Title:  Intracellular delivery vehicles

United States Patent:  6,287,556

Assignee:  The Regents of the University of California (Oakland, CA)

Filed:  December 21, 1999

The invention provides methods and compositions relating to intracellular delivering of agents to eukaryotic cells. The compositions include microbial delivery vehicles such as nonvirulent bacteria comprising a first gene encoding a nonsecreted foreign cytolysin operably linked to a heterologous promoter and a second gene encoding a different foreign agent. The foreign agent may be a nucleic acid or protein, and is frequently bioactive in and therapeutic to the target eukaryote. In addition, the invention provides eukaryotic cells comprising the subject nonvirulent bacteria and nonhuman eukaryotic host organisms comprising such cells.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS OF THE INVENTION

The following preferred embodiments and examples are offered by way of illustration and not by way of limitation.

The subject bacteria comprise a first gene encoding a nonsecreted foreign cytolysin operably linked to a heterologous promoter. A wide variety of foreign (i.e. not native to the microbial delivery vehicle) cytolysins may be used so long as the cytolysin is not significantly secreted by the microbe and facilitates cytosolic delivery of the foreign agent as determined by the assays described below. Exemplary cytolysins include phospholipases (see, e.g., Camilli, A., et al., J. Exp. Med. 173:751-754 (1991)), pore-forming toxins (e.g., an alpha-toxin), natural cytolysins of gram-positive bacteria, such as listeriolysin O (LLO, e.g. Mengaud, J., et al., Infect. Immun. 56:766-772 (1988) and Portnoy, et al., Infect. Immun. 60:2710-2717 (1992)), streptolysin O (SLO, e.g. Palmer M, et al., 1998, Biochemistry 37(8):2378-2383) and perfringolysin O (PFO, e.g. Rossjohn J, et al., Cell 89(5):685-692). Where the target cell is phagosomal, acid activated cytolysins may be advantageously used. For example, listeriolysin exhibits greater pore-forming ability at mildly acidic pH (the pH conditions within the phagosome), thereby facilitating delivery of the liposome contents to the cytoplasm (see, e.g., Portnoy, et al., Infect. Immun. 60:2710-2717 (1992)). Furthermore, natural cytolysins are readily modified to generate mutants which are screened in the assays described below or otherwise known in the art (e.g. Awad MM, et al., Microb Pathog. 1997, 22(5): 275-284) desired activity modifications. In general, the screening assays measure the ability of a candidate cytolysin to confer on a bacterium the ability to render a target cell vacuole permeable to a label (e.g., a fluorescent or radioactive label) that is contained in the vacuole. In a particular example, the invention provides mutations in natural cytolysin wherein highly conserved cysteine residues (e.g., cysteine 460 in PFO, cysteine 486 in LLO) are replaced by conservative amino acid substitutions which are not subject to reduction in order to prepare oxidation/reduction-insensitive cytolysin mutants which exhibit improved lytic activity. Alternatively, mutant cytolysins are selected from naturally occurring mutants by, for example, identifying bacteria which contain cytolysins that are capable of lysing cells over a narrow pH range, preferably the pH range which occurs in phagosomes (pH 5.0-6.0), or under other conditions (e.g., ionic strength) which occur in the targeted phagosomes. Nonsecreted cytolysins may be provided by various mechanisms, e.g. absence of a functional signal sequence, a secretion incompetent microbe, such as microbes having genetic lesions (e.g. a functional signal sequence mutation), or poisoned microbes, etc.

The bacteria also comprise a second gene encoding a foreign agent different from the cytolysin, and the subject methods may be used to deliver a wide variety of such foreign agents for a variety of applications, including diagnosis, therapy including prophylactics such as immunizations (see, e.g. HIV vaccine, Table 1) and treatments such as gene therapy (especially of single gene disorders amenable to localized treatment, see Table 1, below), biosynthesis, etc.; essentially any agent that the microbial host can be engineered to produce. In a particular embodiment, the agent is largely retained by the microbe until lysis within the target cell vacuole. Note that the first and second genes may be the same, i.e. the same nucleic acid encodes both the cytolysin and the foreign agent. For example, in a particular embodiment, the foreign agent is expressed in frame with the cytolysin as a fusion protein. In other embodiments, the microbes are engineered to deliver libraries of agents for screening, e.g. Tenson T, et al., J Biol Chem 1997 Jul 11;272(28):17425-17430.

