysema, and tuberculosis. Alternatively, the treatment may affect a trait, such as fertility, or an immune response associated with an allergen or an infectious agent.

The present invention also provides a pharmaceutically acceptable method for purifying intact killed bacterial cells. The method combines (i) killing live bacterial cells with antibiotics, (ii) cross-flow filtration and/or dead-end filtration, to eliminate free endotoxin, cellular debris, free nucleic acids, bacterial membrane blebs, media contaminants, and (iii) antibody-based sequestration, to eliminate residual free endotoxin.