A wide variety of nucleic acid-based agents may be delivered, including expression vectors, probes, primers, antisense nucleic acids, knockoutin vectors, ribozymes, etc. For example, the subject bacteria are used to deliver nucleic acids which provide templates for transcription or translation or provide modulators of transcription and/or translation by hybridizing to selected endogenous templates, see, e.g. U.S. Pat. No. 5,399,346 for a non-limiting list of genes that can be administered using gene therapy and diseases that can be treated by gene therapy. For example, polynucleotide agents may provide a coding region operably linked to a transcriptional regulatory region finctional in a target mammalian cell, e.g. a human cytomegalovirus (CMV) promoter. In particular, the polynucleotide may encode a transcription factor, whereby expression of the transcription factor in the target cell provides activation or de-activation of targeted gene expression in the target cell. In another example, RNA virus infected cells are targeted by microbes delivering viral RNA-specific ribozymes, e.g. HIV-infected T-cells, leukemia virus infected leukocytes, hepatitis C infected liver cells. In yet another embodiment, labeled probes are delivered which effect in situ hybridization-based diagnostics.

A wide variety of polypeptide-based agents may also be delivered, including antibiotics, insecticides, fungicides, anti-viral agents, anti-protozoan agents, enzymes, anti-cancer agents (e.g. cyclin dependent kinase (CDK) inhibitors such as P16, P21 or P27), antibodies, anti-inflammatory peptides, transcription factors, antigenic peptides, etc. Exemplary therapeutically active polypeptides which can be delivered by the subject invention are described in Nature Biotech 16(2), entire issue, etc. In a particular embodiment, the invention provides for the delivery to antigen-presenting cells of antigenic polypeptides which are presented in association with MHC proteins. In another particular embodiment, both nucleic acids and proteins are delivered together contemporaneously, in the same administration or in the same microbe. In some such applications, the nucleic acids and proteins can act in concert, e.g. an integrating vector and an integrase, and RNA and a reverse transcriptase, a transposon and a transposase, etc.

The subject methods may also be used to deliver a wide variety of other foreign agents that are synthesized by the host microbe. For example, microbes may be selected for, or engineered to contain, biosynthetic machinery to produce any microbiologically producible agent compatible with the subject methods (e.g. sufficiently microbe impermeant to provide effective delivery to the target cell). Preferred such agents are those that are contraindicated for convenient direct (e.g. oral) administration, because of, for example, gut inactivation, toxicity, intolerance, impermeability, etc. In fact, even agents providing significant toxicity to the microbial host find use so long as an effective amount of the agent may be loaded (by synthesis) or maintained in the microbe (see, e.g. LLO toxicity, below).

A wide variety of nonvirulent, non-pathogenic bacteria may be used; preferred microbes are relatively well characterized strains, particularly laboratory strains of E. coli, such as MC4100, MC1061, DH5.alpha., etc. Other bacteria that can be engineered for the invention include well-characterized, nonvirulent, non-pathogenic strains of Listeria monocytogenes, Shigella flexneri, mycobacterium, Salmonella, Bacillus subtilis, etc. In a particular embodiment, the bacteria are attenuated to be nonreplicative, nonintegrative into the host cell genome, and/or non-motile inter- or intra-cellularly. A wide variety of suitable means for microbial attenuation are known in the art. In another particular embodiment, the bacteria are dead or non-viable prior to endocytosis by the target cell or administration to the target organism, obviating any microbial growth or metabolism in the target cell. A wide variety of suitable means for killing or rendering the bacteria nonviable are known in the art, including fixation with organic solvents such as methanol, UV irradiation, heat, freeze-drying, etc. Preferred methods preserve the ability of the microbial membrane and/or wall to retain the cytolysin and the foreign agent. In this embodiment, the first and second genes are sufficiently expressed to load the microbe with an effective amount of the cytolysin and foreign agent prior to microbial cell death. Generally the bacteria contain (i.e. are loaded by expression within the bacteria with) with from about ten to one thousand, preferably from about one hundred to one thousand cytolysin molecules per bacterium.

The microbes of the invention can be used to deliver the foreign agent to virtually any target cell capable of endocytosis of the subject microbe, including phagocytic, non-phagocytic, pathogenic or diseased cells. Exemplary target animal cells include epithelial cells, endothelial cells, muscle cells, liver cells, pancreatic cells, neural cells, fibroblasts, tumor cells, leukocytes such as macrophages, neutrophils, B-cells, T-cells, monocytes, etc., etc. The subject methods generally require microbial uptake by the target cell and subsequent lysis within the target cell vacuole (including phagosomes and endosomes). While phagocytic target cells generally provide for microbial uptake and lysis, for many cell types, it is necessary to provide the bacteria with an invasin to facilitate or mediate uptake by the target cell and an autolysin to facilitate or mediate autolysis of the bacteria within the target cell vacuole. A wide variety of suitable invasins and autolysins are known in the art. For example, both Sizemore et al. (Science, 1995, 270:299-302) and Courvalin et al. (C.R. Acad. Sci. Paris, 1995, 318:1207-12) teach expression of an invasin to effect endocytosis of the bacterium by a target cell and suitable microbial autolysins are described by Cao et al., Infect Immun 1998, 66(6): 2984-2986; Margot et al., J Bacteriol 1998, 180(3):749-752; Buist et al., App Environ Microbiol, 1997, 63(7):2722-2728; Yamanaka et al., FEMS Microbiol Lett,1997, 150(2): 269-275; Romero et al., FEMS Microbiol Lett, 1993, 108(1):87-92; Betzner and Keck, Mol Gen Genet, 1989, 219(3): 489491; Lubitz et al., J. Bacteriol,1984, 159(1):385-387; and Tomasz et al., J. Bacteriol, 1988, 170(12): 5931-5934. Providing the advantage of delayed lysis are temperature-sensitive autolysins, time-sensitive autolysins (see, e.g. Chang et al., 1995, J Bact 177, 3283-3294; Raab et al., 1985, J Mol Biol 19, 95-105; Gerds et al., 1995, Mol Microbiol 17, 205-210) and addiction (poison/antidote) autolysins, (see e.g. Magnuson R, et al., 1996,J Biol Chem. 271(31), 18705-18710; Smith A S, et al., 1997, Mol Microbiol. 26(5), 961-970).

Administration of the microbe to target cells may be in vitro or in vivo according to conventional methodologies. In either case, the methods generally involve growing the microbes, inducing the expression of the first and second genes, and contacting the target cells with an effective amount of bacteria sufficient to effect the desired activity of the foreign agent in the target cell. Immunofluorescense may be used to image and track the contents of the bacteria upon administration to the cells in vivo or in vitro.

In vitro or ex vivo administration generally involves contacting the target cell with an effective amount of the microbes of the invention. Exemplary in vitro administrations are described and/or cited by reference below. In vitro applications include protein delivery (e.g. for functional determiinations, toxin delivery to targeted cells in culture, half-life, degradation and localization determinations), nucleic acid delivery (e.g. DNA to transfected cell lines, genomic libraries to screen and identify specific antigens, i.e. expression cloning, etc.)

In vivo administration generally involves administering a pharmaceutical composition containing a therapeutically effective amount of the microbes of the invention. Generally, the therapeutically effective amount is between about 1 .mu.g and 100 mg/kg, preferably between about 1 .mu.g and 1 mg/kg. The microbes are formulated into a pharmaceutical composition by combination with an appropriate pharmaceutically acceptable excipient in accordance with routine procedures known to one of ordinary skill in the art. The microbes may be used alone or in appropriate association, as well as in combination with other pharmaceutically active compounds. The microbes may be formulated into preparations in solid, semisolid, or liquid form such as tablets, capsules, powders, granules, ointments, solutions, suppositories, and injections, in usual ways for topical, nasal, oral, parenteral, or surgical administration. Administration in vivo can be oral, mucosal, nasal, bronchial, parenteral, subcutaneous, intravenous, intra-arterial, intramuscular, intra-organ, intra-tumoral, surgical or in general by any means typical of a gene therapy administration. Administration will be selected as is appropriate for the targeted host cells. Target cells may also be removed from the subject, treated ex vivo, and the cells then replaced into the subject. Exemplary methods for in vivo administration are described in Shen et al., Proc Natl Acad Sci USA 1995, 92(9):3987-3991; Jensen et al, Immunol Rev 1997, 158: 147-157; Szalay et al., Proc Natl Acad Sci USA 1995, 92(26):12389-12392; Belyi et al, FEMS Immunol Med Microbiol 1996, 13(3): 211-213; Frankel et al., J. Immunol 1995, 155(10):47754782; Goossens et al., Int Immunol 1995, 7(5):797-805; Schafer et al., J. Immunol 1992, 149(1):53-59; and Linde et al., Vaccine 1991, 9(2):101-105.

The foregoing methods and compositions are demonstrated to be effective in a wide variety of exemplary applications. In one application, a K12 strain of E. coli is engineered with a signal sequence deficient LLO gene operably linked to the constitutive tet promoter for expressing the cytolysin in the bacterium and a second gene encoding a truncated BRCA1 cancer antigen, under regulatory control of a trc or tac promoter. The cytolysin and cancer antigen are expressed to maximum levels, the bacteria are then fixed with methanol, and the killed bacteria loaded with the cytolysin and cancer antigen are then injected into solid breast tumors in three weekly injections. At four weeks, a cancer antigen-specific cytotoxic T-cell response (CTL response) and tumor size reduction is detected.

Claim 1 of 42 Claims

What is claimed is:

1. A vaccine comprising a nonvirulent bacterium comprising a first gene encoding a nonsecreted foreign functional cytolysin operably linked to a heterologous promoter which expresses the cytolysin in the bacterium, and a second gene encoding a different foreign antigenic agent.

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